Microbiology and Immunology Department Theses and Dissertations
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Item The Impact of IL-9 on Disease in Murine Models of Systemic Lupus Erythematosus(2026-03) Krishnan, Maya Shraddha; Kaplan, Mark H.; Rhodes, Steven; Richer, Martin; Snell, Laura; Zhou, BaohuaSystemic lupus erythematosus (SLE) is a relapsing-remitting antibody-mediated autoimmune disease characterized by systemic immune complex deposition. A subset of SLE patients has been shown to have elevated CD4+IL-9+ T cells as well as increased secreted IL-9 and Il9 mRNA compared to healthy controls. In other antibody mediated autoimmune models, such as rheumatoid arthritis, type II innate lymphoid cells (ILC2s) secrete increased IL-9 to promote the suppressive response of regulatory T (Treg) cells. As such, the role of IL-9 may be context-dependent and requires further investigation in SLE. This thesis examines standard and novel murine models of SLE to demonstrate that IL-9 exhibits both protective and proinflammatory functions, consistent with IL-9 activity varying with disease progression. Within our model, the use of an IL-9 neutralizing antibody (Ab) for 6 weeks in 6-week-old mice results in an exacerbation of disease and an expansion of immune cell subsets that promote autoimmunity. Anti-IL-9 Ab treatment also results in a loss of Treg cells and IL-9+ ILC2s in the kidney, which suggests that IL-9 promotes a protective response. From 6 to 12 weeks of treatment, the anti-IL-9 Ab treatment does not result in significant disease exacerbation compared to the isotype control. In parallel, we used a chimeric antigen receptor (CAR)-T cell approach, which is a promising treatment option for SLE patients, to test whether a third generation CAR-T cell can be polarized to a CAR-T9 cell and mitigate SLE disease severity. Our preliminary results indicate a reduction in CD19+ B cells and lymphoproliferation in the CAR-T9 cell treated SLE mice. Ultimately, our data suggests that IL-9 has a protective effect at early time points of disease, and this finding could be harnessed for cellular therapies of disease.Item IGFL Unveiled: A Cytokine Saga in Allergic Skin Inflammation(2026-03) Xue, Gloria Regina; Turner, Matthew J.; Kaplan, Mark H.; Zhou, Baohua; Liu, YunlongIGF-Like Family Member (IGFL) proteins are putative cytokines whose functions are undefined. Our investigations discovered IGFL1 and IGFL4 transcripts are increased in human atopic dermatitis (AD) lesions, and complementary experiments demonstrated induction of mouse Igfl in the oxazolone (Oxa)-induced model of allergic skin inflammation. We found Oxa-induced allergic skin inflammation is IGFL-dependent as Igfl-/- mice exhibited marked reductions in clinical, histologic and molecular (Il4 and Il13 transcripts) markers of the disease phenotype. In addition, we discovered IL-4 and IL-13 can each promote Igfl and IGFL4 expression in mouse and human keratinocytes, respectively. Studies with Il4ra-/- mice and AD patients treated with the IL-4Rα neutralizing antibody dupilumab demonstrated that induction of Igfl in the Oxa mouse model and IGFL4 in AD skin lesions require IL-4Rα signaling. Based on these observations, we hypothesized that keratinocyte-derived IGFLs promote allergic skin inflammation via IGFLR1-dependent production of IL-4 and/or IL-13 by leukocyte population(s). Studies with scRNA-seq suggested IGFL promotes Th2 polarization under homeostatic conditions in mouse skin, and pathway analysis of mRNA-seq data from Oxa-induced lesional skin linked IGFL to T cell activation, differentiation, and proliferation. Functional assays confirmed that mouse and human IGFLs enhance Th2 differentiation and reduce IGFLR1 on the plasma membrane suggesting IGFLs mediate these effects through IGFLR1 on T cells. Additional studies revealed mouse IGFL promotes IL-13 production by basophils in the Oxa model. We also demonstrated that IGFLR1 is expressed on mouse and human dendritic cells (DC) and that IGFL stimulation increases mouse BMDC viability, Il4 expression, and DC2 populations, which can promote Th2 responses. These studies suggest allergic skin inflammation is driven by a positive feedback loop in which Th2 cytokines stimulate keratinocytes to produce IGFLs that activate IGFLR1 on T cells, DCs and basophils leading to further Th2 cytokine production.Item Multi-Disciplinary Approaches to Mosquito-Transmitted Virus Prevention: Dissecting the Correlates of Immune Evasion by an Epidemic Zika Virus Isolate and Community Acceptance of New Biorational Larvicide Technologies(2026-03) Carter, Alexandria M.; Richer, Martin; Kaplan, Mark; Katzenellenbogen, Rachel; Snell, Laura; Tilston, Natasha; Turman, JackMosquitoes