Seminal plasma has conventionally been viewed as a transport and survival medium for mammalian sperm; however, its role now extends beyond this process to actively targeting female tissues. Studies in rodents, swine and humans demonstrate that seminal plasma induces molecular and cellular changes within the endometrium or cervix following insemination. Seminal plasma induced alterations to the maternal environment have been theorized to facilitate embryo development, modulate maternal immunity toward the conceptus and potentially improve pregnancy success. It is unknown if bovine seminal plasma modulates the uterine environment following insemination in the cow, where routine use of artificial insemination reduces maternal exposure to seminal plasma. We hypothesize that seminal plasma modulates the expression of inflammatory mediators in the endometrium, altering the maternal environment of early pregnancy. In vitro, seminal plasma altered intact endometrial explant expression of CSF2, IL1B, IL6, IL17A, TGFB1, IFNE, PTGS2, and AKR1C4. Furthermore, endometrial epithelial cell CSF2, CXCL8, TGFB1, PTGS2 and AKR1C4 expression were increased after seminal plasma exposure, while endometrial stromal cell CSF2, IL1B, IL6, CXCL8, IL17A, TGFB1, PTGS2 and AKR1C4 expression were increased following seminal plasma exposure. Endometrial expression of IL1B was increased in the cow 24 h after uterine infusion of seminal plasma, while other evaluated inflammatory mediators remained unchanged. These data indicate that seminal plasma may induce changes in the bovine endometrium in a temporal manner. Understanding the role of seminal plasma in modulating the maternal environment may aid in improving pregnancy success in cattle.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

This study evaluated the performance, gut microbiota, and blood metabolites in broiler chickens fed cranberry and blueberry products for 30 days. A total of 2,800 male day-old broiler Cobb-500 chicks were randomly distributed between 10 diets: control basal diet; basal diet with bacitracin (BACI); four basal diets with 1 and 2% of cranberry (CP1, CP2) and blueberry (BP1, BP2) pomaces; and four basal diets supplemented with ethanolic extracts of cranberry (COH150, COH300) or blueberry (BOH150, BOH300) pomaces. All groups were composed of seven replicates (40 birds per replicate). Cecal and cloacal samples were collected for bacterial counts and 16S rRNA gene sequencing. Blood samples and spleens were analyzed for blood metabolites and gene expressions, respectively. The supplementation of COH300 and BOH300 significantly increased the body weight (BW) during the starting and growing phases, respectively, while COH150 improved (P < 0.05) the overall cumulated feed efficiency (FE) compared to control. The lowest prevalence (P = 0.01) of necrotic enteritis was observed with CP1 and BP1 compared to BACI and control. Cranberry pomace significantly increased the quinic acid level in blood plasma compared to other treatments. At days 21 and 28 of age, the lowest (P < 0.05) levels of triglyceride and alanine aminotransferase were observed in cranberry pomace and blueberry product-fed birds, respectively suggesting that berry feeding influenced the lipid metabolism and serum enzyme levels. The highest relative abundance of Lactobacillaceae was found in ceca of birds fed CP2 (P < 0.05). In the cloaca, BOH300 significantly (P < 0.005) increased the abundances of Acidobacteria and Lactobacillaceae. Actinobacteria showed a significant (P < 0.05) negative correlation with feed intake (FI) and FE in COH300-treated birds, whereas Proteobacteria positively correlated with the BW but negatively correlated with FI and FE, during the growing phase. In the spleen, cranberry products did not induce the release of any pro-inflammatory cytokines but upregulated the expression of several genes (IL4, IL5, CSF2, and HMBS) involved in adaptive immune responses in broilers. This study demonstrated that feed supplementation with berry products could promote the intestinal health by modulating the dynamics of the gut microbiota while influencing the metabolism in broilers.

Keywords: blood metabolites; broilers; cranberry and blueberry pomaces; gut microbiota; immunity; spleen.

