Circulating immune profile in granulomatosis with polyangiitis reveals distinct patterns related to disease activity.

Granulomatosis with polyangiitis (GPA) is an autoimmune disorder characterized by recurrent relapses that can cause severe tissue damage and life-threatening organ dysfunction. Multiple immune cells and cytokines/chemokines are involved in the different stages of the disease. Immune profiling of patients may be useful for tracking disease activity, however, reliable immune signatures for GPA activity are lacking. In this study, we examined circulating immune profiles in GPA patients during active and remission disease states to identify potential immune patterns associated with disease activity. The distribution and phenotypic characteristics of major circulating immune cells, and the profiles of circulating cytokines/chemokines, were studied on cryopreserved peripheral blood mononuclear cells from GPA patients (active, n = 20; remission, n = 20) and healthy controls (n = 20) leveraging a 40-color optimized multicolor immunofluorescence panel (OMIP-69) and in serum using a 46-plex Luminex multiplex assay, respectively. Deep phenotyping uncovered a distinct composition of major circulating immune cells in active GPA and GPA in remission, with the most significant findings emerging within the monocyte compartment. Our detailed analysis revealed circulating monocyte diversity beyond the conventional monocyte subsets. We identified eight classical monocyte populations, two intermediate monocyte populations, and one non-classical monocyte population. Notably, active GPA had a higher frequency of CD45RA+CCR5+CCR6-CCR7+/lowCD127-HLA-DR+CD2- classical monocytes and a lower frequency of CD45RA-CCR5-/lowCCR6-CCR7-CD127-HLA-DR+CD2+/- classical monocytes, which both strongly correlated with disease activity. Furthermore, serum levels of CXCL1, CXCL2, and CCL20, all linked to monocyte biology, were elevated in active GPA and correlated strongly with disease activity. These findings shed light on the circulating immune profile of GPA and may lead to immune signature profiles for assessing disease activity. Monocytes in particular may be studied further as potential markers for monitoring GPA.

Smooth muscle cell-derived Cxcl12 directs macrophage accrual and sympathetic innervation to control thermogenic adipose tissue.

Sympathetic innervation of brown adipose tissue (BAT) controls mammalian adaptative thermogenesis. However, the cellular and molecular underpinnings contributing to BAT innervation remain poorly defined. Here, we show that smooth muscle cells (SMCs) support BAT growth, lipid utilization, and thermogenic plasticity. Moreover, we find that BAT SMCs express and control the bioavailability of Cxcl12. SMC deletion of Cxcl12 fosters brown adipocyte lipid accumulation, reduces energy expenditure, and increases susceptibility to diet-induced metabolic dysfunction. Mechanistically, we find that Cxcl12 stimulates CD301+ macrophage recruitment and supports sympathetic neuronal maintenance. Administering recombinant Cxcl12 to obese mice or leptin-deficient (Ob/Ob) mice is sufficient to boost macrophage presence and drive sympathetic innervation to restore BAT morphology and thermogenic responses. Altogether, our data reveal an SMC chemokine-dependent pathway linking immunological infiltration and sympathetic innervation as a rheostat for BAT maintenance and thermogenesis.

Identification of potential biomarkers of gout through weighted gene correlation network analysis.

