Staphylococcus aureus-specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis-like flares in mice.

BACKGROUND: The contribution of Staphylococcus aureus to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. OBJECTIVES: This study sought to reappraise the main bacterial factors and underlying immune mechanisms by which S aureus triggers AD-like inflammation. METHODS: This study capitalized on a preclinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. RESULTS: The development of S aureus-induced dermatitis depended on the nature of the S aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and nonsecreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor apoptosis-associated speck-like protein containing a CARD domain- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S aureus, and an accumulation of S aureus-specific γδ and CD4+ tissue resident memory T cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis on new bacteria exposures, but also, protected the mice from persistent bacterial colonization. CONCLUSIONS: These data highlight the induction of unique AD-like inflammation, with the generation of proinflammatory but protective tissue resident memory T cells in a context of natural exposure to pathogenic S aureus strains.

Gene profiling in active dermatitis lesions strengthens the diagnosis of allergic contact dermatitis.

BACKGROUND: Distinguishing between allergic and nonallergic forms of Contact Dermatitis (CD) is challenging and requires investigations based on patch-testing. Early detection of allergy biomarkers in active CD lesions could refine and simplify the management of CD patients. OBJECTIVE: To characterize the molecular signatures of active CD lesions. METHODS: We studied the expression of 12 allergy biomarkers by qRT-PCR in active lesions of 38 CD patients. Allergic CD (ACD) was diagnosed based on patch test (PT) results and exposure assessment. Molecular signatures of active lesions, as well as positive PT reactions, were compared with those of reference chemical allergens and irritants. RESULTS: Nineteen of the 38 CD patients reacted positively upon patch-testing and exposure assessment confirmed ACD diagnosis for 17 of them. Gene profiling of active CD lesions revealed 2 distinct molecular patterns: patients harboring signatures similar to reference allergens (n = 23) or irritants (n = 15). Among the 23 patients with an "allergy signature," we found the 17 patients with confirmed ACD, while no culprit allergen was identified for the 6 other patients. Interestingly, the 15 patients without biomarker induction had negative PT, suggesting that they developed nonallergic CD reactions. CONCLUSION: Molecular signatures from active skin lesions may help to stratify CD patients and predict those suffering from ACD.

Topical corticosteroids inhibit allergic skin inflammation but are ineffective in impeding the formation and expansion of resident memory T cells.

BACKGROUND: Tissue-resident memory T (TRM ) cells are detrimental in allergic contact dermatitis (ACD), in which they contribute to the chronicity and severity of the disease. METHODS: We assessed the impact of a standard topical corticosteroid (TCS) treatment, triamcinolone acetonide (TA), on the formation, maintenance and reactivation of epidermal TRM cells in a preclinical model of ACD to 2,4-dinitrofluorobenzene. TA 0.01% was applied at different time points of ACD response and we monitored skin inflammation and tracked CD8+ CD69+ CD103+ TRM by flow cytometry and RNA sequencing. RESULTS: The impact of TA on TRM formation depended on treatment regimen: (i) in a preventive mode, that is, in sensitized mice before challenge, TA transiently inhibited the infiltration of effector T cells and the accumulation of TRM upon hapten challenge. In contrast, (ii) in a curative mode, that is, at the peak of the ACD response, TA blocked skin inflammation but failed to prevent the formation of TRM . Finally, (iii) in a proactive mode, that is, on previous eczema lesions, TA had no effect on the survival of skin TRM , but transiently inhibited their reactivation program upon allergen reexposure. Indeed, specific TRM progressively regained proliferative functions upon TA discontinuation and expanded in the tissue, leading to exaggerated iterative responses. Interestingly, TRM re-expansion correlated with the decreased clearance of hapten moieties from the skin induced by repeated TA applications. CONCLUSIONS: Our results demonstrate that TCS successfully treat ACD inflammation, but are mostly ineffective in impeding the formation and expansion of allergen-specific TRM , which certainly restricts the induction of lasting tolerance in patients with chronic dermatitis.

mTOR Activation Underlies Enhanced B Cell Proliferation and Autoimmunity in PrkcdG510S/G510S Mice.