Understanding of the mechanism for myeloid differentiation provides important insights into the hematopoietic developmental processes. By using an ESC-derived myeloid progenitor cell model, we found that CSF2/GM-CSF triggered macrophage differentiation and activation of the MTOR signaling pathway. Activation or inhibition of the MTOR signaling enhanced or attenuated macrophage differentiation, respectively, suggesting a critical function. We further showed that macroautophagy/autophagy was inhibited with the addition of CSF2. Furthermore, pharmacological inhibition and genetic modification of autophagy enhanced macrophage differentiation and rescued the inhibitory effect on differentiation caused by MTOR inhibition. Thus, the MTOR signaling pathway regulates macrophage differentiation of myeloid progenitors by inhibiting autophagy. Our results provide new insights into the mechanisms for myeloid differentiation and may prove useful for therapeutic applications of hematopoietic and myeloid progenitor cells. Abbreviations: 2-DG: 2-deoxy-D-glucose; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; BM: bone marrow; CQ: chloroquine; ECAR: extracellular acidification rate; ESC: embryonic stem cell; CSF2/GM-CSF: colony stimulating factor 2; CSF3/G-CSF: colony stimulating factor 3; HPC: hematopoietic progenitor cell; ITGAM/CD11b: integrin alpha M; LPS: lipopolysaccharide; MFI: median fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; RPS6KB1/p70S6K1: ribosomal protein S6 kinase, polypeptide 1; shRNA: short hairpin RNA; SQSTM1/p62: sequestosome 1.

Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 ± 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 ± 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 μg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 μg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 μg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1β and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 μg/mL) disturb membrane integrity and kill rapidly human cells (60% after 5 h). This effect is higher in SNP-AmB than in SNP.

Endothelial cells (EC) are key targets during Orientia tsutsugamushi infection. Knowledge of the pro-inflammatory response against O. tsutsugamushi by ECs is limited. The aim of the present study was to characterize the pro-inflammatory transcriptional response during the first 24 hours of infection of the human dermal microvascular endothelial cell line with O. tsutsugamushi Karp by examining five-time points. The transcriptional profiles of 84 genes including cytokines, chemokines, growth factors, and TNF receptor superfamily genes were studied using a RT-PCR array. We identified 40 of the 84 genes that were up or down modulated during the early O. tsutsugamushi infection that differed remarkably from genes of non-infected cells. The modulated genes included: the interleukins (IL-1α/β, IL-4, IL-6, IL-7, IL-10, IL-11, IL-18, and IL-24), chemokines (CXCL8, CCL2/MCP1, CCL5/RANTES, and CCL17), growth factors (NODAL, CNTF, and CSF2/GM-CSF), and TNFSF13B. IL-1β, IL-4, and IL-11 were highly induced at one hour post infection, whereas, CCL17 was profoundly up-regulated and IFNα2 was greatly down-regulated during the entire 24-hour time course. These results provide insight into the early pro-inflammatory response of endothelial cells to O. tsutsugamushi infection and indicate their potential role in the pathophysiology of the host's initial response to O. tsutsugamushi infection.

Oncogenic KRAS has been previously identified to act in a cell-intrinsic manner to modulate multiple biological functions of colorectal cancer (CRC). Here, we demonstrate a cell-extrinsic role of KRAS, where KRAS engages with the tumor microenvironment by functional reprogramming of tumor-associated macrophages (TAMs). In human CRC specimens, mutant KRAS positively correlates with the presence of TAMs. Mutationally activated KRAS in tumor cells reprograms macrophages to a TAM-like phenotype via a combination effect of tumor-derived CSF2 and lactate. In turn, KRAS-reprogrammed macrophages were shown to not only promote tumor progression but also induce the resistance of tumor cells to cetuximab therapy. Mechanistically, KRAS drives the production of CSF2 and lactate in tumor cells by stabilizing hypoxia-inducible factor-1α (HIF-1α), a transcription factor that controls the expression of CSF2 and glycolytic genes. Mutant KRAS increased the production of reactive oxygen species, an inhibitor of prolyl hydroxylase activity which decreases HIF-1α hydroxylation, leading to enhanced HIF-1α stabilization. This cell-extrinsic mechanism awards KRAS a critical role in engineering a permissive microenvironment to promote tumor malignancy, and may present new insights on potential therapeutic defense strategies against mutant KRAS tumors.

