ILC2 activation by keratinocyte-derived IL-25 drives IL-13 production at sites of allergic skin inflammation.

BACKGROUND: Atopic dermatitis skin lesions demonstrate increased expression of IL-25 by keratinocytes and increased numbers of type 2 innate lymphoid cells (ILC2s) that express high levels of IL-25 receptor (IL-25R). IL-13 is expressed in atopic dermatitis skin lesions and plays an important role in pathogenesis of the disease. OBJECTIVE: Our aim was to determine the role of IL-25 and ILC2s in a mouse model of antigen-driven allergic skin inflammation. METHODS: Wild-type mice; mice that express an Il13-driven enhanced green fluorescent protein; and mice that lack IL-25R, IL-25 in keratinocytes, or IL-13 or IL-25R in ILC2s were subjected to acute or chronic epicutaneous sensitization with ovalbumin. Sensitized skin was examined by histology for epidermal thickening. Cellular infiltrates were analyzed for surface markers and intracellular expression of enhanced green fluorescent protein by flow cytometry. Gene expression was quantitated by RT quantitative PCR. RESULT: In both acute and chronic antigen-driven allergic skin inflammation, signaling by keratinocyte-derived IL-25 in ILC2s is important for epidermal hyperplasia, dermal infiltration by CD4+ T cells, and cutaneous expression of Il13 and the IL-13-dependent TH2-cell-attracting chemokines Cc17 and Ccl22. ILCs are the major source of IL-13 in acutely sensitized mouse skin, whereas T cells are its major source in chronically sensitized mouse skin. CONCLUSION: ILC2 activation by IL-25 is essential for IL-13 expression at sites of allergic skin inflammation.

Dabigatran aggravates topoisomerase I peptide-loaded dendritic cells-induced lung and skin fibrosis.

OBJECTIVES: Dysregulated coagulation cascade has been implicated in development of fibrosis in systemic sclerosis (SSc). Thrombin, a key mediator of the coagulation pathway, has both proinflammatory and procoagulant properties. Here, we evaluated the efficacy of oral dabigatran, a direct thrombin inhibitor, on topoisomerase I dendritic cells (TOPOIA DCs)-induced lung and skin fibrosis, an experimental model of SSc. METHODS: Mice were repeatedly immunized with TOPOIA DCs. Dabigatran was administered in food either during the onset of fibrotic (late treatment) or inflammatory (early treatment) phase. RESULTS: Early administration of dabigatran caused an aggravation of pulmonary fibrosis associated with signs of severe perivascular inflammation while late treatment was not protective when compared to the untreated TOPOIA DCs group. Thrombin was increased in lungs of TOPOIA DCs immunized group and, paradoxically, further augmented by administration of dabigatran to immunized mice. As in lungs, early and not late drug administration exacerbated skin fibrosis. Moreover, early dabigatran treatment induced a profibrotic and inflammatory skin gene expression signature with upregulated expression of Col5a1, Timp1, Tweakr, Vwf, Il6, Il33, Il4 and Ifng. CONCLUSIONS: Dabigatran aggravated lung and skin fibrosis in a TOPOIA DCs-induced model of SSc-like disease. Therefore, our results argue against the use of dabigatran to treat patients with SSc.

Human mast cells are major IL-22 producers in patients with psoriasis and atopic dermatitis.

BACKGROUND: Psoriasis is a systemic inflammatory disease in which IL-17 and IL-22 levels are markedly increased in the skin and blood. The prevalent concept, using skin cells that are isolated from psoriatic plaques and examined after cell expansion and in vitro stimulation, is that IL-17 and IL-22 production essentially results from T cells and the rare type 3 innate lymphoid cells. OBJECTIVE: We sought to examine the cellular source of IL-17A and IL-22 at the protein and transcriptional single-cell level immediately after ex vivo skin cell isolation from psoriatic plaques. METHODS: Skin biopsy specimens were collected from patients with psoriasis, as well as from patients with atopic dermatitis. Cell suspensions were prepared by combining mild enzymatic digestion and mechanical dissociation and analyzed for cytokine expression without prior in vitro culture and stimulation. Expression of IL-17 and IL-22 was quantified at the protein and mRNA single-cell level by using flow cytometry. RESULTS: IL-22 is predominantly expressed by CD3(-)c-Kit(+) cells relative to CD3(+) T cells in lesional skin of patients with psoriasis and patients with atopic dermatitis. Strikingly, we identified c-Kit(+)FcεRI(+) mast cells as major IL-22 producers. The proportion of mast cells that produce IL-22 ranges from 20% to 80% in patients with psoriasis or those with atopic dermatitis. Skin mast cells express IL-22 and IL-17 mRNA. Conversely, IL-17-producing T cells outnumber IL-17-producing mast cells, which also express IL-17 receptor. CONCLUSION: Human skin mast cells are previously unrecognized IL-22 producers. We further established that skin mast cells express IL-17. Thus mast cells might play an important role in the physiopathology of chronic inflammatory skin disorders.

