Innate phase production of IFN-γ by memory and effector T cells expressing early activation marker CD69 during infection with Cryptococcus deneoformans in the lungs.

Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.

Differences in clinicopathological characteristics between lipohypertrophy and localized insulin-derived amyloidosis: A scoping review.

Insulin is used as a therapeutic agent in patients with diabetes, and cutaneous lipohypertrophy (LH) and localized insulin-derived amyloidosis (LIDA) are well-known adverse effects associated with insulin injections. The clinical implications, management, assessment methods, and pathological differentiation of LH and LIDA have been recently updated. This review was to update our knowledge of the pathological differentiation, effects of insulin absorption, hypoglycemic events, and recent assessment methods for LH and LIDA. A scoping review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta Analyses extension for Scoping Reviews guidelines. Original studies and case reports in English were also included. PubMed and Scopus databases were searched for keywords to identify papers published up to January 2022. A total of 113 studies were identified through a database search, and 31 were eligible for inclusion in this scoping review. In the 31 studies included in this review, patients with type 2 diabetes had high frequencies of LH and LIDA. LH outcome parameters were assessed using pathological findings and imaging. LIDA is mainly determined by pathological methods, such as hematoxylin and eosin and Congo red staining. Several in vitro and in vivo LIDA models of LIDA have been developed. These results suggest that pathological analysis is required to identify LH and LIDA. It is important to consider LIDA, as it likely influences insulin adsorption and glycemic control. Although several studies have evaluated the LIDA process, little is known about the mechanisms underlying the development of adverse effects associated with insulin injections.

CARD9-mediated macrophage responses and collagen fiber capsule formation caused by textured-type breast implants.

BACKGROUND: An increasing number of women are undergoing breast implantation for cosmetic purposes and for reconstructive purposes after breast excision. The surface morphology of the breast implant is one of the key factors associated with the induction of capsule contraction. The effect of surface morphology on the inflammatory response following implant insertion remains unclear, however. This study conducted comparative analyses to determine the effect of the textured and smooth surface morphology of silicone sheets. METHODS: Each type of silicone sheet was inserted into the subcutaneous pocket below the panniculus carnosus in C57BL/6 mice and mice with genetic disruption of CARD9, Dectin-1, Dectin-2, or Mincle. We also analyzed the collagen fiber capsule thickness, histological findings, and macrophage inflammatory response, including TGF-ß synthesis. RESULTS: We found that textured surface morphology contributed to the formation of collagen fiber capsules and the accumulation of fibroblasts and myofibroblasts, and was accompanied by the accumulation of TGF-ß-expressing macrophages and foreign-body giant cells. CARD9 deficiency attenuated collagen fiber capsule formation, macrophage responses, and TGF-ß synthesis, although the responsible C-type lectin receptors (CLRs) remain to be clarified. CONCLUSIONS: These results suggest that CARD9 may have a strong impact on silicone sheet insertion through the regulation of macrophage responses.

Cutaneous wound healing promoted by topical administration of heat-killed Lactobacillus plantarum KB131 and possible contribution of CARD9-mediated signaling.

Optimal conditions for wound healing require a smooth transition from the early stage of inflammation to proliferation, and during this time alternatively activated (M2) macrophages play a central role. Recently, heat-killed lactic acid bacteria (LAB), such as Lactobacillus plantarum (L. plantarum) have been reported as possible modulators affecting the immune responses in wound healing. However, how signaling molecules regulate this process after the administration of heat-killed LAB remains unclear. In this study, we examined the effect of heat-killed L. plantarum KB131 (KB131) administration on wound healing and the contribution of CARD9, which is an essential signaling adaptor molecule for NF-kB activation upon triggering through C-type lectin receptors, in the effects of this bacterium. We analyzed wound closure, histological findings, and inflammatory responses. We found that administration of KB131 accelerated wound closure, re-epithelialization, granulation area, CD31-positive vessels, and α-SMA-positive myofibroblast accumulated area, as well as the local infiltration of leukocytes. In particular, M2 macrophages were increased, in parallel with CCL5 synthesis. The acceleration of wound healing responses by KB131 was canceled in CARD9-knockout mice. These results indicate that the topical administration of KB131 accelerates wound healing, accompanying increased M2 macrophages, which suggests that CARD9 may be involved in these responses.

Topical Administration of Heat-Killed Enterococcus faecalis Strain KH2 Promotes Re-Epithelialization and Granulation Tissue Formation during Skin Wound-Healing.

