Microbiology and Immunology Department Theses and Dissertations

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https://hdl.handle.net/1805/1660

Find out more about this Program at: http://micro.medicine.iu.edu

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IL-9 Intrinsically Alters Gene Expression and Chromatin Structure in Pulmonary Macrophages to Enhance Lung Tumor Growth
(2025-04) Cannon, Anthony Michael; Kaplan, Mark H.; Jerde, Travis; Brutkiewicz, Randy; Snell, Laura
Tumor-associated macrophages are an abundant, tumor-infiltrating cell population that support tumor progression through mechanisms such as extracellular matrix remodeling, immunosuppression, and metabolic reprogramming. The cytokine milieu is a factor that influences the functional phenotype of TAMs within the tumor microenvironment. Among these, interleukin-9 (IL-9) is a pleiotropic cytokine with both tumor-promoting and tumor-suppressing roles depending on the cancer type and responding cell. Importantly, mechanistic understanding of IL-9 in cancer is largely undefined. The work described in this thesis mechanistically defines the role of IL-9-responsive macrophages in the progression and metastasis of lung tumors. We demonstrate that IL-9 expands lung interstitial macrophage populations and induces the expression of Arginase 1 (ARG1), a critical enzyme in arginine to polyamine metabolism that is associated with poor survival in patients. Therapeutically, targeting ARG1-expressing macrophages using Arg1 siRNA-loaded nanoparticles significantly reduced polyamine levels and tumor burden. Additionally, bulk RNA sequencing and single-nuclei multi-modal ATAC and RNA sequencing in mixed-bone marrow chimeric mice revealed that IL-9 intrinsically reprograms the epigenetic and transcriptomic landscape of lung interstitial macrophages. Dysregulation of key genes, including Pdl1 and several cathepsins (Ctsd, Ctse, and Ctss), was identified and linked to increased tumor growth and an impaired anti-tumor immune response. In summary, this work identifies a critical vii pro-tumor mechanism of IL-9 in lung interstitial macrophages, characterized by dysregulated arginine metabolism, altered macrophage gene expression, and reduced anti-tumor immune responses. These findings underscore the potential of targeting IL-9-mediated pathways in macrophages as a potential therapeutic approach in lung cancer and metastasis.
Longitudinal Keratinocyte Proliferation Induced by Human Papillomavirus 16E6 and NFX1-123 Partnership
(2025-04) Billingsley, Caylin Leann; Katzenellenbogen, Rachel; Androphy, Elliot; Mosley, Amber; Robinson, Christopher
High-risk human papillomaviruses (HPV) cause cervical, anogenital, and head and neck cancers. Despite the availability of preventive HPV vaccines, their poor uptake leaves most men and women at risk for these cancers, many of which remain common globally and others that are increasing domestically. The HPV 16 E6 (16E6) protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 itself has no enzymatic activity and carries out most of its functions through partnerships with host endogenous proteins. Previously, it was demonstrated that 16E6 binds to the host protein NFX1-123, and NFX1-123 expression is increased in HPV 16 positive cervical cancer cell lines and primary cancers compared to normal tissues. In this thesis, we quantify the growth rates of 16E6-expressing keratinocytes with endogenous or overexpressed NFX1-123 (16E6/vec and 16E6/FN123, respectively) levels by measuring population doublings and interrogate whether proteome expression changes occur early or late in longitudinal growth assays that began with higher levels of NFX1-123. Early passage 16E6/vec and 16E6/FN123 cells showed similar growth rates; however, late passage 16E6/FN123 cells had accelerated growth and greater population doublings than the 16E6/vec cells. Mass spectrometry revealed similar proteomes of both cell lines at early passages. In contrast, late passaged cells had significantly higher amounts of differentially expressed protein among 16E6/FN123 and 16E6/vec cells. This indicates a unique proteomic landscape induced by the 16E6/NFX1-123 partnership. Pathway analysis vii showed increased positive telomere regulation, DNA repair, and DNA replication pathways in the late passage 16E6/FN123 cells compared to 16E6/vec. These findings indicate that the 16E6 and NFX1-123 partnership, especially with higher levels of NFX1-123, alters the longitudinal cellular environment in a manner that may initiate a preneoplastic phenotype. Additionally, the work detailed in this dissertation provides the first evidence of NFX1-123 being a direct RNA-binding protein giving new insights into the endogenous roles of the protein.
