C-C Motif) Ligand 2 Research Articles

Anti-bladder cancer and COVID-19 potentials of pachymic acid: clinical features and therapeutic targets

ABSTRACT Pachymic acid, as one of bioactive ingredients in Poria Cocos, has been evidenced preclinically with pharmacological benefits including anti-bladder cancer action. Additionally, pachymic acid has been initiatorily identified in Chinese prescriptions applied for coronavirus disease 2019 (COVID-19) treatment. However, the molecular mechanisms and pharmacological targets associated with mitochondrial functions in pachymic acid against bladder cancer and COVID-19 have not been explored. In this study, network pharmacology and molecular docking detections were employed to identify common, candidate, and core targets that were related to pachymic acid treating bladder cancer COVID-19. Enrichment determinations using core target genes were used to reveal the anti-bladder cancer and COVID-19 molecular mechanisms exerted by pachymic acid. Firstly, all 1150 differentially expressed genes among bladder cancer and COVID-19 were ascertained and a risk score with key genes characterized by different risk groups was established. Furthermore, current study highlighted pachymic acid as a potential compound for anti-bladder cancer and COVID-19. Network pharmacology approaches ascertained ten key targets for pachymic acid against bladder cancer and COVID-19. Molecular docking findings showed that pachymic acid presented effectively binding abilities to key target proteins of chemokine (CC-motif) ligand 2 (CCL2), thrombospondin-1 (THBS1), matrix metallopeptidase 1 (MMP1) in severe acute respiratory syndrome coronavirus 2 ;(SARS-CoV-2). Our study indicates that pachymic acid may be used for treatment of bladder cancer and COVID-19 through increasing tumor immunity and inhibiting SARS-CoV-2 replication based on present bioinformatics findings.

Abstract P397: Preservation Of Renal Microvascular Function In Ischemia-reperfusion Induced Kidney Injury In Rats

Proper renal blood flow and glomerular filtration rate are critical for maintaining normal kidney function by precisely adjusting preglomerular microvascular resistance. Renal ischemia-reperfusion (IR) insult markedly increased mRNA expression of the inflammatory chemokine (C-C motif) Ligand 2 (CCL2) in rat kidney tissues. We hypothesized that depletion of macrophages prevented IR-induced renal autoregulatory impairment via reduction of CCL2 in preglomerular microvessels (PGMV). IR was induced in male rats by 60-minute bilateral renal artery occlusion followed by reperfusion. Some IR rats were treated with the selective macrophage inhibitor, Liposome-encapsulated clodronate (LC) following reperfusion and repeated 72 hours post-IR. Afferent arteriolar autoregulatory reactivity was assessed using the in vitro blood-perfused juxtamedullary nephron preparation in sham, IR, and IR+LC rats on day 7. Afferent arterioles from sham-operated rats exhibited pressure-dependent vasoresponsiveness. The baseline arteriolar diameter averaged 13.4 ± 0.5 μm at renal perfusion pressure (RPP) of 100 mmHg. Diameter increased to 117 ± 4% of baseline at RPP of 65 mmHg and decreased to 69 ± 2% at 170 mmHg, respectively. In contrast, afferent arterioles from IR rats only increased to 104 ± 2% and decreased to 91 ± 5% of baseline at 65 and 170 mmHg, respectively (P < 0.05 vs sham), indicating the impaired autoregulatory capacity in IR rats. LC-treated IR rats, however, markedly improved the pressure-dependent vasoresponsiveness. A decrease in RPP to 65 mmHg resulted in a 124 ± 17% increase in the baseline diameter, whereas increasing RPP to 170 mmHg led to a 74 ± 7% decrease in diameter. Additionally, IR markedly increased CCL2 mRNA expression in isolated PGMV by 10-fold 7 days post-IR. IR also elevated NADPH oxidase subunits (gp91 phox , P67 phox , and P47 phox ). Significantly, LC treatment suppressed mRNA expression of CCL2, gp91 phox , P67 phox , and P47 phox in IR-PGMV. In conclusion, depletion of macrophages preserves afferent arteriolar autoregulatory capacity and reduces PGMV CCL2 mRNA expression in IR rats, suggesting a critical role of macrophages in mediating renal microvascular autoregulatory impairment in IR-induced kidney injury via the increase of CCL2 in PGMV.

