Mice genetically inactivated in interleukin‐17A receptor are defective in long‐term control of Mycobacterium tuberculosis infection

Interleukin-17A (IL-17A), a pro-inflammatory cytokine acting on neutrophil recruitment, is known to play an important role during Mycobacterium tuberculosis infection, but the role of IL-17A receptor signalling in immune defence against this intracellular pathogen remains poorly documented. Here we have analysed this signalling using C57BL/6 mice genetically inactivated in the IL-17 receptor A subunit (IL-17RA^−/−). Although early after infection bacterial growth was controlled to the same extent as in wild-type mice, IL-17RA^−/− mice were defective in exerting long-term control of M. tuberculosis infection, as demonstrated by a progressively increasing pulmonary bacterial burden and shortened survival time. Compared with infected wild-type mice, IL-17RA^−/− mice showed impaired recruitment of neutrophils to the lungs at the early but not the late stage of infection. Pulmonary tumour necrosis factor-α, IL-6 and particularly IL-10 levels were decreased in the absence of IL-17RA signalling, whereas IL-1β was increased. CD4⁺-mediated and γδ-mediated IL-17A production was dramatically increased in IL-17RA^−/− mice (confirming part of their phenotype), whereas production of interferon-γ and expression of the bactericidal enzyme inducible nitric oxide synthase were not affected. Collectively, our data suggest that early but not late neutrophil recruitment is essential for IL-17A-mediated long-term control of M. tuberculosis infection and that a functional interferon-γ response is not sufficient to control M. tuberculosis growth when the IL-17RA pathway is deficient. As treatment of auto-immune diseases with anti-IL-17A antibodies is actually being tested in clinical studies, our data suggest that caution should be taken with respect to possible reactivation of tuberculosis.     

Endogenous interleukin (IL)‐17A promotes pristane‐induced systemic autoimmunity and lupus nephritis induced by pristane

Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A^−/− mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal injury were measured at 7 months. IL-17A production increased significantly 6 days after pristane injection, with innate immune cells, neutrophils (Ly6G⁺) and macrophages (F4/80⁺) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-γ and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4⁺ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A^−/− mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A.     

CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production

It is becoming increasingly clear that the T-cell-mediated immune response is important in many diseases. In this study, we used concanavalin A (Con A) -induced hepatitis to investigate the role of CD49a in the molecular and cellular mechanism of the T-cell-mediated immune response. We found that CD49a^−/− mice had significantly reduced levels of serum alanine aminotransferase and were protected from Con A-induced hepatitis. CD49a deficiency led to decreased production of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) after Con A injection. Furthermore, we found that hepatic CD4⁺ T cells and invariant natural killer T cells up-regulated CD49a expression, along with enhanced activation after Con A injection, leading to production of inflammatory cytokines by these T cells. Blockade of CD49a in vivo ameliorated Con A-induced hepatitis with reduced production of IFN-γ and IL-17A. Hence, CD49a promoted Con A-induced hepatitis through enhancing inflammatory cytokine production (IFN-γ and IL-17A) by CD4⁺ T and invariant natural killer T cells. The protective effect of CD49a blockade antibody suggested a new target therapeutic molecule for intervention of T-cell-mediated liver injury.     

Different immunosuppressive mechanisms in multi‐drug‐resistant tuberculosis and non‐tuberculous mycobacteria patients

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4⁺CD25^+- forkhead box protein 3 (FoxP3)⁺ and CD4⁺CD25⁺CD127⁻ regulatory T (T_reg) cells when compared to NR-TB. Increased numbers of T_reg cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-β was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4⁺/interferon (IFN)-γ⁺ T cells and enhanced CD4⁺CD25⁺FoxP3⁺, CD4⁺CD25⁺CD127⁻ and CD4⁺CD25⁺IL-10⁺ T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4⁺CD25⁺FoxP3⁺ frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4⁺CD25⁺ T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T_reg/T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.     

