CCL20 Expression Research Articles

Expression of key immune genes in polarized porcine monocyte-derived macrophage subsets

Swine are considered one of the most relevant large animal biomedical models since they share many immunological similarities with humans. Despite that, macrophage polarization has not comprehensively investigated in pigs. In this study, porcine monocyte-derived macrophages (moMΦ) were untreated or stimulated with IFN-γ + LPS (classical activation), or by different M2 polarizing stimuli: IL-4, IL-10, TGF-β, or dexamethasone. Expression of key cytokine genes (IL1B2, IL33, IL19, IL22, IL26, CCL17, CCL24, IFNA, IFNB) in macrophage subsets were investigated over time. Expression of the genes encoding the two main enzymes of the arginine pathway (ARG1, NOS2), and molecules related to alternative macrophage polarization in human and mice (MMP9, MRC1, FIZZ1, VEGFA) were also assessed. Stimulation with IFN-γ + LPS triggered up-regulation of IL1B2, IFNB, NOS2, whereas IL-4 triggered upregulation of CCL17, CCL24, CXCR2, and ARG1 expression. IL19 and IL22 expression was enhanced by stimulation with IFN-γ + LPS or TGF-β, but not IL-4, IL-10, or dexamethasone. Our data highlighted some peculiarities in swine, such as induced expression of IL33 after stimulation with IFN-γ + LPS, and no up-regulation of FIZZ1, VEGFA or MMP9 after exposure to any of the M2 polarizing stimuli. A better understanding of porcine macrophage polarization could benefit translational studies using this large animal model.

Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage.

The skin microenvironment created by keratinocytes (KC) influences the stress responses of melanocytes (MC) to UVB insults. This study employed RNA sequencing analysis as well as in vitro and in vivo models to elucidate the underlying mechanisms. Our RNA-Seq analysis revealed a statistically significant upregulation of GCSF and CCL20 genes in UVB-irradiated KC, correlating with the protective effects of KC on MC responses to UVB exposure. Recombinant GCSF and CCL20 exhibited the most pronounced modulation of UVB-induced MC responses. These effects included the attenuation of apoptosis and reduction of ROS formation, along with the upregulation of tyrosinase and tyrosinase-related protein-1, which are involved in the melanogenic pathway. ELISA was also used to confirm that UVB could induce the secretion of GCSF and CCL20 from KC. A similar correlation between GCSF and CCL20 expression in KC and tyrosinase levels in MC was observed in UVB-irradiated mouse skin. Our study provides novel insights into the protective role of GCSF and CCL20 in the paracrine effects of KC on UVB-induced MC damage through the modulation of stress response pathways, the MITF-tyrosinase axis, and the regulation of p53. These findings have implications for the development of pharmacological strategies targeting KC-derived paracrine factors for the prevention of skin photodamage.

Loss of CCL28 and CXCL17 Expression and Increase in CCR1 Expression May Be Related to Malignant Transformation of LGBLEL into Lymphoma.

To investigate the differential expression of the chemokine signaling pathway in lacrimal gland benign lymphoepithelial lesion (LGBLEL) and lacrimal lymphoma, providing insights into the mechanisms underlying malignant transformation and aiding clinical differentiation. Transcriptome analysis was conducted on patients with LGBLEL, lymphoma, and orbital cavernous hemangioma (CH). Three cases of LGBLEL and three cases of lymphoma were randomly selected as control and experimental groups, respectively. A real-time quantitative polymerase chain reaction (RT-qPCR) was used to validate genes associated with the chemokine signaling pathway. Immunohistochemical (IHC) staining and quantitative Western blotting (WB) were performed for precise protein quantification. Transcriptome analysis revealed differential expression of the chemokine signaling pathway between the LGBLEL and lymphoma groups, identifying ten differentially expressed genes: CCL17, VAV2, CXCR5, NRAS, HCK, RASGRP2, PREX1, GNB5, ADRBK2, and CCL22. RT-qPCR showed that, compared to the lymphoma group, the LGBLEL group had significantly higher expression of CCL28, CXCL17, HCK, GNB5, NRAS, and VAV2 (p = 0.001, <0.001, <0.001, <0.001, =0.020, <0.001, respectively) and lower expression of CCR1 (p = 0.002). IHC staining and quantitative analysis confirmed significant differences in protein expression between the groups for CCL28, CCR1, CXCL17, HCK, GNB5, NRAS, and VAV2 (p = 0.003, 0.011, 0.001, 0.024, 0.005, 0.019, and 0.031, respectively). While IHC provided localization, WB offered greater precision. WB revealed that, compared to the lymphoma group, the LGBLEL group exhibited significantly higher expression of CCL28, CXCL17, HCK, GNB5, NRAS, and VAV2 (p = 0.012, 0.005, 0.009, 0.011, 0.008, and 0.003, respectively) and lower expression of CCR1 (p = 0.014). The chemokine signaling pathway plays a role in the malignant transformation of LGBLEL. The decreased expression of CCL28 and CXCL17, coupled with the increased expression of CCR1, may be linked to the progression of LGBLEL into lymphoma.

