Expression of Th17 cell population regulatory cytokines in laryngeal carcinoma - Preliminary study.
ABSTRACT: AIM OF THE STUDY:Aim of the study was to evaluate the potential role of regulatory and proinflammatory cytokines IL-23 and IL-17 as Th17 lymphocyte activity markers in relation to invasiveness in laryngeal cancer. MATERIAL AND METHODS:The immunological analysis was conducted in 50 patients treated for squamous cell laryngeal carcinoma and 30 healthy volunteers as controls. The levels of IL-23 and IL-17 in supernatants of purified peripheral blood mononuclear cell cultures were determined by using the enzyme-linked immunosorbent assay (ELISA). The clinicomorphological criteria included pTNM, stage, G, and the total tumour front grading (TFG) score. RESULTS:Our data demonstrated higher concentrations of IL-23 in patients as compared to controls (p = 0.0001). No statistical difference for IL-17 in these groups was observed. Our study revealed significant dependences in IL-23 expression on pT (p = 0.04), histological differentiation (p = 0.04), and TFG total score (p = 0.02). Advanced tumours (pT3-pT4) with higher grade (G2-G3) and higher invasiveness (> 14 TFG points) were characterised by elevated IL-23 levels in PBMC supernatants. Our data did not indicate a relationship between cytokine levels and three- and five-year survival. However, a tendency towards lower content of IL-23 in PBMC cultures in patients who lived longer than five years after treatment was noted. The relationships between IL-17 level in PBMC cultures and clinicomorphological and prognostic parameters have not been disclosed. CONCLUSIONS:The results of this study suggest the importance of regulatory cytokine IL-23 in determining the aggressive potential of laryngeal carcinomas.
Project description:This study demonstrates that IL-23 stimulates the differentiation of human osteoclasts from peripheral blood mononuclear cells (PBMC). Furthermore, in vivo blockade of endogenous IL-23 activity by treatment with anti-IL-23 antibody attenuates collagen-induced arthritis in rats by preventing both inflammation and bone destruction. IL-23 induced human osteoclastogenesis in cultures of PBMC in the absence of osteoblasts or exogenous soluble-receptor activator of NF-kappaB ligand (RANKL). This IL-23-induced osteoclastogenesis was inhibited by osteoprotegerin, anti-IL-17 antibody, and etanercept, suggesting that RANKL, IL-17, and TNF-alpha are involved. In addition, we found the ratio of production levels of IL-17 to those of IFN-gamma from activated human T cells was elevated at 1 to 10 ng/ml IL-23. The inductive effect of IL-17 and the inhibitory effect of IFN-gamma on osteoclastogenesis indicate that the balance of these two cytokines is particularly important. We also demonstrated that IL-23 administered at a later stage significantly reduced paw volume in rats with collagen-induced arthritis, in a dose-dependent manner. Furthermore, anti-IL-23 antibody reduced synovial tissue inflammation and bone destruction in these rats. These findings suggest that IL-23 is important in human osteoclastogenesis and that neutralizing IL-23 after onset of collagen-induced arthritis has therapeutic potential. Thus, controlling IL-23 production and function could be a strategy for preventing inflammation and bone destruction in patients with rheumatoid arthritis.
Project description:The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was <10% upon 24 h incubation of PBMC with P. aeruginosa biofilms. PBMC incubated for 24 h with preformed P. aeruginosa biofilms were significantly more activated compared to PBMC incubated alone. Interestingly, a marked activation of CD56+CD3- natural killer (NK) cells was observed that reached 60% of NK cells as an average of different donors. In the culture supernatants of PBMC co-cultured with P. aeruginosa biofilms, not only pro-inflammatory (IL-1?, IFN-?, IL-6, and TNF-?) but also anti-inflammatory (IL-10) cytokines were significantly increased as compared to PBMC incubated alone. Furthermore, incubation of biofilms with PBMC, caused a statistically significant increase in the CFU number of P. aeruginosa, as compared to biofilms incubated without PBMC. In order to assess whether PBMC products could stimulate the growth of P. aeruginosa biofilms, we incubated preformed P. aeruginosa biofilms with or without supernatants obtained from the co-cultures of PBMC with biofilms. In the presence of the supernatants, the CFU count of biofilm-derived P. aeruginosa, was two to seven times higher than those of biofilms incubated without supernatants (P < 0.01). Overall, the results obtained shed light on the reciprocal interaction between human PBMC and P. aeruginosa biofilms. P. aeruginosa biofilms induced PBMC activation and cytokine secretion but, in turn, the presence of PBMC and/or PBMC-derived components enhanced the number of P. aeruginosa biofilm associated bacteria. This may indicate a successful bacterial defensive/persistence strategy against immune response.
