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Cannabidiol (CBD) is a bioactive compound isolated from Cannabis plants that has garnered attention within the medical community due to its potent anti-inflammatory properties. To better understand how CBD limits excessive neuroinflammation we administered CBD via oral gavage (20 mg/kg) in a murine model of multiple sclerosis (MS) known as experimental autoimmune encephalomyelitis (EAE). Using single cell RNA sequencing (scRNA Seq) and array-based transcriptomics we were able to delineate how CBD limits excessive inflammation within the central nervous system (CNS) as well as within the intestinal lining in EAE. In-depth scRNA Seq analysis of CNS tissue demonstrated that CBD treatment resulted in a significant reduction in CXCL9, CXCL10 and IL-1β expression within the CNS, leading to inhibited infiltration of inflammatory macrophages. CBD inhibited IL-1β production independent of the classical cannabinoid receptors, CB1 and CB2. CBD treatment also led to induction of Myeloid-derived Suppressor Cells (MDSCs) both in the CNS and periphery. Interestingly, CBD treatment of EAE mice revealed significant suppression of inflammation in the gastrointestinal (GI) tract. The intestinal epithelial cells (IECs) of CBD treated mice demonstrated a transcriptional inhibition of a family of pyroptosis initiators that drive localized inflammation known as gasdermins (GSDMs). Further investigation into the GI tract via 16s sequencing of cecal and fecal contents demonstrated that oral administration of CBD resulted in no significant changes in the intestinal microbiota composition. These findings demonstrate the beneficial effect of CBD treatment on autoimmune neuroinflammation by ablating expression of pro-inflammatory chemoattractants, regulating inflammatory macrophage activity, promoting MDSC expansion, and limiting the systemic low-grade inflammation in the GI tract, culminating in the attenuation of EAE.

This study explores two key questions: (1) How and why the use of touch—defined here as hands-on physical touch—is effective in teaching voice and Alexander Technique, and (2) How might the use of ...

Background and Aims Cannabinoid receptor [CB] activation can attenuate inflammatory bowel disease [IBD] in experimental models and human cohorts. However, the roles of the microbiome, metabolome, and the respective contributions of haematopoietic and non-haematopoietic cells in the anti-colitic effects of cannabinoids have yet to be determined. Methods Female C57BL/6 mice were treated with either cannabidiol [CBD], Δ 9-tetrahydrocannabinol [THC], a combination of CBD and THC, or vehicle, in several models of chemically induced colitis. Clinical parameters of colitis were assessed by colonoscopy, histology, flow cytometry, and detection of serum biomarkers; single-cell RNA sequencing and qRT-PCR were used to evaluate the effects of cannabinoids on enterocytes. Immune cell transfer from CB2 knockout mice was used to evaluate the contribution of haematopoietic and non-haematopoietic cells to colitis protection. Results We found that THC prevented colitis and that CBD, at the dose tested, provided little benefit to the amelioration of colitis, nor when added synergistically with THC. THC increased colonic barrier integrity by stimulating mucus and tight junction and antimicrobial peptide production, and these effects were specific to the large intestine. THC increased colonic Gram-negative bacteria, but the anti-colitic effects of THC were independent of the microbiome. THC acted both on immune cells via CB2 and on enterocytes, to attenuate colitis. Conclusions Our findings demonstrate how cannabinoid receptor activation on both immune cells and colonocytes is critical to prevent colonic inflammation. These studies also suggest how cannabinoid receptor activation can be used as a preventive and therapeutic modality against colitis.