cause millions of human deaths through transmission of pathogens, including arboviruses. Mechanistic studies dissecting arboviral evolution and immune evasion in conjunction with community public health approaches integrating novel mosquito-prevention technologies are necessary to prevent arboviral infections. This thesis examined the differing immune response to pre-epidemic and epidemic Zika virus isolates, their impact on hematopoiesis, and community attitudes towards new biorational mosquito larvicide technology, including RNA-interference based insecticides. We previously demonstrated that an epidemic isolate of Zika virus, ZIKVBR, induces a reduced CD8 T cell response and increased neutralizing antibody response compared to a pre-epidemic isolate, ZIKVCDN. These isolates differ by 13 amino acid (AA) changes, so we compared the immune responses of other ZIKV isolates sharing some but not all of these AA changes to determine which residues contribute to the altered immune response seen during ZIKVBR infection. All isolates tested induced a similar immune response to ZIKVCDN infection, narrowing down the AA residues responsible for the decreased CD8 T cell response induced by ZIKVBR. We compared how ZIKVBR and ZIKVCDN infection impact hematopoiesis, revealing that ZIKVBR infection alters hematopoietic capacity and mature cell differentiation in the bone marrow. Finally, we examined community compatibility with new biorational larvicides by examining public knowledge, attitudes, and practices surrounding mosquito control using the Diffusion of Innovation model. We found that individuals reporting greater mosquito nuisance, desire to decrease mosquito numbers, and knowledge of mosquito breeding habits were more likely to support new larvicide technology. Therefore, educating the public on mosquito breeding habits and communicating bite risk reduction, not arboviral risk, may increase support for new larvicide technologies. Overall, this thesis helps dissect ZIKV’s effect on the immune response and hematopoiesis and how community practices and attitudes support new mosquito-prevention techniques. This may ultimately help prevent mosquito-transmitted illnesses and improve human health.Item TFR Cells Suppress Gut Commensal-Specific IgA Via IL-10(2026-02) Abdi, Abdullahi Mohamed; Dent, Alexander; Cook-Mills, Joan; Nelson, David; Wan, JunImmunoglobulin A (IgA) is the predominant antibody (Ab) at mucosal surfaces, playing a critical role in intestinal defense and regulation of gut commensals. A large amount of IgAcoated gut commensals is generated via T cell-independent pathways. However, highaffinity, antigen-specific IgA Abs are produced through T cell help in the germinal centers of gut-associated lymphoid tissues. Through this pathway, T follicular helper (TFH) cells drive affinity maturation by selecting B cells that produce high-affinity Abs, including IgA. Concurrently, T follicular regulatory (TFR) cells, derived from regulatory T cells (Tregs), also regulate this process. TFR cells suppress or enhance Ab responses depending on the context, but their role in gut IgA responses remains unclear. Based on our preliminary data, we hypothesized that TFR cells regulate high-affinity IgA responses through Treg-derived suppressive pathways such as IL-10. To test this, we employed a food allergy model by priming WT controls (Foxp3Cre) and TFR-deficient (Foxp3Cre.Bcl6fl/fl; Bcl6FC) mice with peanut or ovalbumin plus cholera toxin to induce food allergy, collected fecal samples, measured fecal IgA-bound bacteria, and sorted IgA-bound/unbound bacteria for performing 16S sequencing. Additionally, we used adoptive transfer experiments and direct IgA binding assays to probe mechanisms by which TFR cells suppress commensalspecific IgA. We found that Bcl6FC mice exhibited a sustained increase in IgA-coated bacteria following food allergy induction, along with increased abundance of Clostridia, Prevotellaceae, and Desulfovibrionaceae members in the IgA+ fraction. Adoptive transfer of WT TFR cells into Bcl6FC mice restored IgA coating levels to those observed in WT controls. Next, transfer of IL-10-deficient TFR cells failed to suppress the elevated IgA coating observed in Bcl6FC mice, whereas CTLA-4-deficient TFR cells retained this suppressive capacity. Our findings elucidate how anti-commensal IgA is regulated in the gut, with broad implications for immune-mediated conditions including food allergies, autoimmunity, cancer and vaccine development.Item Identification of Determinants that Mediate Chlamydia Muridarum Tissue Tropism(2025-12) Jacobs, Kaylee Rae; Nelson, David; Jordan, Stephen; Richer, Martin; Tilston-Lunel, NatashaBacteria