IL-17F mRNA Research Articles

The effect of phototherapy on the dynamics of cytokine mRNA expression in the affected skin of patients with mycosis fungoides

Introduction. The role of the cytokine environment and immune deregulation in the pathogenesis of mycosis fungoides is unquestionable. Despite the fact that one of the methods of therapy for the early stages of mycosis fungoides is phototherapy (PUVA, UVB-311 nm), the effect of ultraviolet radiation on the lymphoproliferative substrate and pathogenetic links in mycosis fungoides has not been fully studied. Aim: to evaluate the effect of NB-UVB and PUVA therapy on the dynamics of cytokine mRNA expression in the affected skin of patients with mycosis fungoides. Material and Methods. A comparative non-randomized study of the cytokine mRNA expression dynamics in the affected skin and of the effectiveness of phototherapy was carried out in 28 patients with early stage of mycosis fungoides. The IL4, IL17A, IL17F, and IL22 mRNA expression was determined relative to the endogenous control GAPDH using the real-time reverse transcription PCR (RT-PCR). Evaluation of the effectiveness of NB-UVB and PUVA therapy was carried out using a BSA score (skin lesion area) and a modified severity-weighted assessment tool (mSWAT) score. Results. The study included 28 patients with early stages (IA–IIA) of mycosis fungoides; 9 patients received NB-UVB and 19 received PUVA therapy. 3.71-fold decrease in mSWAT (p < 0.008), and 3-fold decrease in BSA scores (p < 0.013) were observed in the NB-UVB-treated group. In the PUVA-treated group 3.47- and 2.19-fold lower scores of mSWAT (p < 0.001) and BSA (p < 0.001) were found. There were no significant differences in the expression of the studied cytokines in the NB-UVB-treated group; however, a significant 19 and 72 % increase in IL17F (p = 0.003) and IL22 (p = 0.021) was revealed afterPUVA therapy. Correlation analysis has shown a weak correlation between IL4 and IL17A (r = 0.43, p < 0.027), and IL17F (r = 0.43, p < 0.028) before the treatment. Under the influence of phototherapy, the formation of a cytokine network in the affected skin was observed: there were positive associations between IL4 and IL17A (r = 0.73, p < 0.001), IL17F (r = 0.7, p < 0.001) and IL22 (r = 0.43, p < 0.024); IL17A and IL17F (r = 0.78, p < 0.001); IL22 and IL17A (r = 0.63, p < 0.001) and IL17F (r = 0.66, p < 0.001). In the PUVA-treated group a high negative correlation between IL17A and mSWAT (r = -0.79415, p =0.010586), BSA (r = -0.75432, p = 0.018849) were found. Conclusion: The positive correlations between IL4, IL17A, IL17F and IL22 in the affected skin of patients with mycosis fungoides may underlie the positive effect of phototherapy.

Cadmium Exposure Is Associated with Behavioral Deficits and Neuroimmune Dysfunction in BTBR T+ Itpr3tf/J Mice

Autism spectrum disorders (ASD) are neurobehavioral disabilities characterized by impaired social interactions, poor communication skills, and restrictive/repetitive behaviors. Cadmium is a common heavy metal implicated in ASD. In this study, we investigated the effects of Cd exposure on BTBR T+ Itpr3tf/J (BTBR) mice, an ASD model. We looked for changes in repetitive behaviors and sociability through experiments. We also explored the molecular mechanisms underlying the effects of Cd exposure, focusing on proinflammatory cytokines and pathways. Flow cytometry measured IL-17A-, IL-17F-, IL-21-, TNF-α-, STAT3-, and RORγt-expressing CD4+ T cells from the spleens of experimental mice. We then used RT-PCR to analyze IL-17A, IL-17F, IL-21, TNF-α, STAT3, and RORγ mRNA expression in the brain. The results of behavioral experiments showed that Cd exposure significantly increased self-grooming and marble-burying in BTBR mice while decreasing social interactions. Cd exposure also significantly increased the number of CD4+IL-17A+, CD4+IL-17F+, CD4+IL-21+, CD4+TNF-α+, CD4+STAT3+, and CD4+RORγt+ cells, while upregulating the mRNA expression of the six molecules in the brain. Overall, our results suggest that oral exposure to Cd aggravates behavioral and immune abnormalities in an ASD animal model. These findings have important implications for ASD etiology and provide further evidence of heavy metals contributing to neurodevelopmental disorders through proinflammatory effects.