Interleukin‐17A deficiency ameliorates streptozotocin‐induced diabetes

Interleukin-17 (IL-17) is a cytokine with critical functions in multiple autoimmune diseases. However, its roles in type I diabetes and the underlying mechanisms remain to be fully elucidated. In the current study, we investigated the impact of IL-17 deficiency on streptozotocin (STZ) -induced diabetes. Il-17^−/− mice exhibited attenuated hyperglycaemia and insulitis after STZ treatment compared with control mice. The Il-17^−/− mice had fewer CD8⁺ cells infiltrating the pancreas than wild-type controls after STZ injection. Wild-type mice showed increased percentage and number of splenic CD8⁺ cells and decreased Gr1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSC) after STZ treatment, but Il-17^−/− mice maintained the percentages and numbers of splenic CD8⁺ cells and MDSC, suggesting that IL-17 is implicated in STZ-induced cellular immune responses in the spleen. We further purified the MDSC from spleens of STZ-treated mice. Il-17^−/− MDSC showed increased ability to suppress CD8⁺ cell proliferation in vitro compared with wild-type MDSC. Transfer of MDSC to diabetic mice showed that MDSC from Il-17^−/− mice could ameliorate hyperglycaemia. Moreover, recipients with MDSC from Il-17^−/− mice had a decreased percentage of CD8⁺ cell in the spleen compared with recipients with MDSC from wild-type mice. These data suggest that IL-17 is required in splenic MDSC function after STZ delivery. In summary, our study has revealed a pathogenic role of IL-17 in an STZ-induced diabetes model with important implications for our understanding of IL-17 function in autoimmune diseases.     

Specific overexpression of tumour necrosis factor‐α‐induced protein (TNFAIP)9 in CD14+CD16− monocytes in patients with rheumatoid arthritis: comparative analysis with TNFAIP3

The tumour necrosis factor (TNF)-α-induced proteins (TNFAIP)9 and TNFAIP3 play an important pathogenic role in murine arthritis. To clarify their pathophysiological roles in patients with rheumatoid arthritis (RA), we examined their expression and localization in peripheral blood mononuclear cells (PBMC). TNFAIP9 and TNFAIP3 mRNA expression was determined in PBMC of RA patients and healthy subjects (control). Flow cytometry was used to analyse the main TNFAIP9- and TNFAIP3-expressing cell populations. TNFAIP9 and TNFAIP3 mRNA expression levels were examined in vitro on CD14⁺ cells stimulated with TNF-α and lipopolysaccharide (LPS). The expression levels of TNFAIP9 and TNFAIP3 mRNA were also measured before and 12 weeks after treatment with tocilizumab and abatacept. TNFAIP9 expression was significantly higher, while TNFAIP3 expression was lower in PBMC of RA (n = 36) than the control (n = 24) (each P < 0·05). TNFAIP9 was expressed on CD14⁺ cells, especially in human leucocyte antigen D-related (HLA-DR)⁺CD14^brightCD16⁻cells, while TNFAIP3 was expressed mainly on CD3⁺ T cells. TNF-α and LPS induced TNFAIP9 and TNFAIP3 in human CD14⁺monocytes in vitro. Treatment with tocilizumab (n = 13), but not abatacept (n = 11), significantly reduced TNFAIP9 mRNA expression in PBMC, which was associated with reduction in the number of circulating CD14^bright monocytes. The expression of TNFAIP9 in CD14⁺ cells was specifically elevated in patients with RA, regulated by TNF-α and LPS, and suppressed by tocilizumab, while TNFAIP3 in PBMC showed different localization and induction patterns.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

Higher levels of interleukin IL‐17 and antigen‐specific IL‐17 responses in pulmonary sarcoidosis patients with Löfgren's syndrome

