Asthma is a complex disorder with multiple phenotypes which can influence its severity and response to treatment. The T_HIL17 pathway genes with asthma and atopy markers.

Genotyping was performed using a commercial panel in 1245 participants of SCAALA cohort. The study included 91 SNVs in IL-17 pathway genes. Logistic regressions for asthma and atopy markers were performed using PLINK 1.9. In silico analyses were performed using rSNPbase, RegulomeDB, and Gtex portal for in silico gene expression.

Results and discussion

The T allele of rs1974226 in IL17A was positively associated with asthma (OR: 1.37; 95% CI 1.02-1.82). Also, the T allele of rs279548 was positively associated with asthma (OR: 1.30; 95% CI 1.02-1.64), atopy (OR: 1.62; 95% CI 1.05-2.50) and increased expression of the IL17RC in lung and whole blood tissues. The others genetic variants in the ILThe genetic variants in IL-17-related genes are associated with the atopic asthma phenotype and IgE production.

Colorectal cancer (CRC) is one of the most common tumor entities. In patients with inflammatory bowel diseases, the development of colitis-associated colon cancer is considered a dangerous long-term complication. IL-17A and the transcription factor retinoic acid receptor-related orphan receptor γt (RORγt) play fundamental roles in the pathogenesis of inflammatory bowel diseases; in human studies, we detected a dense infiltration of RORγt-dependent CD4(+) IL17A(+) T helper (Th)17 cells in specimens of CRC, ulcerative colitis, and ulcerative colitis-associated colorectal cancer. However, the mechanistic role of RORγt(+) hematopoietic cells in colitis-associated tumorigenesis remains unclear. To investigate colitis-associated colon tumorigenesis, we conducted studies in the AOM+DSS mouse model that revealed the importance of RORγt for colon tumor progression. In the absence of RORγt-dependent Th17 lymphocytes, mice showed signs of intense chronic colitis, but developed significantly fewer macroscopic tumor nodules. The reduction of tumor development in RORγt(-/-) mice was not due to reduced colon tumor initiation. However, the proliferation rate of tumor cells was reduced in the absence of RORγt-dependent Th17 cells and tumor cells showed pronounced signs of senescence-associated epigenetic and lysosomal changes. These results indicate an important role for the immunological milieu in colitis-associated cancer, which is shaped in-part by RORγt-dependent Th17 lymphocytes that support CRC growth.

OBJECTIVE

Our previous study demonstrated using an oral gavage model that Porphyromonas gingivalis could induce various inflammatory changes linked to periodontitis-associated systemic diseases by altering gut microbiota. A ligature-induced periodontitis model is similar to human periodontitis in various aspects: in both cases, alveolar bone resorption depends on oral bacterial load, and gingival tissue becomes infiltrated with inflammatory cells. Therefore, this model may be suitable for the analysis of bacterial burden and gingival tissue inflammation with changes related to systemic diseases.

METHODS

Periodontal tissue destruction was induced by a 2 wk ligature placement around the bilateral maxillary second molar. We analyzed the expression profile of various genes in several tissues, levels of systemic inflammatory markers and induction of insulin resistance. In addition, we studied changes in gut microbiota composition and bacterial load in the oral cavity.

RESULTS

Two weeks after ligature placement gingival inflammation was significantly induced with a disrupted gingival epithelial barrier and alveolar bone resorption accompanied by increased bacterial burden in the oral cavity. Gene expression analysis of the gingival tissue of ligated mice demonstrated that interleukin (Il)1b was significantly elevated and Il6 and Il17a tended to be higher in ligated mice than in untreated mice. Although serum IL-6 was significantly elevated and serum amyloid A tended to be higher in ligated compared to untreated mice, endotoxin levels did not differ between the two groups. Among the genes whose expressions are closely related to glucose and lipid metabolisms, only phosphoenolpyruvate carboxykinase 1 (Pck1) and acetyl-coenzyme A carboxylase alpha (Acaca) showed significant changes following ligature placement in the liver, with the former upregulated and the latter downregulated. However, insulin sensitivity did not change following ligature placement. Furthermore, ligature placement weakly affected the composition of gut microbiota and gene expression in the intestines.

