The microbiota in spondyloarthritis. Matthew Stoll MD,PhD,MSCS University of Alabama at Birmingham June 4, 2017

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1 The microbiota in spondyloarthritis Matthew Stoll MD,PhD,MSCS University of Alabama at Birmingham June 4, 2017

2 Microbiota in arthritis A youth does not get gout before sexual intercourse. Hippocrates, ~ 400 BCE Nuki, Arthritis Res Ther 2006;8 Suppl 1:S1

3 19 th Century Observations It is by no means an infrequent occurrence for gonorrhea to produce a rheumatic and painful affection of the joints certain it is that gonorrhea produces opthalmia and rheumatism, and when not a single drop of matter has been applied to the eye. Astley Cooper, Lancet 1824;3:301

4 Why study the microbiota in spondyloarthritis? Reactive arthritis: clearly triggered by microbes Lessons from inflammatory bowel disease Clinical and genetic overlap with SpA Altered microbial populations Microbial immunologic targets Clues from animal models

5 Gut microbes in animal models of SpA Feature Rat model 1 Mouse model 2 Background 33-3 line B10.BR Transgene HLA-B27, β2m HLA-B27, β2m Clinical features Germ free state Germ free with defined bacteria Colitis, arthritis, spondylitis Enthesitis, ankylosis of ankles and tarsal joints Decreased colitis and arthritis DESEP No DESEP-B Yes No disease Lactobacillus no Mixture of 10 anaerobes Yes DESEP Streptococcus faecum Escherichia coli 1 Rath, Jrnl Clin Invest 1996;98:945 Streptococcus avium 2 Sinkorova, Human Immunol 2008;69:845 Eubacterium contortum Peptostreptococcus productus DESEP-B: DESEP + bacteroides vulgatus

6 Spondylitis microbiota pre NGS era Stool specimens obtained from 15 pts with AS and 15 controls Used PCR / DGGE to look for global differences Followed by assessments for individual differences Stebbings, Rheumatol 2002;41:1395

7 Gut microbiota in AS: 2002 Spondylitis Controls Stebbings, Rheumatology 2002;41:1395

8 PCR identified subtle differences Bacterial group Patients Controls Bacteroides Prevotella 12.5 (2.5) 16.7 (6.2) Bacteroides 16.7 (2.6) 21.1 (3.5) Bacteoides vulgatus 3.7 (1.2) 4.1 (1.3) Bifidobacterium 7.2 (2.8) 4.9 (1.1) Atopobium-Eggerthella-Collinsella 3.4 (0.9) 2.4 (1.5) Eubacterium rectale Clostridium coccoides 24.5 (4.0) 23.2 (3.3) Clostridium leptum 2.4 (0.8) 5.6 (2.4) Stebbings, Rheumatol 2002;41:1395

9 Mucosal microbiota in AS: 2015 Ileal biopsies obtained from 9 TNFinhibitor naïve AS patients and 9 controls 5/9 subjects had intestinal inflammation 16S sequencing performed Costello, Arth Rheumatol 2015;67:686

10 The AS microbiota is distinct Red: AS Blue: HC Proteobacteria Firmicutes Bacteroidetes Costello, Arth Rheumatol 2015;67:686

11 Mucosal microbiota in newly diagnosed SpA: 2017 Ileal and colonic biopsies obtained on 27 patients with treatment-naïve SpA and 15 controls 14/27 had inflammation Study primarily evaluated microbiota as function of disease activity among the SpA patients Tito, Arth Rheumatol 2017;69:114

12 Dialister may be associated with extent of inflammation in SpA ASDAS Not inflamed Acute Chronic Tito, Arth Rheumatol 2017;69:114 Dialister abundance ASDAS

13 Additional associations Inflamed tissue: Decreased Bacteroides Increased Prevotella and Lachnospiraceae Findings not significant after corrections for multiple comparisons

14 Fecal microbiota in PsA: 2015 Fecal specimens obtained from 16 newonset PsA patients and 28 controls 16S sequencing performed Scher, Arth Rheum 2015;67:128

15 Decreased diversity in PsA Scher, elife 2013;2:e01202

16 Decreased abundance of multiple rare bacteria in PsA Scher, Arth Rheum 2015;67:128

17 Why study pediatric SpA? Opportunity to capture early disease Diagnostic delay of ~ 10 years for AS Feldtkeller, Rheumatol Int 2003;23:61 Radiographic changes may begin at age 8 Brophy, Jrnl Rheumatol 2002;29:1236 BASRI Age

