Mycobacterium tuberculosis, the etiologic agent of tuberculosis, has infected one third of the world’s population and is one of the leading global infectious disease threats. Treatment is challenging, lasting upwards of six months even for drug sensitive infections, and the proliferation of multi and extensively drug resistant forms of tuberculosis underscore the urgent need for knowledge of the mechanisms driving pathogenesis of this disease. The ability of the innate immune system to combat infection involves activation of pattern recognition receptors (PRRs) that detect evolutionarily conserved pathogen-associated molecular patterns, including nucleic acids and lipoproteins. Activation of PRRs, including Toll-like receptors (TLRs), induces secretion of inflammatory and immunomodulatory cytokines that instruct adaptive immune pathways for T cell differentiation. For intracellular pathogens like M. tuberculosis, a Th1 response is required, whereas a Th2 response is beneficial for the control of extracellular parasites, but is associated with active tuberculosis disease. This dissertation seeks to understand modulators of the initial immune response to M.�tuberculosis through characterization of innate immune pathways induced by distinct bacterial ligands. The first study explores a mechanism by which Th1 or Th2 cytokines alter the vitamin D-dependent antimicrobial pathway in response to M.�tuberculosis 19kD lipoprotein, and illustrates the importance of the Th1 cytokine IFN-g in guiding an antimicrobial response. Next, is a comparison of the immune response induced by the M.�tuberculosis-derived ligands: 19kD lipoprotein and purified tRNA. M. tuberculosis tRNA was found to induce a gene network inducing secretion of key Th1 cytokines including IL-12p70 and IFN-g, which are necessary for host defense against TB.

Leprosy is a human disease caused by the intracellular pathogen Mycobacterium leprae. By investigating leprosy, we discovered a novel pathway involving NOD2-mediated induction of interleukin-32 (IL-32) triggering the differentiation of M. leprae infected monocytes into CD1+ dendritic cells (DCs) with professional antigen presenting cell function. These IL-32-derived DCs were able to “cross-present” antigen, that is capture exogenous antigen via the endocytic pathway and then present processed peptides via MHC class I to CD8+ T cells. It was also discovered that key components of the NOD2->IL-32->CD1+ DC pathway were highly expressed only in lesions of leprosy patients with cell-mediated immunity. We hypothesized that cross-presentation of M. leprae antigens by IL-32-derived DCs resulted in the activation of MHC class I-restricted CD8+ T cells. To test our hypothesis, we established CD8+ T cell clones from leprosy patients to putatively secreted M. leprae antigens. IL-32-derived DCs were able to cross-present exogenous recombinant M. leprae protein as well as live M. leprae to the CD8+ T cell clones. We conclude that IL-32-induced cross-presentation may play a role in CD8+ T cell responses in leprosy.

Understanding host-pathogen interactions between microbes and the innate immune system will provide insight into the host defense pathways and microbial virulence factors. The macrophage (MΦ) is a sentinel of the innate immune system and serves as the first line of defense against microbial infection. The MΦ provides protection to the host by i) rapidly recognizing harmful pathogens, ii) internalizing pathogens to contain the infection and iii) clearing the pathogen from the host before the onset of disease. Here, we study effector pathways of the MΦ that combat invading pathogens in the host and determine their effects on the invading pathogen.

MΦ are a phenotypically heterogeneous immune subset that provides different functions in host defense. Previous studies in our laboratory have found that interleukin-15 (IL-15) induces a MΦ differentiation program in primary human monocytes (IL-15 MΦ) that express the vitamin D metabolism pathway. Vitamin D supplementation to these specialized immune subsets exhibited an antimicrobial response against mycobacteria in vitro. However, clinical trials that supplemented tuberculosis (TB) patients with vitamin D as an adjuvant have largely been unsuccessful in vivo. Therefore, we investigate whether vitamin D status prior to the onset of microbial infection would contribute to host defense. Our data demonstrates that vitamin D status during IL-15 MΦ differentiation bestows the capacity to mount an antimicrobial response against Mycobacterium leprae. These data suggest that future clinical trials that assess the relationship between vitamin D supplementation to mycobacterial infection will determine if vitamin D can provide prophylactic effects that are therapeutically beneficial.