Background: Although hyperuricemia is not always associated with acute gouty arthritis, uric acid is a significant risk factor for gout. Therefore, we investigated the specific mechanism of uric acid activity. Methods: Using the gout-associated transcriptome dataset GSE160170, we conducted differential expression analysis to identify differentially expressed genes (DEGs). Moreover, we discovered highly linked gene modules using weighted gene coexpression network analysis (WGCNA) and evaluated their intersection. Subsequently, we screened for relevant biomarkers using the cytoHubba and Mcode algorithms in the STRING database, investigated their connection to immune cells and constructed a competitive endogenous RNA (ceRNA) network to identify upstream miRNAs and lncRNAs. We also collected PBMCs from acute gouty arthritis patients and healthy individuals and constructed a THP-1 cell gout inflammatory model, RT-qPCR and western blotting (WB) were used to detect the expression of C-X-C motif ligand 8 (CXCL8), C-X-C motif ligand 2 (CXCL2), and C-X-C motif ligand 1 (CXCL1). Finally, we predicted relevant drug targets through hub genes, hoping to find better treatments. Results: According to differential expression analysis, there were 76 upregulated and 28 downregulated mRNAs in GSE160170. Additionally, WGCNA showed that the turquoise module was most strongly correlated with primary gout; 86 hub genes were eventually obtained upon intersection. IL1ß, IL6, CXCL8, CXCL1, and CXCL2 are the principal hub genes of the protein-protein interaction (PPI) network. Using RT-qPCR and WB, we found that there were significant differences in the expression levels of CXCL8, CXCL1, and CXCL2 between the gouty group and the healthy group, and we also predicted 10 chemicals related to these proteins. Conclusion: In this study, we screened and validated essential genes using a variety of bioinformatics tools to generate novel ideas for the diagnosis and treatment of gout.

Identification of a prognostic signature based on five ferroptosis-related genes for diffuse large B-cell lymphoma.

BACKGROUND: Therapies for diffuse large B-cell lymphoma (DLBCL) are limited due to the diverse gene expression profiles and complicated immune microenvironments, making it an aggressive lymphoma. Beyond this, researches have shown that ferroptosis contributes to tumorigenesis, progression, and metastasis. We thus are interested to dissect the connection between ferroptosis and disease status of DLBCL. We aim at generating a valuable prognosis gene signature for predicting the status of patients of DLBCL, with focus on ferroptosis-related genes (FRGs). OBJECTIVE: To examine the connection between ferroptosis-related genes (FRGs) and clinical outcomes in DLBCL patients based on public datasets. METHODS: An expression profile dataset for DLBCL was downloaded from GSE32918 (https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=gse32918), and a ferroptosis-related gene cluster was obtained from the FerrDb database (http://www. zhounan.org/ferrdb/). A prognostic signature was developed from this gene cluster by applying a least absolute shrinkage and selection operator (LASSO) Cox regression analysis to GSE32918, followed by external validation. Its effectiveness as a biomarker and the prognostic value was determined by a receiver operator characteristic curve mono factor analysis. Finally, functional enrichment was evaluated by the package Cluster Profiler of R. RESULTS: Five ferroptosis-related genes (FRGs) (GOP1, GPX2, SLC7A5, ATF4, and CXCL2) associated with DLBCL were obtained by a multivariate analysis. The prognostic power of these five FRGs was verified by TCGA (https://xenabrowser.net/datapages/?dataset=TCGA.DLBC.sampleMap%2FHiSeqV2_PANCAN&host=https%3A%2F%2Ftcga.xenahubs.net&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A44) and GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse 32918) datasets, with ROC analyses. KEGG and GO analyses revealed that upregulated genes in the high-risk group based on the gene signature were enriched in receptor interactions and other cancer-related pathways, including pathways related to abnormal metabolism and cell differentiation. CONCLUSION: The newly developed signature involving GOP1, GPX2, SLC7A5, ATF4, and CXCL2 has the potential to serve as a prognostic biomarker. Furthermore, our results provide additional support for the contribution of ferroptosis to DLBCL.

PAK1 Promotes Inflammation Induced by Sepsis through the Snail/CXCL2 Signaling Pathway.

