Characterization of human epithelial resident memory regulatory T cells.

Human resident memory regulatory T cells (Tregs) exist in the normal, noninflamed skin. Except one, all previous studies analyzed skin Tregs using full-thickness human skin. Considering that thick dermis contains more Tregs than thin epidermis, the current understanding of skin Tregs might be biased toward dermal Tregs. Therefore, we sought to determine the phenotype and function of human epidermal and epithelial Tregs. Human epidermis and epithelium were allowed to float on a medium without adding any exogenous cytokines and stimulations for two days and then emigrants from the explants were analyzed. Foxp3 was selectively expressed in CD4+CD103- T cells in the various human epithelia, as it is highly demethylated. CD4+CD103-Foxp3+ cells suppressed proliferation of other resident memory T cells. The generation and maintenance of epithelial Tregs were independent of hair density and Langerhans cells. Collectively, immune-suppressive CD4+CD103-Foxp3+ Tregs are present in the normal, noninflamed human epidermis and mucosal epithelia.

Revisiting the Experimental Methods for Human Skin T-Cell Analysis.

Tissue-resident memory T cells exist in both the epidermis and the dermis in human skin. To analyze these cells, the skin needs to be incubated with dispase II to separate the two layers, that is, the epidermis and the dermis. The next step varies among researchers; the subsequent enzymatic digestion of the two layers is popular, whereas the spontaneous migration method can also be done. Scraping of these layers to yield skin T cells may reduce antigen modulation. This study aimed to determine each method's limitations. Dispase II incubation itself cleaves T-cell antigens. Therefore, further enzymatic digestion with collagenases strongly cleaves antigens. The scraping method yields skin T cells that are affected by dispase II as it is. However, skin T-cell yield is low. The spontaneous migration method recovers and/or upregulates antigens with T-cell activation and loses ∼20% of T cells in the floating sheets. However, there was no prominent bias regarding CD103 expression between emigrants and the remaining T cells in the sheets. There were 104 and 105 CD3+ T cells per 1 cm2 of the epidermis and upper dermis, respectively. Collectively, each method has strengths and limitations to analyze both the epidermal and dermal T cells.

Intracellular TLRs of Mast Cells in Innate and Acquired Immunity.

Mast cells (MCs) distribute to interface tissues with environment, such as skin, airway, and gut mucosa, thereby functioning as the sentinel against invading allergens and pathogens. To respond to and exclude these external substances promptly, MCs possess granules containing inflammatory mediators, including heparin, proteases, tumor necrosis factor, and histamine, and produce these mediators as a consequence of degranulation within minutes of activation. As a delayed response to external substances, MCs de novo synthesize inflammatory mediators, such as cytokines and chemokines, by sensing pathogen- and damage-associated molecular patterns through their pattern recognition receptors, including Toll-like receptors (TLRs). A substantial number of studies have reported immune responses by MCs through surface TLR signaling, particularly TLR2 and TLR4. However, less attention has been paid to immune responses through nucleic acid-recognizing intracellular TLRs. Among intracellular TLRs, human and rodent MCs express TLR3, TLR7, and TLR9, but not TLR8. Some virus infections modulate intracellular TLR expression in MCs. MC-derived mediators, such as histamine, cysteinyl leukotrienes, LL-37, and the granulocyte-macrophage colony-stimulating factor, have also been reported to modulate intracellular TLR expression in an autocrine and/or paracrine fashion. Synthetic ligands for intracellular TLRs and some viruses are sensed by intracellular TLRs of MCs, leading to the production of inflammatory cytokines and chemokines including type I interferons. These MC responses initiate and facilitate innate responses and the subsequent recruitment of additional innate effector cells. MCs also associate with the regulation of adaptive immunity. In this overview, the expression of intracellular TLRs in MCs and the recognition of pathogens, including viruses, by intracellular TLRs in MCs were critically evaluated.

Neutrophil Extracellular Traps in Skin Diseases.

Neutrophils are the primary innate immune cells, and serve as sentinels for invading pathogens. To this end, neutrophils exert their effector functions via phagocytosis, degranulation, reactive oxygen species generation, and neutrophil extracellular trap (NET) release. Pathogens and pathogen-derived components trigger NET formation, leading to the clearance of pathogens. However, NET formation is also induced by non-related pathogen proteins, such as cytokines and immune complexes. In this regard, NET formation can be induced under both non-sterile and sterile conditions. NETs are enriched by components with potent cytotoxic and inflammatory properties, thereby occasionally damaging tissues and cells and dysregulating immune homeostasis. Research has uncovered the involvement of NETs in the pathogenesis of several connective tissue diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and ANCA-associated vasculitis. In dermatology, several skin diseases clinically develop local or systemic sterile pustules and abscesses. The involvement of neutrophils and subsequent NET formation has recently been elucidated in these skin diseases. Therefore, this review highlights the NETs in these neutrophil-associated diseases.

Neutrophils initiate and exacerbate Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8+ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, which likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.

Joint contractures responsive to immunosuppressive therapy in a girl with childhood-onset systemic sclerosis double-seropositive for rare anti-nucleolar autoantibodies: a case report.