Gut Microbiome Dysbiosis Exacerbates Malaria Severity in Ugandan Children
(2025-03) Bednarski, Olivia Joanna; Schmidt, Nathan W.; John, Chandy C.; Kaplan, Mark H.; Sankar, Uma; Tran, Tuan M.
Malaria is a life-threatening infectious disease caused by the parasite Plasmodium. Sub-Saharan Africa faces the greatest disease burden and most deaths from severe malaria (SM) occur in children <5 years old. The pathogenesis of SM is not fully known, but murine studies implicate the gut microbiome. It remains unknown if gut microbiota contributes to SM pathogenesis in children. To address this knowledge gap, we sequenced the gut bacteria populations in stool samples from Ugandan children <5 years-old with SM and community children. Our analysis revealed that children with SM have gut bacteria dysbiosis, defined by an increased relative abundance of potentially pathogenic Enterobacteriaceae. Enterobacteriaceae thrive in the presence of inflammation and use diverse nutrients to support growth, and several features of SM associated with gut bacterial dysbiosis, including excess uric acid. Elevated blood uric acid levels during SM from hemolysis and impaired renal clearance likely increases levels of intestinal uric acid, which supports the growth of uricase producing bacteria such as Enterobacteriaceae. Furthermore, Enterobacteriaceae associated with multiple complications of SM such as coma, severe anemia, acidosis, acute kidney injury (AKI), and intestinal damage. Most positive blood cultures from children with SM grew Enterobacteriaceae. Moreover, increased Enterobacteriaceae abundance independently predicted post-discharge mortality in children with SM. The SM complication of excess intravascular hemolysis known as blackwater fever (BWF) also associates with post-discharge mortality, and its incidence has sharply increased in Eastern Uganda. In our cohort, 24% of children with SM reported BWF. This coincided with a significantly greater relative abundance of Enterobacteriaceae bacteria that can cause hemolysis. Our analysis identified 8% of stool samples from children with SM were positive for bacterial Shiga toxin genes, which may contribute to intravascular hemolysis through hemolytic uremic syndrome. This study identified gut dysbiosis during SM that associates with worse clinical complications and post-discharge mortality in a cohort of Ugandan children, suggesting that the gut microbiome may contribute to SM pathogenesis in humans.
Antibodies and Risk of Clinical Malaria in an Area of Low Malaria Transmission in Western Kenya
(2024-12) Odhiambo, Eliud Isaac Onyango; John, Chandy; Dent, Alexander; Cook-Mills, Joan; Schmidt, Nathan; Jerde, Travis
Children and adults are at risk for clinical malaria in areas of low malaria transmission. Antibody or antibody effector mechanism correlates of immunity in these areas, and their differences by age, are poorly characterized. To address this research gap, we evaluated the relationship of Plasmodium falciparum-specific antibody levels and merozoite opsonic phagocytosis to the risk of clinical malaria in a case-control study nested within a cohort of 5,753 individuals in a Kenyan highland area with low malaria transmission. Plasma samples were collected in 2007, and individuals evaluated over 10-year follow-up for risk of clinical malaria. Individuals who developed clinical malaria (cases) were matched by village and age to those who did not (controls). We evaluated total IgG, IgG1, IgG3, IgA, and IgM antibodies to Plasmodium falciparum antigens by cytometric bead assay, and opsonic phagocytosis (OP) by flow cytometry. Antibody and OP levels were compared to risk of clinical malaria in three age groups (< 5 years, 5-14 years and  15 years), adjusting for multiple markers of malaria exposure. Antibody and OP levels increased with age. In children < 5 years old, higher levels of total IgG and IgG1 to the P. falciparum antigen GLURP-R2 and total IgG and IgG3 to the P. falciparum antigen MSP-2 were associated with reduced risk of clinical malaria, but OP levels were not associated with risk of clinical malaria. Conversely, in children 5-14 years old and individuals ≥ 15 years old, higher antibody levels to multiple antigens as well as higher OP levels were associated with increased risk of clinical malaria. In this low transmission area, antibody and OP levels in individuals ≥ 5 years old may serve as markers of malaria risk that add to known risk markers in this area such as insecticide-treated net use, elevation and proximity to forest.
