Differential responses of human colonic ILCs to gut commensal bacteria altered during HIV infection

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1 Differential responses of human colonic ILCs to gut commensal bacteria altered during HIV infection Moriah J. Castleman, Ph.D. Laboratory of Dr. Cara Wilson University of Colorado Anschutz Medical Campus Department of Medicine/Division of Infectious Disease October 18, 2017

2 HIV-1 pathogenesis in gut: Microbial Translocation Lumen Gram - Gram + Epithelium Lamina propria Innate Inflammation HIV-1 CD4 T Circulation Lymphatics

3 Epithelial barrier protection and damage Protection Damage Proliferation Internalization of tight junctions Increased permeability IL-10 IL-22 Survival IFNγ Bacteria Breach Antimicrobial peptide Mucus Upregulates TNFα receptor Apoptosis Non-T cell mucosal producers of IL-22 identified as Group 3 ILCs (ILC3s). Non- T cells mucosal producers of IFNγ identified as Group 1 ILCs (ILC1, NK cells).

4 Innate lymphoid cells (ILCs) Groups Adaptive Innate Group 2 Group 3 Homeostatic function in human gut? In context of HIV-1 infection? Group 1 Walker et. al. (2013) Nature Immunology.

5 Inflammatory colonic ILCs during untreated HIV-1 infection Untreated HIV infected subjects have increased frequency of colonic IFNγ+ ILCs This same ILC subset primarily produced IL-22 in uninfected subjects (characterized as NKp44+ CD56-)(Dillon et. al. JAIDS, 2017) IFNγ+ ILCs positively correlated with colonic mucosa-associated dysbiotic bacteria Proteobacteria family ( in HIV infection) Prevotella species ( in HIV infection) Homeostatic IL-22+? HIV-1 Commensal Bacteria Pro-inflammatory IFNγ+

Microbial Induction of ILC response Murine studies suggest that gut mucosal ILCs respond to bacteria Little is known about how ILCs respond to bacteria in humans Objective: 1.

6 Microbial Induction of ILC response Murine studies suggest that gut mucosal ILCs respond to bacteria Little is known about how ILCs respond to bacteria in humans Objective: 1. To determine the cytokine response of human colonic ILCs to gut bacteria 2. To understand the contribution of ILCs to inflammation during HIV infection Image from Eberl et. al. (2015) Science, Satoh-Takayama et. al. (2008), Sanos et. al. (2009), Sano et. al. (2015), Qui et. al. (2013), Zelante et. al. (2013), Esin et. al. (2008), Crellin et. al. (2010)

Methodology: Ex vivo model of human colonic tissue immune cell

Propria (LPMC) Two step isolation procedure Culture LPMCs ±

IFNγ *Golgi Plug added for last 4 hours Multi-color flow

7 Methodology: Ex vivo model of human colonic tissue immune cell function Macroscopically normal discarded surgical tissue Lamina Propria (LPMC) Two step isolation procedure Culture LPMCs ± Bacteria 20 hours Collect and stain intracellular for IL-22 and IFNγ *Golgi Plug added for last 4 hours Multi-color flow cytometer Howe et. al. Clinical Immunology (2009), Dillon et. al. J Immunol (2010)

8 Methodology: Gut Intestinal Bacteria of Clinical Relevance Bacteria Species Ruminococcus bromii (Rb) Acinetobacter junii (Aj) Prevotella stercorea (Ps) Bifidobacterium infantis (Bi) Salmonella typhimurium (St) Clinical Significance Gram positive, abundance in HIV+ Gram negative, abundance in HIV+ Gram negative, abundance in HIV+ Gram positive, Probiotic Gram negative, Pathogen, HIV+ have increased risk for bacteremia 2 Dillon et al Mucosal Immunology (2014), Dillon et. al. Retrovirology (2016) CDC, NIH HIVMA, AIDS Info. (2017)

9 Induction of IL-22 in ILC3s by Gut Bacteria Bacteria Bacteria Cell Wall %IL-22+ of ILC3s * * *** *** ** % IL-22+ of the ILC 3s * Bi Rb Aj Ps St - LTA LPS Gram Positive Bacteria Gram Negative Bacteria Gram Positive Bacteria Gram Negative Bacteria Bi: Bifidobacterium infantis Aj: Acinetobacter junii LTA: Lipoteichoic Acid LPS: Lipopolysaccharide Rb: Ruminococcus bromii Ps: Prevotella stercorea St: Salmonella typhimurium Whole bacteria (N=6-9) Cell Wall Component (N=3) Error: SEM

10 Induction of IFNγ in ILC3s by Gram Negative Gut Bacteria Bacteria Bacteria Cell Wall 10 * 10 % IFN γ + of ILC3s % IFN γ + of the ILC3s Bi Rb Aj Ps St - LTA LPS Gram Positive Bacteria Gram Negative Bacteria Gram Positive Bacteria Gram Negative Bacteria Bi: Bifidobacterium infantis Aj: Acinetobacter junii LTA: Lipoteichoic Acid LPS: Lipopolysaccharide Rb: Ruminococcus bromii Ps: Prevotella stercorea St: Salmonella typhimurium Whole bacteria (N=6-9) Cell Wall Component (N=3) Error: SEM

11 Induction of IFNγ in NK by Gram Negative Gut Bacteria Bacteria Bacteria Cell Wall 30 ** * ** 30 % IFN γ + of NKs % IFN γ + of NKs Bi Rb Aj Ps St 0 - LTA LPS Gram Positive Bacteria Gram Negative Bacteria Gram Positive Bacteria Gram Negative Bacteria Bi: Bifidobacterium infantis Aj: Acinetobacter junii LTA: Lipoteichoic Acid LPS: Lipopolysaccharide Rb: Ruminococcus bromii Ps: Prevotella stercorea St: Salmonella typhimurium Whole bacteria (N=6-9) Cell Wall Component (N=3) Error: SEM

12 Differential Response of Human Colonic ILCs to Gut Bacteria Lumen Gram + Gram - Epithelium Lamina propria 1 IL-22 ILC3 2 NK/ ILC1 IFNγ 3 Inflammation HIV-1 CD4 T Circulation Lymphatics

13 Clinical Significance & Future Directions During untreated HIV-1 infection where Gram negative bacteria are increased in abundance and translocate across epithelial barrier Group 3 ILCs produce IL-22 in response to bacteria, but not enough to restore epithelial barrier function Group 1 ILCs produce IFNγ likely contributing to further barrier damage Bacteria of the human colon may direct mucosal ILC function during HIV-1 pathogenesis. How is the response mediated? How does HIV-1 effect the bacteria-induced cytokine response?

14 Acknowledgments Cara Wilson Steph Dillon Jay Liu Jon Kibbie (#O-08) Martin McCarter Dan Frank Edward Barker Andrew Cogswell R01 AI T32 AI007405

Intestinal Microbiota in Health and Disease

Intestinal Microbiota in Health and Disease February 27, 2015 Master s Course in Gastroenterology Prof. Kathy McCoy 1 Overview Overview of Gut Microbiota Microbiota in Health Microbiota in Disease 2 Gut