Elevated cholesteryl ester transfer and phospholipid transfer proteins aggravated psoriasis in imiquimod-induced mouse models

BackgroundPsoriasis is a chronic inflammatory skin disorder related to dyslipidemia, with decreased high-density lipoprotein (HDL). Various cell types express phospholipid transfer protein (PLTP) as well as cholesteryl ester transfer protein (CETP). Their elevated levels among transgenic (Tg) mice led to reduced HDL and a higher risk of atherosclerosis (AS). This study examined whether elevated CETP and PLTP could aggravate psoriasis in a psoriasis vulgaris mouse model.MethodsThe back skins of CETP-Tg, PLTP-Tg, and C57BL/6 male mice, aged six to 8 weeks, were shaved for imiquimod cream (IMQ) (5%) treatment for five consecutive days. The clinical pathological parameters were rated independently using the modified target lesion psoriasis severity score. The skin sections stained with hematoxylin-eosin were scored by the Baker score. Epidermal thickening and differentiation and inflammatory factor infiltration were determined by immunohistochemistry. Inflammatory cytokine levels were measured using quantitative reverse transcription-polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA) kits. This work employed SPSS Statistics Version to conduct statistical analyses.ResultsIn this study, CETP-Tg and PLTP-Tg mice had higher clinical and histological scores than wild-type (WT) mice. Immunohistochemistry of the epidermis and dermis revealed a high proportion of proliferating cell nuclear antigen (PCNA) positivity within psoriatic skin lesions of CETP-Tg and PLTP-Tg mice compared with WT mice. Interferon-α (IFN-α), interleukin-1β (IL-1β), IL-6, IL-17A, IL-17F, IL-22, and IL-23p19 mRNA levels increased within CETP-Tg and PLTP-Tg mice compared with WT counterparts. In comparison with WT mice, plasma tumor necrosis factor-α (TNF-α) levels, rather than IL-6 levels, were increased in CETP-Tg and PLTP-Tg mice.ConclusionsElevated CETP and PLTP aggravate psoriasis in a imiquimod-induced mouse model.

IL-38 Gene Deletion Worsens Murine Colitis.

IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn’s disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1β precursor into active IL-1β. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.

Probiotics alleviate inflammatory bowel disease in mice by regulating intestinal microorganisms-bile acid-NLRP3 inflammasome pathway.

The objective of the present study was to elucidate the mechanism of intestinal microorganisms- bile acid- NLRP3 inflammasome regulation in mice with inflammatory bowel disease treated with probiotics. The abnormal activation of NLRP3 inflammasome is the main pathogenic factor that leads to the development of chronic colitis in IL-10-/- mice. In this study, we divided the IL-10-/- and wild-type mice on a C57BL/6 background into 3 groups: control group (wt mice, n=10), IBD group (IL-10-/- mice, n=10), and probiotic group (IL-10-/- mice treated with probiotics, n=10). The analyses included mRNA levels of cytokines and protein expression of NLRP3 inflammasome and NOD2, as well as colorimetric determination of Wnt, Notch and BMP activity in colon tissue and fresh colon mass. The fresh colon mass was increased in the IBD mice when compared with the control and the probiotic groups (P<0.05). The histological score of the proximal colon in the IBD group was higher than in two other groups (P<0.05). The probiotic group showed lower levels of IFN-γ, IL-17F, IL-1α and IL-25 mRNA compared to the IBD group (P<0.05). The main components of NLRP3 inflammasome (NLRP3, ASC, caspase-1 and IL-1β) and NOD2 were increased in IBD group compared to the control, and decreased after probiotic treatment (P<0.05). FXR, TGR5, vitamin D, and CAR were all increased in IBD group compared to the control and probiotic groups (P<0.05). In conclusion, probiotics modulated the intestinal microbial-bile acid-NLRP3 inflammation in IBD mice.

