Production Of Th17-related Cytokines Research Articles

Discordant effects of Janus kinases inhibition ex-vivo on inflammatory responses in colonic compared to ileal mucosa.

Janus kinase (JAK) inhibitors are used for treating inflammatory bowel diseases (IBD). We aimed to identify molecular effects of JAK inhibition in human intestinal mucosa, considering IBD location and phenotype. Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), and non-IBD controls (NC) were assessed for phosphorylated signal transducers and activators of transcription (p-STAT) levels and Inflammatory genes expression panel in response to ex-vivo JAK inhibitor (tofacitinib). Cytokine production by lamina propria lymphocytes in response to tofacitinib was assessed. Human intestinal organoids were used to investigate JAK inhibitors' effects on iNOS expression. Explants were collected from 68 patients (UC=20; CD=20; NC=28). p-STAT1\3\5 inhibition rates varied, being higher in colonic compared to ileal explants. p-STAT1\3 inhibition rates negatively correlated with CRP levels. While significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including NOS2. JAK inhibition significantly decreased Th1\Th2\Th17-related cytokine production from lamina propria lymphocytes. Various JAK inhibitors reduced IFN-γ-induced increase in iNOS expression in organoids. Site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying personalized drug effects in patients with IBD.

Immunomodulation of Ethyl pyruvate on gestational diabetes mellitus in Mice; The impact on Th17/Treg balance

Gestational diabetes mellitus (GDM) is recognized as one of the most common diseases among pregnant women and inflammatory responses can be a major reason for its induction and development. T helper 17 (Th17)/regulatory T cells (Tregs) imbalance resulting in the increased levels of pro-inflammatory and decreased levels of anti-inflammatory cytokines has been showed as major mechanisms involved in the pathogenesis of GDM. There are various treatment options, but none of them are completely therapeutic. Ethyl pyruvate (EP) is a stable derivate of pyruvate that showed anti-oxidant and anti-inflammatory properties in an in-vivo and in-vitro models. To examine the therapeutic efficacy of EP in GDM, mice were mated and EP (100 mg/kg) was administered intraperitoneally to C57BL/6 mice. EP-treated mice exhibited improved symptoms of GDM by decreased blood glucose levels and body-weight and increased insulin levels and insulin sensitivity. Furthermore, EP could significantly attenuate the impairments to offspring, including birth size and birth weight. The inflammatory responses were also decreased by EP through regulating the production of Th17-related cytokines, such as interleukin (IL)− 17 and IL-21. The levels of other inflammatory cytokines were also inhibited, including IL-1β, IL-6, and tumor necrosis factor (TNF)-α. In addition, it was found that EP increased the population of Tregs and Treg-related cytokines, IL-10 and transforming Growth Factor-β TGF-β, in GDM mice. In conclusion, EP could modulate GDM in mice and might be a potential therapeutic strategy candidate for the treatment of patients with GDM.

Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-β) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

Dendritic cells drive profibrotic inflammation and aberrant T cell polarization in systemic sclerosis.

SSc is a devastating autoimmune disease characterized by fibrosis and obliterative vasculopathy affecting the skin and visceral organs. While the processes mediating excessive extracellular matrix deposition and fibroblast proliferation are clear, the exact link between autoimmunity and fibrosis remains elusive. Th17 cells have been proposed as critical drivers of profibrotic inflammation during SSc, but little is known about the immune components supporting their pathogenic role. Our aim was to determine cytokine responses of stimulated monocyte-derived dendritic cells (Mo-DCs) and to determine how they influence T-cell cytokine production in SSc. Dendritic cells (DCs) activate and shape T cell differentiation by producing polarizing cytokines. Hence, we investigated the cytokine responses of monocyte-derived DCs (Mo-DCs) from patients with limited cutaneous SSc (lcSSc), diffuse cutaneous SSc (dcSSc) and healthy controls (HCs) after stimulation with toll-like receptor (TLR) agonists. Also, using co-culture assays, we analysed T cell subpopulations after contact with autologous TLR-activated Mo-DCs. In general, we observed an increased production of Th17-related cytokines like IL-1β, IL-17F, IL-21 and IL-22 by SSc compared with HC Mo-DCs, with variations between lcSSc vs dcSSc and early- vs late-stage subgroups. Noticeably, we found a significant increment in IL-33 production by Mo-DCs in all SSc cases regardless of their clinical phenotype. Strikingly, T cells displayed Th2, Th17 and dual Th2-Th17 phenotypes after exposure to autologous TLR-stimulated Mo-DCs from SSc patients but not HCs. These changes were pronounced in individuals with early-stage dcSSc and less significant in the late-stage lcSSc subgroup. Our findings suggest that functional alterations of DCs promote immune mechanisms favouring the aberrant T cell polarization and profibrotic inflammation behind clinical SSc heterogeneity.