Project description:BACKGROUND:Familial eosinophilia (FE) is a rare autosomal dominant inherited disorder characterized by the presence of lifelong peripheral eosinophilia (>1500/?L). Mapped to chromosome 5q31-q33, the genetic cause of FE is unknown, and prior studies have failed to demonstrate a primary abnormality in the eosinophil lineage. OBJECTIVE:The aim of this study was to identify the cells driving the eosinophilia in FE. METHODS:Microarray analysis and real-time PCR were used to examine transcriptional differences in peripheral blood mononuclear cells (PBMC), and in purified cell subsets from affected and unaffected family members belonging to a single large kindred. Cytokine levels in serum and PBMC culture supernatants were assessed by suspension array multiplexed immunoassays. RESULTS:Whereas IL-5 mRNA expression was significantly increased in freshly isolated PBMC from affected family members, this was not accompanied by increased mRNA expression of other Th2 cytokines (IL-4 or IL-13). Serum levels of IL-5 and IL-5 receptor ?, but not IgE, were similarly increased in affected family members. Of note, IL-5 mRNA expression was significantly increased in purified CD3+ CD4+, CD14+, CD19+, and ILC2 cells from affected family members, as were IL-5 protein levels in supernatants from both stimulated PBMC and ILC2 cultures. CONCLUSIONS:These data are consistent with the hypothesis that the eosinophilia in FE is secondary to dysregulation of IL-5 production in PBMC (and their component subsets).
Project description:BACKGROUND: Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25(+)FOXP3(+) regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples. METHODOLOGY/PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3(+), TGF-? in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4(+)CD25(+)FOXP3(+) T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-? which was further confirmed by increase in TGF-? in culture supernatants (p<0.003). Furthermore, TGF-? in iTreg cells was associated with phosphorylation of STAT5A. TGF-? was seen in CD25(+) cells of the CD4(+) but not that of CD8(+) T cell lineage in leprosy patients. iTregs did not show intracellular IFN-? or IL-17 in lepromatous leprosy patients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that FOXP3(+) iTregs with TGF-? may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.
Project description:Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-?, interferon gamma (IFN-? and transforming growth factor beta (TGF-? were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection.
Project description:During chronic inflammation, immune cells, notably Th17 cells, infiltrate the inflammatory site and interact with local mesenchymal cells. Applied to rheumatoid arthritis (RA), the aim is to study the interactions between synoviocytes and peripheral blood mononuclear cells (PBMC) with a focus on the Th17 pathway and to identify a mechanism which leads to high IL-17 secretion with an interest on podoplanin.PBMC from healthy donors and RA patients were co-cultured with RA synoviocytes during 48 h, in the presence or not of phytohemagglutinin. An antibody against podoplanin was used in co-culture. Cytokine production (IL-6, IL-1? and IL-17) was measured by ELISA and cell staining (CD3, CD4, IL-17 and podoplanin) by flow cytometry.In control conditions, IL-6 and IL-1? production was increased in PBMC-synoviocyte co-culture compared to PBMC alone (p?=?0.02). No additional effect was observed with PBMC activation. Flow cytometry analysis showed no difference in the percentage of Th17 cells in activated PBMC alone or with synoviocytes (p?=?0.4), indicating that Th17 differentiation requires only T cell activation. Conversely, IL-17 production was highly increased in co-cultures with activated PBMC vs. activated PBMC alone (p?=?0.002). Transwell experiments confirm that cell-cell contact was critical for IL-17 secretion. The incubation of either PBMC or synoviocytes with an anti-podoplanin antibody decreased IL-17 secretion by 60 % (p?=?0.008).Interactions between resting PBMC and synoviocytes are sufficient to induce IL-6 and IL-1? production. Both PBMC activation and cell interactions are needed to induce a high IL-17 secretion. Podoplanin contributes at the level of both lymphocytes and synoviocytes.
Project description:Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. ?? T cells are an important innate source of IL-17. Both IL-17+ ?? T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.In this study, we investigated the crosstalk between ?? T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ ?? T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.We report here that IL-17+ ?? T cells but not naïve ?? T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of ?? T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of ?? T cells through IL-1? and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both ?? T cells and microglia, but did not require the expression of TLRs by ?? T cells. Similarly to cytokine-primed IL-17+ ?? T cells, IL-17+ ?? T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ ?? T cells required a direct cell-cell contact between T cells and neurons.Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of ?? T cells towards neurotoxic IL-17+ ?? T cells.