Increased mucosal IL-12 expression is associated with relapse of ulcerative colitis

BackgroundThe role of IL-12/23 in the pathogenesis of ulcerative colitis (UC) is unclear. We analyzed mucosal IL-12/23 expression and its relationship with endoscopic severity, histological activity, and UC relapse.MethodsRectal biopsies were collected from 70 UC patients with clinical remission. IL-12, IL-23, IFN-γ, IL-17A, and IL-17F mRNA expression was measured by real-time PCR. Endoscopic severity and histological activity were evaluated using the Mayo endoscopic subscore (MES) and the Geboes score, respectively.ResultsThe longest follow-up period was 51 months. Thirty-four patients relapsed during the study period. Samples from these subsequently relapsed patients formed the “relapse” group, while those from patients that did not relapse formed the “remission” group. IL-12 (P = 0.0003) and IL-23 (P = 0.014) mRNA expression was significantly higher in the relapse than the remission group. Expression of IL-23 (P = 0.015) but not IL-12 (P = 0.374) was correlated with MES. However, in patients with an MES of 0 and 1, IL-12 expression was statistically higher in the relapse than the remission group (P = 0.0015, P = 0.0342). IL-12 and IL-23 expression did not vary significantly between histologically active and inactive mucosa; both were higher in histologically inactive patients in the remission group (IL-12: P = 0.0002, IL-23: P = 0.046).ConclusionsRectal IL-12 and IL-23 expression was elevated in the relapse group, but IL-12 was more strongly associated with UC relapse, irrespective of endoscopic severity and histological activity. Mucosal IL-12 was elevated in patients with deep mucosal healing. Our results suggest an important role of IL-12 in UC pathogenesis and the molecular mechanism of UC relapse.

Abstract 1636: Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B)

Abstract Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and predicted to become the leading cause of liver transplant in 2020 and of HCC in the next decades. There is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis due to this changing in HCC etiology. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: We used in the whole study ASV-B mice: a transgenic mouse model (C57BL/6J) spontaneously developing a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20). Livers were characterized for angiogenesis and immune populations using immunostaining (IHC and IF), and qRT-PCR (LightCycler, Roche). To mimic NASH, ASV-B mice were exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. A second experiment was performed on ASV-B and C57BL/6J wild type mice, using control and Nash-inducing regimen, to confirm our first results. Results: ASV-B model showed an increase in liver volume and angiogenesis, HCC livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expressions, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weights. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and green regimens. Interestingly, by microscopic analysis, we observed liver steatosis and inflammation, in 100%, and 75% of the mice, respectively. These results indicate that the green regimen is the most suitable to induce NASH-underlyning disease in our transgenic HCC model. At the conference, we will show in the diet-variants, the immune landscape of the livers and the results of our ongoing second experiment. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants mimicking NASH characteristics that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Philippe Bonnin, Benoit Rousseau, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Armand de Gramont, Eric Raymond, Sandrine Faivre, Valérie Paradis, Clarisse Eveno. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1636.

Molecular and Cellular Responses to the TYK2/JAK1 Inhibitor PF-06700841 Reveal Reduction of Skin Inflammation in Plaque Psoriasis

