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The microbiome and HLA-B27 associated inflammatory disease.
Dr. Mark Asquith
Division of Rheumatology and Arthritic DiseasesDepartment of MedicineOregon Health & Science University
Disclosures
Mark Asquith, PhD, has no ACCME-defined commercial relationships.
~ 1-3kg
Biomass Cell content
Geneticcontent
23,000 host genes
2 – 10 million
microbialgenes
~ 300g
~ 2kg
~ 200g
~ 1.4kg
Spondyloarthropathies (SpAs)
AnkylosingSpondylitis
Acute Anterior Uveitis
Reactive Arthritis Psoriasis
HLA-B27 is very prevalent amongst individuals with SpAs….
Pathogenic role of HLA-B27
Molecular mechanism still remains enigmatic.
- Arthritogenic peptide theory: HLA-B27 may bind unique peptides of self or foreign origin.
- Molecular mimicry hypothesis: Antibodies specific for foreign antigens cross-react with HLA-B27.
- Aberrant processing: Misfolding of HLA-B27 may trigger aberrant antigen presentation.
- ER stress: Misfolding of HLA-B27 may trigger inflammatory ER stress responses.
The gut and spondyloarthropathy (SpA)
• Over half of AS patients exhibit subclinical bowel inflammation.
• Development of reactive arthritis following enteric infection (e.g. Salmonella, Shigella and Campylobacter spp.).
• Germ-free or Abx-treated HLA-B27 transgenic rats do not develop disease.
• AS patients exhibit an altered or dysbiotic gut microbiome
• Approx. 5% of IBD patients progress to SpA.
CD103+
DC
FoxP3+
TR
Tr1
IL-10
TGFβ VitA
IL-6
TNF-αIL-1β
CD103-
DC
IL-23
Th1Th17
INFLAMMATION REGULATION
Disrupted immune homeostasis drives the development of intestinal inflammation.
Intestinal microbes that induce inflammatory and anti-inflammatory responses is are increasingly well defined…
Th17
FoxP3+VE TREG
Segmented Filamentous Bacteria (SFB)
Ivanov et al. Cell 2010
Clostridial spp.
GF GF+C
Atarashi et al. Nature 2013
INFLAMMATION REGULATION
The paradigm of microbial dysbiosis
INFLAMMATION REGULATION
The paradigm of microbial dysbiosis
What is the impact of HLA-B27 expression on the intestinal microbiota?
6 wks
16 wks
WT HLA-B27/β2m
Isolate bacterial DNA from stool, skin biopsy, other tissue site…
Amplify 16s rRNA genes and sequence. Bacterial DNA pool that is amplified will contain unique 16s rRNA genes for distinct bacterial spp.
Align DNA sequences to a reference database of known 16s rRNA genes.Perform taxonomic identification and phylogenetic analysis
16s rRNA sequencing overview
16
WT HLA-B27+
HLA-B27 transgenic rats exhibit a distinct intestinal microbiota to WT controls.
Asquith et al.A & R 2016
Burkholderiales alcaligenaceae sutterella
Alphaproteobacteria RF32Erysipelotrichaceae clostridiumClostridiales lachnospiraceae roseburiaBacteroidales S247Bacteroidales bacteroidaceae bacteroidesClostridiales lachnospiraceae blautiaVerrucomicrobiaceae akkermansia
ClostridialesClostridiales rumminococceae oscillospira
Clostridiales rumminococceae rumminococcusClostridiales lachnospiraceae rumminococcus
Turicibacteraceae turicibacterClostridiales clostridiaceae other
Clostridiales lachnospiraceae butyrivibrioClostridiales rumminococcaeae other
HLA B27 > WT WT > HLA B27
HLA-B27 dependent dysbiosis
CECAL CONTENTS
UHPLC/MS and GC/MSMETABOLOMICS
6 and 16 weeks
Does HLA-B27 expression modify the intestinal
metabolome?
GENOTYPE: WT
16 WKS (DISEASED)6 WKS (PRE-DISEASE)
B27+ WT B27+
HLA-B27 expression profoundly alters the intestinal metabolome
Asquith et al. A&R 2017
Does HLA-B27 expression impact the intestinal microbiota of humans?
ROUTINE COLONOSCOPY (COLON CANCER SCREENING)
BLOOD SAMPLE
COLLECT ILEUM, CECUM, RIGHT COLON (asc.), LEFT COLON (desc.), RECTUM AND STOOL
MICROBIOTA ANALYSIS 16s rRNA gene sequencing
HLA-B genotyping
16s rRNA gene community profiling of the intestinal microbiota
N = 11B27+
N = 85B27-
ILEAL BIOPSIES
16s rRNA gene sequencing
HLA-B27+ve individuals exhibit a distinct gut microbiota to those that
are HLA-B27-ve
P < 0.05PERMANOVA
What is the impact of HLA-B27 expression on intestinal immunity?