CKAP2 Regulated by TFDP1 Promotes Metastasis and Proliferation of Colorectal Cancer through Affecting the Tumor Microenvironment.

The current pathological and physiological evaluation system for colorectal cancer (CRC) is limited; thus, effective biological targets to diagnose and treat this disease are urgently needed. In this study, we used qRT-PCR for detecting mRNA levels of genes. The levels of protein were identified by western blot, immunohistochemistry, and immunofluorescence assays. In addition, functional experiments were used to evaluate the role of CKAP2 in CRC cells and HUVECs. Bioinformatics analysis was employed to predict the binding relationship of CKAP2 and TFDP1, which was confirmed through dual luciferase reporter assay and immunoprecipitation assay. Furthermore, we injected HCT116 cells into mice flanks, and we injected Luciferase-labeled HCT116 cells into mice tail vein. HE staining was used to detect tumor nodules. As a result, high CKAP2 expression was found in CRC cells and tissues. CKAP2 silencing reduced CRC cell migration, invasion, proliferation, and epithelial-mesenchymal transition. Moreover, CKAP2 expression was positively associated with M2 macrophage level. CKAP2 promoted protein expression of CD86, CD206, IL-1β, and CCL17. Moreover, CKAP2 promoted the proliferation of HUVECs and angiogenesis via affecting the tumor microenvironment (TME). We also found that CKAP2 could interact with TFDP1. The inhibitory impacts of TFDP1 downregulation on CRC cell' proliferation, migration, and invasion were reversed via CKAP2 overexpression. In vivo silencing of CKAP2 repressed tumor growth and metastasis. Overall, CKAP2 was positively regulated by TFDP1, which promoted tumorigenesis and metastasis in CRC.

Brainstem transcriptomic changes in male Wistar rats after acute stress, comparing the use of duplex specific nuclease (DSN)

In this work, we have analyzed the transcriptomic changes in the brainstem of male Wistar rats 2 h after an acute stress exposure. We performed duplex-specific nuclease normalization of cDNA libraries and compared the results back-to-back for the first time. Based on our RNAseq data, we selected reference genes for RT-qPCR that are best suited for acute stress experiments. Most genes were upregulated. We detected a massive shift in neuropeptide Crh, Trh,Cga, Tshb, Uts2b, Tac4, Lep and neuropeptide receptor Hcrtr1, Sstr5, Bdkrb2, Crhr2 signaling, as well as glutamate Grin3b, Grm2 and GABA Gpr156, acetylcholine Chrm4,Chrne, adrenergic Adra2b receptors expression. A strong increase in the expression of intermediate filaments Krt83/Krt86/Krt80/Krt84/Krt87/Krt4/Krt76 and motor proteins Myo7a, Klc3 was detected. Remarkably, in the absence of astrocyte activation, we also observed signs of microglial activation at this time point. Both expression of anti-inflammatory cytokines Il13, Ccl24 and pro-inflammatory cytokine receptors Il9r, Il12rb1, Tnfrsf14, Tnfrsf13c, Tnfrsf25, Tnfrsf1b were increased. In the Wnt signaling pathway, we observed increased expression of ligands-receptors Wnt1, Wnt11, Ror2 and also negative regulators Notum, Sfrp5, Sost. RNAseq results after DSN treatment correlated at a high level with RNAseq results without DSN, but there was a proportion of genes that shifted their logFC values. They are mostly rare transcripts TPM 1–10 with higher 0.5–0.9 GC content.

Construction of circRNA-mediated ceRNA network and immunoassay for investigating pathogenesis of COPD.

