rheumatoid arthritis and the mucosal origins hypothesis...

Seropositive rheumatoid arthritis (RA), which occurs in individuals who have both genetic and familial risk factors for this disease1–4, begins with a prolonged period (several years) of serologically detectable RA- related autoimmunity associated with the presence of systemic inflammatory biomarkers but the absence of clinical or histologically defined inflammatory arthritis5–9. Serum autoantibodies present at this time include multiple isotypes of both anticitrullinated protein antibodies (ACPAs) and Fc domain- recognizing rheu-matoid factors (RFs) in patterns highly predictive of the future onset of joint disease5,6,9,10. This period can be defined retrospectively as the ‘preclinical’ period of RA in patients who eventually develop the disease11, and as an ‘at- risk’ period for individuals who are sero-positive and/or have familial or clinical risk factors but have not yet developed arthritis when studied in a cross- sectional or longitudinal manner. This preclinical con-dition is followed by the development of early clinically detectable inflammatory arthritis and/or classified RA, the latter of which proceeds as a chronic arthritis and

disease process that can also involve the lung and other target organs.

Data from epidemiological and translational studies suggest that mucosal environmental exposures and/or dysbiosis have causal roles during the at- risk period in the development of RA2,12–16. However, despite support for a ‘mucosal origins hypothesis’— that is, that RA development begins at mucosal sites and then transitions to involve the synovial joints — these studies are limited in that they do not enable identification of the specific mucosal processes present at the initial time point or points during which such exposures would have influ-enced the concurrent autoimmune phenotypes as well as subsequent disease evolution and arthritis develop-ment. In this regard, it is often assumed that a key factor in early RA development is the loss of self- tolerance to citrullinated self- antigens. However, studies of at- risk individuals as well as healthy individuals strongly sug-gest that mucosal, and probably systemic, immuno-globulin A (IgA) isotype ACPA and RF generation is a common finding and is associated with local mucosal

DysbiosisA general term used to indicate a change in the normal bacterial ecology, usually with a potential for association with a disease state.

Rheumatoid arthritis and the mucosal origins hypothesis: protection turns to destructionV. Michael Holers1*, M. Kristen Demoruelle1, Kristine A. Kuhn1, Jane H. Buckner2, William H. Robinson3, Yuko Okamoto1, Jill M. Norris4 and Kevin D. Deane1

Abstract | Individuals at high risk of developing seropositive rheumatoid arthritis (RA) can be identified for translational research and disease prevention studies through the presence of highly informative and predictive patterns of RA-related autoantibodies, especially anti-citrullinated protein antibodies (ACPAs), in the serum. In serologically positive individuals without arthritis, designated ACPA positive at risk , the presence of mucosal inflammatory processes associated with the presence of local ACPA production has been demonstrated. In other at-risk populations, local RA-related autoantibody production is present even in the absence of serum autoantibodies. Additionally , a proportion of at-risk individuals exhibit local mucosal ACPA production in the lung, as well as radiographic small-airway disease, sputum hypercellularity and increased neutrophil extracellular trap formation. Other mucosal sites in at-risk individuals also exhibit autoantibody production, inflammation and/or evidence of dysbiosis. As the proportion of individuals who exhibit such localized inflammation-associated ACPA production is substantially higher than the likelihood of an individual developing future RA , this finding raises the hypothesis that mucosal ACPAs have biologically relevant protective roles. Identifying the mechanisms that drive both the generation and loss of externally focused mucosal ACPA production and promote systemic autoantibody expression and ultimately arthritis development should provide insights into new therapeutic approaches to prevent RA.

1Division of Rheumatology, University of Colorado–Denver, Aurora, CO, USA.2Benaroya Research Institute, Seattle, WA, USA.3Division of Immunology and Rheumatology, Stanford University, Stanford, CA, USA.4Department of Epidemiology, Colorado School of Public Health, Aurora, CO, USA.

*e- mail: [email protected]

https://doi.org/10.1038/ s41584-018-0070-0

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mailto:[email protected]

mailto:[email protected]

https://doi.org/10.1038/s41584-018-0070-0

https://doi.org/10.1038/s41584-018-0070-0

inflammation17,18. These findings suggest that the most important early event in the preclinical development of RA is not loss of tolerance to self-antigens but instead loss of the mucosal barrier function and systemic spread of an IgG ACPA response. With those issues in mind and to address the mucosal origins hypothesis in the context of findings in individuals at risk of developing RA in the future, this Review describes the available evidence linking the presence of RA- related autoimmunity in specific at- risk populations with the concurrent pres-ence of local mucosal inflammation and autoantibody production, mucosal dysbiosis and/or systemic immune dysregulation consistent with mucosal inflammation.

Development of seropositive RARA is an autoimmune disease that evolves over decades and manifests as both locally joint- destructive and sys-temic illness4,19. The disease is characterized by wide-spread inflammation as well as local joint- based signs and symptoms ranging from arthralgias, a term that has been specifically defined for research purposes to encompass seven factors (symptom duration <1 year, symptoms of metacarpophalangeal (MCP) joints, morn-ing stiffness duration ≥ 60 minutes, most severe symp-toms in early morning, first- degree relative (FDR) with RA, difficulty with making a fist and a positive squeeze test of MCP joints)20, to swelling, pain and inflamma-tory synovitis that are clinically detectable through techniques such as physical examination, imaging and synovial biopsy (reviewed elsewhere19,21). Osteopenia and local erosions commonly occur in RA, probably through inflammatory and remodelling processes that alter relative rates of bone formation and degradation by osteoblasts and osteoclasts, respectively22.

RA exhibits a prevalence of 0.5–0.8% in the general population, and an ~3–5-fold increase in FDRs (reviewed elsewhere23), which seems to be owing to both genetic and environmental influences24–26. Patients receive a diagnosis of RA according to the 1987 revised ACR or 2010 ACR/EULAR classification criteria27,28. In some patients, RA first appears clinically as an undifferentiated inflammatory arthritis29,30.

The term ‘RA’ encompasses two major subsets of disease, seropositive and seronegative, that exhibit overlapping but individually distinct pathogenic mechanisms and clinical courses (reviewed else-where21,26,31). Seropositive individuals exhibit substan-tial overlap of expression of two primary autoantibody systems: ACPAs, which are antibodies towards a post- translational modification (citrullination) that is found in an antigenic form on fibrinogen, vimentin, type II collagen (CII), histones and enolase32,33, and antibodies to the Fc domains of self- immunoglobulin molecules, designated RFs34. Additional anti- modified protein antibodies (AMPAs) (reviewed elsewhere35), such as anti- carbamylated protein antibodies36 and antibodies to malondialdehyde–acetaldehyde- modified epitopes37, have been identified in patients with RA. However, the specificity of these AMPAs for RA, and at what point in the transition from preclinical to clinically apparent disease AMPAs develop, are less well defined38,39.

RA can be driven by a variety of genetic and environ-mental factors that lead to a heterogeneous disease process with variable autoantibody positivity. In terms of genetic factors, many genes are associated with RA, including high- risk HLA- DR alleles containing the shared epitope (SE)40 and PTPN22 (ref.41), as well as many other genes with low relative risk variants42. Systemic targeting of citrullinated proteins is probably facilitated by the preferential interactions of peptides bearing citrulline with SE- containing HLA- DR mol-ecules43–45. Heterogeneity in RA disease phenotypes is also likely to be influenced by environmental factors25,46. In addition, epigenetic changes associated with RA affect lymphocytes, fibroblast- like synoviocytes and other cell populations3,4,47,48.

In patients who eventually develop seropositive RA, there is a prolonged asymptomatic preclinical period without evidence of joint involvement that is character-ized by the presence of serologically detectable ACPAs, measured through clinical tests for anti- CCP (cyclic citrullinated peptide) antibodies, and RFs; this evolv-ing process typically occurs 3–5 years prior to the onset of clinically apparent and classified disease (reviewed elsewhere49) (fig. 1). Shortly before the onset of clini-cally apparent arthritis, systemic levels of cytokines and chemokines increase in a heterogeneous manner, with regard to both the number and the pattern of indi-vidual mediators that are increased in an individual patient10. An increase in epitope spreading also occurs, encompassing a wide variety of citrullinated targets9,50; avidity maturation of autoantibodies is also present51.

In addition, effector functions of ACPAs evolve dur-ing the development of RA51,52. For example, altered ACPA Fc domain galactosylation and fucosylation53, as well as excessive Fab (antigen- binding fragment) glyco-sylation54, are common findings in patients with RA and in patients who are transitioning from the arthralgia state to inflammatory arthritis and classified RA; these processes probably have important roles in autoanti-body selection and/or pathogenicity. Notably, studies have suggested that IL-23 and T helper 17 (TH17) cells regulate the β- galactoside α2,6-sialyltransferase 1 acti-vity in B cells that controls the extent of glycosylation

Key points

•Patients who eventually develop seropositive rheumatoid arthritis (RA) pass through a periodofRA-relatedautoantibodypositivitythatisassociatedwithincreasedlevelsofcytokinesandchemokines.

•Variousmucosalprocessescaninfluencethedevelopmentofsystemicimmunityand autoimmunity.

•IndividualswhoareathighriskofthefuturedevelopmentofRAdemonstrateevidenceofchronicsystemicandmucosalinflammation.

•Ratherthanreflectingalossofcitrullinatedantigentolerance,immunoglobulinA(IgA)anti-citrullinatedproteinantibodies(ACPAs)arenormallyproducedlocally;a systemicIgGresponselikelyresultsfromlossofexternallyfocusedcompartmentalization.

•Ongoingstudiesarelinkingthedevelopmentofmucosaldysbiosis,inflammationand autoantibodyproductiontothenextstagesofdevelopmentofsystemicautoimmunity.

•Novelautoimmune-promotingprocessesarelikelytobeidentifiedthatfunctionprimarily,orexclusively,inthepreclinicalperiodofRAandcouldbethetargetsfor newpreventionstrategies.

Shared epitopeA set of HLA antigens that are preferentially found in patients with rA and that are characterized by conserved amino acids within the peptide- binding groove.

Epitope spreadingA process in which the epitopes (target antigen shapes or sequences recognized by B and T cell receptors) that are distinct from and non- cross-reactive with the initial target epitope become major targets of an ongoing immune response.

Avidity maturationA process in which the accumulated strength of multiple affinities of individual non- covalent binding interactions between antigens and antibodies increases over time.

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of immunoglobulin molecules and their pathogenic potential and that changes in these cytokines drive the disease- enhancing changes in secreted autoan-tibodies during the clinical transition to arthritis55. Importantly, autoantibodies that demonstrate effector functions related to osteoclastogenesis56 and joint pain secondary to chemokine release57 have also been iden-tified in patients with RA, although the point during disease development at which these autoantibodies appear is less certain. Perhaps most intriguingly rela-tive to mucosal studies, IgA ACPA and RF isotypes long precede the development of classified RA58, although the relative timing of the appearance of each serum RA- related autoantibody isotype is not yet clearly defined. In addition, in patients with RA, RF contains a high proportion of secretory component, which is nec-essary to transport IgA across the mucosal surface59, although the time point of appearance of this feature is again uncertain.

