Masahiro Tachi

Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

ABSTRACT Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.

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.

Suppressive effects of diltiazem and verapamil on delayed rectifier K+-channel currents in murine thymocytes

BACKGROUNDnLymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and these channels play crucial roles in the lymphocyte activation and proliferation. Since diltiazem and verapamil, which are highly lipophilic Ca(2+) channel blockers (CCBs), exert relatively stronger immunomodulatory effects than the other types of CCBs, they would affect the Kv1.3-channel currents in lymphocytes.nnnMETHODSnEmploying the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of these drugs on the channel currents and the membrane capacitance.nnnRESULTSnBoth diltiazem and verapamil significantly suppressed the peak and the pulse-end currents of the channels, although the effects of verapamil were more marked than those of diltiazem. Both drugs significantly lowered the membrane capacitance, indicating the interactions between the drugs and the plasma membranes.nnnCONCLUSIONSnThis study demonstrated for the first time that CCBs, such as diltiazem and verapamil, exert inhibitory effects on Kv1.3-channels expressed in lymphocytes. The effects of these drugs may be associated with the mechanisms of immunomodulation by which they decrease the production of inflammatory cytokines.

Olopatadine inhibits exocytosis in rat peritoneal mast cells by counteracting membrane surface deformation.

Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm) in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

Salicylate inhibits thrombopoiesis in rat megakaryocytes by changing the membrane micro-architecture.

Background/Aims: Salicylate causes drug-induced immune thrombocytopenia. However, some clinical studies indicate the presence of additional mechanisms in the drug-induced thrombocytopenia, by which the platelet production from megakaryocytes may directly be affected. Since salicylate is amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membrane, it can induce some structural changes in the megakaryocyte membrane surface and thus affect the process of thrombopoiesis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of salicylate on the membrane capacitance in rat megakaryocytes. Taking electron microscopic imaging of the cellular surface, we also examined the effects of salicylate on the membrane micro-architecture of megakaryocytes. Results: Salicylate significantly decreased the membrane capacitance of megakaryocytes, indicating the decreased number of invaginated plasma membranes, which was not detected by the fluorescent imaging technique. As shown by electron microscopy, salicylate actually halted the process of pro-platelet formation in megakaryocytes. Conclusion: This study demonstrated for the first time that salicylate inhibits the process of thrombopoiesis in megakaryocytes, as detected by the decrease in the membrane capacitance. Salicylate-induced changes in the membrane micro-architecture are thought to be responsible for its effects.

Usefulness of targeting lymphocyte Kv1.3-channels in the treatment of respiratory diseases

T lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes. Patch-clamp studies revealed that the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. Using selective channel inhibitors in experimental animal models, in vivo studies further revealed the clinically relevant relationship between the channel expression and the development of chronic respiratory diseases, in which chronic inflammation or the overstimulation of cellular immunity in the airways is responsible for the pathogenesis. In chronic respiratory diseases, such as chronic obstructive pulmonary disease, asthma, diffuse panbronchiolitis and cystic fibrosis, in addition to the supportive management for the symptoms, the anti-inflammatory effects of macrolide antibiotics were shown to be effective against the over-activation or proliferation of T lymphocytes. Recently, we provided physiological and pharmacological evidence that macrolide antibiotics, together with calcium channel blockers, HMG-CoA reductase inhibitors, and nonsteroidal anti-inflammatory drugs, effectively suppress the Kv1.3-channel currents in lymphocytes, and thus exert anti-inflammatory or immunomodulatory effects. In this review article, based on the findings obtained from recent in vivo and in vitro studies, we address the novel therapeutic implications of targeting the lymphocyte Kv1.3-channels for the treatment of chronic or acute respiratory diseases.

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

OBJECTIVEnChronic 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.nnnMETHODnOpen 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.nnnRESULTSnOur 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.nnnCONCLUSIONnThese 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.

In vivo corrosion behaviour of magnesium alloy in association with surrounding tissue response in rats

Biodegradable magnesium (Mg) alloys are the most promising candidates for osteosynthesis devices. However, their in vivo corrosion behaviour has not been fully elucidated. The aim of this study was to clarify the influence of the physiological environment surrounding Mg alloys on their corrosion behaviour. A Mg-1.0Al alloy with a fine-grained structure was formed into plates using titanium (Ti) as a control. These plates were implanted into the subperiosteum in the head, subcutaneous tissue of the back, and in the muscle of the femur of rats for 1, 2 and 4 weeks. The volumes of the remaining Mg alloy and of the insoluble salt deposition and gas cavities around the Mg alloy were determined by microtomography, and the volume losses were calculated. Then, the tissue response around the plates in each implantation site was examined histopathologically, and its relation to the respective volume loss was analyzed. These analyses determined that the Mg alloy was corroded fastest in the head, at an intermediate level in the back, and slowest in the femur. The insoluble salt deposition at the Mg alloy surface had no influence on the volume loss. Gas cavities formed around the Mg alloy at all implantation sites and decreased after 4 weeks. Histopathological examination revealed that the Mg alloy exhibited good biocompatibility, as was seen with Ti. In addition, vascularized fibrous capsules formed around the plates and became mature with time. Notably, the volume loss in the different anatomical locations correlated with capsule thickness. Together, our results suggest that, to facilitate the successful clinical application of Mg alloys, it will be necessary to further comprehend their interactions with specific in vivo environments.

Mast cell involvement in the progression of peritoneal fibrosis in rats with chronic renal failure

Peritoneal fibrosis is a serious complication in patients with end stage renal disease (ESRD), especially those undergoing long‐term peritoneal dialysis therapy. Since the peritoneum is a major site of mast cell accumulation, and since mast cells are known to facilitate the progression of organ fibrosis, they would also contribute to the pathogenesis of peritoneal fibrosis. The aim of this study was to reveal the involvement of mast cells in the progression of peritoneal fibrosis in chronic renal failure.

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.