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Background: Sepsis survivors exhibit immune dysregulation that contributes to poor long-term outcomes. Phenotypic and functional alterations within the myeloid compartment are believed to be a contributing factor. Here we dissect the cellular and transcriptional heterogeneity of splenic CD11b + Ly6C high myeloid cells that are expanded in mice that survive the cecal ligation and puncture (CLP) murine model of polymicrobial sepsis to better understand the basis of immune dysregulation in sepsis survivors., Methods: Sham or CLP surgeries were performed on C57BL/6J and BALB/c mice. Four weeks later splenic CD11b + Ly6C high cells from both groups were isolated for phenotypic (flow cytometry) and functional (phagocytosis and glycolysis) characterization and RNA was obtained for single-cell RNA-seq (scRNA-seq) and subsequent analysis., Results: CD11b + Ly6C high cells from sham and CLP surviving mice exhibit phenotypic and functional differences that relate to immune function, some of which are observed in both C57BL/6J and BALB/c strains and others that are not. To dissect disease-specific and strain-specific distinctions within the myeloid compartment, scRNA-seq analysis was performed on CD11b + Ly6C high cells from C57BL/6J and BALB/c sham and CLP mice. Uniform Manifold Approximation and Projection from both strains identified 13 distinct clusters of sorted CD11b + Ly6C high cells demonstrating significant transcriptional heterogeneity and expressing gene signatures corresponding to classical-monocytes, non-classical monocytes, M1- or M2-like macrophages, dendritic-like cells, monocyte-derived dendritic-like cells, and proliferating monocytic myeloid-derived suppressor cells (M-MDSCs). Frequency plots showed that the percentages of proliferating M-MDSCs (clusters 8, 11 and 12) were increased in CLP mice compared to sham mice in both strains. Pathway and UCell score analysis in CLP mice revealed that cell cycle and glycolytic pathways were upregulated in proliferating M-MDSCs in both strains. Notably, granule protease genes were upregulated in M-MDSCs from CLP mice. ScRNA-seq analyses also showed that phagocytic pathways were upregulated in multiple clusters including the classical monocyte cluster, confirming the increased phagocytic capacity in CD11b + Ly6C high cells from CLP mice observed in ex vivo functional assays in C57BL/6J mice., Conclusion: The splenic CD11b + Ly6C high myeloid populations expanded in survivors of CLP sepsis correspond to proliferating cells that have an increased metabolic demand and gene signatures consistent with M-MDSCs, a population known to have immunosuppressive capacity., (© 2024. The Author(s).)

Lymphocyte antigen 6 complex locus K (LY6K) has been demonstrated to play a significant role in cancers and identified as a therapeutic biomarker for head and neck squamous cell carcinoma. However, the role of LY6K in oral squamous cell carcinoma (OSCC) has not been explored. The current study discovered that LY6K was aberrantly upregulated in OSCC cell lines and tissues and that high LY6K expression significantly correlated with poorer survival of OSCC patients. Through stable knockdown of LY6K, we found that the growth, colony formation, migration, and invasion of OSCC cells were substantially suppressed. In addition, tumor growth and lung metastasis in vivo were effectively inhibited by LY6K depletion. Mechanically, LY6K binds with CAV-1 and activates CAV-1-mediated MAPK/ERK signaling to exert its oncogenic effects on OSCC. In addition, LY6K expression in OSCC was discovered to be regulated by FTO-mediated RNA N6-methyladenosine (m 6 A) modification in an IGF2BP1-dependent manner. Generally, LY6K expression was upregulated by FTO-mediated demethylation in OSCC, which promoted the tumorigenesis and metastasis of OSCC via activating the CAV-1-mediated ERK1/2 signaling pathway., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

The immune response gene 1 (IRG1) and its metabolite itaconate are implicated in modulating inflammation and oxidative stress, with potential relevance to sepsis-induced myocardial dysfunction (SIMD). This study investigates their roles in SIMD using both in vivo and in vitro models. Mice were subjected to lipopolysaccharide (LPS)-induced sepsis, and cardiac function was assessed in IRG1 knockout (IRG1-/-) and wild-type mice. Exogenous 4-octyl itaconate (4-OI) supplementation was also examined for its protective effects. In vitro, bone marrow-derived macrophages and RAW264.7 cells were treated with 4-OI following Nuclear factor, erythroid 2 like 2 (NRF2)-small interfering RNA administration to elucidate the underlying mechanisms. Our results indicate that IRG1 deficiency exacerbates myocardial injury during sepsis, while 4-OI administration preserves cardiac function and reduces inflammation. Mechanistic insights reveal that 4-OI activates the NRF2/HO-1 pathway, promoting macrophage polarization and attenuating inflammation. These findings underscore the protective role of the IRG1/itaconate axis in SIMD and suggest a therapeutic potential for 4-OI in modulating macrophage responses., (© 2024. The Author(s).)

Approximately 50% of patients diagnosed with ovarian cancer harbor tumors with mutations in BRCA1, BRCA2, or other genes involved in homologous recombination repair (HR). The presence of homologous recombination deficiency (HRD) is an approved biomarker for poly-ADP-ribose polymerase inhibitors (PARPis) as a maintenance treatment following a positive response to initial platinum-based chemotherapy. Despite this treatment option, the development of resistance to PARPis is common among recurrent disease patients, leading to a poor prognosis. In this study, we conducted a comprehensive analysis using publicly available datasets to elucidate the molecular mechanisms driving PARPi resistance in BRCA1-deficient ovarian cancer. Our findings reveal a central role for the interferon (IFN) pathway in mediating resistance in the context of BRCA1 deficiency. Through integrative bioinformatics approaches, we identified LY6E, an interferon-stimulated gene, as a key mediator of PARPi resistance, with its expression linked to an immunosuppressive tumor microenvironment (TME) encouraging tumor progression and invasion. LY6E amplification correlates with poor prognosis and increased expression of immune-related gene signatures, which is predictive of immunotherapy response. Interestingly, LY6E expression upon PARPi treatment resistance was found to be dependent on BRCA1 status. Gene expression analysis in the Orien/cBioPortal database revealed an association between LY6E and genes involved in DNA repair, such as Rad21 and PUF60, emphasizing the interplay between DNA repair pathways and immune modulation. Moreover, PUF60, Rad21, and LY6E are located on chromosome 8q24, a locus often amplified and associated with the progression of ovarian cancer. Overall, our study provides novel insights into the molecular determinants of PARPi resistance and highlights LY6E as a promising prognostic biomarker in the management of HRD ovarian cancer. Future studies are needed to fully elucidate the molecular mechanisms underlying the role of LY6E in PARPi resistance.

