The role of endocannabinoids in regulating host-parasitic interactions, particularly in the lungs, has previously not been well-characterized. The murine-infecting model for human hookworm, N. brasiliensis, has a spike in endocannabinoid production during the infectious larval stage, indicating the potential role for these lipid-derived molecules in staging an infection. The rates of endocannabinoid production decreases when the nematode transitions to the mandatory lung-infectious stage. At the same time, endocannabinoids have been shown to have an ameliorating effect on the immune system in the gut of mammals. In this project, we use mice lacking the genes for the mammalian endocannabinoid receptors, Cnr1 (CB1KO) and Cnr2 (CB2KO), in order to isolate the role of endocannabinoid signaling in the lung immune response.

The first chapter focuses on a survey of the small excreted/ secreted molecules that are involved in host and parasite interactions. The second chapter focuses on the immune response to the pathogenic nematode, in which macrophages and eosinophils are co-cultured with live nematode larvae. We found that macrophages isolated from the N. brasiliensis-infected lungs of CB1KO mice, but not CB2KO mice, had increased Relmα expression, indicating an increase in macrophage alternative activation. Both lung macrophages and eosinophils from CB1KO mice exhibited excessive binding to N. brasiliensis larvae in co-culture. This indicates that endocannabinoid signaling is important for regulating the immune response to the nematode in the lung. This chapter informs the exploration of host-nematode interactions in vivo, in which CB1KO mice produced more eosinophils in response to a Nippo infection. The third chapter of this project employs a bioinformatics approach to explore the effect of the host immune system on the gene expression of the nematode. The fourth chapter summarizes these findings. This project implicates a role for the endocannabinoid system in regulating immune cell-helminth interactions in the lungs and is the premise for the exploration of endocannabinoids as a target to regulate the immune response to pulmonary helminth infection.

Resistin-like molecules (RELM), a family of mammalian-conserved secreted proteins, have immunoregulatory effects in helminth infection, acting as a negative feedback loop to limit excess inflammation at the expense of increased parasite burden. This is through downregulation of the Th2 cytokines, IL-4 and IL-13, that are essential for alternative activation of macrophages and clearance of helminths. However, it is unclear how the human RELM proteins regulate immunity to helminth infections and inflammation. In this thesis, we have combine data from human studies and a transgenic mouse model, where human resistin and its regulatory region was inserted in mice (hRetnTg+). Human patients infected with filarial nematodes or soil-transmitted helminths had higher levels of circulating hResistin, and hResistin was associated with higher parasite burden. To assess whether hResistin directly increases helminth infection, we infected hRetnTg+ mice with the murine hookworm, Nippostrongylus brasiliensis (Nb), which revealed that hResistin is upregulated by macrophages and we identified IL-4/STAT6 signaling as a novel pathway for hResistin expression. Human resistin expression during helminth infection contributes to increased parasite burden and prolonged infection. RNA-seq of whole lung RNA demonstrated that hRetnTg+ mice had increase proinflammatory cytokines and toll-like receptor (TLR) signaling. In human patients infected with helminths, hResistin is correlated with proinflammatory cytokines, suggesting that hResistin contributes to increased helminth infection through the upregulation of Th1 cytokines. Structural interaction predictions revealed that hResistin binds to TLR4 in the same binding pocket as MD-2, the adaptor protein that mediates lipopolysaccharide(LPS)-TLR4 interaction. Given that LPS-TLR4 interaction initiates septic shock, we hypothesized that hResistin may protect the host from endotoxic shock by outcompeting LPS binding to TLR4 and blocking subsequent proinflammatory cytokine production. Competitive cell binding assays confirmed that hResistin does indeed outcompete LPS for binding to TLR4, and thereby decreases Th1 cytokines in human PBMC. In a mouse model of LPS-induced sepsis, hResistin expression or exogenous administration resulted in protection against endotoxic shock, which was associated with a decrease in proinflammatory cytokines that promote sepsis progression. Finally, we show that Nb-induced hResistin reduces mortality from sepsis. In summary, we have identified hResistin as a key regulator of inflammatory responses in helminth infections and sepsis.

Parasitic worms infect billions of people worldwide. In humans, these infections and inflammation are chronic and debilitating. Therefore, it is imperative to explore host factors that regulate immunity and promote parasite clearance and host protection. In this thesis we discuss two such host factors; resistin like molecules and endocannabinoids.

We describe here that during infection with the rodent hookworm Nippostrongylus brasiliensis (Nb), a host molecule named RELM alpha dampens Th2 inflammatory responses. We use bone marrow (BM) chimera technology to show that RELM alpha from BM-derived cells and not non BM cells dampens Th2 immunity and prevents worm clearance. We show that of the BM cells, CD11c+ lung macrophages are the dominant source of RELM alpha. Next, we employ a macrophage-worm co-culture system and found that RELM alpha impairs the ability of macrophages to attach to and kill worms. By conducting gene expression analysis, we show that RELM alpha decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing.