Identifying early immune alterationsStudies in the preclinical time period have primar-ily relied on two experimental approaches. The first approach has been to utilize annotated sera that have been collected previously for other purposes — for example, from blood banks and population studies5,6,8,60

as well as the US Department of Defense Serum Repository7. Despite the inherent limitations of patients not being studied and classified with regard to arthritis status at the same time as the sample acquisition, many insights have been generated from these approaches. However, another approach is to perform studies of individuals with an ACPA- positive at- risk status, which is a method that has increasingly helped to define how the earliest immunological abnormalities develop and what might be the early ‘drivers’ of disease. These indi-viduals are recruited through many different means, including recruitment of FDRs, who are themselves at higher risk of developing disease61,62, outreach at com-munity health fairs or other events to identify serum ACPA- positive at- risk individuals63 and the creation of referral networks to specifically identify patients in primary care with arthralgias suggestive of preclinical RA who are subsequently followed for development of inflammatory arthritis and classified RA64. Individuals who are at risk of future RA can be defined on the basis of increased risk as an FDR, which enables one to study phenotypes and epidemiological factors in real time prior to the development of serological positivity for ACPAs or RFs. Thus, within each study it is important to define the basis for increased risk in the population or populations being studied and the control popula-tion or populations against which findings are com-pared. Finally, ongoing population recruitment efforts include screening of biobanks and electronic health records, social media outreach and the increasing num-ber of referrals to rheumato logists of patients without inflammatory arthritis or arthralgias who are found to be ACPA positive.

This heterogeneity in sources of samples might lead to some differences in the findings from retrospective studies. Nevertheless, a number of important observa-tions have been made using these approaches (Box 1). Key findings in serum samples from at- risk populations include the presence of increased levels of chemokines, cytokines and inflammatory biomarkers62,65,66, altered T cell phenotypes67,68 and increased numbers of autoan-tibodies61,65,69–71. Importantly, many of these biomarker patterns mirror those found in the serum from retro-spectively studied populations and strongly support the idea that these individuals are indeed in a preclinical period of RA development.

Stage- specific environmental factorsThe potential roles of environmental exposures in the development of RA have been reviewed extensively23,25. With regard to the at- risk population, however, very few long- term prospective studies have been performed to understand how stage- specific exposures influence the initial preclinical development of RA- related auto-immunity and subsequent clinical and/or immune transitions. Nevertheless, a number of informative cross- sectional and short- term longitudinal studies have been performed in such individuals (TABLe 1).

A well- accepted association in RA is the relationship between heavy smoking and the risk of development of seropositive RA, with the predominant risk being in patients with the SE26. The exact stage- specific timing of

Fig. 1 | Factors relevant to preclinical RA pathogenesis. Various factors contribute to the preclinical stages of development of rheumatoid arthritis (RA). Shown in the middle of this figure is the EUL AR recommended terminology for defining the various disease stages in the natural history of RA and those stages corresponding to patients in the preclinical stages (when defined retrospectively) or ‘at risk’ (when defined cross- sectionally or longitudinally). Examples of the epidemiological and genetic factors, and features of systemic immune dysregulation that are under investigation are listed in boxes above; the study of these various features in the preclinical period could help to understand the causal drivers of preclinical disease. Past studies of these populations have included retrospective analysis of serum samples and biomarkers in patients in the preclinical stages of disease. ACPAs, anticitrullinated protein antibodies; CTL A4, cytotoxic T lymphocyte protein 4; HPA , hypothalamic–pituitary–adrenal; PTPN22, protein tyrosine phosphatase non- receptor type 22; PUFAs, polyunsaturated fatty acids; RF, rheumatoid factor ; STAT4, signal transducer and activator of transcription 4; TRAF1–C5, TNF receptor- associated factor 1–complement component 5.


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smoking effects is not well understood, but several stud-ies have provided insights. For example, initial studies of at- risk individuals in a well- studied cohort evaluated in real time revealed that heavy smoking is associated with asymptomatic RF positivity72. In a Swedish twin study, heavy smoking imparted an increased risk of being ACPA positive without RA, and this increased risk had no major relationship to the SE in the preclin-ical period73. In addition, in a large ( > 40,000 individ-uals) population study in the Netherlands, a positive ACPA status was associated with smoking as well as with older age and joint symptoms74. Further support for a role of smoking exposure in the future develop-ment of RA was provided by a Swedish population study in which smoking was associated with an increased risk of development of both seropositive and seronegative RA75. Similarly, in a Japanese non- RA cohort, a history of smoking was associated with a positive RA- related autoantibody status, but interestingly only when both ACPAs and RFs were present76. In a further analysis of the same population, ACPAs and RFs were shown to associate with each other; smoking was associated with a higher level of ACPAs, but the presence of the SE was not associated with an ACPA- positive status, and no interaction was found between the SE, smoking and autoantibody positivity77. Thus, smoking seems to have an important role in the initial development of ACPAs, but does not have a clear relationship to SE status, which itself seems to have a more important role in the transition to classified RA.

With regard to other factors, a history of birth control pill use is inversely associated with RF positivity72. These results are consistent with findings from patients with classified RA23,25 and suggest that this exposure has a key early role in preclinical RA.

Conversely, particulate air pollution is not associated with RA- related autoimmunity in at- risk individuals78, in contrast to the positive association seen in patients

with classified RA79, suggesting that this exposure has a later role in the development of RA, perhaps pro-moting the transition from RA- related autoantibodies alone to the initial synovitis. Additional results from the Nurses Health Studies have suggested that an increased BMI in conjunction with an expanded ACPA reper-toire is associated with increased risk of developing RA and accelerated transition to arthritis80. Studies from the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) have also suggested that exposure to silica increases the risk of future development of RA81. Associations between RA development and other occu-pational exposures, hormonal factors, dietary intake or alcohol use have previously varied, and the roles of these exposures in the preclinical stages of diseases are less well understood (reviewed elsewhere25).

An intriguing finding is the inverse relationship between the presence of RA- related autoantibodies in at- risk populations and the intake of omega-3 fatty acids as well as the presence of lipid biomarkers that measure levels of these key anti- inflammatory mole-cules. Notably, both established and emerging evidence demonstrates that polyunsaturated fatty acids (PUFAs) derived from the omega-3 fatty acid biosynthesis pathway might have an important role in RA treat-ment and prevention82,83. Omega-3 PUFA supplemen-tation in patients with classified RA has been shown to reduce signs and symptoms of inflammation and lead to decreased use of other therapeutic agents82,83. Population- based approaches have demonstrated that increased intake of fish rich in omega-3 PUFAs and direct supplementation with PUFAs are associated with a reduced risk of developing RA84. Increased omega-3 intake as well as increased levels of the red blood cell (RBC) omega-3 fatty acid pathway products eicosap-entaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with a decreased risk of RA- related autoantibody positivity in individuals with a genetic risk of developing RA85. Specifically, the likelihood of anti- CCP2 positivity in patients was inversely associ-ated with total percentage of omega-3 PUFAs in RBCs (OR = 0.47; 95% CI 0.24–0.92, for a 1 s.d. increase); anti- CCP2-positive individuals were less likely than CCP2-negative demographically matched controls to report omega-3 supplement use (OR = 0.14; 95% CI 0.03–0.68). Intriguingly, the effect was strongest in individuals with the SE86. In further observational studies of ACPA- positive individuals without synovitis at baseline, lower levels of RBC omega-3 PUFAs were associated with an increased risk of progression to inflammatory arthritis87.

Mucosal origins hypothesisThe mucosal origins hypothesis suggests that the disease process in patients who eventually develop RA originates at one or more sites in the mucosa. The theory histor-ically derived from an early suggestion that RA was an infectious illness. Later suggestions included the pos-sibility that RA developed through cross-reactivity to bacterial, viral and/or mycobacterial antigens carried in organisms such as Proteus (reviewed elsewhere88). Furthermore, the finding that Porphyromonas gingivalis,

Box 1 | At-risk population findings supportive of the mucosal origins hypothesis

Serological•Increasedlevelsof,andexpandedepitoperecognitionby,IgAandIgGACPAs

•Increasedlevelsofcytokinesandchemokines

•IncreasedC-reactiveproteinlevels

•IncreasedlevelsofantibodiestoPorphyromonas gingivalis

Peripheral blood cell phenotypes•IncreasedproportionofcirculatingIgA-encodingplasmablasts

•IncreasedcitH3,stimulatedIL-17andIL-2levelsanddecreasedstimulatedATMandPFKFB3levels

Findings at mucosal sites•High-resolutionCTofthelungdemonstratessmall-airwaydiseases

•IncreasedlevelsofRA-relatedautoantibodiesinsputumofat-riskindividuals

•HypercellularityandincreasedNETremnantsinsputumofat-riskindividuals

•PeriodontalproductionofACPAsinpatientswithperiodontitis

•CervicovaginalACPAproduction

ACPA,anti-citrullinatedproteinantibody;ATM,ataxiatelangiectasiamutated;citH3,citrullinatedhistone;IgA,immunoglobulinA;IgG,immunoglobulinG;NET,neutrophilextracellulartrap;PFKFB3,6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase3.


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a major cause of periodontitis, produces an enzyme capable of citrullinating proteins suggested another means by which mucosal dysbiosis could drive or influ-ence disease development89. Nevertheless, the hypothesis remains unproven, although evidence from an increas-ing number of studies is consistent with a major role for the microbiome in RA development.

The microbiome and autoimmunityIncreasing evidence from both murine and human translational research studies points to the important role of the microbiome (present in the intestine and other mucosal sites as well as epithelial sites) in mod-ulating the immune, as well as autoimmune, response (reviewed elsewhere90). Food intake and other environ-mental exposures influence the development and main-tenance of microbial kingdoms, including bacterial, fungal, helminth and viral organisms. Mucosal expo-sure to specific microbial antigens and the metabolic products of biosynthesis and energy generation or uti-lization by microbes has a major effect on the immune system2,91.

Studying the microbiome in humans has many challenges90, including lack of direct access to most mucosal sites, difficulty in isolating and expanding many species in a manner that replicates the local mucosal ecosystem, an incomplete understanding of microbial genotype–phenotype relationships, sub-stantial changes introduced to the microbiome by food exposures such as dietary emulsifiers and noncaloric sweeteners, long- term imprinting by the fetal microbi-ome whose effects cannot be discerned years later, cul-tural differences around food and other exposures that affect microbial communities and the rapid changes that can ensue following alterations in the environment (perhaps best demonstrated by the effects of chang-ing food intake on the microbial community in the gastrointestinal tract90).

Crosstalk with the local immune system. The local mucosal immune system can interact with, respond to and constrain the microbiome in a number of ways (fig. 2), including induction and inhibition mechanisms that are strongly influenced by cells of the adaptive immune system as well as distinct types of mononu-clear phagocytes90. An important feature of the cross-talk process between the immune system and the micro biome is the mucosal barrier, which is created not only by interepi thelial cell tight junctions but also by the integrity of the mucus layer (reviewed elsewhere92). Additional components of the mucosal barrier include antimicrobial peptides (encompassing the microbi-cidal protein RegIIIγ and related families93), antibodies, macro phages, dendritic cells, regulatory T (Treg) cells and TH17 cells. Neutrophils are also important in mucosal defence from infection; these cells are attracted to the lumen through polarized secretion of IL-8 by epithelial cells as well as bioactive lipids, especially hepoxilin A3 (reviewed elsewhere94).