Background: Lymphocyte antigen 9 (LY9) participates in the development of several tumors and diseases but has not been reported yet in lung adenocarcinoma (LUAD)., Methods: First, we analyzed the expression and prognostic value of LY9 in pan-cancer, including LUAD. Additionally, we conducted a correlation analysis of LY9 expression in LUAD with immune cell infiltration using the TIMER database and the CIBERSORT algorithm, and with immune checkpoints using the GEPIA database. Also, we constructed a potential ceRNA network for LY9. Furthermore, we explored LY9-related pathways by Gene Set Enrichment Analysis (GSEA). Finally, validation of differential expression at the mRNA level was obtained from the GEO database. We collected LUAD tissues for Quantitative Real-time PCR (qRT-PCR) to verify the expression of LY9, CD8, and CD4 and calculated the correlation between them. We also conducted immunohistochemistry (IHC) to verify the protein expression of LY9., Results: Results showed that LY9 was highly expressed in various tumors, including LUAD. Besides, patients with high LY9 expression presented longer overall survival (OS) and more multiple lymphocyte infiltrations. The expression of LY9 in LUAD strongly and positively correlates with multiple immune cell infiltration and immune checkpoints. The functional enrichment analysis indicated that LY9 was involved in multiple immune-related pathways and non-small cell lung cancer. Moreover, a ceRNA regulatory network of LINC00943-hsa-miR-141-3p-LY9 might be involved. Finally, GSE68465 dataset confirmed differential expression of LY9 mRNA levels in LUAD and the qRT-PCR results verified LY9 had a strong and positive correlation with CD4 and CD8 T cells. Unfortunately, IHC did not detect the expression of LY9 protein level in tumor tissues and WB experiments validated the protein expression of LY9 in the OCI-AML-2 cell line., Conclusions: Therefore, we hypothesized that LY9 could serve as a potential, novel prognostic biomarker for LUAD and could predict immunotherapy efficacy at the mRNA level., Competing Interests: The authors declare there are no competing interests., (©2024 Deng et al.)

SLURP1 and SLURP2 are both small secreted members of the Ly6/u-PAR family of proteins and are highly expressed in keratinocytes. Loss-of-function mutations in SLURP1 lead to a rare autosomal recessive palmoplantar keratoderma (PPK), Mal de Meleda (MdM), which is characterized by diffuse, yellowish palmoplantar hyperkeratosis. Some individuals with MdM experience pain in conjunction with the hyperkeratosis that has been attributed to fissures or microbial superinfection within the affected skin. By comparison, other hereditary PPKs such as pachyonychia congenita and Olmsted syndrome show prevalent pain in PPK lesions. Two mouse models of MdM, Slurp1 knock-out and Slurp2X knock-out, exhibit robust PPK in all four paws. However, whether the sensory experience of these animals includes augmented pain sensitivity remains unexplored. In this study, we demonstrate that both models exhibit hypersensitivity to mechanical and thermal stimuli as well as spontaneous pain behaviors in males and females. Anatomical analysis revealed slightly reduced glabrous skin epidermal innervation and substantial alterations in palmoplantar skin immune composition in Slurp2X knock-out mice. Primary sensory neurons innervating hindpaw glabrous skin from Slurp2X knock-out mice exhibit increased incidence of spontaneous activity and mechanical hypersensitivity both in vitro and in vivo. Thus, Slurp knock-out mice exhibit polymodal PPK-associated pain that is associated with both immune alterations and neuronal hyperexcitability and might therefore be useful for the identification of therapeutic targets to treat PPK-associated pain., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αβ-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27 + Ly6C - cells convert into CD27 + Ly6C + cells, and these CD27 + Ly6C + cells control cancer progression in mice, while the CD27 + Ly6C - cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27 + Ly6C + cells and human Vδ2 + cells, while IL-27 is dispensable for mouse CD27 + Ly6C - cell and human Vδ1 + cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology., (© 2024. The Author(s).)

Background: Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively., Objective: To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype., Materials and Methods: We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c)., Results: Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3., Conclusion: These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability., (© 2022 American Society of Andrology and European Academy of Andrology.)

Colorectal cancer (CRC) poses a significant global health challenge with high morbidity and mortality rates. This study investigates the role of LY6G6D, a member of the LY6/uPAR superfamily, in CRC. Employing a bioinformatic approach, we analyzed LY6G6D expression across different cancer types, compared it with known oncogenes in CRC, explored the involved genomic alterations, and assessed associated clinicopathological characteristics. LY6G6D exhibited aberrant expression, particularly elevated in CRC adenocarcinoma and highly specific to tumor tissues when compared with other oncogenes, despite its comparatively low frequency of genomic alteration. Subsequently, tumor immune infiltration analysis revealed distinct associations, primarily indicating a negative correlation, suggesting immune down-regulation. Survival analysis in context of LY6G6D was conducted with Kaplan-Meier (KM) curves, indicating a 10% risk of disease recurrence in the case of elevated expression. Additionally, we constructed a 3D protein model of LY6G6D through ab-inito approach. The protein model was validated, followed by conservation analysis and active site identification. Active site identification of LY6G6D's final predicted model revealed some similar sites that were estimated to be conserved. Target-guided drug molecules were collected and molecular docking was executed, proposing Cardigin (Digitoxin) and Manzamine A as potential therapeutic candidates. In conclusion, LY6G6D emerges as a significant biomarker for diagnostic and therapeutic applications in CRC, highlighting its multifaceted role in tumorigenesis. The proposed drugs present avenues for further investigations.