The chronic respiratory condition known as chronic obstructive pulmonary disease (COPD) was one of the main causes of death and disability worldwide. This study aimed to explore and elucidate new targets and molecular mechanisms of COPD by constructing competitive endogenous RNA (ceRNA) networks. GSE38974 and GSE106986 were used to select DEGs in COPD samples and normal samples. Cytoscape software was used to construct and present protein-protein interaction (PPI) network, mRNA-miRNA co-expression network and ceRNA network. The CIBERSORT algorithm and the Lasso model were used to screen the immune infiltrating cells and hub genes associated with COPD, and the correlation between them was analyzed. COPD cell models were constructed in vitro and the expression level of ceRNA network factors mediated by hub gene was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In this study, 852 differentially expressed genes were screened in the GSE38974 dataset, including 439 upregulated genes and 413 downregulated genes. Gene clustering analysis of PPI network results was performed using the Minimum Common Tumor Data Element (MCODE) in Cytoscape, and seven hub genes were screened using five algorithms in cytoHubba. CCL20 was verified as an important hub gene based on mRNA-miRNA co-expression network, GSE106986 database validation and the analysis of ROC curve results. Finally, we successfully constructed the circDTL-hsa-miR-330-3p-CCL20 network by Cytoscape. Immune infiltration analysis suggested that CCL20 can co-regulate immune cell migration and infiltration through chemokines CCL7 and CXCL3. In vitro experiments, the expression of circDTL and CCL20 was increased, while the expression of hsa-miR-330-3p was decreased in the COPD cell model. By constructing the circDTL-hsa-miR-330-3p-CCL20 network, this study contributes to a better understanding of the molecular mechanism of COPD development, which also provides important clues for the development of new therapeutic strategies and drug targets.

The mechanism by which intestinal flora metabolites regulate ILC2s transformation and intestinal immunity through the metabolite-sensitive receptor Ffar2.

The objective of this article was to investigate how the metabolites produced by the intestinal flora regulate the transformation of ILC2s and intestinal immunity via the receptor Ffar2, which is sensitive to metabolites. Forty male C57BL/6 mice with wild-type characteristics (6-7 weeks in age) and 20 FFAR2-/- mice were acquired from The Jackson Laboratory. The mice were kept in a controlled environment without any disease-causing agents, with the help of air conditioning and a 12-hour cycle of light and darkness. Throughout the experiment, every mouse was provided with unrestricted availability of both food and water. All protocols were performed following the Regulations of Animal Welfare and the recommendations for Animal Testing. According to the research protocol, the mice were categorized into three groups, each consisting of 20 mice. FFAR2-/- mice in the FFAR2-/- group were reared in conventional environments. Male C57BL/6 mice with wild-type characteristics were reared in conventional conditions in the wild-type group. In the group where wild-type mice were inhibited with Ffar2, a total of 20 mice with the wild-type phenotype were chosen to receive intraperitoneal injection of an FFAR2 antagonist at a dosage of 5 mg/kg per day for a continuous period of 3 weeks. qRT-PCR was used to detect the expression of FFAR2 mRNA in the mucosal tissue of the mouse colon. High-throughput sequencing was used to conduct an examination of metabolites in the intestinal tract of mice. Flow cytometry was used to test the quantity of ILC2s. ELISA was used to measure the levels of IL-5 and IL-13 in cellular mouse intestinal mucosal tissues. Flow cytometry was used to detect the quantity of CD4+ and CD8+ T cells. CCL20 and CCL25 expression was analyzed by Western blotting. The level of FFAR2 mRNA expression was higher in the wild-type group than the FFAR2-/- group (P < 0.05), while it was lower in the wild-type + Ffar2 inhibition group than the wild-type group (P < 0.05). No variations were observed in the composition of metabolites and levels of major SCFAs in the gut microbiota of mice across all groups (P > 0.05). The quantity of CRTH2+ and ST2+ cells in the wild-type category exceeded that in the FFAR2-/- category (P < 0.05), whereas the quantity of CRTH2+ and ST2+ cells in the wild-type + Ffar2 inhibition category was lower than that in the wild-type category (P < 0.05). The concentrations of IL-5 and IL-13 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), while the wild-type + Ffar2 inhibition group exhibited lower levels of IL-5 and IL-13 than the wild-type group (P < 0.05). The count of CD4+ T cells and CD8+ T cells in the wild-type group showed an increase in comparison to the FFAR2-/- group (P < 0.05), whereas the count of CD4+ T cells and CD8+ T cells in the wild-type + Ffar2 inhibition group exhibited a decrease in comparison to the wild-type group (P < 0.05). The levels of protein expression for CCL20 and CCL25 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), whereas the wild-type + Ffar2 inhibition group exhibited lower protein expression of CCL20 and CCL25 than the wild-type group (P < 0.05). Ffar2 has a significant regulatory function in the conversion of ILC2s, consequently impacting the immune response in the intestines. Ffar2, a crucial receptor for metabolites produced by the intestinal flora, plays a vital function in controlling the conversion of ILC2s and the overall immune response in the intestines.