Broad responses to chemical adducts shape the natural antibody repertoire in early infancy.

Natural antibodies are an integral part of innate humoral immunity yet their development and polyreactive nature are still enigmatic. Here, we show that characteristic monoclonal natural antibodies recognize common chemical moieties or adducts, supporting the view that polyreactive antibodies may often correspond to anti-adduct antibodies. We next examined the development of immunoglobulin M (IgM) and IgG to 81 ubiquitous adducts from birth to old age. Newborn IgM only reacted to a limited number of consensus determinants. This highly restricted neonatal repertoire abruptly diversified around 6 months of age through the development of antibodies to environmental antigens and age-driven epigenetic modifications. In contrast, the IgG repertoire was diverse across the entire life span. Our studies reveal an unrecognized component of humoral immunity directed to common adducts. These findings set the ground for further investigations into the role of anti-adduct B cell responses in homeostatic functions and pathological conditions.

Differential Changes in Inflammatory Mononuclear Phagocyte and T-Cell Profiles within Psoriatic Skin during Treatment with Guselkumab vs. Secukinumab.

Cellular sources of IL-23 and IL-17A driving skin inflammation in psoriasis remain unclear. Using high-dimensional unsupervised flow cytometry analysis, mononuclear phagocytes and T cells were examined in the same lesions of patients before and during guselkumab (IL-23p19 blocker) or secukinumab (IL-17A blocker) treatment. Among CD11c+HLA-DR+ mononuclear phagocytes, CD64brightCD163-CD14brightCD1c-CD1a‒ inflammatory monocyte‒like cells were the predominant IL-23-producing cells and, together with CD64-CD163-CD14-IL-23p19-TNF-α+ inflammatory dendritic cell‒like cells, were increased in lesional compared with those in nonlesional skin taken from the same patient. Within T cells, CD8+CD49a+ and/or CD103+ tissue-resident memory T cells, CD4+CD25+FoxP3+ regulatory T cells, and CD4+CD49a-CD103- T cells were increased. Moreover, CD4+CD49a-CD103- T cells and the relatively rare CD8+ memory T cells equally contributed to IL-17A production. Both treatments decreased the frequencies of inflammatory monocyte‒like, inflammatory dendritic cell‒like, and CD4+CD49a-CD103- T cells. In contrast, guselkumab reduced memory T cells while maintaining regulatory T cells and vice versa for secukinumab. Neither drug modified the frequencies of IL-17A+IL‒17F+/- CD4+ or CD8+ T cells. This study reveals the identity of the major IL-23+ mononuclear phagocyte and IL-17+ T-cell subsets in psoriatic skin lesions and paves the way for a better understanding of the mode of action of drugs targeting the IL-23/IL-17A pathway in psoriasis.

Persistence of Inflammatory Phenotype in Residual Psoriatic Plaques in Patients on Effective Biologic Therapy.

Many psoriasis patients treated with biologics do not achieve total skin clearance. These patients possess residual plaques despite ongoing biologic treatment. To elucidate mechanisms of plaque persistence despite overall good drug response, we studied 50 subjects: psoriasis patients with residual plaques treated with one of three different biologics, untreated patients, and healthy controls. Skin biopsies from all subjects were characterized using three methods: mRNA expression, histology, and FACS of hematopoietic skin cells. Although all three methods provided evidence of drug effect, gene expression analysis revealed the persistence of key psoriasis pathways in treated plaques, including granulocyte adhesion and diapedesis, T helper type17 activation pathway, and interferon signaling with no novel pathways emerging. Focal decreases in parakeratosis and keratinocyte proliferation and differential reduction in IL-17 producing CD103- T cells, but no change in CD103+ tissue-resident memory T cells were observed. Of note, antitumor necrosis factor increased the interferon signaling pathway already present. Interestingly mast cells were the dominant source of IL-22 in all psoriasis subjects. These data suggest that while subtle differences can be observed in drug-treated plaques, underlying biologic mechanisms are similar to those present in untreated psoriatic lesions.