Similarly to vitamin D, the bioactive form of vitamin A, all-trans retinoic acid (ATRA), triggers an antimicrobial responses against M. tuberculosis in vitro. However, high ATRA levels in humans’ results in severe or even fatal side effects in vivo. Therefore, we investigate how the immune system regulates ATRA production at the site of TB disease. Our data demonstrates that dendritic cells express the vitamin A metabolism pathway, which converts the circulatory form of vitamin A, retinol, into ATRA. The dendritic cells subsequently release ATRA and induce vitamin A-dependent antimicrobial responses in neighboring monocytes and MΦ. Interestingly, this immune model has provided insight into the site of TB disease by showing that the dendritic cell-mediated retinol metabolism pathway is significantly diminished in the lung of active TB patients relative to normal lung. These data demonstrate a novel transcellular effector pathway between dendritic cells and MΦ that contributes to the host defense against microbial infection.

Toxoplasma gondii is capable of infecting any nucleated cell in vitro, but MΦ are the first immune subset infected by T. gondii in mice in vivo. It is well known that both interferon-gamma-induced responses and MΦ functions are critical to controlling T. gondii infections in mice in vivo. Interferon-gamma induces the expression of two different families of immune loading proteins called the immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that function to clear the parasite from the MΦ. However, T. gondii is equipped with secretory organelles called the rhoptries that inject ROP proteins into MΦ to modulate host cell functions. We have found a novel rhoptry pseudokinase effector, ROP54. Disruption of ROP54 demonstrates a 100-fold decrease in virulence in mice in vivo and increased GBP2 protein loading onto the parasite containing vacuole in vitro. Immunoprecipitation of ROP54 demonstrates that none of the known ROP effector proteins formed a complex with the pseudokinase, which suggests it may be a divergent effector protein. Collectively these data show that ROP54 is a novel virulence factor that evades a IFN-gamma-mediated immune response in MΦ and may be a potential drug target for the development of novel therapeutics.

The data presented in this thesis evaluates the efficiency of immune pathways against disease causing pathogens. The host-pathogen interaction from these models provides insight into microbial infection, which may help in the development of novel therapeutics and identification of drug targets. These data contributes to the importance of micronutrient supplementation and how it can help contain the spread of mycobacterial-related diseases. Additionally we found a novel ROP effector protein that may be a potential drug target in toxoplasma infection.

The host immune system is required for protection against disease caused by intracellular pathogens, such as mycobacteria. Activated host cells induce a variety of defense mechanisms that coordinate in order to eliminate mycobacteria. Stimulation of toll-like receptors (TLRs) as part of the innate immune system, and T-cell release of IFN-γ as part of the adaptive immune response are both associated with protection against mycobacterial disease progression. However, the various host defense pathways mediated by these two signals in human macrophages still need to be defined. Ligation of TLR2/1 with mycobacteria derived lipoproteins has been shown to trigger anti-mycobacterial activity, with the induction of antimicrobial peptides via a vitamin D-dependent pathway. Here, IFN-γ activation of human macrophages was shown to also induce killing of mycobacteria via a vitamin D-dependent antimicrobial pathway. In addition, IL-32, an IFN-γ inducible protein, was shown to mediate antimicrobial activity against mycobacteria by also inducing the vitamin D pathway.

Since activation via TLR2/1 and IFN-γ converge on at least one common antimicrobial pathway, we further studied the TLR2/1 and IFN-γ inducible gene network by RNA sequencing. Bioinformatics analysis identified “defense response” as a common TLR2/1 and IFN-γ gene ontology term. A subset of genes with previously reported direct or indirect antimicrobial activity was also identified in the common dataset. The role of S100A12, a TLR2/1 and IFN-γ common gene, was further investigated in the context of leprosy, a disease caused by Mycobacterium leprae, in which the clinical manifestations correlate with immune response. S100A12 was more highly expressed in tuberculoid leprosy (T-lep), which is associated with resistance to infection, compared to lepromatous leprosy (L-lep), which is associated with susceptibility to infection. S100A12 was sufficient to kill mycobacteria and was required for the TLR2/1 and IFN-γ inducible antimicrobial activity against M. leprae in human macrophages. Our data further define mechanisms for TLR2/1 and IFN-γ mediated antimicrobial response against mycobacteria.

