Yuumi Nakamura

NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense

Intestinal phagocytes transport oral antigens and promote immune tolerance, but their role in innate immune responses remains unclear. Here we found that intestinal phagocytes were anergic to ligands for Toll-like receptors (TLRs) or commensals but constitutively expressed the precursor to interleukin 1β (pro-IL-1β). After infection with pathogenic Salmonella or Pseudomonas, intestinal phagocytes produced mature IL-1β through the NLRC4 inflammasome but did not produce tumor necrosis factor (TNF) or IL-6. BALB/c mice deficient in NLRC4 or the IL-1 receptor were highly susceptible to orogastric but not intraperitoneal infection with Salmonella. That enhanced lethality was preceded by impaired expression of endothelial adhesion molecules, lower neutrophil recruitment and poor intestinal pathogen clearance. Thus, NLRC4-dependent production of IL-1β by intestinal phagocytes represents a specific response that discriminates pathogenic bacteria from commensal bacteria and contributes to host defense in the intestine.

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells

Atopic dermatitis is a chronic inflammatory skin disease that affects 15–30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca2+) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in KitW-sh/W-sh mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells.

Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin‐derived cultured mast cells (FSMC) and bone marrow‐derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and FcϵRI aggregation in bone marrow‐derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN‐deficient (OPN–/–) and ‐sufficient (OPN+/+) mice without significant differences in yield, purity, granularity, and viability. Using OPN–/– FSMC, we found that recombinant OPN augmented IgE‐mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin αv). IgE‐mediated passive cutaneous anaphylaxis was significantly reduced in OPN–/– mice compared with OPN+/+ mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell‐related pathological conditions.

3,4-Methylenedioxy-β-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome

Background: The NLRP3 inflammasome is a critical component of the innate immune system, and its malfunction contributes to the pathogenesis of inflammatory diseases. Results: Nitrostyrene specifically inhibits NLRP3 activation. Conclusion: Nitrostyrene blocks the assembly of NLRP3 inflammasome by inhibiting NLRP3 ATPase activity. Significance: We identify a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation. The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.

Critical Role for Mast Cells in Interleukin-1β-Driven Skin Inflammation Associated with an Activating Mutation in the Nlrp3 Protein

Cryopyrin-associated periodic syndromes (CAPS) are caused by aberrant interleukin-1β (IL-1β) production induced by mutations in the NLRP3 protein in humans, but the mechanisms involved remain poorly understood. Using a mouse model, we show a role for the indigenous microbiota and mast cells (MCs) in skin disease associated with mutant Nlrp3 protein. Unlike normal cells, MCs expressing mutant Nlrp3 produced IL-1β in response to lipopolysaccharide or tumor necrosis factor-α (TNF-α). In neonatal mice, the microbiota induced TNF-α and IL-1β and promoted skin disease. MC deficiency greatly reduced disease in Nlrp3 mutant mice, and reconstitution of MC-deficient mice with mutant MCs restored skin disease, which required the expression of IL-1β in MCs. Surprisingly, neutralization of TNF-α abrogated IL-1β production and skin disease in neonatal Nlrp3 mutant mice, but not in affected adult mice. Thus, the microbiota and MCs initiate cellular events leading to dysregulated IL-1β production and skin inflammation in neonatal mice with the CAPS-associated Nlrp3 mutation.

Significant Virus Replication in Langerhans Cells following Application of HIV to Abraded Skin: Relevance to Occupational Transmission of HIV

The cellular events that occur following occupational percutaneous exposure to HIV have not been defined. In this study, we studied relevant host cellular and molecular targets used for acquisition of HIV infection using split-thickness human skin explants. Blockade of CD4 or CCR5 before R5 HIV application to the epithelial surface of skin explants completely blocked subsequent HIV transmission from skin emigrants to allogeneic T cells, whereas preincubation with C-type lectin receptor inhibitors did not. Immunomagnetic bead depletion studies demonstrated that epithelial Langerhans cells (LC) accounted for >95% of HIV dissemination. When skin explants were exposed to HIV variants engineered to express GFP during productive infection, GFP+ T cells were found adjacent to GFP+ LC. In three distinct dendritic cell (DC) subsets identified among skin emigrants (CD1a+langerin+DC-specific intercellular adhesion molecule grabbing non-integrin (SIGN)− LC, CD1a+langerin−DC-SIGN− dermal DC, and CD1a−langerin−DC-SIGN+ dermal macrophages), HIV infection was detected only in LC. These results suggest that productive HIV infection of LC plays a critical role in virus dissemination from epithelium to cells located within subepithelial tissue. Thus, initiation of antiretroviral drugs soon after percutaneous HIV exposure may not prevent infection of LC, which is likely to occur rapidly, but may prevent or limit subsequent LC-mediated infection of T cells.

