Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells.

Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

Gap Junctions and Breast Cancer Dormancy.

Breast cancer (BC) relapse, despite clinical advancement, remains one of the biggest issues in the field. Intercellular communication, specifically via connexin (Cx)-mediated gap junctions (GJs), play a key role in the long-term survival of these, treatment-resistant breast cancer stem cells (CSCs), allowing for relapse. Both basic and clinical evidence reveal dual roles for GJs, in tumor suppression, generally referred to as dormancy, and progression and metastasis. GJ intercellular communication (GJIC) can be mediated by multiple types of Cxs, depending on the organ to which the BC cells metastasize. This review expands on the differential expression of Cx-mediated GJIC between CSCs and niche cells within a given microenvironment.

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell-tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

The role of oligodendrocyte gap junctions in neuroinflammation.

Gap junctions (GJs) provide channels for direct cell-to-cell connectivity serving the homeostasis in several organs of vertebrates including the central (CNS) and peripheral (PNS) nervous systems. GJs are composed of connexins (Cx), which show a highly distinct cellular and subcellular expression pattern. Oligodendrocytes, the myelinating cells of the CNS, are characterized by extensive GJ connectivity with each other as well as with astrocytes. The main oligodendrocyte connexins forming these GJ channels are Cx47 and Cx32. The importance of these channels has been highlighted by the discovery of human diseases caused by mutations in oligodendrocyte connexins, manifesting with leukodystrophy or transient encephalopathy. Experimental models have provided further evidence that oligodendrocyte GJs are essential for CNS myelination and homeostasis, while a strong inflammatory component has been recognized in the absence of oligodendrocyte connexins. Further studies revealed that connexins are also disrupted in multiple sclerosis (MS) brain, and in experimental models of induced inflammatory demyelination. Moreover, induced demyelination was more severe and associated with higher degree of CNS inflammation in models with oligodendrocyte GJ deficiency, suggesting that disrupted connexin expression in oligodendrocytes is not only a consequence but can also drive a pro-inflammatory environment in acquired demyelinating disorders such as MS. In this review, we summarize the current insights from human disorders as well as from genetic and acquired models of demyelination related to oligodendrocyte connexins, with the remaining challenges and perspectives.

IL-22 Down-Regulates Cx43 Expression and Decreases Gap Junctional Intercellular Communication by Activating the JNK Pathway in Psoriasis.

The roles of IL-22 in the pathomechanisms of psoriasis have been well demonstrated. Gap junctional intercellular communication (GJIC) is widely known for its involvement in multiple biological and pathological processes such as growth-related events, cell differentiation, and inflammation. Here, we show that IL-22 significantly decreased GJIC and down-regulated Cx43 expression in HaCaT cells. Cx43 overexpression markedly inhibited the proliferation of and increased GJIC in HaCaT cells, but the silencing of Cx43 exerted the opposite effects. Additionally, Cx43 overexpression effectively rescued the IL-22-induced decrease in GJIC in HaCaT cells. The IL-22-induced down-regulation of Cx43 expression and decrease in GJIC can be significantly blocked by the JNK inhibitor SP600125 and by the overexpression of IL-22RA2 (which specifically binds to IL-22 and inhibits its activity), but not by the NF-κB inhibitor BAY11-7082, in HaCaT cells. Furthermore, the IL-22-induced down-regulation of Cx43 expression mediated by the JNK signaling pathway was confirmed in a mouse model of IL-22-induced psoriasis-like dermatitis. Similarly, Cx43 expression was significantly lower in the lesional skin than in the nonlesional skin of patients with psoriasis. These results suggest that IL-22 decreases GJIC by activating the JNK signaling pathway, which down-regulates Cx43 expression; this process is a possible pathomechanism of keratinocyte hyperproliferation in psoriasis.

Increased expression and functionality of the gap junction in peripheral blood lymphocytes is associated with hypertension-mediated inflammation in spontaneously hypertensive rats.

BACKGROUND: Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs). METHODS: The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A). RESULTS: The percentage of CD4+ T cells and the CD4+/CD8+ ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8+ and CD4+CD25+ T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes. CONCLUSIONS: Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation.

The role of connexin and pannexin containing channels in the innate and acquired immune response.

Connexin (Cx) and pannexin (Panx) containing channels - gap junctions (GJs) and hemichannels (HCs) - are present in virtually all cells and tissues. Currently, the role of these channels under physiological conditions is well defined. However, their role in the immune response and pathological conditions has only recently been explored. Data from several laboratories demonstrates that infectious agents, including HIV, have evolved to take advantage of GJs and HCs to improve viral/bacterial replication, enhance inflammation, and help spread toxicity into neighboring areas. In the current review, we discuss the role of Cx and Panx containing channels in immune activation and the pathogenesis of several infectious diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40-60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.

Connexins and their channels in inflammation.

Inflammation may be caused by a variety of factors and is a hallmark of a plethora of acute and chronic diseases. The purpose of inflammation is to eliminate the initial cell injury trigger, to clear out dead cells from damaged tissue and to initiate tissue regeneration. Despite the wealth of knowledge regarding the involvement of cellular communication in inflammation, studies on the role of connexin-based channels in this process have only begun to emerge in the last few years. In this paper, a state-of-the-art overview of the effects of inflammation on connexin signaling is provided. Vice versa, the involvement of connexins and their channels in inflammation will be discussed by relying on studies that use a variety of experimental tools, such as genetically modified animals, small interfering RNA and connexin-based channel blockers. A better understanding of the importance of connexin signaling in inflammation may open up towards clinical perspectives.