Autosomal recessive PRKCD deficiency has previously been associated with the development of systemic lupus erythematosus in human patients, but the mechanisms underlying autoimmunity remain poorly understood. We introduced the Prkcd G510S mutation that we previously associated to a Mendelian cause of systemic lupus erythematosus in the mouse genome, using CRISPR-Cas9 gene editing. PrkcdG510S/G510S mice recapitulated the human phenotype and had reduced lifespan. We demonstrate that this phenotype is linked to a B cell-autonomous role of Prkcd. A detailed analysis of B cell activation in PrkcdG510S/G510S mice shows an upregulation of the PI3K/mTOR pathway after the engagement of the BCR in these cells, leading to lymphoproliferation. Treatment of mice with rapamycin, an mTORC1 inhibitor, significantly improves autoimmune symptoms, demonstrating in vivo the deleterious effect of mTOR pathway activation in PrkcdG510S/G510S mice. Additional defects in PrkcdG510S/G510S mice include a decrease in peripheral mature NK cells that might contribute to the known susceptibility to viral infections of patients with PRKCD mutations.

Epicutaneous allergen immunotherapy induces a profound and selective modulation in skin dendritic-cell subsets.

BACKGROUND: Epicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses. OBJECTIVE: To date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy. METHODS: We capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVA+ skDCs throughout the course of EPIT. RESULTS: Our results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVA+ Langerhans cells progressively lost their capacity to prime CD4+ TEFF cells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4+ TEFF cells or in reactivating TMEM cells in vitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses. CONCLUSIONS: Our results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

Unique molecular signatures typify skin inflammation induced by chemical allergens and irritants.

BACKGROUND: Skin exposure to chemicals may induce an inflammatory disease known as contact dermatitis (CD). Distinguishing the allergic and irritant forms of CD often proves challenging in the clinic. METHODS: To characterize the molecular signatures of chemical-induced skin inflammation, we conducted a comprehensive transcriptomic analysis on the skin lesions of 47 patients with positive patch tests to reference contact allergens and nonallergenic irritants. RESULTS: A clear segregation was observed between allergen- and irritant-induced gene profiles. Distinct modules pertaining to the epidermal compartment, metabolism, and proliferation were induced by both contact allergens and irritants; whereas only contact allergens prompted strong activation of adaptive immunity, notably of cytotoxic T-cell responses. Our results also confirmed that: (a) unique pathways characterize allergen- and irritant-induced dermatitis; (b) the intensity of the clinical reaction correlates with the magnitude of immune activation. Finally, using a machine-learning approach, we identified and validated several minimal combinations of biomarkers to distinguish contact allergy from irritation. CONCLUSION: These results highlight the value of molecular profiling of chemical-induced skin inflammation for improving the diagnosis of allergic versus irritant contact dermatitis.

Inhibitory checkpoint receptors control CD8+ resident memory T cells to prevent skin allergy.

BACKGROUND: Tissue-resident memory T (Trm) cells are detrimental in patients with numerous chronic inflammatory diseases, including allergic contact dermatitis (ACD). OBJECTIVES: We sought to analyze the contribution of Trm cells to the chronicity and severity of ACD and to define the local parameters regulating their development and functions. METHODS: We used an experimental model of ACD (ie, contact hypersensitivity to 2,4-dinitrofluorobenzene) that is mediated by CD8+ T cells. RESULTS: Our data show that early effector T cells accumulated in the skin during the acute contact hypersensitivity reaction and gave rise to epidermal CD8+ Trm cells expressing a specific set of inhibitory checkpoint receptors (ICRs), such as programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin domain 3 (TIM-3). Those Trm cells remained in the epidermis for several weeks and mediated the eczema exacerbations, which developed on allergen re-exposure without the contribution of circulating specific T cells. Furthermore, allergen-induced Trm cell reactivation was constrained because treatment with ICR antagonists dramatically enhanced the magnitude and severity of eczema exacerbations. Finally, we show that the persistence of the allergen in the epidermis for long periods of time was responsible for both the development and maintenance of epidermal Trm cells, as well as the sustained expression of ICRs. CONCLUSION: Although CD8+ Trm cells are key for the pathophysiology of ACD, intrinsic mechanisms control their reactivation to prevent damaging immunopathology. Developing strategies targeting the reactivation of skin Trm cells in situ through their ICRs should open new perspectives for the treatment of ACD.

