Thermoneutral Housing Protects from Fibrosis in IL-10 dependent manner - Interstitial lung disease (ILD) is a chronic pulmonary disorder characterized by aberrant healing and unchecked collagen deposition in the lung interstitium with subsequent respiratory failure. Connective tissue diseases (CTD) are a heterogenous group of rheumatic diseases affecting collagen, elastin and other connective tissue. ILD is a primary complication of connective tissue disease that is associated with high morbidity and mortality. Compared to other forms of ILD, CTD-ILD occurs in younger, primarily female, patients. The role of immune dysregulation, specifically lymphocytes, in CTD-ILD cannot be denied, as administration of immunosuppressors as a treatment for CTD-ILD is a mainstay for clinicians. However, our understanding of T cell biology and their dysregulation in all forms of ILD, including CTD-ILD, are incompletely understood. Thermal regulation is a critical yet understudied variable in pre-clinical models of lung fibrosis. Mice housed within their thermoneutral zone (TN) (29ºC-34ºC), the ambient temperature at which they do not expend energy to maintain their core temperature, are immunologically unique from those housed below thermoneutrality, or in so called cold stress (CS). Mice under cold stress are reported to have higher levels of beta adrenergic (B2AR) signaling, through increased catecholamines, influencing the immunologic milieu. Our preliminary data demonstrate that TN housed mice are protected from lung fibrosis in the bleomycin model (BLM) compared to CS exposed mice. IL-10 is a key cytokine involved in thermoregulation and a potent mediator of T cell activity. We have found that IL-10 deficient mice are not protected in TN conditions and exhibit an increase in the profibrotic cytokine IL-17 and IL-17 producing cell types. Our hypothesis is that decreased beta adrenergic signaling in TN and the absence of the regulatory cytokine IL-10 results in the increase of dysregulated profibrotic IL-17 producing cells. Our approach will utilize the BLM lung fibrosis model at CS and TN, with T cell isolations, functional assays, chromatin studies and RNA sequencing. We will test our hypotheses through two specific aims: Aim 1: Determine the cellular sources of IL-10 in TN housed mice and evaluate NE signaling on IL-10 producing cells. We will interrogate the cellular source of IL-10 production in TN BLM treated mice by flow cytometry analysis. Adoptive transfer of CD8+IL-10+ cells into IL-10 KO mice at TN will reveal if CD8+IL-10+ are uniquely protective by evaluating weight loss, survival and lung collagen content. We will also utilize pharmacological inhibitors/agonists of the B2AR pathway to understand the confounding effect of NE signaling on IL-10 immunosuppression. Aim 2: Determine the impact of IL-17 in TN and mechanism of IL-17 regulation by IL- 10 in TN. We will employ flow cytometry, IL-17 neutralization, ATAC-sequencing and RNA-sequencing to determine the contribution and epigenetic regulation of IL-17 by IL-10 in fibrosis modeling in TN conditions. TN housing conditions may more accurately reflect human immune responses in lung fibrosis for more effective research into potential treatment for CTD-ILD.
