Microglia and astrocytes are the main CNS glial cells responsible for the neuroinflammatory response, where they release a plethora of cytokines into the CNS inflammatory milieu. The TAM (Tyro3, Axl, Mer) receptors and their main ligand Gas6 are regulators of this response, however, the underlying mechanisms remain to be determined. We investigated the ability of Gas6 to modulate the CNS glial inflammatory response to lipopolysaccharide (LPS), a strong pro-inflammatory agent, through a qPCR array that explored Toll-like receptor signalling pathway-associated genes in primary cultured mouse microglia. We identified the Csf2 gene, encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), as a major Gas6 target gene whose induction by LPS was markedly blunted by Gas6. Both the Csf2 gene induction and the suppressive effect of Gas6 on this were emulated through measurement of GM-CSF protein release by cells. We found distinct profiles of GM-CSF induction in different glial cell types, with microglia being most responsive during inflammation. Also, Gas6 markedly inhibited the LPS-stimulated nuclear translocation of NF-κB p65 protein in microglia. These results illustrate microglia as a major resident CNS cellular source of GM-CSF as part of the neuroinflammatory response, and that Gas6/TAM signalling inhibits this response through suppression of NF-κB signalling.

Keywords: GM-CSF; Gas6; NF-κB; TAM receptors; astrocytes; glial cells; lipopolysaccharide; microglia; neuroinflammation; primary culture.

Obesity is correlated with increased incidence of breast cancer metastasis; however, the mechanisms underlying how obesity promotes metastasis are unclear. In a diet-induced obese mouse model, obesity enhanced lung metastasis in both the presence and absence of primary mammary tumors and increased recruitment of myeloid lineage cells into the lungs. In the absence of tumors, obese mice demonstrated increased numbers of myeloid lineage cells and elevated collagen fibers within the lung stroma, reminiscent of premetastatic niches formed by primary tumors. Lung stromal cells isolated from obese tumor-naïve mice showed increased proliferation, contractility, and expression of extracellular matrix, inflammatory markers and transforming growth factor beta-1 (TGFβ1). Conditioned media from lung stromal cells from obese mice promoted myeloid lineage cell migration in vitro in response to colony-stimulating factor 2 (CSF2) expression and enhanced invasion of tumor cells. Together, these results suggest that prior to tumor formation, obesity alters the lung microenvironment, creating niches conducive to metastatic growth.

Keywords: breast cancer; collagen; lung fibroblasts; macrophages; metastasis; obesity.

Background and aims: The immune compartment is critical for maintaining tissue homeostasis. A weak immune response increases susceptibility to infection, but immune hyperactivation causes tissue damage, and chronic inflammation may lead to cancer development. In the stomach, inflammation damages the gastric glands and drives the development of potentially pre-neoplastic metaplasia. Glucocorticoids are potent anti-inflammatory steroid hormones that are required to suppress gastric inflammation and metaplasia. However, these hormones function differently in males and females. Here, we investigate the impact of sex on the regulation of gastric inflammation.

Methods: Endogenous glucocorticoids and male sex hormones were removed from mice by adrenalectomy and castration, respectively. Mice were treated with 5α-dihydrotestosterone (DHT) to test the effects of androgens on regulating gastric inflammation. Single-cell RNA sequencing of gastric leukocytes was used to identify the leukocyte populations that were the direct targets of androgen signaling. ILC2s were depleted by treatment with CD90.2 antibodies.

Results: We show that adrenalectomized female mice develop spontaneous gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) but that the stomachs of adrenalectomized male mice remain quantitatively normal. Simultaneous depletion of glucocorticoids and sex hormones abolished the male-protective effects and triggered spontaneous pathogenic gastric inflammation and SPEM. Treatment of female mice with DHT prevented gastric inflammation and SPEM development when administered concurrent with adrenalectomy and also reversed the pathology when administered after disease onset. Single cell-RNAseq of gastric leukocytes revealed that type 2 innate lymphoid cells (ILC2s) expressed abundant levels of both the glucocorticoid receptor (Gr) and androgen receptor (Ar). We demonstrated that DHT treatment potently suppressed the expression of the proinflammatory cytokines Il13 and Csf2 by ILC2s. Moreover, ILC2 depletion protected the stomach from SPEM development.

Conclusions: Here, we report a novel mechanism by which glucocorticoids and androgens exert overlapping effects to regulate gastric inflammation. Androgen signaling within ILC2s prevents their pathogenic activation by suppressing the transcription of proinflammatory cytokines. This work revealed a critical role for sex hormones in regulating gastric inflammation and metaplasia.

Keywords: Gastric; ILC2; Macrophage; SPEM; Stomach.