Sarcoidosis is a granulomatous disorder of unknown aetiology. The presence of Mycobacterium tuberculosis catalase-peroxidase (mKatG) in sarcoidosis tissue has been reported. T helper type 1 (Th1) responses against mKatG have previously been observed. However, little is known about interleukin (IL)-17 and Th17 responses in sarcoidosis. Here, we investigated the levels of IL-17 and frequencies of IL-17-producing cells responding to mKatG in sarcoidosis patients with different prognosis. Peripheral blood and bronchoalveolar lavage (BAL) cells were obtained from sarcoidosis patients with or without Löfgren''s syndrome (often associated with spontaneous recovery), and also stratified according to human leucocyte antigen (HLA) type. Cells producing IL-17 and interferon (IFN)-γ after stimulation with mKatG were enumerated by enzyme-linked immunospot (ELISPOT). The level of IL-17 in the BAL fluid of sarcoidosis patients and healthy controls was measured by quantitative immuno-polymerase chain reaction (qIPCR). We also performed flow cytometry and immunohistochemistry for further characterization of IL-17 expression. Patients with Löfgren''s syndrome had a higher frequency of IL-17-producing cells responding to mKatG in BAL fluid compared to patients without Löfgren''s syndrome (P < 0·05). The HLA-DR3⁺ sarcoidosis patients with Löfgren''s syndrome (known to have a particularly good prognosis) also had a clearly higher level of IL-17 in BAL fluid compared to healthy controls and sarcoidosis patients without Löfgren''s syndrome (P < 0·01) and (P < 0·05), respectively. No such difference between patient groups was observed with regard to IFN-γ and not with regard to either cytokine in peripheral blood. These findings suggest that IL-17-producing cells may be a useful biomarker for the prognosis of sarcoidosis and play a role in the spontaneous recovery typical of patients with Löfgren''s syndrome.     

Detection and significance of TregFoxP3+ and Th17 cells in peripheral blood of non-small cell lung cancer patients

Introduction

The aim of this study was to explore the relationships between TregFoxP3⁺ cells and Th17 cells and occurrence of lung cancer.

Material and methods

The proportions of TregFoxP3⁺ and Th17 cells, the expression of FoxP3 and RORγt mRNA, and the levels of related cell factors such as transforming growth factor-β (TGF-β), interleukin IL-17 (IL-17) and IL-23 were determined respectively by flow cytometry analysis, real-time-polymerase chain reaction (PCR), and ELISA in peripheral blood of 18 healthy people and 26 patients with non-small cell lung cancer (NSCLC).

Results

The levels of TregFoxP3⁺ and Th17, expression of FoxP3 and RORγt mRNA, and ratios of TregFoxP3⁺/Th17 and FoxP3/RORγt in peripheral blood with NSCLC were higher than those in healthy controls (p < 0.05). The proportion of Th17 cells from NSCLC patients was positively correlated with that of TregFoxP3⁺ (r = 0.81, p < 0.05). The receiver-operating characteristic (ROC) curve demonstrates that the increased level of TregFoxP3⁺/Th17 in the peripheral blood may be a useful indicator in early diagnosis of non-small cell lung carcinoma. The TregFoxP3⁺/Th17 and FoxP3/RORγt levels for patients in stage IV were higher than those of patients in stages I, II, and III (p < 0.05). The levels of TGF-β, IL-17, and IL-23 were higher in NSCLC patients than those in healthy controls.

