BRG1 defines a genomic subset of inflammatory genes transcriptionally controlled by the glucocorticoid receptor

Glucocorticoids (such as Dexamethasone) are commonly used immunomodulatory drugs with potent anti-inflammatory effects, whose mechanisms of action remain incompletely understood. They bind to the Glucocorticoid Receptor (GR), a nuclear hormone receptor that acts as a transcription factor to directly control the expression of inflammatory genes. To elucidate the complex molecular mechanisms employed by GR during the suppression of innate immune responses, we have performed proteomics, ChIP-seq, ATAC-seq, RNA-seq and bioinformatics together with genetic and pharmacological loss of function studies in primary mouse macrophages. We found that GR interacts with the ATP-dependent SWI/SNF chromatin remodeling complex to regulate a specific subset of target genes. Here we show that the central catalytic subunit BRG1 is required not only for the transcriptional activation of classical GR target genes such as Fkbp5 or Klf9, but also for the transcriptional repression of cytokines and chemokines such as Ccl2, Cxcl10 or Il1a. We demonstrate that loss of BRG1 activity leads to reduced histone deacetylase (HDAC) function, and consequently increased histone acetylation, at these repressive GR binding sites. Altogether, our findings suggest that GR interacts with BRG1 to assemble a functional co-repressor complex at a defined fraction of macrophage cis-regulatory elements. These results may indicate additional non-classical, remodeling-independent functions of the SWI/SNF complex and may have implications for the development of future immunomodulatory therapies.

Melatonin-primed ADMSCs elicit an efficacious therapeutic response in improving high-fat diet induced non-alcoholic fatty liver disease in C57BL/6J mice

Background Stem cells are widely used for therapy including treatment of liver damage. Adipose-derived mesenchymal stem cells (ADMSCs) administered to treat fatty liver are known to improve liver function but their use is restricted due to a poor success rate. This study investigates efficacy of melatonin-primed ADMSCs (Mel. MSCs) in experimentally induced non-alcoholic fatty liver disease (NAFLD). Results MSCs treated with LPS showed prominent DCFDA fluorescence as compared to the untreated cells. Also, the JC-1 staining had accounted for higher intensity of green monomer and a weak fluorescence of red dimer indicating weaker mitochondrial membrane potential. But melatonin co-treatment could make necessary corrective changes as evidenced by reverse set of results. The overall cell survival was also found to be improved following melatonin treatment as evidenced by the MTT assay. Also, the antioxidant (Nrf2 and Ho-1) and anti-inflammatory genes (Il-4 and Il-10) showed a decrement in their mRNA levels following LPS treatment whereas the pro-inflammatory genes (Tnf-α, Il-6, Tlr-4, and Lbp) showed a reciprocal increment in the said group. Melatonin co-treatment accounted for an improved status of antioxidant and anti-inflammatory genes as evidenced by their mRNA levels. High-fat high-fructose diet (HFFD) fed C57BL/6J mice recorded higher serum AST and ALT levels and fatty manifestation in histology of liver along with lowered mRNA levels of antioxidant (Nrf2, Catalase, and Gss) genes and Hgf. These set of parameters showed a significant improvement in HFFD + Mel.MSC group. Conclusion A significant improvement in viability of MSCs was recorded due to lowered intracellular oxidative stress and improves mitochondrial membrane potential. Further, melatonin-primed MSCs accounted for a significant decrement in fatty manifestations in liver and an improved physiological status of NAFLD in HFFD fed C57BL/6J mice. Taken together, it is hypothesized that melatonin priming to MSCs prior to its use can significantly augment the success of stem cell therapy.

EZH2 Might Affect Macrophage Chemotaxis and Anti-Inflammatory Factors by Regulating CCL2 in Dental Pulp Inflammation