The IL-23/T helper type 17 cell axis is a target for psoriasis. The TYK2/Janus kinase 1 inhibitor PF-06700841 will directly suppress TYK2-dependent IL-12 and IL-23 signaling and Janus kinase 1-dependent signaling in cells expressing these signaling molecules, including T cells and keratinocytes. This clinical study sought to define the inflammatory gene and cellular pathways through which PF-06700841 improves the clinical manifestations of psoriasis. Patients (n= 30) with moderate-to-severe psoriasis were randomized to once-daily 30 mg (n= 14) or 100 mg (n= 7) PF-06700841 or placebo (n= 9) for 28 days. Biopsies were taken from nonlesional and lesional skin at baseline and weeks 2 and 4. Changes in the psoriasis transcriptome and genes induced by IL-17 in keratinocytes were evaluated with microarray profiling and reverse transcriptase-PCR. Reductions in IL-17A, IL-17F, and IL-12B mRNA were observed as early as 2 weeks and approximately 70% normalization of lesional gene expression after 4 weeks. Immunohistochemistry showed significant decreases in markers of keratinocyte activation, epidermal thickness, KRT16 and Ki-67 expression, and immune cell infiltrates CD3+/CD8+ (T cells) and CD11c (dendritic cells) after 2 weeks of treatment, corresponding with improvement in histologic score. PF-06700841 improves clinical symptoms of chronic plaque psoriasis by inhibition of proinflammatory cytokines that require TYK2 and Janus kinase 1 for signal transduction.

Interleukin 17A and IL-17F Expression and Functional Responses in Rheumatoid Arthritis and Peripheral Spondyloarthritis.

Targeting the interleukin 17 (IL-17) axis is efficacious in psoriasis and spondyloarthritis (SpA), but not in rheumatoid arthritis (RA). We investigated potential differences in tissue expression and function of IL-17A and IL-17F in these conditions. mRNA expression of cytokines and their receptors was assessed by quantitative PCR in psoriasis skin samples, in SpA and RA synovial tissue (ST) samples and in fibroblast-like synoviocytes (FLS). Cytokines were measured in synovial fluid (SF) and FLS supernatants by ELISA. FLS were stimulated with IL-17A or IL-17F cytokines supplemented with tumor necrosis factor (TNF), or with pooled SF from patients with SpA or RA. Levels of IL-17A (P = 0.031) and IL-17F (P = 0.017) mRNA were lower in psoriatic arthritis ST compared to paired psoriasis skin samples. The level of IL-17A mRNA was 2.7-fold lower than that of IL-17F in skin (P = 0.0078), but 17.3-fold higher in ST (P < 0.0001). In SF, the level of IL-17A protein was 37.4-fold higher than that of IL-17F [median 292.4 (IQR 81.4-464.2) vs median 7.8 (IQR 7.7-8.7) pg/mL; P < 0.0001]. IL-17A and IL-17F mRNA and protein levels did not differ in SpA compared to RA synovitis samples, and neither were the IL-17 receptors IL-17RA and IL-17RC, or the TNF receptors TNFR1 and TNR2, differentially expressed between SpA and RA ST, nor between SpA and RA FLS. SpA and RA FLS produced similar amounts of IL-6 and IL-8 protein upon stimulation with IL-17A or IL-17F cytokines, supplemented with 1 ng/ml TNF. Pooled SpA or RA SF samples similarly enhanced the inflammatory response to IL-17A and IL-17F simulation in FLS. The IL-17A/IL-17F expression ratio is higher in SpA synovitis compared to psoriasis skin. Expression of IL-17A and IL-17F, and the functional response to these cytokines, appear to be similar in SpA and RA synovitis.

Protein and mRNA Expression Levels of Interleukin-17A, -17F and -22 in Blood and Skin Samples of Patients with Mycosis Fungoides.

This study investigated the expression of interleukin (IL)-17A, -17F and -22 in mycosis fungoides. Blood samples were collected from 50 patients with mycosis fungoides and 50 healthy controls. Skin samples were obtained from 26 patients with mycosis fungoides and 5 healthy controls. Protein levels of IL-17A, -17F and -22 were measured in serum by multiplex enzyme-linked immunosorbent assay, and mRNA expression levels were measured in blood and skin samples by real-time quantitative reverse transcription PCR. Both IL-17A and IL-17F mRNA expression levels were significantly lower in blood of patient swith mycosis fungoides in comparison with healthy controls. IL-22 serum levels and expression levels of IL-22 mRNA in skintissue,weresignificantlyincreasedinpatients with mycosis fungoides in comparison with healthy controls. These results suggest that low levels of IL-17A and IL-17F in mycosis fungoides may be connected to impaired immune surveillance contributing to tumourigenesis. Upregulation of IL-22 may play a role in the establishment of the tumour microenvironment in mycosis fungoides.

