Increased IL-22 in cerebrospinal fluid of neuro-behçet's disease patients.

Remitting-Relapsing Multiple Sclerosis (RRMS) and Neuro-Behçet Disease (NBD) are two chronic neuro-inflammatory disorders leading to brain damage and disability in young adults. Herein, we investigated in these patients the cytokine response by beads-based multiplex assays during the early stages of these disorders. Cytokine investigations were carried out on treatment-naive patients suffering from RRMS and NBD recruited at the first episode of clinical relapse. Our findings demonstrate that Cerebrospinal Fluid (CSF) cells from NBD patients, but not RRMS, secrete significant high levels of IL-22 which is associated with elevated IL-22 mRNA expression. We also observed an increase in IL-22 levels in the definite NBD subgroup as compared to the probable NBD one, indicating a clear relationship between elevated IL-22 levels and diagnostic certainty. Interestingly, we found no correlation of IL-22 secretion between CSF and serum arguing about intrathecal release of IL-22 in the CNS of NBD patients. Moreover, we showed by correlogram analysis that this cytokine doesn't correlate with IL-17A, IL-17F and IL-21 suggesting that this cytokine is secreted by Th22 cells and not by Th17 cells in the CSF of NBD patients. Finally, we found elevated levels of IL-6 and a positive correlation between IL and 6 and IL-22 in the CSF of NBD. In conclusion, these results suggest that IL-6 contributes to the production of IL-22 by T cells leading to the exacerbation of inflammation and damage within the CNS of NBD patients.

Increased T-bet/GATA-3 and ROR-γt /Foxp3 Ratios in Cerebrospinal Fluid as Potential Criteria for Definite Neuro-Behçet's Disease.

When the central nervous system (CNS) is the primary affected site in an initial attack of Behçet's disease (BD), the differential diagnosis is particularly challenging. Some cases remain unclassified or qualified as probable neuro-Behçet's disease (NBD). Several cytokines are involved in the immunopathogenesis of this disease; however, studies establishing the differential cytokine pattern between probable and definite NBD are scarce. Twenty-eight parenchymal NBD patients, diagnosed according to the International Consensus Recommendation (ICR) criteria and classified into definite (D-NBD; n = 17) and probable (P-NBD; n = 11), were sampled at their first neurological symptoms, and compared with healthy control subjects (n = 20). Oligoclonal bands (OCB) of IgG were detected by isoelectric focusing on agarose, and immunoblotting of matched serum and cerebrospinal fluid (CSF) sample pairs. T cell cytokines (INF-γ, IL-4, IL-17, and IL-10) and transcription factors related to Th1, Th2, Th17, and T regulatory populations (respectively T-bet, GATA-3, ROR-γt, and Foxp3) were studied by quantitative RT-PCR in peripheral blood mononuclear cells (PBMCs) and CSF cells. Inflammatory cytokines such as IL-6, TNF-α, and IL-1ß were also analyzed. CSF OCB pattern 2 was present in only 1 out of 28 neuro-Behçet's patients who belonged to the P-NBD group. Two D-NBD patients had OCB in CSF showing pattern 4. In the D-NBD CSF samples, IL-17 and IL-10 expressions were significantly elevated compared to P-NBD. Moreover, D-NBD patients had increased levels of T-bet/GATA-3 and ROR-γt/Foxp3 ratios compared to P-NBD. Furthermore, a significant increase of CSF IL-6 in D-NBD, compared to P-NBD and the controls, was found. In addition to the increased IL-6 level, the data obtained suggest the existence in D-NBD patients of a significantly disrupted balance between Th17 effector and T regulatory cells, as reflected by the enhanced ROR-γt/Foxp3 ratio. This could be considered as an additional criterion for definite neuro-Behçet's disease.

Longitudinal and Comparative Analysis of Gut Microbiota of Tunisian Newborns According to Delivery Mode.

