The role of the immune system in early-onset schizophrenia: identifying immune characteristic genes and cells from peripheral blood.

BACKGROUND: Early-onset schizophrenia (EOS) is a type of schizophrenia (SCZ) with an age of onset of < 18 years. An abnormal inflammatory immune system may be involved in the occurrence and development of SCZ. We aimed to identify the immune characteristic genes and cells involved in EOS and to further explore the pathogenesis of EOS from the perspective of immunology. METHODS: We obtained microarray data from a whole-genome mRNA expression in peripheral blood mononuclear cells (PBMCs); 19 patients with EOS (age range: 14.79 ± 1.90) and 18 healthy controls (HC) (age range: 15.67 ± 2.40) were involved. We screened for differentially expressed genes (DEGs) using the Limma software package and modular genes using weighted gene co-expression network analysis (WGCNA). In addition, to identify immune characteristic genes and cells, we performed enrichment analysis, immune infiltration analysis, and receiver operating characteristic (ROC) curve analysis; we also used a random forest (RF), a support vector machine (SVM), and the LASSO-Cox algorithm. RESULTS: We selected the following immune characteristic genes: CCL8, PSMD1, AVPR1B and SEMG1. We employed a RF, a SVM, and the LASSO-Cox algorithm. We identified the following immune characteristic cells: activated mast cells, CD4+ memory resting T cells, resting mast cells, neutrophils and CD4+ memory activated T cells. In addition, the AUC values of the immune characteristic genes and cells were all > 0.7. CONCLUSION: Our results indicate that immune system function is altered in SCZ. In addition, CCL8, PSMD1, AVPR1B and SEMG1 may regulate peripheral immune cells in EOS. Further, immune characteristic genes and cells are expected to be diagnostic markers and therapeutic targets of SCZ.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy.

BACKGROUND: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated. METHODS: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining. RESULTS: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control. CONCLUSIONS: Hypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma.

During cancer evolution, tumor cells attract and dynamically interact with monocytes/macrophages. To find biomarkers of disease progression in human melanoma, we used unbiased RNA sequencing and secretome analyses of tumor-macrophage co-cultures. Pathway analysis of genes differentially modulated in human macrophages exposed to melanoma cells revealed a general upregulation of inflammatory hallmark gene sets, particularly chemokines. A selective group of chemokines, including CCL8, CCL15, and CCL20, was actively secreted upon melanoma-macrophage co-culture. Because we previously described the role of CCL20 in melanoma, we focused our study on CCL8 and CCL15 and confirmed that in vitro both chemokines contributed to melanoma survival, proliferation, and 3D invasion through CCR1 signaling. In vivo, both chemokines enhanced primary tumor growth, spontaneous lung metastasis, and circulating tumor cell survival and lung colonization in mouse xenograft models. Finally, we explored the clinical significance of CCL8 and CCL15 expression in human skin melanoma, screening a collection of 67 primary melanoma samples, using multicolor fluorescence and quantitative image analysis of chemokine-chemokine receptor content at the single-cell level. Primary skin melanomas displayed high CCR1 expression, but there was no difference in its level of expression between metastatic and nonmetastatic cases. By contrast, comparative analysis of these two clinically divergent groups showed a highly significant difference in the cancer cell content of CCL8 (p = 0.025) and CCL15 (p < 0.0001). Kaplan-Meier curves showed that a high content of CCL8 or CCL15 in cancer cells correlated with shorter disease-free and overall survival (log-rank test, p < 0.001). Our results highlight the role of CCL8 and CCL15, which are highly induced by melanoma-macrophage interactions in biologically aggressive primary melanomas and could be clinically applicable biomarkers for patient profiling. © 2024 The Pathological Society of Great Britain and Ireland.

Serum profiling identifies CCL8, CXCL13, and IL-1RA as markers of active disease in patients with systemic lupus erythematosus.