18 Microbiota in juvenile SpA Fecal specimens obtained from 27 children with enthesitis-related arthritis and 13 pediatric healthy controls 17 were on TNF inhibitors, 3 on conventional DMARDs alone 16S sequencing performed Stoll, Arth Res Ther 2014;16:486

19 Microbiota in ERA: Alabama Decreased Faecalibacterium prausnitzii in children with ERA Small patient cluster identified by PCoA Fecal abundance of F. prausnitzii Principal coordinates analysis Fraction of total bacteria ERA Controls Blue: patients Red: controls Stoll, Arth Res Ther 2014;16:486

20 Clusters are defined by Bacteroides and Akkermansia The two clusters had similar abundance of F. Prausnitzii The subjects who clustered with the controls tended to have high abundance of Akkermansia (> 1% in 7/17 vs 0/8) The subjects forming their own cluster had high Bacteroides abundance (41% vs 13%) Stoll, Arth Res Ther 2014;16:486

21 Microbiota in ERA: India Fecal specimens obtained from 33 children with ERA and 14 healthy controls All naïve to TNFi as well as to DMARDs within prior six weeks 16S sequencing performed Aggarwal, Clin Exp Immunol 2016;187:480

22 Microbiota in ERA: India Aggarwal, Clin Exp Immunol 2016;187:480

23 Variability within studies Study Population Source of material Stebbings 2002 Costello 2015 Tito 2017 Scher 2015 Technology Key findings AS Fecal DGGE Decreased Bacteroides TNFi-naïve AS Treatmentnaïve SpA Treatmentnaïve PsA Biopsy 16S seq. Increased diversity, Decreased Veillonellaceae Prevotellaceae Biopsy 16S seq. Dialister associated with inflammation. Decreased Bacteroides Fecal 16S seq. Decreased diversity and abundance of rare bacteria Stoll 2014 ERA Fecal 16S seq. Increased Bacteroides, Decreased F. praunitzii Aggarwal 2016 TNFi-naïve ERA Fecal 16S seq. Increased Bacteroides, Decreased Prevotella

24 Variability within studies Study Population Source of material Stebbings 2002 Costello 2015 Tito 2017 Scher 2015 Technology Key findings AS Fecal DGGE Decreased Bacteroides TNFi-naïve AS Treatmentnaïve SpA Treatmentnaïve PsA Biopsy 16S seq. Increased diversity, Decreased Veillonellaceae Prevotellaceae Biopsy 16S seq. Dialister associated with inflammation. Decreased Bacteroides Fecal 16S seq. Decreased diversity and abundance of rare bacteria Stoll 2014 ERA Fecal 16S seq. Increased Bacteroides, Decreased F. praunitzii Aggarwal 2016 TNFi-naïve ERA Fecal 16S seq. Increased Bacteroides, Decreased Prevotella

25 What could be driving these differences in the results? Age of onset Disease duration Diagnosis (AS, USpA, PsA, ERA) Treatment Fecal versus biopsy Technology Geography Random variation

26 Two parallel studies conducted Treatment-naïve children with ERA 30 with ERA versus 19 controls Long-standing adult SpA 11 with SpA vs 10 controls AS (n = 6), PsA (n = 2), USpA, ReA, IBD-arthritis TNFi (n = 4), DMARDs (n = 5), none (n = 2) No antibiotic use for three months

27 Clustering by diagnosis in ERA Blue: patients Red: controls Permanova test: F = 1.45, p = 0.046

28 Not in adult SpA Blue: patients Red: controls

29 Taxonomic assessment in ERA Pairwise comparisons revealed minimal differences between the groups Especially after adjustment for multiple comparisons At the phylum level, decreased Actinobacteria in ERA (3.3 vs 9.0%, p < 0.002) Focused on organisms of interest based upon previous work Faecalibacterium prausnitzii Bacteroides