Microbiota colonization is a dynamic process that impacts the health status during an individual's lifetime. The composition of the gut microbiota of newborns is conditioned by multiple factors, including the delivery mode (DM). Nonetheless, the DM's influence remains uncertain and is still the subject of debate. In this context, the medical indication and the emergency of a cesarean delivery might have led to confounding conclusions regarding the composition and diversity of the neonatal microbiome. Herein, we used high-resolution shotgun sequencing to decipher the composition and dynamics of the gut microbiota composition of Tunisian newborns. Stool samples were collected from 5 elective cesarean section (ECS) and 5 vaginally delivered (VD) newborns at the following time points: Day 0, Day 15, and Day 30. The ECS and VD newborns showed the same level of bacterial richness and diversity. In addition, our data pointed to a shift in microbiota community composition during the first 2 weeks, regardless of the DM. Both ECS and VD showed a profile dominated by Proteobacteria, Actinobacteria, and Firmicutes. However, ECS showed an underrepresentation of Bacteroides and an enrichment of opportunistic pathogenic species of the ESKAPE group, starting from the second week. Besides revealing the intestinal microbiota of Tunisian newborns, this study provides novel insights into the microbiota perturbations caused by ECS.

B Cells Specific CpG Induces High IL-10 and IL-6 Expression In Vitro in Neuro-Behçet's Disease.

Remitting-RelapsingMultiple Sclerosis (RRMS) and Neuro-Behçet Disease (NBD) are two chronic neuroinflammatory disorders leading to neurological damage. Herein, we investigated in these patients the IL-10-producing cells during the early stages of these disorders. Cellular and molecular investigations were carried out on treatment naive patients suffering from RRMS and NBD recruited at the first episode of clinical relapse. Our findings demonstrate that CSF-B cells from NBD patients, but not RRMS, are the major source of intrathecal IL-10 as compared to T-CD4 cells. Moreover, we showed a lower expression of TGF-ß and IL35, in the CSF cells of NBD patients as compared to the control group. Specific in vitro CpG stimulation of peripheral blood B cells from NBD patients resulted in a concomitant early mRNA expression of IL6 and IL10 but was limited to IL10 for RRMS patients. Furthermore, mRNA expression of IL-6 and IL-10 receptors was assessed and intriguingly IL6ST receptor subunit was significantly lower in NBD CSF, but not RRMS while IL10RB was increased in both. Deciphering the role of increased IL-10-producing B cells and IL10RB despite relapsing disease as well as the discordant expression of IL6 and IL6ST may pave the way for a better understanding of the pathophysiology of these neuro-inflammatory disorders.

Leishmania Parasites Differently Regulate Antioxidant Genes in Macrophages Derived From Resistant and Susceptible Mice.

Macrophage-Leishmania interactions are central to parasite growth and disease outcome. Macrophages have developed various strategies to fight invaders, including oxidative burst. While some microorganisms seem to survive and even thrive in an oxidative environment, others are susceptible and get killed. To counter oxidative stress, macrophages switch the expressions of cytoprotective and detoxifying enzymes, which are downstream targets of the nuclear factor erythroid 2-related factor 2 (Nrf2), to enhance cell survival. We have explored the transcription of NRF2 and of its target genes and compared the effect of the parasite on their transcription in bone marrow-derived macrophages (BMdMs) from Leishmania-resistant and Leishmania-susceptible mice. While heme oxygenase 1 (HO-1) transcription is independent of the genetic background, the transcription of glutathione reductase (Gsr) and of cysteine/glutamate exchange transporter (Slc7a11), involved in glutathione accumulation, was differentially regulated in BMdMs from both mouse strains. We also show that, except for HO-1, known to favor the survival of the parasite, the transcription of the selected genes, including Gsr, CD36, and catalase (CAT), was actively repressed, if not at all time points at least at the later ones, by the parasite, especially in Balb/c BMdMs. Consistent with these results, we found that the silencing of NRF2 in this study increases the survival and multiplication of the parasite.

Differential Gene Expression Patterns in Blood and Cerebrospinal Fluid of Multiple Sclerosis and Neuro-Behçet Disease.