Sepsis is a severe syndrome characterized by organ dysfunction, resulting from a systemic imbalance in response to infection. PAK1 plays a critical role in various diseases. The present study aimed to explore and delineate the mechanism of PAK1 in inflammation induced by sepsis. Bioinformatics analysis was performed to assess PAK1, snail, and CXCL2 expression in the whole blood of septic patients and the pathways enriched with PAK1. To simulate the sepsis model, THP-1 cells were stimulated with lipopolysaccharide. Gene expression was evaluated using qRT-PCR, while cell viability was assessed using CCK-8 assay. Cell apoptosis was tested with flow cytometry. Expression of inflammatory factors in cells following different treatments was analyzed using the enzyme linked immunosorbent assay (ELISA). Dual-luciferase and chromatin immunoprecipitation assays were conducted to verify the binding relationship between PAK1 and the snail. Mouse models of cecal ligation and puncture were established, and hematoxylin and eosin staining and ELISA were employed to detect the infiltration levels of inflammatory cells and the expression of related protective factors in lung, liver, and kidney tissues. The results demonstrated upregulation of PAK1, snail, and CXCL2 in the whole blood of septic patients, with PAK1 being enriched in the chemokine-related pathway. Knockdown of PAK1 significantly promoted the apoptosis of LPS-stimulated THP-1 cells and inhibited the expression of inflammatory factors. PAK1 upregulated the expression of the snail, which in turn promoted the expression of CXCL2. Thus, PAK1 mediated the sepsis-induced inflammatory response through the snail/CXCL2 pathway. In conclusion, PAK1 played a role in promoting inflammation induced by sepsis through the snail/CXCL2 axis, thereby providing a potential therapeutic target for the management of sepsis.

Acute stress transiently activates macrophages and chemokines in cervical lymph nodes.

Acute restraint stress (RS) is routinely used to study the effects of psychological and/or physiological stress. We evaluated the impact of RS on cervical lymph nodes in rats at molecular and cellular levels. Male Sprague-Dawley rats were subjected to stress by immobilization for 30, 60, and 120 min (RS30, RS60, and RS120, respectively) and compared with rats of a no-stress control (C) group. The expression of genes encoding chemokines CXCL1/CXCL2 (Cxcl1 and Cxcl2) and their receptor CXCR2 (Cxcr2) was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and microarray analyses. Immunohistochemistry and in situ hybridization were performed to determine the expression of these proteins and the macrophage biomarker CD68. Microarray analysis revealed that the expression of 514 and 496 genes was upregulated and downregulated, respectively, in the RS30 group. Compared with the C group, the RS30 group exhibited a 23.0-, 13.0-, and 1.6-fold increase in Cxcl1, Cxcl2, and Cxcr2 expression. Gene Ontology analysis revealed the involvement of these three upregulated genes in the cytokine network, inflammation, and leukocyte chemotaxis and migration. RT-qPCR analysis indicated that the mRNA levels of Cxcl1 and Cxcl2 were significantly increased in the RS30 group but were reverted to normal levels in the RS60 and RS120 groups. Cxcr2 mRNA level was significantly increased in the RS30 and RS120 groups compared with that in the C group. RS-induced CXCL1-immunopositive cells corresponded to B/plasma cells, whereas CXCL2-immunopositive cells corresponded to endothelial cells of the high endothelial venules. Stress-induced CXCR2-immunopositive cells corresponded to macrophages. Psychological and/or physiological stress induces an acute stress response and formation of an immunoreactive microenvironment in cervical lymph nodes, with the CXCL1/CXCL2-CXCR2 axis being pivotal in the acute stress response.

M2 tumor-associated macrophages and CXCL2 induce lipid remodeling in hepatocellular carcinoma cell lines.

Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors, but its pathogenesis remains incompletely elucidated. Recently, many studies indicated that lipid remodeling plays an important role in the occurrence and development of HCC. Furthermore, lipids have been proven to be indispensable mediators in promoting communication between tumor cells and extracellular matrix in the tumor microenvironment. Thus, this study aims to comprehensively investigate the process of lipid remodeling during HCC metastasis based on the LC-electrospray ionization-MS (LC-ESI-MS) combined with multiple reaction monitoring technology. M2 tumor-associated macrophages and the recombinant human protein CXCL2 were used to simulate the tumor microenvironment. After co-incubating SMMC7721 and MHCC97-H cell lines with M2 tumor-associated macrophages or the recombinant human protein CXCL2 for 48 h, LC-ESI-MS was used to quantify the levels of two major classes of lipid molecules, namely, glycerophospholipids and sphingolipids. Our results suggest that lipid remodeling in the tumor microenvironment may promote the migration and invasion of HCC cell lines.