CONCLUSIONS

The results suggest that increased oral commensals and gingival inflammation have limited roles in the pathological changes to adipose and liver tissues, which are important organs whose dysfunctions contribute to the development of periodontitis-related systemic diseases.

Chronic pelvic pain syndrome (CPPS) is the most common form of prostatitis, accounting for 90-95% of all diagnoses. It is a complex multi-symptom syndrome with unknown etiology and limited effective treatments. Previous investigations highlight roles for inflammatory mediators in disease progression by correlating levels of cytokines and chemokines with patient reported symptom scores. It is hypothesized that alteration of adaptive immune mechanisms results in autoimmunity and subsequent development of pain. Mouse models of CPPS have been developed to delineate these immune mechanisms driving pain in humans. Using the experimental autoimmune prostatitis (EAP) in C57BL/6 mice model of CPPS we examined the role of CD4+T-cell subsets in the development and maintenance of prostate pain, by tactile allodynia behavioral testing and flow cytometry. In tandem with increased CD4+IL17A+ T-cells upon EAP induction, prophylactic treatment with an anti-IL17 antibody one-day prior to EAP induction prevented the onset of pelvic pain. Therapeutic blockade of IL17 did not reverse pain symptoms indicating that IL17 is essential for development but not maintenance of chronic pain in EAP. Furthermore we identified a cytokine, IL7, to be associated with increased symptom severity in CPPS patients and is increased in patient prostatic secretions and the prostates of EAP mice. IL7 is fundamental to development of IL17 producing cells and plays a role in maturation of auto-reactive T-cells, it is also associated with autoimmune disorders including multiple sclerosis and type-1 diabetes. More recently a growing body of research has pointed to IL17's role in development of neuropathic and chronic pain. This report presents novel data on the role of CD4+IL17+ T-cells in development and maintenance of pain in EAP and CPPS.

BACKGROUND

In contrast to anti-TNF-α antibodies, anti-IL-17A antibodies lacked clinical efficacy in a trial with patients suffering from Crohn's disease. We therefore analyzed how IL-17A modulates the inflammatory response elicited by TNF-α in intestinal epithelial cells (IEC).

METHODS

Target mRNA levels in IEC and colonic biopsies were assessed by RNA microarray and quantitative real-time PCR. Signaling pathways were analyzed using receptor neutralization and pharmacological inhibitors. Target protein levels were determined by immunoblotting.

RESULTS

Microarray analysis demonstrated that IL-17A alone is a weak inducer of gene expression in IEC (29 regulated transcripts), but significantly affected the TNF-α-induced expression of 547 genes, with strong amplification of proinflammatory chemokines and cytokines (>200-fold increase of CCL20, CXCL1, and CXCL8). Interestingly, IL-17A differentially modulated the TNF-α-induced expression of several inflammatory bowel disease susceptibility genes in IEC (increase of JAK2 mRNA, decrease of FUT2, ICAM1, and LTB mRNA). Negative regulation of ICAM-1 by IL-17A was verified on protein level. The significance of these findings is emphasized by inflamed lesions of patients with inflammatory bowel disease demonstrating significant correlations (P < 0.01, Rho, 0.57-0.85) for JAK2, ICAM1, and LTB mRNA with IL17A and TNF mRNA.

CONCLUSIONS

Our study demonstrates the modulation of inflammatory bowel disease susceptibility gene mRNA in IEC as a novel important property of IL-17A. Given the weak impact of sole IL-17A stimulation on IEC target gene expression, our study provides an important explanation for the lack of clinical efficacy of sole IL-17A neutralization, but suggests a beneficial effect of combined IL-17A/TNF-α that is currently in clinical development.