Inflammatory demyelinating disorders of the central nervous system are debilitating conditions of the young adult, here we focus on multiple sclerosis (MS) and neuro-Behçet disease (NBD). MS is an autoimmune disorder of the central nervous system. NBD, a neurological manifestation of an idiopathic chronic relapsing multisystem inflammatory disease, the behçet disease. The diagnosis of MS and NBD relies on clinical symptoms, magnetic resonance imaging and laboratory tests. At first onset, clinical and imaging similarities between the two disorders may occur, making differential diagnosis challenging and delaying appropriate management. Aiming to identify additional discriminating biomarker patterns, we measured and compared gene expression of a broad panel of selected genes in blood and cerebrospinal fluid (CSF) cells of patients suffering from NBD, MS and non inflammatory neurological disorders (NIND). To reach this aim, bivariate and multivariate analysis were applied. The Principal Analysis Component (PCA) highlighted distinct profiles between NBD, MS, and controls. Transcription factors foxp3 in the blood along with IL-4, IL-10, and IL-17 expressions were the parameters that are the main contributor to the segregation between MS and NBD clustering. Moreover, parameters related to cellular activation and inflammatory cytokines within the CSF clearly differentiate between the two inflammatory diseases and the controls. We proceeded to ROC analysis in order to identify the most distinctive parameters between both inflammatory neurological disorders. The latter analysis suggested that IL-17, CD73 in the blood as well as IL-1ß and IL-10 in the CSF were the most discriminating parameters between MS and NBD. We conclude that combined multi-dimensional analysis in blood and CSF suggests distinct mechanisms governing the pathophysiology of these two neuro-inflammatory disorders.

Discriminative expression of CD39 and CD73 in Cerebrospinal fluid of patients with Multiple Sclerosis and Neuro-Behçet's disease.

Treg-mediated immune suppression involves many molecular mechanisms including the cleavage of inflammatory extracellular ATP to adenosine by CD39 ectoenzyme. In the peripheral blood of Multiple Sclerosis (MS) patients, it has been suggested that CD39+ Treg cells have the potential to suppress pro-inflammatory IL-17 secreting cells. Herein, we studied cellular phenotype and mRNA expression of CD39 and CD73 ectoenzymes in the Cerebrospinal fluid (CSF) of MS patients and another neuro-inflammatory disease: the Neuro-behçet's disease (NBD). Using qRT-PCR, we assessed mRNA expression of CD39 and CD73 as well as anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNF-α, IL-1ß) cytokines in patients Peripheral blood mononuclear cells (PBMCs) and CSF of 28 relapsing-remitting multiple sclerosis (RRMS), 20 NBD and 22 controls with non inflammatory neurological disorders (NIND). The most substantial result in the CSF was the higher expression of CD39 in both RRMS and NBD patients compared to NIND. While, the expression of CD73 in CSF samples of NBD was low. In RRMS samples, we detected a significant positive correlation of both CD39 and CD73 with IL-10 expression. Moreover, results by flow cytometry revealed a high percentage of CD39 Treg cells in RRMS CSF. CD39 was preferentially expressed on B cells of NBD. Regarding inflammatory response, we showed a significant increase of IL-6 mRNA expression in NBD patients CSF while in RRMS this increase concerned TNF-α. These results bring evidence that CD39 correlates positively with an anti-inflammatory IL-10 response in RRMS. In contrast, no such association was observed in CSF of NBD patients and CD39 was preferentially expressed on B cells.

Cerebrospinal fluid IL-10 as an early stage discriminative marker between multiple sclerosis and neuro-Behçet disease.

Multiple Sclerosis (MS) and Neuro-Behçet's Disease (NBD) are two recurrent disorders affecting the central nervous system (CNS) by causing inflammation and irreversible damage. Inaugural clinical symptoms for both diseases might be very similar and definitive diagnosis could be delayed. The present study aimed to find out possible differences at early stages in the transcription factors/cytokines expression profiles in blood and cerebrospinal fluid (CSF) of MS and NBD patients which could be useful discriminative markers. Cytokines and transcription factors related to Th1, Th2, Th17 and T regulatory populations were studied by quantitative RT-PCR simultaneously in PBMCs and CSF, from 40 patients presenting a first episode of clinical features related to CNS inflammation and 22 controls with non inflammatory neurological diseases enrolled mainly for severe headache. The follow up of 12 months did allow a definitive diagnosis of remitting relapsing MS (RRMS) in 21 patients and of NBD in the other 19 among those with CNS inflammation compared to controls. In initial blood samples, T-bet was significantly increased in NBD patients only while IFN-γ was elevated in patients who evolved into RRMS or NBD. IL-17a, GATA-3 and IL-4 were significantly lower in RRMS patients than in the NBD group. In initial CSF samples, ROR-γt, IL-17a and IFN-γ were significantly elevated in patients compared to controls. The most striking finding was the significant increase of CSF IL-10 that we did observe in NBD patients only. Thus, we propose CSF IL-10 as a predictive marker to help clinicians discriminating between these two neurological disorders.

Direct alloreactivity is more susceptible to regulation by natural regulatory T cells than indirect alloreactivity.

