•Elevated frequencies of skin basophils producing IL-4 in AD and not psoriasis.•Skin and circulating ILC2 were increased in AD relative to psoriasis.•Basophils correlated positively to skin ILC2 and ...inversely to blood ILC2.•T cells expressing IL-4 and IL-13 prior to stimulation also infiltrated AD skin.•Skin and not blood better reflects innate and adaptive type 2 immune response of AD.
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.
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.
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.
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.
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.
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.
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.
Our aim was to determine the role of IL-25 and ILC2s in a mouse model of antigen-driven allergic skin inflammation.
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.
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.
ILC2 activation by IL-25 is essential for IL-13 expression at sites of allergic skin inflammation.
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Inflammatory bowel diseases are associated with dysregulated immune responses in the intestinal tissue. Four molecularly identified macrophage subsets control immune homeostasis in healthy gut. ...However, the specific roles and transcriptomic profiles of the phenotypically heterogeneous CD14
macrophage-like population in inflamed gut remain to be investigated in Crohn's disease (CD). Here we identified two phenotypically, morphologically and functionally distinct colonic HLADR
SIRPα
CD14
subpopulations that were further characterized using single-cell RNA-sequencing (scRNAseq) in CD. Frequencies of CD64
CD163
cells selectively augmented in inflamed colon and correlated with endoscopic score of disease severity. IL-1β and IL-23-producing CD64
CD163
cells predominated over TNF-α-producing CD64
CD163
cells in lesions. Purified "inflammatory monocyte-like" CD163
, but not "macrophage-like" CD163
cells, through IL-1β, promoted Th17/Th1 but not Th1 responses in tissue memory CD4
T cells. Unsupervised scRNAseq analysis that captures the entire HLADR
SIRPα
population revealed six clusters, two of which were enriched in either CD163
or CD163
cells, and best defined by TREM1/FCAR/FCN1/IL1RN or CD209/MERTK/MRCI/CD163L1 genes, respectively. Selected newly identified discriminating markers were used beyond CD163 to isolate cells that shared pro-Th17/Th1 function with CD163
cells. In conclusion, a molecularly distinct pro-inflammatory CD14
subpopulation accumulates in inflamed colon, drives intestinal inflammatory T-cell responses, and thus, might contribute to CD disease severity.
Compelling evidence suggests that the epithelial cell-derived cytokine thymic stromal lymphopoietin (TSLP) may initiate asthma or atopic dermatitis through a dendritic cell-mediated T helper (Th)2 ...response. Here, we describe how TSLP might initiate and aggravate allergic inflammation in the absence of T lymphocytes and immunoglobulin E antibodies via the innate immune system. We show that TSLP, synergistically with interleukin 1 and tumor necrosis factor, stimulates the production of high levels of Th2 cytokines by human mast cells (MCs). We next report that TSLP is released by primary epithelial cells in response to certain microbial products, physical injury, or inflammatory cytokines. Direct epithelial cell-mediated, TSLP-dependent activation of MCs may play a central role in "intrinsic" forms of atopic diseases and explain the aggravating role of infection and scratching in these diseases.
Inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are driven by an abnormal immune response to commensal microbiota in genetically susceptible hosts. ...In addition to epithelial and stromal cells, innate and adaptive immune systems are both involved in IBD immunopathogenesis. Given the advances driven by single-cell technologies, we here reviewed the immune landscape and function of mononuclear phagocytes in inflamed non-lymphoid and lymphoid tissues of CD and UC patients. Immune cell profiling of IBD tissues using scRNA sequencing combined with multi-color cytometry analysis identifies unique clusters of monocyte-like cells, macrophages, and dendritic cells. These clusters reflect either distinct cell lineages (nature), or distinct or intermediate cell types with identical ontogeny, adapting their phenotype and function to the surrounding milieu (nurture and tissue imprinting). These advanced technologies will provide an unprecedented view of immune cell networks in health and disease, and thus may offer a personalized medicine approach to patients with IBD.
We used a candidate gene approach to identify a set of SNPs, located in a predicted regulatory region on chromosome 1q44 downstream of NLRP3 (previously known as CIAS1 and NALP3) that are associated ...with Crohn's disease. The associations were consistently replicated in four sample sets from individuals of European descent. In the combined analysis of all samples (710 father-mother-child trios, 239 cases and 107 controls), these SNPs were strongly associated with risk of Crohn's disease (Pcombined = 3.49 × 10−9, odds ratio = 1.78, confidence interval = 1.47-2.16 for rs10733113), reaching a level consistent with the stringent significance thresholds imposed by whole-genome association studies. In addition, we observed significant associations between SNPs in the associated regions and NLRP3 expression and IL-1β production. Mutations in NLRP3 are known to be responsible for three rare autoinflammatory disorders. These results suggest that the NLRP3 region is also implicated in the susceptibility of more common inflammatory diseases such as Crohn's disease.
The drug targets IL23 and IL12 regulate pathogenicity and plasticity of intestinal Th17 cells in Crohn's disease (CD) and ulcerative colitis (UC), the two most common inflammatory bowel diseases ...(IBD). However, studies examining Th17 dysregulation in mesenteric lymph nodes (mLNs) of these patients are rare. We showed that in mLNs, CD could be distinguished from UC by increased frequencies of CCR6
CXCR3
RORγ
Tbet
CD4
(Th17) memory T cells enriched in CD62L
effector memory T cells (T
), and their differentially expressed molecular profile. Th17 T
cells (expressing
, and
) displayed a higher pathogenic/cytotoxic (
, and
) gene signature in CD relative to UC, while non-pathogenic/regulatory genes (
) were more elevated in UC. In both CD and UC, IL12 but not IL23, augmented IFNγ expression in Th17 T
and switched their molecular profile toward an ex-Th17 (Th1
)-biased transcriptomic signature (increased
, and decreased
), suggesting that Th17 plasticity occurs in mLNs before their recruitment to inflamed colon. We propose that differences observed between Th17 cell frequencies and their molecular profile in CD and UC might have implications in understanding disease pathogenesis, and thus, therapeutic management of patients with IBD.
Th17 cells are implicated in host defence and autoimmune diseases. CD28/B7 co-stimulation is involved in the induction and progression of autoimmune diseases, but its role in controlling murine Th17 ...cell fate remains to be clarified. We here report that soluble anti-CD28 mAb suppressed the differentiation of anti-CD3-stimulated naïve CD4(+) T cells into IL-17-producing cells. CD28 co-stimulation reduced the frequency of proliferating cells that produce IL-17. We provide evidence for an IL-2 and IFN-gamma-dependent mechanism of CD28-mediated IL-17 suppression. CD28 blockade of Th17 development was correlated with a decrease rather than an increase in the percentage of Foxp3(+) T cells. In APC/T cell co-cultures, mature dendritic cells (DC) were less efficient than immature DC in their ability to support Th17 cell differentiation, while CTLA4-Ig, an agent blocking CD28/B7 and CTLA4/B7 interactions, facilitated both murine and human Th17 differentiation. This study identifies the importance of B7 co-stimulatory molecules in the negative regulation of Th17 development. These unexpected results caution targeting the CD28/B7 pathways in the treatment of human autoimmune diseases.
Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune ...system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides.
In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease.
Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.
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