Macrophage Inflammatory Proteins (MIPs) Contribute to Malignant Potential of Colorectal Polyps and Modulate Likelihood of Cancerization Associated with Standard Risk Factors.

Better understanding of molecular changes leading to neoplastic transformation is prerequisite to optimize risk assessment and chemopreventive and surveillance strategies. Data on macrophage inflammatory proteins (MIPs) in colorectal carcinogenesis are scanty and their clinical relevance remains unknown. Therefore, transcript and protein expression of CCL3, CCL4, CXCL2, and CCL19 were determined in 173 and 62 patients, respectively, using RT-qPCR and immunohistochemistry with reference to polyps' characteristics. The likelihood of malignancy was modeled using probit regression. With the increasing malignancy potential of hyperplastic-tubular-tubulo-villous-villous polyps, the expression of CCL3, CCL4, and CCL19 in lesions decreased. CCL19 expression decreased also in normal mucosa while that of CXCL2 increased. Likewise, lesion CCL3 and lesion and normal mucosa CCL19 decreased and normal CXCL2 increased along the hyperplasia-low-high dysplasia grade. The bigger the lesion, the lower CCL3 and higher CXCL2 in normal mucosa. Singular polyps had higher CCL3, CCL4, and CCL19 levels in normal mucosa. CCL3, CCL4 and CXCL2 modulated the likelihood of malignancy associated with traditional risk factors. There was no correlation between the protein and mRNA expression of CCL3 and CCL19. In summary, the polyp-adjacent mucosa contributes to gaining potential for malignancy by polyps. MIPs may help in specifying cancerization probability estimated based on standard risk factors.

CXCR2-Blocking Has Context-Sensitive Effects on Rat Glioblastoma Cell Line Outgrowth (S635) in an Organotypic Rat Brain Slice Culture Depending on Microglia-Depletion (PLX5622) and Dexamethasone Treatment.

In glioblastoma (GBM), the interplay of different immune cell subtypes, cytokines, and/or drugs shows high context-dependencies. Interrelations between the routinely applied dexamethasone (Dex) and microglia remain elusive. Here, we exploited rat organotypic brain slice co-cultures (OBSC) to examine the effects on a rat GBM cell line (S635) outgrowth resulting from the presence of Dex and pretreatment with the colony-stimulating factor receptor 1 (CSF1-R) inhibitor PLX5622: in native OBSC (without PLX5622-pretreatment), a diminished S635 spheroid outgrowth was observable, whereas Dex-treatment enhanced outgrowth in this condition compared to PLX5622-pretreated OBSC. Screening the supernatants of our model with a proteome profiler, we found that CXCL2 was differentially secreted in a Dex- and PLX5622-dependent fashion. To analyze causal interrelations, we interrupted the CXCL2/CXCR2-axis: in the native OBSC condition, CXCR2-blocking resulted in increased outgrowth, in combination with Dex, we found potentiated outgrowth. No effect was found in the PLX5622-pretreated. Our method allowed us to study the influence of three different factors-dexamethasone, PLX5622, and CXCL2-in a well-controlled, simplified, and straight-forward mechanistic manner, and at the same time in a more realistic ex vivo scenario compared to in vitro studies. In our model, we showed a GBM outgrowth enhancing synergism between CXCR2-blocking and Dex-treatment in the native condition, which was levelled by PLX5622-pretreatment.

Identification and Validation of CXCL2 as a Key Gene for Childhood Obesity.

