Wataru Fujii

The intestinal microbiota is essential for maintaining intestinal homeostasis by regulating mucosal immune responses. Identifying bacterial traits that support stable host interactions is therefore crucial for developing next-generation mucosal probiotics. RNase T2 is a conserved ribonuclease with species-specific functions. In Escherichia coli, RNase T2 (RNase I) suppresses biofilm formation, a key process for intestinal colonization. We previously showed that an RNase I-deficient E. coli (Δrna) strain stably colonizes the mouse intestine. Here, we analyzed the immunogenic properties of the Δrna strain. Heat-killed Δrna induced an early IL-1β and TNF-α-skewed cytokine response in RAW264.7 macrophages, with reduced IL-6 expression compared to wild type. In mice, Δrna colonization caused no intestinal tissue injury but enhanced IL-6 and IL-22 expression in the cecum and colon. Selected TLR-specific reporter assays identified TLR5-dependent signaling driven by increased FliC expression. These findings show that RNase I deficiency modulates immune activation while maintaining non-pathogenic mucosal interactions.

Salty taste is mediated through two distinct transduction pathways: the amiloride‐sensitive and amiloride‐insensitive (AI). Transmembrane channel‐like 4 (TMC4), a voltage‐dependent chloride channel, has recently been identified as playing a critical role in AI‐mediated salt taste responses. Although its functional properties have been demonstrated, the specific taste cell types that express TMC4 have not been fully characterized. To investigate the cellular localization of TMC4, we generated Tmc4 ‐EGFP knock‐in mice and performed immunohistochemical analyses of the taste papillae. By performing multiple fluorescent immunostaining with cell‐type‐specific markers, such as KCNQ1 (type I–III), Gustducin and PLCβ2 (type II), and AADC (type III), together with EGFP, we found that over 98% of EGFP‐positive cells overlapped with the signal of all three taste cell type markers in the circumvallate papillae of Tmc4 ‐EGFP mice. In the fungiform papillae, 95.9% of EGFP‐positive cells colocalized with KCNQ1. These results demonstrate that TMC4 is broadly expressed across all three taste cell types, and suggest the possible involvement of multiple, functionally distinct taste cell populations in AI‐mediated salty taste transduction.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7 Mb and 101.2 Mb on rat chromosome 9. Nucleotide sequence analysis identified 2 bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour‐derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra‐tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.

Lipocalin-type PG D synthase (L-PGDS) has two roles: it can be a PGD synthase, or it can be a carrier protein of hydrophobic small molecules. In this study, we investigated the dual roles of L-PGDS in acute lung injury by using L-PGDS–deficient and point-mutated mice, which lack PGD₂ producibility but maintain lipocalin ability. Hydrochloride (HCl) administration (0.1 M intratracheally for 6 h) caused hemorrhage and dysfunction in the wild-type (WT) mouse lung. These symptoms were accompanied by an increase in PGD₂ production. Both deficiency and point mutation of L-PGDS aggravated the HCl-induced hemorrhage and dysfunction. Although both the gene modifications decreased PGD₂ production, only L-PGDS–deficient mice, but not point mutation mice, lacked protein expressions of L-PGDS in the lungs. In the WT mice, HCl administration caused pulmonary edema, indexed as an increase in lung water content and protein leakage in bronchoalveolar lavage fluid. L-PGDS deficiency and point mutation similarly aggravated edema formation. HCl administration also stimulated mucin production and bronchoalveolar lavage fluid leukocyte infiltration in the WT mouse lungs. Of interest, L-PGDS deficiency, but not point mutation, exacerbated these manifestations. Consistently, only L-PGDS deficiency increased the mRNA expression of IL-33, which stimulates mucin production in the inflamed lung. These results show that L-PGDS attenuated HCl-induced acute lung injury progresses in two different ways: L-PGDS produced PGD₂, which inhibited pulmonary edema formation, whereas its lipocalin ability decreased mucin formation and inflammatory cell infiltration in the inflamed lung. Key Points Point mutation of L-PGDS cysteine 65 decreases PGD₂ production in vivo. L-PGDS attenuates pulmonary edema formation via PGD₂-dependent way. L-PGDS reduces leukocyte infiltration and mucin production via a PGD₂-independent way.

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions ofDLEC1are still unclear. This study investigated the critical role ofDlec1in spermatogenesis and male fertility in mice.Dlec1was significantly expressed in testes, with dominant expression in germ cells. We disruptedDlec1in mice and analysed its function in spermatogenesis and male fertility.Dlec1deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids inDlec1^−/−mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet–Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14⁺ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14⁺ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not leukopenia or hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL. Inflammatory responses are crucial in limiting pathogen infections, whereas they often have detrimental effects to the hosts during infectious diseases. Thus, exploring molecules involved in inflammatory responses and targeting those molecules may contribute to development of novel interventions for symptom management during infectious diseases. Although an inflammation-related protein, MRP14, is associated with a wide range of inflammatory diseases, the functions remain elusive. Therefore, we explored the roles of MRP14 in a protozoan disease called leishmaniasis because its pathology is known to be immune-mediated. Interestingly, in two distinct forms of leishmaniases, i.e., cutaneous leishmaniasis caused by Leishmania major and visceral leishmaniasis caused by L. donovani, the molecule showed distinct roles in protection/pathology; although the molecule was protective against L. major infection, depletion of the molecule resulted in easing the pathology during L. donovani infection. These may prove the complexity of MRP14, but at the same time support understanding of the mechanisms behind the complexity.

CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.