Arabinoxylan (AX) extracted from wheat bran has attracted much attention due to its immunomodulatory activity. However, the molecular mechanisms underlying this activity remain unclear. In this study, we conducted a comprehensive transcriptional study to investigate genetic changes related to AX and identified 2325 differentially expressed genes (DEGs). Gene Ontology classification revealed that the DEGs were mainly enriched in a series of immune-related processes. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that immune-related pathways were significantly enriched in top 20 pathways, including the nuclear factor-kappa B (NF-κB) signaling pathway and the TNF signaling pathway. Validation using quantitative polymerase chain reaction analysis revealed dynamic changes in the mRNA levels of immune-related Cd40, Csf1, Csf2, Fas, IL-1β, IL-6, IL-5, Irf1, and Tnfaip3, which were significantly up-regulated in the AX-treated group. Moreover, AX treatment led to the up-regulation of the nuclear translocation of NF-κB and its upstream target proteins such as PDK1, Akt, IκB-α, and GSK-3β. The dataset compiled from this study provides valuable information for further research on the complex molecular mechanisms associated with AX and the identification of target genes.

Keywords: Arabinoxylan; Immunomodulatory; Nuclear factor-kappa B; Transcriptome analyses.

Background: Endometrial scratching (ES) has been proposed as a potential treatment for implantation improvement in unexplained repeated implantation failure (uRIF) patients, however, little is known about its exact molecular mechanisms.

Objective: This randomized controlled trial (RCT) was conducted on twenty uRIF patients to investigate the expression of innate and adaptive immune signaling genes after ES.

Methods: Ten uRIF patients in the intervention (twice endometrial sampling in follicular and luteal phases) and 10 uRIF patients in the control group (only luteal phase sampling) were randomly enrolled. Gene expression analysis with innate and adaptive immune response PCR-array kit between intervention and control groups were performed.

Results: Among innate immune-associated genes, a significant decrease was observed in the expression of APCS, CPR, CCL2, NLRP3, HLA-A, TLR3 and TLR4 in the intervention group. In adaptive immune-related genes, the expression level of CD80, CD86, CXCR3, IFNγ, IFNα1, IFNβ, MBL2, CCR6, CCR8 and IL17A were decreased and CSF2, GATA3, and IL4 increased significantly in the intervention group (P < 0.05). Of 14 uRIF patients, five live birth (35.71 %) was achieved.

Conclusion: ES in uRIF patients may exert positive effects on the endometrial preparation which increases its receptivity for embryo implantation by modulating the expression of an array of immune signaling pathway genes.

Keywords: Endometrial scratching; Inflammasome; Innate and adaptive immune response; Unexplained repeated implantation failure.

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4^-/-) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4^+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4^-/- mice. CD4⁺ T cells were expanded after mating in Tlr4^+/+ but not Tlr4^-/- mice, with failure to expand peripheral CD25⁺FOXP3⁺ NRP1^- or thymic CD25⁺FOXP3⁺ NRP1⁺ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.

During 2020, understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) became a scientific priority due to the devastating effects of the COVID-19. Many researchers have studied the effect of this viral infection on lung epithelial transcriptomes and deposited data in public repositories. Comprehensive analysis of such data could pave the way for development of efficient vaccines and effective drugs. In the current study, we obtained high-throughput gene expression data associated with human lung epithelial cells infected with respiratory viruses such as SARS-CoV-2, SARS, H1N1, avian influenza, rhinovirus and Dhori, then performed comparative transcriptome analysis to identify SARS-CoV-2 exclusive genes. The analysis yielded seven SARS-CoV-2 specific genes including CSF2 [GM-CSF] (colony-stimulating factor 2) and calcium-binding proteins (such as S100A8 and S100A9), which are known to be involved in respiratory diseases. The analyses showed that genes involved in inflammation are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 and S100A9 in COVID-19 disease. In conclusion, our analysis revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.

Objective---: Arteriovenous fistulae (AVF) placed for hemodialysis have high flow rates that can stimulate left ventricular (LV) hypertrophy. LV hypertrophy generally portends poor cardiac outcomes, yet clinical studies point to superior cardiac-specific outcomes for patients with AVF when compared to other dialysis modalities. We hypothesize that AVF induce physiologic cardiac hypertrophy with cardioprotective features.

Methods---: 9-11 week C57Bl/6 male and female mice were treated with sham laparotomy or an aortocaval fistula via a 25Ga needle. Cardiac chamber size and function were assessed with serial echocardiography, and cardiac CTA. Hearts were harvested at 5 weeks post-operatively, and collagen content assessed with Masson's trichrome. Bulk mRNA sequencing was performed from LV of sham and AVF mice at 10 days. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (Qiagen) to identify affected pathways and predict downstream biological effects.