BACKGROUND

Previous studies have revealed an association between interleukin 17A (IL17A) polymorphisms and the prevalence of rheumatoid arthritis (RA) in Japanese and Caucasian patients. We hypothesized that IL17A polymorphisms might also affect RA susceptibility in the Chinese population.

METHODS

We studied IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms in 615 RA patients and 839 controls in a Chinese population. Genotyping was performed using a custom-by-design 48-Plex SNP scan™ Kit.

RESULTS

Our results indicated that IL17A rs4711998 A/G and IL17A rs8193037 G/A polymorphisms were not associated with RA, and IL17A rs2275913 G/A and IL17A rs3819024 A/G variant alleles decrease the risk of RA, while IL17A rs3819025 G/A and IL17A rs8193036 C/T variant alleles increase the risk of RA.

CONCLUSIONS

These findings suggest that IL17A polymorphisms may be associated with RA. Future larger studies with other ethnic populations are required to confirm current findings.

Primary osteoarthritis (OA) is the most common type of a joint disease. It has a polygenic risk inheritance pattern and affects older people. The etiology of this disease is not fully understood. The aim of this study was to investigate the associations between polymorphisms in pro-inflammatory interleukin-17 (IL17A and IL17F) and anti-inflammatory Toll-like Receptor 10 (TLR10) genes with the risk for development of advanced stage hip and knee primary OA in the Croatian population. A total of 500 OA patients and 597 controls were genotyped for IL17A SNP (rs2275913), IL17F SNPs (rs763780 and rs1889570), and TLR10 (rs11096957) genes. The allelic and genotypic frequencies of IL17F SNP (rs763780) showed statistically significant differences in comparisons of controls with hip-but not knee-OA patients. The major allele (T) of rs763780 was associated with the lower risk for developing hip OA (p = 7.9 × 10-4 , OR = 0.45, 95%CI = 0.27-0.74), whereas the minor allele (C) was associated with susceptibility to hip OA (p = 7.9 × 10-4 , OR = 2.24, 95%CI = 1.35-3.72). The genotype T/T was associated with the protection to hip OA (p = 3.9 × 10-4 , OR = 0.41, 95%CI = 0.24-0.70), and, lastly, the genotype T/C was associated with the higher risk to acquiring hip OA (p = 2.6 × 10-4 , OR = 2.50, 95%CI = 1.47-4.25). TLR10 SNP rs11096957 was found significantly associated with predisposition to hip OA (p = 0.04, OR = 1.41, 95%CI = 1.02-1.94) but not knee OA. Our findings suggest that hip OA in Croatian population might have a different genetic risk regarding the IL17 and TLR10 gene locus than knee OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1684-1693, 2018.

Adoptive T-cell transfer therapy is an FDA- approved treatment for leukemia that relies on the ex vivo expansion and reinfusion of a patient's immune cells, which can be engineered with a chimeric antigen receptor (CAR) for more efficient tumor recognition. Type 17 T cells, controlled transcriptionally by RORγ, have been reported to mediate potent antitumor effects superior to those observed with conventionally expanded T cells. Here, we demonstrate that addition of a synthetic, small-molecule RORγ agonist during ex vivo expansion potentiates the antitumor activity of human Th17 and Tc17 cells redirected with a CAR. Likewise, ex vivo use of this agonist bolstered the antitumor properties of murine tumor-specific CD4+ and CD8+ T cells. Expansion in the presence of the RORγ agonist enhanced IL17A production without compromising IFNγ secretion in vitroIn vivo, cytokine neutralization studies revealed that IFNγ and IL17A were required to regress murine melanoma tumors. The enhanced antitumor effect of RORγ agonist treatment was associated with recovery of more donor T cells in the tumor and spleen; these cells produced elevated levels of cytokines months after infusion and expressed markers of long-lived stem and central memory cells such as Tcf7 and CD62L. Conversely, untreated cells mainly exhibited effector phenotypes in the tumor. Cured mice previously treated with agonist-primed T cells were protected from tumor rechallenge. Collectively, our work reveals that in vitro treatment with a RORγ agonist generates potent antitumor Type 17 effector cells that persist as long-lived memory cells in vivoSignificance: RORγ agonists can be used in vitro during T-cell expansion to enhance the efficacy of adoptive cell therapy (e.g., CAR-T) and to provide long-term protection against tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3888/F1.large.jpg Cancer Res; 78(14); 3888-98. ©2018 AACR.