Evaluation of the CCL17/CCL22-CCR4 axis in pediatrics with B-cell acute lymphoblastic leukemia before and after a chemotherapy course

AimsAcute lymphoblastic leukemia (ALL) is the most common type of pediatrics cancer. Chemokines exert different roles in leukemia process through leukocyte recruitment and regulation of disease severity. Due to the prominent roles of chemokine/receptor axes, this study aimed to measure the blood expression levels of CCR4 and their ligands in pediatrics with B-cell ALL (B-ALL). We also evaluated the impact of cytotoxic chemotherapy on this axis. Material and MethodThirty children suffering from B-ALL were included in the study and followed up for 30 days after completion of a chemotherapy course. The blood sampling was performed before and after chemotherapy. 30 healthy donors have also entered the study as control subjects. The mRNA expression of CCL17, CCL22 and CCR4 genes was determined by quantitative real-time PCR. The frequency of the peripheral blood mononuclear cells expressing CCR4 (CCR4 + PBMCs) was also evaluated by the flow cytometry method. Moreover, we evaluated the association of the CCL17/CCL22-CCR4 axis with some diagnostic, prognostic and predictive biomarkers in ALL patients. ResultsThere was overexpression of the CCL17/CCL22-CCR4 axis along with lactate dehydrogenase (LDH) in pediatrics with B-ALL compared to healthy controls. After induction of chemotherapy, the blood expression levels of the CCL17/CCL22-CCR4 axis have reached the levels of healthy controls. The findings for the blood expression levels of CCR4 were also confirmed using flow cytometry. ConclusionThe CCL17/CCL22-CCR4 axis can be used as a novel predictive and prognostic biomarker in B-ALL.

Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide.

This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues.

Nasal vaccination elicits a humoral immune response that provides protection from airborne pathogens1, yet the origins and specific immune niches of antigen-specific IgA-secreting cells in the upper airways are unclear2. Here we define nasalglandular acinar structures and the turbinates as immunological niches that recruit IgA-secreting plasma cells from the nasal-associated lymphoid tissues (NALTs)3. Using intact organ imaging, we demonstrate that nasal vaccination induces B cell expansion in the subepithelial dome of the NALT, followed by invasion into commensal-bacteria-driven chronic germinal centres in a T cell-dependent manner. Initiation of the germinal centre response in the NALT requires pre-expansion of antigen-specific T cells, which interact with cognate B cells in interfollicular regions. NALT ablation and blockade of PSGL-1, which mediates interactions with endothelial cell selectins, demonstrated that NALT-derived IgA-expressing B cells home to the turbinate region through the circulation, where they are positioned primarily around glandular acinar structures. CCL28 expression was increased in the turbinates in response to vaccination and promoted homing of IgA+ B cells to this site. Thus, in response to nasal vaccination, the glandular acini and turbinates provide immunological niches that host NALT-derived IgA-secreting cells. These cellular events could be manipulated in vaccine design or in the treatment of upper airway allergic responses.

CCL28 promotes progression of hepatocellular carcinoma through PDGFD-regulated MMP9 and VEGFA pathways

Hepatocellular carcinoma (HCC) remains a major global health concern with limited therapeutic options and poor prognosis. Chemokines have emerged as critical regulators in the progression and metastasis of HCC. This study aims to investigate the mechanisms involved in CCL28-promoted progression of HCC and provide novel therapeutic targets for HCC treatment. Relationship between CCL28 expression and HCC progression were investigated by bioinformatic analysis and immunohistochemical staining assays. CCK-8, Transwell, and colony formation assay were conducted to explore the impact of CCL28 on the growth, migration and invasion of HCC cells. Quantitative real-time PCR and western blotting assays were performed to learn potential molecular mechanisms underlying the transformation of HCC driven by CCL28. The results showed that there was a direct link between increased CCL28 levels and the advancement of HCC, leading to a worse outcome. CCL28 significantly augmented malignant transformation of HCC cells, containing proliferation, migration, invasion, and clonogenicity, via activation of PDGFD-regulated MMP9 and VEGFA pathways. CCL28 emerges as a pivotal contributor to HCC tumorigenesis, propelling HCC development through the PDGFD signaling pathway. Our findings unveil potential therapeutic targets for HCC treatment.

