Hideki Amano

Background/Aim: Hepatic steatosis is a significant independent risk factor for liver surgery because of its vulnerability to ischemia-reperfusion (IR) injury. Invariant natural killer T (iNKT) cells contribute to IR injury in healthy liver. We previously reported that activated iNKT cells mitigate liver IR injury and facilitate liver repair by interacting with macrophages. This study aimed to assess the role of activated iNKT cells in IR injury in steatotic livers. Materials and Methods: Male C57/BL6 mice were fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks before liver IR. iNKT cells were activated by intraperitoneal injection of α-galactosylceramide (α-GC) into HFD-fed mice. This study assessed liver injury, cytokine levels, and immune cell accumulation. Results: HFD-fed mice exhibited increased levels of liver injury, pro-inflammatory mediators, and macrophages compared to those of ND-fed mice. Administration of α-GC to HFD-fed mice enhanced liver IR injury that was associated with increased numbers of iNKT cells and pro-inflammatory macrophages compared with those in the vehicle-treated group. Additionally, liver repair was delayed in α-GC-treated HFD-fed mice, as demonstrated by the increased necrotic area and decreased proliferating cell nuclear antigen expression. This was accompanied by reduced levels of anti-inflammatory mediators and reparative macrophages. Pro-inflammatory cytokine levels were increased in activated hepatic iNKT cells co-cultured with macrophages isolated from HFD-fed mice. Conclusion: The activation of hepatic iNKT cells aggravates steatotic liver IR injury by upregulating pro-inflammatory mediators and macrophages, while suppressing anti-inflammatory mediators and reparative macrophages.

Aim: TAS5315 is a Bruton tyrosine kinase (Btk) inhibitor in development for autoimmune and allergic diseases, including rheumatoid arthritis (RA) and chronic spontaneous urticaria (CSU). Two clinical studies evaluated the pharmacology and safety of single and multiple oral doses of TAS5315.Methods: Two phase 1 studies (single ascending‐dose [SAD] and multiple ascending‐dose [MAD]) assessed the pharmacokinetics (including effect of food), pharmacodynamics (Btk occupancy, inhibition of basophil activation) and safety of TAS5315 (up to 8 mg/day) in healthy males.Results: TAS5315 showed linear pharmacokinetics over a 0.01‐8 mg dose range; maximum plasma concentration and area under the plasma concentration‐time curve were reduced by ~40% by food. TAS5315 had dose dependent effects on Btk and basophil activation. In the SAD study, doses ≥2 mg resulted in mean Btk occupancy of almost 100% at 2 and 6 h, and >80% at 24 h, post‐administration. TAS5315 1‐8 mg/day inhibited basophil activation (mean change from baseline −55% to −89%). TAS5315 was generally tolerable. Although it dose‐dependently reduced platelet aggregation (over 2‐8 mg in both studies) and prolonged bleeding time (1‐8 mg in the MAD study), no relationship between these effects and clinical symptoms was observed. All adverse drug reactions were mild and resolved without treatment; no noteworthy safety concerns were observed in either study.Conclusion: These data indicate TAS5315 has potential as a novel therapeutic for immunological diseases associated with aberrant Btk signalling, including RA and CSU. Further evaluation of TAS5315 is warranted.

