Cutaneous innate immune tolerance is mediated by epigenetic control of MAP2K3 by HDAC8/9.

The skin typically tolerates exposure to various microbes and chemicals in the environment. Here, we investigated how the epidermis maintains this innate immune tolerance to stimuli that are recognized by Toll-like receptors (TLRs). Loss of tolerance to TLR ligands occurred after silencing of the histone deacetylases (HDACs) HDAC8 and HDAC9 in keratinocytes. Transcriptional analysis identified MAP2K3 as suppressed by HDAC8/9 activity and a potential key intermediary for establishing this tolerance. HDAC8/9 influenced acetylation at H3K9 and H3K27 marks in the MAP2K3 promoter. Proteomic analysis further identified SSRP1 and SUPT16H as associated with HDAC8/9 and responsible for transcriptional elongation of MAP2K3. Silencing of MAP2K3 blocked the capacity of HDAC8/9 to influence cytokine responses. Relevance in vivo was supported by observations of increased MAP2K3 in human inflammatory skin conditions and the capacity of keratinocyte HDAC8/9 to influence dendritic cell maturation and T cell proliferation. Keratinocyte-specific deletion of HDAC8/9 also increased inflammation in mice after exposure to ultraviolet radiation, imiquimod, or Staphylococcus aureus These findings define a mechanism for the epidermis to regulate inflammation in the presence of ubiquitous TLR ligands.

Caerulin-induced pro-inflammatory response in macrophages requires TRAF3-p38 signaling activation.

Acute pancreatitis is a common threat to human health. Caerulin provokes severe inflammations, causing injuries to surrounding pancreatic cells. TNF receptor-associated factor 3 (TRAF3) is a highly versatile regulator of immune response. The current study aims to understand the potential effect of TRAF3 on caerulin-induced pro-inflammatory responses. In the primary-cultured mouse bone marrow-derived macrophages (BMDMs), caerulin induced TRAF3 protein stabilization, which formed a complex with mitogen-activated protein kinase kinase 3 (MKK3) to mediate downstream p38 activation. Lentiviral shRNA-mediated TRAF3 stable knockdown significantly attenuated caerulin-induced MKK3-p38 activation and production of several key pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-17. Remarkably, TRAF3 knockdown in caerulin-stimulated BMDMs also alleviated cytotoxicity to Panc02 and primary mouse pancreatic cells. Thus, TRAF3 is required for caerulin-induced p38 activation and macrophage-mediated pro-inflammatory responses. TRAF3 expression in macrophages could be a novel therapeutic target protein for the treatment of acute pancreatitis.

Macrophage deficiency of miR-21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis.

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR-21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR-21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR-21 expression influences foam cell formation, sensitivity to ER-stress-induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR-21 in macrophages increases the expression of the miR-21 target gene, MKK3, promoting the induction of p38-CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post-translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR-21 in atherogenesis.

Bacillus anthracis' lethal toxin induces broad transcriptional responses in human peripheral monocytes.

BACKGROUND: Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system. RESULTS: Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT. CONCLUSION: We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte's normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

MAPK kinase 3 specifically regulates Fc epsilonRI-mediated IL-4 production by mast cells.

Mast cells play a central role in allergic inflammation and are activated through cross-linking of FcεRI receptor-bound IgE, initiating a signaling cascade resulting in production of biologically potent mediators. Signaling pathways in the regulation of specific mediators remain incompletely defined. In this study, we examined the role of MAPK kinase 3 (MKK3) in IgE-dependent mast cell activation. In an in vivo model of passive cutaneous anaphylaxis, MKK3-deficient mice showed a deficit in late-phase IgE-dependent inflammation. To characterize the mechanism of this deficiency, we cultured bone marrow-derived mast cells (BMMCs) from wild-type and MKK3-deficient mice. We found that FcεRI-mediated mast cell activation induced rapid MKK3 phosphorylation by 5 min, diminishing slowly after 6 h. In MKK3-deficient BMMCs, phosphorylation of p38 was reduced at early and later time points. Among 40 cytokines tested using a protein array, IL-4 was the only cytokine specifically downregulated in MKK3-deficient BMMCs. Reduced IL-4 expression was seen in the local skin of MKK3-deficient mice following passive cutaneous allergic reaction. Furthermore, early growth response-1 (Egr1) bound to the promoter of IL-4 in FcεRI-activated mast cells, and Egr1 transcription factor activity was diminished in MKK3-deficient BMMCs. Finally, mast cell-deficient mice reconstituted with MKK3-deficient BMMCs displayed a significantly impaired late-phase allergic inflammatory response. Thus, mast cell MKK3 signaling contributes to IgE-dependent allergic inflammation and is a specific regulator of FcεRI-induced IL-4 production.

Tissue-specific activities of an immune signaling module regulate physiological responses to pathogenic and nutritional bacteria in C. elegans.

Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.

MyD88-mediated stabilization of interferon-gamma-induced cytokine and chemokine mRNA.

The MyD88 adaptor protein is critical in Toll-like receptor and interleukin 1 receptor (IL-1R) signaling, but has not been linked to interferon-gamma receptor (IFN-gammaR) signaling. Here we demonstrate that MyD88 increased the half-life but not the synthesis of IFN-gamma-induced mRNA transcripts encoding tumor necrosis factor and IFN-gamma-inducible protein 10. IFN-gamma stimulation triggered a physical association between the IFN-gammaR1 and MyD88. Transcript stabilization required activation of mixed-lineage kinase 3 and p38 mitogen-activated protein kinase and the presence of an adenine-uridine-rich element in the transcript's 3' untranslated region. These results demonstrate a MyD88-dependent post-transcriptional mechanism through which IFN-gamma can enhance the expression of genes encoding proinflammatory molecules.