The identification of targets that are expressed on the cell membrane is a main goal in cancer research. The Lymphocyte Antigen 6 Family Member G6D ( LY6G6D ) gene codes for a protein that is mainly present on the surface of colorectal cancer (CRC) cells. Therapeutic strategies against this protein like the development of T cell engagers (TCE) are currently in the early clinical stage. In the present work, we interrogated public genomic datasets including TCGA to evaluate the genomic and immunologic cell profile present in tumors with high expression of LY6G6D . We used data from TCGA, among others, and the Tumor Immune Estimation Resource (TIMER2.0) platform for immune cell estimations and Spearman correlation tests. LY6G6D expression was exclusively present in CRC, particularly in the microsatellite stable (MSS) subtype, and was associated with left-side tumors and the canonical genomic subgroup. Tumors with mutations of APC and p53 expressed elevated levels of LY6G6D. This protein was expressed in tumors with an inert immune microenvironment with an absence of immune cells and co-inhibitory molecules. In conclusion, we described clinical, genomic and immune-pathologic characteristics that can be used to optimize the clinical development of agents against this target. Future studies should be performed to confirm these findings and potentially explore the suggested clinical development options.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1 GFP and Ccr2 RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6C high classical and Ly6C low non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1 GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2 RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Background: Mal de Meleda is an autosomal recessive palmoplantar keratoderma, with SLURP1 identified as the pathogenic gene responsible. Although over 20 mutations in SLURP1 have been reported, only the mutation c.256G > A (p.G87R) has been detected in Chinese patients. Here, we report a novel heterozygous SLURP1 mutation in a Chinese family., Methods: We assessed the clinical manifestations of two Chinese patients with Mal de Meleda and collected specimens from the patients and other family members for whole-exome and Sanger sequencing. We used algorithms (MutationTaster, SIFT, PolyPhen-2, PROVEAN, PANTHER, FATHMM, mCSM, SDM and DUET) to predict the pathogenetic potential of the mutation detected. We also employed AlphaFold2 and PyMOL for protein structure analysis., Results: Both patients displayed the typical manifestation of palmoplantar keratoderma. In Proband 1, we detected a novel compound heterozygous mutation (c.243C > A and c.256G > A) in exon 3 of SLURP1. Proband 2 was an adult female born to a consanguineous family and carried a homozygous mutation (c.211C > T). Algorithms indicated both mutations to be probably disease causing. We used AlphaFold2 to predict the protein structure of these mutations and found that they cause instability, as shown by PyMOL., Conclusions: Our study identified a novel compound heterozygous mutation (c.243C > A and c.256G > A) in a Chinese patient with Mal de Meleda that has the potential to cause instability in protein structure. Moreover, this study expands on the existing knowledge of SLURP1 mutations and contributes to knowledge of Mal de Meleda., (© 2023. The Author(s).)

Ly108 (SLAMF6) is a homophilic cell surface molecule that binds SLAM-associated protein (SAP), an intracellular adapter protein that modulates humoral immune responses. Furthermore, Ly108 is crucial for the development of natural killer T (NKT) cells and CTL cytotoxicity. Significant attention has been paid towards expression and function of Ly108 since multiple isoforms were identified, i.e., Ly108-1, Ly108-2, Ly108-3, and Ly108-H1, some of which are differentially expressed in several mouse strains. Surprisingly, Ly108-H1 appeared to protect against disease in a congenic mouse model of Lupus. Here, we use cell lines to further define Ly108-H1 function in comparison with other isoforms. We show that Ly108-H1 inhibits IL-2 production while having little effect upon cell death. With a refined method, we could detect phosphorylation of Ly108-H1 and show that SAP binding is retained. We propose that Ly108-H1 may regulate signaling at two levels by retaining the capability to bind its extracellular as well as intracellular ligands, possibly inhibiting downstream pathways. In addition, we detected Ly108-3 in primary cells and show that this isoform is also differentially expressed between mouse strains. The presence of additional binding motifs and a non-synonymous SNP in Ly108-3 further extends the diversity between murine strains. This work highlights the importance of isoform awareness, as inherent homology can present a challenge when interpreting mRNA and protein expression data, especially as alternatively splicing potentially affects function.

Aim: Sca-1 + CD31 - cells are resident cardiac progenitor cells, found in many mammalian tissues including the heart, and able to differentiate into cardiomyocytes in vitro and in vivo. Our previous work indicated that heart-derived Sca-1 + CD31 - cells increased the Nr1d1 mRNA level of Nr1d1 with aging. However, how Nr1d1 affects the senescence of Sca-1 + CD31 - cells., Methods: Overexpression and knockdown of Nr1d1 in Sca-1 + CD31 - cells and mouse cardiac myocyte (MCM) cell lines were performed by lentiviral transduction. The effects of Nr1d1 abundance on cell differentiation, proliferation, apoptosis, cell cycle, and transcriptomics were evaluated. Moreover, binding of Nr1d1 to the promoter region of Nr4a3 and Serpina3 was examined by a luciferase reporter assay., Results and Conclusions: Upregulation Nr1d1 in young Sca-1 + CD31 - cells inhibited cell proliferation and promoted apoptosis. However, depletion of Nr1d1 in aged Sca-1 + CD31 - cells promoted cell proliferation and inhibited apoptosis. Furthermore, Nr1d1 was negatively associated with cell proliferation, promoting apoptosis and senescence-associated beta-galactosidase production in MCMs. Our findings show that Nr1d1 stimulates Serpina3 expression through its interaction with Nr4a3 . Nr1d1 may therefore act as a potent anti-aging receptor that can be a therapeutic target for aging-related diseases.

Background: The development of inflammatory bowel diseases is thought to be multifactorial, but the exact steps in pathogenesis are poorly understood. In this study, we investigated involvement of the activation of STAT1 signal pathway in the pathogenesis of an acute colitis model., Methods: A dextran sulfate sodium-induced acute colitis model was established by using wild-type C57BL/6 mice and STAT1-deficient mice. Disease indicators such as body weight loss and clinical score, induction of cytokines, chemokines, and inflammatory cells were evaluated in the acute colitis model., Results: Disease state was significantly improved in the acute colitis model using STAT1-deficient mice compared with wild-type mice. The induction of Ly6c-highly expressing cells in colorectal tissues was attenuated in STAT1-deficient mice. IL-6, CCL2, and CCR2 gene expressions in Ly6c-highly expressing cells accumulated in the inflamed colon tissues and were significantly higher than in Ly6c-intermediate-expressing cells, whereas TNF-α and IFN-α/β gene expression was higher in Ly6c-intermediate-expressing cells. Blockade of CCR2-mediated signaling significantly reduced the disease state in the acute colitis model., Conclusions: Two different types of Ly6c-expressing macrophages are induced in the inflamed tissues through the IFN-α/β-STAT1-mediated CCL2/CCR2 cascade and this is associated with the pathogenesis such as onset, exacerbation, and subsequent chronicity of acute colitis., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Plasmacytoid and conventional dendritic cells (pDC and cDC) are generated from progenitor cells in the bone marrow and commitment to pDCs or cDC subtypes may occur in earlier and later progenitor stages. Cells within the CD11c + MHCII -/lo Siglec-H + CCR9 lo DC precursor fraction of the mouse bone marrow generate both pDCs and cDCs. Here we investigate the heterogeneity and commitment of subsets in this compartment by single-cell transcriptomics and high-dimensional flow cytometry combined with cell fate analysis: Within the CD11c + MHCII -/lo Siglec-H + CCR9 lo DC precursor pool cells expressing high levels of Ly6D and lacking expression of transcription factor Zbtb46 contain CCR9 lo B220 hi immediate pDC precursors and CCR9 lo B220 lo (lo-lo) cells which still generate pDCs and cDCs in vitro and in vivo under steady state conditions. cDC-primed cells within the Ly6D hi Zbtb46 - lo-lo precursors rapidly upregulate Zbtb46 and pass through a Zbtb46 + Ly6D + intermediate stage before acquiring cDC phenotype after cell division. Type I IFN stimulation limits cDC and promotes pDC output from this precursor fraction by arresting cDC-primed cells in the Zbtb46 + Ly6D + stage preventing their expansion and differentiation into cDCs. Modulation of pDC versus cDC output from precursors by external factors may allow for adaptation of DC subset composition at later differentiation stages., (© 2022. The Author(s).)