Involvement of CCL2 and CH25H Genes and TNF signaling pathways in mast cell activation and pathogenesis of chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU), a mast cell-driven disease, substantially affects the quality of life. While genetics affect CSU susceptibility and severity, the specific genetic factors associated with mast cell activation in CSU remain elusive. We aimed to identify key genetic factors and investigate their roles in CSU pathogenesis. Two gene expression datasets from the Gene Expression Omnibus were merged and validated using principal component analysis and boxplots. The merged dataset was subjected to limma and weighted gene co-expression network analyses. Genes whose expression correlated highly with CSU were identified and analyzed using Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. As GSEA, GO, and KEGG analyses highlighted the importance of chemokine (C-C motif) ligand 2 (CCL2) and cholesterol 25-hydroxylase (CH25H) gene and tumor necrosis factor (TNF) signaling pathways in CSU; the three corresponding genes were knocked down in human mast cell line-1 (HMC-1), followed by incubation with thrombin to mimic CSU pathogenesis. CCL2, CH25H, and TNF knockdown reduced excitability and cytokine production in HMC-1. Our findings suggest that genes involved in the CCL2, CH25H, and TNF pathways play crucial roles in CSU pathogenesis, providing insights into potential therapeutic targets for CSU treatment.

Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

Role of interleukin-36γ induced by ultraviolet radiation in chronic actinic dermatitis.

Chronic actinic dermatitis (CAD) is an immune-mediated photodermatosis characterized by a high eosinophil count and total immunoglobulin E (IgE) in the peripheral blood of patients. At present, however, the reasons for their elevation remain unclear. The current study aimed to detect changes in inflammatory cytokines in CAD and explore their role in this disease. Enzyme-linked immunosorbent assay and Luminex assay were conducted to measure inflammatory factor levels. Immunohistochemical analysis and quantitative real-time polymerase chain reaction were performed to evaluate the expression levels of interleukin-36γ (IL-36γ), IL-8, chemokine (C-C motif) ligand 17 (CCL17), and CCL18. CCK8 kits were used to assess cell proliferation. Immunofluorescence was used to detect nuclear factor κB (NF-κB) p65 nuclear translocation. Western blot analysis was performed to detect the protein expression level of phosphorylated NF-κB (p-NF-κB) p65. Hematoxylin and eosin and Masson trichrome staining were applied to observe histological changes in a chronic photo-damaged mouse model. Eosinophils, total IgE, IL-36γ, IL-8, tumor necrosis factor α, CCL17, and CCL18 were elevated in CAD. Of note, IL-36γ promoted the proliferation of eosinophilic cells (EOL-1) and the production of IgE in peripheral blood mononuclear cells. IL-36γ also promoted the production of IL-8 and CCL18 in immortalized human keratinocytes (HaCaT cells), while ultraviolet radiation (UVR)-induced IL-36γ via activation of the NF-κB signaling pathway. IL-36γ was involved in the pathogenesis of CAD and UVR contributed to the production of IL-36γ, which may provide a novel therapeutic target for CAD.

