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Inflammatory mediators such as cytokines and chemokines are crucially involved in the development of abdominal aortic aneurysm (AAA). Here we report that CaCl 2 application into abdominal aorta induces AAA with intra-aortic infiltration of macrophages as well as enhanced expression of chemokine (C-C motif) ligand 3 (CCL3) and MMP-9. Moreover, infiltrating macrophages express C-C chemokine receptor 5 (CCR5, a specific receptor for CCL3) and MMP-9. Both Ccl3 −/− mice and Ccr5 −/− but not Ccr1 −/− mice exhibit exaggerated CaCl 2 -inducced AAA with augmented macrophage infiltration and MMP-9 expression. Similar observations are also obtained on an angiotensin II-induced AAA model. Immunoneutralization of CCL3 mimics the phenotypes observed in CaCl 2 -treated Ccl3 −/− mice. On the contrary, CCL3 treatment attenuates CaCl 2 -induced AAA in both wild-type and Ccl3 −/− mice. Consistently, we find that the CCL3–CCR5 axis suppresses PMA-induced enhancement of MMP-9 expression in macrophages. Thus, CCL3 can be effective to prevent the development of CaCl 2 -induced AAA by suppressing MMP-9 expression. Inflammatory cytokines and chemokines are involved in the development of abdominal aortic aneurysm (AAA). Here the authors show that CCL3 prevents the development of CaCl 2 -induced AAA by suppressing MMP-9 expression.

We investigated the dynamics of the gene expression of M1 and M2 macrophage markers during skin wound healing in mice. Expression of M1-macrophage markers, such as Il12a, Tnf, Il6, Il1b, and Nos2 was upregulated after wounding and peaked at 1 or 3 days after injury, and that of M2-macrophage markers such as Mrc1, Cd163, Ccl17, Arg, and Tgfb1, peaked at 6 days after injury. Consistent with these findings, using triple-color immunofluorescence analysis revealed that F4/80 + CD80 + M1 macrophages were more abundant than F4/80 + CD206 + M2 macrophages on day 3 in mouse wound specimens, and that M2 macrophages were prominently detected in day 6 wounds. For application in forensic practice, we examined macrophage polarization using human wound specimens. The average ratios of CD68 + iNOS + M1 macrophages to CD68 + CD163 + M2 macrophages (M1/M2 ratios) were greater than 2.5 for the wounds aged 2–5 days. Out of 11 wounds aged 1–5 days, five samples had the M1/M2 ratios of > 3.0. These observations propose that the M1/M2 ratios of 3.0 would indicate a wound age of 1–5 days as the forensic opinion. This study showed that M1 and M2 macrophages in human skin wound might be a promising marker for wound age determination.

The potential role of macrophages in pulmonary fibrosis (PF) prompted us to evaluate the roles of CX3CR1, a chemokine receptor abundantly expressed in macrophages during bleomycin (BLM)-induced PF. Intratracheal BLM injection induced infiltration of leukocytes such as macrophages into the lungs, which eventually resulted in fibrosis. CX3CR1 expression was mainly detected in the majority of macrophages and in a small portion of α-smooth muscle actin-positive cells in the lungs, while CX3CL1 was expressed in macrophages. BLM-induced fibrotic changes in the lungs were reduced without any changes in the number of leukocytes in Cx3cr1 −/− mice, as compared with those in the wild-type (WT) mice. However, intrapulmonary CX3CR1 + macrophages displayed pro-fibrotic M2 phenotypes; lack of CX3CR1 skewed their phenotypes toward M1 in BLM-challenged lungs. Moreover, fibrocytes expressed CX3CR1, and were increased in BLM-challenged WT lungs. The number of intrapulmonary fibrocytes was decreased in Cx3cr1 −/− mice. Thus, locally-produced CX3CL1 can promote PF development primarily by attracting CX3CR1-expressing M2 macrophages and fibrocytes into the lungs.