Conserved Residues in Murine Papillomavirus E2 Regulate the Viral Life Cycle
(2024-12) Gonzalez, Jessica Kay; Androphy, Elliot J.; Dong, X. Charlie; Katzenellenbogen, Rachel; Robinson, Christopher
Papillomaviruses (PVs) are small, non-enveloped DNA viruses that infect the stratified epithelia. Once an infection is initiated, the virus must successfully navigate the three stages of its life cycle: establishment, maintenance, and vegetative amplification. A major mechanism of regulating this viral program is post-translational modification on the viral E2 protein, which is responsible for orchestrating viral transcription, replication, and genome partitioning. The hypothesis underscoring this work is that residues in E2 are highly conserved across PV types because they serve some structural or functional purpose for the virus. A targeted mutant library was generated in E2 from murine papillomavirus (MmuPV1) to investigate conserved residues that have been shown to be post-translationally modified in the E2 of other PVs, including BPV-1 and high-risk HPV-31. In the transactivation domain (TAD) tyrosine 102 and the lysine 112/113 motif were modified to their constitutively modified (phosphorylated and acetylated, respectively) or unmodified states, while cysteine 307 in the DNA binding and dimerization domain (DBD) was mutated to a less-reactive serine or DNA binding defective phenylalanine mutant. We characterized how mutation at each of these conserved sites alters E2 function using a battery of in vitro assays to assess for transcription and replication ability. We also studied how each mutant contributes to disease progression using an immunocompromised mouse model assessing cutaneous disease. We demonstrate that mutants which fail to replicate transiently in vitro will also fail to induce proliferative wart formation, establishing a predictive link between in vitro and in vivo experiments. Taken together, our findings suggest that modifications on conserved residues in E2 act as molecular switches that regulate E2 activity throughout the cellular and viral life cycle.
Immunological and Social Determinants of Asthma: From Cytokine Signaling to Air Pollution Disparities
(2024-10) Cheung, Cherry Cheuk Lam; Kaplan, Mark H.; Cook, Nathan; Cook-Mills, Joan; Yang, Kai
Asthma is a chronic respiratory disease characterized by airway inflammation and hyperresponsiveness, impacting 262 million individuals globally. This heterogeneous condition results from a complex interplay of genetic, environmental, and social factors. The pathophysiology involves dysregulated immune responses, particularly through cytokine signaling, and is exacerbated by environmental pollutants and social determinants of health (SDOH). This thesis aims to (1) elucidate novel cytokine signaling pathways involved in asthma, specifically a potential type II IL-9 receptor complex, and (2) evaluate the impact of California's Assembly Bill 617 (AB 617) on reducing air pollution and asthma disparities in disadvantaged communities. The research employs molecular biology techniques, including flow cytometry, proximity ligation assay, and RNA sequencing, to investigate IL-9 signaling in airway epithelial cells. It also involves a policy analysis of AB 617's initial effectiveness in reducing fine particulate matter (PM2.5) levels and asthmaassociated emergency room (ER) visits through environmental monitoring and hospital records. The study identifies a novel type II IL-9 receptor complex composed of IL-9Rα and IL-13Rα1, suggesting new therapeutic targets for asthma management. Policy analysis reveals limited initial success of AB 617 in reducing air pollution and asthma incidence, highlighting the need for enhanced regulatory measures and community engagement. Understanding the molecular mechanisms of IL-9 signaling and addressing environmental and social determinants are crucial for comprehensive asthma management. Integrating scientific research with policy interventions can improve health outcomes and reduce disparities in asthma prevalence and severity.