Abstract 91: Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B)

Abstract Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and is predicted to become the leading cause of HCC in the next decades. Therefore, there is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: To mimic NASH, ASV-B, a transgenic mouse model (C57BL/6J) that spontaneously develops a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20) was exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. RNA was extracted from frozen livers at W20 for 40 immune markers analysis using qRT-PCR (LightCycler, Roche). Immune populations were assessed using automated immunohistochemistry (IHC) (Bond Max, Leica). Results: ASV-B model shows an increase in liver volume and angiogenesis, ASV-B livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expression, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weight. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and the green regimens. At the conference, we will show the morphologic changes of the livers using HPS staining and the immune landscape in the livers of the diet-variants. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants aiming at mimicking NASH that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Benoit Rousseau, Patricia Hainaud, Philippe Bonnin, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Valérie Paradis, Armand de Gramont, Eric Raymond, Evelyne Dupuy, Clarisse Eveno, Sandrine Faivre. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 91.

Characterization of IL-17-producing T helper cells-like autoreactive T cells in aged mice.

IL-17-producing T helper cells (Th17) are attracting attention as a new CD4-positive subset of T cells, reported to be responsible for various autoimmune diseases through stimulation of the release of inflammatory cytokines from target cells. However, most investigations of Th17 mediation of autoimmune diseases have focused on the experimental autoimmune models derived from young animals, with few studies that have analyzed physiological factors such as aging. The present study analyzed autoreactive T cells established in a syngeneic mixed lymphocyte culture (sMLC) from aged mice and examined their similarity with Th17. IL-17-producing autoreactive CD4-intermediate T cells were observed in the sMLC; these expressed several stem cell markers or an immunosuppressive receptor PD-1 on the cell surface and so seemed to be different to typical Th17 cells. RT-PCR analysis revealed that purified Th17-like cells also expressed Il17a, Il17f, Il23r, Rorc and Tdt mRNA, but not Rag1 or Rag2 mRNA. These findings that it is likely that Th17-like cells are involved in autoimmune responses in aged mice.

CD44 Assists the Topical Anti-Psoriatic Efficacy of Curcumin-Loaded Hyaluronan-Modified Ethosomes: A New Strategy for Clustering Drug in Inflammatory Skin.

Background: Psoriasis is a common chronic inflammatory skin disease. Its treatment is challenged by the limited amount of drug reaching the inflamed skin. The overexpressed CD44 protein in inflamed psoriatic skin can serve as a potential target of novel active-targeting nanocarriers to increase drug accumulation in the skin.Methods: Hyaluronic acid (HA) was linked to propylene glycol-based ethosomes by covalent binding to develop a novel topical drug delivery carrier (HA-ES) for curcumin. An imiquimod-induced psoriasis mouse model was established, and curcumin delivery and anti-psoriatic efficacy using HA-ES were compared with those using plain ethosomes (ES).Results: The HA gel network formed on the surface of HA-ES reduced the leakage and release of poorly water-soluble curcumin. Compared with ES, transdermal curcumin delivery was significantly enhanced by using HA-ES as vehicles; the cumulative transdermal amount and the amount retained in the skin in vitro after 8 h were, respectively, 1.6 and 1.4 times those observed with ES, as well as 3.1 and 3.3 times those observed with a curcumin propylene glycol solution (PGS), respectively. The in vivo psoriatic skin retention of curcumin with HA-ES was 2.3 and 4.0 times that of ES and PGS, respectively. CD44 expression in imiquimod-induced psoriasis-like inflamed skin was 2.7 times that in normal skin. Immunostaining revealed similar results, suggesting that the specific adhesion of HA-ES to CD44 increased drug accumulation in the skin. After topical administration to mice, the HA-ES group showed an alleviation of inflammation symptoms; lower TNF-α, IL-17A, IL-17F, IL-22, and IL-1β mRNA levels; and lower CCR6 protein expression compared to the ES and PGS groups.Conclusion: We demonstrated increased topical drug delivery of curcumin to inflamed tissues using HA-ES targeting the highly expressed CD44 protein. This innovative strategy could be applied for the development of topical drug delivery systems targeting inflamed skin.