The contribution of natural CD4(+)CD25(+) regulatory T cells (nTregs) in controlling graft rejection and the mechanism used remain controversial. Using the duality of the 2.102 TCR Ag recognition, we were able to study, for the first time to our knowledge, the involvement of nTregs in the two pathways of allorecognition in a murine adoptive transfer model in which TCR-transgenic nTregs were or were not depleted before transplantation. We show that nTregs used at a physiological ratio were able to delay graft rejection after direct alloreactivity by controlling proliferation and differentiation of alloreactive CD4(+) conventional T cells in draining lymph nodes. In contrast, similar results were found in the indirect alloreactivity pathway only when nTregs were used in high numbers. In the latter pathway, nTregs used at a physiological ratio failed to delay graft rejection and to control proliferation of conventional T cells. These results support recent therapeutic approaches aimed at producing and using in vitro Ag-specific Foxp3(+) nTregs to control graft rejection in transplantation. Finally, late inhibition of Th1 differentiation was shown in indirect alloreactivity, but this suppression could also be mediated by Foxp3(+)-induced Tregs.

Autoimmune diabetes onset results from qualitative rather than quantitative age-dependent changes in pathogenic T-cells.

Diabetogenic T-cells can be detected in pre-diabetic nonobese diabetic (NOD) mice after transfer in NOD-SCID recipients. Here we demonstrate that 6-week-old pre-diabetic NOD mice, >2 months before disease onset, already harbor pathogenic T-cells in equal numbers to overtly diabetic animals. The delay in diabetes appearance is explained by the presence of regulatory CD4+ CD25+ T-cells that control diabetogenic effectors and that are, in our hands, transforming growth factor (TGF)-beta-dependent. Our present results suggest, however, that diabetes onset is only partly explained by a decline in this regulatory T-cell activity. Another major factor appears to be the progressive resistance of diabetogenic cells to TGF-beta-dependent mediated inhibition. We propose that progression to overt disease correlates with the pathogenic T-cell's escape from TGF-beta-dependent T-cell-mediated regulation.

Leishmania major amastigotes induce p50/c-Rel NF-kappa B transcription factor in human macrophages: involvement in cytokine synthesis.

Invasion of a host by pathogens is frequently associated with activation of nuclear factor kappa B (NF-kappaB), which is implicated in various aspects of immune function required for resistance to infection. However, pathogens may also subdue these mechanisms to secure their survival. Here we describe the effect of Leishmania major infection on NF-kappaB transcription factor activation in both promonocytic human cell line U937 and fresh human monocytes. Infection by L. major amastigotes blocked nuclear translocation of a phorbol-12 myristate-13 acetate (PMA)-induced p50/p65 NF-kappaB complex in PMA-treated differentiated U937 cells and triggered expression of p50- and c-Rel-containing complexes in both U937 cells and fresh human monocytes. These p50/c-Rel complexes, triggered by direct cell-parasite interactions, were detectable within 30 min after the interaction and were transcriptionally active. The NF-kappaB inhibitor caffeic acid phenethyl ester inhibited production of both tumor necrosis factor alpha and interleukin-10 (IL-10) induced by Leishmania amastigotes in differentiated U937 cells. Similar results for IL-10 induction were observed with amastigote-infected human monocytes. Our results indicate that L. major amastigotes activate NF-kappaB by specifically inducing p50- and c-Rel-containing complexes which are likely involved in the regulation of cytokine synthesis.

TGF-beta-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes.

CD3-specific antibodies have the unique capacity to restore self-tolerance in established autoimmunity. They induce long-term remission of overt diabetes in nonobese diabetic (NOD) mice and in human type I diabetes. The underlying mechanisms had been unclear until now. Here we report that treatment with CD3epsilon-specific antibodies induces transferable T-cell-mediated tolerance involving CD4+CD25+ cells. However, these CD4+CD25+ T cells are distinct from naturally occurring regulatory T cells that control physiological autoreactivity. CD3-specific antibody treatment induced remission in NOD Cd28-/- mice that were devoid of such regulatory cells. Remission of diabetes was abrogated by coadministration of a neutralizing transforming growth factor (TGF)-beta-specific antibody. The central role of TGF-beta was further suggested by its increased, long-lasting production by CD4+ T cells from tolerant mice. These data explain the intriguing tolerogenic effect of CD3-specific antibodies and position them as the first clinically applicable pharmacological stimulant of TGF-beta-producing regulatory CD4+ T cells.