This study aims to identify the key genes and their regulatory networks by bioinformatics, increasing understanding of childhood obesity. The data comes from the GEO and Immport database. The immune microenvironment was explored in GSE104815. Key genes were identified by intersection of DEGs with the immune gene set. Enrichment analysis revealed gene-related functions and correlation analysis explored the relationship. Regulatory networks were constructed based on miRcode, TarBase and TargetScan databases. GSE29718 was used to validate our findings. Intercellular communication and cell differentiation trends were further explored using single-cell data from GSE153643. Based on our research, the immune microenvironment in the obese group showed higher immune infiltration. We found 962 DEGs and CXCL2 was identified as the key gene. The co-regulatory network of lncRNA-miRNA-mRNA suggested that obtaining TM4SF19-AS1, GUSBP11, AC105020.1, LINC00189, COL4A2-AS2, VIPR1-AS1 and LINC00242 may regulate CXCL2 (r > 0.9 and P < 0.01). Differential expression of CXCL2 was validated in GSE29718 (P < 0.05) and CXCL2 was identified as a biomarker for childhood obesity (AUC = 0.885). GSVA enrichment analysis revealed many pathways of high group obtaining the TNF-α signaling via NF-κB pathway and interferon γ response pathway. In GSE153643, 11 cell types were identified and CXCL2 was highly expressed in monocyte, macrophage, endothelial cell and pericyte. In CXCL2 high expressing macrophages, there was a tendency for cells to polarize toward M1 macrophages (P < 0.05). In summary, we identified CXCL2 as a potential biomarker of childhood obesity. The development of childhood obesity may be associated with the activation of immune infiltration of macrophage M1 polarization by CXCL2 expression.

SHCBP1 Promotes the Proliferation of Breast Cancer Cells by Inhibiting CXCL2.

Breast cancer has the characteristics of high metastasis and recurrence and ranks first in incidence and mortality among female malignant tumors. Shc SH2-domain binding protein 1 (SHCBP1) is an important protein in intracellular signal transduction and cell division, but the role of SHCBP1 in breast cancers remains elusive. Here, we found that SHCBP1 deficiency inhibited the proliferation of breast cancer cells. Mechanistically, SHCPB1 significantly downregulates the mRNA level of CXCL2, which in turn activates the AKT and ERK signaling, while inactivates the p21 and p27 signaling. In addition, overexpression of SHCPB1 downregulates the protein levels of p21 and p27, which could be completely reversed by restoration of CXCL2 expression. Moreover, we analyzed the expression of both SHCPB1 and CXCL2, and found that SHCPB1 is highly expressed in breast cancer cells or tissues from breast cancer patients compared to normal breast cells or adjacent normal tissues, while CXCL2 is lowly expressed in breast cancer cells or tissues. Collectively, our study reveals that SHCBP1 plays an oncogenic role in breast cancer tumorigenesis partially through inhibiting the inflammatory response and ultimately activating the proliferation of breast cancers.

MicroRNA­27a­3p regulates the proliferation and chemotaxis of pulmonary macrophages in non­small cell lung carcinoma tissues through CXCL2.

The present study aimed to investigate microRNA (miRNA)-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with early non-small cell lung carcinoma (NSCLC) and its regulatory effect on the infiltration of pulmonary macrophages into cancer tissues and invasion of NSCLC cells. Blood specimens were withdrawn from 36 patients with NSCLC and 29 healthy subjects. NSCLC tissues and cancer-adjacent tissues were both obtained from patients with NSCLC; furthermore, certain tissue samples were used to extract macrophages. The levels of miRNA-27a-3p and C-X-C motif ligand chemokine 2 (CXCL2) mRNA were detected by reverse transcription-quantitative PCR and the levels of CXCL2 protein were measured by ELISA and western blot analysis. A dual-luciferase reporter assay was performed to determine the interactions between miRNA and mRNA. An MTT assay was employed to examine the viability of transfected cells and macrophages and a Transwell assay was performed to assess chemotaxis. The differential expression of miRNA-27a-3p in NSCLC tissues, pulmonary macrophages and peripheral blood indicated that miRNA-27a-3p exerted different roles in these specimens. CXCL2 was upregulated in NSCLC tissues at both transcriptional and translational levels. In addition, the untranslated region of CXCL2 was confirmed to be directly targeted by miRNA-27a-3p prior to its transcriptional activation. Furthermore, miRNA-27a-3p regulated CXCL2 expression, thereby affecting the proliferation of human pulmonary macrophages. The present study highlights that miRNA-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with NSCLC is downregulated, while its target gene CXCL2 is upregulated. miRNA-27a-3p may regulate the viability and chemotaxis of macrophages in tumor tissues of patients with NSCLC through CXCL2 and is expected to become a genetic marker of this disease.