CD4+ and CD8+ T cells also continuously survey anti-gens in this region, the responses to which are regulated by cytokines, including IL-17, IL-10, IL-22, IL-23, IL-6 and TNF family members, in addition to other popula-tions such as Treg cells. In addition, specialized mucosal- associated invariant T (MAIT) cells, the innate immune functions and activation state of which are intimately tied to MHC class I- related gene protein (MR1) lig-ands that are upregulated during mucosal infection and inflammation, are present and produce IL-17 and TH1 cytokines95. Type 3 innate lymphoid cells contribute to the mucosal immune response through production of IL-22, a cytokine that stimulates production of RegIII lectins in response to dysbiosis96 and controls epithe-lial cell fucosylation to provide a carbohydrate source utilized by certain microbial subsets97. Also present are mucosal homing receptor98 α4β7 integrinhigh CD4+ T cells that are CC- chemokine receptor 5 (CCR5)high

Table 1 | Environmental factors affecting the natural history of RA

Environmental exposure Patients with RA Individuals at risk of developing RA and/or with preclinical RA

Effect? Predominant direction of risk

Effect? Predominant direction of risk

Smoking Yes ↑ Yes ↑Omega-3 fatty acid supplementation Yes ↓ Yes ↓Oral contraceptive use Yes ↓ Yes ↓Breastfeeding Yes ↓ Uncertain Uncertain

Earlier age at menarche Yes Variable Uncertain Uncertain

BMI Yes ↑ Yes ↑Decaffeinated coffee consumption Yes ↑ Uncertain Uncertain

Silica dust, solvents, mineral oil and textile dust

Yes ↑ Yes ↑

Physical workload Yes ↑ Uncertain Uncertain

Proximity to road and/or air pollution Yes ↑ No NA

Ultraviolet light Yes ↓ Uncertain Uncertain

Alcohol Yes ↓ Uncertain Uncertain

NA , not applicable; RA , rheumatoid arthritis.


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and CXC- chemokine receptor 4 (CXCR4)low and that undergo antigen- specific activation in the development of mucosal inflammation99.

With regard to antibodies, IgA is the major Ig iso-form produced at most mucosal surfaces, and although circulating blood levels of this immunoglobulin are low, IgA is the most highly produced immunoglobulin iso-form in the body100. Importantly, both T celldependent and T cell- independent means regulate the production of this mucosal isoform101. IgA exists as monomeric and polymeric forms of IgA1 and IgA2 sub- isotypes as well as polymeric IgA linked by the J chain. The IgA Fc recep-tor (FcαRI) is found on neutrophils, monocytes, eosino-phils and a subset of dendritic cells, and IgA functions to increase the respiratory burst in immune cells and inhibit bacterial adhesion100.

Several contrasting roles have been ascribed to the IgA isotype. Its primary role is to recognize and neu-tralize pathogens and exotoxins at the mucosal surface and in that way to regulate the microbiome, maintain homeostasis and protect intestinal and other barriers from pathogens. A key mechanism is to block infection and invasion of the mucosa through inhibiting trans-port across the epithelium and enabling clearance by

peristalsis in the gut102. IgA plasmablasts are primarily generated in Peyer’s patches and can circulate to secrete antibodies at other mucosal sites. Although IgA is viewed primarily in the context of mucosal immunity, when present systemically at high levels in immune complexes it can damage target tissues, including the joint103.

In another sense, secreted IgA antibodies can be considered to be anti- inflammatory as they are typically not strongly complement activating and are resistant themselves to proteolytic degradation104. Thus, the proinflammatory versus anti- inflammatory roles of IgA must be considered to be context- dependent and prob-ably coexisting. Beyond IgA, there are prominent roles for IgM antibodies as well as IgG antibodies and the bidi-rectional transport of both isotypes by the neonatal Fc receptor (FcRn) in the regulation of the microbiome and control of local pathogens, perhaps most prominently in the cervicovaginal fluid (reviewed elsewhere105). Notably, bacteria that penetrate the mucosal barrier are recog-nized by locally produced IgA isotype antibodies that are typically polyreactive106; if these bacteria avoid neu-tralization and elimination, they can more readily cross the epithelial layer and have antigens be presented by dendritic cells107.

α βγ

β

Fig. 2 | Local immune system and microbial factors that interact in the mucosal environment. Illustrated are components of the bidirectional regulation between the luminal microbiome and the cells within the mucosal interstitium, as well as within mucosa- associated lymphoid tissue (MALT) and lymph nodes (such as the Peyer’s patches and mesenteric lymph nodes of the gut), with several cell types contributing individually to produce protective factors. Specific examples include mucosal- associated invariant T (MAIT) cells that produce IL-17 , whereas type 3 innate lymphoid (ILC3) cells are responsible for producing IL-22. In addition, circulating CD4+ and CD8+ T cells survey antigens in this region, the responses to which are regulated by regulatory T (Treg) cells as well as cytokines, including IL-17 , IL-10, IL-22, IL-23, IL-6 and TNF family members. Antimicrobial peptides, such as microbicidal protein RegIIIγ, have important roles in limiting barrier breaks, as do immunoglobulin A (IgA) antibodies, macrophages, dendritic cells and T helper 17 (TH17) cells. Neutrophils play key roles in mucosal protection from infection, and α4β7 integrinhigh CD4+ T cells preferentially migrate to these sites. IELs, intraepithelial lymphocytes; TCR , T cell receptor ; TFH, T follicular helper cell; TGFβ, transforming growth factor- β.


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In addition to bacteria, viruses and other species can profoundly influence the mucosal immune response90. Examples include bacteriophages as well as DNA and RNA viruses108. Other organisms, including mycobacte-ria, fungi and helminths, can have key roles in regulating local and systemic immune responses90. To date, little information is known regarding the potential influence of the non- bacterial microbiome on the preclinical initiation or evolution of autoimmune diseases.

Another important feature of the mucosal immune response is the regulatory effect of the microbiome on local cells, as demonstrated by the generation of bac-terial metabolites that modulate differentiation and effector functions. For example, the microorganism- derived short- chain fatty acids butyrate, propionate and acetate all strongly affect the differentiation of mucosal Treg cells109.

Evidence of disease modulation from experimental models. Several murine models of arthritis have been studied to investigate what changes occur in the mucosal microbi-ome during the evolution of experimental arthritis and how these changes can affect the development of sys-temic autoimmunity and synovitis. Together, these stud-ies strongly support a role for microbiome influences on arthritis development through several mechanisms.

Important pathogenic roles for the ACPA response have been demonstrated in the CII- induced arthritis (CIA) model, along with the potential benefits (in terms of clinical and autoimmune outcomes) of modulating the ACPA response using tolerogenic approaches to decrease the level of pathogenic ACPAs110. In one study, researchers demonstrated the presence of dysbiosis in the CIA model; furthermore, transfer of faecal material from the arthritic mice, but not from the mice that did not develop arthritis in the same experiment, acceler-ated the development of arthritis in recipient germ- free mice111. Additionally, increases in levels of IL-17 and splenic CD8 and TH17 cells were seen with this proce-dure, in contrast to decreases in numbers of dendritic cells, B cells and Treg cells. Another study demonstrated that although modulation of the microbiota through the use of broad- spectrum antibiotics prior to the induction of CIA resulted in a modest reduction in disease sever-ity, antibiotic use during the late phase of CIA resulted in nearly complete suppression of arthritis112. The effect of antibiotic treatment during the late phase of CIA was in part owing to substantial impairment of the ability of anti- CII antibodies to activate complement ex vivo; by contrast, the reduction in disease severity in mice treated with antibiotics prior to CIA induction was asso-ciated with decreased levels of circulating inflammatory cytokines and anti- CII antibodies along with delayed IL-17 A and IL-22 production in the intestine.

Perhaps most notably, these data establish that the microbiome can exert profoundly different effects dur-ing the preclinical to clinical evolution of arthritis, a finding that is probably quite germane to the study of the potential roles of the human microbiome during ini-tiation of autoimmunity and through the multiple stages of the natural history of RA. In addition, the effects of mucosal immune cell interactions with the micro biome

might not be limited to local effects — subsets of lympho cytes in the mucosa are also known to recirculate and populate other sites and in that process influence the immune and autoimmune response in other tissues113. Indeed, it might be that ‘licensing’ of T cells and B cells to become pathogenic must occur through either local activation or passage of these cells through mucosal sites, especially the gut but perhaps also the lung, after which they can then elaborate their pathogenic potential in target tissues during autoimmune disease processes.

Other models of RA have demonstrated features that highlight the differing roles of the microbiome in human disease. For example, the K/BxN T cell recep-tor (TCR) transgenic mouse is a model in which CD4+ T cell- driven autoantibody recognition of the ubiqui-tous autoantigen glucose-6-phosphate isomerase (GPI) results in an immune- complex-driven arthritis114. In a germ- free environment, transgenic mice exhibit delayed onset and reduced severity of arthritis. However, seg-mented filamentous bacteria are capable of modulating the development of autoantibodies to GPI by activating and recruiting TH17 and T follicular helper cells within the intestine115,116. In addition, studies of DR4 and/or DQ8 humanized mice have demonstrated that treatment with a human commensal organism, Prevotella histicola, suppressed the development of CIA117. Mechanistically, this treatment altered CD103+ dendritic cells, myeloid suppressors and Treg cells in the intestinal mucosa, result-ing in suppression of antigen- specific TH17 responses as well as increases in the levels of antimicrobial peptides and tight junction proteins.

Inflammation in at- risk populationsSeveral systemic immune abnormalities suggest the presence of ongoing mucosal inflammation in individ-uals at risk of developing RA (fig. 3). One of these abnor-malities is the predominance of IgA isotype ACPAs and RFs in retrospectively studied preclinical samples58 as well as the presence of serum IgA ACPAs and RFs in at- risk populations18. More compelling, however, are data obtained from an analysis of plasmablasts from at- risk populations using new technologies118. During an immune response, peripheral blood plasmablasts arise from both naive and memory B cells that are acti-vated by inciting antigens, including those at mucosal sites. To evaluate these cells, plasmablasts derived from RA- related autoantibody- positive at- risk indi-viduals as well as matched controls and patients with early RA were sequenced and characterized utilizing a barcode- enabled single- cell antibody methodology119. Notably, although total plasmablast levels were similar in RA- related autoantibody- positive at- risk individuals and controls, a markedly increased frequency of IgA- positive as compared with IgG- positive plasmablasts was observed in the at- risk individuals compared with the other groups119.

To analyse these cells in further detail, the research-ers performed paired heavy- chain and light- chain rep-ertoire sequencing on plasmablasts from a subgroup of at- risk patients and generated phylogenetic trees of paired IgG and IgA sequences118–120. From these data, clonal families of antibodies were identified that use


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the same heavy- chain V and J gene segments, as well as the same light- chain V and J gene segments, con-sistent with their interrelatedness during development. Combined IgG–IgA phylogenetic trees were also gener-ated to demonstrate the relationship between sequences across these isotypes; remarkably, a substantial number of dual IgG–IgA cross- isotype clonal families of vary-ing sizes were observed119. Because of the relationship between IgA responses and the mucosa as well as the isotype changes being present in plasmablasts, which are continuously being generated, these data are con-sistent with an ongoing immune response that has a pronounced mucosal origin and/or contribution. Other studies of at- risk individuals have also demonstrated findings consistent with a state of chronic immune stimulation and inflammation121, which, in the absence of another tissue or organ site of origin, is a finding consistent with mucosal inflammation. For example, in one study, peripheral blood cells from at- risk individ-uals exhibited unique phenotypes compared with that of control populations, which included increased levels of citrullinated histone H3 (citH3) as well as increased stimulated production of IL-2 and TH17 cytokines but decreased TH2 cytokine expression121. With regard to the underlying immune mechanisms of these particu-lar findings, the abnormal features seem to be owing to the impaired induction of protein tyrosine phosphatase non- receptor type 22 (PTPN22) in T cells. PTPN22 is a phosphatase that also suppresses citrullination independently of its phosphatase activity and when expressed as an RA- associated variant in neutrophils promotes accelerated NeTosis122.