Embryokines are molecules secreted by the mother that regulate embryonic development. Among these molecules in cattle are colony stimulating factor 2 (CSF2) and dickkopf-related protein 1 (DKK1). Here, we evaluated actions of CSF2 and DKK1 alone or in combination on characteristics of embryos produced in vitro in the presence of serum. A total of 70 beef cows from 4 farms were subjected to oocyte retrieval on 1 to 4 occasions. Within each farm, donors were randomly allocated to 1 of 4 treatment groups (vehicle, CSF2, DKK1, CSF2 + DKK1). Embryos from a given donor were always exposed to the same treatment. Treatments were added to the culture medium on d 5 after insemination, and blastocyst stage embryos were transferred to recipient females 2 d later. Treatment did not affect the percent of oocytes or cleaved embryos that developed to the blastocyst stage or the percent of recipients that became pregnant after embryo transfer. However, calves derived from embryos treated with DKK1 were smaller at birth, regardless of CSF2 treatment. Results indicate no effects of addition of CSF2 or DKK1 to culture of embryos produced in vitro with serum-containing medium on development to the blastocyst stage or competence to establish pregnancy after transfer to recipients. The fact that embryos cultured with DKK1 resulted in calves with reduced birth weight illustrates the potential ability of this embryokine to program postnatal phenotype. Results support the concept that properties of the offspring can be programmed as early as the preimplantation period.

OBJECTIVE

Breast cancer patients' response to treatment is highly dependent on the primary tumor molecular features, with triple-negative breast tumors having the worst prognosis of all subtypes. According to the molecular features, tumors stimulate the microenvironment to induce distinct immune responses, baseline immune activation being associated with higher likelihood of pathologic response. In this study, we investigated the deconvolution of the immunological status of triple-negative tumors in comparison with luminal tumors and the association with patients' clinicopathological characteristics.

METHODS

Gene expression of 84 inflammatory molecules and their receptors were analyzed in 40 peripheral blood samples from patients with Her2- primary breast cancer tumors. We studied the association of triple-negative phenotype with age, clinical stage, tumor size, lymph nodes, and menopausal status.

RESULTS

We observed that more patients with estrogen (ER)/progesterone (PR)-negative tumors had grade III, while more patients with ER/PR-positive tumors had grade II tumors. Gene expression analysis revealed a panel of 14 genes to have differential expression between the two groups: several interleukins: IL13, IL16, IL17C and IL17F, IL1A, IL3; interleukin receptors: IL10RB, IL5RA; chemokines: CXCL13 and CCL26; and cytokines: CSF2, IFNA2, OSM, TNSF13.

CONCLUSIONS

The expression levels of these genes have been previously shown to be associated with reduced immunological status; indeed, the triple-negative breast cancer patients presented with lower counts of lymphocytes and eosinophils than the ER/PR-positive ones. These results contribute to a better understanding of the possible role of antitumor immune responses in mediating the clinical outcome.