Programmed cell death ligand 1 (PD‐L1) on tumor cells suppresses anti‐tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD‐L1 disruption in ovarian cancer. PD‐L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome editing. PD‐L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD‐L1‐KO ID8‐inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD‐L1‐KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4+ T cells, CD8+ T cells, NK cells and CD11c+ M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD‐L1‐KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon‐γ, tumor‐necrosis factor‐α, interleukin (IL)‐2, IL‐12a, CXCL9 and CXCL10 was significantly stronger, while that of IL‐10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD‐L1‐KO ID8 groups. These results indicate that CRISPR/Cas9‐mediated PD‐L1 disruption on tumor cells promotes anti‐tumor immunity by increasing tumor‐infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD‐L1‐targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer. We established a programmed cell death ligand 1 (PD‐L1) knockout ovarian cancer cell line using the clustered regularly interspaced short palindromic repeats/Cas9 system. We demonstrated that the complete disruption of PD‐L1 on tumor cells promoted anti‐tumor immunity by increasing tumor‐infiltrating lymphocytes and modulating cytokine/chemokine production within the tumor microenvironment, thereby suppressing ovarian cancer progression.

Acute ischemic heart disease (AIHD) is a major cause of sudden cardiac death, yet postmortem diagnosis is difficult, particularly in early-phase ischemic injury or decomposed bodies. We investigated the diagnostic value of two molecular biomarkers—8-hydroxy-2′-deoxyguanosine (8-OHdG), indicating oxidative DNA damage, and N-terminal pro-brain natriuretic peptide (NT-proBNP), reflecting cardiac wall stress—in 67 human forensic autopsy cases (33 AIHD, 34 non-AIHD). Immunohistochemistry revealed significantly greater and more diffuse nuclear 8-OHdG expression in AIHD cardiomyocytes compared with controls. Postmortem blood NT-proBNP levels were also significantly higher in AIHD cases (2389.1 ± 499.6 pg/mL) than in non-AIHD cases (896.1 ± 190.1 pg/mL). No significant correlations were found between 8-OHdG expression and NT-proBNP levels, contraction band necrosis, postmortem interval, age, or sex, suggesting that these markers reflect distinct pathological processes—oxidative injury and hemodynamic stress. Both markers demonstrated postmortem stability, and their combined evaluation improved diagnostic confidence in cases with inconclusive histology. This dual-biomarker approach may enhance the accuracy of AIHD diagnosis in forensic practice and provide complementary insights into the molecular mechanisms underlying sudden cardiac death.

Heme oxygenase-1 (HO-1), an inducible stress-response protein, exerts anti-oxidant and anti-apoptotic effects. However, its significance in forensic diagnosis of acute ischemic heart diseases (AIHD) such as myocardial infarction (MI) is still unknown. We examined the immunohistochemical expression of HO-1 in the heart samples to discuss their forensic significance to determine acute cardiac ischemia. The heart samples were obtained from 23 AIHD cases and 33 non-AIHD cases as controls. HO-1 positive signals in cardiomyocyte nuclear were detected in 78.2% of AIHD cases, however, that were detected in only 24.2% control cases with statistical difference between AIHD and non-AIHD groups. In contrast to HO-1 protein expression, there was no significant difference in the appearance of myoglobin pallor regions and leukocyte infiltration in the hearts between AIHD and non-AIHD groups. From the viewpoints of forensic pathology, intracardiac HO-1 expression would be considered a valuable marker to diagnose AIHD as the cause of death.