METHODS

The objective was to examine the inhibitory effects of citrus fruit bioactive compounds on BxPC-3 and PANC-1 human pancreatic cancer cells, focusing on the antiproliferative mechanism of action of the flavonoid apigenin related to the glycogen synthase kinase-3β/nuclear factor kappa B signaling pathway.

RESULTS

Flavonoids, limonoids, phenolic acids, and ascorbic acid were tested for cytotoxic effects on BxPC-3 and PANC-1 cells; apigenin was the most potent (IC50 = 23 and 12 μM for 24 and 48 h for BxPC-3 and IC50 = 71 and 41 μM for 24 and 48 h for PANC-1). Apigenin induced pancreatic cell death through inhibition of the glycogen synthase kinase-3β/nuclear factor kappa B signaling pathway. Apigenin arrested cell cycle at G2 /M phase (36 and 32% at 50 μM for BxPC-3 and PANC-1, respectively) with concomitant decrease in the expression of cyclin B1. Apigenin activated the mitochondrial pathway of apoptosis (44 and 14% at 50 μM for BxPC-3 and PANC-1, respectively) and modified the expression of apoptotic proteins. Apigenin highly upregulated the expression of cytokine genes IL17F (114.2-fold), LTA (33.1-fold), IL17C (23.2-fold), IL17A (11.3-fold), and IFNB1 (8.9-fold) in BxPC-3 cells, which potentially contributed to the anticancer properties.

CONCLUSIONS

Flavonoids have a protective role in pancreatic cancer tumorigenesis.

OBJECTIVE

To investigate the effects of adalimumab on the induction of complete endoscopic healing and normalization of mucosal cytokine gene expression in patients with active Crohn's disease.

METHODS

A prospective, single-center study including 77 patients. All were examined by endoscopy before initiation of adalimumab induction therapy with a minimum of six adalimumab injections. Patients were treated until documentation of complete endoscopic healing. Biopsies for measurements of mRNA expression levels of interleukin(IL)-17A (IL17A), IL23, interferon-gamma (IFNG), tumor necrosis factor-alpha (TNF), IL10 and Forkhead Box P3 (FOXP3), as well as for immunohistochemistry (IHC) were sampled at pre- and post-treatment endoscopy, and from 17 control patients.

RESULTS

Complete endoscopic healing was achieved in 27.3% after 10 weeks of treatment, documented by endoscopy at week 12. Cumulative endoscopic healing after 52 weeks was 44.2%. Complete endoscopic healing led to a significant reduction in mRNA expression levels for all cytokines except IL10. Elevated expression of TNF and IL-17A persisted in 52% and 76%, respectively, of patients with complete endoscopic remission. Pre-treatment cytokine gene expression levels did not predict response to adalimumab therapy.

CONCLUSIONS

Adalimumab induces accumulated complete endoscopic healing in 44% of patients after 52 weeks of therapy. Normalization of mucosal gene expression of cytokines does not occur in all patients with endoscopy-verified healed mucosa. Inclusion of normalized mucosal cytokine expression into the concept of mucosal healing could have an impact on long-term clinical outcome.

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.

OBJECTIVE

Indirubin (IR) is a bisindole compound extracted from the leaves of Chinese herb Indigo Naturalis. Indigo Naturalis has been widely used in traditional Chinese medicine to treat inflammatory and autoimmune diseases. Psoriasis is a chronic immune-mediated inflammatory skin disease in which γδ T cells play an important role. This study aims to determine the immunoregulatory effects and the underlying mechanisms of Indirubin in psoriasis-related inflammatory responses.