Interleukin 24 (IL24) has been documented to be highly expressed in several cancers, but its role in laryngeal squamous cell carcinoma (LSCC) remains unclarified. In this study, to reveal the function and its clinical significance of IL24 in LSCC, multiple detecting methods were used comprehensively. IL24 protein expression was remarkably higher in LSCC (n= 49) than non-cancerous laryngeal controls (n= 26) as detected by in-house immunohistochemistry. Meanwhile, the IL24 mRNA expression was also evaluated based on high throughput data from Gene Expression Omnibus, The Cancer Genome Atlas, ArrayExpress and Oncomine databases. Consistently with the protein level, IL24 mRNA expression level was also predominantly upregulated in LSCC (n= 172) compared to non-cancerous laryngeal tissues (n= 81) with the standard mean difference (SMD) being 1.25 and the area under the curve (AUC) of the summary receiver operating characteristic (sROC) being 0.89 (95% CI = 0.86-0.92). Furthermore, the related genes of IL24 and the differentially expressed genes (DEGs) of LSCC were intersected and sent for Gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the protein-protein interaction (PPI) analyses. In the GO annotation, the top terms of biological process (BP), cellular component (CC) and molecular function (MF) were extracellular matrix organization, extracellular matrix, cytokine activity, respectively. The top pathway of KEGG was ECM-receptor interaction. The PPI networks indicated the top hub genes of IL24-related genes in LSCC were SERPINE1, TGFB1, MMP1, MMP3, CSF2, and ITGA5. In conclusion, upregulating expression of IL24 may enhance the occurrence of LSCC, which owns prospect diagnostic ability and therapeutic significance in LSCC.

Keywords: Interleukin 24; RNA-sequencing; immunohistochemistry; laryngeal squamous cell carcinoma; microarray.

Exposure to particulate matter < 2.5 μm (PM2.5) is associated with a variety of airway diseases. Although studies have demonstrated that high doses of PM2.5 cause cytotoxicity and changes to gene expression in bronchial epithelial cells, the effect of lower doses and repeated exposure to PM2.5 are less well studied. Here, we treated BEAS-2B cells with varying doses of PM2.5 for 1-7 days and examined the expression of a variety of genes implicated in airway disorders. At high doses, PM2.5 increased the expression of IL6, TNF, TSLP, CSF2, PTGS2, IL4R, and SPINK5. Other genes such as ADAM33, ORMDL3, DPP10 and CYP1A1, however, were increased by PM2.5 at much lower doses (≤1 μg/cm2). Repeated exposure to PM2.5 at 1 or 5 μg/cm2 every day for 7 days increased the sensitivity and magnitude of change for all of the aforementioned genes. Genes such as IL13 and TGFB1, increased only when cells were repeatedly exposed to PM2.5. Treatment with an antioxidant, or inhibitors to aryl hydrocarbon receptor or NF-κB attenuated the effect of PM2.5. These data demonstrate that PM2.5 exerts pleiotropic actions that differ by dose and duration that affect a variety of genes important to the development of airway disease.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

This study aimed at investigating the role of RAD18 in the regulation of glioblastoma development as well as the underlying mechanisms. The human glioblastoma U251 and U87MG cells were transfected with siRNAs specifically targeting RAD18, and the effects of knockdown of RAD18 on the viability, apoptosis, migration, and invasion of U251 and U87MG cells were investigated. Transcriptome sequencing of the siRNA-RAD18-tranfected and siRNA-NC-transfected U251 cells was performed, followed by bioinformatic analyses for sequencing data. The results showed that knockdown of RAD18 significantly inhibited cell viability, promoted apoptosis, and suppressed migration and invasion of U251 and U87MG cells. Bioinformatic analyses of sequencing data identified 1,051 differentially expressed genes (DEGs) (369 up- and 682 downregulated genes) in the siRNA-RAD18-transfected U251 cells compared with siRNA-NC-transfected U251 cells. Eleven DEGs, including nerve growth factor (NGF), colony-stimulating factor 2 (CSF2), matrix metallopeptidase 1 (MMP1), platelet-derived growth factor receptor α (PDGFRA), and heme oxygenase 1 (HMOX1), were identified as the hub nodes in protein-protein interaction (PPI) network. Moreover, the aforementioned 11 hub genes were significantly enriched in PI3K-Akt signaling pathway and GO functions associated with the extracellular region. Notably, quantitative real-time polymerase chain reaction further confirmed that the expression levels of NGF, CSF2, HMOX1, and MMP1 were significantly downregulated, while that of PDGFRA was markedly upregulated in the siRNA-RAD18-transfected U251 cells than in the siRNA-NC cells. In conclusion, the knockdown of RAD18 may inhibit glioblastoma development by regulating the expression of the aforementioned key DEGs.

