Cytomegalovirus promotes intestinal macrophage-mediated mucosal inflammation through induction of Smad7.
ABSTRACT: Intestinal macrophages in healthy human mucosa are profoundly down-regulated for inflammatory responses (inflammation anergy) due to stromal TGF-? inactivation of NF-?B. Paradoxically, in cytomegalovirus (CMV) intestinal inflammatory disease, one of the most common manifestations of opportunistic CMV infection, intestinal macrophages mediate severe mucosal inflammation. Here we investigated the mechanism whereby CMV infection promotes macrophage-mediated mucosal inflammation. CMV infected primary intestinal macrophages but did not replicate in the cells or reverse established inflammation anergy. However, CMV infection of precursor blood monocytes, the source of human intestinal macrophages in adults, prevented stromal TGF-?-induced differentiation of monocytes into inflammation anergic macrophages. Mechanistically, CMV up-regulated monocyte expression of the TGF-? antagonist Smad7, blocking the ability of stromal TGF-? to inactivate NF-?B, thereby enabling MyD88 and NF-?B-dependent cytokine production. Smad7 expression also was markedly elevated in mucosal tissue from subjects with CMV colitis and declined after antiviral ganciclovir therapy. Confirming these findings, transfection of Smad7 antisense oligonucleotide into CMV-infected monocytes restored monocyte susceptibility to stromal TGF-?-induced inflammation anergy. Thus, CMV-infected monocytes that recruit to the mucosa, not resident macrophages, are the source of inflammatory macrophages in CMV mucosal disease and implicate Smad7 as a key regulator of, and potential therapeutic target for, CMV mucosal disease.
Project description:In physiological conditions, the activity of the intestinal immune system is tightly regulated to prevent tissue-damaging reactions directed against components of the luminal flora. Various factors contribute to maintain immune homeostasis and diminished production and/or function of such molecules trigger and/or propagate detrimental signals, which can eventually lead to chronic colitis and colon cancer. One such a molecule is transforming growth factor-β1 (TGF-β1), a cytokine produced by many inflammatory and non-inflammatory cells and targeting virtually all the intestinal mucosal cell types, with the down-stream effect of activating intracellular Smad2/3 proteins and suppressing immune reactions. In patients with inflammatory bowel diseases (IBD), there is defective TGF-β1/Smad signaling due to high Smad7, an inhibitor of TGF-β1 activity. Indeed, knockdown of Smad7 with a specific antisense oligonucleotide restores endogenous TGF-β1 activity, thereby inhibiting inflammatory pathways in patients with IBD and colitic mice. Consistently, mice over-expressing Smad7 in T cells develop severe intestinal inflammation in various experimental models. Smad7 expression is also upregulated in colon cancer cells, in which such a protein controls positively intracellular pathways that sustain neoplastic cell growth and survival. We here review the role of TGF-β1 and Smad7 in intestinal immunity, inflammation, and cancer.
Project description:Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.
Project description:Smad7, a negative regulator of TGF-? signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells. Smad7 deficiency in DCs promotes TGF-? responsiveness and the co-inhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs. In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro. The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-? and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention.
Project description:Cytomegalovirus (CMV) has been ubiquitously detected within high-grade gliomas, but its role in gliomagenesis has not been fully elicited.Glioblastoma multiforme (GBM) tumors were analyzed by flow cytometry to determine CMV antigen expression within various glioma-associated immune populations. The glioma cancer stem cell (gCSC) CMV interleukin (IL)-10 production was determined by ELISA. Human monocytes were stimulated with recombinant CMV IL-10 and levels of expression of p-STAT3, VEGF (vascular endothelial growth factor), TGF-?, viral IE1, and pp65 were determined by flow cytometry. The influence of CMV IL-10-treated monocytes on gCSC biology was ascertained by functional assays.CMV showed a tropism for macrophages (M?)/microglia and CD133+ gCSCs within GBMs. The gCSCs produce CMV IL-10, which induces human monocytes (the precursor to the central nervous system M?s/microglia) to assume an M2 immunosuppressive phenotype (as manifested by downmodulation of the major histocompatibility complex and costimulatory molecules) while upregulating immunoinhibitory B7-H1. CMV IL-10 also induces expression of viral IE1, a modulator of viral replication and transcription in the monocytes. Finally, the CMV IL-10-treated monocytes produced angiogenic VEGF, immunosuppressive TGF-?, and enhanced migration of gCSCs.CMV triggers a feedforward mechanism of gliomagenesis by inducing tumor-supportive monocytes.
Project description:Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4(+)Foxp3(+)) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-? by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.
Project description:Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-?1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ?-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-?1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-?1 to the TGF-? receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-?1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-?1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.
Project description:Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated whether NEC occurs in the preterm intestine due to incomplete noninflammatory differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products.We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from explanted human fetal and adult intestinal tissues. Transforming growth factor-? (TGF-?) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-? signaling on NEC-like inflammatory mucosal injury.Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-?, particularly the TGF-?(2) isoform. NEC was associated with decreased tissue expression of TGF-?(2) and decreased TGF-? bioactivity. In mice, disruption of TGF-? signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-?(2) was protective.Intestinal macrophages progressively acquire a noninflammatory profile during gestational development. TGF-?, particularly the TGF-?(2) isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally administered TGF-?(2) protected mice from experimental NEC-like injury.
Project description:Crohn's disease and ulcerative colitis, the major forms of inflammatory bowel diseases (IBD) in man, are complex diseases in which genetic and environmental factors interact to promote an excessive mucosal immune response directed against normal components of the bacterial microflora. There is also evidence that the pathologic process is due to defects in counter-regulatory mechanisms, such as those involving the immunosuppressive cytokine transforming growth factor (TGF)-β1. Indeed, studies in human IBD tissues and murine models of colitis have documented a disruption of TGF-β1 signalling marked by a block in the phosphorylation of Smad3, a signalling molecule associated with the activated TGF-β receptor, due to up-regulation of Smad7, an intracellular inhibitor of Smad3 phosphorylation. Knock-down of Smad7 with a specific antisense oligonucleotide restores TGF-β1/Smad3 signalling, thus resulting in a marked suppression of inflammatory cytokine production and attenuation of murine colitis. These findings together with the demonstration that Smad7 antisense oligonucleotide is not toxic when administered in mice have paved the way for the development of a Smad7 antisense oligonucleotide-based pharmaceutical compound that is now ready to enter the clinics. In this article we review the available data supporting the pathogenic role of Smad7 in IBD and discuss whether and how Smad7 antisense therapy could help dampen the ongoing inflammation in IBD.
Project description:OBJECTIVE: Although Smad3 has been considered as a downstream mediator of transforming growth factor-? (TGF-?) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease. RESEARCH DESIGN AND METHODS: Protective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. RESULTS: We found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1? [IL-1?], tumor necrosis factor-? [TNF-?], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-?/Smad2/3 and nuclear factor-?B (NF-?B) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-?/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-?B/p65-driven renal inflammation including IL-1?, TNF-?, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats. CONCLUSIONS: Smad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication.
Project description:BACKGROUND:In Crohn's disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-?1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. METHODS:Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. RESULTS:Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-?-secreting cells. CONCLUSIONS:Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.