Association between CCR2 and CCL2 expression and NET stimulation in adult-onset Still’s disease

Adult-onset Still’s disease (AOSD) is a systemic inflammatory disease characterized by the activation of monocyte-derived cells and the release of neutrophil extracellular traps (NET). C–C motif ligand (CCL) 2 is a chemoattractant that interacts with the C–C motif chemokine receptor (CCR) 2, resulting in monocyte recruitment and activation. CCL2 and CCR2 were measured with enzyme-linked immunosorbent assay (ELISA) at the serum level, and using immunohistochemical staining at the skin and lymph node tissues levels. THP-1 cell lysates were analyzed using western blot and ELISA after NET stimulation in patients with AOSD. Serum CCL2 level was higher in patients with AOSD than in patients with rheumatoid arthritis and healthy controls (HCs). In patients with AOSD, the percentage of CCL2-positive inflammatory cells in the skin tissues and CCR2-positive inflammatory cells in the lymph nodes increased, compared to that in HCs and in patients with reactive lymphadenopathy, respectively. NET induced in patients with AOSD enhanced the secretion of CCR2, higher CCR2 expression in monocytes, and the levels of interleukin (IL)-1β, IL-6, and IL-18 from THP-1 cells. Our findings suggest that upregulation of the CCL2–CCR2 axis may contribute to the clinical and inflammatory characteristics of AOSD.

Immune-checkpoint proteins, cytokines, and microbiome impact on patients with cervical insufficiency and preterm birth.

Microenvironmental factors, including microbe-induced inflammation and immune-checkpoint proteins that modulate immune cells have been associated with both cervical insufficiency and preterm delivery. These factors are incompletely understood. This study aimed to explore and compare interactions among microbiome and inflammatory factors, such as cytokines and immune-checkpoint proteins, in patients with cervical insufficiency and preterm birth. In particular, factors related to predicting preterm birth were identified and the performance of the combination of these factors was evaluated. A total of 220 swab samples from 110 pregnant women, prospectively recruited at the High-Risk Maternal Neonatal Intensive Care Center, were collected between February 2020 and March 2021. This study included 63 patients with cervical insufficiency receiving cerclage and 47 control participants. Endo- and exocervical swabs and fluids were collected simultaneously. Shotgun metagenomic sequencing for the microbiome and the measurement of 34 immune-checkpoint proteins and inflammatory cytokines were performed. First, we demonstrated that immune-checkpoint proteins, the key immune-regulatory molecules, could be measured in endocervical and exocervical samples. Secondly, we identified significantly different microenvironments in cervical insufficiency and preterm birth, with precise cervical locations, to provide information about practically useful cervical locations in clinical settings. Finally, the presence of Moraxella osloensis (odds ratio = 14.785; P = 0.037) and chemokine CC motif ligand 2 levels higher than 73 pg/mL (odds ratio = 40.049; P = 0.005) in endocervical samples were associated with preterm birth. Combining M. osloensis and chemokine CC motif ligand 2 yielded excellent performance for predicting preterm birth (area under the receiver operating characteristic curve = 0.846, 95% confidence interval = 0.733-0.925). Multiple relationships between microbiomes, immune-checkpoint proteins, and inflammatory cytokines in the cervical microenvironment were identified. We focus on these factors to aid in the comprehensive understanding and therapeutic modulation of local microbial and immunologic compositions for the management of cervical insufficiency and preterm birth.

Severe udder cleft dermatitis lesion transcriptomics points to an impaired skin barrier, defective wound repair and a dysregulated inflammatory response as key elements in the pathogenesis.

This study is the first to investigate the transcriptomic changes occurring in severe udder cleft dermatitis lesions (UCD) in Holstein-Friesian cows. An examination of the gene expression levels in natural UCD lesions and healthy udder skin through RNA Seq-Technology provided a deeper insight into the inflammatory pathways associated with this disease. A clear distinction between the gene expression patterns of UCD lesions and healthy skin was shown in the principal component analysis. Genes coding for inflammatory molecules were upregulated such as the chemokines C-X-C motif ligand 2 (CXCL2), 5 (CXCL5) and 8 (CXCL8), and C-C motif ligand 11 (CCL11). Moreover, the genes coding for the multifunctional molecules ADAM12 and SLPI were amongst the highest upregulated ones, whereas the most downregulated genes included the ones coding for keratins and keratin-associated molecules. Predominantly inflammatory pathways such as the chemokine signaling, cytokine receptor interaction and IL-17 signaling pathway were significantly upregulated in the pathway analysis. These results point towards a fulminant, dysregulated inflammatory response concomitant with a disruption of the skin barrier integrity and a hampered wound repair mechanism in severe UCD lesions.