30 Higher B. fragilis in children with newly diagnosed ERA

31 Lower abundance of Bacteroides in adult SpA patients Bacteroides Bacteroides fragilis

32 Summary of findings: adults Study Stebbings 2002 Costello 2015 Tito 2017 Scher 2015 Stoll, unpublished data Population Source of material Technology Key findings AS Fecal DGGE Decreased Bacteroides TNFi-naïve AS Treatmentnaïve SpA Treatmentnaïve PsA Treatmentexposed SpA Biopsy 16S seq. Increased diversity, Decreased Veillonellaceae Prevotellaceae Biopsy 16S seq. Dialister associated with inflammation. Decreased Bacteroides Fecal 16S seq. Decreased diversity and abundance of rare bacteria Fecal 16S seq. Decreased Bacteroides

33 Summary of findings: pediatrics Study Populati on Source of material Technology Key findings Stoll 2014 ERA Fecal 16S seq. Increased Bacteroides, Decreased F. praunitzii Aggarwal 2016 Stoll, unpublished data TNFinaïve ERA Fecal 16S seq. Increased Bacteroides, Decreased Prevotella ERA Fecal 16S seq. Increased B. fragilis, Decreased A2-165 strain of F. praunitzii

34 Similar findings with other categories of arthritis Pediatrics Increased abundance Stoll (2014); ERA Tejesvi (2016); ojia, pjia Aggarwal (2017); ERA Muller (2017); pjia Stoll (preliminary data); ERA Decreased abundance None Adults Zhang et al. (2016); RA Stebbings (2002); AS Vaahtovuo (2008); RA Scher (2013); RA Maeda (2016); RA Tito (2017); SpA Stoll (preliminary data); SpA

35 And in IBD Abundance of Bacteroides in patients with IBD Metaanalysis of early (adult) studies Zhou, Biomed Res Int 2016;2016:

36 And in IBD Studies using NGS technology Increased abundance Decreased abundance Pediatrics Kolho (2015); CD Gevers (2014); CD Adults Abundance of Bacteroides in patients with IBD Le Page (2011); UC Tyler (2013); UC Mar (2016); UC Takahasi (2016); CD

37 Polysaccharide A on B. fragilis inhibits development of Th17 cells Colonic LP lymphocytes Round, Science 2011;332:974

38 Increase in Bacteroides precedes onset of Type I IDDM Abundance of Bacteroidetes Abundance of B. ovatus Timepoint Controls Cases Baseline 1.74% 1.53% Onset 0.62% 9.94% Onset Giongo, ISME 2011;5:82

39 Bacteroides may contribute to type I DM in developed nations Abundance of fecal Bacteroides Cytokine production in human PBMCs Endotoxin tolerance in human PBMCs Onset of diabetes in NOD mice Vatanen, Cell 2016;165:842

40 Summary of Bacteroides findings Anti-inflammatory organism Especially B. fragilis Exposure in early childhood is associated with a variety of autoimmune diseases Adult exposure appears to be protective Thus, in pediatric autoimmune diseases, increased abundance of Bacteroides may be a marker of altered immunologic development Illustration of hygiene hypothesis

41 Immunogenicity of gut bacteria Most of the bacteria identified to date as relevant to SpA may affect general immune function, but are not necessarily directly pathogenic Other studies have focused on immunogenicity of specific organisms

42 Altered immunologic reactivity to commensal organisms in IBD Serologic expression cloning Pelleted bacteria from mouse cecum Phage DNA library expressed in E. coli Probed with colitic serum Lodes, J Clin Invest 2004;113:1296 Targan, Gastroenter 2005;128:2020

43 Anti-flagellin T cells drive inflammatory response in mice Adoptive transfer into scid mice In vitro polarization Adoptive transfer Pathology Scores Histopathology scores Lodes, J Clin Invest 2004;113:1296 Feng, J Immunol 2011;186:6313

44 Other target antigens in CD Plevy, Inflam Bowel Dis 2013;19:1139

45 Increased reactivity to IBDassociated antigens in SpA Wallis, Arth Res Ther 2013;15:R166

46 Or perhaps not Riente, Jrnl Rheumatol 2004;31:920

47 Summary of findings Study ASCA IgA ASCA IgG panca I2 OmpC CBir Hoffman (2003) ND ND ND ND Torok (2004) ± ND ND ND Riente (2004) ND ND ND ND Aydin (2008) ND ND ND ND Mundwiler (2009) ND Wallis (2013) ND Adapted from Stoll, Clin Immunol 2015;159:134

48 Are other enteric antigens targeted? Where do you begin to look? In India, Salmonella-induced ReA is common Could Salmonella be behind other forms of SpA?