The Imbalance in Th17 and Treg Cells in Polycystic Ovarian Syndrome Patients with Autoimmune Thyroiditis

ABSTRACT The ratio of T helper (Th) 17 and T regulatory (Treg) cells in patients with polycystic ovary syndrome complicated with autoimmune thyroiditis (PCOS-AIT) remains unreported. The study aimed to determine the Th17/Treg cell paradigm in PCOS-AIT patients. In peripheral blood mononuclear cells from PCOS patients and controls, the percentages of Th17 and Treg cells were measured by flow cytometry, the mRNA levels of a Th17-related transcription factor (ROR-γt) and a Treg-specific transcription factor (Foxp3) were determined by qRT-PCR, and the levels of Th17-related cytokines and Treg-related cytokines were measured by ELISA. Additionally, to examine the effect of testosterone on the Th17/Treg cell balance in vitro, cultured PCOS-AIT CD4+ T cells were treated with 10 μM testosterone for 24 h, and the Th17/Treg cell proportions and expression of Th17/Treg cell-associated transcription factors and cytokines were analyzed by flow cytometry, qRT-PCR, and ELISA. The Th17 cell percentage, Th17/Treg cell ratio, and expression of Th17-related ROR-γt and IL-17 were significantly higher in peripheral blood mononuclear cells from PCOS-AIT patients than in those from controls. In CD4+ T cells derived from PCOS-AIT patients, testosterone significantly decreased the Th17 cell percentage, Th17/Treg ratio, mRNA level of ROR-γt, and production of Th17-related cytokines and increased the Treg cell percentage, mRNA level of Foxp3, and secretion of Treg-related cytokines. The Th17/Treg cell imbalance favoring proinflammatory Th17 cells is involved in the pathogenesis of PCOS-AIT. Targeting the Th17/Treg cell axis may have therapeutic potential in the treatment of PCOS–AIT.

Specific inhibition of IL-6 receptor attenuates inflammatory bone loss in experimental periodontitis.

Periodontal pathogenesis takes into consideration that disease results from a complex inflammatory immune response. Among the major cytokines related to periodontal damage, interleukin (IL)-6 enhances a cascade of tissue destruction. Tocilizumab (TCZ) is a humanized monoclonal anti-human IL-6 receptor that inhibits IL-6-mediated proinflammatory activity. This study aimed to elucidate whether TCZ inhibits the deleterious effect of ligature-induced periodontitis. Experimental ligature-induced periodontitis was treated with systemic administration of TCZ intraperitoneally in three different concentration dosages (2mg/kg, 4mg/kg, and 8mg/kg. Euthanasia occurred at 7 and 14 days after the initiation of the study. Local changes in the alveolar bone were measured by bone volume, the ratio of bone volume, and trabecular thickness using microcomputed tomography. Attachment loss and inflammatory infiltrate were evaluated by histology. Immune response was analyzed focusing on the Th17 pattern. TCZ inhibited alveolar bone resorption and attachment loss in 7 and 14 days for all dosage groups in comparison to controls (P < 0.05). Besides, TCZ induced lower expression of inflammatory infiltrate (P<0.05) and less production of Th17-related cytokines (P<0.05) and RANKL (P<0.05). The inhibition of IL-6-mediated proinflammatory activity by IL-6R blocking reduced alveolar bone resorption and attachment loss supported by the modulation of the Th17 periodontal response. Considering the inflammatory status, modulatory therapy may be a promising approach to periodontal disease.