LUMEN
MUCOSA
EPITHELIAL MONOLAYER
MUCUS LAYER
ANTI-MICROBIALPEPTIDES
SECRETORYIgA
B
BB
B
BB
B
B
B CELLS
B
BB
B
BB
B
B
Th17
Th17
Th17
Th17
Th17
Th17Th17
Th17 CELLS
TIGHT JUNCTIONPROTEINS
B
BB
B
BB
B
B
Th17
Th17
Th17
Th17
Th17
Th17Th17
Early dysregulation of innate immunity in HLA-B27 rats
WT HLA-B27/β2m
*
*
Huang et al. Dis Markers. 2017; 2017: 7574147
S100A8 is also upregulated in Spondyloarthritis patients
Dysregulated CD4+ Th17 responses in HLA-B27 rats
WTHLA-B27/β2m
FECAL PELLET
extract bacteri
a
stain with
Anti-IgA
IgA+ bacteria
SYTO BC
anti
-IgA
SYTO9 (nucleic acid)
anti-IgABV421
WT HLA-B27/β2m
Asquith et al. A & R. 2016
IgA- bacteria
The frequency of IgA coated fecal bacteria is greatly expanded in HLA-B27/β2m animals and increased in animals with arthritis.
WTHLA-B27/β2m
WTHLA-B27/β2m
Intestinal isotype switching in HLA-B27 transgenic rats
FECAL PELLET
extract bacteria
stain with Anti-IgA
Sort IgA+ and IgA- bacteria
by FACS
16s sequencing
New approaches….
IgA coated(IgA+ve)
IgA coated(IgA-ve)
A
B
C
A
B
C
IgA NEG
IgA POS
IgA+ bacteria
IgA- bacteria
SYTO BC
anti
-IgA
20%
60%
20%
50%
30%
20%
Frequency in each fraction determined by 16s rRNAgene sequencing..IgA COATING INDEX:
= frequency in IgA+ve/frequency in IgA-ve
E.g.A = 50/20 = 2.5 (more IgA coated)
B = 30/60 = 0.5 (less IgA coated)
C = 20/20 = 1 (no difference)
WTWT B27f_Verrucomicrobiaceae.g_Akkermansiaf_Veillonellaceae.g_Phascolarctobacteriumf_Turicibacteraceae.g_Turicibacterf_Streptococcaceae.g_Treponemaf_Spirochaetaceae.g_Treponemaf_S24.7.g_f_Ruminococcaceae.g_Ruminococcusf_Ruminococcaceae.g_Oscillospiraf_Ruminococcaceae.g_f_Rikenellaceae.g_f_Prevotellaceae.g_Prevotellaf_Porphyromonadaceae.g_Parabacteroidesf_Peptococcaceae.g_rc4.4f_Lactobacillaceae.g_Lactobacillusf_Lachnospiraceae.g_Roseburiaf_Lachnospiraceae.g_Doreaf_Lachnospiraceae.g_Coprococcusf_Lachnospiraceae.g_Blautiaf_Lachnospiraceae.g_Anaerostipesf_Lachnospiraceae.g_Ruminococcusf_Lachnospiraceae.g_f_Helictobacteraceae.g_f_Erysipelotrichaceae.g_Holdemaniaf_Erysipelotrichaceae.g_Coprobacillusf_Erysipelotrichaceae.g_Allobaculumf_Erysipelotrichaceae.g_Eubacteriumf_Erysipelotrichaceae.g_f_Enterobacteriaceae.g_f_Desulfovibrionaceae.g_Desulfovibriof_Desulfovibrionaceae.g_f_Dehalobacteriaceae.g_Dehalobacteriumf_Coriobacteriaceae.g_Adlercreutziaf_Clostridiaceae.g_f_Christensenellaceae.g_f_Bacteroidaceae.g_Bacteroidesf_Anaeroplasmataceae.g_Anaeroplasmaf_Alcaligenaceae.g_Sutterellaf_Paraprevotellaceae.g_YRC22f_Paraprevotellaceae.g_Prevotellaf_Mogibacteriaceae.g_
Negative IgA Index
Positive IgA Index
SIGNIFICANCE
MAGNITUDE
p__Firmicutes;c__Clostridia;o__Clostridiales;f__Clostridiaceae;Other
p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__[Paraprevotellaceae];g__[Prevotella]
p__Spirochaetes;c__Spirochaetes;o__Spirochaetales;f__Spirochaetaceae;g__Treponema
p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__;g__
p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__
p__Proteobacteria;c__Deltaproteobacteria;o__Desulfovibrionales;f__Desulfovibrionaceae;g__
p__Proteobacteria;c__Deltaproteobacteria;o__Desulfovibrionales;f__Desulfovibrionaceae;g__Desulfovibrio
p__Verrucomicrobia;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Verrucomicrobiaceae;g__Akkermansia