Introduction: Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease that presents a challenge for clinicians. To identify potential biomarkers for diagnosis and disease activity in SLE, we investigated a selected yet broad panel of cytokines and autoantibodies in patients with SLE, healthy controls (HC), and patients with other autoimmune diseases (AIDs). Methods: Serum samples from 422 SLE patients, 546 HC, and 1223 other AIDs were analysed within the frame of the European PRECISESADS project (NTC02890121). Cytokine levels were determined using Luminex panels, and autoantibodies using different immunoassays. Results: Of the 83 cytokines analysed, 29 differed significantly between patients with SLE and HC. Specifically, CCL8, CXCL13, and IL-1RA levels were elevated in patients with active, but not inactive, SLE versus HC, as well as in patients with SLE versus other AIDs. The levels of these cytokines also correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores, among five other cytokines. Overall, the occurrence of autoantibodies was similar across SLEDAI-2K organ domains, and the correlations between autoantibodies and activity in different organ domains were weak. Discussion: Our findings suggest that, upon validation, CCL8, CXCL13, and IL-1RA could serve as promising serum biomarkers of activity in SLE.

Pathogenic Th2 Cytokine Profile Skewing by IFN-γ-Responding Vitiligo Fibroblasts via CCL2/CCL8.

PURPOSE: Vitiligo is a T cell-mediated skin depigmentation disease. Though treatments arresting disease progression and inducing repigmentation are available, the efficacy of these options is often limited and poorly sustained. How stromal signals contribute to the interferon-γ-dominant skin niches is unclear. This study aims to determine how fibroblasts participate in the IFN-γ-dominant vitiligo niche. PATIENTS AND METHODS: Mouse vitiligo models were established. Fibroblasts from control and vitiligo mice were extracted for RNA sequencing. In vitro IFN-γ stimulation was performed to verify the JAK-STAT pathway by qPCR and Western blot. T cell polarization with chemokines was measured by flow cytometry. Protein levels in tissues were also examined by IHC. RESULTS: The vitiligo mouse model recapitulates the human CD8-IFN-γ pathway. RNA sequencing revealed elevated chemokine CCL2 and CCL8 in vitiligo fibroblast, which may be regulated by the JAK-STAT signaling. Such phenomenon is verified by JAK inhibitor peficitinib in vitro. Moreover, CCL2 addition into the naïve T polarization system promoted type 2 cytokines secretion, which represents a hallmark of vitiligo lesions. CONCLUSION: Dermal fibroblasts, a principal constituent of skin structure, respond to IFN-γ by skewing T cells towards a type 2 cytokine profile via CCL2 and CCL8, which can be abrogated by JAK inhibitor peficitinib.

CCL2, CCL8, CXCL12 chemokines in resectable non-small cell lung cancer (NSCLC).

BACKGROUND: Complex networks of chemokines are part of the immune reaction targeted against tumor cells. Chemokines influence cancer growth. It is unclear whether the concentrations of chemokines at the time of NSCLC (non-small cell lung cancer) diagnosis differ from healthy controls and reflect the extent of NSCLC. AIMS: To compare chemokine concentrations (CCL2, CCL8, CXCL12) in the plasma of patients with resectable NSCLC to those without cancer. To determine whether the chemokine concentrations differ relative to the stage of disease. METHODS: Sixty-nine patients undergoing surgery for proven/suspected NSCLC were enrolled. They underwent standard diagnostic and staging procedures to determine resectability, surgery was performed. Forty-two patients were diagnosed with NSCLC, while 27patients had benign lung lesions and functioned as the control group. Chemokine concentrations in peripheral blood were assessed using ELISA. Parametric statistics were used for the analysis of results. RESULTS: There were no differences in plasma chemokine concentrations in NSCLC patients compared to controls. CXCL12 concentrations correlated positively with tumor extent expressed as clinical stage, (mean values: stage I 5.08 ng/mL, SEM 0.59; stage II and IIIA 7.82 ng/mL; SEM 1.06; P=0.022). Patients with NSCLC stages II+IIIA had significantly higher CXCL12 concentrations than controls (mean values: stage II+IIIA 7.82 ng/mL; SEM 1.06; controls 5.3 ng/mL; SEM 0.46; P=0.017). CONCLUSION: CXCL12 was related to tumor growth and could potentially be used as a biomarker of advanced disease.

Inflammatory chemokine (C-C motif) ligand 8 inhibition ameliorates peritoneal fibrosis.