Mammalian macrophages differ in their basal gene expression profiles and response to the toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS). In human macrophages, LPS elicits a temporal cascade of transient gene expression including feed forward activators and feedback regulators that limit the response. Here we present a transcriptional network analysis of the response of sheep bone marrow-derived macrophages (BMDM) to LPS based upon RNA-seq at 0, 2, 4, 7, and 24 h post-stimulation. The analysis reveals a conserved transcription factor network with humans, and rapid induction of feedback regulators that constrain the response at every level. The gene expression profiles of sheep BMDM at 0 and 7 h post LPS addition were compared to similar data obtained from goat, cow, water buffalo, horse, pig, mouse and rat BMDM. This comparison was based upon identification of 8,200 genes annotated in all species and detected at >10TPM in at least one sample. Analysis of expression of transcription factors revealed a conserved transcriptional millieu associated with macrophage differentiation and LPS response. The largest co-expression clusters, including genes encoding cell surface receptors, endosome-lysosome components and secretory activity, were also expressed in all species and the combined dataset defines a macrophage functional transcriptome. All of the large animals differed from rodents in lacking inducible expression of genes involved in arginine metabolism and nitric oxide production. Instead, they expressed inducible transporters and enzymes of tryptophan and kynurenine metabolism. BMDM from all species expressed high levels of transcripts encoding transporters and enzymes involved in glutamine metabolism suggesting that glutamine is a major metabolic fuel. We identify and discuss transcripts that were uniquely expressed or regulated in rodents compared to large animals including ACOD1, CXC and CC chemokines, CD163, CLEC4E, CPM, CSF1, CSF2, CTSK, MARCO, MMP9, SLC2A3, SLC7A7, and SUCNR1. Conversely, the data confirm the conserved regulation of multiple transcripts for which there is limited functional data from mouse models and knockouts. The data provide a resource for functional annotation and interpretation of loci involved in susceptibility to infectious and inflammatory disease in humans and large animal species.

Keywords: LPS; conservation; feedback; macrophage; network; species; transcriptome.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prothrombotic state and a lack of host anti-tumor immune responsiveness. Linking these two key features, we previously demonstrated that tumor-derived coagulation activity promotes immune evasion. Specifically, thrombin-protease-activated receptor-1 (PAR1) signaling in mouse PDAC cells drives tumor growth by evading cytotoxic CD8a⁺ cells.

Methods: Syngeneic mixed cell tumor growth, transcriptional analyses, and functional tests of immunosuppressive response genes were employed to identify cellular and molecular immune evasion mechanisms mediated by thrombin-PAR-1 signaling in mouse PDAC tumor cells.

Results: Elimination of tumor cell PAR1 in syngeneic graft studies increased cytotoxic T lymphocyte (CTL) infiltration and decreased tumor-associated macrophages in the tumor microenvironment. Co-injection of PAR1-expressing and PAR1-knockout (PAR-1^KO ) tumor cells into immunocompetent mice resulted in preferential elimination of PAR-1^KO cells from developing tumors, suggesting that PAR1-dependent immune evasion is not reliant on CTL exclusion. Transcriptomics analyses revealed no PAR1-dependent changes in the expression of immune checkpoint proteins and no difference in MHC-I cell surface expression. Importantly, thrombin-PAR1 signaling in PDAC cells upregulated genes linked to immunosuppression, including Csf2 and Ptgs2. Functional analyses confirmed that both Csf2 and Ptgs2 are critical for PDAC syngeneic graft tumor growth and overexpression of each factor partially restored tumor growth of PAR1^KO cells in immunocompetent mice.