Conclusions

The results suggest that ratios of Treg/Th17 correlate with the stage of NSCLC.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

A sharp decrease of Th17, CXCR3+-Th17, and Th17.1 in peripheral blood is associated with an early anti-IL-17-mediated clinical remission in psoriasis

Psoriasis—an immune-mediated skin disease—implicates in its pathophysiology by circulating pro-inflammatory cell populations, cytokines, and their interactions with the epidermis. The direct effect of approved anti-interleukin- (IL-)17A and anti-IL-17R biologic therapy on immunophenotyping of peripheral blood mononuclear lymphocytes’ (PBMCs) relative sub-population frequencies in psoriasis patients has not yet been described. Using multiparameter flow cytometry we examined T-cell subpopulations characterized by CCR6, CCR4, and CXCR3 chemokine receptor surface expression at baseline and after initiation of biologic therapy in PBMCs collected from 30 psoriasis patients. Increased CD3⁺CD4⁺CXCR3⁺, CD3⁺CD4⁺CCR6⁺CCR4⁺CXCR3⁺(CXCR3⁺-Th17), and CD3⁺CD4⁺CCR6⁺CCR4^-CXCR3⁺(Th17.1) cell populations were observed in patients with psoriasis in comparison to healthy individuals (n = 10). IL-17 therapeutic blockade decreased CD3⁺CD4⁺CCR6⁺, CD3⁺CD4⁺CXCR3⁺, CD3⁺CD4⁺CCR6^-CXCR3⁺(Th1), CD3⁺CD4⁺CCR6⁺CCR4⁺(Th17), CD3⁺CD4⁺CCR6⁺CCR4⁺CXCR3⁺(CXCR3⁺-Th17), and CD3⁺CD4⁺CCR6⁺CCR4^-CXCR3⁺(Th17.1) cell populations in responding psoriasis patients. Moreover, CD3⁺CD4^-CCR6⁺, CD3⁺CD4^-CXCR3⁺, CD3⁺CD4^-CCR6⁺CCR4⁺(Tc17), and CD3⁺CD4^-CCR6^-CXCR3⁺(Tc1) percentages were also inhibited. Modulation of the same cell sub-populations was also assessed in patients treated with methotrexate (n = 4), apremilast (n = 4), and anti-IL-23 biologic treatment (n = 4). In our study, the levels and functional capacity of peripheral pro-inflammatory Th1, Th17, and additional CCR6⁺T cell sub-gated populations from psoriasis patients that were treated with anti-IL-17 or anti-IL-17R targeted biologic therapy were explored for the first time. Our data clearly demonstrate that early anti-IL-17 mediated clinical remission is accompanied by a significant decrease of Th1, Th17, CXCR3⁺-Th17, and Th17.1 cells.     

Exogenous interleukin‐33 targets myeloid‐derived suppressor cells and generates periphery‐induced Foxp3+ regulatory T cells in skin‐transplanted mice

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4⁺ T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3⁺ regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4⁺ T-cell expansion and conversion of CD4⁺ Foxp3⁻ T cells into CD4⁺ Foxp3⁺ Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3⁺ Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.     

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

Rabbit anti-thymocyte globulins (rATG) induce CD4⁺CD25⁺forkhead box P3 (FoxP3⁺) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4⁺CD25⁺FoxP3⁺CD127^−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25^neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25⁺ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4⁺CD25⁺FoxP3⁺CD127^−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3⁺T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3⁺ T cells. The rATG tacrolimus-induced CD25⁺ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4⁺CD25⁺FoxP3⁺CD127^−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25⁺ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25⁺ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4⁺CD25⁺ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.     

A shift towards a T cell cytokine deficiency along with an anti-inflammatory/regulatory microenvironment may enable the synthesis of anti-FVIII inhibitors in haemophilia A patients

