JAK1/3 inhibition preserves epidermal morphology in full-thickness 3D skin models of atopic dermatitis and psoriasis.

BACKGROUND: Janus kinase (JAK) inhibition may be a promising new treatment modality for inflammatory (skin) diseases. However, little is known about direct effects of kinase inhibitors on keratinocyte differentiation and function as well as skin barrier formation. OBJECTIVE: Our aim was to address the direct impact of kinase inhibition of the JAK1/3 pathways by tofacitinib on keratinocyte immune function and barrier formation in atopic dermatitis (AD) and psoriasis. METHODS: 3D skin equivalents of both diseases were developed and concurrently pretreated with tofacitinib. To induce AD, 3D skin equivalents were stimulated with recombinant human IL-4 and IL-13. Psoriasis-like conditions were induced by incubation with IL-17A, IL-22 and tumour necrosis factor α (TNFα). The activation of signal transducer and activator of transcription (STAT)1, STAT3 and STAT6 was assessed by Western blot analysis. Microarray analysis and quantitative real-time PCR were used for gene expression analysis. RESULTS: Tofacitinib pretreatment preserved epidermal morphology and reduced STAT3 and STAT6 phosphorylation of AD-like and STAT3 phosphorylation of psoriasis-like culture conditions in 3D skin models compared to sham-controls. Filaggrin expression was fully maintained in the AD-like models, but only partially in psoriasis-like conditions after pretreatment with tofacitinib. In addition, tofacitinib upregulated DSC1, FLG and KRT1. Using gene expression analysis, downregulation of POSTN and IL24 was observed in AD-like conditions, whereas downregulation of IL20 and IL1B was observed in psoriasis-like conditions. CONCLUSION: JAK1/3 inhibition counteracted cytokine-induced AD- and psoriasis-like epidermal morphology and enhanced keratinocyte differentiation in 3D skin models. This effect was more pronounced in the AD-like models compared to the psoriasis-like 3D skin models.

Interleukin-4 protects from chemotherapy-induced peripheral neuropathy in mice modal via the stimulation of IL-4/STAT6 signaling.

PURPOSE: To investigate the possible role of IL-4 signaling pathway in vincristine-induced peripheral neuropathy. METHODS: The mouse model of vincristine-induced peripheral neuropathy and interleukin (IL)-4 knockout mice were utilized to investigate the possible role of IL-4 signaling pathway in vincristine-induced peripheral neuropathy. Vincristine induced increased sensitivity to mechanical stimulation was measured by von Frey hair test 7 and 14 days after intraperitoneal administration of 0.1 mg/kg vincristine in mice. Relative expression levels of cytokines were detected by quantitative real-time PCR. STAT6 expression following vincristine treatment was assessed with western blotting. RESULTS: We discovered that IL-4/STAT6 signaling was down-regulated in vincristine-treated mice. Deletion of IL-4 in mice increased the sensitivity to mechanical allodynia. IL-4 knockout mice also produced more pro-inflammatory cytokines, including IL-1ß and TNF-α. Notably, co-administration of exogenous recombination IL-4 significantly prevented vincristine-induced mechanical allodynia. CONCLUSION: Anti-inflammatory cytokine IL-4 protects rodent model from vincristine-induced peripheral neuropathy via the stimulation of IL-4/STAT6 signaling and inhibition of the pro-inflammatory cytokines.

Interleukin-4 protects from chemotherapy-induced peripheral neuropathy in mice modal via the stimulation of IL-4/STAT6 signaling

Abstract Purpose: To investigate the possible role of IL-4 signaling pathway in vincristine-induced peripheral neuropathy. Methods: The mouse model of vincristine-induced peripheral neuropathy and interleukin (IL)-4 knockout mice were utilized to investigate the possible role of IL-4 signaling pathway in vincristine-induced peripheral neuropathy. Vincristine induced increased sensitivity to mechanical stimulation was measured by von Frey hair test 7 and 14 days after intraperitoneal administration of 0.1 mg/kg vincristine in mice. Relative expression levels of cytokines were detected by quantitative real-time PCR. STAT6 expression following vincristine treatment was assessed with western blotting. Results: We discovered that IL-4/STAT6 signaling was down-regulated in vincristine-treated mice. Deletion of IL-4 in mice increased the sensitivity to mechanical allodynia. IL-4 knockout mice also produced more pro-inflammatory cytokines, including IL-1β and TNF-α. Notably, co-administration of exogenous recombination IL-4 significantly prevented vincristine-induced mechanical allodynia. Conclusion: Anti-inflammatory cytokine IL-4 protects rodent model from vincristine-induced peripheral neuropathy via the stimulation of IL-4/STAT6 signaling and inhibition of the pro-inflammatory cytokines.

Tomato paste alters NF-κB and cancer-related mRNA expression in prostate cancer cells, xenografts, and xenograft microenvironment.

