scholarly journals Connexin-43 dependent ATP release mediates macrophage activation during peritonitis

2018 ◽  
Author(s):  
Michel Dosch ◽  
Joël Zindel ◽  
Fadi Jebbawi ◽  
Nicolas Melin ◽  
Daniel Sanchez-Taltavull ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Macrophage Activation ◽  
Purinergic Receptor ◽  
Atp Release ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Autocrine Signaling ◽  
Cx43 Expression ◽  
Pulmonary Macrophages ◽  
And Control

ABSTRACTPeritonitis is the consequence of bacterial spillage into a sterile environment by gastrointestinal hollow-organ perforation that may lead to fulminant sepsis. Outcome of peritonitis-induced sepsis critically depends on macrophage activation by extracellular ATP release and associated para- and autocrine signaling via purinergic receptors. Mechanisms that mediate and control ATP release, however, are poorly understood. Here we show that TLR-2 and -4 agonists trigger ATP release via Connexin-43 (CX43) hemichannels in peritoneal macrophages leading to poor survival during sepsis. In humans, CX43 expression was upregulated on macrophages isolated from the peritoneal cavity in patients with intraperitoneal infection but not in healthy controls. Using a murine caecal ligation and puncture (CLP) model, we identified increased CX43 expression in activated infiltrating peritoneal, hepatic and pulmonary macrophages. Conditional MAC-CX43 KO Lyz2cre/creCx43flox/flox mice were developed to specifically assess the CX43 impact in macrophages. Both macrophage-specific CX43 deletion (using Lyz2cre/creCx43flox/flox mice) or pharmacological CX43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP. This was ultimately resulting in increased survival in Lyz2cre/creCx43flox/flox mice and after pharmacological blockade. Specific inhibition of the purinergic receptor P2RY1 abrogated CX43 elicited cytokine responses. In conclusion, inhibition of autocrine ATP signaling via CX43 on macrophages and P2RY1 improves sepsis outcome in experimental peritonitis.Brief SummaryConnexin-43-mediated ATP release from macrophages in response to TLR-4 and -2 agonists modulates autocrine activation of macrophages in a P2Y1-dependent manner, ultimately determining sepsis survival.

scholarly journals Connexin-43-dependent ATP release mediates macrophage activation during sepsis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michel Dosch ◽  
Joël Zindel ◽  
Fadi Jebbawi ◽  
Nicolas Melin ◽  
Daniel Sanchez-Taltavull ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Macrophage Activation ◽  
Purinergic Receptors ◽  
Atp Release ◽  
Healthy Controls ◽  
Sepsis Model ◽  
Hepatic Macrophages ◽  
Hollow Organ ◽  
Sepsis Survival ◽  
Autocrine Signalling

Bacterial spillage into a sterile environment following intestinal hollow-organ perforation leads to peritonitis and fulminant sepsis. Outcome of sepsis critically depends on macrophage activation by extracellular ATP-release and associated autocrine signalling via purinergic receptors. ATP-release mechanisms, however, are poorly understood. Here, we show that TLR-2 and −4 agonists trigger ATP-release via Connexin-43 hemichannels in macrophages leading to poor sepsis survival. In humans, Connexin-43 was upregulated on macrophages isolated from the peritoneal cavity in patients with peritonitis but not in healthy controls. Using a murine peritonitis/sepsis model, we identified increased Connexin-43 expression in peritoneal and hepatic macrophages. Conditional Lyz2cre/creGja1flox/flox mice were developed to specifically assess Connexin-43 impact in macrophages. Both macrophage-specific Connexin-43 deletion and pharmacological Connexin-43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP, ultimately resulting in increased survival. In conclusion, inhibition of autocrine Connexin-43-dependent ATP signalling on macrophages improves sepsis outcome.

scholarly journals Myoendothelial gap junctions mediate regulation of angiopoietin-2-induced vascular hyporeactivity after hypoxia through connexin 43-gated cAMP transfer

2017 ◽  
Vol 313 (3) ◽  
pp. C262-C273 ◽  
Author(s):  
Jing Xu ◽  
Guangming Yang ◽  
Tao Li ◽  
Liangming Liu
Keyword(s):  
Gap Junctions ◽  
Connexin 43 ◽  
Vascular Endothelial Cell ◽  
Inos Expression ◽  
Glycyrrhetic Acid ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Cx43 Expression ◽  
Vascular Hyporeactivity ◽  
Angiopoietin 2

Angiopoietin-2 (Ang-2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS) in a vascular endothelial cell (VEC)-specific and Ang-2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided coculture model with VECs and VSMCs from Sprague-Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved, and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang-2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang-2 effects were suppressed by the MEGJ inhibitor 18α-glycyrrhetic acid (18-GA), Tie2 siRNA, or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang-2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang-2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang-2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang-2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.