The human body is constantly exposed to a myriad of bacterial organisms, and in

most cases, colonization of the host by these commensal bacteria is harmless and may even

be beneficial. However, a subset of bacteria are pathogenic causing debilitating health that

can lead to fatal diseases. Many microbes deploy strategies to evade and impair the host

response, further contributing to disease severity. In response to infectious agents, the host

can control and subvert infections by triggering both innate and adaptive immunity. The

microenvironment in which an infections occurs greatly dictates the strength, quality, and

duration of immune responses. Herein, we demonstrate mechanisms for triggering

protective immune responses during Mycobacterium leprae infection. Specifically, treatment

of M. leprae-infected CD1a+ Langerhans cells, a dendritic cells subset localized in the skin and mucosa, with IFN-g induces autophagy, leading to increased antimicrobial activity and

antigen presentation to T cells. Furthermore, T cells secrete elevated amounts of IFN-g

following antigenic activation, providing an amplification loop to further augment effective

host immunity. As M. leprae predominantly infect and reside in macrophages, we also

investigated mechanisms of macrophage activation. Our data reveals that treatment of M.

leprae-infected monocyte-derived-macrophages with IL-26, a T cell cytokine, results in

reduced viability of intracellular bacteria. IL-26 may have dual roles in antimycobacterial

defense, one which involves binding to bacterial bacilli and directly reducing its viability; and

the other involving activation of infected macrophages, increasing bacterial traffic to the

lysosomes, where they are degraded.

Acne vulgaris is a great example to study the interaction among the environment, microbes, and host immune response. Acne vulgaris is a highly prevalent chronic inflammatory skin disease thought to be caused by increased in sebum secretion, bacterial colonization by the ubiquitous bacterium Cutibacterium acnes (C. acnes), and host inflammatory response. As a major factor implicated acne pathology, sebum and its composition, squalene, are unique to humans. Sebaceous lipids accumulate in the follicular duct are oxidized by C. acnes lipase leading to bacterial proliferation. The direct cause-and-effect relationship between the bacteria has been difficult to establish given that C. acnes is a ubiquitous bacterium and that there was no quantitative difference in the number of bacteria between subjects with and without acne. Recent genomic and phenotypic analyses on acne clinical samples revealed different phylotypes of C. acnes coexist in the pilosebaceous unit with other Cutibacterium spp. included C. granulosum and C. humerusii. Studies also showed strains phylotype IA1 are more abundant with the skin on acne patients (CA) and phylotype II are more associated with healthy skin (CH). However, the mechanisms underlying the lipid and bacterial interaction to cause the onset of inflammation and subsequent development of acne are still not understood.

This dissertation begins with a detailed analysis using single cell RNA-seq to deconstruct the genetic and molecular profiles of individual cells to study differences in lesional and non-lesional skin of acne patients. Particularly we were interested in changes within the same cell types to understand how normal skin becomes inflamed acne skin. We identified that a TREM2 macrophage subpopulation, expressing a lipid gene metabolism program, is associated with acne lesions. We further discovered that squalene induces TREM2+ macrophages in vitro to have enhanced phagocytosis but minimal antimicrobial activity against C. acnes. As a result, the squalene induced TREM2+ macrophage perpetuates inflammation linking the excess lipid production in acne to inflammation in acne.

Next, we investigated clinical strains of C. acnes, CA and CH, to understand how the microbiome interact in acne lesions. These strains differ in their ability to trigger inflammation with CA induces higher pro-inflammatory cytokine secretion compared to CH in cell culture and mouse infection model. However, the virulence factor that accounts for the differences in immune response between CA and CH are still unknown. In this study, we showed that C. acnes activates the innate immune system through RNA species that are usually reserved for viral detection. Interestingly, RNA species from CA and CH have different bioanalyzer profiles and can trigger distinct immune response as seen with live bacteria. We also showed that CA RNA stimulate the immune response through a TLR-8/IL-18/IL-12p40 pathway. Our in vitro data correlates well with our scRNA-seq data showing the abundance of IFN- and TLR-8 in acne lesions.

