Plasmacytoid dendritic cell function in pathogenic vs. non-pathogenic HIV and SIV infection

by

Rebecca A. Botelho

ABSTRACT

The role of plasmacytoid dendritic cells (pDC) in HIV disease has been studied extensively but a clear consensus on the impact of pDC function on disease progression has not been reached. This thesis studied cases of pathogenic and non-pathogenic lentiviral infection in humans and non-human primates. Pathogenic HIV infection in humans and SIV infection in non-natural hosts is associated with a generalized, nonspecific immune activation and inflammation which persists during chronic infection and is associated with a progressive decline in CD4+ T cells, leading to immune deficiency and eventually development of AIDS. By contrast, nonpathogenic HIV and SIV infections are characterized by limited bystander immune activation and non-specific inflammation.

Plasmacytoid dendritic cells (pDCs) are an essential component of the innate immune response to viral infection and help to shape the innate and adaptive immune antiviral response. Multiple studies point to the possibility that pDC number and/or function may be different in the setting of pathogenic vs. non-pathogenic HIV and SIV infections. pDC functionality consists of maturation into antigen-presenting cells (APCs), secretion of type-I IFNs (most notably IFNα), and, as more recently characterized, production of the immunosuppressive enzyme, indoleamine-2,3-deoxygenase (IDO). All of these functions have discreet effects on the development of the antiviral immune response, and all can be induced through stimulation across various receptors expressed by pDCs.

Because of the wide array of pDC functionality that exists, the potential for important differences in pDC function in pathogenic vs. nonpathogenic infections in humans and non-human primates are considerable. This thesis work examined whether intrinsic differences in pDC function (possibly influenced by genetics) might be associated with differences in HIV disease status. This work also examined whether intrinsic differences existed in pDC function between natural and non-natural hosts of SIV infection, who undergo non-pathogenic and pathogenic infection courses, respectively, and are thought to be a good model for pathogenic vs. non-pathogenic HIV infection in humans. Overall, this work concludes that while significant differences exist in pDC function between natural and non-natural SIV hosts, few, if any, significant differences exist in pDC function between HIV+ human patients with different disease outcomes.

Immune activation and inflammation are predictive of a rapid pace of HIV disease progression, and host immunoregulatory factors that blunt immune activation and inflammation may contribute to delayed disease progression.

We hypothesized that host factors controlling HIV-associated inflammation might be protective during the course of disease and that amongst such factors, heme oxygenase-1 (HO-1) was a good candidate as an important stress-response enzyme. It is the rate-limiting enzyme that initiates heme degradation and maintains cellular homeostasis during stress through its depletion of pro-oxidant heme, via generation of cytoprotective carbon monoxide, and biliverdin.

The experiments discussed in this thesis aim to explain how the activity of HO-1 controls harmful immune activation in HIV-positive individuals primarily through its function in blood monocyte populations.

We show that HO-1 plays an important role in dampening nonspecific T cell activation in murine and human studies. Pharmacological alteration of HO-1 in mice as well as in circulating immune cells of humans leads to altered T cell proliferation, maturation, and survival profiles. This discovery sets the important groundwork for establishing HO-1 pharmacological agents as potential therapies for HIV disease.

Next, we show that inter-individual genetic variability in the HO-1 promoter region influences its transcriptional regulation. We conducted a candidate genotyping analysis of the HO-1 promoter polymorphisms during HIV-disease to show that African Americans with greater HO-1 GTn polymorphic repeats had higher plasma CD14 levels and viral loads on and off therapy, respectively.

HO-1 is regulated to a variable extent in the blood monocyte populations as defined by CD14 and CD16 expression, and we show that these circulating monocytes differentially predict HIV disease parameters.

Lastly, we determined how pharmacological inhibition of HO-1 influences virological and immunological consequences during pathogenic SIV disease. We administered the HO-1 inhibitor, tin mesoporphyrin (SnMP), to AGMs infected with the simian immunodeficiency virus (SIV). Treated animals had higher peak viral load during seroconversion, higher T cell activation, and higher absolute CD4+ T cell counts.

The results from this thesis show the importance of HO-1 activity in regulating homeostatic T cell activity as well as T cell function during inflammatory conditions such as HIV infection.