Background: The impaired osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) is a major cause of bone remodeling imbalance and osteoporosis. The bicaudal C homologue 1 (BICC1) gene is a genetic regulator of bone mineral density (BMD) and promotes osteoblast differentiation. The purpose of this study is to explore the probable function of BICC1 in osteoporosis and osteogenic differentiation of aged BMSCs., Methods: We examined the GSE116925 microarray dataset obtained from the Gene Expression Omnibus (GEO) database. The GEO2R algorithm identified differentially expressed genes (DEGs) in Sca-1+ BMSCs from young (3 months old) and old (18 months old) mice. Then, to identify the most crucial genes, we used pathway enrichment analysis and a protein-protein interaction (PPI) network. Furthermore, starBase v2.0 was used to generate the regulatory networks between BICC1 and related competing endogenous RNAs (ceRNAs). NetworkAnalyst was used to construct TF-gene networks and TF-miRNA-gene networks of BICC1 and ceRNA. Furthermore, we investigated the Bicc1 expression in aged Sca-1-positive BMSCs., Result: We detected 923 DEGs and discovered that epidermal growth factor receptor (EGFR) was the top hub gene with a high degree of linkage. According to the findings of the PPI module analysis, EGFR was mostly engaged in cytokine signaling in immune system and inflammation-related signaling pathways. 282 ceRNAs were found to interact with the BICC1 gene. EGFR was not only identified as a hub gene but also as a BICC1-related ceRNA. Then, we predicted 11 common TF-genes and 7 miRNAs between BICC1 and EGFR. Finally, we found that BICC1 mRNA and EGFR mRNA were significantly overexpressed in aged Sca-1-positive BMSCs., Conclusion: As a genetic gene that affects bone mineral density, BICC1 may be a new target for clinical treatment of senile osteoporosis by influencing osteogenic differentiation of BMSCs through EGFR-related signaling. However, the application of the results requires support from more experimental data., Competing Interests: The authors declare that no potential conflicts of interest exist., (Copyright © 2022 Zhongshuang Liu et al.)

Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a , also known as Stem Cell Ag-1 and T cell-activating protein , is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S , a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S , which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses., (Copyright © 2022 The Authors.)

Background & Aims: Sca-1 is a surface marker for murine hematopoietic stem cells (HSCs) and type-I interferon is a key regulator for Lin - Sca-1 + HSCs expansion through Ifnar/Stat-1/Sca-1-signaling. In this study we aimed to characterize the role and regulation of Sca-1 + cells in pancreatic regeneration., Methods: To characterize Sca-1 in vivo, immunohistochemistry and immunofluorescence staining of Sca-1 was conducted in normal pancreas, in cerulein-mediated acute pancreatitis, and in Kras-triggered cancerous lesions. Ifnar/Stat-1/Sca-1-signaling was studied in type-I IFN-treated epithelial explants of adult wildtype, Ifnar - /- , and Stat-1 - / - mice. Sca-1 induction was analyzed by gene expression and FACS analysis. After isolation of pancreatic epithelial Lin - Sca-1 + cells, pancreatosphere-formation and immunofluorescence-assays were carried out to investigate self-renewal and differentiation capabilities., Results: Sca-1 + cells were located in periacinar and periductal spaces and showed an enrichment during cerulein-induced acute pancreatitis (23.2/100 μm 2  ± 4.9 SEM) and in early inflammation-mediated carcinogenic lesions of the pancreas of Kras G12D mice (35.8/100 μm 2  ± SEM 1.9) compared to controls (3.6/100 μm 2  ± 1.3 SEM). Pancreatic Lin - Sca-1 + cells displayed a small population of 1.46% ± 0.12 SEM in FACS. In IFN-β treated pancreatic epithelial explants, Sca-1 expression was increased, and Lin - Sca-1 + cells were enriched in vitro (from 1.49% ± 0.36 SEM to 3.85% ± 0.78 SEM). Lin - Sca-1 + cells showed a 12 to 51-fold higher capacity for clonal self-renewal compared to Lin - Sca-1 - cells and generated cells express markers of the acinar and ductal compartment., Conclusions: Pancreatic Sca-1 + cells enriched during parenchymal damage showed a significant capacity for cell renewal and in vitro plasticity, suggesting that corresponding to the type I interferon-dependent regulation of Lin - Sca-1 + hematopoietic stem cells, pancreatic Sca-1 + cells also employ type-I-interferon for regulating progenitor-cell-homeostasis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Monocytes play a crucial role in maintaining homeostasis and mediating a successful innate immune response. They also act as central players in diverse pathological conditions, thus making them an attractive therapeutic target. Within the bone marrow, monocytes arise from a committed precursor termed Common Monocyte Progenitor (cMoP). However, molecular mechanisms that regulate the differentiation of cMoP to various monocytic subsets remain unclear. Herein, we purified murine myeloid precursors for deep poly-A-enriched RNA sequencing to understand the role of alternative splicing in the development and differentiation of monocytes under homeostasis. Our analyses revealed intron retention to be the major alternative splicing mechanism involved in the monocyte differentiation cascade, especially in the differentiation of Ly6C hi monocytes to Ly6C lo monocytes. Furthermore, we found that the intron retention of key genes involved in the differentiation of murine Ly6C hi to Ly6C lo monocytes was also conserved in humans. Our data highlight the unique role of intron retention in the regulation of the monocytic differentiation pathway., (© 2021 John Wiley & Sons Ltd.)