Acute Respiratory Distress Syndrome (ARDS) causes up to 40% mortality in humans and is difficult to treat. ARDS is also one of the major triggers of mortality associated with coronavirus-induced disease (COVID-19). We used a mouse model of ARDS induced by Staphylococcal enterotoxin B (SEB), which triggers 100% mortality, to investigate the mechanisms through which &Delta, 9-tetrahydrocannabinol (THC) attenuates ARDS. SEB was used to trigger ARDS in C3H mice. These mice were treated with THC and analyzed for survival, ARDS, cytokine storm, and metabolome. Additionally, cells isolated from the lungs were used to perform single-cell RNA sequencing and transcriptome analysis. A database analysis of human COVID-19 patients was also performed to compare the signaling pathways with SEB-mediated ARDS. The treatment of SEB-mediated ARDS mice with THC led to a 100% survival, decreased lung inflammation, and the suppression of cytokine storm. This was associated with immune cell apoptosis involving the mitochondrial pathway, as suggested by single-cell RNA sequencing. A transcriptomic analysis of immune cells from the lungs revealed an increase in mitochondrial respiratory chain enzymes following THC treatment. In addition, metabolomic analysis revealed elevated serum concentrations of amino acids, lysine, n-acetyl methionine, carnitine, and propionyl L-carnitine in THC-treated mice. THC caused the downregulation of miR-185, which correlated with an increase in the pro-apoptotic gene targets. Interestingly, the gene expression datasets from the bronchoalveolar lavage fluid (BALF) of human COVID-19 patients showed some similarities between cytokine and apoptotic genes with SEB-induced ARDS. Collectively, this study suggests that the activation of cannabinoid receptors may serve as a therapeutic modality to treat ARDS associated with COVID-19.

Summary Intestinal disequilibrium leads to inflammatory bowel disease (IBD), and chronic inflammation predisposes to oncogenesis. Antigen-presenting dendritic cells (DCs) and macrophages can tip the equilibrium toward tolerance or pathology. Here we show that delta-9-tetrahydrocannabinol (THC) attenuates colitis-associated colon cancer and colitis induced by anti-CD40. Working through cannabinoid receptor 2 (CB2), THC increases CD103 expression on DCs and macrophages and upregulates TGF-β1 to increase T regulatory cells (Tregs). THC-induced Tregs are necessary to remedy systemic IFNγ and TNFα caused by anti-CD40, but CB2-mediated suppression of APCs by THC quenches pathogenic release of IL-22 and IL-17A in the colon. By examining tissues from multiple sites, we confirmed that THC affects DCs, especially in mucosal barrier sites in the colon and lungs, to reduce DC CD86. Using models of colitis and systemic inflammation we show that THC, through CB2, is a potent suppressor of aberrant immune responses by provoking coordination between APCs and Tregs., Graphical Abstract, Highlights • THC can prevent the development of colitis-associated colon cancer in mice • CB2 activation on immune cells dampens disease-mediated oncogenic IL-22 • THC attenuates anti-CD40-induced colitis through Tregs and APCs • THC activation of CB2 on APCs reduces pro-inflammatory cytokine secretion, Immunology; Cancer

Background/Objectives Obesity is a pandemic disorder that is characterized by accumulation of adipose tissue and chronic-low grade inflammation that is driven primarily by adipose tissue macrophages (ATMs). While ATM polarization from pro-(M1)to anti-(M2) inflammatory phenotype influences insulin sensitivity and energy expenditure, the mechanisms of such a switch are unclear. In the current study we identified epigenetic pathways including microRNAs (miR) in ATMs that regulate obesity-induced inflammation. Subjects/Methods Male C57BL/6J mice were fed normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks to develop lean and diet-induced obese mice respectively. Transcriptome microarrays, microRNA microarrays, and meDIP-Seq were performed on ATMs isolated from visceral fat. Pathway analysis and bone marrow derived macrophage (BMDM) transfections further allowed computational and functional analysis of miRNA-mediated ATM polarization. Results ATMs from HFD-fed mice were skewed towards M1 inflammatory phenotype. Concurrently, the expression of miRs 30a-5p, 30c-5p, and 30e-5p was downregulated in ATMs from HFD mice when compared to mice fed NCD. The miR-30 family was shown to target Delta-like-4, a Notch1 ligand, whose expression was increased in HFD ATMs. Inhibition of miR-30 in conditioned BMDM triggered Notch1 signaling, pro-inflammatory cytokine production, and M1 macrophage polarization. In addition, DNA hypermethylation was observed in mir30-associated CpG islands suggesting HFD downregulates miR-30 through epigenetic modifications. Conclusions HFD-induced obesity downregulates miR-30 by DNA methylation thereby inducing Notch1 signaling in ATMs and their polarization to M1 macrophages. These findings identify miR-30 as a regulator of pro-inflammatory ATM polarization and suggest miR-30 manipulation could be a therapeutic target for obesity-induced inflammation.