Mechanisms of Rostellularia procumbens (L.) Nees on treating chronic glomerulonephritis explored by network pharmacology, RNA-seq, and in vitro experiments.

The purpose of this study was to demonstrate the in vitro anti-nephritis activity of Rostellularia procumbens (L.) Nees (R. procumbens) extract and to make a preliminary investigation of its anti-nephritis mechanism. A prediction network was built that describes the relationship between R. procumbens and CGN. Then, the potential targets for R. procumbens against CGN were imported into the DAVID database for Gene Ontology (GO) biological annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A lipopolysaccharide (LPS)-stimulated rat mesangial cell HBZY-1 model in vitro was used to examine the anti-inflammatory activity of R. procumbens extract. RNA-seq was utilized to investigate differentially expressed genes (DEGs) and enriched signaling pathways between groups. Finally, qPCR was used for the validation analysis of the experimental results. The results of network pharmacology showed that R. procumbens exerts its therapeutic effect on CGN through the AGE-RAGE signaling pathway in diabetic complications, PI3K-Akt, IL-17 signaling pathway, and so on. R. procumbens n-butanol extract (J-NE) can effectively relieve inflammation in HBZY-1. The results of KEGG pathway enrichment suggest that J-NE attenuated CGN was associated with the IL-17 signaling pathway, and the results of RNA-seq were consistent with network pharmacology. Targets enriched in the IL-17 signaling pathway, including Chemokine (C-C motif) ligand 7 (CCL7), Lipocalin 2 (LCN2), Chemokine (C-C motif) ligand 2 (CCL2), and Chemokine (C-X-C motif) ligand 1 (CXCL1), have been identified as crucial targets attenuating CGN by J-NE. R. procumbens is a promising pharmacological candidate for the treatment of CGN in the present era.

Traditional Chinese medicine Kuan-Sin-Yin decoction inhibits cell mobility via downregulation of CCL2, CEACAM1 and PIK3R3 in hepatocellular carcinoma cells

Ethnopharmacological relevanceKuan-Sin-Yin (KSY) is a traditional Chinese medical decoction, designed based on the classic Si-Jun-Zi-Tang decoction and used clinically to improve the synergic effects of energy promotion, liver function and cancer related symptom and quality of life. However, the anti-hepatocellular carcinoma (HCC) function of KSY is unclear. Aim of the studyThis study aimed to investigate the anti-mobility activity of KSY on HCC cells and elucidate its molecular mechanism. Materials and methodsTwo malignancy hepatocellular carcinoma cells, Mahlavu and SK-Hep-1, were used for the test of cell proliferation via alarm blue assay. The wound healing and Transwell assays were used to determine the anti-mobility activity of KSY in HCC cells. Cell morphology was analyzed via confocal microscopy. The genomic profile of KSY-treated HCC cells was analyzed by microarray. The potential signaling pathways and bio-functions of KSY-mediated genes were analyzed by ingenuity pathway analysis (IPA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the messenger RNA (mRNA) level of indicated gene. ResultsKSY did not affect cell viability of HCC cells but significantly inhibited cell migration and invasion in those HCC Mahlavu and SK-Hep-1 cells. In parallel, KSY induced changes in morphology of HCC cells via re-modulating actin cytoskeleton. KSY upregulated 1270 genes but reduced 1534 genes in Mahlavu cells. KSY regulated various gene networks which controlled cell migration, invasion and movement. Specifically, KSY reduced expression of chemokine (C-C motif) ligand 2 (CCL2), which is correlated to cell mobility, and concomitantly downregulated mRNA levels of phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) and CEA cell adhesion molecule 1 (CEACAM1). ConclusionThese findings indicated that regulation of CCL2-mediated PIK3R3 and CEACAM1 may be involved in KSY inhibited cell mobility. Moreover, KSY may be a potential a Chinese decoction for reducing cell mobility.