Despite the clinical relevance of anti-factor VIII (FVIII) antibodies (anti-FVIII inhibitors) impairing haemostatic activity of haemophilia A (HA) patients, the immunological mechanisms underlying their production are unknown. Aiming to understand more clearly the immune response in patients with [HAα-FVIII(+)] and without [HAα-FVIII(−)] anti-FVIII inhibitors, we have characterized the cytokine pattern of peripheral blood leucocytes, using an in vitro stimulation of whole blood samples with plasma-derived (pFVIII) or recombinant FVIII (rFVIII). The results highlighted decreased levels of tumour necrosis factor (TNF)-α⁺ neutrophils with higher interleukin (IL)-5/TNF-α ratio in HAα-FVIII(+). All HA samples displayed decreased levels of IL-10⁺ monocytes when compared to the blood donor (BD) samples. HAα-FVIII(+) showed lower levels of TNF-α⁺ monocytes and increased IL-10/TNF-α ratio. Analysis of adaptive immunity revealed increased levels of interferon (IFN)-γ⁺, TNF-α⁺ and IL-4⁺ T-cells, from both CD4⁺ and CD8⁺ T cells, in HAα-FVIII(−) when compared to BD. Moreover, increased frequency of IL-10⁺ B cells and higher levels of α-FVIII IgG1 were observed in HAα-FVIII(−). Basal levels of cytokine⁺ B-cells, similar to BD, and higher levels of α-FVIII IgG4 are major features in HAα-FVIII(+). The global cytokine profile demonstrated a major anti-inflammatory/regulatory pattern in HAα-FVIII(+), confirmed by the in vitro stimuli with pFVIII or rFVIII. The polarized anti-inflammatory/regulatory immune response in HAα-FVIII(+) and the mixed pattern with a bias towards an inflammatory cytokine profile, modulated by IL-4 in HAα-FVIII(−), may be the key element to drive the development of distinct subclasses of anti-FVIII antibodies. These finding have implications for the design of safe and effective therapeutic protocols to control inhibitors synthesis in HA patients.     

Engagement of Toll-like receptor 2 enhances interleukin (IL)-17+ autoreactive T cell responses via p38 mitogen-activated protein kinase signalling in dendritic cells

Functional analysis of single Toll-like receptors (TLRs) in vivo is necessary to understand how they shape the ocular inflammation involved in uveitis. In this study we explored the role and mechanisms of TLR-2 agonists on the autoreactive T helper type 17 (Th17) response in experimental autoimmune uveitis (EAU). Treatment by peptidoglycan (PGN), a specific TLR-2 agonist, remarkably increased mRNA levels of Th17-lineage genes interleukin (IL)-17A, IL-21 and RAR-related orphan receptor (ROR)γt and promoted antigen-specific Th17 response in EAU mice. A mixture of PGN and interphotoreceptor retinoid-binding protein peptide (IRBP_161–180) could effectively induce EAU in the absence of complete Freund''s adjuvant (CFA). PGN treatment also enhanced the pathogenic activities of activated antigen-specific Th17 cells in vivo. PGN significantly increased the production of IL-1β, IL-6 and IL-23 of dendritic cells (DCs) and enhanced their ability to promote IL-17⁺ uveitogenic T cells. Enhanced immunostimulatory activities of PGN-DCs depend upon p38 activation. Inhibition of p38 mitogen-activated protein kinase (MAPK) activity dramatically decreased IL-17 gene expression and antigen-specific Th17 responses stimulated by PGN-DCs. Our findings suggest that PGN treatment dramatically promotes the IL-17⁺ uveitogenic T cell responses via enhancing the immunostimulatory activities of DCs. This effect may be mediated, at least in part, by activation of the p38 signalling pathway in DCs.     

Interleukin-10- and Transforming Growth Factor β-Independent Regulation of CD8+ T Cells Expressing Type 1 and Type 2 Cytokines in Human Lymphatic Filariasis

