Keratinocyte Growth Factor Improves Epithelial Structure and Function in a Mouse Model of Intestinal Ischemia/Reperfusion

Background

Intestinal ischemia/reperfusion (I/R) induces the desquamation of the intestinal epithelium, increases the intestinal permeability, and in patients often causes fatal conditions including sepsis and multiple organ failure. Keratinocyte growth factor (KGF) increases intestinal growth, although little is known about KGF activity on intestinal function after intestinal I/R. We hypothesized that KGF administration would improve the intestinal function in a mouse model of intestinal I/R.

Methods

Adult C57BL/6J mice were randomized to three groups: Sham, I/R group and I/R+KGF group. Mice were killed on day 5, and the small bowel was harvested for histology, wet weight, RNA and protein content analysis. Epithelial cell (EC) proliferation was detected by immunohistochemistry for PCNA, and apoptosis was determined by TUNEL staining. The expressions of Claudin-1 and ZO-1 were detected by immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance (TER).

Results

KGF significantly increased the intestinal wet weight, contents of intestinal protein and RNA, villus height, crypt depth and crypt cell proliferation, while KGF resulted in the decrease of epithelial apoptosis. KGF also stimulated the recovery of mucosal structures and attenuated the disrupted distribution of TJ proteins. Moreover, KGF attenuated the intestinal I/R-induced decrease in TER and maintained the intestinal barrier function.

Conclusion

KGF administration improves the epithelial structure and barrier function in a mouse model of intestinal I/R. This suggests that KGF may have clinical applicability.     

The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2

Background

To investigate the regulation of K17 expression by the pro-inflammatory cytokine IL-22 in keratinocytes and its important role in our previously hypothesized “K17/T cell/cytokine autoimmune loop” in psoriasis.

Materials and Methods

K17 expression was examined in the IL-22-treated keratinocytes by real-time quantitative PCR, ELISA, Western blot and Immunofluorescence. In addition, the signaling pathways involved in K17 regulation were investigated with related inhibitors and siRNAs. In addition, K17 expression was examined in the epidermis of IL-22-injected mouse skin.

Results

IL-22-induced K17 expression was confirmed in keratinocytes and the epidermis of IL-22-injected mouse skin at both mRNA and protein levels, which is an important complement to the autoimmune loop. We further investigated the regulatory mechanisms and found that both STAT3 and ERK1/2 were involved in the up-regulation of K17 expression induced by IL-22.

Conclusion

IL-22 up-regulates K17 expression in keratinocytes in a dose-dependent manner through STAT3- and ERK1/2-dependent mechanisms. These findings indicated that IL-22 was also involved in the K17/T cell/cytokine autoimmune loop and may play an important role in the progression of psoriasis.     

The Role of Interleukin-1 Receptor-Associated Kinases in Vogt-Koyanagi-Harada Disease

Purpose

To explore whether IRAK1 and IRAK4 are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.

Methods

Thirty-nine VKH patients and thirty-two healthy controls were included in this study. The mRNA levels of IRAK1 and IRAK4 from active VKH patients, inactive VKH patients, and normal controls in peripheral blood mononuclear cells (PBMCs) were detected using real-time quantitative PCR. CD4⁺T cells were purified from PBMCs obtained from active VKH patients and normal controls. The effect of IRAK1/4 inhibition on CD4⁺T cell proliferation following stimulation with IL-18 or IL-1β was measured using a modified MTT assay. CD4⁺T cell expression of IFN-γ and IL-17 were detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). The effect of IRAK1/4 inhibition on NF-κB, STAT1, and STAT3 activation was detected by FCM.

Results

The mRNA levels of IRAK1 and IRAK4 were both significantly increased in active VKH patients compared to inactive VKH patients and healthy controls. No difference in the IRAK1 or IRAK4 mRNA level could be detected between inactive patients and healthy controls. After incubation with IRAK1/4 inhibitor, the proliferation of CD4⁺T cells was inhibited both in the active VKH patients and in the healthy controls. IRAK1/4 inhibition was also associated with a decreased expression of IFN-γ and IL-17. Phosphorylation of NF-κB, STAT1, and STAT3 in CD4⁺T from healthy controls was significantly decreased after inhibition of IRAK1/4.