Second, we explored how endocannabinoid signaling influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that function in feeding behavior and metabolism. Following Nb infection, we detected elevated levels of endocannabinoids 2-AG and AEA in the worm infected mouse lung and intestine. To test how endocannabinoids influence helminth infection, we utilized pharmacological inhibitors of cannabinoid receptors. We found inhibition of CB1R resulted in higher worm and eggs burdens which was correlated with decreased amounts of the Th2 cytokine IL-5 in the host. Interestingly, bioinformatic analysis revealed putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. Additionally, we found that all lifecycle stages of Nb produce endocannabinoids. Therefore, we report for the first time that helminth and host-derived endocannabinoids promote host immune responses and reduce parasite burden.

Collectively, these studies demonstrate two mechanisms of immunomodulation during helminth infection. In the first mechanism, BM-derived RELM alpha dampens immune responses and inhibits macrophage killing of worms. In the second mechanism, we describe how host and parasite-derived endocannabinoids promote host immunity by upregulating Th2 immunity which drives parasite clearance. In summary, we have identified key regulators of immune responses in helminth infection.

Macrophage-derived proteins play critical roles throughout all stages of the immune response to a variety of pathogens. Depending on the immunological challenge and the subsequent cytokines produced in response, macrophages will become classically (IFNγ) or alternatively activated (IL-4), with an entire spectrum of macrophage phenotypes existing between these two classifications. Upon activation, macrophages will produce a diverse array of proteins and cytokines to guide the immune response depending on the stimulus. Resistin-like molecule alpha (RELMα) is an example of such a protein that is secreted by alternatively activated M2-macrophages. Our laboratory has previously identified a variety of roles for RELMα in vivo and in vitro. However, knowledge gaps remain with regards to potential cell-extrinsic versus cell-intrinsic differences of immune regulating proteins on cell behavior and function. These include whether there is a difference in how a cell behaves when it expresses the protein or whether it encounters it extracellularly, or whether the function differs dependent on the cell-type, given that RELM is expressed by immune and non-immune cells. To begin addressing these questions, new tools are needed that allow overexpression in cell lines and primary cells. In the first part of this thesis, I sought to generate a RELMα over-expressing lentivirus to be later utilized in the efficient transduction of both cell lines and primary cells.

Community-based research has the potential to improve the possibility of reducing certain health disparities. The COVID-19 pandemic brought health disparities to the forefront, many of which existed before and were exacerbated by it. It then becomes the responsibility of researchers to engage within a community-based framework to use these opportunities and address the factors driving these disparities. Research conducted at the UC Riverside School of Medicine on COVID-19 in Riverside County has identified such disparities in the population, where Hispanic individuals were more likely to be severely infected with COVID-19. When opportunity arises in which community-engaged research should be performed, we offer the methodology and framework that was used in planning for our community-engaged research symposium that fostered a rich environment of academic and community collaboration. In the second part of my thesis, I will go into the community-engaged research I performed that allowed for the successful execution of a community-focused COVID-19 symposium.

Preclinical models of infection, such as murine infection models and transgenic mice, provide critical knowledge for the treatment and diagnosis of infections of significant public health concern. In particular, the mucosal barrier, such as the lung and intestine are susceptible to multiple infectious pathogens given their exposure to the external environment. The goal of this dissertation was to investigate the function of RELMα and CX3CR1 in mucosal infections with parasitic helminths and influenza virus, as these are pathogens of significant health concern worldwide. RELMα is a mouse protein, which has orthologs in humans such as resistin and RELMβ. First, we tested the function of RELMα and investigated factors that may regulate its expression in infection. RELMα accelerated resolution of inflammation and tissue repair in infection with mouse intestinal helminth Heligmosomoides polygyrus. We also discovered that choline kinase signaling was critical for optimal RELMα expression: when mice were infected with H. polygyrus and injected with choline kinase alpha inhibitor, RSM-932A, alternative activation of macrophages and RELMα expression was downregulated in RSM-932A-injected mice. Previous studies have reported RELMα expression in type 2 cytokine inflammation, however, the functional relevance of RELMα in other infectious contexts, such as viral infections, is unclear. RELMα was induced by IAV infection in the BALF and airway epithelial cells. RELMα induced influenza infections in mouse lung epithelial cells in vitro and in vivo, suggesting that RELMα expressed in lung epithelial cells promotes influenza infection of neighboring cells. In the last chapter of this thesis, we broadened our research scope to investigate the function of myeloid-derived cells in helminth infection by targeting CX3CR1, a critical chemokine receptor for monocyte recruitment from the blood vessel to the site of infection. CX3CR1 deficiency induced a more inflammatory response but lower parasite burden in the lung. CX3CR1-deficient monocytes were more cytotoxic and active than WT monocytes, demonstrating the regulatory function of CX3CR1 in monocytes. Collectively, these studies demonstrate diverse functions of RELMα and CX3CR1 in different infectious diseases and identify putative downstream targets by which RELMα and CX3CR1 modulate the immune response and tissue repair.