Colitis, an inflammatory bowel disease, is caused by a variety of factors, but luminal microbiota are thought to play crucial roles in disease development and progression. Indole is produced by gut microbiota and is believed to protect the colon from inflammatory damage. In the current study, we investigated whether indole-3-carbinol (I3C), a naturally occurring plant product found in numerous cruciferous vegetables, can prevent colitis-associated microbial dysbiosis and attempted to identify the mechanisms. Treatment with I3C led to repressed colonic inflammation and prevention of microbial dysbiosis caused by colitis, increasing a subset of gram-positive bacteria known to produce butyrate. I3C was shown to increase production of butyrate, and when mice with colitis were treated with butyrate, there was reduced colonic inflammation accompanied by suppression of Th17 and induction of Tregs, protection of the mucus layer, and upregulation in Pparg expression. Additionally, IL-22 was increased only after I3C but not butyrate administration, and neutralization of IL-22 prevented the beneficial effects of I3C against colitis, as well as blocked I3C-mediated dysbiosis and butyrate induction. This study suggests that I3C attenuates colitis primarily through induction of IL-22, which leads to modulation of gut microbiota that promote antiinflammatory butyrate.

Obesity is characterized by chronic low-grade inflammation that contributes to development of cardiometabolic disorders. Cannabinoid receptor 1 (CB1) antagonists attenuate diet-induced obesity (DIO) and related inflammation, although the precise anti-inflammatory mechanisms involved have not been fully explored. In the current study we used a mouse model of DIO intervention to determine the microRNA (miRNA, miR)-mediated anti-obesity and anti-inflammatory effects of the CB1 antagonist, AM251. DIO mice that were fed high-fat diet (HFD) for 12 weeks were treated with AM251 (10 mg/kg) for an additional 4 weeks. HFD + AM251 mice experienced rapid and prolonged weight loss and reduced inflammatory M1 adipose tissue macrophage (ATM) infiltration. To investigate miRNA-mediated regulation of ATMs, F4/80+ cells from stromal vascular fractions (SVF) of epididymal fat were subjected to miR microarray analysis. Several miRs were differentially expressed in AM251-treated mice that were independent of calorie restriction. Prominently, miR-30e-5p was upregulated in ATMs from HFD + AM251 mice while the miR-30e-5p target, DLL4, was downregulated. Consistent with a decrease in DLL4-Notch signaling, fat storage and pro-inflammatory cytokine/chemokine expression was reduced following AM251 treatment. Furthermore, we found that AM251-treated macrophages can suppress DLL4-mediated Th1 polarization in CD4+ T cells. Together these data demonstrate that blocking CB1 receptors leads to upregulation of miR-30e-5p and down regulation of DLL4 in ATMs, which in turn suppress DLL4-Notch signaling-induced polarization of inflammatory Th1 cells and adipocyte energy storage. This combined effect of ATMs and T cells leads to an anti-inflammatory state and attenuation of DIO. These data support therapeutic potential of miR-30 in the treatment of cardiometabolic disorders.

Although cannabinoid receptor 1 (CB1) antagonists have been shown to attenuate diet-induced obesity (DIO) and associated inflammation, the precise molecular mechanisms involved are not clear. In the current study, we investigated the role of microRNA (miR) in the regulation of adipose tissue macrophage (ATM) phenotype following treatment of DIO mice with the CB1 antagonist SR141716A. DIO mice were fed high-fat diet (HFD) for 12 weeks and then treated daily with SR141716A (10 mg/kg) for 4 weeks while continuing HFD. Treated mice experienced weight loss, persistent reduction in fat mass, improvements in metabolic profile, and decreased adipose inflammation. CB1 blockade resulted in down-regulation of several miRs in ATMs, including the miR-466 family and miR-762. Reduced expression of the miR-466 family led to induction of anti-inflammatory M2 transcription factors KLF4 and STAT6, whereas down-regulation of miR-762 promoted induction of AGAP-2, a negative regulator of the neuroimmune retention cues, Netrin-1 and its coreceptor UNC5B. Furthermore, treatment of primary macrophages with SR141716A up-regulated KLF4 and STAT6, reduced secretion of Netrin-1, and increased migration toward the lymph node chemoattractant CCL19. These studies demonstrate for the first time that CB1 receptor blockade attenuates DIO-associated inflammation through alterations in ATM miR expression that promote M2 ATM polarization and macrophage egress from adipose tissue. The current study also identifies additional novel therapeutic targets for diet-induced obesity and metabolic disorder.