2′-fucosyllactose inhibits imiquimod-induced psoriasis in mice by regulating Th17 cell response via the STAT3 signaling pathway

Psoriasis is a chronic immune-mediated inflammatory cutaneous disorder with Th17 cells and Th17-related cytokines playing an important role in its development. 2′-FL (2′-fucosyllactose), which makes up about 30% of all HMOs (human milk oligosaccharides) in blood type secretor positive maternal milk, plays an essential role in supporting aspects of immune development and regulation. To explore the immunomodulatory effect of 2′-FL in psoriasis, we employed the imiquimod (IMQ)-induced psoriasis-like mouse model. Our data showed that mice administered with 2′-FL exhibited attenuated skin damage and inflammation, characterized by significantly decreased erythema and thickness and reduced recruitment of pro-inflammatory cytokines, when compared to control mice. The alleviated skin inflammation in 2′-FL treated mice was associated with a reduced proportion of Th17 cells and decreased production of Th17-related cytokines. Furthermore, we have demonstrated that 2′-FL reduced the phosphorylation of STAT3 in the skin tissue from mice with IMQ stimulation, which could account for the decreasing recruitment of Th17 cells. In vitro studies showed that 2′-FL inhibited differentiation of Th17 cells, phosphorylation of STAT3, and RORγt mRNA levels in T cells under Th17 polarization. Our results indicate that 2′-FL ameliorates IMQ-induced psoriasis by inhibiting Th17 cell immune response and Th17-related cytokine secretion via modulation of the STAT3 signaling pathway.

A novel self-regulatory mechanism of Th17 cells controls autoimmune uveitis through interleukin-24

Abstract The Th17 response is critical in driving inflammation and is associated with many autoimmune diseases. However, clinical trials targeting the Th17 signature cytokine, IL-17A in some autoimmune diseases, including uveitis, have been disappointing. We investigated the role of IL-17A in determining the pathogenicity of uveitogenic Th17 cells. Unexpectedly, IL-17A deficiency in these cells did not reduce the severity of uveitis in recipient mice. We found that IL-17A deficient Th17 cells produced elevated amounts of other Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. RNA-seq analysis and the follow up in-vitro experiments revealed that IL-17A exerts a negative feedback on Th17 cells by inducing them to produce IL-24, which in turn suppresses the production of Th17-related cytokines in an autocrine fashion. In vivo results showed that IL-24 treatment of recipients ameliorated Th17-induced adoptive EAU, and conversely, silencing IL-24 expression in retina-specific Th17 cells increased their pathogenicity, supporting the relevance of this pathway in vivo. Mechanistic studies confirmed that IL-17A activates the NFkB signalling pathway in Th17 cells, whereas site mutagenesis at the NFkB binding sites In IL-24 promoter abrogates the IL-17A-induced IL-24 expression. In vitro experiments implicated SOCS 1 and 3 in the downstream effects of IL-24. Importantly, we also observed a similar inhibitory IL-17A/IL-24 circuit in polarized human Th17 cells. We conclude that IL-17A exerts a negative feedback on Th17 cells by inducing autocrine IL-24, which limits their expression of other Th17-lineage cytokines and dampens their pathogenicity.

Interleukin-35 inhibits alveolar bone resorption by modulating the Th17/Treg imbalance during periodontitis.

T lymphocytes play a central role during the pathogenesis of periodontitis, and the imbalance between the pathogenic T-helper type 17 (Th17) and protective T-regulatory (Treg) lymphocytes determines the tooth-supporting alveolar bone resorption. Interleukin (IL)-35 is a novel anti-inflammatory cytokine with therapeutic properties in diseases whose pathogenesis is associated with the Th17/Treg imbalance; however, its role during periodontitis has not been established yet. This study aimed to elucidate whether IL-35 inhibits the alveolar bone resorption during periodontitis by modulating the Th17/Treg imbalance. Mice with ligature-induced periodontitis were treated with locally or systemically administrated IL-35. As controls, periodontitis-affected mice without IL-35 treatment and non-ligated mice were used. Alveolar bone resorption was measured by micro-computed tomography and scanning electron microscopy. The Th17/Treg pattern of the immune response was analysed by qPCR, ELISA, and flow cytometry. IL-35 inhibited alveolar bone resorption in periodontitis mice. Besides, IL-35 induced less detection of Th17 lymphocytes and production of Th17-related cytokines, together with higher detection of Treg lymphocytes and production of Treg-related cytokines in periodontitis-affected tissues. IL-35 is beneficial in the regulation of periodontitis; particularly, IL-35 inhibited alveolar bone resorption and this inhibition was closely associated with modulation of the periodontal Th17/Treg imbalance.

Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells.

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.

The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT.

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

CD30L/CD30 protects against psoriasiform skin inflammation by suppressing Th17-related cytokine production by Vγ4+ γδ T cells

Psoriasis is a common, autoimmune, chronic inflammatory skin disease. It has been demonstrated that cutaneous T17 cells play an important pro-inflammatory role in the pathogenesis of psoriasis, through the production of various Th17-related cytokines. Our previous studies have demonstrated that CD30L/CD30 signal plays a pivotal role in the differentiation of CD4+ Th17 cells and Vγ6+γδ T17 cells in the gut-associated lymphoid tissues of mouse. However, its effect on the pathogenesis of psoriasis is unknown. Here, we fully prove that CD30L/CD30 signaling plays a novel protective role in the development of psoriasis in mice, through selective inhibition of CCR6 expression and Th17-related cytokine synthesis in the Vγ4+γδ T17 cell subset. Meanwhile, treatment with agonistic anti-CD30 mAb had a significant therapeutic effect on our psoriasis mouse model. Therefore, the CD30L/CD30 signaling pathway is an ideal target for antibody therapy, which may become a new approach for the immunobiological treatment of psoriasis.

Pharmacological inhibitory profile of TAK-828F, a potent and selective orally available RORγt inverse agonist

Retinoic acid-related orphan receptor γt (RORγt) is a key master regulator of the differentiation and activation of IL-17 producing CD4+ Th17, CD8+ Tc17 and IL-17/IFN-γ co-producing cells (Th1/17 cells). These cells play critical roles in the pathogenesis of autoimmune diseases such as inflammatory bowel disease and multiple sclerosis. Thus, RORγt is an attractive target for the treatment of these diseases. We discovered TAK-828F, an orally available potent and selective RORγt inverse agonist. The inhibitory effect on the activation and differentiation of Th17 cells by TAK-828F was evaluated in mouse and human primary cells. TAK-828F inhibited IL-17 production from mouse splenocytes and human peripheral blood mononuclear cells dose-dependently at concentrations of 0.01–10 μM without affecting the production of IFN-γ. Additionally, TAK-828F strongly inhibited Th17, Tc17 and Th1/17 cells’ differentiation from naive T cells and memory CD4+ T cells at 100 nM without affecting Th1 cells’ differentiation. In addition, TAK-828F improved Th17/Treg cells’ population ratio by inhibiting Th17 cells’ differentiation and up-regulating Treg cells. Furthermore, TAK-828F, at 100 nM, reduced the production of Th17-related cytokines (IL-17, IL-17F and IL-22) without affecting IFN-γ production in whole blood. These results demonstrate that TAK-828F has the potent and selective inhibitory activity against RORγt both in mouse and human cells. Additionally, oral administration of TAK-828F showed promising efficacy in naive T cell transfer mouse colitis model. TAK-828F may provide a novel therapeutic option to treat immune diseases by inhibiting Th17 and Th1/17 cells’ differentiation and improving imbalance between Th17 and Treg cells.

