Mechanisms of Mucosal Dysfunction in HIV Infection and Potential Therapeutic Strategies

Thesis (Ph.D.)--University of Washington, 2017-06HIV and pathogenic SIV infection are characterized by chronic gastrointestinal (GI) mucosal dysfunction, which contributes to morbidities and mortality. This includes damage to the epithelial barrier, loss of Th17 cells, microbial translocation and local and systemic inflammation and immune activation. The mechanisms underlying mucosal damage have not been fully elucidated. The broad purpose of this thesis was to address several unanswered questions relating to mechanisms and potential therapies for GI mucosal dysfunction in HIV infection. We aimed to address three main objectives: 1) longitudinally assess the kinetics of GI neutrophil accumulation and reduced neutrophil antimicrobial function in relation to mucosal dysfunction, immune activation, and Th17 cell depletion in acute SIV infection; 2) investigate increased neutrophil lifespan as a mechanism for GI neutrophil accumulation in chronic, treated HIV infection and a potential role for HIV altered mucosal bacteria; and 3) assess the safety and efficacy of fecal microbial transplantation (FMT) as a potential therapeutic for mucosal dysfunction in SIV infection. The role of neutrophils in HIV gastrointestinal mucosal dysfunction is one major theme of the studies described herein. Neutrophils are important for containment of pathogens, but can also contribute to tissue damage due to their release of reactive oxygen species and other potentially harmful effector molecules. Here, we demonstrate that immune alterations such as Th17 loss, prolonged intestinal neutrophil accumulation, and decreased neutrophil functionality occur after the onset of peripheral and mucosal T cell activation and evidence of microbial translocation, and are thus unlikely to be drivers of initial damage. We additionally demonstrate that neutrophils are recruited to the site of lentivirus infection, maintain antimicrobial function in acute infection, and may therefore participate in the antiviral immune response. In chronic treated HIV infection, we report that reduced homeostatic neutrophil apoptosis contributes to neutrophil accumulation in the gastrointestinal tissues, thus implicating neutrophil lifespan as a new therapeutic target for mucosal inflammation in HIV infection. Microbial dysbiosis in HIV infection is a second major underlying theme of this thesis. During HIV infection, the delicate balance of healthy bacterial communities is perturbed, and this dysbiosis associates with T cell activation and disease progression. Here, we report differential effects of HIV-altered mucosal bacteria on neutrophil survival, suggesting that dysbiosis may contribute to GI neutrophil accumulation in treated HIV. We also provide evidence that Lactobacillus species increase neutrophil apoptosis and decrease neutrophil frequency in vitro, which could have important therapeutic implications for reducing neutrophil-driven inflammation in HIV and other chronic inflammatory conditions. Finally, we demonstrate the safety of FMT in primates infected with lentivirus and examine the relationships between alterations to the microbiome and immunological parameters. We observed increased Th17 and Th22 cells as well as decreased activation of CD4+ T cells post-FMT, and these changes correlated most strongly across all sampling time points with lower abundance genera in the colon. Taken together, the data presented in this thesis improves our understanding of the mechanisms contributing to mucosal dysfunction and potential therapies that can be further investigated to improve overall health in HIV-infected individuals

The Dual Role of Neutrophils in HIV Infection

We summarize what is known about neutrophils in HIV infection, focusing on their potential roles in HIV protection, acquisition, and pathogenesis. Recent studies have demonstrated that neutrophil-associated proteins and cytokines in genital tissue pre-infection associate with HIV acquisition. However, recent in vivo assessment of highly exposed seronegative individuals and in vitro studies of anti-HIV functions of neutrophils add to older literature evidence that neutrophils may be important in a protective response to HIV infection. Neutrophils are important for containment of pathogens but can also contribute to tissue damage due to their release of reactive oxygen species, proteases, and other potentially harmful effector molecules. Overall, there is a clear evidence for both helpful and harmful roles of neutrophils in HIV acquisition and pathogenesis. Further study, particularly of tissue neutrophils, is needed to elucidate the kinetics, phenotype, and functionality of neutrophils in HIV infection to better understand this dichotomy

Zonulin as a biomarker and potential therapeutic target in multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) occurs during or recently following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and is characterized by persistent fever, inflammation, and severe illness requiring hospitalization. The majority of patients with MIS-C also present with gastrointestinal (GI) symptoms, including abdominal pain, vomiting, and diarrhea. In this issue of the JCI, Yonker, Gilboa, and colleagues identified zonulin as a biomarker of GI permeability in children with MIS-C and present the results of an intriguing proof-of-concept study indicating that zonulin may represent a potential therapeutic target for MIS-C treatment and prevention. Their findings suggest that intestinal mucosal dysfunction and epithelial barrier breakdown may represent a biological mechanism underlying the development of MIS-C in SARS-CoV-2–infected children

Antibiotic treatment disrupts bacterial communities in the colon and rectum of simian immunodeficiency virus-infected macaques

Antibiotic therapies are known to disrupt gastrointestinal (GI) bacterial communities. HIV and pathogenic simian immunodeficiency virus (SIV) infections have also been associated with disrupted GI bacterial communities. We administered a combination antibiotic therapy to six SIV-infected rhesus macaques and collected colon biopsies, stool samples and rectal swabs before and after antibiotics, and evaluated the bacterial communities at each sample site using high-throughput 16S rRNA gene sequencing. The colon mucosa and stool samples displayed different bacterial communities, while the rectal swabs showed a mixture of the mucosal and stool-associated bacteria. Antibiotics disrupted the native bacterial communities at each sample site. The colon mucosa showed depleted abundances of the dominant Helicobacteraceae, while we found depleted abundances of the dominant Ruminococcaceae sp. in the stool. The rectal swabs showed similar trends as the colon mucosa, but were more variable. After the antibiotic treatment, there were increased abundances of similar taxa of facultative anaerobic bacteria, including Lactobacillaceae and Enterobacteriaceae at each sample site

