CXCL10 mRNA Expression Research Articles

Endometrial Inflammation at the Time of Insemination and Its Effect on Subsequent Fertility of Dairy Cows.

Simple SummaryA detailed understanding of cellular and molecular mechanisms in the bovine uterus is crucial to explain and avoid subfertility in dairy cows. Therefore, we examined the effect of inflammation in the bovine uterus in cows with no clinical signs of disease at the time of artificial insemination (AI) on subsequent pregnancy outcome. In a total of 71 healthy dairy cows, uterine cytology samples were collected by cytobrush technique within 10 min after insemination. Endometrial inflammation was investigated at the cellular and mRNA expression levels. All factors with a significant effect on fertility in our study were related to uterine polymorphonuclear neutrophil (PMN) migration, i.e., the first line of uterine defense. Cows with a proportion of ≥1% PMN had a 1.8-fold increased chance of pregnancy within 150 days postpartum compared to cows with fewer PMNs. From our results, we conclude that a certain level of inflammation at the molecular and cellular levels before the stimulus of AI might be favorable for cows’ fertility.Our objective was to investigate the level of endometrial immune response at artificial insemination (AI) and to relate it to subsequent fertility. From 71 healthy cows, endometrial cytobrush samples were taken at the first AI for cytological and mRNA analyses. Total RNA isolated from the cytobrushes was used for reverse transcription qPCR for selected transcripts. Animals were grouped into pregnant (PREG; n = 32) and non-pregnant (non-PREG; n = 39) cows following their first AI. The mRNA abundance of the neutrophil-related factor CEACAM1 and the chemokine CXCL5 was 1.2- (p = 0.03) and 2.0-fold (p = 0.04) greater in PREG than in non-PREG cows, respectively. Animals were further subdivided according to the number of inseminations until pregnancy (PREG1, n = 32; PREG2-3, n = 19) and in repeat breeder cows (RBC, n = 13). CEACAM1 and CXCL8 mRNA expression was 1.7- (p = 0.01) and 2.3-fold (p = 0.03) greater in PREG1 than in RBC, respectively. Cox regression showed that cows with PMN ≥ 1% had a 1.8-fold increased chance of pregnancy within 150 days postpartum compared with cows with fewer PMNs. We conclude that a certain level of inflammation before the stimulus of AI might be beneficial for subsequent fertility.

Hepatic steatosis precedes pregnancy in the BPH/5 preeclampsia‐like mouse model

BPH/5 mice represent a superimposed model of preeclampsia that spontaneously develop maternal and fetal characteristics of preeclamptic pregnancies. In women, the development of preeclampsia is associated with pre-existing dysregulation of lipid metabolism including hepatic steatosis. The study objective was tocharacterize the basal hepatic health of the female BPH/5 mouse to determine if hepatic steatosis precedes the preeclampsia-like syndrome. We hypothesized that non-pregnant female BPH/5 mice have an excess of hepatocellular lipid that promotes intrahepatic pro-inflammatory cytokine production. Blood and liver samples were collected from 8–10-week-old BPH/5 non-pregnant females (n=10) and 8–10-week-old C57Bl/6 non-pregnant females (n=6). Liver and body weight were recorded. Biochemical markers of hepatocellular function (albumin, ALT, BUN, and bilirubin) were quantified. Hepatic RNA was isolated, and qPCR was used to evaluate pro-inflammatory markers. H&E staining was used to evaluate hepatic histomorphology and lipid accumulation. There was no significant difference in albumin, ALT, BUN, or bilirubin between BPH/5 and C57Bl/6 mice. There was significantly higher hepatic CXCL-2, CXCL-10, IL-1ß, TLR2, and TLR7 mRNA expression in BPH/5 versus C57Bl/6 mice (p<0.05). There was a significantly greater liver weight in BPH/5 mice compared to C57Bl/6 mice (p=0.05). The histologic microsteatosis score was significantly increased in the BPH/5 mice (2.5) compared to C57Bl/6 mice (0.98, p=0.025). The BPH/5 mouse has basal hepatic steatosis and a pro-inflammatory hepatic phenotype that precedes pregnancy. Dysregulated lipid metabolism in the liver may contribute to the development of preeclampsia.

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H2 O2 or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection.

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

Evaluation of CCL21 role in post-knee injury inflammation and early cartilage degeneration.

