Masaya Matsuda

Sphingomyelin synthase 2 (SMS2) produces sphingomyelin from ceramide. In this study, SMS2 deficiency reduced airway accumulation of glycosylated mucins, such as MUC5AC, in a murine model of asthma, prompting investigation of the molecular mechanisms in mucin-forming epithelial cells. In the cell model, SMS2 silencing promoted the conversion of ceramide to glucosylceramide, a precursor of complex glycosphingolipids. This alteration reduced MUC5AC glycosylation, suggesting the potential for biochemical competition between sphingolipids and MUC5AC for glycosylation. Supplying C₆-ceramide, which increases biochemical glycosylation for sphingolipids, reduced glycosylated MUC5AC, but this effect was absent in glucosylceramide synthase-silenced cells. These findings indicate that SMS2 deficiency alters sphingolipid metabolism, reducing MUC5AC glycosylation and maturation, underscoring the role of sphingolipid dynamics in modulating mucus production.

Group 2 innate lymphoid cells (ILC2s) produce large amounts of IL-5, IL-13, and amphiregulin, which are involved in the development of lung fibrosis. Activation of ILC2s is mediated by phosphorylation of STAT5. Although STAT5 has tyrosine and serine phosphorylation sites, the mechanisms responsible for phosphorylating serine residues and their significance in ILC2s remain unclear. The present study demonstrated that cyclin-dependent kinase (CDK) 8 and its paralog CDK19 (CDK8/19) were crucial for the activation of ILC2s in OVA-induced asthmatic BALB/c mice by phosphorylating serine residues of STAT5, leading to lung fibrosis. The following results were obtained: (1) The development of lung fibrosis and the number of ILC2s were significantly ameliorated by the CDK8/19 inhibitor, AS3334366, in OVA-induced asthmatic mice. Consistently, lung fibrosis did not develop in OVA-induced asthmatic ILC2-deficient (Il7rCre/+ Rorafl/fl) mice. (2) IL-33 markedly up-regulated the expression of IL-2 receptor chains, CD25 and CD132, in lung ILC2s. IL-2 significantly augmented the expression of ST2, the high-affinity receptor for IL-33. Furthermore, simultaneous stimulation with IL-33 and IL-2 resulted in the upregulation of CDK8/19 expression in ILC2s. (3) The combined treatment with IL-33 and IL-2 synergistically enhanced the proliferation and cytokine production of ILC2s. Conversely, both these effects, along with the phosphorylation of serine residues in STAT5, were suppressed by AS3334366 in a concentration-dependent manner. The present study is the first to report the involvement of CDK8/19 in the activation of ILC2s via the phosphorylation of STAT5, which led to the development of lung fibrosis.

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid‐resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL‐33, thymic stromal lymphopoietin (TSLP), and/or IL‐7 with or without dexamethasone (10 nM), the pan‐JAK inhibitor, delgocitinib (1–10 000 nM), and/or the Bcl‐xL inhibitor, navitoclax (1–100 nM), followed by the detection of viable and apoptotic cells. The anti‐apoptotic factor, Bcl‐xL was detected in ILC2s by flow cytometry. As a steroid‐resistant asthma model, ovalbumin (OVA)‐sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 μg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3–30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL‐7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL‐7 was performed in the presence of IL‐33, the proliferative response exhibited steroid resistance. Steroid‐resistant ILC2 proliferation was suppressed by delgocitinib in a concentration‐dependent manner. (2) The culture with IL‐33, TSLP, and IL‐7 induced the overexpression of Bcl‐xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose‐dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up‐regulating Bcl‐xL in ILC2s. The inhibition of JAKs and Bcl‐xL has potential as pharmacotherapy for steroid‐resistant asthma, particularly that mediated by ILC2s.

Regulated necrosis, termed necroptosis, represents a potential therapeutic target for refractory cancer. Ceramide nanoliposomes (CNLs), considered potential chemotherapeutic agents, induce necroptosis by targeting the activating protein mixed lineage kinase domain-like protein (MLKL). In the present study, we examined the potential of pronecroptotic therapy using CNLs for refractory triple-negative breast cancer (TNBC), for which there is a lack of definite and effective therapeutic targets among the various immunohistological subtypes of breast cancer. MLKL mRNA expression in tumor tissues was significantly higher in TNBC patients than in those with non-TNBC subtypes. Similarly, among the 50 breast cancer cell lines examined, MLKL expression was higher in TNBC-classified cell lines. TNBC cell lines were more susceptible to the therapeutic effects of CNLs than the non-TNBC subtypes of breast cancer cell lines. In TNBC-classified MDA-MB-231 cells, the knockdown of MLKL suppressed cell death induced by CNLs or the active substance short-chain C₆-ceramide. Accordingly, TNBC cells were prone to CNL-evoked necroptotic cell death. These results will contribute to the development of CNL-based pronecroptotic therapy for TNBC.

Ceramide is a lipid that regulates cell death including apoptosis and necroptosis and acts as a powerful tumor suppressor. These characteristics have supported the development of ceramide-based cancer therapeutics. In the present study, ceramide mimetics (RT-25, RT-71 and YM-07) were designed and chemically synthesized, and their potential as necroptosis-inducing chemotherapeutic reagents was examined. RT-71 and YM-07, but not RT-25, potently decreased cell viability in multiple cancer cell lines. The ranges of IC50 values for RT-71 and YM-07 were 8.4–40.8 µM and 4.4–23.7 µM, respectively. Cleavage of PARP, an apoptosis marker, was not observed in MCF-7 cells treated with the mimetics. To determine the effects on necroptotic cell death, we used siRNAs specific to mixed lineage kinase domain-like protein (MLKL). MLKL knockdown significantly suppressed the cell death induced by YM-07 but not RT-71. These results suggest that YM-07 exhibits pro-necroptotic activity. Taken together, these findings indicate ceramide mimetic YM-07 as a lead compound for developing MLKL-activating necroptotic reagents. Our results help establish a foundation for pro-necroptotic therapy for cancer.

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.