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

BackgroundIt has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure.MethodOverexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC–MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter.ResultsOur study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells.ConclusionWe reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

Evaluation of the activity of cardiac glycosides on RORγ and RORγT nuclear receptors

Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium–potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on RORγ and RORγT. RORγT is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on RORγ, as confirmed in the RORγ-HepG2 reporter cell line. The overexpression of both the RORγ and RORγT isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by RORγ, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the RORγ/RORγT receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on RORγ/RORγT nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.

Activation of the α7nAChR by GTS-21 mitigates septic tubular cell injury and modulates macrophage infiltration

The most common and serious complication among hospitalized and critically ill patients is sepsis-associated acute kidney damage (S-AKI), which raises the risk of comorbidities and is linked to a high mortality rate. Cholinergic anti-inflammatory pathway (CAP), an anti-inflammatory pathway mediated by the vagus nerve, acetylcholine, and α7 nicotinic acetylcholine receptors (α7nAChRs), offers new perspectives for the treatment of S-AKI. In this study, we investigated the role of CAP and α7nAChR in kidney injury by employing an LPS-induced septic kidney injury mouse model and GTS-21 intervention. C57BL/6 mice were injected with LPS, with or without GTS-21, in different subgroups. Kidney function was assessed by plasma creatinine, histology, and markers of kidney injury 24 h after intervention. The results demonstrated that GTS-21 could inhibit the systemic inflammatory response and directly protect the tubular cell injury from LPS. To explore the novel gene involved in this response, RNA sequencing of the renal proximal tubular epithelial cell (HK-2), pretreated with LPS and GTS-21, was conducted. The results indicate that GTS-21 administration reduces LPS-induced cytokines and chemokines secretion by HK-2, including CCL20, a potent chemokine attracting monocytes/macrophages. Furthermore, a macrophage transmigration assay revealed that GTS-21 inhibits macrophage transmigration by downregulating the expression of CCL20 in HK-2 cells. In conclusion, GTS-21, as an α7nAChR agonist, emerges as a noteworthy and versatile treatment for S-AKI. Its dual function of directly protecting renal tubular cells and regulating inflammatory responses represents a major advancement in the treatment of sepsis-induced AKI. This finding might pave the way for novel approaches to improving patient outcomes and reducing death rates in sepsis-related complications.

Enhancing hepatocellular carcinoma management: prognostic value of integrated CCL17, CCR4, CD73, and HHLA2 expression analysis

PurposeHepatocellular carcinoma (HCC) is a critical global health concern, with existing treatments benefiting only a minority of patients. Recent findings implicate the chemokine ligand 17 (CCL17) and its receptor CCR4 as pivotal players in the tumor microenvironment (TME) of various cancers. This investigation aims to delineate the roles of CCL17 and CCR4 in modulating the tumor’s immune landscape, assessing their potential as therapeutic interventions and prognostic markers in HCC.Methods873 HCC patients post-radical surgery from 2008 to 2012 at Zhongshan Hospital, Fudan University were retrospectively examined. These individuals were stratified into a training cohort (n = 354) and a validation cohort (n = 519). Through immunohistochemical analysis on HCC tissue arrays, the expressions of CCL17, CCR4, CD73, CD47, HHLA2, and PD-L1 were quantified. Survival metrics were analyzed using the Cox model, and a prognostic nomogram was devised via R software.ResultsThe investigation confirmed the presence of CCL17 and CCR4 within the cancerous and stromal compartments of HCC tissues, associating their heightened expression with adverse clinical markers and survival outcomes. Notably, the interplay between CD73 and CCR4 expression in tumor stroma highlighted a novel cellular entity, CCR4 + CD73 + stromal cells, impacting overall and relapse-free survival. A prognostic nomogram amalgamating these immunological markers and clinical variables was established, offering refined prognostic insights and aiding in the management of HCC. The findings suggest that reduced CCR4 and CCR4 + CD73 + cell prevalence may forecast improved outcomes post-TACE.ConclusionThis comprehensive evaluation of CCR4, CCL17, and associated markers introduces a nuanced understanding of the HCC immunological milieu, proposing CCR4 + CD73 + stromal cells as critical to HCC pathogenesis and patient stratification.