The expression of some chemokines and chemokine receptors is induced during the development of post-traumatic osteoarthritis (PTOA), but their involvement in the pathogenesis of the disease is unclear. The goal of this study was to test whether CCL21 and CXCL13 play a role in PTOA development. For this purpose, we evaluated the expression profiles of the chemokines Ccl21 and Cxcl13, matrix metalloproteinase enzymes Mmp3 and Mmp13, and inflammatory cell markers in response to partial medial meniscectomy and destabilization (MMD). We then assessed the effect of local administration of CCL21 neutralizing antibody on PTOA development and post-knee injury inflammation. The mRNA expression of both Ccl21 and Cxcl13 was induced early post-surgery, but only Ccl21 mRNA levels remained elevated 4 weeks post-surgery in rat MMD-operated knees compared to controls. This suggests that while both CXCL13 and CCL21 are involved in post-surgery inflammation, CCL21 is necessary for development of PTOA. A significant increase in the mRNA levels of Cd4, Cd8 and Cd20 was observed during the first 3 days post-surgery. Significantly, treatment with CCL21 antibody reduced post-surgical inflammation that was accompanied by a reduction in the expression of Mmp3 and Mmp13 and post-MMD cartilage degradation. Our findings are consistent with a role for CCL21 in mediating changes in early inflammation and subsequent cartilage degeneration in response to knee injury. Our results suggest that targeting CCL21 signaling pathways may yield new therapeutic approaches effective in delaying or preventing PTOA development following injury.

Combination of Ginsenosides Rb2 and Rg3 Promotes Angiogenic Phenotype of Human Endothelial Cells via PI3K/Akt and MAPK/ERK Pathways.

Shexiang Baoxin Pill (SBP) is an oral formulation of Chinese materia medica for the treatment of angina pectoris. It displays pleiotropic roles in protecting the cardiovascular system. However, the mode of action of SBP in promoting angiogenesis, and in particular the synergy between its constituents is currently not fully understood. The combination of ginsenosides Rb2 and Rg3 were studied in human umbilical vein endothelial cells (HUVECs) for their proangiogenic effects. To understand the mode of action of the combination in more mechanistic detail, RNA-Seq analysis was conducted, and differentially expressed genes (DEGs), pathway analysis and Weighted Gene Correlation Network Analysis (WGCNA) were applied to further identify important genes that a play pivotal role in the combination treatment. The effects of pathway-specific inhibitors were observed to provide further support for the hypothesized mode of action of the combination. Ginsenosides Rb2 and Rg3 synergistically promoted HUVEC proliferation and tube formation under defined culture conditions. Also, the combination of Rb2/Rg3 rescued cells from homocysteine-induced damage. mRNA expression of CXCL8, CYR61, FGF16 and FGFRL1 was significantly elevated by the Rb2/Rg3 treatment, and representative signaling pathways induced by these genes were found. The increase of protein levels of phosphorylated-Akt and ERK42/44 by the Rb2/Rg3 combination supports the notion that it promotes endothelial cell proliferation via the PI3K/Akt and MAPK/ERK signaling pathways. The present study provides the hypothesis that SBP, via ginsenosides Rb2 and Rg3, involves the CXCR1/2 CXCL8 (IL8)-mediated PI3K/Akt and MAPK/ERK signaling pathways in achieving its proangiogenic effects.

The Anti-inflammatory Protein TSG-6 Induced by S. aureus Regulates the Chemokine Function of Endothelial Cells In Vitro by Inhibiting the Chemokine-Glycosaminoglycan Interaction