Peritoneal fibrosis (PF) is an irreversible complication of peritoneal dialysis (PD) that leads to loss of peritoneal membrane function. We investigated PD effluent and serum levels and the tissue expression of chemokine (C-C motif) ligand 8 (CCL8) in patients with PD. Additionally, we investigated their association with PF in a mouse model. Eighty-two end-stage renal disease (ESRD) patients with PD were examined. CCL8 levels were measured via enzyme-linked immunosorbent assays in PD effluents and serum and analyzed with peritoneal transport parameters. Human peritoneal mesothelial cells (hPMCs) were obtained from the PD effluents of 20 patients. Primary cultured hPMCs were treated with recombinant (r) transforming growth factor (TGF)-ß, and CCL8 expression was assessed via western blotting. As the duration of PD increased, the concentration of CCL8 in PD effluents significantly increased. Correlations between peritoneal transport parameters and dialysate CCL8 levels were observed. Western blotting analysis showed that CCL8 was upregulated via rTGF-ß treatment, accompanied by increases in markers of inflammation, fibrosis, senescence, and apoptosis in hPMCs after induction of fibrosis with rTGF-ß. Anti-CCL8 monoclonal antibody (mAb) treatment suppressed the rTGF-ß-induced increase in all analyzed markers. Immunohistochemical analysis revealed that CCL8 along with fibrosis- and inflammation-related markers were significantly increased in the PF mouse model. Functional blockade of CCL8 using a CCR8 inhibitor (R243) abrogated peritoneal inflammation and fibrosis in vivo. In conclusion, high CCL8 levels in PD effluents may be associated with an increased risk of PD failure, and the CCL8 pathway is associated with PF. CCL8 blockade can ameliorate peritoneal inflammation and fibrosis.

Iron accelerates Fusobacterium nucleatum-induced CCL8 expression in macrophages and is associated with colorectal cancer progression.

Accumulating evidence suggests that high levels of Fusobacterium nucleatum in colorectal tumor tissues can be associated with poor prognosis in patients with colorectal cancer (CRC); however, data regarding distinct prognostic subgroups in F. nucleatum-positive CRC remain limited. Herein, we demonstrate that high-iron status was associated with a worse prognosis in patients with CRC with F. nucleatum. Patients with CRC presenting elevated serum transferrin saturation exhibited preferential iron deposition in macrophages in the tumor microenvironment. In addition, F. nucleatum induced CCL8 expression in macrophages via the TLR4/NF-κB signaling pathway, which was inhibited by iron deficiency. Mechanistically, iron attenuated the inhibitory phosphorylation of NF-κB p65 by activating serine/threonine phosphatases, augmenting tumor-promoting chemokine production in macrophages. Our observations indicate a key role for iron in modulating the NF-κB signaling pathway and suggest its prognostic potential as a determining factor for interpatient heterogeneity in F. nucleatum-positive CRC.

CCL8 as a promising prognostic factor in diffuse large B-cell lymphoma via M2 macrophage interactions: A bioinformatic analysis of the tumor microenvironment.

Backgrounds: Prior investigations of the tumor microenvironment (TME) of diffuse large B-cell lymphoma (DLBCL) have shown that immune and stromal cells are key contributing factors to patients' outcome. However, challenges remain in finding reliable prognostic biomarkers based on cell infiltration. In this study, we attempted to shed some light on chemokine C-C motif chemokine ligand 8 (CCL8) in DLBCL via interaction with M2 macrophages. Methods: The Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm was applied to evaluate immune and stromal scores from transcriptomic profiles of 443 DLBCL samples from The Cancer Genome Atlas (TCGA) and GSE10846 datasets. Immune cell infiltration (ICI) clusters were obtained based on different immune cell infiltrations of each sample, and gene clusters were derived through differentially expressed genes (DEGs) between the distinct ICI clusters. Five immune-related hub genes related to overall survival (OS) and clinical stages were obtained by COX regression analysis and protein-protein interaction (PPI) network construction then verified by quantitative real-time PCR (qPCR) and immunofluorescence staining in the FFPE tissues. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and TIMER websites were employed to explore the biological functions of CCL8-related DEGs. Uni- and multivariable Cox regression analyses were performed to analyze CCL8 as an independent prognostic risk factor in GSE10846 and were verified in other independent GEO cohorts. Results: A higher stromal score was associated with favorable prognosis in DLBCL. Patients in the ICI B cluster and gene B clusters had a better follow-up status with a higher programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA4) expression. Most of ICI-related DEGs were enriched for immune-related signaling pathways. Five hub genes with a distinct prognosis association were identified, including CD163, which is a biomarker of M2 macrophages, and CCL8. Abundant M2 macrophages were discovered in the high-CCL8 expression group. The functional analysis indicated that CCL8 is a key component of immune-related processes and secretory granule groups. Cox regression analysis and data from other GSE datasets yielded additional evidence of the prognostic value of CCL8 in DLBCL. Conclusions: CCL8 has been implicated in macrophage recruitment in several solid tumors, and only a few reports have been published on the role of CCL8 in the pathogenesis of hematological malignancies. This article attempted to find out TME-related genes that associated with the survival in DLBCL patients. CCL8 was identified to be involved in immune activities. Importantly, a series of bioinformatics analysis indicated that CCL8 might become an effective target for DLBCL, which interacts with M2 macrophage and immune checkpoint. The potential related mechanisms need to be further elucidated.