Results---: Mice with AVF had similar body weight and wet lung mass, but increased cardiac mass compared to sham-operated mice. AVF increased cardiac output while preserving LV systolic and diastolic function, as well as indices of right heart function; all 4 cardiac chambers were enlarged, with slight decrement in relative LV wall thickness. Histology showed preserved collagen density within each of the 4 chambers without areas of fibrosis. RNA sequencing captured 19,384 genes, of which 857 were significantly differentially expressed, including transcripts from extracellular matrix-related genes, ion channels, metabolism, and cardiac fetal genes. Top upstream regulatory molecules predicted include activation of angiogenic (Vegf, Akt1), pro-cardiomyocyte survival (Hgf, Foxm1, Erbb2, Lin9, Areg), and inflammation-related (CSF2, Tgfb1, TNF, Ifng, Ccr2, IL6) genes, as well as the inactivation of cardiomyocyte antiproliferative factors (Cdkn1a, FoxO3, α-catenin). Predicted downstream effects include reduction to heart damage, and increased arrhythmia, angiogenesis, and cardiogenesis. There were no significant sex-dependent differences in the AVF-stimulated cardiac adaptation.

Conclusions---: AVF stimulate adaptive cardiac hypertrophy in wild-type mice without heart failure or pathological fibrosis. Transcriptional correlates suggest AVF-induced cardiac remodeling has some cardioprotective, although also arrhythmogenic features.

Keywords: Arteriovenous fistulae; cardiac hypertrophy; cardiac remodeling; cardioprotection.

A prelabor rupture of membranes (PROM) and its subtypes, preterm PROM (pPROM) and term PROM (tPROM), are associated with disturbances in the hemostatic system and angiogenesis. This study was designed to demonstrate the role of single nucleotide polymorphisms (SNPs), localized in CSF2 (rs25881), FLT1 (rs722503), TFPI (C-399T) and TLR9 (rs352140) genes, in PROM. A population of 360 women with singleton pregnancy consisted of 180 PROM cases and 180 healthy controls. A single-SNP analysis showed a similar distribution of genotypes in the studied polymorphisms between the PROM or the pPROM women and the healthy controls. Double-SNP TT variants for CSF2 and FLT1 polymorphisms, CC variants for TLR9 and TFPI SNPs, TTC for CSF2, FLT1 and TLR9 polymorphisms, TTT for FLT1, TLR9 and TFPI SNPs and CCCC and TTTC complex variants for all tested SNPs correlated with an increased risk of PROM after adjusting for APTT, PLT parameters and/or pregnancy disorders. The TCT variants for the CSF2, FLT1 and TLR9 SNPs and the CCTC for the CSF2, FLT1, TLR9 and TFPI polymorphisms correlated with a reduced risk of PROM when corrected by PLT and APTT, respectively. We concluded that the polymorphisms of genes, involved in hemostasis and angiogenesis, contributed to PROM.

Keywords: angiogenesis; genotyping; hemostasis; pPROM; pregnancy; prelabor rupture of membranes (PROM); restriction fragment length polymorphism (RFLP); single nucleotide polymorphism (SNP); tPROM.

Purpose: The purpose of this study was to elucidate the effects of interleukin (IL)-38 on experimental autoimmune uveitis (EAU) and its underlying mechanisms.

Methods: Mice with EAU were treated with IL-38, and the retinas and cervical draining lymph nodes (CDLNs) were analyzed by flow cytometry. Single-cell RNA sequencing (scRNA-seq) was conducted to analyze the immune cell profiles of CDLNs from normal, EAU, and IL-38-treated mice.

Results: Administration of IL-38 attenuated EAU symptoms and reduced the proportion of T helper 17 (Th17) and T helper 1 (Th1) cells in the retinas and CDLNs. In scRNA-seq analysis, IL-38 downregulated the IL-17 signaling pathway and reduced the expression of Th17 cell pathogenicity-related genes (Csf2 and Il23r), findings which were also confirmed by flow cytometry. In vitro, IL-38 reduced the granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation function of IL-23 and inhibited IL-23R expression in Th17 cells. Moreover, when co-cultured with Th17 cells, IL-38 prevented IL-23 production in antigen-presenting cells (APCs).

Conclusions: Our data demonstrate the therapeutic effect of IL-38 on EAU, and suggest that the effect of IL-38 may be caused by dampening of the GM-CSF/IL-23R/IL-23 feedback loop between Th17 cells and APCs.

Epidemiological studies and work in animal models indicate that immune activation may be a risk factor for autism spectrum disorders (ASDs). We measured levels of 60 cytokines and growth factors in 869 maternal mid-gestational (MMG) and 807 child cord blood (CB) plasma samples from 457 ASD (385 boys, 72 girls) and 497 control children (418 boys, 79 girls) from the Norwegian Autism Birth Cohort. We analyzed associations first using sex-stratified unadjusted and adjusted logistic regression models, and then employed machine learning strategies (LASSO + interactions, Random Forests, XGBoost classifiers) with cross-validation and randomly sampled test set evaluation to assess the utility of immune signatures as ASD biomarkers. We found prominent case-control differences in both boys and girls with alterations in a wide range of analytes in MMG and CB plasma including but not limited to IL1RA, TNFα, Serpin E1, VCAM1, VEGFD, EGF, CSF1, and CSF2. MMG findings were most striking, with particularly strong effect sizes in girls. Models did not change appreciably upon adjustment for maternal conditions, medication use, or emotional distress ratings. Findings were corroborated using machine learning approaches, with area under the receiver operating characteristic curve values in the test sets ranging from 0.771 to 0.965. Our results are consistent with gestational immunopathology in ASD, may provide insights into sex-specific differences, and have the potential to lead to biomarkers for early diagnosis.