In the same study of at- risk individuals, attenuated phosphatase activity of PTPN22 in T cells was found to result in aberrant expression of IL-2, ataxia telangi-ectasia mutated (ATM) and 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase 3 (PFKFB3), which is associated with a hyperproliferative phenotype121. Furthermore, diminished non- phosphatase activity of PTPN22 led to hypercitrullination mediated by pepti-dylarginine deiminases and reduction of expression of TH2 cytokines. Notably, these T cell phenotypic changes identified in at- risk patients closely mirror results obtained in patients with longstanding RA — these studies showed that patients with active arthritis also exhibit pronounced changes in the metabolic activity of CD4+ T cells in which the cells do not metabolize glucose as efficiently as CD4+ T cells from healthy indi-viduals, resulting in less intracellular ATP and increased sensitivity to apoptosis123,124. Mechanistically, the defect was related to a lower induction of PFKFB3, with result-ant metabolic shifts towards a hypoglycolytic phenotype in which the T cells divert glucose towards the pentose phosphate pathway, produce more NADPH (a reduced form of NADP) and consume more intracellular reac-tive oxygen species (ROS)123. In aggregate, these changes leave the T cells energy deprived, apoptosis sensitive and autophagy resistant. Subsequent studies demon-strated that insufficient activation of ATM (owing to ROS depletion) drove T cells towards a hyperinflam-matory TH17 and TH1 lineage and enabled bypassing of the G2/M checkpoint and hyperproliferation124. Notably, correction of the ROS changes reversed these defects both in vitro and in an in vivo model124. Finally, one additional finding in support of the relevance of chronic immune activation at this early point in disease is the increased type I interferon score noted in individuals who subsequently transition from a late preclinical phase to classified RA64.

In summary, several lines of evidence from peripheral blood and serum studies strongly suggest the presence of ongoing inflammation, probably mucosal in origin, in at- risk individuals and an immune response that is asso-ciated with a state of heightened immune reactivity. As the at- risk individuals do not show evidence of arthritis, as assessed by MRI, physical examination and even syn-ovial biopsy, the presence of an asymptomatic chronic mucosal process associated with these peripheral blood changes is feasible.

Tissue- specific mucosal inflammationStudies in at- risk populations that demonstrate the concurrent presence of inflammation and RA- related autoantibody production at various mucosal sites sup-port the mucosal origins hypothesis in RA development. This section highlights available evidence regarding site- specific changes in early RA as well as what is known in the same organ in at- risk populations.

Intestinal biomarkers. Seminal studies in patients with new- onset RA demonstrated a relative expansion of faecal Prevotella copri in a substantial number of patients13. Although no microbiome- based replication studies have been published, findings in a separate study

Fig. 3 | Immune alterations in at- risk individuals. Findings from the study of at- risk individuals demonstrate the presence of immune alterations, both systemically and within local mucosal sites, within these individuals that are consistent with the presence of chronic mucosal inflammation. The figure shows findings derived from at- risk populations, but not from studies of patients with classified rheumatoid arthritis (RA), as it is unknown whether those factors have a role in preclinical RA. ACPA , anticitrullinated protein antibody ; ATM, ataxia telangiectasia mutated; HRCT, high- resolution CT; IgA , immunoglobulin A ; NET, neutrophil extracellular trap; PFKFB3, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase 3; P. gingivalis, Porphyromonas gingivalis; RF, rheumatoid factor.

NETosisA unique form of neutrophil extracellular trap (NeT)-associated cell death characterized by nuclear condensation and extrusion of chromatic and granular contents in a manner especially conducive to capturing pathogens.


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demonstrated an increased IgG and IgA humoral and TH1 cell immune response to this organism in a sub-set of patients with established RA125. Another study of patients with established RA identified alterations in multiple strains of faecal and oral bacteria as compared with non- RA control individuals15, changes that par-tially normalized following drug treatment. In a separate cohort study, an expansion of rare bacterial species was found in patients with established RA; these species were suggested to be relevant because of their ability to mod-ify experimental RA16. Notably, whether the changes at these sites in patients with active disease are causal or secondary to inflammation or drug therapy is unknown. In addition, it is unclear why the microbiome findings differ from one research group to another.

No comprehensive studies of the faecal or intestinal microbiome have been reported in populations at risk of RA. However, such studies are underway, and prelimi-nary results suggest the presence of local ACPA produc-tion and dysbiosis in these individuals (K.A.K., K.D.D. and V.M.H., unpublished observations).

Lung biomarkers. In addition to the risk factors described above, such as smoking, that have been largely identi-fied in epidemiological studies of patients with RA, and the demonstration that ACPAs and RFs are produced in inducible bronchus-associated lymphoid tissue (iBALT) in patients with chronic RA126, compelling studies of early RA have supported the presence of substantial pulmonary inflammation in conjunction with arthri-tis. Findings from these studies include the presence of histological structural abnormalities as well as inflam-mation and ACPA production of a similar nature to that in the joint in lung biopsy samples and bronchoalveolar lavage fluid127,128. Microbiome studies of the broncho-alveolar lavage of patients with early RA demonstrated that the microbiota was considerably less diverse and abundant than that in matched healthy controls and that the changes were quite similar to those in patients with sarcoidosis12. The dysbiosis could be attributed to the reduced presence of several genera and taxa. In addi-tion, several clades correlated with the levels of autoan-tibodies both systemically and in the bronchoalveolar lavage, and the genus Pseudonocardia was the only taxa over- represented.

Whether these same dysbiotic changes are seen in at- risk individuals has not yet been reported. However, two techniques have assessed the presence of lung abnormal-ities and local RA- related autoantibody production in at- risk populations. First, high- resolution CT (HRCT) findings demonstrated the presence of small- airway abnormalities (bronchial wall thickening, bronchiec-tasis, bronchiolitis and air trapping) in the absence of arthritis in two- thirds of at- risk individuals, findings that were consistent with airway inflammation; joints were assessed and negative for inflammatory arthritis by both clinical examination and (in a subset of individuals) by MRI129. Notably, the presence of these small- airway abnormalities was not dependent on a history of cur-rent or prior smoking, which suggests that other aeti-ological factors must be involved. In addition, patients were not aware of any pulmonary problems and did

not demonstrate evidence of lung abnormalities by pulmonary function testing.

The second technique utilized in at- risk popula-tions has been induced sputum collection, a validated methodology that assesses changes in biomarkers derived from the small airways17,18. This technique demonstrated the presence of IgA and IgG RA- related autoantibodies in the lung of at- risk individuals (which in this study comprised FDRs of patients with RA) even in the absence of serum ACPAs or RFs18. Analysis of the ratios of ACPA and RF levels to the total levels of mucosal IgA and IgG antibodies, and comparing those with the serum ratios of each, demonstrated that the RA-related autoantibodies were produced locally. Importantly, local IgA anti- CCP antibody generation was also closely associated with the concurrent pres-ence of elevated levels of inflammatory cells (neutro-phils and macrophages) and increased endogenous neutrophil extracellular trap (NET) formation in the sputum, as assessed by the detection and quantifica-tion of NET remnants17. In aggregate, ~25% of at- risk FDRs and individuals with serum autoantibody pos-itivity seemed to demonstrate the presence of local RA- related autoantibody production. Furthermore, a subset of at- risk individuals demonstrated systemic ACPAs but no pulmonary inflammation, sugges-ting that a separate mucosal site is involved in those individuals in the generation of systemic ACPAs.

In addition, sputum studies comparing individ-ual ACPA reactivities identified an increased number of sputum ACPA peptide reactivities in patients with RA compared with at- risk patients130. Although cross- sectional, this finding suggests that epitope spreading, which is well described in the blood during the preclini-cal period leading to RA onset, also occurs at mucosal sites. Furthermore, in serum anti- CCP-positive patients with lung disease but no joint disease, lymphoid aggre-gates consistent with iBALT were highly prevalent, fur-ther supporting RA- related immune reactivity in the lung in at- risk individuals131.

Periodontal biomarkers. Periodontal inflammation, chronic periodontitis, P. gingivalis infection and anti-body reactivity to citrullinated enolase have been asso-ciated with RA and RA severity (reviewed elsewhere132). Although the relationship between RA and periodontitis has been challenged through population- based studies, such as studies of the EIRA cohort133, substantial data support a close relationship between the ACPA response, the specific P. gingivalis peptidyl arginine deiminase enzyme and RA risk.

Other factors implicated in linking RA to periodonti-tis and ACPA production include an elevated abundance of Anaeroglobus geminatus in the periodontal fluid in patients with new- onset RA that correlated with the presence of ACPAs and RFs and an increase in Prevotella and Leptotrichia species in patients with new- onset RA irrespective of the presence of periodontitis14. In another study, the presence of Aggregatibacter actinomycet-emcomitans was associated with periodontitis in vivo, and this strain demonstrated the capacity for inducing hypercitrullination ex vivo in neutrophils134.


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In contrast to lung studies, understanding of the relationship between the RA at- risk state and periodon-titis is more limited, as studies of that population are just being completed. Prior studies have demonstrated an increased prevalence of antibodies to P. gingivalis in at- risk populations135. In addition, initial metagenome sequencing reports from the periodontium of at- risk patients have begun to emerge, although no conclu-sions have been made available136. A study of patients with periodontitis without RA demonstrated the pres-ence of citrullinated proteins as well as peptidyl arginine deiminase enzymes 2 and 4 in the periodontal fluid of a subset of patients; both of these components correlated with the level of inflammation and with ACPA levels in these patients137.

Nevertheless, no comprehensive studies of at- risk populations have been reported that can address the longitudinal relationships between local dysbiosis in the periodontium and the future development of RA. Thus, it remains uncertain whether, and in which direc-tion, a causal relationship might exist in the at- risk and established RA populations.

Other mucosal site biomarkers: salivary, urinary and cervicovaginal. Comprehensive studies of other mucosal sites have not been reported in at- risk patients but are underway. In a small cohort of patients with RA, ~20% of individuals expressed increased salivary IgA ACPA levels relative to healthy individuals138. Notably, in that setting, IgA anti- CCP antibody positivity was found to be inversely associated with several poor patient out-comes, thus suggesting that the anti- inflammatory, pro-tective functions of the IgA isotype described above are the predominant effect of expression at this site. To our knowledge, there are no similar studies of at- risk popu-lations, although in a small set of samples there were marked differences between ACPA levels in induced sputum and saliva of at- risk patients17, suggesting that the two sites operate independently and consistent with the understanding that sputum ACPA levels reflect lung and not oral sources. Additional studies demon-strated that IgA RF was likely to be produced locally within the saliva and tears in patients with RA, but no studies of at- risk populations are available139.

With regard to urinary biomarkers, it is uncertain whether urinary ACPAs are present in patients with RA or at- risk populations as this has not been studied to our knowledge. Urinary tract infections have been postu-lated to have a role in the development of RA, perhaps most notably infection with Proteus species (reviewed elsewhere140), but no studies in at- risk populations have emerged that directly address this relationship. However, opportunities to study the urinary microbiome are increasing141.

Finally, the cervicovaginal mucosal site has been evaluated with regard to the mucosal immune response and susceptibility to transmission of infections such as HIV142. Of interest, similar to findings discussed above demonstrating ACPAs in the sputum of individuals at risk of RA, antigen- specific antibodies for HIV can be found in cervicovaginal fluid in the absence of serum antibodies, consistent with a local- limited protective

antibody response143. Although published studies of cervicovaginal- specific biomarkers and ACPA produc-tion are lacking, our group has identified increased levels of anti-CCP IgG antibodies in the cervicovaginal fluid of premenopausal women with RA as well as in some indi-viduals in the at- risk state144. A portion of healthy controls in that study were also identified who were serum anti- CCP negative but had high levels of cervicovaginal fluid anti-CCP antibodies that strongly correlated with signs of local inflammation as measured by increased levels of cytokines. These findings further support the hypothesis that localized and externally focused ACPA production occurs at mucosal sites in response to inflammation and that identifying the mechanisms leading to a loss of externally focused ACPA production will improve our understanding of the development of systemic ACPAs in individuals who develop RA.