Circulating inflammatory proteins associate with response to immune checkpoint inhibition therapy in patients with advanced melanoma.

BACKGROUND: Inflammation can modulate tumour growth and progression, and influence clinical response to treatment. We investigated the potential of circulating inflammatory proteins for response stratification of immune checkpoint inhibitor (ICI) therapy for advanced melanoma. METHODS: Study subjects were 87 patients with unresectable stage III or IV cutaneous melanoma from the multiple centres across the United Kingdom (UK) and the Netherlands (NL) who received ipilimumab, nivolumab, or pembrolizumab, or a combination of ipilimumab and nivolumab. Serum samples were collected before and during ICI therapy at follow-up visits scheduled every third week over a 12-week period. We performed targeted quantification of 92 proteins involved in inflammation and tested for association of their pre-treatment and on-treatment levels, as well as longitudinal changes, with overall response rate, progression-free survival, and overall survival. FINDINGS: We observed consistently higher pre-treatment levels of interleukin-6 (IL-6), hepatocyte growth factor (HGF), and monocyte chemotactic protein 2 (MCP-2), in non-responders compared to responders (meta-analysis p=3.31 × 10-4, 2.29 × 10-4, and 1.02 × 10-3, respectively). Patients' stratification according to the median value of IL-6, HGF, and MCP-2 highlighted a cumulative negative effect of pre-treatment levels of the three proteins on response (p=1.13 × 10-2), with overall response rate among patients presenting with combined elevated IL-6, HGF, and MCP-2 levels being three-fold lower (26.7%) compared to patients with none of the three proteins elevated (80.0%, p=9.22 × 10-3). Longitudinal data analysis showed that on-treatment changes in circulating inflammatory proteins are not correlated with response. INTERPRETATION: Our findings are in line with an increasing body of evidence that the pro-inflammatory cytokine IL-6 can influence response to ICI in advanced melanoma, and further support a role of circulating HGF and MCP-2 levels as prognostic biomarkers as suggested by previous smaller studies. Inflammatory proteins may serve as predictive biomarkers of ICI response and valuable targets for combination therapy. FUNDING: This work was supported by the Seerave Foundation and Dutch Cancer Society.

Dexamethasone attenuated thoracic aortic aneurysm and dissection in vascular smooth muscle cell Tgfbr2-disrupted mice with CCL8 suppression.

NEW FINDINGS: What is the central question of this study? What is the relationship of chemokine (C-C motif) ligand 8 (CCL8) to thoracic aortic aneurysm and dissection (TAAD) formation in postnatal mice with vascular smooth muscle cell (VSMC) Tgfbr2 disruption, and is dexamethasone a potential treatment? What is the main finding and its importance? CCL8 was associated with the formation of TAAD in VSMC Tgfbr2-disrupted mice. Dexamethasone reduced TAAD formation and inhibited mitogen-activated protein kinase (p-p38) and nuclear factor-κB (p-p65) signalling pathways. CCL8 might be an important promoter of aortic inflammation. Dexamethasone provided potential therapeutic effects in TAAD treatment. ABSTRACT: Aortic inflammation plays a vital role in initiation and progression of thoracic aortic aneurysm and dissection (TAAD). Disturbance of the transforming growth factor-ß (TGF-ß) signalling pathway is believed to be one of the pathogenic mechanisms of TAAD. Initially, Myh11-CreERT2 .Tgfbr2f/f male mice were used to build a TAAD mouse model, and bioinformatic analyses revealed enriched inflammatory signal pathways and upregulated chemokine (C-C motif) ligand 8 (CCL8). So we hypothesized that vascular smooth muscle cell (VSMC) Tgfbr2 disruption in postnatal mice results in aortic inflammation associated with CCL8 secretion. Real-time quantitative PCR and serum enzyme-linked immunosorbent assay (ELISA) results confirmed that CCL8 expression began to increase after VSMC Tgfbr2 disruption. Next, we cultured mouse thoracic aortas ex vivo, and observed that the protein expression of CCL8 in culture supernatants was increased by ELISA. Subsequently, the co-localization of CCL8 with α-smooth muscle actin or CD68 was found to be significantly increased by immunofluorescence. Then, dexamethasone (DEX) was used to treat TAAD in VSMC Tgfbr2-disrupted mice; the results of histochemical, immunofluorescence and immunohistochemical staining indicated that DEX therapy reduced CCL8 secretion, inflammatory cell recruitment, aortic medial thickening, elastic fibre fragmentation, extracellular matrix degradation and contractile apparatus impairment, and thereby ameliorated TAAD formation. Western blotting showed that mitogen-activated protein kinase and nuclear factor-κB signalling pathways in aorta were overactivated after VSMC Tgfbr2 disruption, but inhibited by DEX therapy. Altogether, CCL8 might be an important promoter in TAAD formation of VSMC Tgfbr2-disrupted mice, and DEX provided potential therapeutic effects in TAAD treatment.

