CCL2 Expression Research Articles

Identifying methamphetamine use predictors in HIV infection: Immune-dopaminergic signatures in peripheral leukocytes and the role of COMT genotype

The pursuit of translational biomarkers is complex due to the heterogeneous human pathophysiology, but critical for disease diagnosis, prognosis, monitoring therapeutic efficacy, and for patient stratification. In HIV-associated neurocognitive impairment (NCI), biomarkers that delineate the trajectory of neuropathogenesis and neurocognitive sequelae are critical, particularly considering confounders such as substance use, including Methamphetamine (METH). METH use is a significant health concern among persons living with HIV (PWH), aggravating cognitive deficits and neuroinflammation despite of antiretrovirals, introducing elements in the microenvironment that are fundamentally differerent in relation to non-METH users, such as high levels of dopamine (DA) affecting HIV-innate immune targets. Yet, current biomarkers do not detect these differences. We hypothesized that predefined DA-induced signatures detectable in peripheral blood leukocytes, can distinguish HIV+ METH users compared to HIV-negative or PWH that are non METH users. The elevated expression of CD8A, CREBBP, CCL5, and combinations of dopaminergic pathway transcripts clustered METH users with detectable CSF viral load and major depressive disorder (MDD), indicating neuroimmune-mechanistic links. Cathecol-o-methyltransferase (COMT) gene polymorphisms affecting DA metabolism improved the identification of PWH using METH with biomarkers. The results indicate that underlying immunedopaminergic mechanisms provide signatures and genotypes that can identify PWH that are METH users and their attributes.

Impact of photobiomodulation therapy on pro-inflammation functionality of human peripheral blood mononuclear cells – a preliminary study

Research into the efficacy of photobiomodulation therapy (PBMT) in reducing inflammation has been ongoing for years, but standards for irradiation methodology still need to be developed. This study aimed to test whether PBMT stimulates in vitro human peripheral blood mononuclear cells (PBMCs) to synthesize pro-inflammatory cytokines, including chemokines. PBMCs were irradiated with laser radiation at two wavelengths simultaneously (λ = 808 nm in continuous emission and λ = 905 nm in pulsed emission). The laser radiation energy was dosed in one dose as a whole (5 J, 15 J, 20 J) or in a fractionated way (5 J + 15 J and 15 J + 5 J) with a frequency of 500, 1,500 and 2,000 Hz. The surface power densities were 177, 214 and 230 mW/cm2, respectively. A pro-inflammatory effect was observed at both the transcript and protein levels for IL-1β after PBMT at the energy doses 5 J and 20 J (ƒ=500 Hz) and only at the transcript level after application of PBMT at energy doses of 20 J (ƒ= 1,500; ƒ=2,000 Hz) and 5 + 15 J (ƒ=500 Hz). An increase in CCL2 and CCL3 mRNA expression was observed after PBMT at 5 + 15 J (ƒ=1,500 Hz) and 15 + 5 J (ƒ=2,000 Hz) and CCL3 concentration after application of an energy dose of 15 J (frequency of 500 Hz). Even though PBMT can induce mRNA synthesis and stimulate PBMCs to produce selected pro-inflammatory cytokines and chemokines, it is necessary to elucidate the impact of the simultaneous emission of two wavelengths on the inflammatory response mechanisms.