Conclusions: Our results provide novel insight into the mechanisms of a previously unrecognized pathway coupling coagulation to PDAC immune evasion by identifying PAR1-dependent changes in the tumor microenvironment, a PAR1-driven immunosuppressive gene signature, and Csf2 and Ptgs2 as critical PAR1 downstream targets.

Keywords: cancer; cytotoxic T lymphocyte; immune evasion; immunosuppression; pancreatic ductal adenocarcinoma; protease activated receptor 1; thrombin.

BACKGROUND

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to promote the development and survival of human and mouse preimplantation embryos; however, the mechanism of action of GM-CSF in embryos is not defined.

METHODS

Mouse blastocysts were cultured from zygote stage in vitro with and without recombinant mouse GM-CSF (rmGM-CSF), and in vivo developed blastocysts were flushed from Csf2 null mutant and wild-type mice. The effect of GM-CSF on blastocyst expression of stress response and apoptosis genes was evaluated by microarray, qPCR and immunochemistry.

RESULTS

Microarray analysis of the gene transcription profile showed suppression of stress response and apoptosis gene pathways in blastocysts exposed to rmGM-CSF in vitro. qPCR analysis confirmed that rmGM-CSF inhibited expression of heat shock protein (HSP) and apoptosis pathway genes Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5 in in vitro blastocysts. Immunocytochemical analysis of HSP 1 (HSPA1A/1B; HSP70), BAX, BCL2 and TRP53 (p53) in in vitro blastocysts showed that HSPA1A/1B and BCL2 proteins were less abundant when embryos were cultured with rmGM-CSF. BAX and TRP53 were unchanged at the protein level, but Bax mRNA expression was reduced after GM-CSF treatment. In in vivo developed blastocysts, Csf2 null mutation caused elevated expression of Hsph1 but not other stress response genes.

CONCLUSIONS

We conclude that GM-CSF inhibits the cellular stress response and apoptosis pathways to facilitate embryo growth and survival, and the protective effects of GM-CSF are particularly evident in in vitro culture media, whereas in vivo other cytokines can partly compensate for absence of GM-CSF.

Sepsis is a systemic inflammatory state that occurs in response to infection and significantly increases mortality in combination with acute kidney injury (AKI). Macrophages accumulate in the kidney after injury and undergo a transition from a proinflammatory (M1) phenotype to an alternatively activated (M2) phenotype that is required for normal repair. However, the specific signals that regulate the transition from the M1 to M2 phenotype in vivo are unknown. Here, we found an unexpected role of Colony stimulating factor 2 (Csf2) in controlling macrophage transition in vitro and in a mouse model of sepsis induced by cecal ligation and puncture (CLP). We first co-cultured human M1 macrophages with HK-2 cells and characterized cytokine/chemokine profiles via Luminex. Of the cytokines and chemokines that were overexpressed in medium from M1 macrophages cocultured with human kidney-2 (HK-2) cells compared with that from M1 macrophages cultured alone, Csf2 and IL6 showed the greatest increases. Csf2 was exclusively secreted by HK-2 cells but not by M1 macrophages. Furthermore, recombinant human Csf2 protein promoted transition of M1 macrophages to the M2 phenotype in a dose and time-dependent manner. The apoptosis and reactive oxygen species (ROS) release induced by M1 macrophages in HK-2 cells was attenuated after exposure to exogenous Csf2. In addition, the switch from the proinflammatory M1 phenotype to the M2 phenotype occurred via the p-Stat5 pathway, which was activated by Csf2. Importantly, we found that intraperitoneal injection of a Csf2-neutralizing antibody after CLP aggravated kidney injury and suppressed tubular proliferation, subsequently decreasing survival. However, administration of recombinant mouse Csf2 protein could rescue mice with sepsis. Together, our results indicate that Csf2 plays critical roles in regulating macrophage transition via activation of p-STAT5. These data form a foundation upon which new therapeutic strategies can be designed to improve the therapeutic efficacy of cytokine-based treatments for sepsis-induced AKI.