Lymphatic filariasis is known to be associated with diminished CD4⁺ Th1 and elevated CD4⁺ Th2 responses to parasite-specific antigens. The roles of cytokine-expressing CD8⁺ T cells in immune responses to filarial infections are not well defined. To study the roles of CD8⁺ T cells expressing type 1, type 2, and type 17 cytokines in filarial infections, we examined the frequencies of these cells in clinically asymptomatic, patently infected (INF) individuals, directly ex vivo and in response to parasite or nonparasite antigens; these frequencies were compared with the results for individuals with filarial lymphedema (i.e., clinical pathology [CP]) and those without active infection or pathology (i.e., endemic normal [EN]). INF individuals exhibited significant decreases in the frequencies of CD8⁺ T cells expressing tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin-22 (IL-22) at baseline and/or in response to filarial antigens, compared with CP and EN individuals. In contrast, the same individuals exhibited significant increases in the frequencies of CD8⁺ T cells expressing IL-4, IL-5, IL-9, IL-13, and IL-21, compared with CP and/or EN individuals. Curative treatment resulted in significantly increased frequencies of CD8⁺ T cells expressing IL-2 and significantly decreased frequencies of CD8⁺ T cells expressing type 2 cytokines. Finally, the regulation of these responses appears to be independent of IL-10 and transforming growth factor β (TGF-β), since blockade of IL-10 or TGF-β signaling did not significantly alter the frequencies of type 1 or type 2 cytokine-expressing CD8⁺ T cells. Our findings suggest that alterations in the frequencies of cytokine-expressing CD8⁺ T cells are characteristic features of lymphatic filarial infections.     

Dietary gluten alters the balance of pro‐inflammatory and anti‐inflammatory cytokines in T cells of BALB/c mice

Several studies have documented that dietary modifications influence the development of type 1 diabetes. However, little is known about the interplay of dietary components and the penetration of diabetes incidence. In this study we tested if wheat gluten is able to induce differences in the cytokine pattern of Foxp3⁺ regulatory T cells, as well as Foxp3⁻ T cells, isolated from intestinal mucosal lymphoid tissue and non-mucosal lymphoid compartments in BALB/c mice. The gluten-containing standard diet markedly changed the cytokine expression within Foxp3⁻ T cells, in all lymphoid organs tested, towards a higher expression of pro-inflammatory interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-2. In Foxp3⁺ regulatory T cells, gluten ingestion resulted in a mucosal increase in IL-17 and IL-2 and an overall increase in IFN-γ and IL-4. The gluten-free diet induced an anti-inflammatory cytokine profile with higher proportion of transforming growth factor-β (TGF-β)⁺ Foxp3⁻ T cells in all tested lymphoid tissues and higher IL-10 expression within non-T cells in spleen, and a tendency towards a mucosal increase in TGF-β⁺ Foxp3⁺ regulatory T cells. Our data shows that the gluten-containing standard diet modifies the cytokine pattern of both Foxp3⁻ T cells and Foxp3⁺ regulatory T cells towards a more inflammatory cytokine profile. This immune profile may contribute to the higher type 1 diabetes incidence associated with gluten intake.     

Presence,function, and regulation of IL-17F-expressing human CD4+ T cells

The pro-inflammatory cytokine IL-17A has been implicated in the immunopathology of inflammatory arthritis. IL-17F bears 50% homology to IL-17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL-17F⁺ CD4⁺ T cells, and how IL-17F may contribute to inflammation. Upon culture of healthy donor CD4⁺ T cells with IL-1β, IL-23, anti-CD3, and anti-CD28 mAb, both IL-17A and IL-17F-expressing cells were detected. In comparison to IL-17A⁺IL-17F⁻ CD4⁺ T cells, IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cells contained lower proportions of IL-10-expressing and GM-CSF-expressing cells and higher proportions of IFN-γ-expressing cells. Titration of anti-CD28 mAb revealed that strong co-stimulation increased IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cell frequencies, whereas IL-17A⁺IL-17F⁻ CD4⁺ T cell frequencies decreased. This was partly mediated via an IL-2-dependent mechanism. Addition of IL-17A, IL-17F, and TNF-α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL-6 and IL-8, which was reduced to a larger extent by combined blockade of IL-17A and IL-17F than blockade of IL-17A alone. Our data indicate that IL-17A and IL-17F are differentially regulated upon T cell co-stimulation, and that dual blockade of IL-17A and IL-17F reduces inflammation more effectively than IL-17A blockade alone.