Inflammation by Toxoplasma gondii Infection Induces Prostatic Hyperplasia and Accompanying Urinary Dysfunction
(2024-08) Stanczak, Emily F.; Arrizabalaga, Gustavo; Bauer, Margaret; Jerde, Travis; Nakshatri, Harikrishna
Benign prostatic hyperplasia is the non-cancerous enlargement of the prostate. This syndrome develops as men age and affects 50% of men by the age of 50 and 80% of men by the age of 80. BPH is associated with a pattern of symptoms and pathology in patients that can be painful, problematic, and lower the quality of life. BPH is a multifactorial disease which may develop due to lifestyle choices, genetics, metabolic disorder, and potentially infection of the organ. Interestingly, previous work from our research group showed that the common parasite T. gondii can infect and establish a chronic infection in the prostate of mice resulting in histological hyperplasia and glandular nodule formation, reminiscent of that observed in men with BPH. This leads to the hypothesis that T. gondii contributes to BPH in humans. In this work, we investigated whether there is a correlation between T. gondii infection and BPH using a cohort of age matched BPH diagnosed and non-BPH diagnosed control donors. My data show that men diagnosed with BPH are significantly more likely to have evidence of T. gondii infection than undiagnosed controls. Additionally, men with antibodies against T. gondii and BPH had significantly more severe pathology in several categories including and most notably in epithelial and glandular nodules seen only in a steroid hormone model of the disease. Moreover, I have determined that mice infected with parasites demonstrated abnormal urination pattern behavior indicating lower urinary tract dysfunction. Based on these results, we conclude that T. gondii can contribute to the development and severity of BPH and BPH pathology in humans. In addition, T. gondii can potentially be used as a model for BPH and BPH-like symptoms and pathology in mice and other model organisms.
IL-33 Mediated Th2 Effector Functions are Suppressed in Tregs by Bcl6 and Regulated by Sex
(2024-08) Lee, Kyu Been; Dent, Alexander; Richer, Martin; Robinson, Christopher; Yang, Kai
Allergic airway inflammation (asthma) is a prevalent and uncurable disease worldwide, affecting many individuals’ quality of life. Although asthma does not form from a singular cause, one primary mediator comes from the exposure to environmental allergens and the improper activation of the T cell subset: T helper 2 (Th2) cells. Th2 cells produce pro-inflammatory cytokines and promote the activation and recruitment of various pro-inflammatory cells into the lung, causing greater damage and inflammatory responses in the organ. Th2 cell’s activation is regulated by another T cell subset, Regulatory T (Treg) cells, by expressing anti-inflammatory cytokines and downregulating the inflammatory response. On the contrary, the release of interleukin-33 (IL-33) from damaged lung epithelial cells transitions Tregs into Th2-like Tregs (ST2+ Tregs) which release both pro-and anti-inflammatory cytokines and cannot suppress the inflammatory disease. However, transcriptional repressor protein B cell lymphoma 6 (Bcl6) provides Tregs a stable follicular phenotype and suppresses the ST2+ Treg transition. Preliminary data revealed that Bcl6 repressive function is dependent on mouse sex, in which Tregs of male mice are more resistant to the ST2+ Treg phenotype than those of female mice. However, the removal of Bcl6 also removed the sex-dependent suppression against the ST2+ Treg transition. The project therefore sought to further confirm and answer whether Bcl6 suppressed the ST2+ Treg phenotype in a sex-dependent manner, ultimately leading to a sex-biased asthma prevalence and severity. We utilized quantitative polymerase chain reaction (qPCR) and next-generation sequencing techniques to uncover which genes Bcl6 regulates, how IL-33 affects chromatin accessibility/gene expression, and what relation sex hormones have with Bcl6 in the expression of Th2 cytokines from Tregs. Currently, we have discovered that estrogen-like chemicals in common cell culturing media may be acting on the estrogen receptor of Tregs and causing differential gene expressions based on media conditions. We also determined that Bcl6 is acting independently of mouse sex to suppress Th2 genes in Tregs, contrary to preliminary findings. Overall, we have obtained insight on the role of the estrogen receptor and Bcl6’s mechanism of suppression in relation to sex.