Nicotiana benthamiana-derived dupilumab-scFv reaches deep into the cultured human nasal epithelial cells and inhibits CCL26 expression

Plants offer a cost-effective and scalable pharmaceutical platform devoid of host-derived contamination risks. However, their medical application is complicated by the potential for acute allergic reactions to external proteins. Developing plant-based protein therapeutics for localized diseases with non-invasive treatment modalities may capitalize on the benefits of plant proteins while avoiding their inherent risks. Dupilumab, which is effective against a variety of allergic and autoimmune diseases but has systemic responses and injection-related side effects, may be more beneficial if delivered locally using a small biological form. In this study, we engineered a single-chain variable fragment (scFv) of dupilumab, termed Dup-scFv produced by Nicotiana benthamiana, and evaluated its tissue permeability and anti-inflammatory efficacy in air–liquid interface cultured human nasal epithelial cells (HNECs). Despite showing 3.67- and 17-fold lower binding affinity for IL-4Ra in surface plasmon resonance assays and cell binding assays, respectively, Dup-scFv retained most of the affinity of dupilumab, which was originally high, with a dissociation constant (KD) of 4.76 pM. In HNECs cultured at the air–liquid interface, Dup-scFv administered on the air side inhibited the inflammatory marker CCL26 in hard-to-reach basal cells more effectively than dupilumab. In addition, Dup-scFv had an overall permeability of 0.8% across cell layers compared to undetectable levels of dupilumab. These findings suggest that plant-produced Dup-scFv can be delivered non-invasively to cultured HNESc to alleviate inflammatory signaling, providing a practical approach to utilize plant-based proteins for topical therapeutic applications.

CCL17 and CCL19 are markers of disease progression in alveolar echinococcosis

ObjectivesAlveolar echinococcosis (AE) represents one of the deadliest helminthic infections, characterized by an insidious onset and high lethality. MethodsThis study utilized the Gene Expression Omnibus (GEO) database, applied Weighted Correlation Network Analysis (WGCNA) and Differential Expression Analysis (DEA), and employed the Matthews Correlation Coefficient (MCC) to identify CCL17 and CCL19 as key genes in AE. Immunohistochemistry and immunofluorescence co-localization techniques were used to examine the expression of CCL17 and CCL19 in liver tissue lesions of AE patients. Additionally, a mouse model of multilocular echinococcus larvae infection was developed to study the temporal expression patterns of these genes, along with liver fibrosis and inflammatory responses. ResultsThe in vitro model simulating echinococcal larva infection mirrored the hepatic microenvironment post-infection with multilocular echinococcal tapeworms. Quantitative RT-PCR analysis showed that liver fibrosis occurred in AE patients, with proximal activation and increased expression of CCL17 and CCL19 over time post-infection. Notably, expression peaked during the late stages of infection. Similarly, F4/80, a macrophage marker, exhibited corresponding trends in expression. Upon stimulation of normal hepatocytes by vesicular larvae in cellular experiments, there was a significant increase in CCL17 and CCL19 expression at 12 h post-infection, mirroring the upregulation observed with F4/80. ConclusionCCL17 and CCL19 facilitate macrophage aggregation via the chemokine pathway and their increased expression correlates with the progression of infection, suggesting their potential as biomarkers for AE progression.

Fusobacterium nucleatum to promote esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of macrophages to the tumour micro environment.