A phase 2a trial of brepocitinib for cicatricial alopecia

BackgroundCicatricial alopecias (CA) are chronic, progressive scarring hair-loss conditions. Molecular dysregulation is not fully understood, hindering treatment development. Th1/IFNγ signaling and JAK dysregulation has shown involvement, providing rationale for this phase 2a trial with TYK2/JAK1 inhibitor brepocitinib. MethodsRandomized, placebo-controlled phase 2a trial spanning 52 weeks. Adults (18≥years of age) with lichen planopilaris, frontal fibrosing alopecia, or central centrifugal cicatricial alopecia diagnosis were randomized 3:1 to brepocitinib 45mg daily or placebo for 24 weeks, after which all patients received brepocitinib for another 24 weeks, with a safety follow up 4 weeks later. Lesional scalp biopsies were collected at baseline, week 24, and week 48. Co-primary endpoints were changes in lesional expression of CCL5, changes in lesional expression of fibrosis-related markers, and safety at week 24. ResultsPatients receiving brepocitinib showed significant downregulation in CCL5 expression at week 24 (p=0.004). Enrichment analysis of a subset of fibrosis markers showed trending upregulation in placebo patients (p<0.1). Brepocitinib was well tolerated and improved clinical severity scores. LimitationsSingle-dose regimen, small placebo group. ConclusionBrepocitinib significantly reduces CCL5 expression and was well tolerated at week 24, meeting co-primary endpoints. Brepocitinib reduces inflammatory biomarker expression and improves clinical severity, while maintaining favorable safety profile.

Effects of chemokine-binding protein in visceral ovine aphthae on immune regulation response.

ORFV of the family poxvirus，which produces a pustular dermatitis both in humans and animals.,Previous studies have found an fatal case caused by the infection of ORFV in the viscera. However, the mechanisms of ORFV how to infect the viscera remain unknown. Our sequencing results revealed that the CBP of the visceral infection strain lacked a 24-base pair segment at position 217 comparison to the oral infection strain. Subsequently, we successfully packaged the recombinant adenoviruses pAd-CBP-K and pAd-CBP-N in HEK-293A cells and carried it to infect lymphocytes. RT-PCR analysis showed that the CBP protein was expressed in lymphocytes, and pAd-CBP-N group exhibited a significantly higher CBP expression level compared to the pAd-CBP-K group. At 4, 8, and 12hours post-infection, both pAd-CBP-K and pAd-CBP-N were found to downregulate the expression of MIP-1 and CCL-5 in the supernatant of lymphocytes. However, the expression of IL-2, IL-6, IL-8, IL-12, INF-γ, and TNF-α showed a significant up-regulation. Furthermore, the inflammatory factors relative expression levels of IL-2, IL-6, IL-8, IL-8, IL-12, IFN-γ and TNF-α were significantly up-regulated in the both group. Interestingly, a significant increase in the expression of IL-6, IL-8 and TNF-α were detected in the pAd-CBP-N group at both 8 and 12hours compared to pAd-CBP-N. Taken together, these findings showed that CBP can regulate the expression of free chemokines and activate the expression of inflammatory factors, and provide a basis for follow-up research.

CCL2 may contribute to the damage of fallopian tube epithelium in women with tubal endometriosis

Research QuestionHow does tubal endometriosis (TEM) impact the morphology and ultrastructure of the fallopian tube epithelium? What are the underlying pathophysiological mechanisms of TEM? DesignEpithelial samples of the fallopian tubes from patients with (TEM group) and without (non-TEM group) TEM were collected. The morphological characteristics, ultrastructure, ciliated cell percentage, and ciliary beat frequency (CBF) were assessed via hematoxylin-eosin staining, electron microscopy, and high-speed video microscopy. mRNA microarray analysis was conducted to identify differentially expressed genes (DEGs) in the fallopian tube epithelium, which were further validated using qPCR, immunohistochemistry, and Western blotting. The effects of recombinant CCL2 protein on primary human fallopian tube epithelial cells (FTEC) were examined in vitro. ResultsPatients with TEM exhibited significant abnormalities in the morphology and ultrastructure of the fallopian tube epithelium with reduced percentage of ciliated cells and CBF, compared to the non-TEM patients. Among the 765 DEGs identified in the fallopian tube epithelium, 512 genes were upregulated and 253 genes were downregulated in the TEM group. qPCR, immunohistochemistry, and Western blotting validation confirmed a significant upregulation of CCL2 expression in the TEM group. In vitro experiments revealed that recombinant CCL2 protein significantly reduced ciliated cell differentiation, length of cilia, and CBF in FTEC, suggesting a role for CCL2 in the development of the TEM-related pathological phenotype. ConclusionsThese findings indicate that CCL2 may contribute to the pathological phenotype associated with TEM and could be a crucial signaling molecule in the damage of fallopian tube epithelium in patients with TEM.

Downregulation of chemokine (C‑C motif) ligand 5 induced by a novel 8‑hydroxyquinoline derivative (91b1) suppresses tumor invasiveness in esophageal carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a particularly aggressive form of cancer with high mortality. In the present study, a novel 8‑hydroxyquinoline derivative (91b1) was investigated for its anticancer activities in ESCC along with its associated mechanisms. The in vitro cytotoxic effect of 91b1 were evaluated across five ESCC cell lines using MTS assay, with cisplatin serving as a comparative standard. Changes in gene expression profile were identified by cDNA microarray and further validated by qualitative PCR and immunostaining. Additionally, protein levels of the most notably downregulated target in archival ESCC samples were also studied. 91b1 demonstrated comparable anticancer effect with cisplatin. Notably, chemokine ligand 5 (Ccl5) was identified as the most substantially downregulated gene, with its suppression at both mRNA and protein expression in ESCC cells, exhibiting a dose‑dependent manner. The recombinant human protein of CCL5 enhanced the invasion of ESCC cells using the Transwell assay. The upregulation of CCL5 protein was also detected in 50% of ESCC cell lines. CCL5 was also overexpressed in 76.9% of ESCC specimens. The overall results indicated that 91b1 could effectively induce anticancer effect on ESCC cells through downregulating CCL5 expression with suppression of tumor invasion. Overall, these findings suggested that 91b1 effectively inhibited ESCC cell proliferation and tumor invasion by downregulating CCL5 expression, highlighting its potential as a therapeutic agent for ESCC treatment.

Wnt/B-catenin activation and TP53 mutations associate with distinct immune profiles in advanced thyroid cancer.

Anaplastic thyroid carcinomas (ATCs) and poorly differentiated thyroid carcinomas (PDTCs) exhibit distinct immune-related gene expression profiles. Most ATCs are characterized by active immune interactions (hot or altered immunosuppressed immunophenotypes), while PDTCs are largely immunologically inert (cold immunophenotypes). This study aimed to elucidate the mechanisms driving these divergent immunological fates, focusing on the Wnt/β-catenin pathway and TP53 mutations. Our data reveal that ATCs frequently harbor TP53 mutations (83.3%), which correlate with a hot immunophenotype, characterized by high expression of β-catenin-regulated cytokine CCL4 and recruitment of CD103+ dendritic cells. Conversely, PDTCs, with a lower incidence of TP53 mutations (12.5%), often exhibit a cold immunophenotype. In cold cancers and PDTCs, β-catenin is overexpressed suggesting that Wnt/β-catenin pathway activation drives immune exclusion through CCL4 downregulation.Further analysis indicated that loss of p53 function is inversely correlated with β-catenin expression. P53-mutated cancers showed significantly higher expression of CCL4 and densities of CD103+ dendritic cells compared to their p53-wild-type counterparts. Additionally, p53-mutated ATCs expressed a higher number of immune-related genes, supporting the role of p53 loss in activating immune responses in cancer. Our study indicates a potential correlation between the activation of the Wnt/β-catenin pathway and the development of cold thyroid cancers, which may be mediated by the suppression of CCL4 expression. Concurrently, mutations in the p53 gene appear to be linked with the occurrence of hot thyroid cancers. While these associations are compelling, they are based on observational data. Experimental research is necessary to determine the causal relationships underlying these findings.