Tomatoes may protect against prostate cancer development, possibly through targeting signaling pathways such as nuclear factor-κB (NF-κB). We investigated whether tomato paste could modulate NF-κB activity and cancer-related gene expression in human derived prostate cancer cells (PC3) and PC3 xenografts. PC3-cells were stably transduced with an NF-κB-luciferase construct, and treated with tomato extracts or vehicle control. Nude mice bearing PC3 xenografts were fed a Western-like diet with or without 10% tomato paste for 6.5 wk. The tomato diet significantly inhibited TNFα stimulated NF-κB activity in cultured PC3 cells, and modulated the expression of genes associated with inflammation, apoptosis, and cancer progression. Accumulation of lycopene occurred in liver, xenografts, and serum of mice fed tomato diet. Tomato paste in the diet did not affect tumor size in mice; however, there was a trend toward inhibition of NF-κB activity in the xenografts. The effect of tomato on gene expression was most prominent in the xenograft microenvironment, where among others NFKB2, STAT3, and STAT6 showed higher expression levels after tomato treatment. Our findings support biological activity of tomatoes in cancer-related inflammation.

The flavone eupatilin inhibits eotaxin expression in an NF-κB-dependent and STAT6-independent manner.

The CC chemokine eotaxin contributes to epithelium-induced inflammation in airway diseases such as asthma. Eupatilin (5,7-dihydroxy-3',4',6'-trimethoxyflavone), a bioactive component of Artemisia asiatica Nakai (Asteraceae), is reported to inhibit the adhesion of eosinophils to bronchial epithelial cells. However, little is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelium-induced inflammation. In this study, we investigated the effect of eupatilin on expression of eotaxin-1 (CCL11), a potent chemoattractant for eosinophils. Eupatilin significantly inhibited eotaxin expression in bronchial epithelial cells stimulated with TNF-α, while NF-κB and IκBα kinase (IKK) activities declined concurrently. Eupatilin also inhibited mitogen-activated protein kinase (MAPK) activity; however, all of these anti-inflammatory activities were reversed by MAPK overexpression. In contrast, eupatilin did not affect the signal transducer and activator of transcription 6 (STAT6) signalling in bronchial epithelial cells stimulated with IL-4. Furthermore, eupatilin significantly attenuated TNF-α-induced eosinophil migration. These results suggest that the eupatilin inhibits the signalling of MAPK, IKK, NF-κB and eotaxin-1 in bronchial epithelial cells, leading to inhibition of eosinophil migration.

The effect of recombinant sTGFß1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and signal transduction in a mouse model of schistosome disease.

This study was designed to investigate the effect of recombinant sTGFß1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and the expression of SMAD3 and STAT6. The proteins sTGFß1RII and sIL13Rα2 were expressed in Escherichiacoli, purified using affinity chromatography and characterized by Western blotting. Female BALB/C mice (48) were randomly divided into eight groups and infected with Schistosoma japonicum. Five weeks after infection, test groups were injected with the recombinant proteins at different doses. Eight weeks after infection, lung and hepatic tissue samples were obtained and stained with hematoxylin and eosin (HE) and Masson's trichrome. Immunohistochemical staining was used to detect the expression of SMAD3 and STAT6. The recombinant proteins sTGFß1RII and sIL13Rα2 were successfully expressed, purified, and characterized. The granuloma area, hepatic hydroxyproline (HYP) level and hepatic fibrosis of the protein therapeutic groups were significantly smaller than those of the positive control group (P<0.01). Treatment with sTGFß1RII was more effective when the protein was administered for 4weeks rather than 2 (P<0.01). Hepatic fibrosis in the groups using a low dose of protein sTGFß1 was lower that of the combination group (P<0.05). The expression level of STAT6 was significantly lower in groups treated with sIL13Rα2 than in groups not treated with the protein (P<0.01). The recombinant proteins TGFß1RII and sIL13Rα2 were able to decrease granuloma area and hepatic fibrosis in schistosomiasis japonica, and also reduced the expression of the signal transduction proteins SMAD3 and STAT6. The proteins were more effective when used in combination than when applied singly.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

Peptide YY attenuates STAT1 and STAT3 activation induced by TNF-alpha in acinar cell line AR42J.