Pericytes facilitate blood-brain barrier (BBB) integrity; however, the mechanisms involved remain unclear. Hence, using co-cultures of human cerebral microvascular endothelial cells (ECs) and vascular pericytes (PCs) in different spatial arrangements, as well as PC conditioned media, we investigated the impact of PC-EC orientation and PC-derived soluble factors on EC barrier function. We provide the first evidence that barrier-inducing properties of PCs require basolateral contact with ECs. Gene expression analysis (GEA) in ECs co-cultured with PCs versus ECs alone showed significant upregulation of 38 genes and downregulation of 122 genes. Pathway enrichment analysis of modulated genes showed significant regulation of several pathways, including transforming growth factor-β and interleukin-1 regulated extracellular matrix, interferon and interleukin signaling, immune system signaling, receptor of advanced glycation end products (RAGE), and cytokine-cytokine receptor interaction. Transcriptomic analysis showed a reduction in molecules such as pro-inflammatory cytokines and chemokines, which are known to be induced during BBB disruption. Moreover, cytokine proteome array confirmed the downregulation of key pro-inflammatory cytokines and chemokines on the protein level. Other molecules which influence BBB and were favorably modulated upon EC-PC co-culture include IL-18 binding protein, kallikrein-3, CSF2 CSF3, CXCL10, CXCL11 (downregulated) and IL-1-R4; HGF, PDGF-AB/BB, PECAM, SERPIN E1 (upregulated). In conclusion, we provide the first evidence that (1) basolateral contact between ECs and PCs is essential for EC barrier function and integrity; (2) in ECs co-cultured with PCs, the profile of BBB disrupting pro-inflammatory molecules and cytokines/chemokines is downregulated; (3) PCs significantly modulate EC mechanisms known to improve barrier function, including TGF-β regulated ECM pathway, anti-inflammatory cytokines, growth factors and matrix proteins. This human PC-EC co-culture may serve as a viable in vitro model for investigating BBB function and drug transport.

Keywords: blood–brain barrier; co-culture; conditioned media; hCMEC/D3; micro array; orientation; pericyte; transcriptome.

The toxicity and disposal concerns of organic solvents used in conventional extraction purposes has entailed the need for greener alternatives. Among such techniques, supercritical fluid extraction (SFE) has gained popularity by yielding extracts of high purity in a much faster manner. Carbon dioxide (CO₂) is generally preferred as a supercritical solvent because of its lower temperature requirements, better diffusivity and easy removal. The present study describes the characterization of supercritical CO₂ extracts of Indian variety of Cordyceps sinensis (CS)- a high-altitude medicinal mushroom widely revered in traditional medicine for its extensive anti-hypercholesterolemic, anti-inflammatory, anti-proliferative and energy-enhancing properties. Experimental parameters viz. 300 and 350 bar of extraction pressure, 60°C of temperature, 0.4°L/h CO₂ of flow rate and use of 1% (v/v) of ethanol as entrainer were optimized to prepare three different extracts namely, CSF1, CSF2 and CSF3. High-performance thin-layer chromatography (HPTLC) was used for assessing the quality of all the extracts in terms of cordycepin, the pivot biomarker compound in CS. Characterization by HPTLC and GC-MS confirmed the presence of flavonoids and nucleobases and, volatile organic compounds (VOCs), respectively. The chromatographic data acquired from metabolite profiling were subjected to chemometric analysis in an open source R studio which illustrated interrelatedness between CSF1 and CSF2 in terms of two major principal components. i.e. Dim 1 and Dim 2 whose values were 40.33 and 30.52% in variables factor map plotted using the HPTLC-generated retardation factor values. The factor maps based on retention times of the VOCs exhibited a variance of Dim 1 = 43.95% and Dim 2 = 24.85%. Furthermore, the extracts demonstrated appreciable antibacterial activity against Escherichia coli and Salmonella typhi by generation of reactive oxygen species (ROS), protein leakage and efflux pump inhibition within bacterial pathogens. CSFs were elucidated to be significantly cytoprotective (p < 0.05) in a simulated hypobaric hypoxia milieu (0.5% oxygen). CSF2 showed the best results by effectively improving the viability of human embryonic kidney (HEK 293) cells to 82.36 ± 1.76% at an optimum dose of 100 µg/ml. Levels of hypoxia inducible factor-1 alpha (HIF-1α) were modulated four-fold upon supplementation with CSF2. The results collectively evinced that the CSF extracts are substantially bioactive and could be effectively utilized as mycotherapeutics for multiple bioeffects.

Keywords: Cordyceps sinensis (Berk) Sacc.; GC-MS; HPTLC; hypobaric hypoxia (HH); metabolomics; supercritical fluid extract.