The aim of this study was to explore the effect of the anti-inflammatory protein TSG-6 induced by Staphylococcus bacteria on the regulation of chemokine function in endothelial cells by inhibiting the chemokine-glycosaminoglycan interaction. To cultivate human umbilical vein endothelial cells and Staphylococcus aureus bacteria, respectively, after the experiment is divided into the control group, S. aureus bacteria-induced group, S. aureus bacteria glycosaminoglycans about 1 mg/L sugar group, S. aureus bacteria glycosaminoglycans about 5 mg/L sugar group, and S. aureus bacteria glycosaminoglycans about 10 mg/L sugar group, E-selectin; intercellular adhesion molecule-1 (ICAM-1); monocyte chemoattractant protein-1 (MCP-1); interleukin-8 (IL-8) expression level; chemokine CXCL9, CXCL10, and CXCL11 mRNA and protein expression level; and TSG mRNA and protein expression level were determined in each cell; the endothelial cell proliferation and vascular endothelial cell function indicators were analyzed. The expression levels of E-selectin, ICAM-1, IL-8, MCP-1, and chemokines CXCL9, CXCL10, and CXCL11 mRNA and protein in each group at 6, 12, and 24 h were significantly different (P < 0.05). TSG mRNA and protein expression levels, endothelial cell proliferation ability, and vascular endothelial cell function were also significantly different (P < 0.05). The expression levels of E-selectin, ICAM-1, IL-8, MCP-1, endothelial cell proliferation ability, and vascular endothelial cell function in the Staphylococcus aureus-induced group were lower than those in the control group and the Staphylococcus aureus glycosaminoglycan group, and the mRNA and protein expression levels of chemokines CXCL9, CXCL10, and CXCL11, and TSG mRNA and protein expression levels were higher. With the increase of glycosaminoglycan concentration, the above indexes were improved. The anti-inflammatory protein TSG-6 induced by S. aureus can regulate the chemokine function of endothelial cells by inhibiting the chemokine-glycosaminoglycan interaction.

MicroRNA-424 regulates the expression of CX3CL1 (fractalkine) in human microvascular endothelial cells during Rickettsia rickettsii infection

Cytokines and chemokines trigger complex intracellular signaling through specific receptors to mediate immune cell recruitment and activation at the sites of infection. CX3CL1 (Fractalkine), a membrane-bound chemokine also capable of facilitating intercellular interactions as an adhesion molecule, contributes to host immune responses by virtue of its chemoattractant functions. Published studies have documented increased CX3CL1 expression in target tissues in a murine model of spotted fever rickettsiosis temporally corresponding to infiltration of macrophages and recovery from infection. Because pathogenic rickettsiae primarily target vascular endothelium in the mammalian hosts, we have now determined CX3CL1 mRNA and protein expression in cultured human microvascular endothelial cells (HMECs) infected in vitro with Rickettsia rickettsii. Our findings reveal 15.5 ± 4.0-fold and 12.3 ± 2.3-fold increase in Cx3cl1 mRNA expression at 3 h and 24 h post-infection, coinciding with higher steady-state levels of the corresponding protein in comparison to uninfected HMECs. Since CX3CL1 is a validated target of microRNA (miR)-424-5p (miR-424) and our earlier findings demonstrated robust down-regulation of miR-424 in R. rickettsii-infected HMECs, we further explored the possibility of regulation of CX3CL1 expression during rickettsial infection by miR-424. As expected, R. rickettsii infection resulted in 87 ± 5% reduction in miR-424 expression in host HMECs. Interestingly, a miR-424 mimic downregulated R. rickettsii-induced expression of CX3CL1, whereas an inhibitor of miR-424 yielded a converse up-regulatory effect, suggesting miR-424-mediated regulation of CX3CL1 during infection. Together, these findings provide the first evidence for the roles of a host microRNA in the regulation of an important bifunctional chemokine governing innate immune responses to pathogenic rickettsiae.

Naoxintong accelerates diabetic wound healing by attenuating inflammatory response

Context Naoxintong (NXT), a prescribed traditional Chinese medicine, widely used in cerebrovascular and cardiovascular diseases, could be effective in diabetic wounds. Objective This study evaluates the wound healing activity of NXT by employing an excisional wound splinting model. Materials and methods NXT was dissolved in saline and given daily by gavage. Wounds were induced at the dorsum of non-diabetic (db/+) and diabetic (db/db) mice and treated with saline or 700 mg/kg/d NXT for 16 days. Wound closure was measured every four days. Extracellular matrix (ECM) remodelling, collagen deposition, leukocyte infiltration and expression of Col-3, CK14, CXCL1, CXCL2, MPO, Ly6G, CD68, CCR7, CD206, p-JAK1, p-STAT3 and p-STAT6 was analysed. Results NXT significantly accelerated rate of wound closure increased from 70% to 84%, accompanied by up-regulation of collagen deposition and ECM at days 16 post-injury. Moreover, NXT alleviated neutrophil infiltration, accompanied by down-regulation of CXCL1 and CXCL2 mRNA expression. In addition, NXT markedly augmented neutrophil efferocytosis. In diabetic wounds, the levels of M1 marker gene (CCR7) increased, while M2 marker gene (CD206) decreased, demonstrating a pro-inflammatory shift. Application of NXT increased M2 macrophage phenotype in db/db mice. Mechanistically, NXT treatment increased expression level of p-STAT3 and p-STAT6 at days 3 post-injury, indicating NXT mediated macrophages towards M2 phenotype and alleviated inflammation in diabetic wounds by activation of STAT3 and STAT6. Conclusions Our study provides evidence that NXT accelerates diabetic wound healing by attenuating inflammatory response, which provides an important basis for use of NXT in the treatment of chronic diabetic wound healing.

Exploration of prognostic biomarkers and therapeutic targets in the microenvironment of bladder cancer based on CXC chemokines.

Bladder cancer (BLCA) has a high rate of morbidity and mortality, and is considered as one of the most malignant tumors of the urinary system. Tumor cells interact with surrounding interstitial cells, playing a key role in carcinogenesis and progression, which is partly mediated by chemokines. CXC chemokines exert anti-tumor biological roles in the tumor microenvironment and affect patient prognosis. Nevertheless, their expression and prognostic values patients with BLCA remain unclear. We used online tools, including Oncomine, UALCAN, GEPIA, GEO databases, cBioPortal, GeneMANIA, DAVID 6.8, Metascape, TRUST (version 2.0), LinkedOmics, TCGA, and TIMER2.0 to perform the relevant analysis. The mRNA levels of C-X-C motif chemokine ligand (CXCL)1, CXCL5, CXCL6, CXCL7, CXCL9, CXCL10, CXCL11, CXCL13, CXCL16, and CXCL17 were increased significantly increased, and those of CXCL2, CXCL3, and CXCL12 were decreased significantly in BLCA tissues as assessed using the Oncomine, TCGA, and GEO databases. GEO showed that high levels of CXCL1, CXCL6, CXCL10, CXCL11, and CXCL13 mRNA expression are associated significantly with the poor overall survival (all p < 0.05), and similarly, those of CXCL2 and CXCL12 in the TCGA database (p < 0.05). The predominant signaling pathways involving the differentially expressed CXC chemokines are cell cycle, chemokine, and cytokine-cytokine receptor interaction. Moreover, transcription factors such as Sp1 transcription factor (SP1), nuclear factor kappa B subunit 1 (NFKB1), and RELA proto-oncogene, NF-KB subunit (RELA) were likely play critical roles in regulating CXC chemokine expression. LYN proto-oncogene, src family tyrosine kinase (LYN) and LCK proto-oncogene, src family tyrosine kinase (LCK) were identified as the key targets of these CXC chemokines. MicroRNAs miR200 and miR30 were identified as the main microRNAs that interact with several CXC chemokines through an miRNA-target network. The expression of these chemokines is closely associated with the infiltration of six categories of immune cells. We explored the CXC chemokines superfamily-based biomarkers associated with BLCA prognosis using public databases, and provided possible chemokine targets for patients with BLCA.

Cancer-associated fibroblasts in gastric cancer affect malignant progression via the CXCL12-CXCR4 axis.

Background: Cancer-associated fibroblasts (CAFs) are principal constituents of the tumor microenvironment (TME) and play a critical role in tumor progression. The CXCL12/CXCR4 axis regulates multiple facets of the TME. The aim of this study was to determine the relationship between CXCL12 expression in CAFs and the malignant progression of gastric cancer (GC).Methods: In the GEO (Gene Expression Omnibus) database, we performed transcriptome analysis on paired gastric cancer RNA sequencing samples, and scRNA analysis was performed on advanced malignant GC samples from the scRNA sequencing data set. Fibroblast cells were co-cultured with GC cells, and invasion, migration, epithelial-mesenchymal transformation (EMT) were determined. After blocking the expression of fibroblast CXCL12, cells were co-cultured with a GC cell line. Detection of GC cell line invasion, migration, EMT and CXCR4, Wnt5a and β-Catenin expression levels was performed. Primary CAFs and gastric normal fibroblasts were isolated and CXCL12 mRNA and protein expression were determined. In addition, a cohort of 285 GC cases was established, protein expression was evaluated immunohistochemically, and prognostic results were analyzed.Results: GC transcriptome analysis suggested that cytokine-cytokine receptor interaction and the Wnt signaling pathway in GC tissues were significantly up-regulated. scRNA analysis of advanced malignant GC samples showed that severe intestinal metaplasia (SIM) in GC specimens of different malignant grades had obvious fibroblast clusters compared to non-atrophic gastritis (NAG) and early gastric cancer (EGC). In the SIM group, fibroblast cluster, CXCL12, CXCR4, and Wnt5a were overexpressed. Co-culturing with fibroblast cells significantly increased the invasion, migration, and EMT of GC cells, and blocking CXCL12 in CAFs disturbed the expression of Wnt5a and β-catenin. In our cohort of GC patients, high CXCL12 expression in CAFs significantly correlated with histological grade (P = 0.012) and TNM stage (P = 0.014), as well as with poor overall survival (p = 0.0107).Conclusion: High expression of CXCL12 in CAFs in a GC microenvironment can affect the migration, invasion, and EMT of GC cells. Furthermore, it can cause poor prognosis in patients with GC.

The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression

We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1β and IL-34 were investigated by RT-qPCR and ELISA. Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1β induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.

Changes in histidine decarboxylase expression influence extramedullary hematopoiesis in postnatal mice.

Histidine decarboxylase (HDC), histamine synthase, is expressed in hematopoietic stem cells and in lineage-committed progenitors in the bone marrow (BM). However, the role of histamine in hematopoiesis is not well described. To evaluate the role of histamine in hematopoiesis, we analyzed the changes in HDC expression at hematopoietic sites, the BM, spleen, and liver of 2-, 3-, and 6-week-old wild-type mice. We also performed morphological analyses of the hematopoietic sites using HDC-deficient (HDC-KO) mice. In wild-type adults, HDC expression in the BM was higher than that in the spleen and liver and showed an age-dependent increase. Histological analysis showed no significant change in the adult BM and spleen of HDC-KO mice compared to wild-type mice. In the liver, HDC expression was temporarily increased at 3 weeks and decreased at 6 weeks of age. Morphological analysis of the liver revealed more numerous hematopoietic colonies and megakaryocytes in HDC-KO mice compared to wild-type mice at 2 and 3 weeks of age, whereas no changes were observed in adults. Most of these hematopoietic colonies consisted of B220-positive B-lymphocytes and TER119-positive erythroblasts and were positive for the cell proliferation marker PCNA. Notably, these hematopoietic colonies declined in HDC-KO mice upon N-acetyl histamine treatment. A significant increase in the expression of hematopoiesis-related cytokines, Il3, Il7, Epo, Gcsf, and Cxcl12 mRNA was observed in the liver of 3-week-old HDC-KO mice compared to wild-type mice. These results suggest that histamine-deficiency may maintain an microenvironment suitable for hematopoiesis by regulating hematopoiesis-related cytokine expression in the liver of postnatal mice.

Mitochondrial DNA drives noncanonical inflammation activation via cGAS\u2013STING signaling pathway in retinal microvascular endothelial cells

BackgroundPathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood–retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli.MethodsRMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H2O2), or 25 mM d-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA.ResultsPathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation.ConclusionsPathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING–TBK1 signaling pathway.Ba7zzwjjDYYV6NH7beoBySVideo . (MP4 37490 kb)

Mechanisms underlying the production of chemokine CXCL11 in the reaction of renal tubular epithelial cells with CD4+ and CD8+ T cells

AimTo study the release mechanism of C-X-C motif chemokine 11 (CXCL11) and other chemokines after the co-cultivation of CD4+ and CD8+ T cells with the renal tubular epithelial cells (RTEC) in the process of allograft renal transplantation rejection. MethodsThe Human CD4+, CD8+ T cells were obtained from the blood of volunteers and kidney transplantation (Ktx) patients, and co-cultured with renal tubular epithelial cells (RTEC) in vitro. RT-PCR was run for detecting the mRNA transcription of CXCL11, IFN-induced protein of 10 (CXCL10), and IL-6 in cells after RTEC was stimulated with IFN-γ or co-cultured with CD4+ and CD8+ T cells. The concentration of CXCL11, CXCL10 and IL-6 in the culture medium was detected by Multiplex Assay after RTEC was stimulated with IFN-γ or co-cultured with CD4+ and CD8+ T cells. IFN-γ receptor antibody was used for interfering with the above reaction and the blocking effect was observed. Western blot was used for protein expression analysis. Finally, we applied renal biopsies from kidney transplantation patients with and without rejection to verify the results of the above experiments by using RT-PCR and Western blot. ResultsThe mRNA expression of CXCL11 and CXCL10 were significantly increased after RTEC was stimulated with IFN-γ or co-cultured with CD4+ and CD8+ T cells. Multiplex Assay showed that the concentration of CXCL11 and CXCL10 in the supernatant were significantly increased in a time-dependence fashion after stimulation RTEC by IFN-γ. Anti-IFN-γ receptor1 (anti-IFN-γR1) antibody could reduce the production of CXCL11 and CXCL10 in this situation. The concentration of CXCL11 and CXCL11 in the supernatant was significantly increased with a time-dependent effect after the co-culture of CD4+ and CD8+ T cells with RTEC. The anti-IFN-γR1 blocked this effect. Our study showed that the expression levels of CXCL11 and CXCL10 were upgraded in the biopsies of patients with renal transplant rejection comparatively to pre-transplant biopsies, both at mRNA and protein levels. ConclusionsRTEC and T cells can stimulate each other during the acute rejection of allogeneic kidney transplantation and secret CXCL11，CXCL10 and other chemokines. IFN-γ plays a key role in this process.

Abstract 1587: Stress keratin 17 overexpression downregulates CD8+ T cell infiltration in papillomavirus-associated disease

Abstract High-risk human papillomaviruses (HPVs) cause 5% of human cancers. Despite the availability of HPV vaccines, there remains a strong urgency to find ways to treat persistent HPV infections, as current HPV vaccines are not therapeutic for individuals already infected. It has not well understood how papillomaviruses evade host immune surveillance to establish long-term infections, which can lead to neoplastic disease including cancer. Using a mouse papillomavirus (MmuPV1) infection model in immunocompetent FVB/N mice, we found that stress keratins are upregulated in mouse papillomavirus-induced papillomas. In mice deficient for stress keratin K17 (K17KO), we observed early regression of papillomas dependent on T-cells. Expression of cellular genes involved in immune response were significantly upregulated in the papillomas arising on the K17KO mice correlating with increased numbers of infiltrating CD8+T cells and upregulation of IFNγ-related genes, including CXCL9 and CXCL10, prior to complete regression. This T cell-dependent regression also required ligand accessibility to CXCR3, a receptor for CXCL9 and CXCL10. By analyzing public available TCGA data, we found an inverse correlation between K17 mRNA expression and CD8A, GZMB and CXCL9 mRNA expression in a cohort of head and neck cancer patients. We validated these observations by antibody immunofluorescence staining of a tissue microarray (TMA) composed of 380 tumor tissues from head and neck cancer patients (HPV+ n=265, HPV- n=49, HPV unknown n=66). We stained TMA with CD8 and K17. Out of 380 cancer specimens, there are n=46 that had either high percentage of CD8+ cells in tumor (Z score&amp;gt;=1.64, n=14) or a high mean fluorescence intensity of K17 (Z score&amp;gt;=1.64, n=33). The heatmap of these 46 samples showed that the high K17 stained tissue and high CD8+ T cell infiltrated tissues are mutually exclusive, consistent with our prior analysis from TCGA mRNA data from head and neck cancer patients. In the process of exploring the mechanism of K17-mediated immunosuppression, we found that mouse papillomas arising from K17 knockout mice lacked nuclear staining for YAP1 whereas a significant amount of nuclear staining for YAP1 was observed in WT FVB papillomas, suggesting K17 may regulate Yap1 activation to mediate downstream immunosuppressive effect. Citation Format: Wei Wang, Ellery Gronski, Paul Lambert. Stress keratin 17 overexpression downregulates CD8+ T cell infiltration in papillomavirus-associated disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1587.

Abstract 6194: Evaluation of the differential expression profile of CXC-receptor-2 ligands for potential biomarker candidates in pancreatic ductal adenocarcinoma

Abstract The major complexity of pancreatic cancer (PC) cases is late clinical presentation and metastasis at the time of detection. Achievement of significant clinical advancement in the treatment of PC disease needs extensive research efforts to discover early detection markers of this disease. In this study, we wanted to analyze the expression profile of different CXC-receptor 2 (CXCR2) ligands, well documented in the literature for their role in inflammation, in pancreatic ductal adenocarcinoma (PDAC) cases for the potential of biomarker candidates. With this perspective, we utilized different online portals to evaluate mRNA expression of different CXCR2 ligands CXCL1-3 and 5-8 in PDAC tumors in comparison with the normal pancreas. Analysis of GEO PDAC microarray dataset, GSE15471 containing 39 pancreatic tumors with corresponding matched normal samples, showed significantly higher expression of CXCL-3,5,6,8 in pancreatic tumors in comparison with their matched normal tissue. Similarly, analysis of next-generation sequenced transcriptomics data on MiPanda portal showed higher expression of CXCL-1,3,5,6,8 in pancreatic tumors of 417 PC patients in comparison with the normal pancreas. Interestingly, only CXCL5 ligand showed association with patient survival data based on the mRNA, miRNA, or lncRNA expression on Oncolnc-TCGA portal. PC patients with lower CXCL5 expression showed significantly higher survival in comparison with patients with higher CXCL5 expression. Next, we evaluated the expression pattern of CXCR2 ligands CXCL1, 3, and 8 in the human PC tissue specimens and using pancreatic tissues derived from disease progression mouse model of PDX-cre-LSL-KRASG12D. Immunohistochemical (IHC) analysis revealed positive staining for CXCL1, and 3 in both in the ducts and the stroma of the human PC tumors and normal pancreas specimens. However, in normal pancreas, the expression was localized only to the acinar cell compartment and pancreatic ducts were negative for expression. Overall the average composite score of CXCL3 IHC was higher in the PC tissue specimens versus the normal pancreas. Furthermore, we observed an increase in the expression of mCXCL1, 3, and 5 in the pre-cancerous lesions of tumors and metastasis derived from PDX-cre-LSL-KrasG12D. However, unlike the human tissues the normal murine pancreas was negative for the expression of mCXCL1 and 5. Lastly, mRNA expression analysis of nine different human PC cell lines showed higher CXCL2, 3, and 5 in cell lines derived from metastatic sites in comparison with cell lines derived from primary tumors. Our observations resonated with CXCL5 protein levels in these cell lines. In conclusion, our data suggest that among different CXCR2 ligands, CXCL5 shows the potential of being used as a biomarker in PC patients. Citation Format: Sugandha Saxena, Abhilasha Purohit, Evie Ehrhorn, Pranita Atri, Caitlin Molczyk, Michelle Varney, Rakesh K. Singh. Evaluation of the differential expression profile of CXC-receptor-2 ligands for potential biomarker candidates in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6194.

Increased Osteoblastic Cxcl9 Contributes to the Uncoupled Bone Formation and Resorption in Postmenopausal Osteoporosis.

IntroductionEstrogen deficiency leads to bone loss in postmenopausal osteoporosis, because bone formation, albeit enhanced, fails to keep pace with the stimulated osteoclastic bone resorption. The mechanism driving this uncoupling is central to the pathogenesis of postmenopausal osteoporosis, which, however, remains poorly understood. We previously found that Cxcl9 secreted by osteoblasts inhibited osteogenesis in bone, while the roles of Cxcl9 on osteoclastic bone resorption and osteoporosis are unclear.Materials and MethodsPostmenopausal osteoporosis mouse model was established by bilateral surgical ovariectomy (OVX). In situ hybridization was performed to detect Cxcl9 mRNA expression in bone. ELISA assay was conducted to assess Cxcl9 concentrations in bone and serum. Cxcl9 activity was blocked by its neutralizing antibody. Micro-CT was performed to determine the effects of Cxcl9 neutralization on bone structure. Cell Migration and adhesion assay were conducted to evaluate the effects of Cxcl9 on osteoclast activity. TRAP staining and Western blot were performed to assess osteoclast differentiation. CXCR3 antagonist NBI-74,330 or ERK antagonist SCH772984 was administered to osteoclast to study the effects of Cxcl9 on CXCR3/ERK signaling.ResultsCxcl9 was expressed and secreted increasingly in OVX mice bone. Neutralizing Cxcl9 in bone marrow prevented bone loss in the mice by facilitating bone formation as well as inhibiting bone resorption. In vitro, Cxcl9 secreted from osteoblasts facilitated osteoclast precursors adhesion, migration and their differentiation into mature osteoclasts. The positive role of osteoblastic Cxcl9 on osteoclasts was eliminated by blocking CXCR3/ERK signaling in osteoclasts. Estrogen negatively regulated Cxcl9 expression and secretion in osteoblasts, explaining the increased Cxcl9 concentration in OVX mice bone.ConclusionOur study illustrates the roles of Cxcl9 in inhibiting bone formation and stimulating bone resorption in osteoporotic bone, therefore providing a possible therapeutic target to the treatment of postmenopausal osteoporosis.