Racial differences in serum chemokines in prostate cancer patients.

BACKGROUND: This study aimed to understand the differential levels of inflammatory chemokines in association with higher prostate cancer incidence and mortality in African American (AA) men than in Caucasians (CA). METHODS: The authors used a chemokine assay to simultaneously measure 40 chemokines and cytokines levels in the serum of preoperative prostate cancer patients and healthy controls of AA and CA races. Selected chemokines (CXCL2, CXCL5, and CCL23) serum level was validated in 211 serum samples from prostate cancer patients and healthy controls. Differential expression of CXCL5 and CCL23 was analyzed using immunohistochemistry in a representative cohort of prostate tumor tissues of AA and CA races. RESULTS: Race-specific comparisons from 211 serum samples showed significantly higher levels of CXCL2 (control: 3104.0 pg/mL vs. cancer: 2451.0 pg/mL) and CXCL5 (control: 5189.0 pg/mL vs. cancer: 5459.0 pg/mL) in AA men than in CAs (CXCL2; control: 1155.0 pg/mL vs. cancer: 889.3 pg/mL, and CXCL5; control: 1183.0 pg/mL vs. cancer: 977.5 pg/mL). CCL23 differed significantly within and between the races with a lower level in AA cancer cases (454.5 vs. 966.6 pg/mL) than healthy controls (740.5 vs. 1263.0 pg/mL). Patient age, prostate-specific antigen, or Gleason scores were not significantly associated with these chemokines. Immunostaining for CXCL5 and CCL23 in a representative cohort of archival prostate tissues displayed significantly higher CXCL5 in prostate tumors than in adjacent benign tissues, whereas CCL23 was nondetectable in most of the analyzed tumor tissues. CONCLUSION: Lower levels of CCL23 in AA prostate cancer patient sera and tumor tissues and high CXCL2 and CXCL5 may contribute to aggressive prostate cancer, as often seen in AA men. The disproportionate levels of serum chemokines associated with race warrant further exploration to improve equitability in precision oncology to benefit prostate cancer patients.

CXCR2 Is Deregulated in ALS Spinal Cord and Its Activation Triggers Apoptosis in Motor Neuron-Like Cells Overexpressing hSOD1-G93A.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by progressive depletion of motor neurons (MNs). Recent evidence suggests a role in ALS pathology for the C-X-C motif chemokine receptor 2 (CXCR2), whose expression was found increased at both mRNA and protein level in cortical neurons of sporadic ALS patients. Previous findings also showed that the receptor inhibition is able to prevent iPSC-derived MNs degeneration in vitro and improve neuromuscular function in SOD1-G93A mice. Here, by performing transcriptional analysis and immunofluorescence studies, we detailed the increased expression and localization of CXCR2 and its main ligand CXCL8 in the human lumbar spinal cord of sporadic ALS patients. We further investigated the functional role of CXCR2/ligands axis in NSC-34 motor neuron-like cells expressing human wild-type (WT) or mutant (G93A) SOD1. A significant expression of CXCR2 was found in doxycycline-induced G93A-SOD1-expressing cells, but not in WT cells. In vitro assays showed CXCR2 activation by GROα and MIP2α, two murine endogenous ligands and functional homologs of CXCL8, reduces cellular viability and triggers apoptosis in a dose dependent manner, while treatment with reparixin, a non-competitive allosteric CXCR2 inhibitor, effectively counteracts GROα and MIP2α toxicity, significantly inhibiting the chemokine-induced cell death. Altogether, data further support a role of CXCR2 axis in ALS etiopathogenesis and confirm its pharmacological modulation as a candidate therapeutic strategy.

