One of the sex-dependent observations in our study was related to lymphoid aggregate formation in the lung

Taken together, our results indicate sex-dependent protective effects associated with elevated tissue levels of ω-3 fatty acids in reducing the number of infiltrating immune cells into the lung, lower pro-inflammatory cytokine levels and reduced overall histopathology of the lung. The results identified herein model a long-term dietary intake of high ω-3 PUFA and reduced intake of ω-6 PUFA that achieves an ideal ω-6:ω-3 PUFA ratio throughout the body as reviewed before . This was achieved through the use of the Fat-1 transgenic mouse model; these mice express the Fat-1 gene from C. elegans encoding an ω-3 FA desaturase, converting ω-6 PUFA to ω-3 PUFA, leading to tissue ω-6:ω-3 PUFA ratios of ∼1:1 . This model is advantageous because it overcomes several issues that limit diet- and supplementation based experimental strategies; current clinical and preclinical studies have no standardization in terms of dose, duration, or source and quality of ω-3 PUFA, and each of these factors has implications on outcomes . These inconsistencies are considered leading factors for discrepancies in ω-3 PUFA study outcomes . The Fat-1 mouse thus provides a preclinical model for assessing how elevated tissue levels of ω-3 PUFA and reduced ω-6 PUFA can influence health outcomes by minimizing variation based on supplementation, intake and absorption and distribution of ω-3 rich fatty acids. Using this model, we have found genotype and sex-specific differences in infiltrating cells , proinflammatory cytokines and lung histopathology . The decreases in infiltrating PMNs and cytokines were consistent with each other within the Fat-1 male sex. Among the swine farm workers, sex-specific differences in lung function and TLR gene polymorphisms, which is a toll like receptor activated by swine farm dust, have been reported before . Gao et al. found that lung function is worse in males with the TLR9 gene polymorphism as compared to those males without the polymorphism,stackable planters and females with the TLR2 gene polymorphism exhibit better functioning lungs as compared to those females without the polymorphism in swine farm full-time workers .

Another study by Senthilselvan et al. found increased plasma levels of TNF-α in males without any TLR4 gene polymorphisms , and in females with the TLR4 gene polymorphism following 5 hours of swine farm exposure in naïve healthy subjects . We are not the first ones to report sex-specific differences in Fat-1 mice; a recent study investigating the role of elevated tissue levels of ω-3 fatty acids in obesity-associated post-traumatic osteoarthritis also reported sex-specific differences in Fat-1 transgenic mice . It appears that such sex-dependent differences are disease model-specific where one sex in the Fat-1 transgenic background exhibits a greater response than the other sex. Another study reported on sex-and age-specific differences of Resolvin D1 levels in the retina. This study found sex-dependent differences in retinal levels of RvD1 in aged mice as compared to young mice , with aged male mice showing a larger decrease in RvD1 levels . Sexdependent changes in the expression of genes involved in fatty acid synthesis, steroids and drug metabolizing enzymes have been identified before . Some of those genes encode for enzymes involved in the metabolism of ω-3 and ω-6 PUFA, thus identifying sex-dependent differences can inform pharmacokinetics, bio-availability and treatment options related to ω-3 and ω-6 PUFA-derived lipid mediators. The sEH inhibitor TPPU was used in this study as an indicator that to the inflammation resolving epoxide metabolites of ω-3 lipids might be a partial explanation for their beneficial effect. It is attractive to consider sEH inhibitors or mimics of ω-3 fatty acid epoxides as a prophylactic or therapeutic agent . Such mimics and sEH inhibitors are in clinical development by several companies but none are available on the market . However, there are a number of sEH inhibitors from natural sources that are commercially available such as Maca . Male sex in the Fat-1 transgenic mouse had lesser number of aggregates as compared to their corresponding WT controls, which was further reduced in the presence of TPPU .

While we observed a similar trend for the female mice these differences did not reach significance. Lymphoid aggregates, which can be composed of T cells, B cells and dendritic cells play different roles in different disease models such as COPD and tuberculosis. For example, in a chronic cigarette smoke-induced murine COPD model, lymphoid aggregates are associated with adverse outcomes and thus pathological, whereas in a tuberculosis model induced by the Mycobacterium tuberculosis, it is found to be a host defense mechanism . To our knowledge sex-differences in lymphoid aggregate formation in the lung has not been fully explored. A recent study found that females are more susceptible to lymphoid aggregate formation as compared to males, and this was further confirmed by ovariectomy in a smoke-induced COPD model . This is consistent with the previous reports indicating that women are more susceptible to develop COPD than men . Overall, our results are consistent with the previously published literature on the effects of agricultural dust in males; and the improved histopathological effects seen in the male sex are novel. We observed an elevated alveolar cellularity , in wild-type female mice as compared to male mice. A study investigated the differences in alveolar macrophage proteome between males and females, and found several proteins associated with inflammation and interact with estrogen receptor to be expressed higher in females than males . Another study looking at sex-related differences in lung inflammation showed a higher baseline for lung histology score and number of infiltrating cells into the lungs in females compared to males in sham-operated controls . Both studies are consistent with the histological finding we observed in female wild-type mouse lungs. Because males responded TPPU treatment better, this might be related to differences in fatty acid metabolism between males and females given fatty acid epoxides regulate alveolar cell influx, as shown in a study demonstrating that pharmacological modulation of fatty acid epoxides affect inflammatory cell influx to the lungs . In addition, SPMs also modulate macrophage chemotaxis, trans-endothelial migration and cytokine release from macrophages as reviewed by Haworth and Levy . More studies are needed to dissect differences in fatty acid metabolism between males and females. It is well accepted that sex-specific differences that result in different biological responses between males and females stem from sex hormones. These differences affect storage and distribution of lipids thereby affect free fatty acid availability between sexes.