Lactic acid bacteria (LAB) are known to have beneficial effects on immune responses when they are orally administered as bacterial products. Although the beneficial effects of LAB have been reported for the genera Lactobacillus and Lactococcus, little has been uncovered on the effects of the genus Enterococcus on skin wound-healing. In this study, we aimed to clarify the effect of heat-killed Enterococcus faecalis KH2 (heat-killed KH2) strain on the wound-healing process and to evaluate the therapeutic potential in chronic skin wounds. We analyzed percent wound closure, re-epithelialization, and granulation area, and cytokine and growth factor production. We found that heat-killed KH2 contributed to the acceleration of re-epithelialization and the formation of granulation tissue by inducing tumor necrosis factor-α, interleukin-6, basic fibroblast growth factor, transforming growth factor (TGF)-ß1, and vascular endothelial growth factor production. In addition, heat-killed KH2 also improved wound closure, which was accompanied by the increased production of TGF-ß1 in diabetic mice. Topical administration of heat-killed KH2 might have therapeutic potential for the treatment of chronic skin wounds in diabetes mellitus. In the present study, we concluded that heat-killed KH2 promoted skin wound-healing through the formation of granulation tissues and the production of inflammatory cytokines and growth factors.

Deficiency of lung-specific claudin-18 leads to aggravated infection with Cryptococcus deneoformans through dysregulation of the microenvironment in lungs.

Cryptococcus deneoformans is an opportunistic fungal pathogen that infects the lungs via airborne transmission and frequently causes fatal meningoencephalitis. Claudins (Cldns), a family of proteins with 27 members found in mammals, form the tight junctions within epithelial cell sheets. Cldn-4 and 18 are highly expressed in airway tissues, yet the roles of these claudins in respiratory infections have not been clarified. In the present study, we analyzed the roles of Cldn-4 and lung-specific Cldn-18 (luCldn-18) in host defense against C. deneoformans infection. luCldn-18-deficient mice exhibited increased susceptibility to pulmonary infection, while Cldn-4-deficient mice had normal fungal clearance. In luCldn-18-deficient mice, production of cytokines including IFN-γ was significantly decreased compared to wild-type mice, although infiltration of inflammatory cells including CD4+ T cells into the alveolar space was significantly increased. In addition, luCldn-18 deficiency led to high K+ ion concentrations in bronchoalveolar lavage fluids and also to alveolus acidification. The fungal replication was significantly enhanced both in acidic culture conditions and in the alveolar spaces of luCldn-18-deficient mice, compared with physiological pH conditions and those of wild-type mice, respectively. These results suggest that luCldn-18 may affect the clinical course of cryptococcal infection indirectly through dysregulation of the alveolar space microenvironment.

Role of Dectin-2 in the Phagocytosis of Cryptococcus neoformans by Dendritic Cells.

The cell walls and capsules of Cryptococcus neoformans, a yeast-type fungal pathogen, are rich in polysaccharides. Dectin-2 is a C-type lectin receptor (CLR) that recognizes high-mannose polysaccharides. Previously, we demonstrated that Dectin-2 is involved in cytokine production by bone marrow-derived dendritic cells (BM-DCs) in response to stimulation with C. neoformans. In the present study, we analyzed the role of Dectin-2 in the phagocytosis of C. neoformans by BM-DCs. The engulfment of this fungus by BM-DCs was significantly decreased in mice lacking Dectin-2 (Dectin-2 knockout [Dectin-2KO]) or caspase recruitment domain-containing protein 9 (CARD9KO), a common adapter molecule that delivers signals triggered by CLRs, compared to wild-type (WT) mice. Phagocytosis was likewise inhibited, to a similar degree, by the inhibition of Syk, a signaling molecule involved in CLR-triggered activation. A PI3K inhibitor, in contrast, completely abrogated the phagocytosis of C. neoformans. Actin polymerization, i.e., conformational changes in cytoskeletons detected at sites of contact with C. neoformans, was also decreased in BM-DCs of Dectin-2KO and CARD9KO mice. Finally, the engulfment of C. neoformans by macrophages was significantly decreased in the lungs of Dectin-2KO mice compared to WT mice. These results suggest that Dectin-2 may play an important role in the actin polymerization and phagocytosis of C. neoformans by DCs, possibly through signaling via CARD9 and a signaling pathway mediated by Syk and PI3K.

Dectin-2-mediated initiation of immune responses caused by influenza virus hemagglutinin.