p__Firmicutes;c__Clostridia;o__Clostridiales;f__Clostridiaceae;g__
p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__[Paraprevotellaceae];g__YRC22
p__Firmicutes;c__Erysipelotrichi;o__Erysipelotrichales;f__Erysipelotrichaceae;g__Allobaculum
p__Tenericutes;c__Mollicutes;o__Anaeroplasmatales;f__Anaeroplasmataceae;g__Anaeroplasma
p__Firmicutes;c__Bacilli;o__Turicibacterales;f__Turicibacteraceae;g__Turicibacter
p__Firmicutes;c__Clostridia;o__Clostridiales;f__Clostridiaceae;g__Segmented filamentous bacteria
p__Firmicutes;c__Clostridia;o__Clostridiales;f__Veillonellaceae;g__Phascolarctobacterium
p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Helicobacteraceae;Other
p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Blautia
346.9
306.7
198.9
156.6
111.4
102.9
76.0
73.2
56.9
50.6
42.2
39.3
36.4
31.2
29.3
28.6
28.3
1.5
2.4
1.9
2.3
1.9
1.8
1.5
0.8
1.2
1.8
1.6
15.6
1.6
3.4
1.9
1.3
0.62
MICROBIAL OTU ICI TG ICI WT
HLA-B27 expression alters the intestinal IgA repertoire
ROUTINE COLONOSCOPY (COLON CANCER SCREENING)
BLOOD SAMPLE
COLLECT ILEUM, CECUM, RIGHT COLON (asc.), LEFT COLON (desc.), RECTUM AND STOOL
MICROBIOTA ANALYSIS 16s rRNA gene sequencing
HLA-B genotyping
HLA-B27 shapes the intestinal microbiota in humans
Preliminary data (low n!!!) indicates HLA-B27 also modifies the IgA repertoire in HLA-B27+ve individuals.
HLA-B27-ve HLA-B27+ve
f_Verrucomicrobiaceae.g_Akkermansiaf_Veillonellaceae.g_Succiniclasticumf_Veillonellaceae.g_Phasolarctobacteriumf_Veillonellaceae.g_Dialisterf_Veillonellaceae.g_Acidaminococcusf_Streptococcaceae.g_Streptococcusf_Ruminococcaceae.g_unknownf_Ruminococcaceae.g_Ruminoococcusf_Ruminococcaceae.g_Oscillospiraf_Ruminococcaceae,g_Faecalibacteriumf_Ruminococcaceae.g_Butyricicoccusf_Ruminococcaceaef_Rikenellaceaef_Prevotellaceae.g_Prevotellaf_Porphyromonadaceae.g_Porphyromonasf_Porphyromonadaceae.g_Parabacteroidesf_Lactobacillaceae.g_Lactobacillusf_Lachnospiraceae.g_unknownf_Lachnospiraceae.g_Roseburiaf_Lachnospiraceae.g_Lachnospiraf_Lachnospiraceae.g_Lanchnobacteriumf_Lachnospiraceae.g_Doreaf_Lachnospiraceae.g_Coprococcusf_Lachnospiraceae.g_Clostridiumf_Lachnospiraceae.g_Blautiaf_Lachnospiraceae.g_Ruminococcusf_Lachnospiraceaef_Enterobacteriaceae.g_Klebsiellaf_Enterobacteriaceae.g_Escherichiaf_Desulfovibrionaceae.g_Bilophilaf_Coriobacteriaceae.g_Collinsellaf_Clostridiaceae.g_Clostridiumf_Christensenellaceae.g_f_Bifidobacteriaceae.g_Bifidobacteriumf_Bacteroidaceae.g_Bacteroidesf_Alcaligenaceae.g_Sutterellaf_Tissierellaceae.g_WAL_1855Df_Tissierellaceae.g_Peptoniphilusf_Tissierellaceae.g_Finegoldiaf_Tissierellaceae.g_Anaerococcusf_Paraprevotellaceae.g_Prevotellaf_Odoribacteraceae.g_Odoribacterf_Mogibacteriaceae.g_
Negative IgA Index
Positive IgA Index
SIGNIFICANCE
MAGNITUDE
• What impact does HLA-B27 have on intestinal barrier function?
WT B27B27WT
CLAUDIN-4MAADAPI
DIMINISHED TIGHT JUNCTION EXPRESSION IN HLA-B27 RATS
Mid colon5 wks
Microbial translocation – a link from gut to joint?
f_Alicagenesg_suturella
f_Lachnospiriceaeg_roseburia
f_Prevotellaceaeg_prevotella
f_Erysipelotrichaceaeg_coprobacillus
TG WT TG WT TG WT TG WT
TG WT TG WT
f_Verrucomicrobiag_akkermansia
f_Bacteroideceaeg_bacteroides
ANKLE
STEADY STATE.