Promotion of axon regeneration and protection on injured retinal ganglion cells by rCXCL2.

BACKGROUND: In addition to rescuing injured retinal ganglion cells (RGCs) by stimulating the intrinsic growth ability of damaged RGCs in various retinal/optic neuropathies, increasing evidence has shown that the external microenvironmental factors also play a crucial role in restoring the survival of RGCs by promoting the regrowth of RGC axons, especially inflammatory factors. In this study, we aimed to screen out the underlying inflammatory factor involved in the signaling of staurosporine (STS)-induced axon regeneration and verify its role in the protection of RGCs and the promotion of axon regrowth. METHODS: We performed transcriptome RNA sequencing for STS induction models in vitro and analyzed the differentially expressed genes. After targeting the key gene, we verified the role of the candidate factor in RGC protection and promotion of axon regeneration in vivo with two RGC-injured animal models (optic nerve crush, ONC; retinal N-methyl-D-aspartate, NMDA damage) by using cholera toxin subunit B anterograde axon tracing and specific immunostaining of RGCs. RESULTS: We found that a series of inflammatory genes expressed upregulated in the signaling of STS-induced axon regrowth and we targeted the candidate CXCL2 gene since the level of the chemokine CXCL2 gene elevated significantly among the top upregulated genes. We further demonstrated that intravitreal injection of rCXCL2 robustly promoted axon regeneration and significantly improved RGC survival in ONC-injured mice in vivo. However, different from its role in ONC model, the intravitreal injection of rCXCL2 was able to simply protect RGCs against NMDA-induced excitotoxicity in mouse retina and maintain the long-distance projection of RGC axons, yet failed to promote significant axon regeneration. CONCLUSIONS: We provide the first in vivo evidence that CXCL2, as an inflammatory factor, is a key regulator in the axon regeneration and neuroprotection of RGCs. Our comparative study may facilitate deciphering the exact molecular mechanisms of RGC axon regeneration and developing high-potency targeted drugs.

Microglia and Astroglia-The Potential Role in Neuroinflammation Induced by Pre- and Neonatal Exposure to Lead (Pb).

Neuroinflammation is one of the postulated mechanisms for Pb neurotoxicity. However, the exact molecular mechanisms responsible for its pro-inflammatory effect are not fully elucidated. In this study, we examined the role of glial cells in neuroinflammation induced by Pb exposure. We investigated how microglia, a type of glial cell, responded to the changes caused by perinatal exposure to Pb by measuring the expression of Iba1 at the mRNA and protein levels. To assess the state of microglia, we analyzed the mRNA levels of specific markers associated with the cytotoxic M1 phenotype (Il1b, Il6, and Tnfa) and the cytoprotective M2 phenotype (Arg1, Chi3l1, Mrc1, Fcgr1a, Sphk1, and Tgfb1). Additionally, we measured the concentration of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α). To assess the reactivity and functionality status of astrocytes, we analyzed the GFAP (mRNA expression and protein concentration) as well as glutamine synthase (GS) protein level and activity. Using an electron microscope, we assessed ultrastructural abnormalities in the examined brain structures (forebrain cortex, cerebellum, and hippocampus). In addition, we measured the mRNA levels of Cxcl1 and Cxcl2, and their receptor, Cxcr2. Our data showed that perinatal exposure to Pb at low doses affected both microglia and astrocyte cells' status (their mobilization, activation, function, and changes in gene expression profile) in a brain-structure-specific manner. The results suggest that both microglia and astrocytes represent a potential target for Pb neurotoxicity, thus being key mediators of neuroinflammation and further neuropathology evoked by Pb poisoning during perinatal brain development.

Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis.

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.

Serum cytokine and chemokine profiles and disease prognosis in hepatitis B virus-related acute-on-chronic liver failure.