Upregulation of long noncoding RNA (lncRNA) AW112010 has been observed in multiple inflammatory disease models. In line with implication of this lncRNA as a regulator of inflammation, it has also been shown that AW112010 expression decreases upon treatment with anti-inflammatory compounds. Since lncRNAs show abnormal expression in many inflammatory diseases and reactions, elucidating their role in such ailments is necessary. Data from our lab has shown that treatment with aryl hydrocarbon receptor (AhR) ligands induce epigenetic pathway dysregulation, so this study aimed to determine inflammatory genes dysregulated upon treatment with the endogenous AhR ligand, 6-formylindolo[3,2-b] carbazole (FICZ). A murine model of immune cell-mediated liver injury was employed by intravenously injecting 12.5 mg/kg Concanavalin A (ConA). One hour after challenge, mice were treated with 50 μg/kg FICZ via an intraperitoneal injection. Interestingly, analysis of serum collected twenty-four hours after challenge showed that FICZ was able to reduce the level of alanine transaminase (ALT) suggesting a decrease in disease severity. Single cell RNA sequencing was conducted on infiltrating liver mononuclear cells enriched via a percoll gradient. We observed a reduced percentage of T cells upon FICZ treatment and an increase in the percentage of neutrophils. Overall, we observed downregulation of AW112010 in the FICZ-treated group. Specifically, AW112010 was identified as one of the top 20 genes dysregulated in the NK cell cluster and was downregulated in the B cell, CD8+ T cell, and Kupffer cell clusters. Together, these data suggest AW112010 downregulation as a potential mechanism through which FICZ protects mice from severe liver injury.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has anti-inflammatory properties; however the precise immunological mechanisms involved are not clear. Research from our laboratory has established that CBD promotes expansion of immunosuppressive myeloid-derived suppressor cells (MDSC), which are immature leukocytes thought to have stalled their differentiation towards mature myeloid lineages such as macrophages and dendritic cells (DC). In the current study, we aimed to better understand the mechanisms of CBD-altered hematopoiesis. We performed flow cytometry and single cell RNA sequencing (sc-RNAseq) on peritoneal cells from C57BL/6 mice treated with Vehicle (Veh) or CBD (20mg/kg, i.p.). Consistent with our previous studies, CBD promoted accumulation of MDSCs after 24h, which dissipated after 48h. Strikingly, MDSC accumulation was preceded by ablation of macrophages, which was maintained beyond 48h. In addition, accumulation of DCs started at 12h and accelerated through 48h. Similarly, sc-RNAseq of peritoneal cells harvested at 24h showed two distinct populations of cells expressing Adgre1, which encodes F4/80. These two clusters were distinct between Veh- and CBD-treated mice due to their drastically altered transcriptomes. The Veh-distinct cluster was comprised of macrophages expressing complement genes, while the CBD-distinct cluster was comprised of MDSCs expressing Arg1. Furthermore, the DC cluster expressing Ccr2 was enlarged in CBD-treated mice. Together, our results demonstrate that CBD drastically alters hematopoiesis, depleting macrophages and promoting MDSCs and DCs. These findings suggest CBD treatment may be beneficial in various macrophage-driven inflammatory disorders.