Increased frequencies of basophils, type 2 innate lymphoid cells and Th2 cells in skin of patients with atopic dermatitis but not psoriasis.

BACKGROUND: Pathogenesis of atopic dermatitis (AD) involves interaction between type 2 cells that include basophils, mast cells, innate lymphoid type 2 cells (ILC2), and Th2 cells. Levels of IL-4 and IL-13 are elevated in AD patients. OBJECTIVE: Here, we investigated the distribution of type 2 cells and the source of IL-4 and IL-13 in skin and blood of AD relative to psoriasis. METHODS: Lesional skin biopsies and blood were collected from patients. Skin cell suspensions were prepared by mild enzymatic digestion and mechanical dissociation. IL-4 and IL-13 expression was analyzed at single-cell level before or after stimulation using flow cytometry. RESULTS: Frequencies of basophils, ILC2 and Th2 but not mast cells were significantly elevated in skin, and not blood, of AD relative to psoriasis. IL-4 production by circulating basophils and Th2 cells, and IL-13 by ILCs and Th2 cells was similar in both diseases. In contrast, skin T cells expressed IL-4 and IL-13 prior to stimulation in AD when compared to psoriasis. Moreover, skin basophils, which were detected in AD only, expressed IL-4 following stimulation. Interestingly, basophils and ILC2 were positively correlated in skin, whereas skin basophils were inversely correlated with blood ILC2. CONCLUSIONS: Lesional AD skin harbors a distinctive innate and adaptive type 2 profile, which is characterized by basophils producing IL-4, Th2 cells expressing IL-4 or IL-13, and ILC2. This underlies the therapeutic efficacy of targeting IL-4 and IL-13 signaling pathways in AD.

Early-Life Antibiotic Exposure Causes Intestinal Dysbiosis and Exacerbates Skin and Lung Pathology in Experimental Systemic Sclerosis.

Patients with systemic sclerosis (SSc) display altered intestinal microbiota. However, the influence of intestinal dysbiosis on the development of experimental SSc remains unknown. Topoisomerase I peptide-loaded dendritic cell immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was withdrawn (earlyATB) or initiated (adultATB) during adulthood. Topoisomerase I peptide-loaded dendritic cell (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), profibrotic (Il13, Tweakr), and vascular function (Serpine1) gene expression. Remarkably, earlyATB exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13+ cells, mononuclear phagocytes, and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life (earlyATB) being sufficient to cause long-term modification of the microbiota and a shift toward increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T-cell responses were observed in earlyATB and lifelongATB but not adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.

Palmoplantar pustular psoriasis (PPPP) is characterized by activation of the IL-17A pathway.

BACKGROUND: Palmoplantar pustular psoriasis (PPPP) is a variant of psoriasis, which has significant negative impact on quality of life. The cellular and molecular inflammatory pathways involved in PPPP have not been well studied. OBJECTIVE: Study the expression of cytokines and chemokines involved in the IL-17/IL-23 axis in palmoplantar pustular psoriasis and other difficult to treat psoriasis areas (palms, scalp, elbows and lower legs). METHODS: Skin biopsies were performed on a total of 80 patients with PPPP, non-pustular palmoplantar psoriasis (NPPPP), or psoriasis located on elbows, knees and scalp as well as 10 healthy subjects. RT-PCR, immunohistochemistry and flow cytometry on cells extracted from skin biopsies were used to compare PPPP to other forms of psoriasis. RESULTS: There was a significant (p<0.05) increase in the expression of IL-1ß, IL-6, LL-37, IL-19, IL-17A, CXCL1 and CXCL2 in PPPP as compared to NPPPP. However, there was no significant difference in expression of IL-23 in PPPP as compared to NPPPP and other forms of psoriasis. The proportion of IL-22+ but not IL-17A+ mast cells was higher in PPPP as compared to NPPPP (p<0.05). CONCLUSION: These results suggest that the IL-17A pathway may play a more important role in PPPP than in NPPPP.