Stable isotope studies report that ARA and DHA contribute more to blood lipids in women than in men and that there are differences in the conversion rate of fatty acids, for example conversion of ALA to omega-3 fatty acids is higher in women than in men . Also, preclinical studies show that reproductive hormones affect the enzymes involved in the biosynthesis of fatty acids. Another study found that sex-related differences in COPD might be related in part to the increased production of leukotoxin-diol by goblet cell P450 and sEH activities . Overall,stacking pots our results suggest a mechanism that 1) availability of free fatty acids from the elevated tissue levels of ω-3 acids in Fat-1 mice are sex-dependent and 2) females have different conversion rates of fatty acids as compared to males and this might lead to changes in gene expression of chemokines and cytokines, thereby affecting alveolar macrophage recruitment into the lung and thus creating sex-dependent host defense mechanism within the Fat-1 genotype. In addition to dietary strategies aimed at promoting the healthful benefits of ω-3 PUFA, therapeutic strategies leveraging the endogenous repair SPM pathways to promote inflammation resolution and repair hold great clinical promise. For example, many investigations have identified positive outcomes in ameliorating lung inflammation/disease via therapeutic administration of SPM , including our own previous studies identifying beneficial effects of the DHA-derived SPM maresin-1 in reducing the lung inflammatory effects of acute and repetitive organic dust exposure . While SPM can potently inhibit inflammation while promoting tissue repair, these bioactive metabolites are quickly deactivated by subsequent metabolism . Thus, another therapeutic strategy to leverage these endogenous inflammation resolution pathways is to combine ω-3 PUFA supplementation with pharmacologic inhibition of enzymes responsible for the deactivation of SPM. One such strategy has been the use of inhibitors of the sEH enzyme to prevent the deactivation of the cytochrome P450 family of SPM, thereby potentiating their protective effects . Previous studies have found enhanced protective effects of ω-3 PUFA when used in combination with inhibitors of sEH such as TPPU . This enzyme deactivates the epoxide SPM into less active diol forms – DiHDPA. A recent study in a model of metabolic disease identified that a sEH inhibitor enhanced the protective effects identified in Fat-1 mice vs. WT mice , while previous studies also identify beneficial effects of sEH inhibition in murine models of acute lung injury , pulmonary fibrosis , asthma , and COPD . Corroborating these previous reports, when we utilized TPPU in the experiments described herein, we found that addition of TPPU lowered all outcomes examined in the DE-exposed animals, suggesting that TPPU not only enhanced the effects of ω-3 fatty acids as in Fat-1 + DE + TPPU animals, but also showed efficacy independent of the Fat-1 genotype. In addition, we observed a better response in the Fat-1 male sex receiving the TPPU treatment and three-weeks DE exposure. Consistent with our results sex-specific differences have been reported in sEH null mice and sEH activity in other mouse disease models . While our results support a beneficial effect of maintaining a low omega6:omega3 ratio in response to agricultural dust, omega- 6 PUFAs and their metabolites also modulate inflammation and participate in inflammation resolution and tissue homeostasis.

The SPMs derived from arachidonic acid, such as LXA4 and metabolites generated by the P450 pathway have been repeatedly shown to have anti-inflammatory effects . Most surprisingly, the ARA metabolites generated by the COX-2 pathway, such as PGE2 have protective effects in the lung despite their infamous proinflammatory notion, as shown in asthma and allergicairway inflammation . Airway epithelial cells are the major source of PGE2 production in the lung, and PGE2 protects against airway hyperresponsiveness to allergens by inhibiting leukotriene and thromboxane synthesis that cause bronchoconstriction and by reducing eosinophil recruitment, both of which are antiinflammatory effects of PGE2. Similarly, in asthma, it has been shown that the homeostatic balance between the COX and LOX pathways metabolizing ARA are altered due to damaged epithelium in asthmatic airways. Given this, it has been proposed that the dysregulation of these pathways leads to an imbalance between PGE2 and PGD2/LT which is in part responsible for increased bronchoconstriction. Other roles attributed to PGE2 in the lung include inflammatory cell recruitment, eosinophil degranulation, bronchodilation, T-cell recruitment and differentiation and adhesion molecule expression as reviewed before . With regards to sex-related differences in response to environmental stimuli, a study examined changes in gene expression in the lung associated with inflammation and immunity after ozone exposure . This study found increased lung histological scores and increased infiltrating PMN in the female sex as compared to males after ozone exposure. Among control mice exposed to filtered air alone, females displayed about 5% difference in gene expression of genes related to chemokines and cytokines as compared to males. These genes included Cxcl2 and Ccl19 , Myd88 , and C4b , all of which were associated with immune cell adhesion and recruitment. Considering the sex- and genotype-dependent differences in our model, we examined changes in gene expression as well. Since the most significant changes we observed in our model was in the male sex , we focused our gene expression studies to male sex. NanoString gene expression analysis identified differentially regulated pathways consistent with our previous results indicating that immune cell activation, cell proliferation, wound healing and transport pathways were altered following 3-weeks DE exposure. In an In-depth analysis both using STRING database proteinprotein interaction and NanoString advanced analyses, we also identified changes in NFκBIA, response to macrophage colony stimulating factor, immune clearance, and neutrophil aggregation between the two genotypes . In addition, TPPU treatment affected distinct cellular processes such as T-cell differentiation in WT mice and regulation of neutrophil activation in the Fat-1 genotype. This observed effect of TPPU is consistent with a previous report showing modulation of the Th1/Th17 response while elevating regulatory T-cells by TPPU in an arthritis model .