The microbiome and HLA-B27 associated inflammatory disease. · The microbiome and HLA-B27...

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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

~ 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+

FoxP3+

TGFβ VitA

TNF-αIL-1β

CD103-

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…

FoxP3+VE TREG

Segmented Filamentous Bacteria (SFB)

Ivanov et al. Cell 2010

Clostridial spp.

GF GF+C

Atarashi et al. Nature 2013

The paradigm of microbial dysbiosis

What is the impact of HLA-B27 expression on the intestinal microbiota?

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

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?

MUCOSA

EPITHELIAL MONOLAYER

MUCUS LAYER

ANTI-MICROBIALPEPTIDES

SECRETORYIgA

B CELLS

Th17Th17

Th17 CELLS

TIGHT JUNCTIONPROTEINS

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

stain with

Anti-IgA

IgA+ bacteria

SYTO BC

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

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)

IgA NEG

IgA POS

IgA+ bacteria

IgA- bacteria

SYTO BC

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

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

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)

ALTERATION OF THE INTESTINAL MICROBIOTADYSBIOSIS

- OUTGROWTH of mucin degrading bacteria- OUTGROWTH of Th17-inducing bacteria- LOSS of Clostridial Spp.

‘RUNAWAY’ ADAPTIVE IMMUNE RESPONSES

INFLAMMATION, REDUCED BARRIER FUNCTION AND ENHANCED MICROBIAL TRANSLOCATION

5INFLAMMATORY FEEDBACK LOOP OF INFLAMMATION AND ENHANCED

MICROBIAL TRANSLOCATION5INFLAMMATORY FEEDBACK LOOP OF INFLAMMATION AND ENHANCED

MICROBIAL TRANSLOCATION

BACTERIA

flagellin

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 0

SCFA administration significantly attenuates Experimental Autoimmune Uveitis (EAU)

Nakamura et al. Sci Reports. In press.

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

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

The microbiome and HLA-B27 associated inflammatory disease. · The microbiome and HLA-B27...

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