Antigen-presenting cells express pattern recognition receptors (PRRs), which sense pathogen-associated molecular patterns from microorganisms and lead to the induction of inflammatory responses. C-type lectin receptors (CLRs), the representative PRRs, bind to microbial polysaccharides, among which Dectin-2 and Mincle recognize mannose-containing polysaccharides. Because influenza virus (IFV) hemagglutinin (HA) is rich in mannose polysaccharides, Dectin-2 or Mincle may contribute to the recognition of HA. In this study, we addressed the possible involvement of Dectin-2 and Mincle in the viral recognition and the initiation of cytokine production. Interleukin (IL)-12p40 and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) upon stimulation with HA was significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice whereas there was no difference between WT mice and Mincle KO mice. BM-DCs that were treated with Syk inhibitor resulted in a significant reduction of cytokine production upon stimulation with HA. The treatment of BM-DCs with methyl-α-D-mannopyranoside (ManP) also led to a significant reduction in cytokine production by BM-DCs that were stimulated with HA, except for the A/H1N1pdm09 subtype. IL-12p40 and IL-6 synthesis by BM-DCs was completely diminished upon stimulation with HA treated with concanavalin A (ConA)-bound sepharose beads. Finally, GFP expression was detected in reporter cells that were transfected with the Dectin-2 gene, but not with the Mincle gene, when stimulated with HA derived from the A/H3N2 subtype. These data suggested that Dectin-2 may be a key molecule as the sensor for IFV to initiate the immune response and regulate the pathogenesis of IFV infection.

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds.

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell-deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.

Distinct Roles for Dectin-1 and Dectin-2 in Skin Wound Healing and Neutrophilic Inflammatory Responses.

C-type lectin receptors recognize microbial polysaccharides. The C-type lectin receptors such as dendritic cell-associated C-type lectin (Dectin)-1 and Dectin-2, which are triggered by ß-glucan and α-mannan, respectively, contribute to upregulation of the inflammatory response. Recently, we demonstrated that activation of the Dectin-2 signal delayed wound healing; in previous studies, triggering the Dectin-1 signal promoted this response. However, the precise roles of these C-type lectin receptors in skin wound healing remain unclear. This study was conducted to determine the roles of Dectin-1 and Dectin-2 in skin wound healing, with a particular focus on the kinetics of neutrophilic inflammatory response. Full-thickness wounds were created on the backs of C57BL/6 mice, and the effects of Dectin-1 or Dectin-2 deficiency and those of ß-glucan or α-mannan administration were examined. We also analyzed wound closure, histological findings, and neutrophilic inflammatory response, including neutrophil extracellular trap formation at the wound sites. We found that Dectin-1 contributed to the acceleration of wound healing by inducing early-phase neutrophil accumulation, whereas Dectin-2 was involved in prolonged neutrophilic responses and neutrophil extracellular trap formation, leading to delayed wound healing. Dectin-2 deficiency also improved collagen deposition and TGF-ß1 expression. These results suggest that Dectin-1 and Dectin-2 have different roles in wound healing through their different effects on the neutrophilic response.

Effect of CARD9 Deficiency on Neutrophil-Mediated Host Defense against Pulmonary Infection with Streptococcus pneumoniae.

Streptococcus pneumoniae is a major causative bacterium of community-acquired pneumonia. Dendritic cell-associated C-type lectin-2 (dectin-2), one of the C-type lectin receptors (CLRs), was previously reported to play a pivotal role in host defense against pneumococcal infection through regulating phagocytosis by neutrophils while not being involved in neutrophil accumulation. In the present study, to elucidate the possible contribution of other CLRs to neutrophil accumulation, we examined the role of caspase recruitment domain-containing protein 9 (CARD9), a common adaptor molecule for signal transduction triggered by CLRs, in neutrophilic inflammatory response against pneumococcal infection. Wild-type (WT), CARD9 knockout (KO), and dectin-2 KO mice were infected intratracheally with pneumococcus, and the infected lungs were histopathologically analyzed to assess neutrophil accumulation at 24 h postinfection. Bronchoalveolar lavage fluids (BALFs) were collected at the same time point to count the neutrophils and assess the production of inflammatory cytokines and chemokines. Neutrophil accumulation was significantly decreased in CARD9 KO mice, but not in dectin-2 KO mice. Tumor necrosis factor alpha (TNF-α), keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) production in BALFs were also attenuated in CARD9 KO mice, but not in dectin-2 KO mice. Production of TNF-α and KC by alveolar macrophages stimulated with pneumococcal culture supernatants was significantly attenuated in CARD9 KO mice, but not in dectin-2 KO mice, compared to that in each group's respective control mice. In addition, pneumococcus-infected CARD9 KO mice showed larger bacterial burdens in the lungs than did WT mice. These data indicate that CARD9 is required for neutrophil migration after pneumococcal infection, as well as inflammatory cytokine and chemokine production by alveolar macrophages, and suggest that a CLR distinct from dectin-2 may be involved in this response.