Combined Inhibition of SREBP and m-TORC1 Signaling Synergistically Inhibits the Proliferation of B Cell Lymphoma
(2024-06) Zhu, Zhenhan; Luo, Wei; Capitano, Meagan L; Yuan, Xue
Sterol regulatory element-binding protein (SREBP) signaling plays a crucial role in maintaining sterol homeostasis during B cell activation and the proliferation of germinal center B cells. It is unclear whether this pathway can be targeted to effectively treat B cell lymphoma. We discovered that inhibiting SREBP signaling or its downstream target HMG-CoA reductase (HMGCR) using Fatostatin or Simvastatin effectively restrains the proliferation of B cell lymphoma cells. However, B cell lymphoma cells activate the mTORC1-pS6 pathway in response to statin treatment, suggesting a possible mechanism to counteract statin-induced cell cycle arrest. Combining low dose statin treatment with the mTORC1 inhibitor rapamycin demonstrates a synergistic effect in inhibiting B cell lymphoma proliferation, cell cycle progression and lipid raft generation. These findings emphasize the potential of a combined therapy approach targeting both SREBP and mTORC1 as a novel treatment strategy for B cell lymphoma.
Innate Immune Roles of Alpha-Defensin 1-3 in Neutralizing Uropathogenic Escherichia Coli
(2024-05) Canas Kouaifati, Jorge Jose; Hains, David S.; Kaplan, Mark H.; Nelson, David E.; Dent, Alexander L.; Eadon, Michael T.
Urinary tract infections (UTIs) are characterized by microbial colonization of the bladder, ureters or/and kidneys. Host-pathogen interactions compound to drive UTI susceptibility. The host innate immune system is composed of physio-chemical barriers and broad-spectrum potent effector responses that prevent pathogenic host colonization and bystander damage. Uropathogenic Escherichia coli (UPEC) isolates account for the majority of reported UTI cases. Virulence factors that are expressed by CFT073/UPEC heighten pathogenesis by permitting preferential invasion towards kidneys (pyelonephritis). During the acute invasion of the urinary tract tissues, pathogen molecular patterns and host-damage signals represent triggers that lead to host-defense responses. The release of antimicrobial peptides (AMPs) prevents microbial attachment to the epithelium. α-Defensin 1-3 are host-defense AMPs that work by eliciting microbial membrane interactions and inducing immunomodulatory effects. In humans, the DEFA1A3 locus encodes for three α-Defensin peptides (1-3). Chromosome 8 can harbor copy number polymorphism of the DEFA1A3 locus that range from 3-16 copies per diploid genome. Low copy numbers of DEFA1A3 have been associated with increased UTI risk in children (< 5 copies). On the other hand, patients with > 8 per copies per diploid genome have improved antibiotic therapy outcomes. In this thesis, we establish a pipeline to characterize α-Defensin 1-3 mechanisms of action, dissect contributors of expression at the cellular level, and protective DEFA1A3 gene-dose-dependent effects in the pyelonephritis setting. Using a manipulable mouse model expressing transgenic human DEFA1A3 gene copies, we explored dynamics of inducible α-Defensin 1-3 expression in the UPEC-infected kidney. Additionally, in vitro combinations of α-Defensin 1-3 and other host-defense AMPs work in concert to drive diverse cooperative action effects against UPEC. Methods and findings from my research in this thesis improve the current biomedical approaches to study AMP functions. Collectively, my results expand the understanding of DEFA1A3 polymorphic locus as a stratification UTI biomarker and exploration for the pre-clinical evaluation of kidney α-Defensin 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

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