Project description:The neuropeptide substance P (SP) exhibits cytokine-like properties and exerts different effects in autoimmune inflammation. Various immune cells express SP and its neurokinin-1 receptor (NK1R) isoforms. A role for SP has been demonstrated in a number of autoimmune conditions, including multiple sclerosis (MS). In this work, we studied the role of SP and NK1R in human immune cells with a focus on their relationship with IL-12/IL-23 family cytokines and the associated IFN-?/IL-17.(1) To determine the role of SP mediated effects on induction of various inflammatory cytokines in peripheral blood mononuclear cells (PBMC); (2) to investigate the expression of SP and its receptor in T cells and the effects of stimulation with IL-12 and IL-23. Quantitative real-time PCR, flow cytometry, ELISA, promoter studies on PBMC and primary T cells from healthy volunteers, and Jurkat cell line. Treatment with SP significantly increased the expression of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA in human PBMC. Expression of NK1R and SP in T cells was upregulated by IL-23 but a trend was observed with IL-12. The IL-23 effect likely involves IL-17 production that additionally mediates IL-23 effects. Mutual interactions exist with SP enhancing the cytokines IL-23 and IL-12, and SP and NK1R expression being differentially but potentially synergistically regulated by these cytokines. These findings suggest a proinflammatory role for SP in autoimmune inflammation. We propose a model whereby immunocyte derived SP stimulates Th1 and Th17 autoreactive cells migrating to the central nervous system (CNS), enhances their crossing the blood brain barrier and perpetuates inflammation in the CNS by being released from damaged nerves and activating both resident glia and infiltrating immune cells. SP may be a therapeutic target in MS.
Project description:Human peripheral blood contains antigen-presenting cells (APC), including dendritic cells (DC) and monocytes, that may encounter microbes that have translocated from the intestine to the periphery in disease states like HIV-1 infection and inflammatory bowel disease. We investigated the response of DC and monocytes in peripheral blood mononuclear cells (PBMC) to a panel of representative commensal enteric bacteria, including Escherichia coli, Enterococcus sp., and Bacteroides fragilis. All three bacteria induced significant upregulation of the maturation and activation markers CD40 and CD83 on myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). However, only mDC produced cytokines, including interleukin-10 (IL-10), IL-12p40/70, and tumor necrosis factor alpha (TNF-?), in response to bacterial stimulation. Cytokine profiles in whole PBMC differed depending on the stimulating bacterial species: B. fragilis induced production of IL-23, IL-12p70, and IL-10, whereas E. coli and Enterococcus induced an IL-10-predominant response. mDC and monocyte depletion experiments indicated that these cell types differentially produced IL-10 and IL-23 in response to E. coli and B. fragilis. Bacteroides thetaiotaomicron did not induce levels of IL-23 similar to those of B. fragilis, suggesting that B. fragilis may have unique proinflammatory properties among Bacteroides species. The addition of recombinant human IL-10 to PBMC cultures stimulated with commensal bacteria abrogated the IL-23 response, whereas blocking IL-10 significantly enhanced IL-23 production, suggesting that IL-10 controls the levels of IL-23 produced. These results indicate that blood mDC and monocytes respond differentially to innate stimulation with whole commensal bacteria and that IL-10 may play a role in controlling the proinflammatory response to translocated microbes.
Project description:Active liver diseases are characterized by an infiltration of inflammatory immune cells, which interact locally with hepatocytes. Co-cultures between non- and -activated human peripheral blood mononuclear cells (PBMCs) and human hepatoma HepaRG cells were used to determine the role of these cell interactions in the inflammatory response. At the early stage, PBMC-HepaRG cell interactions increased mRNA expression and/or secretion of IL-6, IL-8, CCL-20 and MCP-1, in part through direct cell contact and the induction was higher in PHA-activated conditions. The pro-inflammatory cytokines IL-17 and/or TNFα contributed to the increase of IL-6 and IL-8 secretion. HepaRG cells modulated T cell polarization by increasing Th1 cell transcription factor expression and by reducing CD3+ CD4+ IL-17+ cell frequency when PBMCs were activated with PHA. At a later stage, the presence of HepaRG cells inhibited PHA-induced HLA-DR expression on PBMCs, and PBMC proliferation. In contrast, the presence of skin fibroblasts had no effect of PBMC proliferation induced by PHA. After a first pro-inflammatory phase, PBMC-HepaRG cell interactions may down-regulate the immune response. The PBMC-hepatocyte interactions can thus participate first to the initiation of hepatitis and later to the maintenance of immune tolerance in liver, possibly contributing to chronicity.