BACKGROUND: STAT1 and STAT3, members of the cytoplasmic family of signal transducers and activators of transcription factors (STAT), have been associated with numerous inflammatory pathologies, including inflammatory bowel disease, hepatitis, and acute lung injury. But little is known about their role in the pancreas. Peptide YY (PYY), an inhibitory gastrointestinal hormone, ameliorates pancreatitis in vivo and in vitro. In addition, we have shown that PYY attenuates transcription factors, such as nuclear transcription factor (NF)-kappaB and Smad3/4, which mediate inflammation. We hypothesized that tumor necrosis factor (TNF)-alpha would induce STAT1 and STAT3, and PYY would attenuate their transcription factor binding. STUDY DESIGN: Rat pancreatic acinar cells were treated with recombinant TNF-alpha (200 ng/mL); PYY (3-36; 500 pM) was added 30 minutes post-TNF-alpha treatment. Cells were harvested at 2 hours, and nuclear protein and conditioned media were extracted. Levels of amylase secretion and cytokine production were measured using commercially available kits. STAT transcription factor binding was determined by protein/DNA array analysis and densitometry; results were verified again by electrophoretic mobility shift assay (EMSA) and ELISA-based assay. RESULTS: Amylase production was considerably increased (p < 0.05) as early as 5 minutes after addition of exogenous TNF-alpha and remained elevated for 24 hours. PYY decreased amylase production to control levels. A notable increase (p < 0.05) in the production of cytokines interleukin (IL)-1beta, IL-4, IL-6, IL-10, and TNF-alpha was observed with TNF-alpha treatment; production was reduced with PYY. TNF-alpha substantially upregulated STAT1 and STAT3 (two-fold or greater); PYY downregulated their binding activity to control levels. Results from both the electrophoretic mobility shift assay- and the ELISA-based assays verified STAT1 and STAT3 responses to TNF-alpha and PYY. CONCLUSIONS: In pancreatic acinar cells, TNF-alpha activated STAT1 and STAT3, known mediators of inflammatory cytokines. Interestingly, PYY attenuated their protein/DNA binding, which may have an impact on development of the disease. Additional investigation of STAT proteins and PYY could provide new therapeutic strategies for pancreatitis.

Niflumic acid suppresses interleukin-13-induced asthma phenotypes.

RATIONALE: Chloride channels have been implicated in the regulation of mucus production in epithelial cells. Expression of hCLCA1, a calcium-activated chloride channel, has been reported to be increased in the airway epithelium of patients with asthma. Interleukin (IL)-13 induces the cardinal features of bronchial asthma, and glucocorticoids are not sufficient to suppress IL-13-induced airway hyperresponsiveness or goblet cell hyperplasia. OBJECTIVES: We studied the effects of chloride channel inhibitors in IL-13-induced asthma. METHODS: The effects of niflumic acid (NA), a relatively specific blocker of calcium-activated chloride channel (CLCA), on goblet cell hyperplasia, eosinophil accumulation, and airway hyperresponsiveness were evaluated after IL-13 instillation into the airways. Because IL-13-dependent features rely on JAK/STAT6 signaling, the effect of NA on phosphorylation of JAK2 and STAT6 after IL-13 stimulation was examined in airway epithelial cells in vitro. The expression of the mCLCA family in mouse lung after IL-13 local administration in vivo was analyzed using reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Treatment with NA inhibited not only IL-13-induced goblet cell hyperplasia but also airway hyperresponsiveness and eosinophilic infiltration. NA suppressed the eotaxin levels in bronchoalveolar lavage fluids and overexpression of the MUC5AC gene, a marker of goblet cell hyperplasia, in the lung after IL-13 instillation. NA suppressed JAK2 activation, STAT6 activation, and eotaxin expression in epithelial cells. The expression of mCLCA3 (mouse homolog hCLCA1), but not that of other CLCA family members, was up-regulated by IL-13. CONCLUSIONS: These findings suggest that a chloride channel inhibitor can control IL-13-mediated airway features at least by suppressing JAK/STAT6 activation.

All-trans-retinoic acid suppresses interferon-gamma and tumor necrosis factor-alpha; a possible therapeutic agent for rheumatoid arthritis.

OBJECTIVES: To study the effects of all-trans-retinoic acid (ATRA), we determined the proliferation and cytokine production by peripheral blood mononuclear cells (PBMCs) and CD4+ T cells in healthy volunteers and patients with rheumatoid arthritis (RA), and explored the possibility of using ATRA as a therapeutic agent for autoimmune diseases. METHODS: Proliferation of these cells was determined by modified MTT assay, and expression of CC chemokine receptors 4 (CCR4) and CCR5 was determined by flow cytometry. Production and expression of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and tumor necrosis factor (TNF)-alpha was determined by Enzyme-Linked Immunosorbent Assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The presence of STAT6 protein was determined by Western blot analysis. RESULTS: ATRA did not affect the proliferation or production of IL-2 and IL-4. We did not detect STAT6 protein, and saw no evidence of the differentiation of PBMCs to Th1 or Th2 cells. In contrast, ATRA suppressed the production of IFN-gamma and TNF-alpha significantly. There were no significant differences between the healthy volunteers and RA patients. CONCLUSIONS: ATRA was demonstrated to affect the cytokine production of IFN-gamma and TNF-alpha. ATRA might be useful in the treatment of autoimmune diseases such as RA.