GTPase of the immune-associated protein 5 (Gimap5) has been implicated in immune cell maintenance and development. A mouse model comparable to autoimmune hepatitis in humans was used to study the alterations induced in the gene expression profile of immune cells upon administration of environmental toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In this study, we tested the hypothesis that TCDD is capable of modulating gene expression to ameliorate autoimmune hepatitis severity. Mice were injected intravenously with 12.5 mg/kg concanavalin A (ConA) and treated intraperitoneally one hour after challenge with vehicle or 10 μg/kg TCDD. TCDD-treated mice showed lower levels of liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as compared to vehicle-treated mice. Histopathological analysis revealed a reduction in liver damage in the TCDD-treated group as compared to the vehicle-treated control. Furthermore, flow cytometric analysis of liver-infiltrating mononuclear cells demonstrated that following treatment with TCDD, there was an increase in FoxP3+CD4+ Tregs as well as a decrease in proinflammatory Tbet+ Th1 and RORγt+ Th17 T helper cell subtypes after induction of the disease. Single cell RNA-sequencing was conducted on liver mononuclear cells to further assess the changes induced by TCDD. The gene expression profile showed that TCDD treatment significantly upregulated the expression of vital genes such as Gimap5, a maintenance gene for normal liver function and survival of T cells. In summary, our data suggests that TCDD is capable of inducing Gimap5 expression in autoimmune hepatitis, which in turn modulates the progression of this disease.

Over thirty percent of individuals infected with human immunodeficiency virus type 1 (HIV) worldwide suffer from HIV-associated neurocognitive disorder (HAND). GFAP-gp120 transgenic (Tg) mice serve as an excellent model system for HAND because they manifest the same neuropathological attributes observed in the brains of AIDS patients. In this study, we sought to identify novel genes associated with the GFAP-gp120 neurodegenerative mouse model. Von Frey filament and hot plate tests on 8 month old GFAP-gp120 mice resulted in a significant increase in mechanical and thermal nociception in Tg versus wild-type mice. Neuropathology was confirmed via immunohistochemistry of MAP2, Synaptophysin, Iba-1, and GFAP. The MAP2 and Synaptophysin allowed us to visualize dendritic density and synaptic loss. Infiltration of macrophages was observed with anti-Iba-1 and astrocytosis with anti-GFAP Abs. Additionally, single cell RNA sequencing (scRNASeq) of encephalitogenic mononuclear cells illustrated 10 distinct clusters of cells. These clusters included: astrocytes, neurons, oligodendrocytes, endothelial cells, microglia, and macrophages. Differential expression analysis showed decreased expression of GM42031 in Tg macrophages and microglia. This locus is currently unclassified and thought to be either a long non coding RNA (lncRNA) or a gene for a NYNRIN-like protein. Until now, this particular RNA has not been associated with the GFAP-gp120 disease model. However, there has been some association of GM42031 with macrophage regulation in Parkinson’s disease and colitis models. Together our study provides novel insights into the immunopathology of HAND that may form the basis for development of effective therapeutics.

Although recent studies have established cannabidiol (CBD) as a potent anti-inflammatory agent, the mechanisms governing these processes remain unclear. Our previous research demonstrated that CBD treatment induced expansion of CD11b+Gr-1+MDSC in the peritoneum, which suppressed T cell functions leading to attenuation of staphylococcal enterotoxin B (SEB)-mediated liver inflammation in mice. Using the same model, we characterize the effects of CBD administration in the liver. Briefly, mice were injected intraperitoneally (I.P.) with 50mg/kg of CBD for 4 days (d), followed by SEB challenge on d 3. Flow cytometry analysis revealed a significant reduction in Vβ8+ CD8+ T cells. In agreement with our earlier findings, mice treated with CBD displayed robust expansion of Gr-1+ MDSC in the liver, which was primarily comprised of Ly6CHI MHCIILO leukocytes. As mast cells have been found to play a role in CBD-mediated induction of MDSC via increased peroxisome proliferator-activated receptor gamma (PPARγ) signaling, we examined changes to liver mast cell populations. We discovered two distinct subsets of mast cells, C-KithiFcɛR1a+ and C-Kitlo FcɛR1a+present in the liver. CBD treatment promoted expansion of the C-Kitlo FcɛR1a+ subset. Analysis by miRNA microarray displayed enrichment of miRNAs targeting mast cell growth factor, KIT ligand (KITLG), and significant downregulation of miR-217-5p, which is conserved for the co-activator of PPARγ (PPARγC1A). Increased gene expression of PPARγ was confirmed by single-cell RNA sequencing (sc-RNAseq). Taken together, our results identify CBD induced epigenetic reduction of C-Kit expression and KITLG signaling in mast cells to favor PPARγ signaling as a novel anti-inflammatory mechanism.