Simvastatin inhibits the pro-inflammatory and pro-thrombotic effects of IL-17 and TNF-α on endothelial cells.

OBJECTIVES: Statins are widely used for primary and secondary prevention of coronary atherosclerosis. Simvastatin, besides its lipid lowering properties, has various anti-inflammatory effects. The aim of this study was to assess whether simvastatin modulates the vascular effects of interleukin (IL)-17, an emerging actor in atherosclerosis. METHODS: The effect of simvastatin was assessed in human umbilical vein endothelial cells treated by IL-17 alone or combined with tumour necrosis factor (TNF)-α, with or without mevalonate, an inhibitor of simvastatin. Its effects on IL-17-induced cytokine or chemokine expression were assessed at the mRNA level using qRT-PCR or protein level by ELISA. Its effect on the IL-17-induced pro-thrombotic state and cell invasion was assessed using a lumi-aggregometer and a Matrigel assay, respectively. RESULTS: Simvastatin decreased IL-17-induced IL-6, IL-8, CX3CL-1, RANTES mRNA and CX3CL-1 and CCL20 production. Simvastatin restored the level of IL-33 mRNA which was decreased by IL-17. It reduced the expression of IL-17-induced pro-thrombotic genes such as tissue factor. Simvastatin restored the level of platelet aggregation to normal levels. Simvastatin enhanced the expression of CD39 and thrombomodulin mRNA initially reduced by IL-17 and TNF-α combination. Simvastatin suppressed IL-17-induced endothelial cells invasion. All these effects were reversed by the addition of mevalonate. Finally, simvastatin had an additive effect with infliximab to decrease the effect of the combination of IL-17 and TNF-α on IL-6 mRNA expression. Similar conclusion was obtained with rosuvastatin. CONCLUSIONS: Statins inhibit the pro-inflammatory, thrombotic and pro-aggregation effects of IL-17 on vessels. This provides a new understanding of the beneficial effects of statins in blood vessel inflammation.

IL-17 and tumour necrosis factor α combination induces a HIF-1α-dependent invasive phenotype in synoviocytes.

OBJECTIVES: To examine the effect of interleukin-17 (IL-17) on rheumatoid arthritis (RA) synoviocyte migration and invasiveness. METHODS: IL-17A and tumour necrosis factor α (TNFα)-induced messenger RNA expression in RA synoviocytes was analysed using Affymetrix U133A microarrays. The capacity of IL-17 alone or in combination with TNFα to induce synoviocyte migration and invasion was tested using Boyden and transwell Matrigel invasion chambers. A functional DNA binding assay was used to evaluate the regulation of the key hypoxia-related gene hypoxia-inducible factor 1 (HIF-1α) expression and activation. The role of metalloproteinase 2 (MMP2) in IL-17-induced invasiveness was assessed using small interfering RNA. Hypoxia pathway gene expression was measured in the blood of RA patients and healthy volunteers using Affymetrix microarrays. RESULTS: Among the genes induced by IL-17A in RA synoviocytes, a molecular pattern of inflammation hypoxia-related genes, including CXC chemokine receptor 4 (CXCR4) and MMP2 was identified. Using immunofluorescence microscopy, the expression of CXCR4 was confirmed on synoviocytes. IL-17A and TNFα induced synoviocyte migration and invasion through a CXCR4-dependent mechanism with a synergistic effect. Their combination activated HIF-1α through the nuclear factor κB pathway. IL-17 enhanced invasion through MMP2 induction as demonstrated using siRNA. Finally, hypoxia genes were overexpressed in the blood of RA patients. CONCLUSION: IL-17A, specifically when combined with TNFα may contribute to the progression of RA, notably through their effect on synoviocyte aggressiveness. Part of this effect results from activation of the CXCR4/stromal cell-derived factor 1 and hypoxia-mediated pathways.