within the genus Chlamydia are obligate intracellular pathogens that infect a wide variety of vertebrates. Despite having highly similar genomes, closely related Chlamydia species can have different tissue tropisms. The human pathogen Chlamydia trachomatis exemplifies this; strains in three genetically similar biovars (trachoma, chlamydia, and lymphogranuloma venereum) target different human tissues (ocular epithelia, urogenital epithelia, and the lymphatic system, respectively). Studies using the mouse pathogen Chlamydia muridarum have suggested that tropism is heavily influenced by evasion of host immune responses, particularly, responses mediated by the cytokine interferon-gamma (IFNγ). My goal was to use an unbiased approach to investigate how C. muridarum circumvents immunity. I conducted a tropism screen using a mutagenized C. muridarum library and murine-derived cells from distinct tissues. This screen identified mutants that have reduced ability to proliferate in specific cell types in IFNγ-dependent and independent manners. Some of the mutants had complex phenotypes that could not be linked to single mutations. However, I also identified two isolates whose growth was restricted in murine rectal and oviduct epithelia in an IFNγ- independent manner. Both of these isolates had the same missense mutation in the gene tc0237. I determined that complementation of the mutants with wild type tc0237 was sufficient to reverse their tropism phenotype. Blocking the ability of host cells to respond to infection by treating them with a protein synthesis inhibitor revealed that the factors responsible for restricting tc0237 mutants were cell-line specific rather than a shared characteristic of epithelial cells. Several attempts to localize TC0237 using different approaches were unsuccessful, but bioinformatic analyses revealed that TC0237 likely remains associated with chlamydial cells and could oligomerize. Overall, my findings suggest that TC0237 plays a critical role in C. muridarum immune evasion and show that chlamydial tropism is not solely defined by IFNγ.Item Maternal β-Glucosylceramides Alter Neonate Hematopoiesis and Lung Microbiota(2025-06) Bloodworth, Jeffrey Clay; Cook-Mills, Joan; Brutkiewicz, Randy R.; Ye, Yuzhen; Schmidt, Nathan W.Asthma affects over 25 million Americans and is the most common chronic pulmonary disease in children in the United States. Currently approximately 1 in 14 children are afflicted with asthma in the United States. Between 2008 and 2013, the estimated economic burden of asthma in the United States was $81.9 billion. The prevalence of asthma has steadily increased over the last 40 years, and the 20-year projected economic impact of uncontrolled asthma alone is projected to be about $300 billion from year 2019 to 2038. Offspring born to allergic mothers are three times more likely to develop allergies than offspring born to mothers without allergies. Lipids such as αTocopherol, γ-Tocopherol, and β-Glucosylceramides are known to influence allergen hyperresponsiveness. Elevated β-Glucosylceramides alter dendritic cell populations in neonates, and β-Glucosylceramides are known to signal through a C-type lectin receptor called Mincle. In addition, lung microbial dysbiosis is associated with development of allergic asthma in children. Here we show that maternal β-Glucosylceramides alter hematopoiesis in the neonate by signaling through Mincle in hematopoietic precursors. This causes the expansion of IRF4+ dendritic cells, which are known to induce allergy. Furthermore, we show that the lung microbiota is altered in mouse pups born to allergic mothers and that this dysbiotic microbiota is sufficient to induce allergen hyperresponsiveness in neonates. Β-Glucosylceramides altered lung microbial relative abundance compared to vehicle-treated controls and enhanced biofilm formation in bacteria isolated from mouse lungs. In silico analysis revealed predictions of microbes and metabolites that are important for microbial response to β-Glucosylceramides. This new knowledge is important for our understanding of not only allergy development, as well as immune development in neonates overall. Understanding the mechanism of action of elevated β-Glucosylceramides provides an opportunity to counteract their harmful effects, for instance by dietary α-Tocopherol supplementation during pregnancy and nursing.Item Characterizing the Formation and Functionality of Immune Memory Cells in Response to Plasmodium Infection(2025-05) Fusco, Elizabeth Michelle; Schmidt, Nathan W.; Absalon, Sabrina; Bauer, Margaret; Longtin, Krista; Luo, Wei; Richer, MartinMalaria is an infectious disease caused by Plasmodium