Light-induced silencing of neural activity in Rosa26 knock-in and BAC transgenic mice conditionally expressing the microbial halorhodopsin eNpHR3

An engineered light-inducible chloride pump, Natronomonas pharaonis halorhodopsin 3 (eNpHR3) enables temporally and spatially precise inhibition of genetically defined cell populations in the intact nervous tissues. In this report, we show the generation of new mouse strains that express eNpHR3-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to optically inhibit neuronal activity. In these mouse strains, Cre/Flp recombination induced high levels of opsin expression. We confirmed their light-induced activities by brain slice whole-cell patch clamp experiments. eNpHR3-expressing neurons were optically hyperpolarized and silenced from firing action potentials. In prolonged silencing of action potentials, eNpHR3 was superior to eNpHR2, a former version of the engineered pump. Thus, these eNpHR3 mouse strains offer reliable genetic tools for light-induced inhibiting of neuronal activity in defined sets of neurons

H3K9 Demethylases JMJD1A and JMJD1B Control Prospermatogonia to Spermatogonia Transition in Mouse Germline

Histone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles

Neutrophil S100A9 supports M2 macrophage niche formation in granulomas

慢性炎症「肉芽腫」における好中球の新しい炎症制御系の解明 --M2マクロファージの新たな誘導メカニズム解明--. 京都大学プレスリリース. 2023-02-17.In search of inflammatory Achilles heel. 京都大学プレスリリース. 2023-03-10.Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPβ, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas

Tuning of Sry expression by H3K9 methylation and demethylation

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance

Combined Loss of JMJD1A and JMJD1B Reveals Critical Roles for H3K9 Demethylation in the Maintenance of Embryonic Stem Cells and Early Embryogenesis

Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome

Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs

Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3⁺–basal cells (Tbx3⁺-BCs) and their neighboring cells where Adam8–extracellular signal–regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3⁺-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3⁺-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3⁺-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin

Hematopoietic cell-derived IL-15 supports NK cell development in scattered and clustered localization within the bone marrow

骨髄のNK細胞の分化に造血細胞が産生するIL-15が必須である --2種類の局在を示すNK細胞の新規分化モデル--. 京都大学プレスリリース. 2023-09-20.Natural killer (NK) cells are innate immune cells critical for protective immune responses against infection and cancer. Although NK cells differentiate in the bone marrow (BM) in an interleukin-15 (IL-15)-dependent manner, the cellular source of IL-15 remains elusive. Using NK cell reporter mice, we show that NK cells are localized in the BM in scattered and clustered manners. NK cell clusters overlap with monocyte and dendritic cell accumulations, whereas scattered NK cells require CXCR4 signaling. Using cell-specific IL-15-deficient mice, we show that hematopoietic cells, but not stromal cells, support NK cell development in the BM through IL-15. In particular, IL-15 produced by monocytes and dendritic cells appears to contribute to NK cell development. These results demonstrate that hematopoietic cells are the IL-15 niche for NK cell development in the BM and that BM NK cells are present in scattered and clustered compartments by different mechanisms, suggesting their distinct functions in the immune response

A circulating subset of iNKT cells mediates antitumor and antiviral immunity

新規の循環型iNKT細胞を発見 --抗腫瘍・抗ウイルス感染効果の高い免疫細胞療法の開発への貢献に期待--. 京都大学プレスリリース. 2022-10-24.Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244⁻CXCR6⁻), C1 (CD244⁻CXCR6⁺), or C2 (CD244⁺CXCR6⁺) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell–like features, whereas C1 iNKT cells showed more T cell–like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244⁺CXCR6⁺ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell–like properties distinct from conventional tissue-resident iNKT cells

Characterization of the IL-15 niche in primary and secondary lymphoid organs in vivo.

サイトカインIL-15を産生する細胞の可視化に成功 -免疫系の微小環境の解明に期待-. 京都大学プレスリリース. 2014-01-21.IL-15 is a cytokine critical for development, maintenance, and response of T cells, natural killer (NK) cells, NK T cells, and dendritic cells. However, the identity and distribution of IL-15-expressing cells in lymphoid organs are not well understood. To address these questions, we established and analyzed IL-15-CFP knock-in mice. We found that IL-15 was highly expressed in thymic medulla, and medullary thymic epithelial cells with high MHC class II expression were the major source of IL-15. In bone marrow, IL-15 was detected primarily in VCAM-1(+)PDGFRβ(+)CD31(-)Sca-1(-) stromal cells, which corresponded to previously described CXCL12-abundant reticular cells. In lymph nodes, IL-15-expressing cells were mainly distributed in the T-cell zone and medulla. IL-15 was expressed in some fibroblastic reticular cells and gp38(-)CD31(-) double-negative stromal cells in the T-cell zone. Blood endothelial cells, including all high endothelial venules, also expressed high IL-15 levels in lymph nodes, whereas lymphatic endothelial cells (LECs) lacked IL-15 expression. In spleen, IL-15 was expressed in VCAM-1(+) stromal cells, where its expression increased as mice aged. Finally, IL-15 expression in blood and LECs of peripheral lymphoid organs significantly increased in LPS-induced inflammation. Overall, we have identified and characterized several IL-15-expressing cells in primary and secondary lymphoid organs, providing a unique perspective of IL-15 niche in immune microenvironment. This study also suggests that some stromal cells express IL-7 and IL-15 differentially and suggests a way to functionally classify different stromal cell subsets