Cannabidiol (CBD) is a nonpsychoactive ingredient from Cannabis that has garnered attention in the medical community due to its anti-inflammatory properties and therapeutic potential. CBD has in particular become a popular alternative medicine among individuals with autoimmune disorders due to CBD lacking the side effects and costs associated with immunosuppressants. To better define how CBD inhibits inflammation, we studied the effects of orally administering CBD (20mg/kg) in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Using single cell RNA sequencing on cells isolated from the central nervous system (CNS) of EAE mice, we demonstrated that CBD treatment inhibits neuroinflammation by regulating gene expression and infiltration of myeloid cells. Within the resident and infiltrating myeloid cells of the CNS, there was decreased expression of inflammatory cytokines and chemokines (CXCL9, CXCL10, IL-18) associated with neuroinflammation and CD4+ T cell recruitment, pyroptosis initiators (gasdermin D; Gsdmd), oxide synthesizers (Arg1) and antigen presentation mediators (CD40) in CBD-treatment group. Additionally, CBD yielded an increase in the polymorphonuclear myeloid derived suppressor cells in the CNS, signifying immunotolerance induction at the site of inflammation. In the VEH treated mice, we found CD4+ T cells that express the receptor for IL-18 (IL-18r1) that are absent in CBD treated mice, suggesting CBD affects T cell activity via inhibition of myeloid cell activity. This data shows that CBD treatment ameliorates EAE by inhibiting infiltration, pro-inflammatory cytokine production, antigen presentation, T cell recruitment and pyroptosis of myeloid cells in the CNS.

Diethylstilbestrol (DES) is an endocrine disruptor that was used to prevent adverse effects of pregnancy in women in late 1940s until early 1970s. Its use was banned following significant toxicity and negative effects not only in the mothers but also transgenerationally. Previous studies from our laboratory showed that DES induces thymic atrophy and immunosuppression in mice. In this study, we investigated the molecular mechanisms through which DES triggers thymic atrophy, specifically autophagy. To that end, we treated C57BL/6 mice with DES, and determined expression of two autophagy-related proteins, microtubule-associated protein-1 light chain 3 (LC3) and Beclin-1 (Becn1). We observed that DES-induced thymic atrophy was associated with increased autophagy in thymocytes and significant upregulation in the expression of both Becn1 and LC3. DES also caused downregulation in the expression of miR-30a in thymocytes, and transfection studies revealed that miR-30a targeted Becn1. Upon examination of methylation status of Becn1, we noted hypomethylation of Becn1 in thymocytes of mice exposed to DES. Together, these data demonstrate for the first time that DES induces autophagy in thymocytes potentially through epigenetic changes involving hypomethylation of Becn1 and down-regulation of miR-30a expression.

Obesity is characterized by chronic low-grade inflammation that contributes to development of cardiometabolic disorders. Cannabinoid receptor 1 (CB1) antagonists attenuate diet-induced obesity (DIO) and related inflammation, although the precise anti-inflammatory mechanisms involved have not been fully explored. In the current study we used a mouse model of DIO intervention to determine the microRNA (miR)-mediated anti-obesity and anti-inflammatory effects of the CB1 antagonist AM251. DIO mice that were fed high-fat diet (HFD) for 12 weeks were treated with AM251 (10mg/kg) for an additional four weeks. HFD+AM251 mice experienced rapid and prolonged weight loss and reduced inflammatory M1 adipose tissue macrophage (ATM) infiltration. To investigate miRNA-mediated regulation of ATMs, F4/80+ cells from stromal vascular fractions of epididymal fat were subjected to miR microarray analysis. Several miRs were differentially expressed in AM251-treated mice that were independent of calorie restriction. Prominently, miR-30e-5p was upregulated in ATMs from HFD+AM251 mice while the miR-30e-5p targeting DLL4 was downregulated. Consistent with a decrease in DLL4-Notch signaling, fat storage and pro-inflammatory cytokine/chemokine expression was reduced following AM251 treatment. Furthermore, we demonstrate that AM251-treated macrophages can suppress DLL4-mediated Th1 polarization in CD4+ T cells. Together these data indicate that weight-loss due to AM251 treatment mediates anti-inflammatory effects and amelioration of obesity through miR-30e-5p regulation of DLL4-Notch signaling-induced type 1 inflammation. These data support therapeutic potential of miR-30 in the treatment of cardiometabolic disorders.