Background: The seminal vesicles synthesise bioactive factors that support gamete function, modulate the female reproductive tract to promote implantation, and influence developmental programming of offspring phenotype. Despite the significance of the seminal vesicles in reproduction, their biology remains poorly defined. Here, to advance understanding of seminal vesicle biology, we analyse the mouse seminal vesicle transcriptome under normal physiological conditions and in response to acute exposure to the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or vehicle control daily for five consecutive days prior to collecting seminal vesicle tissue 72 h following the final injection.

Results: A total of 15,304 genes were identified in the seminal vesicles with those encoding secreted proteins amongst the most abundant. In addition to reproductive hormone pathways, functional annotation of the seminal vesicle transcriptome identified cell proliferation, protein synthesis, and cellular death and survival pathways as prominent biological processes. Administration of acrylamide elicited 70 differentially regulated (fold-change ≥1.5 or ≤ 0.67) genes, several of which were orthogonally validated using quantitative PCR. Pathways that initiate gene and protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways. Inflammation was also a key transcriptomic response to acrylamide, with the cytokine, Colony stimulating factor 2 (Csf2) identified as a top-ranked upstream driver and inflammatory mediator associated with recovery of homeostasis. Early growth response (Egr1), C-C motif chemokine ligand 8 (Ccl8), and Collagen, type V, alpha 1 (Col5a1) were also identified amongst the dysregulated genes. Additionally, acrylamide treatment led to subtle changes in the expression of genes that encode proteins secreted by the seminal vesicle, including the complement regulator, Complement factor b (Cfb).

Conclusions: These data add to emerging evidence demonstrating that the seminal vesicles, like other male reproductive tract tissues, are sensitive to environmental insults, and respond in a manner with potential to exert impact on fetal development and later offspring health.

Keywords: Acrylamide; Reproduction; Reproductive toxicant; Seminal vesicle; Transcriptomics.

Interleukin (IL)-13-associated inflammatory response is important for the pathogenesis of allergic rhinitis (AR). Apremilast is a phosphodiesterase-4 (PDE4) inhibitor approved for psoriasis treatment. Here, we investigated the potential effects of Apremilast against IL-13-induced injury in human nasal epithelial cells (hNECs). Firstly, Apremilast ameliorated oxidative stress in IL-13-challenged cells by decreasing the levels of reactive oxygen species (ROS) and the production of malondialdehyde (MDA). Secondly, Apremilast inhibited the expressions of IL-6 and IL-8. Moreover, Apremilast inhibited the expressions of the chemokines colony-stimulating factor 2 (CSF2) and chemokine ligand 11 (CCL11). Interestingly, exposure to IL-13 increased the expressions of mucin 4 and mucin 5AC (MUC5AC), which was ameliorated by treatment with Apremilast. Interestingly, we found that Apremilast inhibited the phosphorylation of c-Jun-N-terminal kinase (JNK). Importantly, Apremilast reduced the levels of c-fos and c-Jun, the two AP-1 subfamilies. The luciferase reporter assay demonstrates that Apremilast reduced the transcriptional activity of activator protein 1 (AP-1). Lastly, we found that Apremilast prevented the activation of nuclear factor kappa-B (NF-κB) by decreasing the levels of nuclear NF-κB p65 and the luciferase activity of the NF-κB reporter. In summary, we conclude that Apremilast possesses a protective effect against IL-13-induced inflammatory response and mucin production in hNECs by inhibiting the activity of AP-1 and NF-κB.

Keywords: Allergic rhinitis; Apremilast; IL-13; NF-κB; mucin.

The growth of viable cattle embryos in culture to stages beyond the hatching blastocyst is of interest to developmental biologists wishing to understand developmental events beyond the first lineage decision, as well as for commercial applications, because a lengthening of the culturing time allows more time for diagnostic tests on biopsies, whereas extended survival can be used as a better assay system for monitoring developmental potential. We here report on a novel extended culture medium for embryo growth until embryonic day (Day) 12. We used a non-invasive morphological characterisation system that scored viability, inner cell mass (ICM) grade, hatching and embryo and ICM diameter. The basal medium was based on published uterine fluid concentrations of amino acids, carbohydrates and electrolytes. Addition of fetal bovine serum was necessary and the additive ITSX greatly improved culture success. We tested the inclusion of a seven-growth factor cocktail consisting of Activin A, Artemin, BMP4, EGF, FGF4, GM-CSF/CSF2 and LIF, as well as omission of individual components of the cocktail. In the context of the growth factor cocktail, Artemin and BMP4 provided the greatest benefit, while FGF omission had more positive than negative effects on embryo characteristics. Lastly, replacement of ITSX by B27-additive led to the most successful culture of embryos, in all media permutations.

Keywords: Bovine; Embryo medium; Growth factors; ICM; Preimplantation embryo; Trophoblast.

CSF1R inhibition combined with STAT5 blockade normalized TAM phenotype and sustained tumor control.