Chemokine (C-C Motif) Ligand 8 and Tubulo-Interstitial Injury in Chronic Kidney Disease.

Kidney fibrosis has been accepted to be a common pathological outcome of chronic kidney disease (CKD). We aimed to examine serum levels and tissue expression of chemokine (C-C motif) ligand 8 (CCL8) in patients with CKD and to investigate their association with kidney fibrosis in CKD model. Serum levels and tissue expression of CCL8 significantly increased with advancing CKD stage, proteinuria level, and pathologic deterioration. In Western blot analysis of primary cultured human tubular epithelial cells after induction of fibrosis with rTGF-ß, CCL8 was upregulated by rTGF-ß treatment and the simultaneous treatment with anti-CCL8 mAb mitigated the rTGF-ß-induced an increase in fibronectin and a decrease E-cadherin and BCL-2 protein levels. The antiapoptotic effect of the anti-CCL8 mAb was also demonstrated by Annexin V/propidium iodide staining assay. In qRT-PCR analysis, mRNA expression levels of the markers for fibrosis and apoptosis showed similar expression patterns to those observed by western blotting. The immunohistochemical analysis revealed CCL8 and fibrosis- and apoptosis-related markers significantly increased in the unilateral ureteral obstruction model, which agrees with our in vitro findings. In conclusion, CCL8 pathway is associated with increased risk of kidney fibrosis and that CCL8 blockade can ameliorate kidney fibrosis and apoptosis.

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

Association between the Genetic Polymorphisms of CCL2, CCL5, CCL8, CCR2, and CCR5 with Chronic Hepatitis C Virus Infection in the Chinese Han Population.

Hepatitis C virus (HCV) infection is a global public health burden. Chronic HCV infection leads to the development of fibrosis, cirrhosis, liver cancer, and liver failure over time. A total of 250 patients with chronic HCV infection and 299 healthy blood donors were recruited. Sixteen candidate single nucleotide polymorphisms (SNPs) in chemokine (C-C motif) ligand 2 (CCL2), CCL5, CCL8, C-C chemokine receptor 2 (CCR2), and CCR5 were genotyped in all participants. The rs1024610 AA genotype was significantly associated with decreased susceptibility to chronic HCV infection. Aspartate aminotransferase (AST) levels, AST/platelet ratio index, and the fibrosis 4 score were significantly lower in the CCL2 rs1024610 T allele and haplotype ATGC carriers. Moreover, expression levels of collagen IV (C-IV) and laminin (LN) were significantly higher in patients with the CCL5 rs2280788 C allele compared to the non-carriers. Similarly, the expression levels of C-IV, LN, and hyaluronic acid were significantly higher in patients with the CCL5 haplotype, TGCT. No significant differences were identified between the SNPs/haplotypes and plasma levels of CCL2, CCL5, CCL8, CCR2, and CCR5 in the healthy controls, and the rs1024610 allele alteration had no effect on CCL2 promoter activity. This is the first study to report an association between CCL2 rs1024610 and the risk of chronic HCV infection in the Chinese Han population. rs1024610 and ATGC haplotype in CCL2 were reasonable candidate markers of liver abnormalities. rs2280788 and TGCT haplotype in CCL5 are likely to play a significant role in liver fibrosis during chronic HCV infection.