TCDD, FICZ, and Resveratrol (RES) are potent ligands of aryl hydrocarbon receptor (AhR). TCDD is well known to cause thymic atrophy but the effects of FICZ and RES have not been studied to date. In the current study, we investigated the effect of AhR ligands in vivo. We chose these AhR ligands for their different origin-TCDD, an environmental contaminant, FICZ, endogenously generated, and RES, found in plant products. To this end, C57BL/6 mice were treated with vehicle (corn oil) or TCDD (10 μg/kg bw) or FICZ (10 μg/kg bw) or RES (100 mg/kg bw) by oral gavage. Three days post treatment, thymi and spleens were harvested and stained with fluorochrome-labelled Abs against various T cell surface markers and analyzed by flow cytometry. We observed significant effect of TCDD on decreasing thymic weight and thymic cellularity, when compared to vehicle. FICZ, on the other hand, had no significant effect on thymus. RES, however, significantly caused increase in thymic weight as well as thymic cellularity. Upon analysis of various T cell populations in the thymus, TCDD caused significant downregulation of almost all the markers (CD3, CD4, CD8, CD25, CD44, and αβTCR) whereas, FICZ and RES had moderate effects on thymic T cell subpopulations. Upon analysis of spleen, all the three AhR ligands had mixed effects. We observed significant effect of TCDD and RES in generation of Tregs, when compared to vehicle. FICZ, on the other hand, suppressed Tregs but significantly increased Th17 cells. Together, our studies suggest that although TCDD, FICZ, and RES are AhR ligands, they had different effects on both thymus and spleen. TCDD and RES had role in inducing immune suppression by generating more Tregs, whereas, FICZ had a role in inflammation by generating more Th17 cells.

The endocannabinoid (eCB) system regulates a variety of physiological processes including inflammation and metabolism. Obesity is characterized by overactivation of the eCB system, chronic low-grade inflammation, metabolic dysfunction, and gut microbial dysbiosis. Blockade of cannabinoid receptor 1 (CB1) ameliorates obesity, however CB1-mediated immune-microbial interactions have not been extensively explored. In the current study, antagonists and genetic ablation of CB1 were used in a mouse model of diet-induced obesity to determine the role of CB1 in modulating inflammation, metabolism, and the gut microbiome. As expected, CB1−/− mice were resistant to high-fat diet (HFD)-induced weight gain and metabolic inflammation was significantly less in CB1−/− mice when compared to wild-type (WT) controls. Lamina propria lymphocytes from CB1−/− mice did not display a Th1 induction after HFD feeding whereas WT mice fed HFD had elevated CD4+T-bet+ cells compared to WT mice fed low-fat diet. Decreased colonic inflammation indicated CB1-dependent alterations in the gut microbiome may contribute to a decreased obese phenotype. Similarly, WT obese mice treated with the CB1 antagonist AM251 experienced weight loss, decreased inflammation, and improvement in metabolic parameters. Interrogation of the gut microbiome by 16S metagenomics revealed CB1-dependent alterations in abundance of bacteria belonging to the Clostridiales order. In addition, functional predictions by PICRUSt analysis revealed decreased abundance of operational taxonomic units belonging to bacterial metabolism and membrane transport pathways. Together these data suggest CB1 regulates metabolism by shaping the immune response and gut microbial community.