Hair follicle stem cells are extensively reprogrammed by the aging process, manifesting as diminished self-renewal and delayed responsiveness to activating cues, orchestrated by both intrinsic microenvironmental and extrinsic macroenvironmental regulators. Dermal white adipose tissue (dWAT) is one of the peripheral tissues directly adjacent to hair follicles (HFs) and acts as a critical macroenvironmental niche of HF. dWAT directly contributes to HF aging by paracrine signal secretion. However, the altered interrelationship between dWAT and HF with aging has not been thoroughly understood. Here, through microdissection, we separated dWAT from the skin of aged mice (18 months) and young mice (2 months) in telogen and depilation-induced anagen for transcriptome comparing. Notably, compared with young dWAT, aberrant inflammatory regulators were recapitulated in aging dWAT in telogen, including substantial overexpressed inflammatory cytokines, matrix metalloproteinases, and prostaglandin members. Nonetheless, with anagen initiation, inflammation programs were mostly abolished in aging dWAT, and instead of which, impaired collagen biosynthesis, angiogenesis, and melanin synthesis were identified. Furthermore, we confirmed the inhibitory effect on hair growth of CXCL1, one of the most significantly upregulated inflammation cytokines in aging dWAT. Besides this, we also identified the under-expressed genes related to Wnt signaling fibroblast growth factor family members and increased BMP signaling in aging dWAT, further unraveling the emerging role of dWAT in aging HFs malfunction. Finally, we proved that relieving inflammation of aging dWAT by injecting high-level veratric acid stimulated HF regenerative behavior in aged mice. Concomitantly, significantly decreased TNF-a, CCL2, IL-5, CSF2, and increased IL10 in dWAT was identified. Overall, the results elaborated on the complex physiological cycling changes of dWAT during aging, providing a basis for the potential regulatory effect of dWAT on aging HFs.

Keywords: aging; dermal white adipose tissue; hair follicle; inflamm-aging; microenvironment.

Background: Coronavirus disease 2019 is characterized by the elevation of a broad spectrum of inflammatory mediators associated with poor disease outcomes. We aimed at an in-silico analysis of regulatory microRNA and their transcription factors (TF) for these inflammatory genes that may help to devise potential therapeutic strategies in the future.

Methods: The cytokine regulating immune-expressed genes (CRIEG) were sorted from literature and the GEO microarray dataset. Their co-differentially expressed miRNA and transcription factors were predicted from publicly available databases. Enrichment analysis was done through mienturnet, MiEAA, Gene Ontology, and pathways predicted by KEGG and Reactome pathways. Finally, the functional and regulatory features were analyzed and visualized through Cytoscape.

Results: Sixteen CRIEG were observed to have a significant protein-protein interaction network. The ontological analysis revealed significantly enriched pathways for biological processes, molecular functions, and cellular components. The search performed in the miRNA database yielded ten miRNAs that are significantly involved in regulating these genes and their transcription factors.

Conclusion: An in-silico representation of a network involving miRNAs, CRIEGs, and TF, which take part in the inflammatory response in COVID-19, has been elucidated. Thus, these regulatory factors may have potentially critical roles in the inflammatory response in COVID-19 and may be explored further to develop targeted therapeutic strategies and mechanistic validation.

Keywords: AHR, Aryl hydrocarbon receptor; ARDS, acute respiratory distress syndrome; BAL, Bronchoalveolar Lavage; CC, Cellular components; CCL, Chemokine (C-C motif) ligands; CCL2, C-C motif chemokine 2; CCL3, C-C motif chemokine 3; CCL4, C-C motif chemokine 4; CCR, CC chemokine receptor; CEBPA, CCAAT/enhancer-binding protein alpha; COVID-19; COVID-19, Coronavirus Disease 2019; CREM, cAMP responsive element modulator; CRIEGs, Cytokine regulating immune expressed genes; CSF2, Granulocyte-macrophage colony-stimulating factor; CSF3, Granulocyte colony-stimulating factor; CXCL10, C-X-C motif chemokine 10; CXCL2, Chemokine (C-X-C motif) ligand 2; CXCL8, Interleukin-8; CXCR, C-X-C chemokine receptor; Cytokine storm; Cytokines; DDIT3, DNA damage-inducible transcript 3 protein; DEGs, Differentially expressed genes; E2F1, Transcription factor E2F1; EGR1, Early growth response protein 1; EP300, Histone acetyltransferase p300; ESR1, Estrogen receptor, Nuclear hormone receptor; ETS2, Protein C-ets-2; FOXP3, Forkhead box protein P3; GO, Gene Ontology; GSEs, Gene Series Expressions; HDAC1, Histone deacetylase 1; HDAC2, Histone deacetylase 2; HSF1, Heat shock factor protein 1; IL-6, interleukin-6; IL10, Interleukin-10; IL17A, Interleukin-17A; IL1B, Interleukin-1; IL2, Interleukin-2; IL6, Interleukin-6; IL7, Interleukin-7; IL9, Interleukin-9; IP-10, Interferon-Inducible Protein 10; IRF1, Interferon regulatory factor 1; Immuno-interactomics; JAK-STAT, Janus kinase (JAK)-signal transducer and activator; JAK2, Tyrosine-protein kinase JAK2; JUN, Transcription factor AP-1; KEGG, Kyoto Encyclopedia of Genes and Genomes; KLF4, Krueppel-like factor 4; MicroRNA, SARS-CoV-2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NFAT5, Nuclear factor of activated T-cells 5; NFKB1, Nuclear factor NF-kappa-B p105 subunit; NFKBIA, NF-kappa-B inhibitor alpha; NR1I2, Nuclear receptor subfamily 1 group I member 2; PDM, peripheral blood mononuclear cell; REL, Proto-oncogene c-Rel; RELA, Transcription factor p65; RUNX1, Runt-related transcription factor 1; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SIRT1, NAD-dependent protein deacetylase sirtuin-1; SP1, Transcription factor Sp1; SPI1, Transcription factor PU.1; STAT1, Signal transducer and activator of transcription 1-alpha/beta; STAT3, Signal transducer and activator of transcription 3; TLR3, Toll-like receptor 3 (TLR3); TNF, Tumor necrosis factor; TNF-α, Tumor Necrosis Factor-Alpha; VDR, Vitamin D3 receptor; XBP1, X-box-binding protein 1; ZFP36, mRNA decay activator protein ZFP36; ZNF300, Zinc finger protein 300, heme oxygenase-1 (HO-1); miEAA, miRNA Enrichment Analysis and Annotation t.