CCL8 deficiency in the host abrogates early mortality of acute graft-versus-host disease in mice with dysregulated IL-6 expression.

Although allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for diverse malignant and nonmalignant diseases, acute graft-versus-host disease (aGVHD) is strongly linked to mortality caused by HSCT. We previously reported that CC chemokine ligand 8 (CCL8) is closely correlated to aGVHD mortality in both humans and mice. To study the role of CCL8 in aGVHD, CCL8 knockout (CCL8-/-) mice were transplanted with fully allogeneic marrow grafts. These mice exhibited a significant reduction in mortality (90.0% vs. 23.4% survival for CCL8-/- vs. wild-type recipients at day 28, p < 0.0001). As a result, apparent prolonged median survival from 9 days in wild-type mice to 45 days in CCL8-/- mice was observed. Acute GVHD pathology and liver dysfunction in CCL8-/- mice were significantly attenuated compared with those in wild-type mice. In association with the reduced mortality, a surge of plasma interleukin (IL)-6 was observed in CCL8-/- recipients with allogeneic marrow, which was significantly increased compared with wild-type mice that received allografts. Donor T-cell expansion and plasma levels of interferon-γ and TNF-α during aGVHD were similar in both types of mice. Collectively, these findings indicate that CCL8 plays a major role in aGVHD pathogenesis with possible involvement of an IL-6 signaling cascade.

Serum levels of NLRC4 and MCP-2/CCL8 in patients with active Crohn's disease.

Crohn's disease (CD) is characterized by malfunction of immune-regulatory mechanisms with disturbed intestinal mucosal homeostasis and increased activation of mucosal immune cells, leading to abnormal secretion of numerous pro- and anti-inflammatory mediators. MCP2/CCL8 is produced by intestinal epithelial cells and macrophages, and is a critical regulator of mucosal inflammation. NLRC4 is expressed in phagocytes and intestinal epithelial cells and is involved in intestinal homeostasis and host defense. However, no study to date has assessed the circulating levels of NLRC4 and MCP2/CCL8 in patients with CD. The study was aimed to investigate the serum levels of MCP2/CCL8 and NLRC4 in patients with active CD. Sixty-nine patients with active CD and 60 healthy participants were included in the study. Serum levels of NLRC4 and MCP2/CCL8 were determined using an enzyme-linked immunosorbent assay. The median serum NLRC4 levels were lower in the patient group than in the controls (71.02 (range, 46.59-85.51) pg/mL vs. 99.43 (range 83.52-137.79) pg/mL) (P < 0.001). The median serum levels of MCP2/CCL8 were decreased in patients with CD (28.68 (range, 20.16-46.0) pg/mL) compared with the controls (59.96 (range, 40.22-105.59) pg/mL) (P < 0.001). Cut-off points of NLRC4 (<81 pg/mL) and MCP2/CCL8 (<40 pg/mL) showed high sensitivity and specificity for identifying active CD. In conclusion, this is the first study to examine circulating levels of MCP2/CCL8 and NLRC4 in patients with active CD. Our results suggest that serum NLRC4 and MCP2/CCL8 levels may be involved in the pathogenesis of CD and may have a protective effect on intestinal homeostasis and inflammation. Serum levels of MCP2/CCL8 and NLRC4 could be used as a diagnostic tool and therapeutic target for CD.

Altered Secretome of Diabetic Monocytes Could Negatively Influence Fracture Healing-An In Vitro Study.

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.

Further Findings Concerning Endothelial Damage in COVID-19 Patients.

Systemic vascular damage with micro/macro-thrombosis is a typical feature of severe COVID-19. However, the pathogenesis of this damage and its predictive biomarkers remain poorly defined. For this reason, in this study, serum monocyte chemotactic protein (MCP)-2 and P- and E-selectin levels were analyzed in 204 patients with COVID-19. Serum MCP-2 and P-selectin were significantly higher in hospitalized patients compared with asymptomatic patients. Furthermore, MCP-2 increased with the WHO stage in hospitalized patients. After 1 week of hospitalization, MCP-2 levels were significantly reduced, while P-selectin increased in patients in WHO stage 3 and decreased in patients in WHO stages 5-7. Serum E-selectin was not significantly different between asymptomatic and hospitalized patients. The lower MCP-2 levels after 1 week suggest that endothelial damage triggered by monocytes occurs early in COVID-19 disease progression. MCP-2 may also predict COVID-19 severity. The increase in P-selectin levels, which further increased in mild patients and reduced in severe patients after 1 week of hospitalization, suggests that the inactive form of the protein produced by the cleavage of the active protein from the platelet membrane is present. This may be used to identify a subset of patients that would benefit from targeted therapies. The unchanged levels of E-selectin in these patients suggest that endothelial damage is less relevant.