Macrophages are innate immune cells that play integral roles in maintenance of adipose tissue homeostasis. Visceral obesity stimulates pro-inflammatory macrophages to infiltrate into adipose tissue causing chronic low-grade inflammation that can lead to insulin resistance and cardiometabolic disorders. Notch signaling is elevated in obesity and serves as a form of communication between macrophages and adipocytes. Blockade of this pathway promotes adipose tissue browning while reducing inflammation and therefore, has therapeutic potential. MicroRNAs (miRNA, miR) are non-coding RNAs that bind the 3′UTR of target mRNAs to repress their translation and miRNA based therapies are presently being developed for clinical purposes. In the current study, we identified differentially expressed miRNAs in adipose tissue macrophages (ATMs) between lean and obese mice by microarray. Array analysis and PCR validation revealed miRNAs -30a-5p, -30c-5p, and -30e-5p were downregulated in obese ATMs suggesting the miR-30 family plays an important role in macrophage phenotype. Pathway analysis demonstrated that miR-30 targeted Notch signaling genes including Delta-like ligand-4 (DLL4), a previously identified therapeutic target for cardiometabolic disorders. It was noted that DLL4 expression was increased on obese ATMs and in vitro miRNA transfection studies further demonstrated that miR-30 modulates Notch signaling-induced inflammation. These data demonstrate for the first time that the miR-30 family may play a critical role in the regulation of DLL4-mediated Notch signaling in ATMs, thereby modulating metabolic inflammation. (Supported in part by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755 and P20GM103641).

Obesity is a major health problem in the U.S. that continues to increase in prevalence. Excessive calorie consumption leads to expansion of adipose tissue through both hypertrophy and hyperplasia. As a result, adipocytes die and macrophages infiltrate into the adipose tissue to repair damage. Activation and infiltration of immune cells contribute to the chronic low-grade inflammation present in obesity. Immune cells express cannabinoid receptors that regulate inflammation. In this study, a mouse model of high fat diet-induced obesity was used to determine the effect the CB1 receptor antagonist AM251 has on adipose tissue inflammation. C57BL6 mice were fed a high fat diet (HFD) for twelve weeks to induce obesity and then given AM251 (10mg/kg/day) oral gavage for four weeks while continuing HFD. Treatment improved metabolic parameters while reducing fat mass and pro-inflammatory cytokine production. To further investigate the anti-inflammatory effects of AM251, infiltrating F4/80+ cells were isolated from epididymal fat and used for microRNA microarray. Nine miRNAs were differentially expressed as a result of AM251 treatment. These miRNAs were analyzed using Ingenuity Pathway Analysis software (Qiagen) and were found to significantly affect the “Inflammatory Disease and Inflammatory Response” pathways. Upregulated miRNAs such as miR-466i-3p were found to target macrophage polarization genes including Pik3r1. The current study suggests that CB1 antagonists may mediate anti-inflammatory effects and amelioration of obesity through regulation of miRNA expression. (Supported in parts by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641, and Veterans Affairs Merit Award BX001357.)

This thesis explores how the ideals and expectations attached to the female body in Australia and Japan affect women’s self-harm and suicidal behaviours. In the majority of cultural contexts, traditional ideas of sex (male/female) and gender (masculine/feminine) still affect the modern constructions of sex and gender and the ideals and expectations prescribed to them. All behaviours have the potential to be performed but the ideals and expectations prescribed to a male or female body can influence the behaviours performed by a ‘good’ man or woman. Consequently, at a foundational level, male suicides have been very differently conceptualised and perceived than female suicides. Depending on the performance, the selfharm and suicidal behaviours some women choose to inscribe upon their bodies can be read as ‘good’; they have complied with feminine ideals. However, since the time of Durkheim, it has been argued that women have been protected from suicide by their ‘inside’ social role and adherence to social ideals. Durkheim’s inside role constructed the female body in frames which evolved into the constructions of beauty, sexuality and marriage considered traditional today. It was implied that complying with the ideals of a ‘good’ woman — being a beautiful and sexually faithful wife – would protect a woman from suicide. Yet, this assumption may neither be relevant nor realistic to the lives of modern Australian and Japanese women. Further, ‘good’ women may still be vulnerable to performing harmful inscriptions upon their bodies.