Keywords: Csf2; acute kidney injury; cytokine-based therapy; macrophage transition; sepsis.

Objective: PD-L1 is an immune checkpoint molecule that regulates immune and inflammatory responses. While cells of periodontal tissues express PD-L1, its presence in GCF is not known. The purpose of this study was to measure the PD-L1 values in GCF and correlate values with the presence of chemokine and cytokine values from periodontally diseased subjects and periodontally healthy subjects.

Results: PD-L1 values (pg/30 s), determined in triplicate using a fluorescent microparticle-based immunoassay ranged from 0.04-31.65 pg/30 s. PD-L1 correlated with 15 out of 22 chemokine and cytokine responses. In 85 healthy sites in 31 subjects, PD-L1 values were negatively correlated with IL6, CXCL8, IL10, and CCL3 values. In 53 diseased sites in 20 subjects, PD-L1 values were positively correlated with CCL11, CSF2, IFNG, IL1A, IL1B, IL2, IL7, IL15, and CCL5 values and negatively correlated with IL12A and IL5 values. Gene ontology (GO) annotations identified roles of PD-L1 in Th1 and Th2 activation and T-cell exhaustion signaling canonical pathways. PD-L1 values were correlated with the expression of chemokines and cytokines, which likely regulates immune cell trafficking and protects the periodontium from uncontrolled immune responses to pathogens and inflammation-induced tissue damage.

Keywords: Cytokine; PD-1; PD-L1; Periodontal disease; Periodontitis.

Brain metastatic papillary thyroid carcinomas (PTCs) are afflicted with unfavorable prognosis; however, the underlying molecular genetics of these rare metastases are virtually unknown. In this study, we compared whole transcript microarray expression profiles of a BRAF mutant, brain metastasis from a PTC, including its technical replicate (TR), with eight non-brain metastatic PTCs and eight primary brain tumors. The top 95 probe sets (false discovery rate (FDR) p-value < 0.05 and fold change (FC)>> 2) that were differentially expressed between the brain metastatic PTC, including the TR, and both, non-brain metastatic PTCs and primary brain tumors were in the vast majority upregulated and comprise, e.g. ROS1, MYBPH, SLC18A3, HP, SAA2-SAA4, CP, CCL20, GFAP, RNU1-120P, DMBT1, XDH, CXCL1, PI3, and NAPSA. Cytokines were represented by 10 members in the top 95 probe sets. Pathway and network analysis (p-value < 0.05 and FC>> 2) identified granulocytes adhesion and diapedesis as top canonical pathway. Most significant upstream regulators were lipopolysaccharide, TNF, NKkB (complex), IL1A, and CSF2. Top networks categorized under diseases & functions were entitled migration of cells, cell movement, cell survival, apoptosis, and proliferation of cells. Probe sets that were significantly shared between the brain metastatic PTC, the TR, and primary brain tumors include CASP1, CASP4, C1R, CC2D2B, RNY1P16, WDR72, LRRC2, ZHX2, CITED1, and the noncoding transcript AK128523. Taken together, this study identified a set of candidate genes and biofunctions implicated in, so far nearly uncharacterized, molecular processes of a brain metastasis from a PTC.

Lung cancer is a heterogeneous and complex disease with the highest incidence and mortality rate. The present study aims at defining the lung cancer phenome specificity of lipidomic profiles, screening target lipid-dependent transcriptional alternations, identifying target lipid-associated target genes, and exploring molecular mechanisms. Lung cancer-specific and lung cancer subtype-specific target lipids palmitic acid (C16:0) and stearic acid (C18:0) were found as target lipids by integrating clinical phenomics, lipidomics, and transcriptomics and exhibited antiproliferative effects in sensitive cells. The metabolism-associated gene ACSL5 or inflammation-associated gene CCL3 was identified in lung adenocarcinoma or small lung cancer cells, respectively. C16:0 or C18:0 could upregulate ACSL5 or CSF2 expression in a time- and dose-dependent pattern, and the deletion of both genes led to the insensitivity of cells. Target lipids increased the expression of PDK4 gene in different patterns and inhibited cell proliferation through alterations of intracellular energy. Thus, our data provide a new strategy to investigate the trans-points between clinical and phenomics and lipidomics and target lipid-associated molecular mechanisms to benefit from the discovery of new therapies.