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324 + epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C + and Ly6C - ) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Olesch, Brunn, Aktay-Cetin, Sirait-Fischer, Pullamsetti, Grimminger, Seeger, Brüne, Weigert and Savai.)

Background: Ovarian cancer is a common gynecological malignant disease in women. Our work aimed to study the specific functions of ZNF252P antisense RNA 1 (ZNF252P-AS1) in ovarian cancer., Methods: ZNF252P-AS1, miR-324-3p, and lymphocyte antigen 6 family member K (LY6K) expression were analyzed by bioinformatics tools in ovarian cancer tissues and was quantified by qRT-PCR in ovarian cancer cells. The effect of ZNF252P-AS1 knockdown, miR-324-3p suppression, and LY6K over-expression on apoptosis, cell viability, invasion, migration, and epithelial to mesenchymal transition (EMT) was determined in vitro by using colony formation and EdU assays, flow cytometry, transwell assay, and Western blot. The interactions between ZNF252P-AS1 and miR-324-3p and between miR-324-3p and LY6K were validated by luciferase assays. The effects of restraining ZNF252P-AS1 in vivo were studied using BALB/c male nude mice., Results: ZNF252P-AS1 and LY6K levels were up-regulated, while miR-324-3p was declined in ovarian cancer tissues and cells. ZNF252P-AS1 knockdown reduced ovarian cancer cell proliferation, invasion, migration, and EMT, whereas promoted its apoptosis. Besides, ZNF252P-AS1 interacted with miR-324-3p and reversely regulated its level, and miR-324-3p was directly bound to LY6K and negatively regulated its expression. Moreover, ZNF252P-AS1 knockdown reversed the effect of miR-324-3p on cancer cell apoptosis, growth, migration, invasion, and EMT. Similar results were discovered in the rescue experiments between miR-324-3p and LY6K. Additionally, mouse models in vivo experiments further validated that ZNF252P-AS1 knockdown distinctly inhibited tumor growth., Conclusion: ZNF252P-AS1 mediated miR-324-3p/LY6K signaling to facilitate progression of ovarian cancer., (© 2021. The Author(s).)

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7 fl/fl Vav Cre/+ , Tcf7 fl/fl CD122 Cre/+ and Tcf7 fl/fl Ncr1 Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7 fl/fl Ncr1 Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes , Ets1 , Gata3 , Ikzf1 , Ikzf2 , Nfil3 , Runx3 , Sh2d1a , Slamf6 , Tbx21 , Tox , and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Wang, Hao, Wang, Yao, Zhang, Zhao, Guo, Yu, Ma, Liu, Chen, Yuan, Kang and Yu.)

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6C hi population, and deplete the homeostatic non-classical Ly6C lo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6C hi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD + ; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pradhan, Yi, Geng and Li.)

The strength of the T cell receptor interaction with self-ligands affects antigen-specific immune responses. However, the precise function and underlying mechanisms are unclear. Here, we demonstrate that naive CD8 + T cells with relatively high self-reactivity are phenotypically heterogeneous owing to varied responses to type I interferon, resulting in three distinct subsets, CD5 lo Ly6C - , CD5 hi Ly6C - , and CD5 hi Ly6C + cells. CD5 hi Ly6C + cells differ from CD5 lo Ly6C - and CD5 hi Ly6C - cells in terms of gene expression profiles and functional properties. Moreover, CD5 hi Ly6C + cells demonstrate more extensive antigen-specific expansion upon viral infection, with enhanced differentiation into terminal effector cells and reduced memory cell generation. Such features of CD5 hi Ly6C + cells are imprinted in a steady-state and type I interferon dependence is observed even for monoclonal CD8 + T cell populations. These findings demonstrate that self-reactivity controls the functional diversity of naive CD8 + T cells by co-opting tonic type I interferon signaling., (© 2021. The Author(s).)

Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is critical for the pathogenesis of Escherichia coli ( E. coli ) K1 meningitis, a severe central nervous system infection of the neonates. However, little is known about how E. coli K1 manipulates α7 nAChR signaling. Here, through employing immortalized cell lines, animal models, and human transcriptional analysis, we showed that E. coli K1 infection triggers releasing of secreted Ly6/Plaur domain containing 1 (SLURP1), an endogenous α7 nAChR ligand. Exogenous supplement of SLURP1, combined with SLURP1 knockdown or overexpression cell lines, showed that SLURP1 is required for E. coli K1 invasion and neutrophils migrating across the blood-brain barrier (BBB). Furthermore, we found that SLURP1 is required for E. coli K1-induced α7 nAChR activation. Finally, the promoting effects of SLURP1 on the pathogenesis of E. coli K1 meningitis was significantly abolished in the α7 nAChR knockout mice. These results reveal that E. coli K1 exploits SLURP1 to activate α7 nAChR and facilitate its pathogenesis, and blocking SLURP1-α7 nAChR interaction might represent a novel therapeutic strategy for E. coli K1 meningitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 He, Wang, Liu, Gao, Long, Chi, Hu, Wan, Gong, Li, Zhen, Zhang, Cao and Huang.)

Calcium oxalate (CaOx) stones are the most common type of kidney stones and are associated with high recurrence, short chain fatty acids (SCFAs), and inflammation. However, it remains uncertain whether SCFAs affect the formation of CaOx stones through immunomodulation. We first performed mass cytometry (CyTOF) and RNA sequencing on kidney immune cells with glyoxylate-induced CaOx crystals (to elucidate the landscape of the associated immune cell population) and explored the role of SCFAs in renal CaOx stone formation through immunomodulation. We identified 29 distinct immune cell subtypes in kidneys with CaOx crystals, where CX3CR1 + CD24 - macrophages significantly decreased and GR1 + neutrophils significantly increased. In accordance with the CyTOF data, RNA sequencing showed that most genes involved were related to monocytes and neutrophils. SCFAs reduced kidney CaOx crystals by increasing the frequency of CX3CR1 + CD24 - macrophages and decreasing GR1 + neutrophil infiltration in kidneys with CaOx crystals, which was dependent on the gut microbiota. GPR43 knockdown by transduction with adeno-associated virus inhibited the alleviation of crystal formation and immunomodulatory effects in the kidney, due to SCFAs. Moreover, CX3CR1 + CD24 - macrophages regulated GR1 + neutrophils via GPR43. Our results demonstrated a unique trilateral relationship among SCFAs, immune cells, and the kidneys during CaOx formation. These findings suggest that future immunotherapies may be used to prevent kidney stones using SCFAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jin, Jian, Chen, Ma, Ma, Liu, Gong, Xiang, Zhu, Shu, Qi, Li and Wang.)