TLR-Activated Gap Junction Channels Protect Mice against Bacterial Infection through Extracellular UDP Release.

Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1ß, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.

Función barrera intestinal y su implicación en enfermedades digestivas / The intestinal barrier function and its involvement in digestive disease

La superficie de la mucosa del tracto gastrointestinal está revestida de células epiteliales que establecen una barrera efectiva, mediante uniones intercelulares, entre el medio interno y el medio externo, impidiendo el paso de sustancias potencialmente nocivas. Sin embargo las células epiteliales también son responsables de la absorción de nutrientes y electrolitos, por lo que se requiere una barrera semipermeable que permita el paso selectivo a ciertas sustancias, mientras que evite el acceso a otras. Para ello, el intestino ha desarrollado la “función barrera intestinal”, un sistema defensivo compuesto por diferentes elementos, tanto extracelulares como celulares, que actúan de forma coordinada para impedir el paso de antígenos, toxinas y productos microbianos y, a la vez, mantiene el correcto desarrollo de la barrera epitelial, el sistema inmunitario y la adquisición de tolerancia hacia los antígenos de la dieta y la microbiota intestinal. La alteración de los mecanismos que componen la función barrera favorece el desarrollo de respuestas inmunitarias exageradas, y, aunque se desconoce su implicación exacta, la alteración de la función barrera intestinal se ha asociado al desarrollo de enfermedades inflamatorias en el tracto digestivo. En esta revisión se detallan los diferentes elementos que componen la función barrera intestinal y las alteraciones moleculares y celulares más características descritas en enfermedades digestivas asociadas a la disfunción de este mecanismo de defensa

The gastrointestinal mucosal surface is lined with epithelial cells representing an effective barrier made up with intercellular junctions that separate the inner and the outer environments, and block the passage of potentially harmful substances. However, epithelial cells are also responsible for the absorption of nutrients and electrolytes, hence a semipermeable barrier is required that selectively allows a number of substances in while keeping others out. To this end, the intestine developed the “intestinal barrier function”, a defensive system involving various elements, both intra- and extracellular, that work in a coordinated way to impede the passage of antigens, toxins, and microbial byproducts, and simultaneously preserves the correct development of the epithelial barrier, the immune system, and the acquisition of tolerance against dietary antigens and the intestinal microbiota. Disturbances in the mechanisms of the barrier function favor the development of exaggerated immune responses; while exact implications remain unknown, changes in intestinal barrier function have been associated with the development of inflammatory conditions in the gastrointestinal tract. This review details de various elements of the intestinal barrier function, and the key molecular and cellular changes described for gastrointestinal diseases associated with dysfunction in this defensive mechanism

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 µM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.

The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-ß during Chlamydia trachomatis infection.

IFN-ß has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-ß expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-ß expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-ß expression. We determined that cGAS is required for IFN-ß expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-ß, coculture of cells depleted for either STING or cGAS rescued IFN-ß expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-ß expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-ß expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

Connexins in renal endothelial function and dysfunction.

The renal endothelium plays a critical role in kidney physiopathology as it is implicated in various processes such as the regulation of vasomotor tone, the control of tissue inflammation and thrombosis. Recent evidence highlights direct implication of renal endothelial dysfunction in the progression of chronic kidney disease. Renal endothelial dysfunction is a multifaceted process in which chemokines, cytokines, prothrombotic factors and adhesion molecules are known to play a crucial role. Apart from paracrine cell-to-cell signaling, the role for gap junction-mediated intercellular communication in renal physiopathology has been recently suggested. Gap junction channels are formed by the hexameric assembly of connexins and directly connect the cytoplasm of adjacent cells. Due to their ability to regulate multiple physiological and pathological signals connexins are currently taking an important place in the list of actors involved in renal endothelial function and dysfunction. In this review we will focus on possible implications of connexins in the physiopathological processes associated with renal vascular endothelium.

Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

Neuron-glial communication mediated by TNF-α and glial activation in dorsal root ganglia in visceral inflammatory hypersensitivity.

Communication between neurons and glia in the dorsal root ganglia (DRG) and the central nervous system is critical for nociception. Both glial activation and proinflammatory cytokine induction underlie this communication. We investigated whether satellite glial cell (SGC) and tumor necrosis factor-α (TNF-α) activation in DRG participates in a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat model of visceral hyperalgesia. In TNBS-treated rats, TNF-α expression increased in DRG and was colocalized to SGCs enveloping a given neuron. These SGCs were activated as visualized under electron microscopy: they had more elongated processes projecting into the connective tissue space and more gap junctions. When nerves attached to DRG (L6-S1) were stimulated with a series of electrical stimulations, TNF-α were released from DRG in TNBS-treated animals compared with controls. Using a current clamp, we noted that exogenous TNF-α (2.5 ng/ml) increased DRG neuron activity, and visceral pain behavioral responses were reversed by intrathecal administration of anti-TNF-α (10 µg·kg(-1)·day(-1)). Based on our findings, TNF-α and SGC activation in neuron-glial communication are critical in inflammatory visceral hyperalgesia.