Accommodating multiple potential normalizations in microbiome associations studies

Abstract Background Microbial communities are known to be closely related to many diseases, such as obesity and HIV, and it is of interest to identify differentially abundant microbial species between two or more environments. Since the abundances or counts of microbial species usually have different scales and suffer from zero-inflation or over-dispersion, normalization is a critical step before conducting differential abundance analysis. Several normalization approaches have been proposed, but it is difficult to optimize the characterization of the true relationship between taxa and interesting outcomes.  Results To avoid the challenge of picking an optimal normalization and accommodate the advantages of several normalization strategies, we propose an omnibus approach. Our approach is based on a Cauchy combination test, which is flexible and powerful by aggregating individual p values. We also consider a truncated test statistic to prevent substantial power loss. We experiment with a basic linear regression model as well as recently proposed powerful association tests for microbiome data and compare the performance of the omnibus approach with individual normalization approaches. Experimental results show that, regardless of simulation settings, the new approach exhibits power that is close to the best normalization strategy, while controling the type I error well.  Conclusions The proposed omnibus test releases researchers from choosing among various normalization methods and it is an aggregated method that provides the powerful result to the underlying optimal normalization, which requires tedious trial and error. While the power may not exceed the best normalization, it is always much better than using a poor choice of normalization

Effects of Fecal Microbial Transplantation on Microbiome and Immunity in Simian Immunodeficiency Virus-Infected Macaques

An altered intestinal microbiome during chronic human immunodeficiency virus (HIV) infection is associated with mucosal dysfunction, inflammation, and disease progression. We performed a preclinical evaluation of the safety and efficacy of fecal microbiota transplantation (FMT) as a potential therapeutic in HIV-infected individuals. Antiretroviral-treated, chronically simian immunodeficiency virus (SIV)-infected rhesus macaques received antibiotics followed by FMT. The greatest microbiota shift was observed after antibiotic treatment. The bacterial community composition at 2 weeks post-FMT resembled the pre-FMT community structure, although differences in the abundances of minor bacterial populations remained. Immunologically, we observed significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4 + T cell activation in gastrointestinal tissues post-FMT. Importantly, the transplant was well tolerated with no negative clinical side effects. Although this pilot study did not control for the differential contributions of antibiotic treatment and FMT to the observed results, the data suggest that FMT may have beneficial effects that should be further evaluated in larger studies. IMPORTANCE Due to the immunodeficiency and chronic inflammation that occurs during HIV infection, determination of the safety of FMT is crucial to prevent deleterious consequences if it is to be used as a treatment in the future. Here we used the macaque model of HIV infection and performed FMT on six chronically SIV-infected rhesus macaques on antiretroviral treatment. In addition to providing a preclinical demonstration of the safety of FMT in primates infected with a lentivirus, this study provided a unique opportunity to examine the relationships between alterations to the microbiome and immunological parameters. In this study, we found increased numbers of Th17 and Th22 cells as well as decreased activation of CD4 + T cells post-FMT, and these changes correlated most strongly across all sampling time points with lower-abundance taxonomic groups and other taxonomic groups in the colon. Overall, these data provide evidence that changes in the microbiome, particularly in terms of diversity and changes in minor populations, can enhance immunity and do not have adverse consequences

Changes in gastrointestinal microbial communities influence HIV-specific CD8+ T-cell responsiveness to immune checkpoint blockade.

ObjectivesThe aim of this study was to examine the relationship between gut microbial communities in HIV-infected individuals on suppressive antiretroviral therapy (cART), and the peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo immune checkpoint blockade (ICB).DesignThirty-four HIV-seropositive, 10 HIV-seronegative and 12 HIV-seropositive receiving faecal microbiota transplant (FMT) participants were included. Gut microbial communities, peripheral and gut associated negative checkpoint receptors (NCRs) and peripheral effector functions were assessed.MethodsBacterial 16s rRNA sequencing for gut microbiome study and flow-based assays for peripheral and gut NCR and their cognate ligand expression, including peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo anti-PD-L1 and anti-TIGIT ICB were performed.ResultsFusobacteria abundance was significantly higher in HIV-infected donors compared to uninfected controls. In HIV-infected participants receiving Fusobacteria-free FMT, Fusobacteria persisted up to 24 weeks in stool post FMT. PD-1 TIGIT and their ligands were expanded in mucosal vs. peripheral T cells and dendritic cells, respectively. PD-L1 and TIGIT blockade significantly increased the magnitude of peripheral anti-HIV-Gag-specific CD8 T-cell responses. Higher gut Fusobacteria abundance was associated with lower magnitude of peripheral IFN-γ+ HIV-Gag-specific CD8 T-cell responses following ICB.ConclusionThe gut colonization of Fusobacteria in HIV infection is persistent and may influence anti-HIV T-cell immunity to PD-1 or TIGIT blockade. Strategies modulating Fusobacteria colonization may elicit a favourable mucosal immune landscape to enhance the efficacy of ICB for HIV cure