With the increasingly early stage lung squamous cell carcinoma (LUSC) being discovered, there is an urgent need for a comprehensive analysis of the prognostic characteristics of early stage LUSC. Here, we developed an immune-related gene signature for outcome prediction of early stage LUSC based on three independent cohorts. Differentially expressed genes (DEGs) were identified using CIBERSORT and ESTMATE algorithm. Then, a 17-immune-related gene (RPRM, APOH, SSX1, MSGN1, HPR, ISM2, FGA, LBP, HAS1, CSF2, RETN, CCL2, CCL21, MMP19, PTGIS, F13A1, C1QTNF1) signature was identified using univariate Cox regression, LASSO regression and stepwise multivariable Cox analysis based on the verified DEGs from 401 cases in The Cancer Genome Atlas (TCGA) database. Subsequently, a cohort of GSE74777 containing 107 cases downloaded from Gene Expression Omnibus (GEO) database and an independent data set consisting of 36 frozen tissues collected from National Cancer Center were used to validate the predictive value of the signature. Seventeen immune-related genes were identified from TCGA cohort, which were further used to establish a classification system to construct cases into high- and low-risk groups in terms of overall survival. This classifier was still an independent prognostic factor in multivariate analysis. In addition, another two independent cohorts and different clinical subgroups validated the significant predictive value of the signature. Further mechanism research found early stage LUSC patients with high risk had special immune cell infiltration characteristics and gene mutation profiles. In conclusion, we characterized the tumor microenvironment and established a highly predictive model for evaluating the prognosis of early stage LUSC, which may provide a lead for effective immunotherapeutic options tailored for each subtype.

Keywords: early stage lung squamous cell carcinoma; gene mutation; immune cell infiltration; immune signature; prognostic model.

OBJECTIVE

Acute Respiratory Distress Syndrome (ARDS) is an important clinical and public health problem. Why some at-risk individuals develop ARDS and others do not is unclear but may be related to differences in inflammatory and cell signaling systems. The Receptor for Advanced Glycation Endproducts (RAGE) and Granulocyte-Monocyte Stimulating Factor (GM-CSF) pathways have recently been implicated in pulmonary pathophysiology; whether genetic variation within these pathways contributes to ARDS risk or outcome is unknown.

METHODS

We studied 842 patients from three centers in Utah and 14 non-Utah ARDS Network centers. We studied patients at risk for ARDS and patients with ARDS to determine whether Single Nucleotide Polymorphisms (SNPs) in the RAGE and GM-CSF pathways were associated with development of ARDS. We studied 29 SNPs in 5 genes within the two pathways and controlled for age, sepsis as ARDS risk factor, and severity of illness, while targeting a false discovery rate of ≤ 5%. In a secondary analysis we evaluated associations with mortality.

RESULTS

Of 842 patients, 690 had ARDS, and 152 were at-risk. Sepsis was the risk factor for ARDS in 250 (30%) patients. When controlling for age, APACHE III score, sepsis as risk factor, and multiple comparisons, no SNPs were significantly associated with ARDS. In a secondary analysis, only rs743564 in CSF2 approached significance with regard to mortality (OR 2.17, unadjusted p = 0.005, adjusted p = 0.15).

CONCLUSIONS

Candidate SNPs within 5 genes in the RAGE and GM-CSF pathways were not significantly associated with development of ARDS in this multi-centric cohort.