Sam68 is an RNA-binding protein with an adaptor role in signal transduction. Our previous work identified critical proinflammatory and apoptotic functions for Sam68, downstream of the TNF/TNFR1 and TLR2/3/4 pathways. Recent studies have shown elevated Sam68 in inflamed tissues from rheumatoid arthritis and ulcerative colitis (UC) patients, suggesting that Sam68 contributes to chronic inflammatory diseases. Here, we hypothesized that deletion of Sam68 is protective against experimental colitis in vivo, via reductions in TNF-associated inflammatory signaling. We used Sam68 knockout (KO) mice to study the role of Sam68 in experimental colitis, including its contributions to TNF-induced inflammatory gene expression in three-dimensional intestinal organoid cultures. We also studied the expression of Sam68 and inflammatory genes in colon tissues of UC patients. Sam68 KO mice treated with an acute course of DSS exhibited significantly less weight loss and histopathological inflammation compared to wild-type controls, suggesting that Sam68 contributes to experimental colitis. Bone marrow transplants showed no pathologic role for hematopoietic cell-specific Sam68, suggesting that non-hematopoietic Sam68 drives intestinal inflammation. Gene expression analyses showed that Sam68 deficiency reduced the expression of proinflammatory genes in colon tissues from DSS-treated mice, as well as TNF-treated three-dimensional colonic organoids. We also found that inflammatory genes, such as TNF, CCR2, CSF2, IL33 and CXCL10, as well as Sam68 protein, were upregulated in inflamed colon tissues of UC patients. This report identifies Sam68 as an important inflammatory driver in response to intestinal epithelial damage, suggesting that targeting Sam68 may hold promise to treat UC patients.

Keywords: Inflammation; Inflammatory bowel disease; KHDRBS1; NF-kappaB; TNF.

Few intravenously administered mesenchymal stromal cells (MSCs) engraft to the injured myocardium, thereby limiting their therapeutic efficacy for the treatment of ischemic heart injury. Here, it is found that irisin pretreatment increases the cardiac homing of adipose tissue-derived MSCs (ADSCs) administered by single and multiple intravenous injections to mice with MI/R by more than fivefold, which subsequently increases their antiapoptotic, proangiogenic, and antifibrotic effects in rats and mice that underwent MI/R. RNA sequencing, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, and loss-of-function studies identified CSF2RB as a cytokine receptor that facilitates the chemotaxis of irisin-treated ADSCs in the presence of CSF2, a chemokine that is significantly upregulated in the ischemic heart. Cardiac-specific CSF2 knockdown blocked the cardiac homing and cardioprotection abilities of intravenously injected irisin-treated ADSCs in mice subjected to MI/R. Moreover, irisin pretreatment reduced the apoptosis of hydrogen peroxide-induced ADSCs and increased the paracrine proangiogenic effect of ADSCs. ERK1/2-SOD2, and ERK1/2-ANGPTL4 are responsible for the antiapoptotic and paracrine angiogenic effects of irisin-treated ADSCs, respectively. Integrin αV/β5 is identified as the irisin receptor in ADSCs. These results provide compelling evidence that irisin pretreatment can be an effective means to optimize intravenously delivered MSCs as therapy for ischemic heart injury.

Keywords: CSF2RB; integrin αV/β5; irisin; ischemic heart injury; mesenchymal stromal cells.