Background: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) has significant morbidity and mortality and is associated with the induction of cytokines/chemokines, which might contribute to the pathogenesis of liver injury. This study aimed to explore the cytokine/chemokine profiles of patients with HBV-ACLF and develop a composite clinical prognostic model. Methods: We prospectively collected blood samples and the clinical data of 107 patients with HBV-ACLF admitted to the Beijing Ditan Hospital. The concentrations of 40-plex cytokines/chemokines were measured in 86 survivors and 21 non-survivors using the Luminex assay. Discrimination between the cytokine/chemokine profiles in different prognosis groups was analyzed using the multivariate statistical techniques of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). An immune-clinical prognostic model was obtained using multivariate logistic regression analysis. Results: The PCA and PLS-DA indicated that cytokine/chemokine profiling could clearly distinguish patients with different prognoses. A total of 14 cytokines, namely, IL-1ß, IL-6, IL-8, IL-10, TNF-α, IFN-γ, CXCL1, CXCL2, CXCL9, CXCL13, CX3CL1, GM-SCF, CCL21, and CCL23, were significantly correlated with disease prognosis. Multivariate analysis identified CXCL2, IL-8, total bilirubin, and age as independent risk factors that constituted the immune-clinical prognostic model, which showed the strongest predictive value of 0.938 compared with those of the Chronic Liver Failure Consortium (CLIF-C) ACLF (0.785), Model for End-Stage Liver Disease (MELD) (0.669), and MELD-Na (0.723) scores (p < 0.05 for all). Conclusion: The serum cytokine/chemokine profiles correlated with the 90-day prognosis of patients with HBV-ACLF. The proposed composite immune-clinical prognostic model resulted in more accurate prognostic estimates than those of the CLIF-C ACLF, MELD, and MELD-Na scores.

Sirt4 deficiency promotes the development of atherosclerosis by activating the NF-κB/IκB/CXCL2/3 pathway.

BACKGROUND AND AIMS: As a member of mitochondrial sirtuins, Sirt4 plays a vital role in cellular metabolism and intracellular signal transduction; however, its effect on atherosclerosis is unclear. This study aimed to explore the effect of Sirt4 on atherosclerosis and its underlying mechanism. METHODS: In vivo, Apoe-/- and Apoe-/-/Sirt4-/- mice were fed a high-fat diet to induce atherosclerosis. In vitro, peritoneal macrophages from two mouse types were extracted and treated with oxidized low-density lipoprotein to establish a cell model, THP-1 cells were used to observe the effect of Sirt4 on the adhesion ability of monocytes. The growth and composition of aortic plaques in two mouse types were analyzed by H&E staining, Oil Red O staining, Dil oxidized low-density lipoprotein, immunohistochemistry, real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Transcriptome analysis and Western blotting were performed to explore the specific mechanism. RESULTS: Sirt4 deficiency aggravated atherosclerosis in mice. In vivo, aortic plaque size, lipid content, and expression of related inflammatory factors in Apoe-/-/Sirt4-/- mice were higher than those in the control group, whereas the content of collagen Ⅰ and smooth muscle actin-α was significantly lower. Sirt4-deficient macrophages exhibited stronger lipid phagocytosis in vitro, and the adhesion ability of monocytes increased when Sirt4 expression decreased. Transcriptome analysis showed that the expression of CXCL2 and CXCL3 in Sirt4-deficient peritoneal macrophages increased significantly, which may play a role by activating the NF-κB pathway. In further analysis, the results in vitro and in vivo showed that the expression of VCAM-1 and pro-inflammatory factors, such as IL-6, TNF-α and IL-1ß, increased, whereas the expression of anti-inflammatory factor IL-37 decreased in Sirt4-deficient peritoneal macrophages and tissues. After blocking the effect with NK-κB inhibitor BAY11-7082, the inflammatory reaction in sirt4 deficient macrophages was also significantly decreased. CONCLUSIONS: This study demonstrates that Sirt4 deficiency promotes the development of atherosclerosis by activating the NF-κB/IκB/CXCL2/3 pathway, suggesting that Sirt4 may exhibit a protective effect in atherosclerosis, which provides a new strategy for clinical prevention and treatment of atherosclerosis.