One limitation to the interpretation of the current studies, especially outside of the lungs, is a lack of understanding on a larger population basis of what the levels of mucosal ACPAs are, how these levels change over time and what additional phenotypes and factors are associated with antibody status. These questions are important areas for future investigation.

Potential disease mechanismsFollowing initial phenotyping studies in populations at risk of RA and the demonstration of substantial sys-temic immune dysregulation, the study of local mucosal sites in at- risk individuals is now starting to accelerate. No comprehensive longitudinal studies of mucosal biomarkers have been reported yet. Nevertheless, the intersection of informative mechanistic studies in other settings with cross- sectional translational human studies in at- risk individuals provides a conceptual framework in which to consider the role of mucosal processes in preclinical RA.

Potential protective role of ACPAs. What is perhaps the most striking finding from initial work is that local mucosal production of IgA and IgG ACPAs and RFs is frequently present in at- risk individuals, ranging from 15–25% of individuals when evaluating a single isotype or autoantibody specificity in cross- sectional studies of the lung in at- risk populations to ~40% when con-sidering all reactivities. In addition, local lung mucosal RA- related autoantibody production is not a fixed phenotype; preliminary studies suggest that individ-uals in the at- risk state both gain and lose anti- CCP antibodies over time (M.K.D. and K.D.D., unpublished observations).

With these observations and the knowledge gained from studies of the protective role of mucosal anti-bodies, the following hypotheses can be put forward (fig. 4). First, local ACPA production does not reflect an inherent loss of tolerance to self- antigens but is rather a hard- wired form of natural IgA production, perhaps T cell- independent, in which ACPAs are normally externally directed into the mucosal fluid where they bind to citrullinated antigens that are released from neutrophils undergoing NETosis and macrophages that are present as part of a chronic inflammatory state


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in a mucosal site. Second, given that multiple mucosal sites have demonstrated the presence of local ACPA production, these findings suggest that RA does not have to begin in a single mucosal region but might instead originate in one of many sites that could vary from patient to patient. This finding could underlie the very heterogeneous manner in which the auto-immune process develops in the preclinical period, with neither specific ACPA peptide reactivities9 nor individual or combinations of cytokines10 being nec-essary or sufficient for the transition to classified RA. Third, it is likely that a major driver in early disease initiation is the loss of the mucosal barrier; thus, development of a systemic IgG ACPA response could reflect an unresolved state of inflammation in the mucosa. This process could be driven by dysbiosis, the nature of which might be similar from one individ-ual to another, or alternatively might vary substantially across at- risk individuals or subsets thereof.

Going forward, to validate this hypothesis, a clear understanding of the relationships between systemic and mucosal B cells and the B cell receptor sequences that encode ACPAs must be generated through paired tissue and peripheral blood studies. These studies would evalu-ate whether the systemic and mucosal ACPA- producing and RF- producing B cells originate from the same clonal families, a finding that would link together their developmental pathways.

T cells and molecular mimicry as drivers of autoimmunity. The strong association of RA with the SE implicates antigen presentation and CD4+ T cells in its pathogen-esis. Studies have demonstrated that CD4+ T cells spe-cific for joint- derived citrullinated peptides are present in increased numbers and with distinct phenotypes in RA44,45. These cells probably have a role in the evolution of RA by both promoting tissue- specific inflammation and providing B cell help for the formation of high- avidity

Fig. 4 | Loss of the mucosal barrier allows systemic spread of ACPA response. Hypothetical model for rheumatoid arthritis (RA) initiation and the mechanism by which chronic mucosal inflammation transitions to systemic autoimmune disease, focusing on the potential roles for normal and dysregulated local mucosal anticitrullinated protein antibody (ACPA) generation. The left panel depicts the reversible production of immunoglobulin A (IgA) ACPAs in individuals as part of normal surveillance or in those individuals who have transient mild inflammation. This process is common and part of normal homeostasis at the mucosal surface. The right panel is the postulated status of individuals in the preclinical RA state in whom persistent inflammation leads to increased ACPA production and the systemic spread of an autoimmune process through multiple potential processes. The drivers of this local and then systemic process could include cytokines, metabolomic changes, ACPAs that cross- react with bacterial antigens or the presence of other antigen- specific or innate T cells that are expanded as they either recognize bacterial antigens or respond to local signals. Regardless of the mechanisms involved, these antibodies and cells begin to circulate as a result of the chronic inflammation and are detectable through serological analysis and the analysis of other biomarkers. IELs, intraepithelial lymphocytes.


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pathogenic ACPAs. Molecular mimicry has been proposed as one source of failed tolerance in RA and provides a framework with which to link the microbiome with genetic risk by identifying CD4+ T cells that respond to microbial and self- antigens that share similar motifs.

Several approaches are being used to address the molecular mimicry hypothesis. For example, motif anal-ysis identified α- viral peptides with a similar motif to CII, a known autoantigen in RA145. A peptide sequence motif (DERAA) contained within HLADRB1*13, a pro-tective allele in RA, has been shown to mediate thymic tolerance in the context of HLA- DQ, but when this molecule and the peptide derived from it are absent, the peptide sequence can be recognized by CD4+ T cells that are not tolerized during development and are thus cross- reactive with DERAA-containing microbially derived epitopes and the self- antigen vinculin146. This finding suggests a mechanism by which an initial fail-ure of thymic tolerance is followed by activation with a microbial antigen that then supports a T cell and B cell response to citrullinated vinculin146. Proteomic tools (such as mass spectroscopy) have also been used to characterize RA- related epitopes through the elution of peptides from HLA class II molecules found in the synovium of patients with RA. This approach has iden-tified novel autoantigens that are cross- reactive with microbial proteins and for which the corresponding T cell and B cell responses are increased in patients with RA125. As we increase our understanding of the micro-bial species that participate in the development of RA and determine which autoantigens are early targets of the immune response, the role of molecular mimicry in the development of RA should be further clarified.

Molecular mimicry and the hard- wired IgA response to citrullinated products of inflammation might both be necessary to develop classified RA. To address this question, regardless of whether the local mucosal ACPA immune response is meant to primarily react with citrul-linated products of dead cells, the potential for molec-ular mimicry as a driver of an expanded pathogenic mucosal and then ACPA immune response, as recently demonstrated in type 1 diabetes147, should be further evaluated. Similarly, B cell molecular mimicry is con-sistent with plasmablast studies in patients with chronic RA, from whom IgG antibodies have been cloned and expressed and found to recognize citrullinated targets as well as P. gingivalis148. Notably, in patients with early RA, IgA ACPAs are associated with smoking whereas IgG ACPAs are associated with the SE, supporting a possible distinction between local and systemic processes149.

To better understand these causal mechanisms, and using a rationale similar to that used for ACPA- reactive B cells, characterizing citrullinated- peptide-reactive sys-temic and mucosal T cells and their interrelationships is essential to further evaluate the potential role of autore-active T cells in the initial and subsequent development of ACPAs and then classified RA.

Other potential interrelated mechanisms. Other interre-lated mechanisms might exist, including mechanisms that modify local ACPA generation and mechanisms by which ACPAs originating at different mucosal sites, or through

distinct inflammatory processes, might influence the sub-sequent arthritis phenotypes. For instance, alternative pri-mary drivers of autoimmunity might have key roles, such as inadequate local T cell immuno regulation influenced by genetic or environmental factors, aberrant expression of adhesion molecules and/or cytokines and chemokines, activation of non- tolerogenic antigen- presenting cells or engagement of non- traditional antigen- presenting cells such as fibroblasts. Additional modulators of the loss of tol-erance and the autoimmune disease process might include metabolomic changes that promote systemic spread of the ACPA response, a physical or physiological break in the mucosal barrier, direct or indirect activation of trans-iting autoimmune cells through enhancement of antigen presentation or alteration of dendritic cell function to expand a response and production of innate immune factors that promote activation, such as lipopolysaccha-ride, high mobility group protein B1 (HMGB1), CpG or other danger- recognition molecules. In addition, acute infection in the presence of chronic local ACPA produc-tion might have an important role in triggering additional pathogenic changes.

An important question in the evolution of RA is how does chronic inflammation relate mechanistically to epi-demiological factors such as smoking or low intake of omega-3 PUFAs? Studies suggest that smoking is asso-ciated with increased levels of citrullinated proteins in the lung150. With regard to the omega-3 PUFA levels and exposures, studies have shown that molecules derived from this pathway (especially bioactive lipid mediators including resolvins and maresins that are biosynthesized in vivo from EPA and DHA) are key factors in resolving or reducing inflammation151. The observation that RBC levels of omega-3 PUFAs are lower in those patients who progress to inflammatory arthritis suggests that these factors are either present in insufficient quanti-ties or are consumed as part of the excessive mucosal inflammation that is present and that these two pro-cesses, local inflammation and a lack of appropriate levels of omega-3 PUFAs, are both necessary for con-tinued evolution of disease. These anti- inflammatory compounds from the omega-3 PUFA pathway decrease inflammation through mechanisms including blocking neutrophil and other cell chemotaxis or recruitment, increasing neutrophil apoptosis and promoting cellular immune switching to regulatory phenotypes (reviewed elsewhere83). Furthermore, the actions of agents such as resolvins have been shown to reduce inflammation at mucosal surfaces in the lung as well as elsewhere (for example, the gut)152,153 and to reduce cell activation and immune- complex-mediated inflammation in a variety of diseases, including IgA nephropathy83,154,155. Importantly, as described above, all of these proinflammatory path-ways might be critical to the initiation and propaga-tion of autoimmunity and the ultimate development of arthritis and thus might be modifiable by increasing intake of omega-3 PUFA levels.

As to the potential influence of mucosal inflam-mation at specific sites on subsequent arthritis generation, distinct synovial subtypes have been identified in RA, including subsets of patients with lymphoid- predominant or myeloid- predominant

Molecular mimicryThe development of autoreactivity commonly considered to be through the recognition of a microbial or other foreign pathogen peptide that is structurally similar to self- antigen.


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synovial infiltrates versus patients with fibrous or low- inflammation synovial features156. The different syn-ovial phenotypes in RA might correlate with different responses to biological therapeutics156. The specific mucosal trigger and IgA plasmablast response, either in general or from specific mucosal sites, might promote a transition to the high- grade synovial inflammatory subtype of RA, whereas other mechanisms might pro-mote transition to the fibrous synovial subtype. It is also striking that what are two distinct autoantibody systems, ACPAs and RFs, can come together and accelerate and amplify joint damage in an experimental model of RA157, again highlighting the possibility that separate mucosal processes drive distinct autoantibody phenotypes and the interconnectedness of the responses drives arthritis.