The aryl hydrocarbon receptor (AHR) is a cytosolic transcription factor that recognizes and induces metabolic enzymes in response to a wide variety of xenobiotics. However, more recently the AHR has been proven critical in a diverse array of biological processes, including organ development, circadian rhythm, and immune response. In particular, AHR activation has been shown to affect T cell differentiation for both inflammatory T cells that produce IL-17 (Th17) and IL-22 as well as regulatory T cells (Treg) involved in tolerance. Given that the balance between inflammatory and immunoregulatory T cells has been implicated in pathological processes in contexts such as HIV infection and autoimmunity, understanding the role AHR and its ligands play in immune cell diffentiation is critical. While environmental AHR ligands can alter T cell differentiation, endogenous ligands are likely to be more relevant in host immune responses. We investigated downstream metabolites of tryptophan as potential AHR ligands because (1) tryptophan metabolites have been implicated in regulating the balance between Th17 and Treg cells and (2) many of the AHR ligands identified thus far are derivatives of tryptophan. We characterized the ability of tryptophan metabolites to bind and activate the AHR and to alter T cell differentiation. We report that the tryptophan metabolite, cinnabarinic acid (CA), is an AHR ligand that stimulates the differentiation of human and mouse T cells producing IL-22. We compare the IL-22-stimulating activity of CA to that of other tryptophan metabolites and define stimulation conditions that lead to CA production from immune cells. Our findings link tryptophan metabolism to AHR activation and define a novel endogenous AHR agonist with potentially broad biological functions.

Immune development in utero and early in life represents a critical period in human health. Until this point, most studies have been conducted using model organisms which may or may not mirror the actual developmental events found in humans. In this dissertation we strove to elucidate and understand the inherent differences between the fetal immune system and the adult, with a particular emphasis on the myeloid compartment. We extended these studies to look at the response to cytokines in utero as well as the potential response to HIV in utero, as well as whether these fetal phenotypes persisted after birth in both the myeloid and lymphoid compartment.

To carry out these studies we developed in vitro flow cytometry assays to phenotypically characterize cell surface markers and the phosphorylation responses present in the JAK/STAT pathways. We also developed a high-throughput qPCR genetic signature to assess umbilical cord blood for fetal and adult transcripts. We have shown that human fetal and adult classical monocytes have distinct baseline transcriptional and signaling programs as well as that transcriptional and signaling differences in fetal monocytes underlie stronger responses to cytokine stimulation. Further, we have identified and validated a genetic signature to query umbilical cord blood for the presence of fetal-like classical monocyte and naïve T cells. And finally we have demonstrated that fetal classical monocytes may more strongly induce HIV restriction factors in response to IFNγ than adult and have a stronger canonical and non-canonical STAT response to IFNα/β. These studies shed light, for the first time, on many of the immune mechanisms at play both during normal development in utero as well as in the case of infection.

Clinical and laboratory studies have demonstrated that helminth infection can ameliorate intestinal inflammation driving inflammatory bowel diseases (IBD). Nonetheless, the cellular and molecular mechanisms mediating the benefits of helminth exposure remain poorly understood. Host immune strategies that have evolved to tolerate chronic helminth infection, including mechanisms for tissue repair and the mitigation of inflammatory pathology, are likely at play. These mechanisms are largely orchestrated by T-helper (TH)2 cells.

The studies described here investigate the immunologic mechanisms underlying helminthic therapy. First, in an ulcerative colitis patient who achieved clinical remission after infecting himself with Trichuris trichiura, we described the induction of a TH2 response in the colonic mucosa following helminth exposure as well as the expression of IL-22, a cytokine known to promote intestinal barrier functions. The severely impaired mucus production in the inflamed colon was restored, suggesting that disease remission was associated with repair of the mucosal barrier. Similarly, in juvenile rhesus macaques suffering from chronic idiopathic colitis, a putative model for IBD, we found that symptomatic improvement following T. trichiura exposure was associated with a mucosal TH2 response and reduced bacterial attachment to the intestinal epithelium.

We also explored the role of retinoic acid (RA), a vitamin A metabolite that regulates T cell function and is vital for mucosal epithelial maintenance, during helminth infection in mice. RA synthesis was induced during Schistosoma mansoni infection and played a critical role in the TH2 response. Alternatively-activated macrophages were found to be an important source of RA synthesis in this context.