Combination of IL-17 and TNFα induces a pro-inflammatory, pro-coagulant and pro-thrombotic phenotype in human endothelial cells.

OBJECTIVE: Cardiovascular events remain the leading cause of death in rheumatoid arthritis (RA). To study the role of cytokines in these observations, the effects of tumour necrosis factor α (TNFα) and interleukin (IL)-17, a classical and a new key player in RA, were assessed in endothelial cell (EC) dysfunction. METHODS: Primary human EC were treated with IL-17 alone or combined with TNFα. mRNA expression was quantified by qRT PCR and Affymetrix microarrays. The role of IL-17 was studied using functional assays of platelet aggregation, EC migration and invasion. RESULTS: IL-17 alone induced 248 pro-inflammatory genes and 9803, when combined with TNFα. IL-17 plus TNFα induced synergistically chemokine genes such as CCL5, IL-8 and cytokine genes such as IL-6. In contrast, IL-17 decreased genes involved in the regulation of inflammation such as IL-33. IL-17 induced EC migration and invasion in synergy with TNFα. Such invasion was inhibited with an antiCXCR4 antibody, indicating the contribution of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis. Supernatants of IL-17-treated EC induced strong platelet aggregation. IL-17 inhibited endothelial CD39/ATPDase expression, an inhibitor of platelet activation. Finally, IL-17 enhanced genes critical for coagulation such as tissue factor and decreased thrombomodulin, leading to a pro-thrombotic state. CONCLUSION: These results indicate that IL-17 specifically when combined with TNFα has major pro-coagulant and pro-thrombotic effects on vessels.

Immature muscle precursors are a source of interferon-ß in myositis: role of Toll-like receptor 3 activation and contribution to HLA class I up-regulation.

OBJECTIVE: To investigate the production of type I interferon (IFN) by myoblasts and to identify its cell source and the link to Toll-like receptor (TLR) and C-type lectin receptor (CLR) expression and function in myositis biopsy sections. METHODS: Production of IFNß was assessed in cultured myoblasts after stimulation with the TLR-3 agonist poly(I-C) or with cytokines involved in Th1 and Th17 differentiation. Expression of HLA class I molecules by myoblasts was analyzed by fluorescence-activated cell sorting after activation of TLR-3 and IFNß neutralization. In muscle biopsy samples from patients with polymyositis or dermatomyositis, expression of IFNß, CD56 (a marker of immature muscle precursors), and HLA class I was analyzed using immunohistochemistry. Inflammatory infiltrates were characterized for the expression of myeloid dendritic cells (DCs), their associated CLRs, and the products of activated DCs, interleukin-12 (IL-12), and IL-23. RESULTS: In cultured myoblasts, stimulation of TLR-3 induced the production of IFNß when combined with IFNγ and up-regulated the expression of HLA class I molecules, which was decreased after IFNß blockade. In myositis biopsy tissues, immature muscle precursors overexpressing HLA class I were identified as a source of IFNß. CLRs associated with myeloid DCs were broadly expressed in inflammatory infiltrates, in association with IL-12 and IL-23, and with immature muscle precursors. CONCLUSION: Immature muscle precursors may represent a local source of IFNß and the target of an immune response involving activated DCs associated with the expression of CLRs and of IL-12 and IL-23, which are implicated in T cell polarization. In turn, such local production of IFNß after TLR-3 activation in the presence of the Th1 cytokine IFNγ may explain HLA class I overexpression in myositis.