The STK Receptor Tyrosine Kinase Regulates the Response of Primary Macrophages to LPS in a Ligand-Independent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3436-3436
Author(s):  
Pamela Correll ◽  
Qingping Liu Liu
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Macrophage Activation ◽  
Endotoxic Shock ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Retroviral Transduction

Abstract We have shown previously that activation of the STK/RON receptor tyrosine kinase expressed on tissue resident macrophages, by it’s ligand macrophage stimulating protein (MSP), results in the inhibition of NFkB activation, inducible nitric oxide synthase (iNOS) expression and TNFa production, as well as the induction of arginase expression, suggesting a role for this receptor in the regulation of classical vs. alternative macrophage activation. Furthermore, mice with a targeted deletion in this receptor exhibit increased sensitivity to endotoxic shock and DTH responses. More recently, we have demonstrated that MSP stimulation of primary peritoneal macrophages inhibits the production of IL-12. In order to map the domains of STK responsible for the inhibition of classical macrophage activation by MSP, we generated mutant forms of the receptor and expressed wild-type and mutant receptors in primary bone marrow derived macrophages by retroviral transduction. Expression of wild-type STK in these primary cells resulted in the ligand-independent reduction in IL-12p40 production in response to LPS stimulation, which was further inhibited by MSP treatment. This is consistent with the lack of a requirement for MSP in regulating responses to endotoxin in vivo. Surprisingly, a kinase dead receptor, which fails to signal in 293T cells, was fully functional in this assay, suggesting that the kinase activity of the receptor is not required for the inhibition of IL-12p40 under these conditions. However, the docking site tyrosines in the c-terminal tail of the receptor are essential for the inhibition of IL-12p40 by STK, suggesting that STK may be phosphorylated by an another kinase in this system. STK/RON has been shown to associate both physically and functionally with a number of other cell-surface receptors including EpoR, IL-3R bc, EGFR, MET as well as a number of integrins and cadherins. We have shown previously that STK regulates the activity of the aMb2 integrin (CR3) in peritoneal macrophages in a PI3K, PKCz-dependent manner. Here we show that STK also physically associates with CR3, as well as CD14, in RAW264.7 cells in the absence of ligand. Both CR3 and CD14 are capable of directly binding to LPS. Thus, we speculate that STK may exist as part of a receptor complex in macrophages and that signalling through STK might be induced directly by LPS. This would provide a means by which STK could temper the response of tissue-resident macrophages to LPS thereby preventing damage to host tissues.

Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys

2002 ◽  
Vol 282 (4) ◽  
pp. F763-F775 ◽  
Author(s):  
Erik M. Schwiebert ◽  
Darren P. Wallace ◽  
Gavin M. Braunstein ◽  
Sandi R. King ◽  
Janos Peti-Peterdi ◽  
...  
Keyword(s):  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Renal Tubules ◽  
Autocrine Signaling ◽  
Polycystic Kidney ◽  
Polycystic Kidneys ◽  
Cyst Volume ◽  
Autosomal Dominant Polycystic Kidney

ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic Ca2+concentration and secretory Cl− transport show P2Y and P2X purinergic receptor-mediated stimulation of Cl− secretion via cytosolic Ca2+-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression.

scholarly journals AAV-BR1 targets endothelial cells in the retina to reveal their morphological diversity and to deliver Cx43.

2021 ◽  
Author(s):  
Elena Ivanova ◽  
Carlo Corona ◽  
Cyril G. Eleftheriou ◽  
Randy F. Stout ◽  
Jakob Korbelin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Connexin 43 ◽  
Expression System ◽  
Morphological Diversity ◽  
Mouse Retina ◽  
Vascular Tree ◽  
Cx43 Expression ◽  
Membrane Contacts ◽  
And Control

Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impariment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.

scholarly journals Role of Connexin 43 in an Inflammatory Model for TMJ Hyperalgesia

2021 ◽  
Vol 2 ◽  
Author(s):  
Fabeeha Ahmed ◽  
Md. Rahman ◽  
Randall Thompson ◽  
David A. Bereiter
Keyword(s):  
Temporomandibular Joint ◽  
Connexin 43 ◽  
Temporomandibular Joint Disorders ◽  
Female Rats ◽  
Dependent Manner ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Junction Protein ◽  
Males And Females