49 Salmonella is immunogenic in Indian patients with SpA Study of patients with ReA 1 SFMC of 20/53 demonstrated proliferative responses to Salmonella 19/20 responded to OMP Confirmatory study of ReA and USpA 2 CD8 proliferative responses to crude OMP lysate detected in 12/16 with ReA and 5/14 with USpA, versus 1/10 with RA 16/17 SpA versus 0/10 RA patients also responded to recombinant OMP 1 Saxena, J Rheumatol 2005;32:86 2 Chaurasia, Clin Exp Immunol 2016;185:210

50 Altered Salmonella reactivity among children with ERA S.I. to OMP in ERA subjects IgG against OMP Singh, J Rheumatol 2011;38:161

51 E. Coli may be immunogenic in SpA Increased Abs to E. coli in AS 1 IgG: culture supernatant, LPS IgA: OMP Increased Ifnγ production after culture of PBMCs with pool of E. coli proteins compared to RA but not HC 2 1 Madhavan, Ann NY Acad Sci 2002;958:408 2 Syrbe, Ann Rheum Dis 2012;71:1573

52 What about Klebsiella? Evidence for involvement 1 Increased humoral response to K. Pneumoniae Proposed cross-reactivity of HLA-B27 and K. pneumoniae Contradictory data; no differences in humoral or T cell reactivity against K. pneumoniae btw patients with AS and unaffected relatives 2 1 Rashid and Ebringer, Clin Rheumatol 2007;26:858 2 Stone, Rheumatol 2004;43:148

53 Antigen array to identify enteric targets in SpA Developed by Dr. Charles Elson (UAB) Slides printed with enteric antigens Flagellin antigens with known links to IBD Randomly selected peptides with no known association with IBD Designed to screen for antigenic associations with IBD Christmann, JACI 2015;136:1378

54 Antigen array in IBD Red: Crohn Disease Blue: Controls Elson, unpublished data

55 Initial array in ERA Red: ERA Blue: Controls Red: ERA Blue: Controls Green: ERA/IBD

56 Elevated P. oralis IgG in ERA Red: ERA Blue: Controls

57 Additional organisms Red: ERA Blue: Controls IgG Red: ERA Blue: Controls IgA

58 Large array study Feature Controls ERA n Age at diagnosis (years; median, IQR) N/A 12.9 ( ) Age at collection (years; median, IQR) 12.5 ( ) 13.7 ( ) Disease duration (months; median, IQR) N/A 0.13 (0 1.1) Males (n, %) 33, 56% 44, 53% Race (n, %) Caucasian African-American Other 39, 66% 15, 25% 5, 8.5% 60, 72% 18, 22% 5, 6.0% HLA-B27+ (n, %) 0 / 6, 0% 23/78, 28% Medicines (n, %) None Conventional DMARDs alone TNFi alone TNFi + conventional DMARDs 59, 100% 46, 55% 15, 18% 4, 4.8% 18, 21.7%

59 ERA patients have increased antibodies against Prevotella Red: ERA Blue: Controls IgG Red: ERA Blue: Controls IgA

60 Increased P. oralis IgA levels in ERA patients

61 Summary SpA patients have increased antigenic reactivity to enteric organisms, including commensal organisms IBD and SpA may have different targets Animal data in IBD indicates that T cells directed against commensal organisms are pathogenic

62 Conclusion Multiple organisms have altered abundance in SpA Decreased abundance of F. prausnitzii Bacteroides may be depleted in adult disease, yet abundant in pediatric disease Dialister may correlate with disease activity Other organisms may stimulate aberrant immune responses Salmonella in endemic areas Enterobacteriaceae Prevotella species

63 Acknowledgements UAB Lou Bridges Randy Cron Maio Danila Peter Eipers Charles Elson Laura Hughes Ranjit Kumar Elliot Lefkowitz Casey Morrow Kathy Pierce Other sites Pamela Weiss Jennifer Weiss Barbara Edelheit Peter Nigrovic Charles Spencer Lynn Punaro Kenneth Schikler Andreas Reiff Funding NIAMS ACR UAB CoA CARRA