IL-17A- versus IL-17F-induced intracellular signal transduction pathways and modulation by IL-17RA and IL-17RC RNA interference in rheumatoid synoviocytes.

OBJECTIVE: The aim of this study was to compare the effects of interleukin (IL)-17A and IL-17F on gene expression and signalling in human rheumatoid arthritis (RA) synoviocytes. METHODS: IL-17A- and IL-17F-induced mRNA expression was analysed using Affymetrix microarrays. IL-6 and IL-8 secretion was evaluated by ELISA. Inhibition of two receptors (IL-17RA and IL-17RC) was achieved by small interfering RNA (saran). The effects on mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1) and nuclear factor κB (NF-κB) expression and activation were evaluated by western blotting, qRT-PCR and DNA binding assay. RESULTS: IL-17A and IL-17F induced a molecular pattern characterised by 27 inflammation-related genes for IL-17F and 165 for IL-17A. Virtually all IL-17A and IL-17F inducible genes were dependent on NF-κB activation, whereas a small number were modulated by p38. IL-17A induced activation of all three MAPKs (ERK, p38 and JNK) and downstream transcription factors AP-1 and p65 NF-κB. IL-17F was less potent but induced activation of p50 NF-κB. IL-17A was more potent at inducing IL-6 secretion than IL-17F, which was inactive alone. IL-17A and, to a lesser extent, IL-17F induced TRAF6 but not MyD88. Inhibition of either IL-17RA or IL-17RC expression via siRNA led to near complete abrogation of IL-6 expression mediated by IL-17A and the combination of IL-17F and tumour necrosis factor α. CONCLUSION: Like IL-17A, IL-17F regulates proinflammatory gene expression by a very similar but not identical signalling pathway involving IL-17RA and IL-17RC.

Genome-wide comparison between IL-17A- and IL-17F-induced effects in human rheumatoid arthritis synoviocytes.

IL-17A is implicated in rheumatoid arthritis (RA) pathogenesis; however, the contribution of IL-17F remains to be clarified. Using microarrays and gene-specific expression assays, we compared the regulatory effects of IL-17A and IL-17F alone or in combination with TNF-alpha on RA synoviocytes. IL-17A and IL-17F expression was studied in osteoarthritis and RA synovium by immunohistochemistry. The comparison between the IL-17A and IL-17F stimulatory effect on RA synoviocytes was assessed at the protein level by ELISA and at the mRNA level by microarrays and real-time RT-PCR. TNFRII expression was studied by real-time RT-PCR and immunofluorescence, and neutralizing Ab was used to analyze its contribution to CCL20 secretion. IL-17A and IL-17F were detected in plasma cell-like cells from RA but not osteoarthritis synovium. In microarrays, IL-17A and IL-17F alone had similar regulatory effects, IL-17F being quantitatively less active. Both cytokines induced a similar expression pattern in the presence of TNF-alpha. Based on a cooperation index, 130 and 203 genes were synergistically induced by IL-17A or IL-17F plus TNF-alpha, respectively. Among these, the new target genes CXCR4, LPL, and IL-32 were validated by real-time RT-PCR. IL-17A and IL-17F up-regulated TNFRII expression, but had no effects on TNFRI, IL-17RA or IL-17RC. TNFRII blockade inhibited the synergistic induction of CCL20 by IL-17A or IL-17F and TNF-alpha. IL-17A and IL-17F are both expressed in RA synovium. In the presence of TNF-alpha, they induced a similar expression pattern in RA synoviocytes. Accordingly, IL-17F appears as a target in Th17-mediated diseases such as RA.

Development of a lab-made microarray for analyzing the genetic diversity of nitrogen fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae.

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.