Temporomandibular joint disorders (TMD) consist of a heterogeneous group of conditions that present with pain in the temporomandibular joint (TMJ) region and muscles of mastication. This project assessed the role of connexin 43 (Cx43), a gap junction protein, in the trigeminal ganglion (TG) in an animal model for persistent inflammatory TMJ hyperalgesia. Experiments were performed in male and female rats to determine if sex differences influence the expression and/or function of Cx43 in persistent TMJ hyperalgesia. Intra-TMJ injection of Complete Freund's Adjuvant (CFA) caused a significant increase in Cx43 expression in the TG at 4 days and 10 days post-injection in ovariectomized (OvX) female rats and OvX females treated with estradiol (OvXE), while TG samples in males revealed only marginal increases. Intra-TG injection of interference RNA for Cx43 (siRNA Cx43) 3 days prior to recording, markedly reduced TMJ-evoked masseter muscle electromyographic (MMemg) activity in all CFA-inflamed rats, while activity in sham animals was not affected. Western blot analysis revealed that at 3 days after intra-TG injection of siRNA Cx43 protein levels for Cx43 were significantly reduced in TG samples of all CFA-inflamed rats. Intra-TG injection of the mimetic peptide GAP19, which inhibits Cx43 hemichannel formation, greatly reduced TMJ-evoked MMemg activity in all CFA-inflamed groups, while activity in sham groups was not affected. These results revealed that TMJ inflammation caused a persistent increase in Cx43 protein in the TG in a sex-dependent manner. However, intra-TG blockade of Cx43 by siRNA or by GAP19 significantly reduced TMJ-evoked MMemg activity in both males and females following TMJ inflammation. These results indicated that Cx43 was necessary for enhanced jaw muscle activity after TMJ inflammation in males and females, a result that could not be predicted on the basis of TG expression of Cx43 alone.

The TRPV4 channel is a novel regulator of intracellular Ca2+ in human esophageal epithelial cells

2011 ◽  
Vol 301 (1) ◽  
pp. G138-G147 ◽  
Author(s):  
Takashi Ueda ◽  
Michiko Shikano ◽  
Takeshi Kamiya ◽  
Takashi Joh ◽  
Shinya Ugawa
Keyword(s):  
Cell Proliferation ◽  
Elevated Temperature ◽  
Epithelial Cells ◽  
Cell Viability ◽  
Connexin 43 ◽  
Transient Receptor Potential ◽  
Atp Release ◽  
Transient Receptor Potential Vanilloid ◽  
Dependent Manner ◽  
Esophageal Epithelial Cells

The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca2+ in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 μM 4α-PDD, whereas higher concentrations (10 μM and 30 μM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1β-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca2+-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.

scholarly journals Author response: Connexin-43-dependent ATP release mediates macrophage activation during sepsis

2019 ◽  
Author(s):  
Michel Dosch ◽  
Joël Zindel ◽  
Fadi Jebbawi ◽  
Nicolas Melin ◽  
Daniel Sanchez-Taltavull ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Macrophage Activation ◽  
Atp Release ◽  
Author Response

scholarly journals Macrophage activation: increased ingestion of IgG-coated erythrocytes after administration of interferon inducers to mice.

1978 ◽  
Vol 147 (2) ◽  
pp. 593-598 ◽  
Author(s):  
S I Hamburg ◽  
R E Manejias ◽  
M Rabinovitch
Keyword(s):  
Macrophage Activation ◽  
Peritoneal Macrophages ◽  
Response Curves ◽  
Polycytidylic Acid ◽  
Vesicular Stomatitis ◽  
Macrophage Populations ◽  
And Control ◽  
Mouse Peritoneal Macrophages

In vitro phagocytosis of IgG-opsonized sheep erythrocytes (EA) was used to measure the in vivo activation of mouse peritoneal macrophages. Uptake of EA as enhanced by the extraperitoneal administration of Newcastle disease virus, vesicular stomatitis virus, tilorone or polyinosinic-polycytidylic acid. Ingestion of EA was similarly stimulated by lipopolysaccharide or killed Corynebacterium parvum. Dose-response curves relating concentrations of IgG to phagocytosis were parallel for both treated and control animals. This indicates that the heterogeneity of the macrophage populations did not change and that the overall populations were activated with respect to phagocytic ability. Numbers of macrophages were not increased (except in C. parvum-treated mice), suggesting that resident, rather than newly recruited macrophages, were activated by the different agents.

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