A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It.

Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses, including the synthesis and secretion of a variety of cytokines. In light of the controversial data in the literature, the main objective of this study was to more in-depth reevaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family in vitro. By reverse transcription quantitative real-time PCR, protein measurement via commercial ELISA, immunohistochemistry (IHC) and immunofluorescence (IF), flow cytometry, immunoblotting, chromatin immunoprecipitation (ChIP), and ChIP-seq experiments, we found that highly pure (>99.7%) populations of human neutrophils do not express/produce IL-17A, IL-17F, IL-17AF, or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils isolated from active psoriatic patients. In contrast with published studies, IL-17A and IL-17F mRNA expression/production was not even found when neutrophils were incubated with extremely high concentrations of IL-6 plus IL-23, regardless of their combination with inactivated hyphae or conidia from Aspergillus fumigatus. Consistently, no deposition of histone marks for active (H3K27Ac) and poised (H3K4me1) genomic regulatory elements was detected at the IL-17A and IL-17F locus of resting and IL-6 plus IL-23-stimulated neutrophils, indicating a closed chromatin conformation. Concurrent experiments revealed that some commercial anti-IL-17A and anti-IL-17B antibodies (Abs), although staining neutrophils either spotted on cytospin slides or present in inflamed tissue samples by IHC/IF, do not recognize intracellular protein having the molecular weight corresponding to IL-17A or IL-17B, respectively, in immunoblotting experiments of whole neutrophil lysates. By contrast, the same Abs were found to more specifically recognize other intracellular proteins of neutrophils, suggesting that their ability to positively stain neutrophils in cytospin preparations and, eventually, tissue samples derives from IL-17A- or IL-17B-independent detections. In sum, our data confirm and extend, also at epigenetic level, previous findings on the inability of highly purified populations of human neutrophils to express/produce IL-17A, IL-17B, and IL-17F mRNAs/proteins in vitro, at least under the experimental conditions herein tested. Data also provide a number of justifications explaining, in part, why it is possible to false positively detect IL-17A+-neutrophils.

Relationship between the methylation status of STAT3 promoter DNA in peripheral blood CD4+ T cells from patients after allo-HSCT and aGVHD

To study the relationship between acute graft versus host disease (aGVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4+ T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors. Serum IL-10, TGF-β1, IL-17A and IL-17F levels were detected by ELISA. Foxp3 cytotoxic T-lymphocyte-associated protein 4 (CTLA4), IL-10, TGF-β1, RORγt, IL-17A and IL-17F mRNA levels in CD4+ T cells were measured by real-time PCR. STAT3 expression levels were detected by real-time PCR and Western blot, and promoter DNA methylation was analyzed by bisulfite sequencing PCR (BSP). Results: IL-10 and TGF-β1 levels were significantly down-regulated, while IL-17A and IL-17F levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. Foxp3, CTLA4, IL-10, TGF-β1 mRNA levels were significantly down-regulated, while RORγt, IL-17A, IL-17F mRNA levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. STAT3 expression was increased, while STAT3 promoter DNA was hypomethylated in patients with aGVHD compared with those without aGVHD. The STAT3 mRNA level was negatively correlated with STAT3 promoter DNA methylation. Conclusion: The imbalance of Treg/Th17 in CD4+ T cells from patients after allo-HSCT is a key factor for triggering aGVHD, and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4+ T cells and contribute to the imbalance.

RORγt-expressing cells attenuate cardiac remodeling after myocardial infarction.