Background: Acetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure. Macrophages are key players in liver restoration following APAP-induced liver injury. Thromboxane A₂ (TXA₂) and its receptor, thromboxane prostanoid (TP) receptor, have been shown to be involved in tissue repair. However, whether TP signaling plays a role in liver repair after APAP hepatotoxicity by affecting macrophage function remains unclear. Methods: Male TP knockout (TP^−/−) and C57BL/6 wild-type (WT) mice were treated with APAP (300 mg/kg). In addition, macrophage-specific TP-knockout (TP^△mac) and control WT mice were treated with APAP. We explored changes in liver inflammation, liver repair, and macrophage accumulation in mice treated with APAP. Results: Compared with WT mice, TP^−/− mice showed aggravated liver injury as indicated by increased levels of alanine transaminase (ALT) and necrotic area as well as delayed liver repair as indicated by decreased expression of proliferating cell nuclear antigen (PCNA). Macrophage deletion exacerbated APAP-induced liver injury and impaired liver repair. Transplantation of TP-deficient bone marrow (BM) cells to WT or TP^−/− mice aggravated APAP hepatotoxicity with suppressed accumulation of macrophages, while transplantation of WT-BM cells to WT or TP^−/− mice attenuated APAP-induced liver injury with accumulation of macrophages in the injured regions. Macrophage-specific TP^−/− mice exacerbated liver injury and delayed liver repair, which was associated with increased pro-inflammatory macrophages and decreased reparative macrophages and hepatocyte growth factor (HGF) expression. In vitro, TP signaling facilitated macrophage polarization to a reparative phenotype. Transfer of cultured BM-derived macrophages from control mice to macrophage-specific TP^−/− mice attenuated APAP-induced liver injury and promoted liver repair. HGF treatment mitigated APAP-induced inflammation and promoted liver repair after APAP-induced liver injury. Conclusions: Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury, which is associated with reduced accumulation of reparative macrophages and the hepatotrophic factor HGF. Specific activation of TP signaling in macrophages may be a potential therapeutic target for liver repair and regeneration after APAP hepatotoxicity.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury that induces cytokine hypersecretion. Receptor activity-modifying protein (RAMP) 1, a subunit of the calcitonin gene-related peptide (CGRP) receptor, regulates the production of cytokines. This study examined the role of RAMP1 signaling during lipopolysaccharide (LPS)-induced acute lung injury (ALI). LPS administration to wild-type (WT) mice depleted alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) and neutrophils. RAMP1-deficient (RAMP1^−/−) mice exhibited higher lung injury scores, cytokine levels, and cytokine-producing neutrophil infiltration. RAMP1-deficient AMs produced more cytokines in response to LPS than WT AMs. Adoptive transfer of RAMP1-deficient AMs to RAMP1^−/− mice increased cytokine levels and neutrophil accumulation compared to the transfer of WT AMs. RAMP1^−/− mice had reduced MDM recruitment and lower pro-inflammatory and reparative macrophage profiles. Cultured bone marrow (BM)-derived RAMP1-deficient macrophages stimulated with LPS showed decreased expression of pro-inflammatory and pro-repairing genes. CGRP administration to WT mice reduced cytokine production and neutrophil accumulation. These findings indicate that RAMP1 signaling mitigates LPS-induced ALI by inactivating AMs and promoting inflammatory and repair activities of MDMs. Targeting RAMP1 signaling presents a potential therapeutic approach for the treatment of ARDS.

Endometriosis is highly dependent on angiogenesis and lymphangiogenesis. Prostaglandin E2, an arachidonic acid metabolite, has been shown to promote the formation of new blood and lymphatic vessels. However, the role of another arachidonic acid metabolite, thromboxane A₂ (TXA₂) in angiogenesis and lymphangiogenesis during endometriosis remains largely unexplored. Using a murine model of ectopic endometrial transplantation, fragments from the endometrium of WT donor mice were transplanted into the peritoneal walls of recipient WT mice (WT→WT), resulting in an increase in both the area and density of blood and lymphatic vessels. Upon transplantation of endometrial tissue from thromboxane prostanoid (TP) receptor (TXA₂ receptor)‑deficient (TP^‑/‑) mice into TP^‑/‑ mice (TP^‑/‑→TP^‑/‑), an increase in implant growth, angiogenesis, and lymphangiogenesis were observed along with upregulation of pro‑angiogenic and lymphangiogenic factors, including vascular endothelial growth factors (VEGFs). Similar results were obtained using a thromboxane synthase (TXS) inhibitor in WT→WT mice. Furthermore, TP^‑/‑→TP^‑/‑ mice had a higher number of F4/80⁺ cells than that of WT→WT mice, with increased expression of genes related to the anti‑inflammatory macrophage phenotype in endometrial lesions. In cultured bone marrow (BM)‑derived macrophages, the levels of VEGF‑A, VEGF‑C, and VEGF‑D decreased in a TP‑dependent manner. Furthermore, TP signaling affected the polarization of cultured BM‑derived macrophages to the anti‑inflammatory phenotype. These findings imply that inhibition of TP signaling promotes endometrial implant growth and neovascularization.