Vitamin D modulates different IL-17-secreting T cell subsets in multiple sclerosis patients

Vitamin D deficiency is an environmental risk factor for MS, a Th17 cell-mediated autoimmune disease that results in demyelination in the CNS. Therefore, we aimed to evaluate the ability of in vitro 1,25(OH)2D in modulating different Th17 cell subsets in MS patients in remission phase. In the present study, the production of Th17-related cytokines (IL-1β, IL-6, IL-17, IL-22), as well as GM-CSF, was significantly higher in cell cultures from MS patients than in healthy subjects (HS). The 1,25(OH)2D reduced all pro-inflammatory cytokines essayed, mainly those released from HS cell cultures. The proportion of both IL-17+IFN-γ+ (CD4+ and CD8+) T cells and IL-17+IFN-γ−CD8+ T cells was positively related with neurological disorders, determined by EDSS score. The addition of 1,25(OH)2D reduced not only these pathogenic T cell subsets but elevated the percentage of IL-10-secreting conventional (FoxP3+CD25+CD127−CD4+) and non-conventional (IL-17+) regulatory-like T cells. Taken together, the results indicate that the active form of vitamin D should benefit MS patients by attenuating the percentage of pathogenic T cells. This effect could be direct and/or indirect, by enhancing classical and non-classical regulatory T cells.

Bone loss in HLA-DRβ1-bearing humanized mice following oral challenge with Porphyromonas gingivalis

Abstract Periodontal disease (PD) is a chronic pro-inflammatory response to oral pathogens such as Porphyromonas gingivalis which culminates in alveolar bone loss and loss of dental structure and function. Like rheumatoid arthritis (RA), periodontal disease is influenced strongly by lifestyle factors such as tobacco use, but also greatly influenced by genetic components, such as the “shared epitope,” which refers to a conserved linear sequence of amino acids in the DRβ1 chain of the HLA-DRα/β heterodimer between amino acids 67 and 74. Aggressive PD (the most destructive form of PD) is also now thought to be associated with specific HLA haplotypes including DRβ1. Using IL-17Frfp and Foxp3gfpreporters engineered into I-A° C57BL/6 mice expressing chimeric mouse/human HLA-DRβ1 (B6.DR1 mice), we find that gingival brushing with P gingivalis results in a rapid Th17 response in the peripheral blood and cervical lymph nodes, which decreases shortly after brushing. Evidence of a chronic infection can be detected in these nodes three months after oral inoculation. In addition to Th17 responses, we also find a concomitant increase in Th17-related cytokine production that parallels the Th17 activity. Furthermore, we measured a brisk production of antibodies directed against P. gingivalis and also against cyclic citrullinated peptides (these ACPAs are widely used as the principle early diagnostic for RA) as well as a loss in trabecular bone that can be detected in peri-articular bone structures, e.g. distal to the alveolar bone structures normally measured in PD models. Our findings suggest that periodontal disease can have far-reaching effects in bone morphology which may be driven by Th17/cytokine/ACPA activity even in the absence of other pathologies.

‘Efferocytosis of infected apoptotic cells inhibits Th17 differentiation via PGE2-EP4 signaling’

Abstract Clearance of infected apoptotic cells (IAC) by dendritic cells (DC) triggers the production of Th17-related cytokines. Moreover, we have found that prostaglandin E2 (PGE2) is also produced during efferocytosis containing microbial products, but the effect of this prostanoid on Th17-differentiation is still unknown. The purpose of this study was to evaluate the role of PGE2, produced during recognition of IAC by DC, on Th17 differentiation. As source of IAC, Raw 264-7 cells were cultured with Escherichia coli during 2h for phagocytosis and apoptosis was induced by UV irradiation. BMDC were co-culture with IAC during 18h, and the supernatant from this co-culture was used to differentiate naïve CD4+ T cells for 4d, plus anti-CD3/anti-CD28. Phagocytosis of IAC by DC promotes the production of high levels of PGE2 (15±0.7 ng/ml), IL-6 (26±0.4 ng/ml), TGF-β (3.5±0.09 ng/ml) and IL-1β (5.4±0.1 ng/ml), when compared to phagocytosis of uninfected-AC. The treatment with COX inhibitor significantly decreased PGE2 level and promotes Th17 cell differentiation at least 4 times higher (16%) than untreated co-cultures (4%). Moreover, adding back exogenous PGE2 decreased drastically the percentage of Th17 cells (2%). Moreover, the treatment of naive T cells with antagonists of PGE2 receptor (EP1/EP2/EP4) suggests that the suppressor effect of this prostanoid on Th17 cell differentiation is mainly mediated by EP4/cAMP/PKA activation pathway. Taken all together, our results show that PGE2 is able to suppress Th17 differentiation in vitro through EP4 signaling. Further studies are needed to clarify how PGE2 can modulate the Th17 differentiation using in vivo model. Financial Support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7.