AimsRetinoic acid receptor-related orphan nuclear receptor γt (RORγt) is a transcriptional factor responsible for IL-17-producing T-cell differentiation. Although it was demonstrated that RORγt plays essential roles in the onset of autoimmune myocarditis, pathophysiological significance of RORγt in cardiac remodeling after myocardial infarction (MI) remains to be fully elucidated.Methods and resultsMI was generated by ligating coronary artery. The expression of RORγt and IL-17A transcripts increased in murine hearts after MI. Additionally, immunohistochemical staining revealed that RORγt-expressing cells infiltrated in the border zone after MI. Flow cytometric analysis showed that RORγt-expressing cells were released from the spleen at day 1 after MI. Though RORγt-expressing cells in spleen expressed γδTCR or CD4, γδTCR+ cells were major population of RORγt-expressing cells that infiltrated into post-infarct myocardium. To address the biological functions of RORγt-expressing cells in infarcted hearts, we used mice with enhanced GFP gene heterozygously knocked-in at RORγt locus (RORγt+/- mice), which physiologically showed reduced expression of RORγt mRNA in thymus. Kaplan-Meier analysis showed that MI-induced mortality was higher in RORγt+/- mice than wild-type (WT) mice. Masson’s trichrome staining demonstrated that cardiac injury was exacerbated in RORγt+/- mice 7 days after MI (Injured area: RORγt+/-; 42.1±6.5%, WT; 34.0±3.7%, circumference of injured myocardium: RORγt+/-; 61.8±4.8%, WT; 49.6±5.1%), accompanied by exacerbation of cardiac function (fractional shortening: RORγt+/-; 32.9±2.9%, WT; 38.3±3.6%). Moreover, immunohistochemical analyses revealed that capillary density in border zone was significantly reduced in RORγt+/- mice after MI, compared with WT mice, associated with the reduced expression of angiopoietin 2. Finally, the mRNA expression of RORγt, IL-17A, IL-17F and IL-23 receptor (IL-23R) mRNA and protein expression of IL-10 were decreased in RORγt+/- hearts.ConclusionsHeterozygous deletion of RORγt gene resulted in aggravated cardiac remodeling, accompanied by reduced capillary density, after MI, suggesting that RORγt-expressing cells contribute to tissue repair in infarcted myocardium.

Anti-Psoriasis Effects and Mechanisms of Α-(8-Quinolinoxy) Zinc Phthalocyanine-Mediated Photodynamic Therapy

Background/Aims: The aim of this study was to determine the anti-psoriasis effects of α-(8-quinolinoxy) zinc phthalocyanine (ZnPc-F7)-mediated photodynamic therapy (PDT) and to reveal its mechanisms. Methods: HaCaT cells were used to observe the influence of ZnPc-F7-PDT on cell proliferation in vitro. The in vivo anti-psoriasis effects of ZnPc-F7-PDT were evaluated using a mouse vagina model, a propranolol-induced cavy psoriasis model and an imiquimod (IMQ)-induced nude mouse psoriasis model. Flow cytometry was carried out to determine T lymphocyte levels. Western blotting was performed to determine protein expression, and a reverse transcription-polymerase chain reaction test was performed to determine mRNA expression. Results: The results showed that ZnPc-F7-PDT significantly inhibited the proliferation of HaCaT cells in vitro; when the light doses were fixed, changing the irradiation time or output power had little influence on the inhibition rate. ZnPc-F7-PDT significantly inhibited the hyperproliferation of mouse vaginal epithelium induced by diethylstilbestrol and improved propranolol- and IMQ-induced psoriasis-like symptoms. ZnPc-F7-PDT inhibited IMQ-induced splenomegaly and T lymphocyte abnormalities. ZnPc-F7-PDT did not appear to change T lymphocytes in the mouse vagina model. ZnPc-F7-PDT down-regulated the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), interleukin (IL)-17A mRNA and IL-17F mRNA, and up-regulated the expression of Bax. Conclusion: In conclusion, ZnPc-F7-PDT exhibited therapeutic effects in psoriasis both in vitro and in vivo and is a potential approach in the treatment of psoriasis. Potential mechanisms of these effects included the inhibition of hyperproliferation; regulation of PCNA, Bcl-2, Bax, IL-17A mRNA and IL-17F mRNA expression; and immune regulation.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Validation of Th17 Cell Differentiation from Peripheral Blood CD4+ T Cells Through Assessment of mRNA Expression and Cytokine Secretion using Microplate Reading and Cellular Imaging