Conclusions

High mRNA levels of IRAK1 and IRAK4 correlated with VKH disease activity. IRAK1 and IRAK4 play a role in the activation and proliferation of CD4⁺T cells and the higher expression observed in VKH may contribute to the pathogenesis of this blinding condition.     

IL-9 Induces CCL11 Expression via STAT3 Signalling in Human Airway Smooth Muscle Cells

Background

Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.

Methodology/Principal Findings

In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.

Conclusion/Significance

Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.     

Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis

Objective

Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. However, the underlying mechanisms of EMT in lung adenocarcinoma, and how this process might be inhibited, remain to be explored. This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.

Methods

Invasion assay and MTT assay was performed to determine cell invasion and cell proliferation. Western blotting, immunofluorescence, real-time PCR, ELISA, and immunohistochemistry were performed to detect the expression of IL-6, E-cadherin, Vimentin, and p-STAT3.

Results

We discovered that IL-6, via STAT3 phosphorylation, could promote lung adenocarcinoma cell invasion via EMT in vitro. This was supported by the inverse correlation between E-cadherin and IL-6 expression, positive correlation between IL-6 and vimentin mRNA expression and between STAT3 phosphorylation and IL-6 expression in tumor tissues. Importantly, metformin inhibited tumor growth and distant metastases in tumor-bearing nude mice and reversed IL-6-induced EMT both in vitro and in vivo. Furthermore, we found that blockade of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.

Conclusions

Collectively, our present results show that enhanced IL-6 expression, via STAT3 phosphorylation, is a mechanism of EMT in lung adenocarcinoma. We found that metformin could inhibit IL-6-induced EMT possibly by blocking STAT3 phosphorylation.     

Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway

Background

Renal cell carcinoma (RCC) is one of the most common kidney cancers and is highly resistant to chemotherapy. Accumulating evidence suggests that interleukin-22 (IL-22) may mediate host defense against varietal pathogens as a proinflammatory and anti-inflammatory cytokine. The purpose of this study is to assess the inhibitory effects of IL-22 on human RCC cell line A498 and to investigate the possible mechanisms underlying the anti-tumor effects of this cytokine.

Methodology

A498 cells, a RCC cell line, were used to assess the inhibitory growth effects of IL-22 using the MTT assay and flow cytometric analysis in vitro. BALB/C nude mice bearing A498 cell xenografts were used to examine the antitumor efficacy of IL-22 in vivo. Western blotting assay was performed to detect the regulation of the intracellular signaling pathway of IL-22.

Principal Findings

We found that IL-22 suppressed the growth of A498 cells in a dose-dependent manner and inhibited the growth of A498 xenografts. We also observed that IL-22 produced a dose-dependent inhibitory effect on A498 cells that involved the induction of G2/M cell cycle arrest without cell apoptosis. Moreover, we showed that the phosphorylation of STAT1 was increased and the phosphorylation of ERK1/2 was attenuated in A498 cells exposed to IL-22. The growth inhibition of A498 cells was partially revised after IL-22 treatment as the expression of STAT1 was knocked down. And inflammatory cytokines, interferon-α and tumor necrosis factor-α (TNF-α) were barely involved in the suppression of A498 cell xenografts treated with IL-22.

Conclusions

IL-22 dose-dependently suppresses RCC cell line A498 cells in vitro and induces growth inhibition of A498 cell-bearing mouse xenografts. These results suggest that the anti-RCC effects of IL-22 are at least partially mediated through regulation of STAT1 signaling pathways and G2/M cell cycle arrest, rather than by inducing apoptosis and inflammatory cytokines.     

Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

Background

The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors γ-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1.

Methodology/Principal Findings

Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells.

Conclusion/Significance

Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.     

Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Background

Regulation of human airway smooth muscle cells (HASMC) by cytokines contributes to chemotactic factor levels and thus to inflammatory cell accumulation in lung diseases. Cytokines such as the gp130 family member Oncostatin M (OSM) can act synergistically with Th2 cytokines (IL-4 and IL-13) to modulate lung cells, however whether IL-17A responses by HASMC can be altered is not known.

Objective

To determine the effects of recombinant OSM, or other gp130 cytokines (LIF, IL-31, and IL-6) in regulating HASMC responses to IL-17A, assessing MCP-1/CCL2 and IL-6 expression and cell signaling pathways.