Severe dermatitis with loss of epidermal Langerhans cells in human and mouse zinc deficiency

Zinc deficiency can be an inherited disorder, in which case it is known as acrodermatitis enteropathica (AE), or an acquired disorder caused by low dietary intake of zinc. Even though zinc deficiency diminishes cellular and humoral immunity, patients develop immunostimulating skin inflammation. Here, we have demonstrated that despite diminished allergic contact dermatitis in mice fed a zinc-deficient (ZD) diet, irritant contact dermatitis (ICD) in these mice was more severe and prolonged than that in controls. Further, histological examination of ICD lesions in ZD mice revealed subcorneal vacuolization and epidermal pallor, histological features of AE. Consistent with the fact that ATP release from chemically injured keratinocytes serves as a causative mediator of ICD, we found that the severe ICD response in ZD mice was attenuated by local injection of soluble nucleoside triphosphate diphosphohydrolase. In addition, skin tissue from ZD mice with ICD showed increased levels of ATP, as did cultured wild-type keratinocytes treated with chemical irritants and the zinc-chelating reagent TPEN. Interestingly, numbers of epidermal Langerhans cells (LCs), which play a protective role against ATP-mediated inflammatory signals, were decreased in ZD mice as well as samples from ZD patients. These findings suggest that upon exposure to irritants, aberrant ATP release from keratinocytes and impaired LC-dependent hydrolysis of nucleotides may be important in the pathogenesis of AE.

The expression of nectin‐1α in normal human skin and various skin tumours

Summary Background A novel cell–cell adhesion system that consists of nectin and afadin has been identified at cadherin‐based cell–cell adherens junctions. Nectin is a Ca2+‐independent homophilic and heterophilic cell adhesion molecule that belongs to the immunoglobulin superfamily. Nectin has recently been shown to serve as an α‐herpesvirus entry and cell–cell spread mediator. In spite of the ubiquitous expression of nectin‐1α, its detailed localization in human skin has not been examined so far.

The Inflammasome, an Innate Immunity Guardian, Participates in Skin Urticarial Reactions and Contact Hypersensitivity

Urticarial rash, one of the clinical manifestations characteristic of cryopyrin-associated periodic syndrome (CAPS), is caused by a mutation in the gene encoding for NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeats containing family, pyrin domain containing 3). This intracellular pattern recognition receptor and its adaptor protein, called apoptosis associated speck-like protein containing a caspase-recruitment and activating domain (ASC), participate in the formation of a multi-protein complex termed the inflammasome. The inflammasome is responsible for activating caspase-1 in response to microbial and endogenous stimuli. From the analysis of cellular mechanisms of urticarial rash in CAPS, we have traced caspase-1 activated IL-1beta in CAPS to a surprising source: mast cells. Recently, two groups have generated gene-targeted mice that harbored Nlrp3 mutations. These mice had very severe phenotypes, with delayed growth and the development of dermatitis, but not urticaria. The reason for the differences in the skin manifestations observed with CAPS and these knock-in mice relates to the findings that the inflammasome also plays a role in contact hypersensitivity, and that IL-18, another cytokine involved with inflammasome-activation of caspase-1, may be a major player in dermatitis development.

Erythema elevatum diutinum with primary Sjögren syndrome associated with IgA antineutrophil cytoplasmic antibody

SIR, Erythema elevatum diutinum (EED) is a rare chronic neutrophilic dermatosis clinically characterized by violaceous plaques and nodules that usually have a symmetrical and acral distribution. The origin of EED remains unknown. However, EED has been reported in association with autoimmune diseases and haematological abnormalities, among which polyclonal IgA gammopathies are the most commonly observed. Moreover, 50% of patients with EED with IgA polyclonal gammopathies go on to develop haematological diseases such as myelodysplasia, multiple myeloma or monoclonal IgA gammopathy. Histopathologically, EED typically shows a leucocytoclastic vasculitis that manifests as mild eosinophilic infiltrates in early lesions, and granulocytic or fibrotic replacement of the dermis in later lesions. Direct immunofluorescence studies reveal perivascular deposits of immune complexes composed of IgA, IgG, IgM, fibrin and complement components. The presence of IgA antineutrophil cytoplasmic antibody (ANCA) has recently been reported to be a useful clinical marker for EED. Here we report the first case of EED with primary Sjögren syndrome (SjS) in which IgA ANCA was detected. A 64-year-old Japanese woman presented at our clinic with a 1-month history of violaceous plaques. Multiple, elastic, soft, violaceous plaques and nodules were observed on the dorsum of her left great toe and both lower legs (Fig. 1a). She had a 16-year history of primary SjS treated with lowdose oral steroids (prednisolone 10 mg daily). Apart from her skin lesions, physical examination was unremarkable. Abnormal laboratory findings included polyclonal IgG (3374 mg dL; normal 870–1700) and IgA (1699 mg dL; normal 110–410) and an elevated erythrocyte sedimentation rate (55 mm in the first hour). C-reactive protein, rheumatoid factor and serum IgG classes (both myeloperoxidaseand proteinase 3-) ANCA were negative. The blood cell count and blood chemistry were within normal range. Clinically, her skin lesions showed typical features of EED. To exclude differential diagnoses (e.g. Sweet syndrome, malignant lymphoma and sarcoidosis), skin biopsy was performed. This showed a dense, predominantly perivascular infiltrate of neutrophils intermingled with a small number of eosinophils in association with perivascular fibrin deposits and swollen endothelial cells. These findings confirmed a leucocytoclastic vasculitis (Fig. 1b, c). Based on the clinical and histopathological findings, we diagnosed this case as EED. Administration of oral