Pathogenic roles and therapeutic potential of the CCL8-CCR8 axis in a murine model of IgG4-related sialadenitis.

BACKGROUND: Our previous studies reveal that CCL18-CCR8 chemokine axis is upregulated in patients of immunoglobulin G4-related disease (IgG4-RD), suggesting that the CCL18-CCR8 axis is implicated in the etiology of IgG4-RD, although whether this axis has a potential as a therapeutic target remains unclear. Our purpose was to clarify the pathogenic roles and therapeutic potential of the murine CCL8 (analog of human CCL18)-CCR8 axis by using an animal model of IgG4-RD (LAT Y136F knockin mice; LAT mice). METHODS: We compared the infiltration of inflammatory cells and the fibrosis of the salivary glands of 6-week-old LAT mice and littermate mice. The expressions of Ccl8 and Ccr8 were also compared. Next, we investigated the therapeutic effects of intravenous administration of anti-CCL8 neutralizing antibody in LAT mice against inflammation and fibrosis of the salivary glands. We also investigated the effects of stimulation with recombinant mouse CCL8 on the collagen production in a mouse fibroblast cell line (NIH/3 T3) in vitro. RESULTS: When compared with the littermates, the LAT mice showed apparent infiltration of inflammatory cells and fibrosis in the salivary glands. The focus and fibrosis score in the salivary glands were significantly higher in the LAT mice than in the littermates. The expression levels of Ccl8 in the spleen and of Ccr8 in the salivary glands were significantly higher in the LAT mice than in the littermates. Anti-CCL8 antibody significantly improved the focus and fibrosis score in the salivary glands of the LAT mice. In vitro, stimulation with recombinant mouse CCL8 significantly increased the expression of collagen and ERK1/2 phosphorylation in NIH/3 T3. CONCLUSION: We clarified the overexpression and therapeutic potential of the mouse CCL8-CCR8 axis in LAT mice, which could play a crucial role in fibrosis via ERK1/2 phosphorylation, as well as the chemotaxis of inflammatory cells. The human CCL18-CCR8 axis might be a novel therapeutic target for IgG4-RD.

Early inflammatory changes and CC chemokine ligand-8 upregulation in the heart contribute to uremic cardiomyopathy.

Uremic cardiomyopathy is a common complication in chronic kidney disease (CKD) patients, accounting for a high mortality rate. Several mechanisms have been proposed to link CKD and cardiac alterations; however, the early cardiac modifications that occur in CKD that may trigger cardiac remodeling and dysfunction remain largely unexplored. Here, in a mouse model of CKD induced by 5/6 nephrectomy, we first analyzed the early transcriptional and inflammatory changes that occur in the heart. Five days after 5/6 nephrectomy, RNA-sequencing showed the upregulation of 54 genes in the cardiac tissue of CKD mice and the enrichment of biological processes related to immune system processes. Increased cardiac infiltration of T-CD4+ lymphocytes, myeloid cells, and macrophages during early CKD was observed. Next, since CC chemokine ligand-8 (CCL8) was one of the most upregulated genes in the heart of mice with early CKD, we investigated the effect of acute and transient CCL8 inhibition on uremic cardiomyopathy severity. An increase in CCL8 protein levels was confirmed in the heart of early CKD mice. CCL8 inhibition attenuated the early infiltration of T-CD4+ lymphocytes and macrophages to the cardiac tissue, leading to a protection against chronic cardiac fibrotic remodeling, inflammation and cardiac dysfunction induced by CKD. Altogether, our data show the occurrence of transcriptional and inflammatory changes in the heart during the early phases of CKD and identify CCL8 as a key contributor to the early cardiac inflammatory state that triggers further cardiac remodeling and dysfunction in uremic cardiomyopathy.