METHODS

BALB/c mice with imiquimod (IMQ)-induced psoriasis-like dermatitis were treated with saline (Model), 1 mg/kg methotrexate (MTX) that serves as a positive control, or 12.5, 25 and 50 mg/kg Indirubin(IR) intragastrically. Keratinocytes proliferation, inflammatory cells infiltration, the expression of inflammatory cytokines and Jak/Stat pathway-related proteins in the skin lesion were examined. The abundance of γδ T cells in lymph nodes and spleen was determined by flow cytometry. The IL-17 expression and secretion, and the activation of Jak3/Stat3 pathways in in vitro cultured γδ T cell were tested.

RESULTS

Indirubin ameliorated keratinocyte proliferation, reduced the infiltration of CD3+ T cells, IL-17 A-producing γδ T cells, and CD11b+ neutrophils, inhibited the mRNA expression of Il1, Il6, Il23, Il17a and Il22, and the protein expression of Jak/Stat pathway-related molecules in the skin lesion. Indirubin also reduced the abundance of γδ T cell and CCR6+ γδ T cells (the major IL-17 A producer) in spleen and lymph nodes. In cultured γδ T cells, Indirubin inhibited the mRNA expression of Il17a and Ifng, and the secretion of IL-17 A, while suppressed the activation of Jak3/Stat3 pathways.

CONCLUSIONS

Indirubin alleviates IMQ-induced psoriasis-like dermatitis mainly through reducing the inflammatory responses mediated by IL-17 A-producing γδ T cells involving Jak3/Stat3 activation. Our results highlighted the novel mechanisms by which Indirubin ameliorates psoriasis-related inflammatory responses, supporting its therapeutic potential.

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in anti-inflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1β (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties.

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.

BACKGROUND

Disrupted bile secretion leads to liver damage characterized by inflammation, fibrosis, eventually cirrhosis, and hepatocellular cancer. As obstructive cholestasis often progresses insidiously, markers for the diagnosis and staging of the disease are urgently needed. To this end, we compiled a comprehensive data set of serum markers, histological parameters and transcript profiles at 8 time points of disease progression after bile duct ligation (BDL) in mice, aiming at identifying a set of parameters that could be used as robust biomarkers for transition of different disease progression phases.

RESULTS

Statistical analysis of the more than 6,000 data points revealed distinct temporal phases of disease. Time course correlation analysis of biochemical, histochemical and mRNA transcript parameters (=factors) defined 6 clusters for different phases of disease progression. The number of CTGF-positive cells provided the most reliable overall measure for disease progression at histological level, bilirubin at biochemical level, and metalloproteinase inhibitor 1 (Timp1) at transcript level. Prominent molecular events exhibited by strong transcript peaks are found for the transcriptional regulator Nr0b2 (Shp) and 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1) at 6 h. Based on these clusters, we constructed a decision tree of factor combinations potentially useful as markers for different time intervals of disease progression. Best prediction for onset of disease is achieved by fibronectin (Fn1), for early disease phase by Cytochrome P450 1A2 (Cyp1a2), passage to perpetuation phase by collagen1α-1 (Col1a1), and transition to the progression phase by interleukin 17-a (Il17a), with early and late progression separated by Col1a1. Notably, these predictions remained stable even for randomly chosen small sub-sets of factors selected from the clusters.

CONCLUSIONS

Our detailed time-resolved explorative study of liver homogenates following BDL revealed a well-coordinated response, resulting in disease phase dependent parameter modulations at morphological, biochemical, metabolic and gene expression levels. Interestingly, a small set of selected parameters can be used as diagnostic markers to predict disease stages in mice with cholestatic liver disease.