The IL-17-independent role of IL-23 in severe asthma (HYP7P.300)

Abstract Asthma is a common respiratory disease affecting approximately 300 million people worldwide with no current preventions or cures. Th17 cells are known to be influential in asthma pathogenesis, especially in asthmatics with severe disease that fail to respond to glucocorticoid therapy. Interleukin-23 is critical for the maintenance and full acquisition of Th17 cells, but very little is known about its direct effects outside of IL-17 signaling. This study seeks to investigate novel mechanisms by which IL-23 regulates allergic airway disease. To do this, BALB/c SCID and Rag2-/-γc-/- mice were treated with adenoviral IL-23 or control and airway disease was assessed 3 days later. Preliminary data show that exogenous IL-23 induced airway neutrophilia and pulmonary expression of genes related to asthma pathogenesis and Th17-related cytokines and chemokines, while it increased airway resistance in response to methacholine and decreased lung compliance in BALB/c SCID mice. Further, IL-23 induced airspace inflammation and production of Th17-related cytokines and chemokines, while not impacting lung function in Rag2-/-γc-/- mice. This work suggests a novel role for IL-23 in airway disease as IL-23 itself could induce inflammation, which was predominately neutrophilic, in mice regardless of immune status. More significantly, IL-23 was also shown to modulate pulmonary expression of extracellular matrix-related proteins in these mice, which is currently under investigation.

Innate immunity in myasthenia gravis thymus: Pathogenic effects of Toll-like receptor 4 signaling on autoimmunity

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4+ thymic epithelial cells (TECs) and CCR4+ dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4+ Th1/Th17 AChR-specific T cells, naïve CD4+CD25+ Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.

Alteration of Cytokine Production during Visceral Larva Migrans by Toxascaris leonina in Mice

To determine alteration of immune responses during visceral larva migrans (VLM) caused by Toxascaris leonina at several time points, we experimentally infected mice with embryonated eggs of T. leonina and measured T-helper (Th) cell-related serial cytokine production after infection. At day 5 post infection (PI), most larvae were detected from the lungs, spleen, intestine, and muscle. Expression of thymic stromal lymphopoietin (TSLP) and CCL11 (eotaxin) showed a significant increase in most infected organs, except the intestine. However, expression of the CXCL1 (Gro-α) gene was most highly enhanced in the intestine at day 14 PI. Th1-related cytokine secretion of splenocytes showed increases at day 28 PI, and the level showed a decrease at day 42 PI. Th2-related cytokine secretion of splenocytes also showed an increase after infection; in particular, IL-5 level showed a significant increase at day 14 PI, and the level showed a decrease at day 28 PI. However, levels of Th17-related cytokines, IL-6 and IL-17A, showed gradual increases until day 42 PI. In conclusion, Th1, Th2, and Th17-related cytokine production might be important in immune responses against T. leonina VLM in experimental mice.

Low sensitivity to glucocorticoid inhibition of in vitro Th17-related cytokine production in multiple sclerosis patients is related to elevated plasma lipopolysaccharide levels

Exogenous glucocorticoid plays an important role in controlling clinical relapses of multiple sclerosis (MS), but the response to this treatment differs among patients. In this study, T-cell proliferation and IL-17 production were less sensitive to hydrocortisone (HC) inhibition in MS patients than healthy individuals, mainly in CD8(+) compartment. Furthermore, in vitro IL-17 production was positively related with neurological disability and its release was proportional to IL-23 and IL-6 productions by LPS-activated monocytes. Interestingly, elevated LPS levels were quantified in the plasma of MS patients, and their levels were directly related to in vivo IL-6 production. Finally, HC-resistance in reducing IL-17 production by polyclonally-activated CD8(+) T cells was particularly observed among MS patients with higher in vivo LPS levels. In summary, the results indicate that T-cells derived from MS patients show an enhanced Th17-like phenotype that is directly associated with neurological disability, resistance to glucocorticoid inhibition and elevated bacterial translocation.