Emmanuel Xystrakis

Ligation of TLR9 induced on human IL-10–secreting Tregs by 1α,25-dihydroxyvitamin D3 abrogates regulatory function

Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3+ T cells in the presence of 1alpha,25-dihydroxyvitamin D3 (1alpha25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4+CD25+ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1alpha25VitD3 in vitro. Furthermore, ingestion of calcitriol (1alpha25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3+CD4+ T cells analyzed directly ex vivo. Stimulation of 1alpha25VitD3-induced IL-10-secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-gamma synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1alpha25VitD3-induced IL-10-secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.

The role of 1α,25-dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL-10+ CD4+ T cells

1α,25‐Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL‐10‐secreting CD4+ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3+ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3+ and IL‐10+ CD4+T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL‐10 at more moderate levels, with little coexpression of these molecules. The Foxp3+ and IL‐10+ T‐cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL‐10. 1α25VitD3 enables the selective expansion of Foxp3+ Treg cells over their Foxp3− T‐cell counterparts. Equally, 1α25VitD3 maintains Foxp3+ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4+Foxp3+ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL‐10+ and Foxp3+ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14hi/CD16neg, CD14hi/CD16pos and CD14lo/CD16pos) have been described in several diseases. The combination of CD14, CD16 and HLA‐DR has been suggested to discriminate monocytes from the CD16pos/HLA‐DRneg NK‐cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA‐DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66bpos neutrophils, CD56pos NKcells, CD19pos B‐cells, and CD3pos T‐cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA‐DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA‐DR expression]. Bland‐Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14hi/CD16pos monocytes was higher (7% vs 4%) whilst the percentage of CD14lo/CD16pos was lower (1.9% vs. 7%) (P ≤ 0.001); HLA‐DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA‐DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody‐fluorochrome “backbone” targeting HLA‐DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody‐fluorochrome “backbone” of HLA‐DR, CD14, and CD16. CD16pos monocytes continue to constitutively express HLA‐DR even in conditions where HLA‐DR is pathologically reduced on CD14hi/CD16neg monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico‐pathological diseases may allow the development of novel biomarkers or therapeutic strategies.

Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease

Associations between vitamin D status and childhood asthma are increasingly reported, but direct causation and mechanisms underlying an effect remain unknown. We investigated the effect of early‐life vitamin D deficiency on the development of murine neonatal allergic airways disease (AAD).

Regulatory T cell therapy as individualized medicine for asthma and allergy.

Purpose of reviewRegulatory T cells have been identified as key players in the maintenance of peripheral tolerance, which prevents inappropriate immune responses to both self-antigens and innocuous allergens. This review aims to provide an update on our current understanding of the therapeutic potential of naturally occurring and adaptive regulatory T cell subsets in allergic and asthmatic disease. Recent findingsEvidence is emerging that regulatory T cells control aberrant immune responses to allergens in health and exhibit impaired function in active disease. These data provide a rationale for developing therapeutic strategies that promote regulatory T cell numbers or function in patients. SummaryPreclinical studies of adoptive transfer of antigen-specific regulatory T cells into mouse models of allergic airway disease ameliorate the inflammatory response and in some studies airway hyperresponsiveness. Although these studies are encouraging this remains an invasive and expensive therapeutic protocol for the treatment of allergic disease in humans and a number of concerns relating to safety and efficacy exist. Existing therapies, both allergen specific immunotherapy and nonspecific treatments such as glucocorticoids, induce IL-10 secreting T regulatory populations in patients. Strategies to combine allergen immunotherapy with add-on treatments or adjuvants with the potential to boost regulatory T cells, safety and efficacy remain a major research focus.

Defective IL-10 expression and in vitro steroid-induced IL-17A in paediatric severe therapy-resistant asthma

Background Understanding of immune mechanisms underpinning asthma has emerged from studies in adults. It is increasingly recognised, both immunologically and in the development of novel therapies, that adult responses cannot be used accurately to predict those of children. Methods Using a well-defined paediatric cohort of severe therapy-resistant asthma (STRA) patients, we investigated cytokine profiles in the airway by analysis of bronchoalveolar lavage fluid. The in vitro capacity of peripheral blood mononuclear cells (PBMCs) for cytokine production was also assessed following polyclonal T cell activation in culture, in the absence or presence of dexamethasone and 1α,25-dihydroxyvitamin D3. Results Children with both moderate and STRA had significantly diminished levels of anti-inflammatory interleukin (IL)-10 in airway lavage samples when compared with non-asthmatic controls (p<0.001). Their PBMCs also demonstrated significantly impaired capacity to secrete IL-10 in culture (p<0.001). Dexamethasone regulated the balance between PBMC IL-10 and IL-13 production, increasing IL-10 secretion (p<0.001) and decreasing IL-13 (p<0.001) but unexpectedly enhanced IL-17A production in all groups—most strikingly in the STRA cohort (p<0.001). The inclusion of the active form of vitamin D, 1α,25-dihydroxyvitamin D3, in culture enhanced dexamethasone-induced IL-10 (p<0.05) without marked effects on IL-13 or IL-17A production. Furthermore, systemic vitamin D status directly correlated with airway IL-10 (r=0.6, p<0.01). Conclusions These findings demonstrate reduced peripheral and local IL-10 synthesis in paediatric asthma, and support therapeutic augmentation of low circulating vitamin D in severe, difficult-to-treat asthma, in order to correct impaired IL-10 levels. Conversely, steroids enhanced IL-17A levels, and therefore any steroid-sparing properties of vitamin D may have additional benefit in STRA.

Impact of Donation Mode on the Proportion and Function of T Lymphocytes in the Liver

Background Liver T-cells respond to the inflammatory insult generated during organ procurement and contribute to the injury following reperfusion. The mode of liver donation alters various metabolic and inflammatory pathways but the way it affects intrahepatic T-cells is still unclear. Methods We investigated the modifications occurring in the proportion and function of T-cells during liver procurement for transplantation. We isolated hepatic mononuclear cells (HMC) from liver perfusate of living donors (LD) and donors after brain death (DBD) or cardiac death (DCD) and assessed the frequency of T-cell subsets, their cytokine secretion profile and CD8 T-cell cytotoxicity function, responsiveness to a danger associated molecular pattern (High Mobility Group Box1, HMGB1) and association with donor and recipient clinical parameters and immediate graft outcome. Results We found that T-cells in healthy human livers were enriched in memory CD8 T-cells exhibiting a phenotype of non-circulating tissue-associated lymphocytes, functionally dominated by more cytotoxicity and IFN-γ-production in DBD donors, including upon activation by HMGB1 and correlating with peak of post-transplant AST. This liver-specific pattern of CD8 T-cell was prominent in DBD livers compared to DCD and LD livers suggesting that it was influenced by events surrounding brain death, prior to retrieval. Conclusion Mode of liver donation can affect liver T-cells with increased liver damage in DBD donors. These findings may be relevant in designing therapeutic strategies aimed at organ optimization prior to transplantation.