Ameloblasts are sensitive cells whose metabolism and function may be affected by inflammatory stimuli. The aim of this study was to evaluate the possible association between polymorphisms in immune response-related genes and molar-incisor hypomineralization (MIH), and their interaction with polymorphisms in amelogenesis-related genes. DNA samples were obtained from 101 nuclear families that had at least 1 MIH-affected child. Eleven single-nucleotide polymorphisms (SNPs) were investigated in immune response genes using TaqMan® technology allele-specific probes. A transmission disequilibrium test was performed to verify overtransmission of alleles in all MIH families, as well as in families only with mild or severe MIH-affected children. Gene-gene interactions between the immune-related and amelogenesis-related polymorphisms were analyzed by determining whether alleles of those genes were transmitted from heterozygous parents more often in association than individually with MIH-affected children. In severe cases of MIH, significant results were observed for rs10733708 (TGFBR1, OR = 3.5, 95% CI = 1.1-10.6). Statistical evidence for gene-gene interactions between rs6654939 (AMELX) and the SNPs rs2070874 (IL4), rs2275913 (IL17A), rs1800872 (IL10), rs1800587 (IL1A), and rs3771300 (STAT1) was observed. The rs2070874 SNP (IL4) was also significantly overtransmitted from heterozygous parents with the rs7526319 (TUFT1) and the rs2355767 (BMP2) SNPs, suggesting a synergistic effect of the transmission of these alleles with susceptibility to MIH. This family-based study demonstrated an association between variation in TGFBR1 and MIH. Moreover, the polymorphisms in immune response and amelogenesis genes may have an additive effect on the risk of developing MIH.

The functional heterogeneity of T cell responses to diverse antigens expressed at different stages of Mycobacterium tuberculosis (Mtb) infection, in particular early secreted versus dormancy related latency antigens expressed later, that distinguish subjects with latent (LTBI), pulmonary (PTB) or extrapulmonary (EPTB) tuberculosis remains unclear. Here we show blood central memory CD4 T-cell responses specific to Mtb dormancy related (DosR) latency, but not classical immunodominant secretory antigens, to clearly differentiate LTBI from EPTB and PTB. The polyfunctionality score integrating up to 31 DosR-specific CD4 T-cell functional profiles was significantly higher in LTBI than EPTB or PTB subjects. Further analysis of 256 DosR-specific T-cell functional profiles identified regulatory IL10 + Th17 cells (IL10+IL17A+IL17F+IL22+) to be significantly enriched in LTBI; in contrast to pro-inflammatory Th17 cells (IFNγ+IL17A+/IL10-) in the blood and lung of EPTB and PTB subjects respectively. A blood polyfunctional, Mtb DosR latency antigen specific, regulatory, central memory response is therefore a novel functional component of T-cell immunity in latent TB and potential correlate of protection.

T helper 17 (Th17) and T regulatory (Treg) cytokines appear to be contributing greatly to colorectal cancer (CRC) development and progression. The aim of the current study was to investigate the expression of Foxp3; IL10; TGFB1; IL17A; IL6 and NOS2 genes in tumor tissue, regional positive lymph nodes and distant metastasis obtained from 26 patients with advanced CRC. Quantitative real-time polymerase chain reaction (qPCR) was performed for mRNA detection by TaqMan gene expression assay. In distant metastasis, IL6 was strongly expressed, over 7.5 fold, followed by Treg-related genes Foxp3; IL10 and TGFB1 in contrast to IL17A and NOS2. The similar pattern of expression was observed in positive regional lymph node in addition to significant down-regulation of NOS2 (RQ = 0.287; p = 0.011) and a trend for the elevation of IL17A. In tumor tissue, Fopx3 was significantly upregulated and Foxp3 mRNA positively correlated with TGFB1 in all investigated tissue types. In tumor tissue, expression of IL17A was correlated with NOS2 (r = 0.68; p = 0.005), while in distant metastasis IL10 was in strong relation with TGFB1 and IL6. In addition, a reverse correlation between IL6 and NOS2 (r = -0.66; p = 0.009), was observed in distant metastasis. The simultaneous expression of given Treg and Th17-related genes found both in the primary tumor and in the regional lymph nodes appears to provide suitable microenvironment sufficient for promoting metastatic growth. The upregulation of Foxp3; IL10, TGFB1 and IL6 might be a transcriptional profile hallmark for colorectal metastases.