Abstract CD4+ T cells signal and regulate an immune response to pathogens after interacting with an antigen-MHC complex. This activation causes the cells to differentiate into distinct phenotypic and functional effectors, collectively referred to as T helper cells (Th). One such differentiated T cell subset includes the pro-inflammatory T helper 17 (Th17) cell subset. These cells can be beneficial to the host during infection, as they amplify ongoing inflammation by inducing expression of tumor necrosis factor-α. CD4+ T cells that differentiate into Th17 cells are uniquely characterized by production of the pro-inflammatory cytokine, IL-17. T cells isolated from peripheral blood mononuclear cells and selected for CD4 are differentiated into Th17 cells using a cocktail of specific antibodies and cytokines. The procedure to create and confirm this differentiation can be labor and time intensive. Here we demonstrate a validated, robust method to differentiate peripheral blood CD4+ T cells into functional Th17 cells using a bead-based activation technology. A novel, cell-imaging multi-mode reader can be used to image phenotypic differences between cells exposed to the antibody/cytokine cocktail and control cells as differentiation proceeds. Creation of fully functioning Th17 cells was then confirmed by assessing IL-17F mRNA levels using a fluorescence RNA in situ hybridization assay, in addition to IL-17 secretion using a homogeneous, bead-based immunoassay technology. The reader was able to perform brightfield and fluorescence imaging steps, as well as laser-based excitation for the secretion immunoassay. The combination provides a comprehensive solution for the creation and validation of this important class of helper CD4+ T cells.

Interleukin-17-producing decidual CD4+ T cells are not deleterious for human pregnancy when they also produce interleukin-4.

BackgroundTrophoblast expressing paternal HLA-C antigens resemble a semiallograft, and could be rejected by maternal CD4+ T lymphocytes. We examined the possible role in human pregnancy of Th17 cells, known to be involved in allograft rejection and reported for this reason to be responsible for miscarriages. We also studied Th17/Th1 and Th17/Th2 cells never investigated before. We defined for the first time the role of different Th17 subpopulations at the embryo implantation site and the role of HLA-G5, produced by the trophoblast/embryo, on Th17 cell differentiation.MethodsCytokine production by CD4+ purified T cell and T clones from decidua of normal pregnancy, unexplained recurrent abortion, and ectopic pregnancy at both embryo implantation site and distant from that site were analyzed for protein and mRNA production. Antigen-specific T cell lines were derived in the presence and in the absence of HLA-G5.ResultsWe found an associated spontaneous production of IL-17A, IL-17F and IL-4 along with expression of CD161, CCR8 and CCR4 (Th2- and Th17-type markers) in fresh decidua CD4+ T cells during successful pregnancy. There was a prevalence of Th17/Th2 cells (producing IL-17A, IL-17F, IL-22 and IL-4) in the decidua of successful pregnancy, but the exclusive presence of Th17 (producing IL-17A, IL-17F, IL-22) and Th17/Th1 (producing IL-17A, IL-17F, IL-22 and IFN-γ) cells was found in the decidua of unexplained recurrent abortion. More importantly, we observed that Th17/Th2 cells were exclusively present at the embryo implantation site during tubal ectopic pregnancy, and that IL-4, GATA-3, IL-17A, ROR-C mRNA levels increased in tubal biopsies taken from embryo implantation sites, whereas Th17, Th17/Th1 and Th1 cells are exclusively present apart from implantation sites. Moreover, soluble HLA-G5 mediates the development of Th17/Th2 cells by increasing IL-4, IL-17A and IL-17F protein and mRNA production of CD4+ T helper cells.ConclusionNo pathogenic role of decidual Th17 cells during pregnancy was observed. Indeed, a beneficial role for these cells was observed when they also produced IL-4. HLA-G5 could be the key feature of the uterine microenvironment responsible for the development of Th17/Th2 cells, which seem to be crucial for successful embryo implantation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12948-016-0039-y) contains supplementary material, which is available to authorized users.