Objectives. We aimed to evaluate the effects of Enhancer of Zeste Homolog 2 (EZH2) on regulation of macrophage migration and expression of anti-inflammatory genes in pulpitis. Methods. Dental pulp inflammation was verified by histology in rat pulpitis model induced by lipopolysaccharide (LPS). Immunohistochemistry staining was used to detect changes of the expression of EZH2 and tumor necrosis factor alpha (TNF-α) in dental pulp inflammation. The expression of EZH2, CCL2, and cluster of differentiation 68 (CD68: macrophage surface marker) was measured by immunofluorescence staining. The effect of EZH2 on microphage migration was assessed by cell migration assay. The expressions of anti-inflammatory cytokine interleukins (IL-4 and IL-10) and transforming growth factor-β (TGF-β) in HDPCs which were treated by EZH2 complex, CCL2 complex, and CCL2 antibody were examined by quantitative real-time polymerase chain reaction (q-PCR). Results. The expression of TNF-α gradually increased in dental pulp inflammation. The expression of EZH2 in dental pulp decreased in 8 hours after LPS stimulation. However, the expression of EZH2 gradually increased in dental pulp after 1 day stimulation by LPS. The results of immunofluorescence staining showed that the expressions of EZH2, CCL2, and CD68 were significantly upregulated in dental pulp inflammation of rats. EZH2 could enhance macrophage migration. And the chemotactic activity of macrophages exposed to supernatants of EZH2-treated HDPCs could be inhibited by CCL2 inhibition. In addition, EZH2 suppressed the expression of anti-inflammatory genes, but CCL2 inhibition reversed the downregulation of anti-inflammatory factors, including IL-4 and TGF-β in HDPCs. Conclusions. EZH2 might affect chemotaxis of macrophages and the expression of anti-inflammatory factors by regulating CCL2. EZH2 plays an important role in the development of dental pulp inflammation, and it might be as a target for treatment of pulpitis.

Development of Exhausted Memory Monocytes and Underlying Mechanisms

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Transcriptome Profiling of Peripheral Blood Mononuclear Cells Reveals COVID-19 Patients Are Not Recovered to Normal After Discharge for 5 Months

BackgroundHighly pathogenic coronavirus disease-2019 (COVID-19) initiated by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has swiftly expanded throughout the world, and the fatality rate is still expanding due to the second wave in 2020 winters. This ongoing epidemic threatens public health with its new strain that emerged in some countries and might cause devastating deaths. Therefore, the host transcriptomic profile from patients during recovery is important for understanding this disease. MethodsWe performed transcriptome profiling of the RNAs isolated from the peripheral blood mononuclear cells (PBMCs) of recovered COVID-19 patients at hospital discharge of three months and five months respectively.ResultsOur results exposed diverse inflammatory genes and cytokine profiles to infection in recovered patients, and emphasize the highly expressed genes in COVID-19 patients like CCL4, CCL3, CXCL9, CXCL16, IL10, CSF2, VEGFA showed a decreasing trend in recovered patients. Furthermore, the integrated analysis predicted that JUN, CTSL, DDIT4, RRAS, BIRC5, CTSZ, CCNB2, CDK1, OAS1/2, IFIT3, RSAD2, and TP53I3 genes may be valuable for the recovery of COVID-19 patients. ConclusionsOur analysis confirms the presence of some inflammatory genes in recovered patients, suggesting COVID-19 patients did not return to their normal expression even after 5-months of discharge. Identification of transcriptome profiling of recovered patients provides useful information regarding its pathogenesis and might help for the development of better treatment for COVID-19.

p38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. In males, microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. In females, the effect of p38α deficiency was divergent, exhibiting a unique transcriptional profile that included an upregulation of tissue protective genes, and a small subset of inflammatory genes that were also upregulated in males. Taken together, these results reveal a p38α-dependent sex-specific molecular pathway in microglia that is protective in CNS autoimmunity in males, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus suggesting design of future sex-specific therapeutic approaches.

Costimulation of TLR8 responses by CXCL4 in Human Monocytes Mediated by TBK1-IRF5 Signaling and Epigenomic Remodeling

CXCL4 regulates responses of immune cells to endosomal TLRs and has been implicated in the pathogenesis of inflammatory and fibrotic diseases. However, mechanisms by which CXCL4 modulates TLR responses, and its functions in monocytes/macrophages, are still unclear. Here we report that CXCL4 changes the profile of the TLR8 response in human monocytes by selectively and dramatically amplifying inflammatory gene transcription and IL-1β production while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKKε and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for a second signal. CXCL4 strongly induced chromatin remodeling in a cooperative and synergistic manner with TLR8 signaling, inducing de novo enhancers associated with inflammatory genes. These findings identify signaling and epigenomic mechanisms that underly synergistic activation of inflammatory genes by CXCL4 and TLR8, provide a new paradigm for modulation of TLR responses that is relevant for cytokine storm, and suggest targeting the TBK1/IKKε-IRF5 axis may be beneficial in inflammatory diseases.

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss)

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.