BACKGROUND

Palmitoleic acid (PA) is a n-7 monounsaturated fatty acid (MUFA) secreted by adipose tissue and related to decreased insulin resistance in peripheral tissues. Evidences have been shown that PA also decreased proinflammatory cytokine expression in cultured macrophages. Although studies have shown that other fatty acids (FAs) modulate several lymphocyte functions, the specific effect of PA on these cells is unknown. The aim of the present study was to evaluate the possible influence of PA on activation and differentiation of human lymphocytes in comparison to oleic acid (OA).

METHODS

Human lymphocytes were isolated from peripheral blood of health men and cultured in the presence of growing concentrations of PA or OA (5 to 200 μM), for 24 h. After that, cells were collected and cytotoxicity evaluated by flow cytometry. Then, we analyzed proliferative capacity in lymphocytes treated with non toxic concentrations of PA and OA (25 and 50 μM, respectively), in the presence or absence of concanavalin A (ConA). The Th1/Th2/Th17 cytokine production was determined by the Cytometric Bead Array. CD28 and CD95 surface expression and T regulatory cell percentage were determined by flow cytometry.

RESULTS

We observed that PA is toxic to lymphocytes above 50 μM. PA promoted a decrease of lymphocyte proliferation stimulated by ConA in both concentrations. PA also decreased CD28 externalization and increased CD95. On the other hand, OA did not alter these parameters. In the same way, PA reduced IL6, IFN-gamma, TNF-alpha and IL17A production in both concentration and IL2 only at 50 μM (in the presence of ConA). OA promoted IFN-gamma reduction in both concentrations and an increase of IL-2, IL4 and IL10 at 25 μM. Both fatty acids decreased the percentage of T regulatory cells.

CONCLUSIONS

In conclusion, PA promoted a suppressive effect on lymphocyte proliferation characterized by a decrease of Th1 and Th17 response, and co-stimulatory molecule (CD28). However, OA increased lymphocyte proliferation through IL2 production and Th2 response. These results also show a more suppressive effect of PA on lymphocytes in comparison to OA.

Regnase-1 and Roquin are RNA binding proteins that are essential for degradation of inflammatory mRNAs and maintenance of immune homeostasis. Although deficiency of either of the proteins leads to enhanced T cell activation, their functional relationship in T cells has yet to be clarified because of lethality upon mutation of both Regnase-1 and Roquin. By using a Regnase-1 conditional allele, we show that mutations of both Regnase-1 and Roquin in T cells leads to massive lymphocyte activation. In contrast, mutation of either Regnase-1 or Roquin affected T cell activation to a lesser extent than the double mutation, indicating that Regnase-1 and Roquin function nonredundantly in T cells. Interestingly, Regnase-1 and Roquin double-mutant mice suffered from severe inflammation and early formation of fibrosis, especially in the heart, along with the increased expression of Ifng, but not Il4 or Il17a Consistently, mutation of both Regnase-1 and Roquin leads to a huge increase in the Th1, but not the Th2 or Th17, population in spleens compared with T cells with a single Regnase-1 or Roquin deficiency. Regnase-1 and Roquin are capable of repressing the expression of a group of mRNAs encoding factors involved in Th1 differentiation, such as Furin and Il12rb1, via their 3' untranslated regions. Moreover, Regnase-1 is capable of repressing Roquin mRNA. This cross-regulation may contribute to the synergistic control of T cell activation/polarization. Collectively, our results demonstrate that Regnase-1 and Roquin maintain T cell immune homeostasis and regulate Th1 polarization synergistically.