BACKGROUND: Systemic sclerosis (SSc; scleroderma) is an autoimmune connective tissue disease that affects the skin and subcutaneous tissue, in addition to the internal organs of the whole body. Onset in childhood is uncommon; however, both patients with childhood-onset and adult-onset SSc are positive for anti-nuclear antibodies (ANAs).Detection of SSc-related anti-nuclear antibodies is often useful for predicting clinical features, disease course, and outcomes. CASE PRESENTATION: A 5-year-old Japanese female manifested gradually progressive abnormal gait disturbance, regression of motor development, Raynaud's phenomenon, and the shiny appearance of the skin of the face and extremities at age 2. On admission, she presented a mask-like appearance, loss of wrinkles and skin folds, puffy fingers, moderate diffuse scleroderma (18/51 of the modified Rodnan total skin thickness score), and contracture in the ankle and proximal interphalangeal joints. Grossly visible capillary hemorrhage on nail fold and severe abnormal capillaroscopy findings including bleeding, giant loop and disappearance of capillaryconsistent with the late phase in SSc. A skin biopsy showed fibrous thickening of the dermis, entrapment of an eccrine sweat glands, and thickened fiber. Chest high-resolution computed tomographic scanning demonstrated patchy areas of ill-defined air-space opacity and consolidation predominantly involving the posterior basilar aspects of the lower lobes presenting withinterstitial lung disease. Positive ANA (1:160 nucleolar and homogeneous nuclear staining by indirect fluorescent antibody technique) and double-seropositive for anti-Th/To and anti-PM-Scl antibodies were identified. She was diagnosed with diffuse cutaneous SSc based on the Pediatric Rheumatology European Society/American College of Rheumatology/European League Against Rheumatism Provisional Classification Criteria for Juvenile Systemic Sclerosis and was successfully treated with immunosuppressive agents, including methylprednisolone pulses and intravenous cyclophosphamide. CONCLUSIONS: We experienced the first case of juvenile SSc with anti-PM-Scl and anti-Th/To antibodies. ILD was identified as a typical feature of patients with these autoantibodies; however, diffuse cutaneous SSc and joint contraction were uncharacteristically associated. The case showed unexpected clinical findings though the existence of SSc-related autoantibodies aids in determining possible organ involvement and to estimate the children's outcome.

Expression of CD1 molecules and colony-stimulating factor 1 receptor in indeterminate cell histiocytosis.

Indeterminate cell histiocytosis (ICH) and Langerhans cell histiocytosis (LCH) are rare histiocyte proliferating disorders with unknown etiologies. However, both tumor cells immunophenotypically share some features of Langerhans cells (LC), thereby expressing CD1a. Recent transcriptome analysis revealed that circulating CD1c+ myeloid dendritic cells are the potential precursor of LCH tumor cells. LC express CD1a as well as CD1c, but not CD1b. We discovered that both tumor cells express CD1c, but not CD1b, similar to LC. Moreover, like LC, both tumor cells express colony-stimulating factor 1 receptor (CSF-1R). Considering the crucial role of the interleukin (IL)-34/CSF-1R axis for the development and survival of LC, CSF-1R on both tumor cells might facilitate their survival and proliferation in situ. These data provide additional evidence to support the fact that ICH and LCH share immunophenotypical features with LC. In addition, we hypothesized that tumor cells in ICH and LCH survive and proliferate through IL-34-mediated CSF-1R signaling.

Downregulation of integrin-αvß6 on keratinocytes in the scar of lichen planopilaris and folliculitis decalvans: Relevance for the disappearance of epidermal Langerhans cells.

Primary cicatricial alopecia (PCA) is a group of poorly understood mechanisms in which the destruction of hair follicles leads to permanent hair loss. Lichen planopilaris (LPP) is a type of lymphocytic PCA and it has been known for epidermal Langerhans cells (LC) to disappear in the scar of LPP. We also found that epidermal LC also disappeared in the scar of folliculitis decalvans (FD), a type of neutrophilic PCA. Of note was that epidermal LC did not disappear in the scar of discoid lupus erythematosus, another type of lymphocytic PCA, suggesting that LC disappearance in the scar was not always a common feature of PCA. We found that the expression of integrin (ITG)-αvß6 in scar epidermis was significantly diminished in LPP and FD, but not in other PCA and disorders accompanied with scar formation. We also found that exogenous interleukin-1ß and α-interferon downregulated ITG-αvß6 expression in normal human epidermal keratinocytes. These data suggest that downregulation of ITG-αvß6 may be one of the causes of LC disappearance in the scar of LPP and FD.

Biotin Is Required for the Zinc Homeostasis in the Skin.

Patients with biotin deficiency present symptoms that are similar to those in patients with acrodermatitis enteropathica (inherent zinc deficiency). However, the association between biotin and zinc deficiency remains unknown. We have previously shown that epidermal keratinocytes of mice fed zinc-deficient (ZD) diets secreted more adenosine triphosphate (ATP) than those of mice fed zinc-adequate (ZA) diets and that epidermal Langerhans cells are absent in ZD mice. Langerhans cells highly express CD39, which potently hydrolyzes ATP into adenosine monophosphate (AMP). Thus, a lack of Langerhans cells in ZD mice leads to non-hydrolysis of ATP, thereby leading to the development of ATP-mediated irritant contact dermatitis. In this study, we examined if biotin-deficient (BD) mice showed the same underlying mechanisms as those in ZD mice. BD mice showed reduced serum zinc levels, disappearance of epidermal Langerhans cells, and enhanced ATP production in the skin. Consequently, irritant contact dermatitis was significantly enhanced and prolonged in BD mice. In conclusion, the findings of our study showed that biotin deficiency leads to zinc deficiency because of which patients with biotin deficiency show similar symptoms as those with acrodermatitis enteropathica.

Anti-Depressant Fluoxetine Reveals its Therapeutic Effect Via Astrocytes.

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.