Therapeutic implicationsAlthough the exact roles of mucosal inflammation in the development of RA are still uncertain, current findings provide an impetus to consider specific mechanism- based therapeutic approaches that follow up on the findings of chronic immune activation and mucosal inflammation in at- risk individuals. For example, although omega-3 PUFA supplementation has not yet been formally tested in a clinical trial in preclinical RA, studies in animals and trials in humans across a range of diseases (including classified RA) have demonstrated that supplementation improves clinical parameters82,83, strongly suggesting that supplementation can result in biological changes affecting the inflammatory process at multiple sites, including the mucosa, and might be able to prevent or delay the devel-opment of clinically apparent RA. Furthermore, although targeting IL-17 is not consistently beneficial in patients with established RA (reviewed elsewhere158), findings in an at- risk population described above121, along with the known role of IL-17 in promoting mucosal inflam-mation159, suggest that therapeutic approaches directed at IL-17, or targeting TH17 cells through IL-12–IL-23, might be particularly effective in preclinical RA. TH17 cells might contribute to the evolution of preclinical RA via pathways and mediators other than IL-17, includ-ing IL-6, IL-21, IL-22 and TNF160, suggesting that these molecules are also therapeutic targets in preclinical RA. Other pathways that have roles in epitope spreading fol-lowing the breaking of tolerance, including B7/CD28 and CD40:CD40L, should also be considered. The success of co- stimulatory pathway modulation in cancer immu-notherapy highlights the importance of these pathways in regulating T cell responses, and we anticipate the involvement of other T cell pathways in the development of autoimmunity161. If so, therapeutically targeting these pathways in preclinical RA could provide a powerful approach to abrogate the development of RA. Of course, if a specific microbial species or metabolomic signal-ling pathway162 is identified as causal in preclinical RA, targeting these processes would be especially effective.

Future directionsTo test the hypotheses laid out above, a number of stud-ies and experimental approaches must continue to be pursued. Longitudinal studies of the microbiome, local mucosal phenotypes at multiple sites and systemic

RA- related autoantibody production are needed to determine whether potential causal relationships exist that could drive relevant phenotypic changes in the preclinical to clinical transitions. In a related fashion, understanding the causes of local mucosal inflamma-tion and local ACPA generation should be a research goal. Additionally, exploring the relationships between local and systemic IgA and IgG ACPA production at the molecular and immunoglobulin sequence level over time will enable understanding of how these two systems are interrelated.

Although it is firmly established that local inflamma-tion in the lung is associated with ACPA production, it is uncertain yet whether biomarkers of mucosal inflamma-tion (such as faecal calprotectin) or responses to bacte-ria (such as increased expression of RegIII proteins) are altered in at- risk individuals. Beyond these considera-tions, although studies of single mucosal sites have been performed in at- risk individuals, a more comprehensive approach to simultaneously sampling multiple mucosal sites would reveal whether changes are unique to one site or are shared across multiple sites and then whether one site is sufficient to lead to systemic spread of ACPA generation. To address interrelationships between local mucosal findings and systemic immune changes, addi-tional studies of T cell antigen- specific or innate immune reactivity should be evaluated, both systemically and at mucosal sites.

To further expand our understanding, a determina-tion of whether other RA- related autoantibodies, includ-ing other AMPAs, are similarly generated locally in the mucosa as part of the hard- wired innate immune system and then spread systemically should be undertaken. Likewise, understanding how systemic immune and metabolomic changes relate to mucosal inflammation and ACPA production will provide additional insights. Finally, it will be important to determine whether this concept of ‘hard- wired mucosal autoimmunity’ having an important role in normal mucosal immune responses is relevant to the initial preclinical stages of other autoimmune diseases, such as systemic lupus erythematosus, in which antibod-ies to intracellular proteins are prominent. It is uncertain why these intracellular antigens that are released during normal inflammatory processes are ‘chosen’ as targets of autoantibodies in these conditions, and if these antibodies are expressed locally in a manner similar to RA at- risk individuals, unresolved mucosal inflammation might play a key early role in systemic spread.

ConclusionsPrior to the onset of the first clinical signs and symp-toms of seropositive RA, most patients will have passed through a prolonged period of serological RA- related autoantibody positivity. Herein, we have reviewed the current understanding of the relationships during this preclinical period between mucosal alterations, including dysbiosis and inflammation, and the initia-tion of local and systemic ACPA production. Mucosal ACPA production in the lung, and probably other sites, is associated with the presence of local inflammation and increased levels of NET formation. Mucosal IgA ACPA production seems to be a hard- wired and


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common phenotype, suggesting that this factor has an important role in the local protective immune response outside of its eventual causal relationship with inflammatory arthritis. Many potential factors in the mucosa, as well as systemic alterations, could influ-ence both the local initiation and systemic expansion

of ACPA expression. Further study of these processes should provide both additional insights into very early disease pathogenesis in RA and the identification of novel therapeutic strategies.

Published online 15 August 2018

1. Catrina, A. I., Svensson, C. I., Malmström, V., Schett, G. & Klareskog, L. Mechanisms leading from systemic autoimmunity to joint- specific disease in rheumatoid arthritis. Nat. Rev. Rheumatol. 13, 79–86 (2017).

2. Catrina, A. I., Deane, K. D. & Scher, J. U. Gene, environment, microbiome and mucosal immune tolerance in rheumatoid arthritis. Rheumatology 55, 391–402 (2016).

3. Viatte, S., Plant, D. & Raychaudhuri, S. Genetics and epigenetics of rheumatoid arthritis. Nat. Rev. Rheumatol. 9, 141–153 (2013).

4. Firestein, G. S. & McInnes, I. B. Immunopathogenesis of rheumatoid arthritis. Immunity 46, 183–196 (2017).

5. Nielen, M. M. J. et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis. Arthritis Rheum. 50, 380–386 (2004).

6. Rantapaa Dahlqvist, S. et al. Antibodies against citrullinated peptides (CCP) predict the development of rheumatoid arthritis. Arthritis Rheum. 48, 2701–2705 (2003).

7. Majka, D. S. et al. Duration of preclinical rheumatoid arthritis- related autoantibody positivity increases in subject with older age at time of disease diagnosis. Ann. Rheum. Dis. 67, 801–807 (2008).

8. Karlson, E. W. et al. Biomarkers of inflammation and development of rheumatoid arthritis in women from two prospective cohort studies. Arthritis Rheum. 60, 641–652 (2009).

9. Sokolove, J. et al. Autoantibody epitope spreading in the preclinical phase predicts progression to rheumatoid arthritis. PLOS One 7, e35296 (2012).

10. Deane, K. D. et al. The number of elevated cytokines and chemokines in preclinical seropositive rheumatoid arthritis predicts time to diagnosis in an age- dependent manner. Arthritis Rheum. 62, 3161–3172 (2010).

11. Gerlag, D. M. et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann. Rheum. Dis. 71, 638–641 (2012).

12. Scher, J. U. et al. The lung microbiota in early rheumatoid arthritis and autoimmunity. Microbiome 4, 60 (2016).

13. Scher, J. U. et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife https://doi.org/10.7554/eLife.01202 (2013).

14. Scher, J. U. et al. Periodontal disease and the oral microbiota in new- onset rheumatoid arthritis. Arthritis Rheum. 64, 3083–3094 (2012).

15. Zhang, X. et al. The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat. Med. 21, 895–905 (2015).

16. Chen, J. et al. An expansion of rare lineage intestinal microbes characterizes rheumatoid arthritis. Genome Med. 8, 43 (2016).

17. Demoruelle, M. K. et al. Anti- citrullinated protein antibodies are associated with neutrophil extracellular traps in the sputum in relatives of rheumatoid arthritis patients. Arthritis Rheum. 69, 1165–1175 (2017).

18. Willis, V. C. et al. Sputa autoantibodies in patients with established rheumatoid arthritis and subjects at- risk for future clinically apparent disease. Arthritis Rheum. 65, 2545–2554 (2013).

19. Lee, D. M. & Weinblatt, M. E. Rheumatoid arthritis. Lancet 359, 903–911 (2001).

20. van Steenbergen, H. W. et al. EULAR definition of arthralgia suspicious for progression to rheumatoid arthritis. Ann. Rheum. Dis. 76, 491–496 (2017).

21. Fuchs, H. A. & Sergent, J. S. in Arthritis and Allied Conditions: A Textbook of Rheumatology (ed. Koopman, W. J.) 1041–1070 (Williams and Wilkins, 1997).

22. Schett, G. & Gravallese, E. M. Bone erosion in rheumatoid arthritis: Mechanisms, diagnosis and treatment. Nat. Rev. Rheumatol. 8, 656–664 (2012).

23. Silman, A. J. Epidemiology of rheumatoid arthritis. APMIS 102, 721–728 (1994).

24. Karlson, E. W. & Costenbader, K. H. Epidemiology: Interpreting studies of interactions between RA risk factors. Nat. Rev. Rheumatol. 6, 72–73 (2010).

25. Karlson, E. W. & Deane, K. D. Environmental and gene- environment interactions and risk of rheumatoid arthritis. Rheum. Dis. Clin. North Am. 38, 405–426 (2012).

26. Klareskog, L., Malmstrom, V., Lundberg, K., Padyukov, L. & Alfredsson, L. Smoking, citrullination and genetic variability in the immunopathogenesis of rheumatoid arthritis. Semin. Immunol. 23, 92–98 (2011).

27. Aletaha, D. et al. Rheumatoid arthritis classification criteria: An American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 62, 2569–2581 (2010).

28. Arnett, F. C. et al. The American Rheumatism Association1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 314–324 (1998).

29. de Rooy, D. P. C., van der Linden, M. P. M., Knevel, R., Huizinga, T. W. & van der Helm- van Mil, A. H. M. Predicting arthritis outcomes- what can be learned from the Leiden Early Arthritis Clinic? Rheumatology 50, 93–100 (2011).

30. van der Helm- van Mil, A. H. M. et al. A prediction rule for disease outcome in patients with recent- onset undifferentiated arthritis: how to guide individual treatment decisions. Arthritis Rheum. 56, 433–440 (2007).

31. Padyukov, L., Silva, C., Stolt, P., Alfredsson, L. & Klareskog, L. A gene- environment interaction between smoking and shared epitope genes in HLA- DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum. 50, 3085–3092 (2004).

32. Schellekens, G. A., de Jong, B. A. W., van den Hoogen, F. H. J., van de Putte, L. B. A. & van Venrooij, W. J. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis- specific autoantibodies. J. Clin. Invest. 101, 273–281 (1998).

33. van Venrooij, W. J. & Pruijn, G. J. M. Citrullination: a small change for a protein with great consequences for rheumatoid arthritis. Arthritis Res. Ther. 2, 249–251 (2000).

34. Bas, S., Genevay, S., Meyer, O. & Gabay, C. Anti- cyclic citrullinated peptide antibodies, IgM and IgA rheumatoid factors in the diagnosis and prognosis of rheumatoid arthritis. Rheumatology 42, 677–680 (2003).

35. Trouw, L. A., Rispens, T. & Toes, R. E. M. Beyond citrullination: other post- translational protein modifications in rheumatoid arthritis. Nat. Rev. Rheumatol. 13, 331–339 (2017).

36. Shi, J. et al. Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage. Proc. Natl Acad. Sci. USA 108, 17372–17377 (2011).

37. Thiele, G. M. et al. Malondialdehyde- acetaldehyde adducts and anti- malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis. Arthritis Rheum. 67, 645–655 (2015).

38. Pecani, A. et al. Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases. Arthritis Res. Ther. 18, 276 (2016).

39. Gan, R. W. et al. Anti- carbamylated protein antibodies are present prior to rheumatoid arthritis and are associated with its future diagnosis. J. Rheumatol. 42, 572–579 (2015).

40. Nepom, G. T. & Nepom, B. S. Prediction of susceptibility to rheumatoid arthritis by human leukocyte antigen phenotyping. Rheum. Dis. Clin. North Am. 18, 785–792 (1992).

41. Begovich, A. B. et al. A missense single- nucleotide polymorphism in a gene encoding a protein tyrosine

phosphatase (PTPN22) is associated with rheumatoid arthritis. Am. J. Hum. Genet. 75, 330–337 (2004).

42. Viatte, S. & Barton, A. Genetics of rheumatoid arthritis susceptibility, severity, and treatment response. Semin. Immunopathol. 39, 395–408 (2017).