INFLAMMATION.
MIGRATION TO EXTRAINTESTINAL
SITES.
MIGRATION TO EXTRAINTESTINAL
SITES.
Increased trafficking of lymphocytes from gut to eye is observed in
Experimental Autoimmune Uveitis (EAU)
EAU (immunized 3 weeks prior)
405nm laser
48 hours
SUMMARY
ALTERED INNATE IMMUNE RESPONSES(E.g. Mucus and Antimicrobial Peptides)
1
ALTERATION OF THE INTESTINAL MICROBIOTADYSBIOSIS
2
2
- OUTGROWTH of mucin degrading bacteria- OUTGROWTH of Th17-inducing bacteria- LOSS of Clostridial Spp.
‘RUNAWAY’ ADAPTIVE IMMUNE RESPONSES
3
INFLAMMATION, REDUCED BARRIER FUNCTION AND ENHANCED MICROBIAL TRANSLOCATION
4
5INFLAMMATORY FEEDBACK LOOP OF INFLAMMATION AND ENHANCED
MICROBIAL TRANSLOCATION5INFLAMMATORY FEEDBACK LOOP OF INFLAMMATION AND ENHANCED
MICROBIAL TRANSLOCATION5INFLAMMATORY FEEDBACK LOOP OF INFLAMMATION AND ENHANCED
MICROBIAL TRANSLOCATION
6
7
BACTERIA
flagellin
LPS
DNA
BACTERIALPRODUCTS
IMMUNE CELLS
MECHANICAL STRESS?VIRAL INFECTION?
ER STRESS RESPONSE?
HLA-B27Adapted from Brown et al.Nat Rev Rheum 2016
Potential translational implications?
Adapted from Lozupone et al.
Nature. 2012489(7415):220-30.
mindbodymicrobiome.com
DIETARY POLYSACCHARIDES (FIBER)
monosaccharidesSCFAs MCFAs
Clostridial spp…
oligoosaccharides
10 wks6 wks
Sodium propionate or buytrate (150 mM) in drinking water
Disease?
SCFA study
HLA-B27/β2m rats
SCFA administration significantly attenuates B27-associated inflammation
C57 NaCL
C57 Prop
B10 Prop
3wk 4wk
Grade 1 Grade 1.5
Grade 0 Grade 0
Grade 3.5
Grade 3.5
Grade 3.5
Grade 0
SCFA administration significantly attenuates Experimental Autoimmune Uveitis (EAU)
Nakamura et al. Sci Reports. In press.
79
Fecal Microbiota Transfer (FMT)
Nood et al. Jan 2013. NEJM.
FMT SCHEMATIC
HLA-B27/β2m rats
AmpicillinVancomycinNeomycinMetronidazole
4wks ad libitum 6wks oral gavage of donor feces(from HC or AS donor pools)
Daily (X3) followed by weekly (x6) FMT
Healthy Control and AS donor feces exhibit differential Th17 inductive potential
FREQUENCY OF CD4+ Th17 cells in the intestinal lamina propria
Longitudinal Analysis of the Intestinal Microbiota
40% develop arthritis by 24 wks of age
60% remain disease free
COLLECT STOOL WEEKLY AND ANALYZE MICROBIOTA BY qRT-PCR.
HLA-B27/β2m rat
Do those animals that go on to develop arthritis have a distinct microbiota to those that remain disease-free?
Early expansion of Akkermansia muciniphila correlates with subsequent arthritis development in HLA-B27+ rats
SUMMARY
• HLA-B27 profoundly alters the intestinal microbiome.
• HLA-B27 expression leads to dysregulated innate and adaptive immune responses.
• HLA-B27 drives changes to the human microbiota, even in healthy individuals.
• Longitudinal analysis of the microbiota may predict disease progression.
• Microbial metabolites may attenuate B27-associated inflammatory disease.
Acknowledgements
OHSUAsquith LabPatrick StaufferSean DavinClaire Mitchell Christina MeteaMatthew Schleisman Dr. Yongseop HanMelanie Alvarado Dr. Erick RiveraMark KlickProf. Jim RosenbaumProf. Phoebe Lin
Prof. Steve PlanckDr. Sarah DiamondDr. Tammy MartinDr. Lisa Karstens
Oregon State UniversityDr. Natalia Shulzhenko
UCSD
Prof. Rob Knight
Dr. Gail Ackermann Justine Debelius
Chris Lauber
NIH/NIAMS
Prof. Robert Colbert
Dr. Tejpal Gill
UT Southwestern/University of Paris
Prof. Joel Taurog/Maxime Breban
University of QueenslandProf. Matt BrownDr. Mary-Ellen Costello