Limited Role of Mincle in the Host Defense against Infection with Cryptococcus deneoformans.

Cryptococcus deneoformans is an opportunistic fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired cell-mediated immune responses such as AIDS. Caspase-associated recruitment domain 9 (CARD9) plays a critical role in the host defense against cryptococcal infection, suggesting the involvement of one or more C-type lectin receptors (CLRs). In the present study, we analyzed the role of macrophage-inducible C-type lectin (Mincle), one of the CLRs, in the host defense against C. deneoformans infection. Mincle expression in the lungs of wild-type (WT) mice was increased in the early stage of cryptococcal infection in a CARD9-dependent manner. In Mincle gene-disrupted (Mincle KO) mice, the clearance of this fungus, pathological findings, Th1/Th2 response, and antimicrobial peptide production in the infected lungs were nearly comparable to those in WT mice. However, the production of interleukin-22 (IL-22), tumor necrosis factor alpha (TNF-α), and IL-6 and the expression of AhR were significantly decreased in the lungs of Mincle KO mice compared to those of WT mice. In in vitro experiments, TNF-α production by bone marrow-derived dendritic cells was significantly decreased in Mincle KO mice. In addition, the disrupted lysates of C. deneoformans, but not those of whole yeast cells, activated Mincle-triggered signaling in an assay with a nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Mincle may be involved in the production of Th22-related cytokines at the early stage of cryptococcal infection, although its role may be limited in the host defense against infection with C. deneoformans.

Production of IL-17A at Innate Immune Phase Leads to Decreased Th1 Immune Response and Attenuated Host Defense against Infection with Cryptococcus deneoformans.

IL-17A is a proinflammatory cytokine produced by many types of innate immune cells and Th17 cells and is involved in the elimination of extracellularly growing microorganisms, yet the role of this cytokine in the host defense against intracellularly growing microorganisms is not well known. Cryptococcus deneoformans is an opportunistic intracellular growth fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired immune responses. In the current study, we analyzed the role of IL-17A in the host defense against C. deneoformans infection. IL-17A was quickly produced by γδT cells at an innate immune phase in infected lungs. In IL-17A gene-disrupted mice, clearance of this fungal pathogen and the host immune response mediated by Th1 cells were significantly accelerated in infected lungs compared with wild-type mice. Similarly, killing of this fungus and production of inducible NO synthase and TNF-α were significantly enhanced in IL-17A gene-disrupted mice. In addition, elimination of this fungal pathogen, Th1 response, and expression of IL-12Rß2 and IFN-γ in NK and NKT cells were significantly suppressed by treatment with rIL-17A. The production of IL-12p40 and TNF-α from bone marrow-derived dendritic cells stimulated with C. deneoformans was significantly suppressed by rIL-17A. In addition, rIL-17A attenuated Th1 cell differentiation in splenocytes from transgenic mice highly expressing TCR for mannoprotein 98, a cryptococcal Ag, upon stimulation with recombinant mannoprotein 98. These data suggest that IL-17A may be involved in the negative regulation of the local host defense against C. deneoformans infection through suppression of the Th1 response.

Novel Toll-Like Receptor 9 Agonist Derived from Cryptococcus neoformans Attenuates Allergic Inflammation Leading to Asthma Onset in Mice.

INTRODUCTION: The enhanced type 2 helper (Th2) immune response is responsible for the pathogenesis of allergic asthma. To suppress the enhanced Th2 immune response, activation of the Th1 immune response has been an alternative strategy for anti-asthma therapy. In this context, effective Th1-inducing adjuvants that inhibit the development of allergic asthma but do not flare the side effects of the primary agent are required in clinical treatment and preventive medicine. OBJECTIVE: In this study, we aimed to determine the regulation of the Th2 type immune response in asthma by a novel immunostimulatory oligodeoxynucleotide (ODN) derived from Cryptococcus neoformans, termed ODN112, which contains a cytosine-guanine (CG) sequence but not canonical CpG motifs. METHODS: Using an ovalbumin-induced asthma mouse model, we assessed the effect of ODN112 on prototypical asthma-related features in the lung and on the Th1/Th2 profile in the lymph nodes and lung of mice treated with ODN112 during sensitization. RESULTS AND CONCLUSION: ODN112 treatment attenuated asthma features in mice. In the bronchial lymph nodes of the lungs and in the spleen, ODN112 increased interferon-γ production and attenuated Th2 recall responses. In dendritic cells (DCs) after allergen sensitization, ODN112 enhanced cluster of differentiation (CD) 40 and CD80 expression but did not alter CD86 expression. Interleukin-12p40 production from DCs was also increased in a Th2-polarizing condition. Our results suggest that ODN112 is a potential Th1-inducing adjuvant during Th2 cell differentiation in the sensitization phase.