Blockage of ATGL-mediated breakdown of lipid droplets in microglia alleviates neuroinflammatory and behavioural responses to lipopolysaccharides

Lipid droplets (LD) are triglyceride storing organelles that have emerged as an important component of cellular inflammatory responses. LD lipolysis via adipose triglyceride lipase (ATGL), the enzyme that catalyses the rate-limiting step of triglyceride lipolysis, regulates inflammation in peripheral immune and non-immune cells. ATGL elicits both pro- and anti-inflammatory responses in the periphery in a cell-type dependent manner. The present study determined the impact of ATGL inhibition and microglia-specific ATGL genetic loss-of-function on acute inflammatory and behavioural responses to pro-inflammatory insult. First, we evaluated the impact of lipolysis inhibition on lipopolysaccharide (LPS)-induced expression and secretion of cytokines and phagocytosis in mouse primary microglia cultures. Lipase inhibitors (ORlistat and ATGListatin) and LPS led to LD accumulation in microglia. Pan-lipase inhibition with ORlistat alleviated LPS-induced expression of IL-1β and IL-6. Specific inhibition of ATGL had a similar action on CCL2, IL-1β and IL-6 expression in both neonatal and adult microglia cultures. CCL2 and IL-6 secretion were also reduced by ATGListatin or knockdown of ATGL. ATGListatin increased phagocytosis in neonatal cultures independently from LPS treatment. Second, targeted and untargeted lipid profiling revealed that ATGListatin reduced LPS-induced generation of pro-inflammatory prostanoids and modulated ceramide species in neonatal microglia. Finally, the role of microglial ATGL in neuroinflammation was assessed using a novel microglia-specific and inducible ATGL knockout mouse model. Loss of microglial ATGL in adult male mice dampened LPS-induced expression of IL-6 and IL-1β and microglial density. LPS-induced sickness- and anxiety-like behaviours were also reduced in male mice with loss of ATGL in microglia. Together, our results demonstrate potent anti-inflammatory effects produced by pharmacological or genetic inhibition of ATGL-mediated triglyceride lipolysis and thereby propose that supressing microglial LD lipolysis has beneficial actions in acute neuroinflammatory conditions.

CCL4 as a potential serum factor in differential diagnosis of central nervous system inflammatory diseases and gliomas.

Computed tomography (CT) scans and magnetic resonance imaging (MRI) are commonly utilized to detect brain gliomas and central nervous system inflammation diseases. However, there are instances where depending solely on medical imaging for a precise diagnosis may result in unsuitable medications or treatments. Pathological analysis is regarded as the definitive method for diagnosing brain gliomas or central nervous system inflammation diseases. To achieve this, a craniotomy or stereotaxic biopsy is necessary to collect brain tissue, which can lead to complications such as cerebral hemorrhage, neurological deficits, cerebrospinal fluid leaks, and cerebral edema. Consequently, the advancement of non-invasive or minimally invasive diagnostic techniques is currently a high priority. This study included samples from four glioma patients and five patients with central nervous system inflammatory diseases, comprising both serum and paired cerebrospinal fluid (CSF). A total of 40 human cytokines were identified in these samples. We utilized a receiver operating characteristic (ROC) analysis to assess the sensitivity and specificity for distinguishing central nervous system inflammation diseases and gliomas. Additionally, we examined the correlation of these factors between serum and CSF in the patients. Ultimately, the identified factors were validated using serum from patients with clinically confirmed gliomas and central nervous system inflammation diseases followed by detection and statistical analysis through ELISA. The levels of serum factors IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, CCL11, and VEGF were found to be significantly higher in gliomas compared with inflammatory diseases of the central nervous system (p < 0.05). Furthermore, a strong correlation was observed between the levels of CCL4 in serum and CSF, with a correlation coefficient of r = 0.92 (95% CI = 0.20-0.99, p = 0.027). We gathered more clinical samples to provide further validation of the abundance of CCL4 expression. A clinical study analyzing serum samples from 19 glioma patients and 22 patients with central nervous system inflammation diseases revealed that CCL4 levels were notably elevated in the inflammatory group compared with the glioma group (p < 0.001). These results suggest that assessing serum CCL4 levels may be useful in distinguishing those patients for clinical diagnostic purposes.

G6pdN126D Variant Increases the Risk of Developing VEGFR (Vascular Endothelial Growth Factor Receptor) Blocker-Induced Pulmonary Vascular Disease.