43. Hill, J. A. et al. The conversion of arginine to citrulline allows for a high- affinity peptide interaction with the rheumatoid arthritis- associated HLA- BRB1*0401 MHC Class II molecule. J. Immunol. 171, 538–541 (2003).

44. Law, S. C. et al. T- cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA- DRB1 shared epitope alleles. Arthritis Res. Ther. 14, R118 (2012).

45. James, E. et al. Citrulline specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy. Arthritis Rheum. 66, 1712–1722 (2014).

46. Klareskog, L. et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA- DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 54, 38–46 (2006).

47. Nakano, K., Whitaker, J. W., Boyle, D. L., Wang, W. & Firestein, G. S. DNA methylome signature in rheumatoid arthritis. Ann. Rheum. Dis. 72, 110–117 (2013).

48. Rhead, B. et al. Rheumatoid arthritis naive T cells share hypermethylation sites with synoviocytes. Arthritis Rheum. 69, 550–559 (2017).

49. Deane, K. D., Norris, J. M. & Holers, V. M. Pre-clinical rheumatoid arthritis: identification, evaluation and future directions for investigation. Rheum. Dis. Clin. North Am. 36, 213–241 (2010).

50. Brink, M. et al. Multiplex analyses of antibodies against citrullinated peptides in individuals prior to the development of rheumatoid arthritis. Arthritis Rheum. 65, 899–910 (2013).

51. Suwannalai, P. et al. Avidity maturation of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 64, 1323–1328 (2012).

52. Trouw, L. A. et al. Anti- cyclic peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways. Arthritis Rheum. 60, 1923–1931 (2009).

53. Rombouts, Y. et al. Anti- citrullinated protein antibodies acquire a proinflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann. Rheum. Dis. 74, 234–241 (2015).

54. Rombouts, Y. et al. Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis. Ann. Rheum. Dis. 75, 578–585 (2016).

55. Pfeifle, R. et al. Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease. Nat. Immunol. 18, 104–113 (2017).

56. Harre, U. et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J. Clin. Invest. 122, 1791–1802 (2012).

57. Wigerblad, G. et al. Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine- dependent mechanism. Ann. Rheum. Dis. 75, 730–738 (2016).

58. Kokkonen, H. et al. Antibodies of IgG, IgA and IgM isotypes against cyclic citrullinated peptide precede the development of rheumatoid arthritis. Arthritis Res. Ther. 13, R13 (2011).

59. Jorgensen, C., Moynier, M., Bologna, C., Youinou, P. & Sany, J. Rheumatoid factor associated with a secretory component in rheumatoid arthritis. Br. Med. J. 34, 236–240 (1995).

60. Aho, K., Heliovaara, M., Maatela, J., Tuomi, T. & Palosuo, T. Rheumatoid factors antedating clinical rheumatoid arthritis. J. Rheumatol. 18, 1282–1284 (1991).

61. Kolfenbach, J. R. et al. A prospective approach to investigating the natural history of preclinical


R e v i e w s


https://doi.org/10.7554/eLife.01202

rheumatoid arthritis (RA) using first- degree relatives of probands with RA. Arthritis Care Res. 61, 1735–1741 (2009).

62. El- Gabalawy, H. S. et al. Familial clustering of the serum cytokine profile in the relatives of rheumatoid arthritis patients. Arthritis Rheum. 64, 1720–1729 (2012).

63. Deane, K. D. et al. Identification of undiagnosed inflammatory arthritis in a community health fair screen. Arthritis Care Res. 61, 1642–1649 (2009).

64. Lübbers, J. et al. The type I IFN signature as a biomarker of preclinical rheumatoid arthritis. Ann. Rheum. Dis. 72, 776–780 (2013).

65. Young, K. A. et al. Relatives without rheumatoid arthritis show reactivity to anticitrullinated protein/peptide antibodies that are associated with arthritis- related traits: studies of the etiology of rheumatoid arthritis. Arthritis Rheum. 65, 1995–2004 (2013).

66. Hughes- Austin, J. et al. Multiple cytokines and chemokines are associated with rheumatoid arthritis- related autoimmunity in first- degree relatives without rheumatoid arthritis: Studies of the Aetiology of Rheumatoid Arthritis (SERA). Ann. Rheum. Dis. 72, 901–907 (2012).

67. Aslam, A. et al. Emergence of proinflammatory autoreactive T- cell responses in preclinical rheumatoid arthritis. Lancet 383, S22 (2014).

68. Hunt, L. et al. T cell subsets: an immunological biomarker to predict progression to clinical arthritis in ACPApositive individuals. Ann. Rheum. Dis. 75, 1884–1889 (2016).

69. Nielen, M. M. J. et al. Increased levels of C- Reactive Protein in serum from blood donors before the onset of rheumatoid arthritis. Arthritis Rheum. 50, 2423–2427 (2004).

70. van de Stadt, L. A. et al. The extent of the anti-citrullinated protein antibody repertoire is associated with arthritis development in patients with seropositive arthralgia. Ann. Rheum. Dis. 70, 128–133 (2011).

71. Arkema, E. V. et al. Anti- citrullinated peptide autoantibodies, human leukocyte antigen shared epitope and risk of future rheumatoid arthritis: a nested case–control study. Arthritis Res. Ther. 15, R159 (2013).

72. Bhatia, S. S. et al. Rheumatoid factor seropositivity is inversely associated with oral contraceptive use in women without arthritis. Ann. Rheum. Dis. 66, 267–269 (2007).

73. Hensvold, A. H. et al. Environmental and genetic factors in the development of anticitrullinated protein antibodies (ACPAs) and ACPA- positive rheumatoid arthritis: an epidemiological investigation in twins. Ann. Rheum. Dis. 74, 375–380 (2015).

74. van Zanten, A. et al. Presence of anticitrullinated protein antibodies in a large population- based cohort from the Netherlands. Ann. Rheum. Dis. 76, 1184–1190 (2017).

75. Hedström, A. K., Stawiarz, L., Klareskog, L. & Alfredsson, L. Smoking and susceptibility to rheumatoid arthritis in a Swedish population- based case- control study. Eur. J. Epidemiol. 33, 415–423 (2018).

76. van Wesemael, T. J. et al. Smoking is associated with the concurrent presence of multiple autoantibodies in rheumatoid arthritis rather than with anticitrullinated protein antibodies per se: a multicenter cohort study. Arthritis Res. Ther. 18, 285 (2016).

77. Terao, C. et al. Effects of smoking and shared epitope on the production of anticitrullinated peptide antibody in a Japanese adult population. Arthritis Care Res. 66, 1818–1827 (2014).

78. Gan, R. W. et al. Relationship between air pollution and positivity of RA- related autoantibodies in individuals without established RA: a report on SERA. Ann. Rheum. Dis. 72, 2002–2005 (2013).

79. Hart, J. E., Laden, F., Puett, R. C., Costenbader, K. H. & Karlson, E. W. Exposure to traffic pollution and increased risk of rheumatoid arthritis. Environ. Health Perspect. 117, 1065–1069 (2009).

80. Tedeschi, S. K. et al. Elevated BMI and antibodies to citrullinated proteins interact to increase rheumatoid arthritis risk and shorten time to diagnosis: a nested case- control study of women in the Nurses’ Health Studies. Semin. Arthritis Rheum. 46, 692–698 (2017).

81. Stolt, P. et al. Silica exposure is associated with increased risk of developing rheumatoid arthritis: results from the Swedish EIRA study. Ann. Rheum. Dis. 64, 582–586 (2005).

82. Dawczynski, C. et al. Docosahexaenoic acid in the treatment of rheumatoid arthritis: A double- blind,

placebo- controlled, randomized cross- over study with microalgae vs. sunflower oil. Clin. Nutr. 37, 494–504 (2017).

83. Souza, P. R. & Norling, L. V. Implications for eicosapentaenoic acid- and docosahexaenoic acid-derived resolvins as therapeutics for arthritis. Eur. J. Pharmacol. 785, 165–173 (2016).

84. Di Giuseppe, D., Crippa, A., Orsini, N. & Wolk, A. Fish consumption and risk of rheumatoid arthritis: a doseresponse meta- analysis. Arthritis Res. Ther. 16, 446 (2014).

85. Gan, R. W. et al. Lower omega-3 fatty acids are associated with the presence of anti- cyclic citrullinated peptide autoantibodies in a population at risk for future rheumatoid arthritis: a nested case- control study. Rheumatology 55, 367–376 (2016).

86. Gan, R. W. et al. Omega-3 fatty acids are associated with a lower prevalence of autoantibodies in shared epitope positive subjects at risk for rheumatoid arthritis. Ann. Rheum. Dis. 76, 147–152 (2017).

87. Gan, R. W. et al. The association between omega-3 fatty acid biomarkers and inflammatory arthritis in an anticitrullinated protein antibody positive population. Rheumatology 56, 2229–2236 (2017).

88. Li, S., Yu, Y., Yue, Y., Zhang, Z. & Su, K. Microbial infection and rheumatoid arthritis. J. Clin. Cell. Immunol. 4, 174 (2013).

89. Lundberg, K., Wegner, N., Yucel- Lindberg, T. & Venables, P. J. Periodontitis in RA- the citrullinated enolase connection. Nat. Rev. Rheumatol. 6, 727–730 (2010).

90. Longman, R. S. & Littman, D. R. The functional impact of the intestinal microbiome on mucosal immunity and systemic autoimmunity. Curr. Opin. Rheum. 27, 381–387 (2015).

91. Scher, J. U., Littman, D. R. & Abramson, S. B. Review: Microbiome in inflammatory arthritis and human rheumatic diseases. Arthritis Rheum. 68, 35–45 (2015).

92. Belkaid, Y. & Hand, T. W. Role of the microbiota in immunity and inflammation. Cell 157, 121–141 (2014).

93. Cash, H. L., Whitham, C. V., Behrendt, C. L. & Hooper, L. V. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313, 1126–1130 (2006).

94. Szabady, R. L. & McCormick, B. A. Control of neutrophil inflammation at mucosal surfaces by secreted epithelial products. Front. Immunol. https://doi.org/10.3389/fimmu.2013.00220 (2013).

95. Howson, L. J., Salio, M. & Cerundolo, V. MR1-restricted mucosal- associated invariant T cells and their activation during infectious diseases. Front. Immunol. https://doi.org/10.3389/fimmu.2015.00303 (2015).

96. Sonnenberg, G. F., Monticelli, L. A., Elloso, M. M., Fouser, L. A. & Artis, D. CD4(+) lymphoid tissue- inducer cells promote innate immunity in the gut. Immunity 34, 122–134 (2011).

97. Goto, Y. et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 345, 1254009 (2014).

98. von Andrian, U. & Mackay, C. R. T- cell function and migration. Two sides of the same coin. New Engl. J. Med. 343, 1020–1034 (2000).

99. Kurmaeva, E. et al. T cell- associated a4b7 but not a4b1 integrin is required for the induction and perpetuation of chronic coliti. Mucosal Immunol. 7, 1354–1365 (2014).

100. Macpherson, A. J., McCoy, K. D., Johansen, F. E. & Brandtzaeg, P. The immune geography of IgA induction and function. Mucosal Immunol. 11, 11–22 (2008).

101. Fagarasan, S., Kawamoto, S., Kanagawa, O. & Suzuki, K. Adaptive immune regulation in the gut: T celldependent and T cell- independent IgA synthesis. Annu. Rev. Immunol. 28, 243–273 (2010).

102. Corthésy, B. Multi- faceted functions of secretory IgA at mucosal surfaces. Front. Immunol. https://doi.org/10.3389/fimmu.2013.00185 (2013).

