Hiromasa Tanno

Wound healing in skin promoted by inoculation with Pseudomonas aeruginosaPAO1: The critical role of tumor necrosis factor‐α secreted from infiltrating neutrophils

Wound healing is promoted by the presence of replicating microorganisms adhering to the wounded tissue, but the precise mechanism is not fully understood. In the present study, using a rat model with full‐thickness dermal wounds, we examined the effect of Pseudomonas aeruginosa inoculation on wound healing and the role of neutrophils infiltrating the wound site. Within 3 days, inoculation with this bacterium had accelerated re‐epithelialization, epidermal cell proliferation, and neo‐vascularization, as well as the local infiltration of neutrophils, which reached a peak at 24 hours. Tumor necrosis factor (TNF)‐α was detected in the wound tissues on the mRNA and protein levels within 24 hours. Flow cytometry and immunohistochemical analyses detected higher levels of TNF‐α in the infiltrating neutrophils in rats inoculated with P. aeruginosa than in uninoculated rats. Neutropenic rats treated with anti‐neutrophil mAb or cyclophosphamide exhibited significant attenuation in re‐epithelialization, epidermal cell proliferation, neo‐vascularization, and TNF‐α synthesis compared with control; administration of TNF‐α reversed these attenuations. These wound‐healing responses were decelerated in rats treated with anti‐TNF‐α mAb, as was the infiltration of neutrophils. These results indicate that inoculation with P. aeruginosa promotes wound healing by inducing the infiltration of neutrophils, which play a critical role as a major source of TNF‐α.

IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil‐derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin‐17A (IL‐17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL‐17A production affected the wound healing in skin. IL‐17A‐knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild‐type (WT) mice. In contrast, the administration of recombinant IL‐17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL‐17A‐administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL‐17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL‐17A may be associated with impaired wound healing in skin.

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

Reconsideration of iodine in wound irrigation: the effects on Pseudomonas aeruginosa biofilm formation

OBJECTIVE Chronic skin wounds are usually colonised with bacteria and subsequent infection may develop. Topical antiseptics are commonly used to control bacterial colonisation. The topical antiseptic, 1% polyvinylpyrrolidone-iodine (PVP-I), that is used on chronic open skin wounds remains controversial in the clinical setting because of its cytotoxicity. Here, we tested 1% PVP-I solution against saline to determine if it reduces bacterial count on the wound surface and within the tissue that may lead to wound reduction. METHOD Open wounds that were created on the backs of Sprague Dawley rats were inoculated with Pseudomonas aeruginosa at the wound surface. Wounds were kept covered except for wound irrigation, two days post-wounding, wounds were irrigated daily using a 10ml syringe and spray tip. RESULTS Our results indicate that 1% PVP-I irrigation resulted in a reduced bacterial count on the wound surface and within the tissue compared with saline irrigation. The 1% PVP-I irrigation promoted wound re-epithelialisation compared with saline irrigation, but it did not reach significance. CONCLUSION These results indicated that irrigation with 1% PVP-I was an effective way to reduce bacterial count on the wound surface, and allow the wound to progress to healing.

Contribution of Invariant Natural Killer T Cells to Skin Wound Healing

In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase.

Neutrophil-derived tumor necrosis factor-α contributes to acute wound healing promoted by N-(3-oxododecanoyl)-l-homoserine lactone from Pseudomonas aeruginosa

BACKGROUND Pseudomonas aeruginosa is frequently isolated from chronic wounds and causes serious infection in immunocompromised hosts. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) is synthesized by an autoinducer synthase encoded by the bacterial lasI gene in P. aeruginosa, which regulates the production of virulence factors and biofilm formation in this bacterium. Recent studies have suggested that 3-oxo-C12-HSL contributes to the modulation of immune responses. However, the effect of this molecule on wound healing in P. aeruginosa infection remains to be elucidated. OBJECTIVE We used an animal model to study the effect of 3-oxo-C12-HSL on wound healing in skin infected with P. aeruginosa. METHODS Wounds were created on the backs of Sprague-Dawley (SD) rats and the P. aeruginosa strain PAO1 (PAO1) or its lasI deletion mutant (ΔlasI) was inoculated onto the wound surface. To examine the biological activity of 3-oxo-C12-HSL, rats were injected intraperitoneally with anti-3-oxo-C12-HSL antiserum or administered 3-oxo-C12-HSL at the wound surface. The wound tissues were harvested for analysis of the healing process and inflammatory response. RESULTS PAO1 inoculation significantly accelerated the wound healing and inflammatory response on day 3 post-wounding. These responses were reversed by inoculation with ΔlasI instead of PAO1 or treatment with anti-3-oxo-C12-HSL antiserum. In contrast, administration of 3-oxo-C12-HSL in the absence of PAO1 significantly promoted these responses, which were suppressed by the anti-TNF-α mAb. CONCLUSION These results strongly suggest that 3-oxo-C12-HSL may be involved in healing wounds infected with P. aeruginosa through induction of inflammatory responses.

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‐1β, 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.

Dectin-2-mediated signaling leads to delayed skin wound healing through enhanced neutrophilic inflammatory response and neutrophil extracellular traps 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.

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.

Promotion of acute-phase skin wound healing by Pseudomonas aeruginosa C4 -HSL.

A Pseudomonas aeruginosa quorum‐sensing system, which produces N‐(3‐oxododecanoyl)‐l‐homoserine lactone (3‐oxo‐C12‐HSL) and N‐butanoyl‐l‐homoserine lactone (C4‐HSL), regulates the virulence factors. In our previous study, 3‐oxo‐C12‐HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C4‐HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C4‐HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague–Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti‐C4‐HSL antiserum or treated with C4‐HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF‐α synthesis. These responses were reversed, except for TNF‐α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co‐treated with PAO1 and anti‐C4‐HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF‐α synthesis, were accelerated when C4‐HSL was administered in the absence of PAO1. This acceleration was not affected by anti‐TNF‐α antibody. These results suggest that C4‐HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation.