P2Y(6) agonist uridine 5'-diphosphate promotes host defense against bacterial infection via monocyte chemoattractant protein-1-mediated monocytes/macrophages recruitment.

Extracellular nucleotides are important messengers involved in series crucial physiological functions through the activation of P2 purinergic receptors. The detailed function and mechanism of the P2Y family in regulating immune response against invaded pathogens still remains unknown. In this study, the activation of purinoreceptor P2Y(6) by UDP was found to play a crucial role in promoting host defense against invaded bacteria through monocytes/macrophages recruitment. The expression level of P2Y(6) was much higher than other purinoreceptors in RAW264.7 cells, bone marrow macrophages, and peritoneal macrophages determined by real-time PCR. The supernatant of UDP (P2Y(6)-specific agonist)-treated RAW264.7 cells exhibited direct chemotaxis to monocytes/macrophages in vitro through Boyden Chambers assay. Meanwhile, the releasing of MCP-1 (MCP-1/CCL2) was enhanced obviously by UDP both in mRNA and protein level. Furthermore, the activation of P2Y(6) receptor by UDP also promotes ERK phosphorylation and AP-1 activation in a concentration- and time-dependent manner in RAW264.7 cells. This UDP-induced activation could be inhibited by P2Y(6) selectivity antagonist (MRS2578), MEK inhibitor (U0126), and MCP-1 blocking Ab, respectively. Moreover, i.p. injection with UDP resulted in a more efficacious clearance of invaded Escherichia coli and lower mortality in peritonitis mouse model. Together, our studies demonstrate that P2Y(6) receptor could be a novel mediator in upregulating innate immune response against the invaded pathogens through recruiting monocytes/macrophages.

Identification and validation of immunotherapy for four novel clusters of colorectal cancer based on the tumor microenvironment.

The incidence and mortality of colorectal cancer (CRC) are increasing year by year. The accurate classification of CRC can realize the purpose of personalized and precise treatment for patients. The tumor microenvironment (TME) plays an important role in the malignant progression and immunotherapy of CRC. An in-depth understanding of the clusters based on the TME is of great significance for the discovery of new therapeutic targets for CRC. We extracted data on CRC, including gene expression profile, DNA methylation array, somatic mutations, clinicopathological information, and copy number variation (CNV), from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) (four datasets-GSE14333, GSE17538, GSE38832, and GSE39582), cBioPortal, and FireBrowse. The MCPcounter was utilized to quantify the abundance of 10 TME cells for CRC samples. Cluster repetitive analysis was based on the Hcluster function of the Pheatmap package in R. The ESTIMATE package was applied to compute immune and stromal scores for CRC patients. PCA analysis was used to remove batch effects among different datasets and transform genome-wide DNA methylation profiling into methylation of tumor-infiltrating lymphocyte (MeTIL). We evaluated the mutation differences of the clusters using MOVICS, DeconstructSigs, and GISTIC packages. As for therapy, TIDE and SubMap analyses were carried out to forecast the immunotherapy response of the clusters, and chemotherapeutic sensibility was estimated based on the pRRophetic package. All results were verified in the TCGA and GEO data. Four immune clusters (ImmClust-CS1, ImmClust-CS2, ImmClust-CS3, and ImmClust-CS4) were identified for CRC. The four ImmClusts exhibited distinct TME compositions, cancer-associated fibroblasts (CAFs), functional orientation, and immune checkpoints. The highest immune, stromal, and MeTIL scores were observed in CS2, in contrast to the lowest scores in CS4. CS1 may respond to immunotherapy, while CS2 may respond to immunotherapy after anti-CAFs. Among the four ImmClusts, the top 15 markers with the highest mutation frequency were acquired, and CS1 had significantly lower CNA on the focal level than other subtypes. In addition, CS1 and CS2 patients had more stable chromosomes than CS3 and CS4. The most sensitive chemotherapeutic agents in these four ImmClusts were also found. IHC results revealed that CD29 stained significantly darker in the cancer samples, indicating that their CD29 was highly expressed in colon cancer. This work revealed the novel clusters based on TME for CRC, which would guide in predicting the prognosis, biological features, and appropriate treatment for patients with CRC.

HIF-1α promotes SARS-CoV-2 infection and aggravates inflammatory responses to COVID-19.

Cytokine storm induced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a major pathological feature of Coronavirus Disease 2019 (COVID-19) and a crucial determinant in COVID-19 prognosis. Understanding the mechanism underlying the SARS-CoV-2-induced cytokine storm is critical for COVID-19 control. Here, we identify that SARS-CoV-2 ORF3a and host hypoxia-inducible factor-1α (HIF-1α) play key roles in the virus infection and pro-inflammatory responses. RNA sequencing shows that HIF-1α signaling, immune response, and metabolism pathways are dysregulated in COVID-19 patients. Clinical analyses indicate that HIF-1α production, inflammatory responses, and high mortalities occurr in elderly patients. HIF-1α and pro-inflammatory cytokines are elicited in patients and infected cells. Interestingly, SARS-CoV-2 ORF3a induces mitochondrial damage and Mito-ROS production to promote HIF-1α expression, which subsequently facilitates SARS-CoV-2 infection and cytokines production. Notably, HIF-1α also broadly promotes the infection of other viruses. Collectively, during SARS-CoV-2 infection, ORF3a induces HIF-1α, which in turn aggravates viral infection and inflammatory responses. Therefore, HIF-1α plays an important role in promoting SARS-CoV-2 infection and inducing pro-inflammatory responses to COVID-19.

[Chronic toxicity test of fangyouling by transdermal administration in rabbits].

OBJECTIVE: To observe the toxicity of fangyouling after one month' s transdermal administration in rabbits and evaluate its security. METHODS: Forty rabbits were randomly divided into 4 groups including a control group and low, middle and high dose groups of fangyouling. The rabbits in the control group were administered with sunflower oil, and the other rabbits were administrated dermally with fangyouling of 50,300 and 2,000 mg/kg respectively once a day for 4 weeks. The general condition, the skin irritation reaction, body weight, food consumption, hematology, blood biochemistry, organ coefficients and histopathological changes of all the rabbits were observed. RESULTS: There was no obvious effect on the general condition in all the rabbits. However, the mild skin irritation was observed in 2 rabbits of the middle dose group and 4 rabbits of the high-dose group. The decreases of body weight and food consumption were noted in the high dose group. No changes were detected of hematology, blood biochemistry or viscera pathological at all dose levels. CONCLUSION: The dose of non-toxic response of fangyouling is 50 mg/kg at this study condition.