G6PD (glucose-6-phosphate-dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension-associated vascular remodeling. Here, we investigated the role of an X-linked G6pd mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub-Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer. CRISPR-Cas9 genome editing was used to generate the G6pd variant (N126D; G6pdN126D) in rats. A single dose of the vascular endothelial cell growth factor receptor blocker sugen-5416 (SU; 20 mg/kg in DMSO), which is currently in a Phase 2/3 clinical trial for cancer treatment, was subcutaneously injected into G6pdN126D rats and their wild-type littermates. After 8 weeks of normoxic conditions, right ventricular pressure and hypertrophy, pulmonary artery remodeling, the metabolic profile, and cytokine expression were assessed. Right ventricular pressure and pulmonary arterial wall thickness were increased in G6PDN126D+SU/normoxic rats. Simultaneously, levels of oxidized glutathione, inositol triphosphate, and intracellular Ca2+ were increased in the lungs of G6PDN126D+SU/normoxic rats, whereas nitric oxide was decreased. Also increased in G6PDN126D+SU/normoxic rats were pulmonary levels of plasminogen activator inhibitor-1, thrombin-antithrombin complex, and expression of proinflammatory cytokines CCL3 (chemokine [C-C motif] ligand), CCL5, and CCL7. Our results suggest G6PDN126D increases inositol triphosphate-Ca2+ signaling, inflammation, thrombosis, and hypertrophic pulmonary artery remodeling in SU-treated rats. This suggests an increased risk of vascular endothelial cell growth factor receptor blocker-induced pulmonary hypertension in those carrying this G6PD variant.

Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1+ monocytes-derived macrophages

ObjectiveWe assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc).MethodsAssociation of NCF1-H90 with SSc was performed in...

Fullerene and fullerene whisker are not carcinogenic to the lungs and pleura in rat long-term study after 2-week intra-tracheal intrapulmonary administration

Fullerene whiskers (FLW)s are thin rod-like structures composed of C60 and C70 fullerene (FL). The shape of FLWs suggests potential toxic effects including carcinogenicity to the lung and pleura, similar to effects elicited by asbestos and multi-walled carbon nanotubes (MWCNT)s. However, no long-term carcinogenic studies of FL or FLW have been conducted. In the present study we investigated the pulmonary and pleural carcinogenicity of FL and FLW. Twelve-week-old male F344 rats were administered 0.25 or 0.5 mg FL, FLW, MWCNT-7, and MWCNT-N by intra-tracheal intra-pulmonary spraying (TIPS). Acute lung lesions and carcinogenicity were analyzed at 1 and 104 weeks after 8 doses/15 days TIPS administration. At week 1, FLW, MWCNT-7, and MWCNT-N significantly increased alveolar macrophage infiltration. Expression of Ccl2 and Ccl3, reactive oxygen species production, and cell proliferation were significantly increased by administration of MWCNT-7 and MWCNT-N but not FL or FLW. At week 104, the incidence of bronchiolo-alveolar adenoma plus adenocarcinoma was significantly increased in the MWCNT-7 and MWCNT-N groups, and the incidence of mesothelioma was significantly increased in the MWCNT-7 group. No significant induction of pulmonary or pleural tumorigenesis was observed in the FL or FLW groups. The number of 8-OHdG-positive cells in the alveolar epithelium was significantly increased in the MWCNT-7 and MWCNT-N groups but not in the FL or FLW groups. FL and FLW did not exert pulmonary or pleural carcinogenicity in our study. In addition, oxidative DNA damage was implicated in MWCNT-induced lung carcinogenesis, suggesting that it may be a useful initial marker of carcinogenicity.