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4 -deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2 -/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2 -/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets., Competing Interests: Declaration of interests I.C., B.A.Z., J.C., and R.S. are presently or were employees of ChemoCentryx during this study. B.T.W., S.G., L.J., M.C., G.B., and E.D.P. are presently or were employees of Biogen during this study. B.T.W. is currently employed by Moderna. C.J.Y. is a Scientific Advisory Board member for and hold equity in Related Sciences and ImmunAI, a consultant for and hold equity in Maze Therapeutics, and a consultant for TReX Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. A U.S. Provisional Patent Application No. 63/177,727 for the use of CCX872 in TBI, spinal cord injury, or stroke was filed April 21, 2021., (Published by Elsevier Inc.)

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C + CD8 + Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5 hi CD8 + cells, which in the case of polyinfection translates into a numerical CD8 + Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C + Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C + CD8 +  Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy., (© 2021. The Author(s).)

Mal de Meleda is an autosomal recessive palmoplantar keratoderma associated with mutations in a gene encoding SLURP-1. SLURP-1 controls growth, differentiation, and apoptosis of keratinocytes by interaction with α7-type nicotinic acetylcholine receptors. SLURP-1 has a three-finger structure with a β-structural core (head) and three prolonged loops (fingers). To determine the role of SLURP-1 mutations, we produced 22 mutant variants of the protein, including those involved in Mal de Meleda pathogenesis. All mutants except R71H, R71P, T52A, R96P, and L98P were produced in the folded form. SLURP-1 reduces the growth of Het-1A keratinocytes; thus, we studied the influence of the mutations on its antiproliferative activity. Mutations in loops I and III led to the protein inactivation, whereas most mutations in loop II increased SLURP-1 antiproliferative activity. Alanine substitutions of R96 and L98 residues located in the protein head resulted in the appearance of additional pro-apoptotic activity. Our results agree with the diversity of Mal de Meleda phenotypes. Using obtained functional data, the SLURP-1/α7 type nicotinic acetylcholine receptor complex was modeled in silico. Our study provides functional and structural information about the role of the SLURP-1 mutations in Mal de Meleda pathogenesis and predicts SLURP-1 variants, which could drive the disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The Lymphocyte antigen-6 (Ly-6) superfamily has been considered to play an important role in the innate immunity of mammals. The functions of Ly-6 proteins are diverse since their low sequence homology. Currently, the function of Ly-6D, a member of Ly-6 family proteins, is completely unknown in teleost. In the present study, we identified and characterized a Ly-6D homologue (named PoLy-6D) from the teleost fish Paralichthys olivaceus and examined its immune function. PoLy-6D possesses a hydrophobic signal peptide, a LU domain including a conserved "LXCXXC" motif in N-terminus and a "CCXXXXCN" motif in C-terminus. Under normal physiological condition, PoLy-6D expression distributes in all the examined tissues, the highest three tissues are successively spleen, head kidney, and blood. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, PoLy-6D expression was induced and the patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoLy-6D (rPoLy-6D) inhibited the lysis of rabbit red blood cells by serum and selectively improved bacterial survival in serum. After serum were treated by antibody of rPoLy-6D, bacteriostatic effect of serum was obviously enhanced. These results indicate the importance of PoLy-6D as a complement regulator. rPoLy-6D possessed the binding activity to multiple bacteria but did not exhibit antimicrobial activities. The interaction between rPoLy-6D and bacteria suggests that PoLy-6D is involved in host clearance of pathogens probably by serving as a receptor for pathogens. Overexpression of PoLy-6D in vivo promoted the host defense against invading E. piscicida. These findings add new insights into the regulation mechanism of the complement system in teleost and emphasize the importance of Ly-6D products for the control of pathogen infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

Objective: Skin inflammation heralds systemic disease in juvenile myositis, yet we lack an understanding of pathogenic mechanisms driving skin inflammation in this disease. We undertook this study to define cutaneous gene expression signatures in juvenile myositis and identify key genes and pathways that differentiate skin disease in juvenile myositis from childhood-onset systemic lupus erythematosus (SLE)., Methods: We used formalin-fixed paraffin-embedded skin biopsy samples from 15 patients with juvenile myositis (9 lesional, 6 nonlesional), 5 patients with childhood-onset SLE, and 8 controls to perform transcriptomic analysis and identify significantly differentially expressed genes (DEGs; q ≤ 5%) between patient groups. We used Ingenuity Pathway Analysis (IPA) to highlight enriched biologic pathways and validated DEGs by immunohistochemistry and quantitative real-time polymerase chain reaction., Results: Comparison of lesional juvenile myositis to control samples revealed 221 DEGs, with the majority of up-regulated genes representing interferon (IFN)-stimulated genes. CXCL10, CXCL9, and IFI44L represented the top 3 DEGs (fold change 23.2, 13.3, and 13.0, respectively; q < 0.0001). IPA revealed IFN signaling as the top canonical pathway. When compared to childhood-onset SLE, lesional juvenile myositis skin shared a similar gene expression pattern, with only 28 unique DEGs, including FBLN2, CHKA, and SLURP1. Notably, patients with juvenile myositis who were positive for nuclear matrix protein 2 (NXP-2) autoantibodies exhibited the strongest IFN signature and also demonstrated the most extensive Mx-1 immunostaining, both in keratinocytes and perivascular regions., Conclusion: Lesional juvenile myositis skin demonstrates a striking IFN signature similar to that previously reported in juvenile myositis muscle and peripheral blood. Further investigation into the association of a higher IFN score with NXP-2 autoantibodies may provide insight into disease endotypes and pathogenesis., (© 2020, American College of Rheumatology.)

Recent advances in genomics unraveled several actionable mutational drivers in lung cancer, leading to promising therapies such as tyrosine kinase inhibitors and immune checkpoint inhibitors. However, the tumors' acquired resistance to the newly-developed as well as existing therapies restricts life quality improvements. Therefore, we investigated the noncoding portion of the human transcriptome in search of alternative actionable targets. We identified an antisense transcript, LY6K-AS, with elevated expression in lung adenocarcinoma (LUAD) patients, and its higher expression in LUAD patients predicts poor survival outcomes. LY6K-AS abrogation interfered with the mitotic progression of lung cancer cells resulting in unfaithful chromosomal segregation. LY6K-AS interacts with and stabilizes 14-3-3 proteins to regulate the transcription of kinetochore and mitotic checkpoint proteins. We also show that LY6K-AS regulates the levels of histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of kinetochore members. Cisplatin treatment and LY6K-AS silencing affect many common pathways enriched in cell cycle-related functions. LY6K-AS silencing affects the growth of xenografts derived from wildtype and cisplatin-resistant lung cancer cells. Collectively, these data indicate that LY6K-AS silencing is a promising therapeutic option for LUAD that inhibits oncogenic mitotic progression.