The findings presented here support the broad conclusion that TH cell responses in the intestinal mucosa elicited by helminth infection, including the canonical TH2 cytokines as well as IL-22, support mucosal barrier functions that avert pathogenic inflammatory responses to commensal bacteria. The innate immune response elicited by helminth infection drives RA synthesis, which is critical for the development of TH2 immunity. These studies provide insights into the mechanisms underlying helminthic therapy, and support the development of IBD therapies that target mucosal repair.

The developing immune system has long been recognized to be uniquely prone to tolerance induction. Historical observations in rodent models, suggest that the tolerogenic properties of the fetus and neonate are linked to the immaturity of the adaptive immune system at this stage of development. However, the adaptive immune system of larger mammals undergoes considerable maturation during fetal development and is not thought to be amenable to tolerance induction at this time. There have, however, been clinical observations that suggest that the human fetus may be subject to tolerance induction under certain conditions, though very little is known about the mechanisms underlying such tolerance. This is in part attributable to a general deficit of knowledge concerning the functional properties of the developing immune system in human beings.

We characterized fetal T cells at early stages of T cell development (10-20 gestational weeks (g.w.)). Our analysis revealed that a high frequency of fetal CD4+ T cells bore the phenotype of regulatory T cells (TReg). Analysis of neonatal cord blood and adult peripheral blood and lymphoid tissues revealed that fetal tissues contained greater frequencies of TReg than neonates or adults.

These observations, coupled with the clinical findings that certain individuals showed tolerance to non-inherited maternal HLA molecules in a transplant setting, led to the hypothesis that fetal TReg could play a role in suppressing T cell responses to maternal alloantigens. A survey of lymph node specimens revealed that maternal cells could be detected within the developing fetal tissues. Fetal T cells were highly responsive to alloantigens yet fetal TReg were capable of suppressing fetal T cell responses to maternal alloantigens. Stimulation of naïve fetal T cells with alloantigens led to the differentiation and expansion of TReg cells, providing a mechanism for how tolerance could be achieved following fetal exposure to foreign antigens in humans.

Ongoing studies have revealed that the tolerogenic properties of the developing fetal immune system may be rooted in changes that occur within the hematopoietic stem cell (HSC) compartment. These findings provide novel insights into the development of immunological tolerance in human beings.

The mammalian gut bacterial microbiome is increasingly recognized as a vital immune organ. Numerous studies in murine models implicate the gut bacterial microbiome not only as being required for the induction and maintenance of various important cellular mediators of immunity, but also as being capable of initiating and/or exacerbating states of chronic inflammation in the gut and the periphery. HIV infection and subsequent disease progression is driven by chronic inflammation, and is concomitantly characterized by a disruption within many mucosal cellular compartments involved in regulating gut microbial community composition, including Paneth cells, macrophages, dendritic cells, and Th17 cells. Previous work from our laboratory has shown a link between Th17 cell depletion and activity of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO1), which produces molecules known as kynurenines that can directly inhibit differentiation of Th17 cells in vitro and in vivo. However, a putative interaction between the gut microbiome and the constellation of immune disruptions in HIV, including chronic inflammation and mucosal immune disruption (perhaps involving the IDO1 pathway), had not yet been described. We hypothesized that HIV infection is associated with a shift in gut microbiome composition, and that this shift could potentiate further mucosal immune disruptions and the chronic inflammation that drives HIV disease progression.

We profiled the gut mucosal-adherent microbiome of 32 human subjects at varying stages of HIV disease. We found that the microbiomes of untreated, infected individuals was significantly different from those that were uninfected, and the extent to which this microbiome differed was proportionally related to markers of chronic inflammation and mucosal immune disruption (IDO1 activity). Furthermore, we found that some gut bacterial taxa that were enriched in HIV infection correlated strongly and positively with IDO1 activity, and representative members of the top such correlate could directly produce kynurenines in vitro. We further sought to identify gut-resident bacteria that may inhibit IDO1 activity, and longitudinally profiled 12 rhesus macaques before and after SIV infection toward that end. We found that fecal abundance of Lactobacillus was negatively correlated with IDO1 activity, and macaques given a Lactobacillus-containing probiotic exhibited diminished IDO1 activity as measured in peripheral blood.