Cisplatin is an effective chemotherapeutic agent widely used for the treatment of various solid tumors. However, cisplatin has an important limitation in its use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Thrombomodulin (TM) is well known not only for its role as a cofactor in the clinically important natural anticoagulation pathway but also for its anti-inflammatory properties. Here, we investigated the effects of TM in cisplatin-induced AKI. In mice intraperitoneally injected with 15 mg/kg cisplatin, TM (10 mg/kg) or PBS was administered intravenously at 24 h after cisplatin injection. TM significantly attenuated cisplatin-induced nephrotoxicity with the suppressed elevation of blood urea nitrogen and serum creatinine, and reduced histological damages. Actually, TM treatment significantly alleviated oxidative stress-induced apoptosis by reducing reactive oxygen species (ROS) levels in cisplatin-treated renal proximal tubular epithelial cells (RPTECs) in vitro. Furthermore, TM clarified cisplatin-induced apoptosis by reducing caspase-3 levels. In addition, TM attenuated the endoplasmic reticulum (ER) stress signaling pathway in both renal tissues and RPTECs to protect the kidneys from cisplatin-induced AKI. These findings suggest that TM is a potential protectant against cisplatin-induced nephrotoxicity through suppressing ROS generation and ER stress in response to cisplatin.

Intense neutrophil infiltration into the liver is a characteristic of acetaminophen-induced acute liver injury. Neutrophil elastase is released by neutrophils during inflammation. To elucidate the involvement of neutrophil elastase in acetaminophen-induced liver injury, we investigated the efficacy of a potent and specific neutrophil elastase inhibitor, sivelestat, in mice with acetaminophen-induced acute liver injury. Intraperitoneal administration of 750 mg/kg of acetaminophen caused severe liver damage, such as elevated serum transaminase levels, centrilobular hepatic necrosis, and neutrophil infiltration, with approximately 50% mortality in BALB/c mice within 48 h of administration. However, in mice treated with sivelestat 30 min after the acetaminophen challenge, all mice survived, with reduced serum transaminase elevation and diminished hepatic necrosis. In addition, mice treated with sivelestat had reduced NOS-II expression and hepatic neutrophil infiltration after the acetaminophen challenge. Furthermore, treatment with sivelestat at 3 h after the acetaminophen challenge significantly improved survival. These findings indicate a new clinical application for sivelestat in the treatment of acetaminophen-induced liver failure through mechanisms involving the regulation of neutrophil migration and NO production.

After the ligation of the inferior vena cava (IVC) of wild-type (WT) mice, venous thrombi formed and grew progressively until 5 days and resolved thereafter. Concomitantly, intrathrombotic gene expression of was enhanced later than 5 days after IVC ligation. IL-6 protein expression was detected mainly in F4/80-positive macrophages in thrombus. When -deficient ( ) mice were treated in the same manner, thrombus mass was significantly larger than in WT mice. Moreover, the recovery of thrombosed IVC blood flow was markedly delayed in compared with WT mice. F4/80-positive macrophages in thrombus expressed proteolytic enzymes such as matrix metalloproteinase ( ) , and urokinase-type plasminogen activator ( ); and their mRNA expression was significantly reduced in mice. Consistently, the administration of anti-IL-6 antibody delayed the thrombus resolution in WT mice, whereas IL-6 administration accelerated thrombus resolution in WT and mice. Moreover, IL-6 enhanced , and mRNA expression in WT-derived peritoneal macrophages in a dose-dependent manner; and the enhancement was abrogated by a specific Stat3 inhibitor, Stattic. Thus, IL-6/Stat3 signaling pathway can promote thrombus resolution by enhancing , and expression in macrophages.

BM-derived endothelial progenitor cells (EPCs) are critical and essential for neovascularization in tissue repair and tumorigenesis. EPCs migrate from BM to tissues via the bloodstream, but specific chemotactic cues have not been identified. Here we show in mice that the absence of CCR5 reduced vascular EPC accumulation and neovascularization, but not macrophage recruitment, and eventually delayed healing in wounded skin. When transferred into Ccr5-/- mice, Ccr5+/+ BM cells, but not Ccr5-/- cells, accumulated in the wound site, were incorporated into the vasculature, and restored normal neovascularization. Consistent with these observations, CCL5 induced in vitro EPC migration in a CCR5-dependent manner. Moreover, expression of VEGF and TGF-β was substantially diminished at wound sites in Ccr5-/- mice, which suggests that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair. Taken together, these data identify the CCL5/CCR5 interaction as what we believe to be a novel molecular target for modulation of neovascularization and eventual tissue repair.