Ageing results in an exacerbated inflammatory response to LPS by resident lung cells

BackgroundAgeing is associated with an increased risk of lung infection and chronic inflammatory lung disease. Innate immune responses are the first line of defence in the respiratory tract, however, age-related changes to innate immunity in the lung are not fully described. Both resident haematopoietic cells, such as alveolar macrophages, and non-haematopoeitic cells, such as epithelial and endothelial cells can contribute to inflammatory and immune responses in the lung. In this study we aimed to determine the impact of ageing on early innate responses of resident cells in the lung.ResultsAged and young mice were inoculated intranasally with lipopolysaccharide (LPS). After 4 h, aged mice recruited higher numbers of neutrophils to the airways and lung. This exacerbated inflammatory response was associated with higher concentrations of chemokines CXCL1, CXCL2 and CCL2 in the airways. Next, precision cut lung slices (PCLS) were stimulated ex vivo with LPS for 16 h. Gene expression of Cxcl2, Tnf and Il1b were all higher in PCLS from aged than young mice and higher levels of secretion of CXCL2 and TNF were detected. To determine which lung cells were altered by age, LPS was intranasally administered to aged and young mice and individual populations of cells isolated by FACS. RT-PCR on sorted cell populations demonstrated higher expression of inflammatory cytokines Cxcl2, Ccl2 and Tnf in epithelial cells and alveolar macrophages and higher expression of Cxcl2 by endothelial cells of aged mice compared to young. These differences in expression of pro-inflammatory cytokines did not correspond to higher levels of Tlr4 expression.ConclusionsAgeing leads to a heightened neutrophilic inflammatory response in the lung after LPS exposure, and higher expression and production of pro-inflammatory cytokines by resident lung cells, including alveolar macrophages, epithelial cells and endothelial cells. The responses of multiple resident lung cell populations are altered by aging and contribute to the exacerbated inflammation in the lung following LPS challenge. This has implications for our understanding of respiratory infections and inflammation in older people.

Eravacycline improves the efficacy of anti-PD1 immunotherapy via AP1/CCL5 mediated M1 macrophage polarization in melanoma

Screening approved library is a promising and safe strategy to overcome the limitation of low response rate and drug resistance in immunotherapy. Accumulating evidence showed that the application of antibiotics has been considered to reduce the effectiveness of anti-PD1 immunotherapy in tumor treatment, however, in this study, an antibiotic drug (Eravacycline, ERV) was identified to improve the efficacy of anti-PD1 immunotherapy in melanoma through screening approved library. Administration of ERV significantly attenuated melanoma cells growth as well as directly or indirectly benefited M1 macrophage polarization. Meanwhile, ERV treatment significantly induced cellular autophagy via damage of mitochondria, leading to up-regulation of ROS production, subsequently, raised CCL5 secretion through elevation AP1 binding to CCL5 promoter via p38 or JNK1/2 activation. Knockdown of Ccl5 expression attenuated ERV triggered M1 macrophage polarization in melanoma cells. Clinical analysis revealed a positive association between high expression of CCL5 and improved prognosis as well as a favorable anti-PD1 therapy in melanoma patients. As expected, application of ERV improved the efficacy of anti-PD1. Overall, our results approved that ERV enhances the efficacy of anti-PD1 immunotherapy in melanoma by promoting the polarization of M1 macrophages, which provided novel therapeutic strategy for improving the effectiveness of melanoma anti-PD1 immunotherapy.

Single-cell analysis identifies distinct macrophage phenotypes associated with prodisease and proresolving functions in the endometriotic niche

Endometriosis negatively impacts the health-related quality of life of 190 million women worldwide. Novel advances in nonhormonal treatments for this debilitating condition are desperately needed. Macrophages play a vital role in the pathophysiology of endometriosis and represent a promising therapeutic target. In the current study, we revealed the full transcriptomic complexity of endometriosis-associated macrophage subpopulations using single-cell analyses in a preclinical mouse model of experimental endometriosis. We have identified two key lesion-resident populations that resemble i) tumor-associated macrophages (characterized by expression of Folr2, Mrc1, Gas6, and Ccl8+) that promoted expression of Col1a1 and Tgfb1 in human endometrial stromal cells and increased angiogenic meshes in human umbilical vein endothelial cells, and ii) scar-associated macrophages (Mmp12, Cd9, Spp1, Trem2+) that exhibited a phenotype associated with fibrosis and matrix remodeling. We also described a population of proresolving large peritoneal macrophages that align with a lipid-associated macrophage phenotype (Apoe, Saa3, Pid1) concomitant with altered lipid metabolism and cholesterol efflux. Gain of function experiments using an Apoe mimetic resulted in decreased lesion size and fibrosis, and modification of peritoneal macrophage populations in the preclinical model. Using cross-species analysis of mouse and human single-cell datasets, we determined the concordance of peritoneal and lesion-resident macrophage subpopulations, identifying key similarities and differences in transcriptomic phenotypes. Ultimately, we envisage that these findings will inform the design and use of specific macrophage-targeted therapies and open broad avenues for the treatment of endometriosis.