An imbalance between blood CD4+CXCR5+Foxp3+ Tfr cells and CD4+CXCR5+Tfh cells may contribute to the immunopathogenesis of rheumatoid arthritis

BackgroundFollicular helper T (Tfh) cells are a subgroup of activated CD4+ T cells which can assist the formation and maintenance of germinal centers. Follicular regulatory T (Tfr) cells are a new class of regulatory T cells which play a major role in suppressing cells in humoral immunity. In contrast to the role of Tfh cells, Tfr cells can inhibit the function of Tfh cells and B cells. Imbalance of blood Tfr/Tfh ratio resulted in the expansion of auto-reactive B cells and auto-antibody production (). However, the effect of Tfr cells and Tfh cells in the pathogenesis of RA (rheumatoid arthritis) is unclear. The purpose of this study was to investigate the function of Tfr cells and Tfh cells in the pathogenesis of RA. MethodsWe recruited 20 patients fulfilled the the American College of Rheumatology diagnosis criteria and 20 healthy controls (HCs). The number of CD4+CXCR5+Foxp3+ Tfr cells and CD4+CXCR5+ Tfh cells in 20 RA patients were measured by flow cytometry analysis. Furthermore, the correlations between the Tfr/Tfh ratio and the characteristic clinical parameters were assessed. The serum levels of IL-21(interleukin-21), CXCL13 (chemokine (C-X-C motif) ligand 13) and TGF-β (Transforming growth factor-β) were measured by ELISA. The formation of ectopic germinal center (GC) of synovial membrane was examined by H&E staining. The transcriptional levels of CXCR5 (C-X-C chemokine receptor type 5), CXCL13, ICOS (inducible co-stimulater) and TGF-β mRNA were also analyzed. In addition, the expression of Bcl-6 (B-cell lymphoma 6), CXCR5, CXCL13 and ICOS in synovial membrane were examined by immunohistochemistry. ResultsRA patients had more Tfh cells in peripheral blood, conversely, the frequency of blood Tfr cells (p < 0.05) and the ratio of Tfr/Tfh were significantly decreased compared to healthy controls (p < 0.05, p < 0.01). Furthermore, the ratio of Tfr/Tfh was negatively correlated with values of ESR (r=-0.57, p < 0.05), RF (r=-0.5275, p < 0.001), CRP (r=-0.4486, p < 0.001), IgG (r=-0.4631, p < 0.05), DAS28 scores (r=-0.5645, p < 0.01), as well as the levels of IL-21(r=-0.7398, p < 0.01), CXCL13 (r=-0.4832, p < 0.05). However, the ratio of Tfr/Tfh was positively with the serum level of TGF-β (r=0.5115, p < 0.05). Higher mRNA expression of CXCR5, CXCL13, ICOS and lower TGF-β mRNA expression were observed in RA patients. The serum expression level of IL-21, CXCL13 was significantly increased and expression of TGF-β was significantly decreased in RA patients. Furthermore, ectopic germinal center formation and higher expression of Bcl-6, CXCR5, ICOS, CXCL13 in the synovial membrane of the joints in RA patients were observed. ConclusionsThe decreased blood CD4+CXCR5+Foxp3+ Tfr cells/CD4+CXCR5+ Tfhcells may be responsible for the immunopathogenesis of RA.

Discovery of triazoloquinoxaline as novel STING agonists via structure-based virtual screening.

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor facilitating innate immune signaling. Activation of STING leads to expression of interferons (IFNs) and pro-inflammatory cytokines which is associated with antiviral and antitumor responses. It is imperative to discovery potent compounds that precisely modulate STING. Herein, we describe the discovery of triazoloquinoxaline 1a as a novel STING agonist via Structure-based Virtual Screening. Specifically, biochemical and cell-based assays suggested that 1a stimulated concentration-dependently mRNA expression of IFNβ, CXCL-10 and IL-6. Furthermore, 1a significantly induced phosphorylation of STING, TANK-binding kinases1 (TBK1) and interferon regulatory factor 3 (IRF3), suggesting the activation of STING and its downstream TBK1-IRF3 signaling axis. In addition, 1a activated secretion of secreted alkaline phosphatase (SEAP) in dose-dependent manner and EC50 was 16.77±3.814μM, which is comparable with EC50 of 2'3'-cGAMP (9.212±2.229μM). These studies revealed that 1a is a promising STING agonist possessing the potential to be further developed for antiviral and antitumor treatment.