e14668 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignant tumor in China. Recent studies have shown that Fusobacterium nucleatum ( F. nucleatum) promotes ESCC progression through various mechanisms. One of them is about changing the tumor micro- environment through cytokine–cytokine receptor interaction. Methods: From January 2022 to June 2023, a total of 34 patients with ESCC from 4th Hospital of Hebei Medical University thoracic surgery were enrolled. All of the patients did not apply any neoadjuvant therapy before and received radical resection. Real-time reverse transcriptase - PCR (RT-PCR) were performed to examine the expressions of F. nucleatum in tumor tissues and para-tumor tissue. According to the CT values of the RT-PCR results, the level of the F. nucleatum infection could be separated in two groups - positive group and negative group. Immunohistochemistry (IHC) staining were used to examine the expressions of chemokine CCL20 in both groups. Immunofluorescence (IF) was characterised homing receptors CCR6 on CD206+ macrophages. In addition, transwell assay was carried on to quantify macrophages migration ability towards chemokines CCL20 in vitro. Results: F. nucleatum DNA positivity was significantly associated with higher expression of chemokine CCL20 and accumulation of CD68+CD206+ macrophages. F. nucleatum DNA positivity was also significantly correlated with worse histopathological grading. Otherwise, there were no significant correlation between F. nucleatum infection and gender, age, tobacco or alcohol use, T or N grade, TNM stage, tumor locations, blood vessel or nerve invasion and tumor size. IHC showed that the expression of CCL20 were higher in F. nucleatum DNA positive tumor tissue. After coculture with F. nucleatum and KYSE30 cell lines in vitro, the CCL20 production by KYSE30 cells were accelerated. In addition, immunofluorescence showed that CCL20 could recruit M2-like macrophages into the tumor environment by bonding CCR6 existed on the cell surface. Furthermore, the migration ability of macrophages was distinctly elevated by CCL20 result from F. nucleatum infection. Conclusions: F. nucleatum promotes esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of M2-like macrophages to the tumor micro- environment and deteriorates the suppression of the local immune micro- environment within the tumor. Such bacteria may be a biomarker to predict the efficacy of immunotherapy in ESCC. [Table: see text]

Lactate activates CCL18 expression via H3K18 lactylation in macrophages to promote tumorigenesis of ovarian cancer.

This study investigates the role of lactate in the genesis and progression of ovarian cancer (OV) and explores the underlying mechanisms. Serum lactate levels show a positive correlation with tumor grade and poor prognosis in patients with OV. Bioinformatics analysis identifies CCL18 as a lactate-related gene in OV. CCL18 is up-regulated in cancerous tissues and positively related to serum lactate levels in OV patients. THP-1 cells are exposed to phorbol-12-myristate-13-acetate for M0 macrophage induction. The results of RT-qPCR and ELISA for M1/M2 macrophage-related markers and inflammatory cytokines show that the exposure of lactate to macrophages induces M2 polarization. Based on the coculture of OV cells with macrophages, lactate-treated macrophages induces a significant increase in the proliferation and migration of OV cells. However, these effects can be reversed by silencing of Gpr132 in macrophages or treatment with anti-CCL18 antibody. Experiments using the xenograft model verify that the oncogenic role of lactate in tumor growth and metastasis relies on Gpr132 and CCL18. ChIP-qPCR and luciferase reporter assays reveal that lactate regulates CCL18 expression via H3K18 lactylation. In conclusion, lactate is a potential therapeutic target for OV. It is involved in tumorigenesis by activating CCL18 expression via H3K18 lactylation in macrophages.

Orally administered yeast-derived β-glucan alleviates mast cell-dependent airway hyperresponsiveness and inflammation in a murine model of asthma.

Particulate β-glucans (WGP) are natural compounds with regulatory roles in various biological processes, including tumorigenesis and inflammatory diseases such as allergic asthma. However, their impact on mast cells (MCs), contributors to airway hyperresponsiveness (AHR) and inflammation in asthma mice, remains unknown. C57BL/6 mice underwent repeated OVA sensitization without alum, followed by Ovalbumin (OVA) challenge. Mice received daily oral administration of WGP (OAW) at doses of 50 or 150 mg/kg before sensitization and challenge. We assessed airway function, lung histopathology, and pulmonary inflammatory cell composition in the airways, as well as proinflammatory cytokines and chemokines in the bronchoalveolar lavage fluid (BALF). The 150 mg/kg OAW treatment mitigated OVA-induced AHR and airway inflammation, evidenced by reduced airway reactivity to aerosolized methacholine (Mch), diminished inflammatory cell infiltration, and goblet cell hyperplasia in lung tissues. Additionally, OAW hindered the recruitment of inflammatory cells, including MCs and eosinophils, in lung tissues and BALF. OAW treatment attenuated proinflammatory tumor necrosis factor (TNF)-α and IL-6 levels in BALF. Notably, OAW significantly downregulated the expression of chemokines CCL3, CCL5, CCL20, CCL22, CXCL9, and CXCL10 in BALF. These results highlight OAW's robust anti-inflammatory properties, suggesting potential benefits in treating MC-dependent AHR and allergic inflammation by influencing inflammatory cell infiltration and regulating proinflammatory cytokines and chemokines in the airways.