Defect of Interferon γ Leads to Impaired Wound Healing through Prolonged Neutrophilic Inflammatory Response and Enhanced MMP-2 Activation.

Interferon (IFN)-γ is mainly secreted by CD4+ T helper 1 (Th1), natural killer (NK) and NKT cells after skin injury. Although IFN-γ is well known regarding its inhibitory effects on collagen synthesis by fibroblasts in vitro, information is limited regarding its role in wound healing in vivo. In the present study, we analyzed how the defect of IFN-γ affects wound healing. Full-thickness wounds were created on the backs of wild type (WT) C57BL/6 and IFN-γ-deficient (KO) mice. We analyzed the percent wound closure, wound breaking strength, accumulation of leukocytes, and expression levels of COL1A1, COL3A1, and matrix metalloproteinases (MMPs). IFN-γKO mice exhibited significant attenuation in wound closure on Day 10 and wound breaking strength on Day 14 after wound creation, characteristics that are associated with prolonged neutrophil accumulation. Expression levels of COL1A1 and COL3A1 mRNA were lower in IFN-γKO than in WT mice, whereas expression levels of MMP-2 (gelatinase) mRNA were significantly greater in IFN-γKO than in WT mice. Moreover, under neutropenic conditions created with anti-Gr-1 monoclonal antibodies, wound closure in IFN-γKO mice was recovered through low MMP-2 expression levels. These results suggest that IFN-γ may be involved in the proliferation and maturation stages of wound healing through the regulation of neutrophilic inflammatory responses.

TNF Receptor-Associated Factor 5 Limits Function of Plasmacytoid Dendritic Cells by Controlling IFN Regulatory Factor 5 Expression.

The physiological functions of TNF receptor-associated factor 5 (TRAF5) in the skin inflammation and wound healing process are not well characterized. We found that Traf5 -/- mice exhibited an accelerated skin wound healing as compared with wild-type counterparts. The augmented wound closure in Traf5 -/- mice was associated with a massive accumulation of plasmacytoid dendritic cells (pDCs) into skin wounds and an enhanced expression of genes related to wound repair at skin sites. In accordance with this result, adoptive transfer of Traf5 -/- pDCs, but not wild-type pDCs, into the injured skin area in wild-type recipient mice significantly promoted skin wound healing. The expression of skin-tropic chemokine receptor CXCR3 was significantly upregulated in Traf5-/- pDCs, and treatment with a CXCR3 inhibitor cancelled the promoted wound healing in Traf5-/- mice, suggesting a pivotal role of CXCR3 in pDC-dependent wound healing. Traf5 -/- pDCs displayed significantly higher expression of IFN regulatory factor 5 (IRF5), which correlated with greater induction of proinflammatory cytokine genes and CXCR3 protein after stimulation with TLR ligands. Consistently, transduction of exogeneous TRAF5 in Traf5-/- pDCs normalized the levels of abnormally elevated proinflammatory molecules, including IRF5 and CXCR3. Furthermore, knockdown of IRF5 also rescued the abnormal phenotypes of Traf5-/- pDCs. Therefore, the higher expression and induction of IRF5 in Traf5-/- pDCs causes proinflammatory and skin-tropic characteristics of the pDCs, which may accelerate skin wound healing responses. Collectively, our results uncover a novel role of TRAF5 in skin wound healing that is mediated by IRF5-dependent function of pDCs.

Dectin-2-Mediated Signaling Leads to Delayed Skin Wound Healing through Enhanced Neutrophilic Inflammatory Response and Neutrophil Extracellular Trap Formation.

