EDCs are commonly thought to bind or interfere with estrogen, androgen, progesterone, thyroid, and retinoid receptors. Oxybenzone is considered to be an endocrine-disrupting chemical and approximately 97% of people in the United States were found to have BP3 in their urine. This thesis will address how BP3 affects the innate immune system, in particular myeloid cells. My Master’s thesis aims to address two main overarching questions. Does BP3 alter macrophage polarization, cytokine/chemokine secretion, the viability in vitro? Does exposure to BP3 in vivo during pregnancy/lactation affect the RNA expression of cytokines and immunosuppressant factors associated with the myeloid population? It is unknown how BP3 impacts immune subpopulations in a neoplastic setting. Additionally, it is important to consider how these effects may contribute to malignant behaviors. My thesis evaluates the effects of BP3 on the Raw 264.7 cell lines as well as tumor tissues from mice exposed to BP3 during pregnancy and lactation. We hypothesized that BP3 exposures induce changes in myeloid cell interactions in the immune system through ER-mediated mechanisms. We anticipated that BP3 would increase the growth and migration of 4T1 cells through indirect signals imparted by myeloid cell populations. We also hypothesized that there will be a decrease in T cell proliferation following BP3 exposure and an alteration in gene expression consistent with a shift from Th1 to Th2. Finally, we expected that BP3 exposure would increase the number of myeloid cells in mouse tumors. Our research shows that oxybenzone appears to enhance the pro-inflammatory state of RAW264.7 cells and may result in the release of unidentified factors that can impact 4T1 cell anchorage-independent cell growth in these pro-inflammatory conditions. BP3 may also impact the metabolic activity of recovering RAW264.7 cells following LPS-induced activity. Additionally, BP3 may impact the release of factors from macrophages that control T cell activation-induced proliferation. By using the p53-/- mouse tumors we found that exposure to 3mg/kg/day BP3 during pregnancy and lactation did alter IDO1 RNA expression but this was not associated with markers of immunosuppressive cell types.

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Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.