IL-10 and TGF-ß Increase Connexin-43 Expression and Membrane Potential of HL-1 Cardiomyocytes Coupled with RAW 264.7 Macrophages.

Cardiac resident macrophages facilitate electrical conduction by interacting with cardiomyocytes via connexin-43 (Cx43) hemichannels. Cx43 is critical for impulse propagation and coordination between muscle contractions. Cardiomyocyte electrophysiology can be altered when coupled with noncardiomyocyte cell types such as M2c tissue-resident macrophages. Using cocultures of murine HL-1 cardiomyocytes and RAW 264.7 macrophages, we examined the hypothesis that cytokine signals, TGF-ß1 and IL-10, upregulate Cx43 expression at points of contact between the two cell types. These cytokine signals maintain the macrophages in an M2c anti-inflammatory phenotype, mimicking cardiac resident macrophages. The electrophysiology of cardiomyocytes was examined using di-8-ANEPPS potentiometric dye, which reflects a change in membrane potential. Greater fluorescence intensity of di-8-ANEPPS occurred in areas where macrophages interacted with cardiomyocytes. Suppressor of cytokine signaling 3 (SOCS3) peptide mimetic downregulated fluorescence of this membrane potentiometric stain. Cx43 expression in cocultures was confirmed by fluorescence microscopy and flow cytometry. Confocal images of these interactions demonstrate the Cx43 hemichannel linkages between the cardiomyocytes and macrophages. These results suggest that TGF-ß1 and IL-10 upregulate Cx43 hemichannels, thus enhancing macrophage-cardiomyocyte coupling, raising the cellular resting membrane potential and leading to a more excitatory cardiomyocyte.

HSV-1-induced SOCS-1 expression in keratinocytes: use of a SOCS-1 antagonist to block a novel mechanism of viral immune evasion.

Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.

Inflammatory cytokines and cell adhesion molecules in a rat model of decompression sickness.

To characterize early blood and tissue markers predictive of decompression sickness (DCS), this study focused on identifying changes in inflammatory mediators during the 24-h period immediately following compression-decompression of female Sprague-Dawley rats. Early blood and tissue markers predictive of DCS include inflammatory cytokines and cell adhesion molecules (CAMs). Increased levels of inflammatory cytokines, especially tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma), were detected in the circulation 6 h after decompression. Increased levels of only IL-6 were observed at 24 h. Compared with control animals maintained at 1 atmospheres absolute pressure ATA (101 kPascal), significant increases in expression of E-selectin, and L-selectin, as well as intercellular adhesion molecule-1 (ICAM-1), were observed immunohistochemically in the lungs and brains of the rats 6 h after exposure to 2 (203 kPascal), 3 (303 kPascal), or 4 (404 kPascal) ATA, followed by rapid decompression. These levels drop by 24 h. In contrast to the observations in brain, greater increases in expression of E-selectin and L-selectin around vessels and connective tissue were seen at 24 h after decompression in the quadriceps of rats exposed to either 3 or 4 ATA. Significant increases in expression of the A(2A) receptor, which modulates inflammation by downregulating production of these cytokines, were detected only in the quadriceps removed at 24 h after decompression from 4 ATA. This study demonstrated that rapid decompression induces the release of mediators of inflammation and resulting tissue inflammation cascades, as well as a protective anti-inflammatory response.

Complexity of Interferon-γ Interactions with HSV-1.

The intricacies involving the role of interferon-gamma (IFN-γ) in herpesvirus infection and persistence are complex. Herpes simplex virus type 1 (HSV-1) uses a variety of receptors to enter cells and is transported to and from the host cell nucleus over the microtubule railroad via retrograde and anterograde transport. IFN-γ exerts dual but conflicting effects on microtubule organization. IFN-γ stimulates production of suppressors of cytokine signaling 1 and 3 (SOCS1 and SOCS3), which are involved in microtubule stability and are negative regulators of IFN-γ signaling when overexpressed. IFN-γ also interferes with the correct assembly of microtubules causing them to undergo severe bundling, contributing to its anti-viral effect. Factors leading to the decision for a replicative virus lytic cycle or latency in the trigeminal ganglion (TG) occur on histone 3 (H3), involve IFN-γ produced by natural killer cells and non-cytolytic CD8(+)T cells, SOCS1, SOCS3, and M2 anti-inflammatory microglia/macrophages maintained by inhibitory interleukin 10 (IL-10). Both M2 microglia and CD4(+)CD25(+)Foxp3(+) Treg cells produce IL-10. Histone deacetylases (HDACs) are epigenetic regulators maintaining chromatin in an inactive state necessary for transcription of IFN-γ-activated genes and their anti-viral effect. Following inhibition of HDACs by stressors such as ultraviolet light, SOCS1 and SOCS3 are acetylated, and chromatin is relaxed and available for virus replication. SOCS1 prevents expression of MHC class 1 molecules on neuronal cells and SOCS3 attenuates cytokine-induced inflammation in the area. A model is presented to unify the effects of IFN-γ, SOCS1, SOCS3, and HSV-1 on H3 and chromatin structure in virus latency or reactivation. HSV-1 latency in the TG is viewed as an active ongoing process involving maintenance of microglia in an M2 anti-inflammatory state by IL-10. IL-10 is produced in an autocrine manner by the M2 microglia/macrophages and by virus-specific CD4(+)Foxp3(+) Treg cells interacting with virus-specific non-cytolytic CD8(+) T cells.

Innate immune response to encephalomyocarditis virus infection mediated by CD1d.

CD1d-reactive natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and/or Th2 cytokines, can activate antigen-presenting cell (APC) interleukin-12 (IL-12) production, and are implicated in the regulation of adaptive immune responses. The role of the CD1d system was assessed during infection with encephalomyocarditis virus (EMCV-D), a picornavirus that causes acute diabetes, paralysis and myocarditis. EMCV-D resistance depends on IL-12-mediated interferon-gamma (IFN-gamma) production. CD1d-deficient mice, which also lack CD1d-reactive NKT cells, were substantially more sensitive to infection with EMCV-D. Infected CD1d knockout mice had decreased IL-12 levels in vitro and in vivo, and indeed were protected by treatment with exogenous IL-12. IFN-gamma production in CD1d knockout mice was decreased compared with that in wild-type (WT) mice in response to EMCV-D in vitro, although differences were not detected in vivo. Treatment with anti-asialo-GM1 antibody, to deplete NK cells, caused a marked increase in susceptibility of WT mice to EMCV-D infection, whereas CD1d knockout mice were little affected, suggesting that NK-cell-mediated protection is CD1d-dependent. Therefore, these data indicate that CD1d is essential for optimal responses to acute picornaviral infection. We propose that CD1d-reactive T cells respond to early immune signals and function in the innate immune response to a physiological viral infection by rapidly augmenting APC IL-12 production and activating NK cells.