Pan-cancer analysis reveals CCL5/CSF2 as potential predictive biomarkers for immune checkpoint inhibitors

BackgroundCurrently, there are no optimal biomarkers available for distinguishing patients who will respond to immune checkpoint inhibitors (ICIs) therapies. Consequently, the exploration of novel biomarkers that can predict responsiveness to ICIs is crucial in the field of immunotherapy.MethodsWe estimated the proportions of 22 immune cell components in 10 cancer types (6,128 tumors) using the CIBERSORT algorithm, and further classified patients based on their tumor immune cell proportions in a pan-cancer setting using k-means clustering. Differentially expressed immune genes between the patient subgroups were identified, and potential predictive biomarkers for ICIs were explored. Finally, the predictive value of the identified biomarkers was verified in patients with urothelial carcinoma (UC) and esophageal squamous cell carcinoma (ESCC) who received ICIs.ResultsOur study identified two subgroups of patients with distinct immune infiltrating phenotypes and differing clinical outcomes. The patient subgroup with improved outcomes displayed tumors enriched with genes related to immune response regulation and pathway activation. Furthermore, CCL5 and CSF2 were identified as immune-related hub-genes and were found to be prognostic in a pan-cancer setting. Importantly, UC and ESCC patients with high expression of CCL5 and low expression of CSF2 responded better to ICIs.ConclusionWe demonstrated CCL5 and CSF2 as potential novel biomarkers for predicting the response to ICIs in patients with UC and ESCC. The predictive value of these biomarkers in other cancer types warrants further evaluation in future studies.

Hsa-miR-3928–3p targets the CCL3/CCR5 axis to induce amniotic epithelial cell senescence involved in labor initiation

IntroductionSenescence in human amniotic epithelial cells (hAECs) and increased sterile inflammation in the amniotic cavity can lead to the initiation of term labor (TL). We investigated the possible roles of hsa-miR-3928–3p and chemokine ligand 3 (CCL3) in labor initiation and the underlying molecular mechanisms. MethodsMicroarray chip screening was used to analyse the differential expression of miRNAs in amniotic fluid exosomes from women in TL and term not-in-labor. The GEO and miRWalk databases were used to identify differential genes, and a dual luciferase assay was used to verify the relationship. Reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence were used to determine the expression and localization of CCL3/CCR5 in fetal membranes. RT-qPCR and western blotting were used to detect the expression of CCL3/CCR5 in hAECs with hsa-miR-3928–3p knockdown/overexpression. Cell counting kit 8, flow cytometry, EdU proliferation, senescence-associated β-galactosidase, and enzyme-linked immunosorbent assays were performed to detect the impact of hsa-miR-3928–3p on hAEC function. Resultshsa-miR-3928–3p expression was downregulated in TL. CCL3 (macrophage inflammatory protein-1α) was identified as a differentially expressed target gene. hsa-miR-3928–3p targeted the 3′ UTR of CCL3. Downregulation of hsa-miR-3928–3p expression increased CCL3 expression. CCL3, via its CCR5 receptor, decreased the proliferation, but increased the senescence, apoptosis rate, secretion of inflammatory factors (IL-8, TNF-α, and IL-6), and expression of senescence-associated protein p21 in hAECs. Discussionhsa-miR-3928–3p negatively regulates CCL3, promoting hAEC senescence through the CCL3-CCR5 axis and inducing signals for labor initiation. These findings provide novel insights for labor initiation in clinical settings.