Abstract: Several genetic loci have been reported to be significantly associated with coronary artery disease (CAD) by multiple genome-wide association studies (GWAS). Nevertheless, the biological and functional effects of these genetic variants on CAD remain largely equivocal. In the current study, we performed an integrative genomics analysis by integrating large-scale GWAS data (N = 459,534) and 2 independent expression quantitative trait loci (eQTL) datasets (N = 1890) to determine whether CAD-associated risk single nucleotide polymorphisms (SNPs) exert regulatory effects on gene expression. By using Sherlock Bayesian, MAGMA gene-based, multidimensional scaling (MDS), functional enrichment, and in silico permutation analyses for independent technical and biological replications, we highlighted 4 susceptible genes (CHCHD1, TUBG1, LY6G6C, and MRPS17) associated with CAD risk. Based on the protein-protein interaction (PPI) network analysis, these 4 genes were found to interact with each other. We detected a remarkably altered co-expression pattern among these 4 genes between CAD patients and controls. In addition, 3 genes of CHCHD1 (P = .0013), TUBG1 (P = .004), and LY6G6C (P = .038) showed significantly different expressions between CAD patients and controls. Together, we provide evidence to support that these identified genes such as CHCHD1 and TUBG1 are indicative factors of CAD., Competing Interests: The authors have no conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown. Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits. Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC ( s MC), MC activation, immune parameters (TNFα, IL1β, IL10, TGFβ, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC ( c MC) from C57BL/6 or Chrna7-Knockout mice. Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and s MC and demonstrated upregulated contacts and increased Chrna7+ s MC in stressed skin, while 5 ng/mL SLURP-1 degranulated c MC. Third, IL1β+ s MC were high in stressed skin, and while SLURP-1 alone had no significant effect on c MC cytokines, it upregulated IL1β in c MC from Chrna7-KO and in IL1β-treated wildtype c MC. In addition, HIF1α+ s MC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in c MC while SLURP-1 upregulated TNFα and IL1β in c MC when HIF1α was blocked. Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1β expression if SLURP-1 is involved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ertle, Rommel, Tumala, Moriwaki, Klein, Kruse, Gieler and Peters.)

Natural killer (NK) cells are innate effectors armed with cytotoxic and cytokine-secreting capacities whose spontaneous antitumor activity is key to numerous immunotherapeutic strategies. However, current mouse models fail to mirror the extensive immune system variation that exists in the human population which may impact on NK cell-based therapies. We performed a comprehensive profiling of NK cells in the Collaborative Cross (CC), a collection of novel recombinant inbred mouse strains whose genetic diversity matches that of humans, thereby providing a unique and highly diverse small animal model for the study of immune variation. We demonstrate that NK cells from CC strains displayed a breadth of phenotypic and functional variation reminiscent of that reported for humans with regards to cell numbers, key marker expression, and functional capacities. We took advantage of the vast genetic diversity of the CC and identified nine genomic loci through quantitative trait locus mapping driving these phenotypic variations. SNP haplotype patterns and variant effect analyses identified candidate genes associated with lung NK cell numbers, frequencies of CD94 + NK cells, and expression levels of NKp46. Thus, we demonstrate that the CC represents an outstanding resource to study NK cell diversity and its regulation by host genetics., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFβ-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C + monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C + monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Heterogeneity among naive adaptive lymphocytes determines their individual functions and fate decisions during an immune response. NK cells are innate lymphocytes capable of generating "adaptive" responses during infectious challenges. However, the factors that govern various NK cell functions are not fully understood. In this study, we use a reporter mouse model to permanently "time stamp" NK cells and type 1 innate lymphoid cells (ILC1s) to characterize the dynamics of their homeostatic turnover. We found that the homeostatic turnover of tissue-resident ILC1s is much slower than that of circulating NK cells. NK cell homeostatic turnover is further accelerated without the transcription factor Eomes. Finally, heterogeneity in NK cell age diversifies NK cell function, with "older" NK cells exhibiting more potent IFN-γ production to activating stimuli and more robust adaptive responses during CMV infection. These results provide insight into how the functional response of an NK cell varies over its lifespan., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Prostate adenocarcinoma undergoes neuroendocrine differentiation to acquire resistance toward antihormonal therapies. The underlying mechanisms have been investigated extensively, among which Sox2 has been shown to play a critical role. However, genetic evidence in mouse models for prostate cancer to support the crucial role of Sox2 is missing. The adult mouse prostate luminal cells contain both castration-resistant Sox2-expressing Sca-1 + cells and castration-responsive Sca-1 - cells. We show that both types of the luminal cell are susceptible to oncogenic transformation induced by loss of function of the tumor suppressor Pten. The tumors derived from the Sca-1 + cells are castration resistant and are more inclined to develop castration-induced neuroendocrine differentiation. Genetic ablation of Sox2 suppresses neuroendocrine differentiation but does not impact the castration-resistant property. This study provides direct genetic evidence that Sox2 is necessary for androgen ablation-induced neuroendocrine differentiation of Pten null prostate adenocarcinoma, corroborates that the lineage status of the prostate cancer cells is a determinant for its propensity to exhibit lineage plasticity, and supports that the intrinsic features of cell-of-origin for prostate cancers can dictate their clinical behaviors.