Background/Aim: Invariant natural killer T (iNKT) cells are involved in the initiation and resolution of inflammatory responses. We previously reported that activated iNKT cells facilitate liver repair after hepatic ischemia reperfusion (I/R) injury by accelerating macrophage polarization during the early phase of hepatic I/R injury. Upon activation with α-galactosylceramide (α-GalCer), iNKT cell numbers transiently decrease before increasing within 72 h of stimulation. In the present study, we examined the role of expanded hepatic iNKT cells in the late phase of hepatic I/R injury. Materials and Methods: iNKT cells were activated by intraperitoneal injection of α-GalCer in male C57/BL6 mice at the induction of hepatic ischemia followed by reperfusion. Results: Numbers of activated hepatic iNKT cells immediately diminished after hepatic I/R and reached minimal levels at 24 h and 48 h post-reperfusion. Numbers of hepatic iNKT cells then increased at 72 h and 96 h post-reperfusion to levels approximately 2-fold higher than in mice that underwent a sham operation. Liver repair as demonstrated by decreased necrotic area and increased expression of proliferating cell nuclear antigen (PCNA) was enhanced in α-GalCer-treated mice at 96 h post-reperfusion. Interleukin (IL)-13 production by proliferating iNKT cells was observed at 96 h post-reperfusion, which was associated with enhanced liver repair and increased numbers of reparative macrophages. Conclusion: Repopulation of hepatic iNKT cells promotes liver repair by stimulating macrophage phenotype switching in the late phase of hepatic I/R injury.

Background/Aim: Microsomal prostaglandin E synthase-1 (mPGES-1) is an enzyme, which catalyzes the final step of prostaglandin E₂ (PGE₂) synthesis. PGE₂ in involved in wound-induced angiogenesis. Regulatory T cells (Tregs) regulate not only immune tolerance but also tissue repair and angiogenesis. We examined whether the mPGES-1/PGE₂ axis contributes to wound-induced angiogenesis and granulation tissue formation through Treg accumulation. Materials and Methods: The dorsal subcutaneous tissues of male mPGES-1-deficient (mPGES-1^−/−) and C57BL/6 wild-type (WT) mice were implanted with polyurethane sponge disks. Angiogenesis was estimated by determining the wet weight of sponge tissues and the expression of proangiogenic factors including CD31, vascular endothelial growth factor (VEGF), and transforming growth factor β (TGF-β) in granulation tissues. Results: Angiogenesis was suppressed in mPGES-1^−/− mice compared with WT mice, which was associated with attenuated forkhead box P3 (Foxp3) expression and Foxp3⁺ Treg accumulation. The number of cells double-positive for Foxp3/TGFβ and Foxp3/VEGF were lower in mPGES-1^−/− mice than in WT mice. Neutralizing Tregs with antibodies (Abs) against CD25 or folate receptor 4 (FR4) inhibited the Foxp3+ Treg angiogenesis and accumulation in WT mice, but not in mPGES-1^−/− mice. The topical application of PGE₂ into the implanted sponge enhanced angiogenesis and accumulation of Tregs expressing TGFβ and VEGF in WT and mPGES-1^−/− mice. Conclusion: Tregs producing TGFβ and VEGF accumulate in wounds and contribute to angiogenesis through mPGES-1-derived PGE₂. mPGES-1 induction may control angiogenesis in skin wounds by recruiting Tregs.

Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E₂ (PGE₂) induces angiogenesis through the prostaglandin E₂ receptor (EP1–4). Among immune cells, regulatory T cells (Tregs), which inhibit immune responses, have been implicated in angiogenesis, and PGE₂ is known to modulate the function and differentiation of Tregs. We hypothesized that mPGES-1/PGE₂-EP signalling could contribute to recovery from ischaemic conditions by promoting the accumulation of Tregs. Wild-type (WT), mPGES-1-deficient (mPges-1^−/−), and EP4 receptor-deficient (Ep4^−/−) male mice, 6–8 weeks old, were used. Hindlimb ischaemia was induced by femoral artery ligation. Recovery from ischaemia was suppressed in mPges-1^−/− mice and compared with WT mice. The number of accumulated forkhead box protein P3 (FoxP3)⁺ cells in ischaemic muscle tissue was decreased in mPges-1^−/− mice compared with that in WT mice. Expression levels of transforming growth factor-β (TGF-β) and stromal cell derived factor-1 (SDF-1) in ischaemic tissue were also suppressed in mPges-1^−/− mice. The number of accumulated FoxP3⁺ cells and blood flow recovery were suppressed when Tregs were depleted by injecting antibody against folate receptor 4 in WT mice but not in mPges-1^−/− mice. Recovery from ischaemia was significantly suppressed in Ep4^−/− mice compared with that in WT mice. Furthermore, mRNA levels of Foxp3 and Tgf-β were suppressed in Ep4^−/− mice. Moreover, the number of accumulated FoxP3⁺ cells in ischaemic tissue was diminished in Ep4^−/− mice compared with that in Ep4^+/+ mice. These findings suggested that mPGES-1/PGE₂ induced neovascularization from ischaemia via EP4 by promoting the accumulation of Tregs. Highly selective EP4 agonists could be useful for the treatment of peripheral artery disease.