Secretome and immune cell attraction analysis of head and neck cancers

Immune checkpoint inhibitors are approved for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) but the response rate is only 13–18%. For an effective antitumor immune response, trafficking of immune cells to the tumor microenvironment (TME) is essential. We aimed to better understand immune cell migration as well as the involved chemokines in HNSCC. A transwell assay was used to study immune cell migration toward TME-conditioned medium. While T cell migration was not observed, conventional dendritic cell (cDC) migration was induced by TME-conditioned media. cDC migration correlated with various proteins in the TME secretome. CCL8, CXCL5, CCL13 and CCL7 were tested in validation experiments and addition of these chemokines induced cDC migration. Using single cell RNA-sequencing, we observed expression of CCL8, CXCL5, CCL13 and CCL7 in cancer-associated fibroblasts (CAFs). Depleting fibroblasts led to reduced cDC migration. Thus CAFs, while often seen as suppressors of antitumor immunity, play a role in attracting cDCs toward the head and neck cancer TME, which might be crucial for effective antitumor immunity and response to therapies. Indeed, we found RNA expression signatures of the indicated chemokines, cDC and CAF subpopulations, to be significantly higher in baseline tumor specimen of patients with a major pathological response to pre-surgical anti-PD-1 treatment compared to non-responding patients.

An atlas of genetic effects on cellular composition of the tumor microenvironment.

Deciphering the composition of the tumor microenvironment (TME) is critical for understanding tumorigenesis and to design immunotherapies. In the present study, we mapped genetic effects on cell-type proportions using single-cell and bulk RNA sequencing data, identifying 3,494 immunity quantitative trait loci (immunQTLs) across 23 cancer types from The Cancer Genome Atlas. Functional annotation revealed regulatory potential and we further assigned 1,668 genes that regulate TME composition. We constructed a combined immunQTL map by integrating data from European and Chinese colorectal cancer (CRC) samples. A polygenic risk score that incorporates these immunQTLs and hits on a genome-wide association study outperformed in CRC risk stratification within 447,495 multiethnic individuals. Using large-scale population cohorts, we identified that the immunQTL rs1360948 is associated with CRC risk and prognosis. Mechanistically, the rs1360948-G-allele increases CCL2 expression, recruiting regulatory T cells that can exert immunosuppressive effects on CRC progression. Blocking the CCL2-CCR2 axis enhanced anti-programmed cell death protein 1 ligand therapy. Finally, we have established a database (CancerlmmunityQTL2) to serve the research community and advance our understanding of immunogenomic interactions in cancer pathogenesis.

Network Pharmacology and Experimental Analysis to Explore the Effect and Mechanism of Modified Buyang Huanwu Decoction in the Treatment of Diabetic Nephropathy.

Preventing and treating diabetic nephropathy (DN) are global challenges due to the complexity and diversity of its causes and manifestations. It is important to find effective medications to treat DN. Gene expression files of DN were downloaded from the GEO database to identify the differentially expressed genes. Network pharmacology and molecular docking were used to explore the possible mechanisms of modified Buyang Huanwu Decoction (mBHD) in treating DN. Biochemical, histopathological, and real-time PCR analyses were conducted in both in vivo and in vitro DN models to investigate the effects of mBHD. A total of 336 active ingredients and 124 potential targets of mBHD associated with DN were identified. Among them, 8 hub genes were found to be important targets for mBHD in treating DN and were significantly correlated with the infiltration status of six immune cells. Partially, the active ingredients of mBHD demonstrated good stability in binding to CASP3 and TP53. mBHD treatment significantly reduced levels of total cholesterol, triglyceride, blood urea nitrogen, serum creatinine, and microalbumin in db/db mice. HE and Masson's staining results showed that mBHD attenuated renal injury in db/db mice. Additionally, mBHD treatment could significantly alter the expression of CASP3, CCL2, TP53, ALB, and HMOX1. mBHD may be involved in the treatment of DN through multiple ingredients, targets, and pathways. In addition, mBHD could alleviate renal injury in db/db mice, possibly involving CASP3, CCL2, TP53, ALB, and HMOX1.