Background: Though bladder cancer has been the subject of many well-powered genome-wide association studies, the mechanisms involving bladder-cancer-associated single nucleotide polymorphisms (SNPs) remain largely unknown. This study focuses on rs798766, rs401681, rs2294008, and rs8102137, which have been associated with bladder cancer and are also cis-acting methylation quantitative loci (mQTL)., Methods: Among 412 bladder cancer cases and 424 controls from the Women's Health Initiative (WHI), we assessed whether the effects of these SNPs on bladder cancer are mediated through proximal DNA methylation changes in pre-diagnostic blood at mQTL-associated CpG sites, which we refer to as natural indirect effects (NIEs). We used a multiple-mediator mediation model for each of the four mQTL adjusted for matching variables and potential confounders, including race/ethnicity, smoking status, and pack-years of smoking., Results: While not statistically significant, our results suggest that substantial proportions of the modest effects of rs401681 (OR NIE  = 1.05, 95% confidence interval (CI) = 0.89 to 1.25; NIE percent = 98.5%) and rs2294008 (OR NIE  = 1.10, 95% CI = 0.90 to 1.33; NIE percent = 77.6%) on bladder cancer risk are mediated through differential DNA methylation at nearby mQTL-associated CpG sites. The suggestive results indicate that rs2294008 may affect bladder cancer risk through a set of genes in the lymphocyte antigen 6 family, which involves genes that bind to and modulate nicotinic acetylcholine receptors. There was no suggestive evidence supporting mediation for rs8102137 and rs798766., Conclusions: Though larger studies are necessary, the methylation changes associated with rs401681 and rs2294008 at mQTL-associated CpG sites may be relevant for bladder carcinogenesis, and this study demonstrates how multi-omic data can be integrated to help understand the downstream effects of genetics variants.

The phenotypic and functional heterogeneity of the mouse prostate epithelial cell lineages remains incompletely characterized. We show that the Sca-1 + luminal cells at the mouse proximal prostate express Sox2. These cells are replicative quiescent, castration resistant, and do not possess secretory function. We use the Probasin-CreER T2 and Sox2-CreER T2 models in concert with a fluorescent reporter line to label the Sca-1 - and Sca-1 + luminal cells, respectively. By a lineage tracing approach, we show that the two luminal cell populations are independently sustained. Sox2 is dispensable for the maintenance of the Sca-1 + luminal cells but is essential for their facultative bipotent differentiation capacity. The Sca-1 + luminal cells share molecular features with the human TACSTD2 + luminal cells. This study corroborates the heterogeneity of the mouse prostate luminal cell lineage and shows that the adult mouse prostate luminal cell lineage is maintained by distinct cellular entities rather than a single progenitor population., (©AlphaMed Press 2020.)

Zika virus (ZIKV) can establish infection in immune privileged sites such as the testes, eye, and placenta. Whether ZIKV infection of white blood cells is required for dissemination of the virus to immune privileged sites has not been definitively shown. To assess whether initial ZIKV replication in myeloid cell populations is critical for dissemination during acute infection, recombinant ZIKVs were generated that could not replicate in these specific cells. ZIKV was cell restricted by insertion of a complementary sequence to a myeloid-specific microRNA in the 3' untranslated region. Following inoculation of a highly sensitive immunodeficient mouse model, crucial immune parameters, such as quantification of leukocyte cell subsets, cytokine and chemokine secretion, and viremia, were assessed. Decreased neutrophil numbers in the spleen were observed during acute infection with myeloid-restricted ZIKV that precluded the generation of viremia and viral dissemination to peripheral organs. Mice inoculated with a nontarget microRNA control ZIKV demonstrated increased expression of key cytokines and chemokines critical for neutrophil and monocyte recruitment and increased neutrophil influx in the spleen. In addition, ZIKV-infected Ly6C hi monocytes were identified in vivo in the spleen. Mice inoculated with myeloid-restricted ZIKV had a decrease in Ly6C hi ZIKV RNA-positive monocytes and a lack of inflammatory cytokine production compared to mice inoculated with control ZIKV. IMPORTANCE Myeloid cells, including monocytes, play a crucial role in immune responses to pathogens. Monocytes have also been implicated as "Trojan horses" during viral infections, carrying infectious virus particles to immune privileged sites and/or to sites protected by physical blood-tissue barriers, such as the blood-testis barrier and the blood-brain barrier. In this study, we found that myeloid cells are crucial to Zika virus (ZIKV) pathogenesis. By engineering ZIKV clones to encode myeloid-specific microRNA target sequences, viral replication was inhibited in myeloid cells by harnessing the RNA interference pathway. Severely immunodeficient mice inoculated with myeloid-restricted ZIKV did not demonstrate clinical signs of disease and survived infection. Furthermore, viral dissemination to peripheral organs was not observed in these mice. Lastly, we identified Ly6C mid/hi murine monocytes as the major myeloid cell population that disseminates ZIKV.

Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized β-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake α-neurotoxins WTX and NTII. Two different topologies of the β-structure were revealed: one large antiparallel β-sheet in Lypd6 and Lypd6b, and two β-sheets in other proteins. α-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the β-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.

The blood-brain barrier (BBB) is a major obstacle for the treatment of central nervous system (CNS) disorders. Significant progress has been made in developing adeno-associated virus (AAV) variants with increased ability to cross the BBB in mice. However, these variants are not efficacious in non-human primates. Herein, we employed various bioinformatic techniques to identify lymphocyte antigen-6E (LY6E) as a candidate for mediating transport of AAV across the human BBB based on the previously determined mechanism of transport in mice. Our results provide insight into future discovery and optimization of AAV variants for CNS gene delivery in humans.

Background: Lymphocyte antigen 6 complex, locus K (LY6K) is a putative oncogene in various cancers. Elevated expression of LY6K is correlated with poor patient prognosis in glioblastoma (GBM). The aim of this study is to advance our understanding of the mechanism by which LY6K contributes to GBM tumor biology., Methods: Bioinformatic data mining was used to investigate LY6K expression in relation to GBM clinical outcome. To understand the role of LY6K in GBM, we utilized patient-derived glioma stemlike cells (GSCs) and U87 cells and employed immunoblotting, immunofluorescent staining, radiation treatment, and orthotopic GBM xenograft models., Results: Our results show that increased expression of LY6K inversely correlates with GBM patient survival. LY6K promotes tumorigenicity in GBM cells both in vitro and in vivo. The mechanism underlying this tumorigenic behavior is enhancement of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Interestingly, we observed that tumor-promoting LY6K-ERK1/2 signaling is mediated by the interaction of LY6K with caveolin-1, rather than through oncogenic receptor tyrosine kinase-mediated signaling. Moreover, association of LY6K with the cell membrane is crucial for its tumorigenic functions. Finally, DNA methylation maintains LY6K silencing, and hypomethylation of the LY6K promoter increases its expression. In GSCs, ionizing radiation leads to demethylation of the LY6K promoter, thereby increasing LY6K expression and GSC resistance to radiation., Conclusions: Our study highlights the importance of the contribution of LY6K to GBM tumor biology and suggests LY6K as a potential membrane target for treating GBM., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)