Problem: A significant rate of spontaneous abortion is observed in cattle pregnancies produced by somatic cell nuclear transfer (SCNT). Major histocompatibility complex class I (MHC-I) proteins are abnormally expressed on the surface of trophoblast cells from SCNT conceptuses.

Method of study: MHC-I homozygous compatible (n = 9), homozygous incompatible (n = 8) and heterozygous incompatible (n = 5) pregnancies were established by SCNT. Eight control pregnancies were established by artificial insemination. Uterine and trophoblast samples were collected on day 35 ±1 of pregnancy, the expression of immune-related genes was examined by qPCR, and the expression of trophoblast microRNAs was assessed by sequencing.

Results: Compared to the control group, trophoblast from MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL1A, IL2, IL10, IL12B, TBX21 and TNF, while GNLY expression was down-regulated. The MHC-I homozygous incompatible treatment group expressed increased levels of IFNG, IL1A, and IL2 while the MHC-I homozygous compatible group did not differentially express any genes compared to the control group. In the endometrium, relative to the control group, MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL10, IL12B, and TNF, while GATA3 expression was down-regulated. The MHC-I homozygous incompatible group expressed decreased amounts of CSF2 transcripts compared with the control group but did not have abnormal expression of any other immune-related genes. MHC-I incompatible pregnancies had 40 deregulated miRNAs compared to control pregnancies and 62 deregulated microRNAs compared to MHC-I compatible pregnancies.

Conclusions: MHC-I compatibility between the dam and fetus prevented an exacerbated maternal immune response from being mounted against fetal antigens. This article is protected by copyright. All rights reserved.

Keywords: cattle; cytokines; gene expression; microRNA; miscarriage; pregnancy; somatic cell nuclear transfer.

Background/purpose: Conditioned media of cultured mesenchymal stem cells (MSCs) contain numerous kinds of secretomes such as cytokines and chemokines. We previously reported that conditioned media of bone marrow-derived MSCs (MSC-CM) promote bone formation. Recently, macrophage phenotype switching from the pro-inflammatory M1 type to the anti-inflammatory M2 type has been reported to be an important phenomenon during tissue regeneration. Some studies reported that this phenotype switching is regulated by secretomes. In this study, macrophage phenotype during bone formation by MSC-CM was investigated.

Materials and methods: Human MSCs (hMSCs) were cultured in serum-free medium and the collected medium was defined as MSC-CM. Macrophage-related gene expressions in hMSCs cultured with MSC-CM were evaluated by quantitative real-time polymerase chain reaction. MSC-CM was implanted and the evaluations by micro-CT and immunohistochemistry were performed using a rat the calvaria bone defect model.

Results: Two and four weeks after implantation, the MSC-CM group demonstrated enhanced bone regeneration. Gene expressions of C-C motif chemokine 2 (CCL2), colony-stimulating factor 2 (CSF2) and CD163 was significantly upregulated in cells exposed to MSC-CM. Immunohistochemical staining revealed that iNOS-positive M1 macrophages were reduced, while CD204-positive M2 macrophages were increased in the MSC-CM group at 72 h after implantation, and the M2/M1 ratio increased only in the MSC-CM group.

Conclusion: MSC-CM enhances macrophage migration and induces M1 to M2 type macrophage switching at an early stage of osteogenesis. Such phenotype switching provides a favorable environment for angiogenesis, cellular migration, and osteogenesis and contributes to MSC-CM-induced early bone formation.

Keywords: Bone regeneration; Conditioned media; Macrophage; Mesenchymal stem cell; Phenotype switching; Secretome.

Abdominal aortic aneurysms (AAAs) elicit massive inflammatory leukocyte recruitment to the aorta. CD4⁺ T cells, which include regulatory T cells (Tregs) and conventional T cells (Tconvs), are involved in the progression of AAA. Tregs have been reported to limit AAA formation. However, the function and phenotype of the Tconvs found in AAAs remain poorly understood. We characterized aortic Tconvs by bulk RNA sequencing and discovered that Tconvs in aortic aneurysm highly expressed Cxcr6 and Csf2. Herein, we determined that the CXCR6/CXCL16 signaling axis controlled the recruitment of Tconvs to aortic aneurysms. Deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF), encoded by Csf2, markedly inhibited AAA formation and led to a decrease of inflammatory monocytes, due to a reduction of CCL2 expression. Conversely, the exogenous administration of GM-CSF exacerbated inflammatory monocyte infiltration by upregulating CCL2 expression, resulting in worsened AAA formation. Mechanistically, GM-CSF upregulated the expression of interferon regulatory factor 5 to promote M1-like macrophage differentiation in aortic aneurysms. Importantly, we also demonstrated that the GM-CSF produced by Tconvs enhanced the polarization of M1-like macrophages and exacerbated AAA formation. Our findings revealed that GM-CSF, which was predominantly derived from Tconvs in aortic aneurysms, played a pathogenic role in the progression of AAAs and may represent a potential target for AAA treatment.

Keywords: CXCR6; GM-CSF; Tconvs; abdominal aortic aneurysm.