103. Aleyd, E., Al, M., Tuk, C. W., van der Laken, C. J. & van Egmond, M. IgA complexes in plasma and synovial fluid of patients with rheumatoid arthritis induce neutrophil extracellular traps via FcαRI. J. Immunol. 197, 4552–4559 (2016).

104. Russell, M. W., Sibley, D. A., Nikolova, E. B., Tomana, M. & Mestecky, J. IgA antibody as a non- inflammatory regulator of immunity. Biochem. Soc. Trans. 25, 466–470 (1997).

105. Baker, K., Lencer, W. I. & Blumberg, R. S. Beyond IgA: the mucosal immunoglobulin alphabet. Mucosal Immunol. 3, 324–325 (2010).

106. Bunker, J. J. et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 358, eaan6619 (2017).

107. Vaishnava, S. et al. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science 334, 255–258 (2011).

108. Virgin, H. W. The virome in mammalian physiology and disease. Cell 157, 142–150 (2014).

109. Smith, P. M. et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569–573 (2013).

110. Kuhn, K. A. et al. Antibodies to citrullinated proteins enhance tissue injury in experimental arthritis. J. Clin. Invest. 116, 961–973 (2006).

111. Liu, X. et al. Role of the gut microbiome in modulating arthritis progression in mice. Sci. Rep. 6, 30594 (2016).

112. Jubair, W. K. et al. Modulation of inflammatory arthritis by gut microbiota through mucosal inflammation and autoantibody generation. Arthritis Rheum. 70, 1220–1233 (2018).

113. Jacques, P. & Elewaut, D. Joint expedition: linking gut inflammation to arthritis. Mucosal Immunol. 1, 364–371 (2008).

114. Ji, H. et al. Arthritis critically dependent on innate immune system players. Immunity 16, 157–168 (2002).

115. Block, K. E., Zheng, Z., Dent, A. L., Kee, B. L. & Huang, H. Gut microbiota regulates K/BxN autoimmune arthritis through follicular helper T but not Th17 cells. J. Immunol. 196, 1550–1557 (2016).

116. Wu, H. J. et al. Gut- residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32, 815–827 (2010).

117. Marietta, E. V. et al. Suppression of inflammatory arthritis by human gut- derived Prevotella histicola in humanized mice. Arthritis Rheum. 68, 2878–2888 (2016).

118. Tan, Y. C. et al. High- throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination. Clin. Immunol. Immunopathol. 151, 55–66 (2014).

119. Kinslow, J. D. et al. IgA plasmablasts are elevated in subjects at risk for future rheumatoid arthritis. Arthritis Rheum. 68, 2372–2383 (2016).

120. Tan, Y. C. et al. Barcode- enabled sequencing of plasmablast antibody repertoires in rheumatoid arthritis. Arthritis Rheum. 66, 2706–2715 (2014).

121. Chang, H. H. et al. A molecular timeline of preclinical rheumatoid arthritis defined by dysregulated PTPN22. JCI Insight https://doi.org/10.1172/jci.insight.90045 (2016).

122. Chang, H. H., Dwivedi, N., Nicholas, A. P. & Ho, I. C. The W620 polymorphism in PTPN22 disrupts its interaction with peptidylarginine deiminase type 4 and enhances citrullination and NETosis. Arthritis Rheum. 67, 2323–2334 (2015).

123. Yang, Z., Fujii, H., Mohan, S. V., Goronzy, J. J. & Weyand, C. M. Phosphofructokinase deficiency impairs ATP generation, autophagy, and redox balance in rheumatoid arthritis T cells. J. Exp. Med. 210, 2119–2134 (2013).

124. Yang, Z. et al. Restoring oxidant signaling suppresses proarthritogenic T cell effector functions in rheumatoid arthritis. Sci. Transl Med. 8, 331ra38 (2016).

125. Pianta, A. et al. Evidence of the immune relevance of Prevotella copri, a gut microbe, in patients With rheumatoid arthritis. Arthritis Rheum. 69, 964–975 (2017).

126. Rangel- Moreno, J. et al. Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis. J. Clin. Invest. 116, 3183–3194 (2006).

127. Reynisdottir, G. et al. Signs of immune activation and local inflammation are present in the bronchial tissue of patients with untreated early rheumatoid arthritis. Ann. Rheum. Dis. 75, 1722–1727 (2016).

128. Ytterberg, A. J. et al. Identification of shared citrullinated immunological targets in the lungs and joints of patients with rheumatoid arthritis. Ann. Rheum. Dis. 74, 1772–1777 (2012).

129. Demoruelle, M. K. et al. Airways abnormalities and rheumatoid arthritis- related autoantibodies in subjects without arthritis: Early injury or initiating site of autoimmunity? Arthritis Rheum. 64, 1756–1761 (2012).


R e v i e w s


https://doi.org/10.3389/fimmu.2013.00220



https://doi.org/10.1172/jci.insight.90045

130. Demoruelle, M. K. et al. Antibody responses to citrullinated and non- citrullinated antigens in the sputum of subjects with and at- risk for rheumatoid arthritis. Arthritis Rheum. 70, 516–527 (2018).

131. Fischer, A. et al. Lung disease with anti- CCP antibodies but not rheumatoid arthritis or connective tissue disease. Respir. Med. 106, 1040–1047 (2012).

132. Bingham, C. O. & Moni, M. Periodontal disease and rheumatoid arthritis: the evidence accumulates for complex pathobiologic interactions. Curr. Opin. Rheumatol. 25, 345–353 (2013).

133. Eriksson, K. et al. Prevalence of periodontitis in patients with established rheumatoid arthritis: a Swedish population based case- control study. PLOS One https://doi.org/10.1371/journal.pone.0155956 (2016).

134. Konig, M. F. et al. Aggregatibacter actinomycetemcomitans- induced hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis. Sci. Transl Med. 8, 369ra176 (2017).

135. Mikuls, T. R. et al. Porphyromonas gingivalis and disease- related autoantibodies in individuals at increased risk of rheumatoid arthritis. Arthritis Rheum. 64, 3522–3530 (2012).

136. Cheng, Z. et al. The subgingival microbiomes in periodontitis and health of individuals with rheumatoid arthritis and at risk of developing rheumatoid arthritis. J. Oral Microbiol. 9, Abstr. 1325216 (2017).

137. Harvey, G. P. et al. Expression of peptidylarginine deiminase-2 and -4, citrullinated proteins and anticitrullinated protein antibodies in human gingiva. J. Periodontal Res. 48, 252–261 (2013).

138. Svärd, A., Kastbom, A., Sommarin, Y. & Skogh, T. Salivary IgA antibodies to cyclic citrullinated peptides (CCP) in rheumatoid arthritis. Immunobiology 218, 232–237 (2013).

139. Otten, H. G. et al. IgA rheumatoid factor in mucosal fluids and serum of patients with rheumatoid arthritis: immunological aspects and clinical significance. Clin. Exp. Immunol. 90, 256–259 (1992).

140. Ebringer, A. & Rashid, T. Rheumatoid arthritis is caused by a Proteus urinary tract infection. APMIS 122, 363–368 (2013).

141. Whiteside, S. A., Razvi, H., Dave, S., Reid, G. & Burton, J. P. The microbiome of the urinary tract — a role beyond infection. Nat. Rev. Urol. 12, 81–90 (2015).

142. Gosmann, C. et al. Lactobacillus- deficient cervicovaginal bacterial communities are associated

with increased HIV acquisition in young south African women. Immunity 46, 29–37 (2017).

143. Tudor, D. et al. HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgGseronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4(+) cell infection: an IgA gene and functional analysis. Mucosal Immunol. 2, 412–426 (2009).

144. Khatter, S. et al. Anti- CCP antibody levels are elevated in cervicovaginal fluid in association with local inflammation in premenopausal women without RA [abstract]. Arthritis Rheum. (2017).

145. Hogeboom, C. Peptide motif analysis predicts alphaviruses as triggers for rheumatoid arthritis. Mol. Immunol. 68, 465–475 (2015).

146. van Heemst, J. et al. Crossreactivity to vinculin and microbes provides a molecular basis for HLA- based protection against rheumatoid arthritis. Nat. Commun. 6, 6681 (2015).

147. Cole, D. K. et al. Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide crossreactivit. J. Clin. Invest. 126, 2191–2204 (2016).

148. Li, S. et al. Autoantibodies from single circulating plasmablasts react with citrullinated antigens and Porphyromonas gingivalis in rheumatoid arthritis. Arthritis Rheum. 68, 614–626 (2016).

149. Svärd, A. et al. Associations with smoking and shared epitope differ between IgA- and IgG- class antibodies to cyclic citrullinated peptides in early rheumatoid arthritis. Arthritis Rheum. 67, 2032–2037 (2015).

150. Makrygiannakis, D. et al. Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells. Ann. Rheum. Dis. 67, 1488–1492 (2008).

151. Serhan, C. N. Pro- resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101 (2014).

152. Campbell, E. L. et al. Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution. FASEB J. 21, 3162–3170 (2007).

153. Uddin, M. & Levy, B. D. Resolvins: natural agonists for resolution of pulmonary inflammation. Prog. Lipid Res. 50, 75–88 (2011).

154. Norling, L. V. et al. Proresolving and cartilage-protective actions of resolvin D1 in inflammatory arthritis. JCI Insight 1, e85922 (2016).

155. Donadio, J. V. & Grande, J. P. The role of fish oil/omega-3 fatty acids in the treatment of IgA nephropathy. Semin. Nephrol. 24, 225–243 (2004).

156. Dennis, G. J. et al. Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics. Arthritis Res. Ther. 16, R90 (2014).

157. Sokolove, J. et al. Rheumatoid factor as a potentiator of anticitrullinated protein antibody mediated inflammation in rheumatoid arthritis. Arthritis Rheum. 66, 813–821 (2015).

158. Kunwar, S., Dahal, K. & Sharma, S. Anti- IL-17 therapy in treatment of rheumatoid arthritis: a systematic literature review and meta- analysis of randomized controlled trials. Rheumatol. Intl 36, 1065–1075 (2016).

159. Yen, D. et al. IL-23 is essential for T cell–mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 1310–1316 (2006).

160. Azizi, G., Jadidi- Niaragh, F. & Mirshafiey, A. Th17 Cells in Immunopathogenesis and treatment of rheumatoid arthritis. Intl. J. Rheum. Dis. 16, 243–253 (2013).

161. van der Vlist, M., Kuball, J., Radstake, T. R. & Meyaard, L. Immune checkpoints and rheumatic diseases: what can cancer immunotherapy teach us? Nat. Rev. Rheumatol. 12, 593–604 (2016).

162. O’Neill, L. A., Kishton, R. J. & Rathmell, J. A guide to immunometabolism for immunologists. Nat. Rev. Immunol 16, 553–565 (2016).

AcknowledgementsThe work of the authors is supported by U01 AI101981 (V.M.H.), T32 AR07534 (V.M.H.), UH2 AR067681 (V.M.H.), UM1 AI110503 (V.M.H.), the Rheumatology Research Foundation (V.M.H. and K.D.), R01 AR051394 (V.M.H.), U19 AI50864 (V.M.H.), K08 DK107905 and Pfizer ASPIRE (K.A.K.). The authors appreciate the early input by R. Thomas to the conception of this Review.

Author contributionsV.M.H. researched data, contributed to discussion of content, wrote, reviewed and edited the manuscript before submis-sion. M.K.D., K.A.K., Y.O., J.M.N. and K.D.D. contributed to discussion of content, and reviewed and edited the manu-script before submission. J.H.B. and W.H.R. reviewed and edited the manuscript before submission.

Competing interestsThe authors declare no competing interests.

Publisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


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