Dendritic cell-associated C-type lectin-2 (i.e., dectin-2) recognizes fungal polysaccharides, including α-mannan. Dectin-2-mediated recognition of fungi, such as Candida albicans, leads to NF-κB activation, which induces production of inflammatory cytokines. However, the role of dectin-2 in skin wound healing remains unclear. In this study, we sought to determine how dectin-2 deficiency and the administration of α-mannan affected the wound healing process. Full-thickness wounds were created on the backs of wild type C57BL/6 and dectin-2-deficient mice. We analyzed wound closure, histological findings, and re-epithelialization. We also examined the neutrophilic inflammatory responses and neutrophil extracellular trap (NET)-osis at the wound sites after administration of α-mannan. The percent wound closure and re-epithelialization was significantly accelerated in dectin-2-knockout mice compared with wild-type mice on days 3 and 5 after wounding. In contrast, administration of α-mannan delayed wound closure in wild-type mice, and these responses were canceled in dectin-2-knockout mice. Furthermore, mice administered α-mannan, neutrophil infiltration was prolonged, and the expression of citrullinated histone, an indicator of NETosis, at the wound sites was accelerated. Administration of a neutrophil elastase inhibitor significantly improved the delayed wound healing caused by α-mannan. These results suggest that dectin-2 may have a deep impact on the skin wound healing process through regulation of neutrophilic responses.

Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α-galactosylceramide (α-GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α-GalCer/LPS-treated mice, the number of CD4+ CD25+ Foxp3+ regulatory T (Treg) cells and the expression of a Treg cell-tropic chemokine, secondary lymphoid-tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α-GalCer/LPS-treated mice. Treatment with anti-IFN-γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α-GalCer/LPS-treated mice, whereas giving recombinant (r)IFN-γ reduced the number of Treg cells in mice treated with LPS alone. IL-10 production was significantly lower in α-GalCer/LPS-treated mice than in mice treated with LPS alone. Giving rIL-10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL-1ß, IL-6, TNF-α, and IFN-γ) and chemokines (including MCP-1, RANTES, IP-10, Mig, MIP-2, and KC) in α-GalCer/LPS-treated mice. Treatment with anti-IFN-γ mAb enhanced IL-10 production in α-GalCer/LPS-treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL-10.

Invariant NKT cells promote skin wound healing by preventing a prolonged neutrophilic inflammatory response.

The wound-healing process consists of the inflammation, proliferation, and remodeling phases. In chronic wounds, the inflammation phase is prolonged with persistent neutrophil infiltration. The inflammatory response is critically regulated by cytokines and chemokines that are secreted from various immune cells. Recently, we showed that skin wound healing was delayed and the healing process was impaired under conditions lacking invariant natural killer T (iNKT) cells, an innate immune lymphocyte with potent immuno-regulatory activity. In the present study, we investigated the effect of iNKT cell deficiency on the neutrophilic inflammatory response during the wound healing process. Neutrophil infiltration was prolonged in wound tissue in mice genetically lacking iNKT cells (Jα18KO mice) than in wild-type (WT) control mice on days 1 and 3 after wounding. MIP-2, KC, and IL-17A were produced at a significantly higher level in Jα18KO mice than in WT mice. In addition, neutrophil apoptosis was significantly reduced in the wound tissue in Jα18KO mice than in WT mice. Treatment with anti-IL-17A mAb, anti-Gr-1 mAb, or neutrophil elastase inhibitor reversed the impaired wound healing in Jα18KO mice. These results suggest that iNKT cells may promote the wound healing process through preventing the prolonged inflammatory response mediated by neutrophils.

Contribution of CARD9-mediated signalling to wound healing in skin.

The inflammatory response after skin injury involves the secretion of a variety of cytokines and growth factors that are necessary for tissue repair. Caspase recruitment domain-containing protein 9 (CARD9) is an essential signalling adaptor molecule for NF-κB activation upon triggering through C-type lectin receptors (CLRs), which are expressed in macrophages and dendritic cells. However, the role of CARD9 in inflammatory responses at the wound site has not been elucidated. In this study, we analysed the role of CARD9 in the healing process of skin wounds. Wounds were created on the backs of wild-type (WT) C57BL/6 mice and CARD9 gene-disrupted (knockout [KO]) mice. We analysed per cent wound closure, and the wound tissues were harvested for analysis of leucocyte accumulation and cytokine and chemokine expressions. CARD9KO mice exhibited significant attenuation of wound closure compared with WT mice on days 5, 7 and 10 postwounding, which was associated with decreased macrophage accumulation and reduced TNF-α, IL-1ß, CCL3 and CCL4 expressions. These results suggest that CARD9 may be involved in the wound-healing process through the regulation of macrophage-mediated inflammatory responses.