parasites. Over 40% of the world lives in malaria endemic regions, and children under the age of 5 in Sub-Saharan Africa face the highest burden of this disease. The clinical symptoms of malaria are caused by the cyclical infection and rupture of red blood cells by Plasmodium, and these parasites are cleared from the blood by the immune system. Plasmodium infection does not induce sterilizing immunity; however, individuals can generate clinical immunity to malaria after repeated exposures, but the factors that regulate this process are poorly understood. An emerging modulator of the immune response is the gut microbiome. We previously identified that the composition of the gut microbiome correlates with the outcome of Plasmodium infections in African children and impacts ability of the immune system to fight a Plasmodium infection in mice. We next investigated how the gut microbiome impacts the immune memory response in mice. We determined that the gut microbiome influences the formation of memory B cells and memory T cells during primary Plasmodium yoelii infection. Furthermore, the gut microbiome governs the ability of these immune memory cells to mount a secondary germinal center (GC) response to a Plasmodium berghei ANKA challenge. Curiously, the gut microbiome did not affect the accumulation of plasma cells (PCs) in the bone marrow following P. yoelii infection, and we observed that antigen-specific PC accumulation was poor. It is hypothesized that PCs are important for protection against reinfection with Plasmodium due to their ability to secrete high-affinity antibodies. We next characterized how P. yoelii infection impacted the generation and maintenance of PCs. We discovered that P. yoelii infection impairs the ability of the GC to produce long-lived PCs (LLPCs). Additionally, P. yoelii alters the composition of the bone marrow, negatively impacting the ability of PCs to engraft in the bone marrow as LLPCs. These defects in the generation and maintenance of P. yoelii-induced LLPCs likely impairs the ability of the immune system to protect against future Plasmodium infections.Item Decreased Natural Killer Cell Function in Pediatric Severe Malaria in Areas of Higher Transmission(2025-05) Turyasingura, Grace; John, Chandy C.; Kaplan, Mark H.; Schmidt, Nathan W.; Absalon, SabrinaNatural killer (NK) cells inhibit Plasmodium falciparum parasite growth through antibody-dependent cellular cytotoxicity (ADCC) in vitro. Research conducted in malaria-endemic regions has demonstrated that memory-like NK cells are elevated in individuals exposed to malaria, exhibit enhanced ADCC activity, and correlate with reduced parasitemia and protection against uncomplicated malaria. However, the role of NK cells in pediatric severe malaria (SM) is not known. To evaluate the NK cell phenotype and function in SM, we used flow cytometry to evaluate CD56 bright, CD56 dim, and CD56 neg NK cell subsets in Ugandan children 6 months – 4 years of age with SM (cerebral malaria (CM), n=11), severe malarial anemia (SMA), n=10) and asymptomatic community children as controls (CC, n=19). Children were enrolled from sites of moderate (Jinja) and low (Kampala) malaria transmission. Analysis revealed that children with SM had a lower proportion of total NK cells and CD56 bright NK cells; however, absolute counts of NK cells per ml did not differ. In addition, LILRB1, an inhibitory receptor, was the only phenotypic marker whose proportions were significantly increased in children with SM compared to CC. Functionally, children with SM had a higher proportion of degranulating (CD107a+, IFN-γ-) memory-like NK cells. However, memory-like NK cell subsets from children with SM had a lower proportion of interferon-γ only (CD107a-, IFN-γ+)-production than CC. In addition, when comparing malaria transmission intensities with NK cell function, NK cells of children with SM in moderate transmission area exhibited a lower proportion of degranulation compared to the area of low transmission. Conversely, in low malaria transmission areas, NK cells of children with SM demonstrated a higher proportion of degranulation compared to CC. These findings elucidate distinct functional differences in NK cells among children with SM in areas of low versus moderate malaria transmission.Item Longitudinal Keratinocyte Proliferation Induced by Human Papillomavirus 16E6 and NFX1-123 Partnership(2025-04) Billingsley, Caylin Leann; Katzenellenbogen, Rachel; Androphy, Elliot; Mosley, Amber; Robinson, ChristopherHigh-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.Item 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, LauraTumor-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.