STAT4 is expressed in neutrophils and promotes antimicrobial immunity.

Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell-independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice were acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 was expressed in neutrophils and activated by IL-12 via a JAK2-dependent pathway. We demonstrate that STAT4 was required for multiple neutrophil functions, including IL-12-induced ROS production, chemotaxis, and production of the neutrophil extracellular traps. Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 resulted in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.

Granzyme A-producing T helper cells are critical for acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4+ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.

AhR Activation by TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) Attenuates Pertussis Toxin-Induced Inflammatory Responses by Differential Regulation of Tregs and Th17 Cells Through Specific Targeting by microRNA.

The Aryl Hydrocarbon Receptor (AhR) is a transcription factor that, when activated by ligand-binding, has been shown to regulate the immune response. Pertussis Toxin (PTX) is a virulence factor found in Bordetella pertussis, a human respiratory pathogen that causes whooping cough. PTX promotes colonization and disease promotion by triggering a heightened inflammatory response. The role of AhR in the regulation of PTX-mediated inflammation has not previously been studied. In the current study, we investigate if AhR activation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a well characterized ligand, can attenuate PTX-mediated systemic inflammation. To that end, C57BL/6 mice were injected intraperitoneally (IP) with PTX twice and treated with TCDD or vehicle (VEH). The PTX+VEH group showed elevated levels of pro-inflammatory cytokines (IL-17A, IL-6, and IFNγ) in serum and increased proportions of CD4+ Th1 and Th17 cells in their spleens. In contrast, the PTX+TCDD group showed significantly lower levels of these inflammatory cytokines and decreased proportions of Th1 and Th17 cells, but increased proportions of Th2 and FoxP3+Tregs when compared to the PTX+VEH group. PTX+TCDD treated mice also showed elevated levels of IL-10, and TFG-b, potent anti-inflammatory cytokines. MicroRNAs (miRs) analysis of CD4+ T cells from the spleens of the PTX+TCDD treated mice revealed significant alterations in their expression and several of these miRs targeted cytokines and signaling molecules involved in inflammation. Specifically, the PTX+TCDD group had a significantly enhanced expression of miR-3082-5p that targeted IL-17, and a decreased expression of miR-1224-5p, which targeted FoxP3. Transfection studies with these miR mimics and inhibitors confirmed the specificity of the target genes. The current study suggests that AhR activation by TCDD suppresses PTX-induced inflammation through miR regulation that triggers reciprocal polarization of Tregs and Th17 cells and also suggests that AhR activation may serve as a treatment modality to suppress heightened inflammation induced during B. pertussis infection.

Cannabinoid Receptor 1 Blockade Attenuates Obesity and Adipose Tissue Type 1 Inflammation Through miR-30e-5p Regulation of Delta-Like-4 in Macrophages and Consequently Downregulation of Th1 Cells.

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.

Role of microRNA in CB1 antagonist-mediated regulation of adipose tissue macrophage polarization and chemotaxis during diet-induced obesity.

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.

Blockade of CB1 cannabinoid receptor alters gut microbiota and attenuates inflammation and diet-induced obesity.

Obesity is characterized by chronic low-grade, systemic inflammation, altered gut microbiota, and gut barrier disruption. Additionally, obesity is associated with increased activity of endocannabinoid system (eCB). However, the clear connection between gut microbiota and the eCB system in the regulation of energy homeostasis and adipose tissue inflammation and metabolism, remains to be established. We investigated the effect of treatment of mice with a cannabinoid receptor 1 (CB1) antagonist on Diet-Induced Obesity (DIO), specifically whether such a treatment that blocks endocannabinoid activity can induce changes in gut microbiota and anti-inflammatory state in adipose tissue. Blockade of CB1 attenuated DIO, inflammatory cytokines and trafficking of M1 macrophages into adipose tissue. Decreased inflammatory tone was associated with a lower intestinal permeability and decreased metabolic endotoxemia as evidenced by reduced plasma LPS level, and improved hyperglycemia and insulin resistance. 16S rRNA metagenomics sequencing revealed that CB1 blockade dramatically increased relative abundance of Akkermansia muciniphila and decreased Lanchnospiraceae and Erysipelotrichaceae in the gut. Together, the current study suggests that blocking of CB1 ameliorates Diet-Induced Obesity and metabolic disorder by modulating macrophage inflammatory mediators, and that this effect is associated with alterations in gut microbiota and their metabolites.

CD44 deletion leading to attenuation of experimental autoimmune encephalomyelitis results from alterations in gut microbiome in mice.

Dysbiosis in gut microbiome has been shown to be associated with inflammatory and autoimmune diseases. Previous studies from our laboratory demonstrated the pivotal role played by CD44 in the regulation of EAE, a murine model of multiple sclerosis. In the current study, we determined whether these effects resulted from an alteration in gut microbiota and the short-chain fatty acid (SCFA) production in CD44 knockout (CD44KO) mice. Fecal transfer from naïve CD44KO but not C57BL/6 wild type (CD44WT) mice, into EAE-induced CD44WT mice, led to significant amelioration of EAE. High-throughput bacterial 16S rRNA gene sequencing, followed by clustering sequences into operational taxonomic units (OTUs) and biochemical analysis, revealed that EAE-induced CD44KO mice showed significant diversity, richness, and evenness when compared to EAE-induced CD44WT mice at the phylum level, with dominant Bacteroidetes (68.5%) and low Firmicutes (26.8%). Further, data showed a significant change in the abundance of SCFAs, propionic acid, and i-butyric acid in EAE-CD44KO compared to EAE-CD44WT mice. In conclusion, our results demonstrate that the attenuation of EAE seen following CD44 gene deletion in mice may result from alterations in the gut microbiota and SCFAs. Furthermore, our studies also demonstrate that the phenotype of gene knock-out animals may be shaped by gut microbiota.

Antibacterial and Biofilm-Disrupting Coatings from Resin Acid-Derived Materials.

We report antibacterial, antibiofilm, and biocompatible properties of surface-immobilized, quaternary ammonium-containing, resin acid-derived compounds and polycations that are known to be efficient antimicrobial agents with minimum toxicities to mammalian cells. Surface immobilization was carried out by the employment of two robust, efficient chemical methods: Copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition click reaction, and surface-initiated atom transfer radical polymerization. Antibacterial and antibiofilm activities against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were strong. Hemolysis assays and the growth of human dermal fibroblasts on the modified surfaces evidenced their biocompatibility. We demonstrate that the grafting of quaternary ammonium-decorated abietic acid compounds and polymers from surfaces enables the incorporation of renewable biomass in an effective manner to combat bacteria and biofilm formation in biomedical applications.