Methods

Cell responses of primary HASMC cultures were measured by the assessment of protein levels in supernatants (ELISA) and mRNA levels (qRT-PCR) in cell extracts. Activation of STAT, MAPK (p38) and Akt pathways were measured by immunoblot. Pharmacological agents were used to assess the effects of inhibition of these pathways.

Results

OSM but not LIF, IL-31 or IL-6 could induce detectable responses in HASMC, elevating MCP-1/CCL2, IL-6 levels and activation of STAT-1, 3, 5, p38 and Akt cell signaling pathways. OSM induced synergistic action with IL-17A enhancing MCP-1/CCL-2 and IL-6 mRNA and protein expression, but not eotaxin-1 expression, while OSM in combination with IL-4 or IL-13 synergistically induced eotaxin-1 and MCP-1/CCL2. OSM elevated steady state mRNA levels of IL-4Rα, OSMRβ and gp130, but not IL-17RA or IL-17RC. Pharmacologic inhibition of STAT3 activation using Stattic down-regulated OSM, OSM/IL-4 or OSM/IL-13, and OSM/IL-17A synergistic responses of MCP-1/CCL-2 induction, whereas, inhibitors of Akt and p38 MAPK resulted in less reduction in MCP-1/CCL2 levels. IL-6 expression was more sensitive to inhibition of p38 (using SB203580) and was affected by Stattic in response to IL-17A/OSM stimulation.

Conclusions

Oncostatin M can regulate HASMC responses alone or in synergy with IL-17A. OSM/IL-17A combinations enhance MCP-1/CCL2 and IL-6 but not eotaxin-1. Thus, OSM through STAT3 activation of HASMC may participate in inflammatory cell recruitment in inflammatory airway disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0164-4) contains supplementary material, which is available to authorized users.     

Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance

Background and aims

Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.

Methods

Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.

Results

The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.

Conclusions

Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.     

Oncostatin M Mediates STAT3-Dependent Intestinal Epithelial Restitution via Increased Cell Proliferation,Decreased Apoptosis and Upregulation of SERPIN Family Members

Objective

Oncostatin M (OSM) is produced by activated T cells, monocytes, and dendritic cells and signals through two distinct receptor complexes consisting of gp130 and LIFR (I) or OSMR-β and gp130 (II), respectively. Aim of this study was to analyze the role of OSM in intestinal epithelial cells (IEC) and intestinal inflammation.

Methods

OSM expression and OSM receptor distribution was analyzed by PCR and immunohistochemistry experiments, signal transduction by immunoblotting. Gene expression studies were performed by microarray analysis and RT-PCR. Apoptosis was measured by caspases-3/7 activity. IEC migration and proliferation was studied in wounding and water soluble tetrazolium assays.

Results

The IEC lines Caco-2, DLD-1, SW480, HCT116 and HT-29 express mRNA for the OSM receptor subunits gp130 and OSMR-β, while only HCT116, HT-29 and DLD-1 cells express LIFR mRNA. OSM binding to its receptor complex activates STAT1, STAT3, ERK-1/2, SAPK/JNK-1/2, and Akt. Microarray analysis revealed 79 genes that were significantly up-regulated (adj.-p≤0.05) by OSM in IEC. Most up-regulated genes belong to the functional categories “immunity and defense” (p = 2.1×10⁻⁷), “apoptosis” (p = 3.7×10⁻⁴) and “JAK/STAT cascade” (p = 3.4×10⁻⁶). Members of the SERPIN gene family were among the most strongly up-regulated genes. OSM significantly increased STAT3- and MEK1-dependent IEC cell proliferation (p<0.05) and wound healing (p = 3.9×10⁻⁵). OSM protein expression was increased in colonic biopsies of patients with active inflammatory bowel disease (IBD).

Conclusions

OSM promotes STAT3-dependent intestinal epithelial cell proliferation and wound healing in vitro. Considering the increased OSM expression in colonic biopsy specimens of patients with active IBD, OSM upregulation may modulate a barrier-protective host response in intestinal inflammation. Further in vivo studies are warranted to elucidate the exact role of OSM in intestinal inflammation and the potential of OSM as a drug target in IBD.