Taken together, the results enhanced our understanding of the impact of squalene, a sebum lipid, and resident microbes on the immunological functions of the skin. This work advanced our knowledge of the environment-microbe-host interplay occurring at the pilosebaceous follicle and introduced a novel way of thinking about the mechanisms underlying the initiation and pathogenesis of acne vulgaris and inflammatory skin conditions.

Human host defense against mycobacteria depends on a functioning immune system. While it is currently established that Th1 cells are major players in host defense against mycobacteria, other cell types such as Th17 cells also correlate strongly with the protective forms of disease. However, the role of Th17 cells in the context of intracellular infection are incompletely understood. Recent work has shown that Th17 cells can secrete an antimicrobial protein IL-26, which can directly lyse extracellular bacteria. Given the established role of antimicrobial peptides against mycobacterial infection, we decided to further investigate IL-26 and determine whether it is antimicrobial against mycobacteria. We find that IL-26 can be secreted by PBMCs and purified T cells in response to IL-1β in the absence of T cell receptor activation. This process is also more rapid than TCR stimulation. Among helper T cells, we show that IL-1RI expression was necessary for this response and identified IL-1RI+ Th17 cells as a cell type that can secrete IL-26 in response to IL-1β. Furthermore, we establish that IL-26 secreted in response to IL-1β is functionally antimicrobial.

We also examined whether IL-26 is antimicrobial against intracellular bacteria. We find that IL-26 is differentially expressed between clinical forms of leprosy, with higher expression in tuberculoid leprosy, the resistant form of the disease, as compared to lepromatous leprosy, the progressive form of the disease. Incubation of IL-26 with M. leprae and attenuated M. tuberculosis strain H37RA led to dose dependent killing, both with bacteria in culture and, importantly, while the bacteria resided within infected macrophages. Additionally, we find that IL-26 treatment of infected macrophages stimulates the autophagy response and enhances phagolysosome fusion in a STING dependent manner. Altogether, this work uncovers a novel mechanism by which Th17 cells contribute to defense against mycobacterial infection.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is a powerful model to study the human immune response because the clinical manifestations of disease present as a spectrum correlating with the level of immune response to the pathogen. Research investigating mechanisms of host defense and M. leprae-induced suppression of antimicrobial pathways continues to provide insight into which immune pathways are essential for containment of mycobacterial infections. Here, we utilized three bioinformatics approaches to identify mechanisms of modulation of host immune responses by mycobacterium and gain a clearer understanding of disease pathogenesis and potential targets of mitigation, including: i) integration of microRNA and mRNA gene expression profiling of disseminated lepromatous leprosy (L-lep) versus self-containing tuberculoid leprosy (T-lep) lesions to identify microRNAs capable of downregulating antimicrobial peptide production, ii) overlap of highly expressed receptors on IL-15-derived M1 and IL-10-derived M2 macrophages (MΦ) with known drivers of MΦ polarization to identify ligands capable of reeducating MΦ phenotype and function, and iii) comparison of transcriptome profiles of M. leprae-infected monocyte-derived MΦ and leprosy skin lesions to identify genes contributing to suppression of host immune responses and bacterial survival.

Here, we provide evidence illustrating the ability of M. leprae to induce specific microRNAs and host genes to alter host immune responses. M. leprae, but not M. tuberculosis, can induce upregulation of microRNA-21, which then targets and downregulates antimicrobial peptide production and mycobacterial killing. Differential cytokine expression coupled with the presence of mycobacterial ligands can alter MΦ polarization correlating to a change in phagocytic function and expression of antimicrobial genes. Lastly, M. leprae can induce expression of the autophagy regulator NUPR1, which is more highly expressed in L-lep lesions. As more insight is gathered on (i) the functional consequences of host genes regulated during mycobacterial infections on the immune response and ii) how these genes are regulated by the pathogens, an opportunity for directed therapeutic intervention may present itself.