#2974 SERUM CHEMOKINE CC-MOTIF LIGAND 17 IS A PREDICTIVE MARKER OF SEVERE COVID-19 IN HAEMODIALYSIS PATIENTS

Abstract Background and Aims Maintenance haemodailaysis (HD) patients are at higher risk for severe coronavirus disease 2019 (COVID-19). Because of a limited number of facilities that can provide inpatient treatment for COVID-19 and HD, it is important to identify HD patients who are at high risk for severe COVID-19. For mild to moderate COVID-19 patients, chemokine CC-motif ligand 17 (CCL17) was reported to be a predictive marker for severe COVID-19; however, the validity of CCL17 among HD patients is unknown. Method This retrospective observational study enrolled 107 HD patients with mild or moderate COVID-19 at hospitalisation (mean age 70.1 ± 15.1 years; 71.0% male). Receiver operating characteristic and logistic regression analyses were used to examine the predictive validity of indices for severe COVID-19 which is defined as partial pressure of oxygen/fraction of inspired oxygen (P/F) ratio <300 or SpO2 <94%. Multi-variate logistic regression models were used to investigate the association of CCL17 level with the development of severe COVID-19. Furthermore, to evaluate the degree of improvement in predicting performance in the pre-existing model with the addition of CCL17 compared to the pre-existing model, we calculated net reclassification improvement (NRI) and the integrated discrimination improvement (IDI). Results During hospitalisation, 32 patients developed severe COVID-19. Serum CCL17 collected at admission exhibited a higher area under the curve value (0.818) compared with that of other indicators including LDH and C-reactive protein for the prediction of severe COVID-19. The optimal cut-off value for CCL17 was 150.5 pg/ml. A multi-variate logistic analysis revealed that CCL17 (above 150.5 pg/ml) was significantly associated with severe COVID-19 (Odds ratio, 0.063; 95% Confidence interval [CI], 0.017–0.227; P < 0.001) even after adjustment for covariates. The addition of the CCL17 to a model consisting of vaccination status, albumin, blood urea nitrogen, C-reacting protein and lactate dehydrogenase, that are variables incorporated in multivariate logistic model, significantly improved classification performance for severe COVID-19 using the NRI (1.16, 95% CI: 0.82–1.50, P < 0.001) and IDI (0.18, 95% CI: 0.09–0.26, P < 0.001). Conclusion CCL17 levels in HD patients with mild or moderate COVID-19 predict risk of developing severe COVID-19.

CDYL knockdown reduces glioma development through an antitumor immune response in the tumor microenvironment

Gliomas are highly prevalent and aggressive brain tumors. Growing evidence shows that epigenetic changes are closely related to cancer development. Here we report the roles of Chromodomain Y-like (CDYL), an important epigenetic transcriptional corepressor in the central nervous system in glioma progression. We found that CDYL was highly expressed in glioma tissues and cell lines. CDYL knockdown decreased cell mobility in vitro and significantly reduced tumor burden in the xenograft mouse in vivo. RNA sequencing analysis revealed the upregulation of immune pathways after CDYL knockdown, as well as chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 12. The immunohistochemistry staining and macrophage polarization assays showed increased infiltration of M1-like tumor-associated macrophages/microglia (TAMs) while decreased infiltration of M2-like TAMs after CDYL knockdown in vivo and in vitro. Following the in situ TAMs depletion or CCL2 antibody neutralization, the tumor-suppressive role of CDYL knockdown was abolished. Collectively, our results show that CDYL knockdown suppresses glioma progression, which is associated with CCL2-recruited monocytes/macrophages and the polarization of M1-like TAMs in the tumor microenvironment, indicating CDYL as a promising target for glioma treatment.

Role of high-temperature requirement serine protease A 2 in rheumatoid inflammation

BackgroundHigh-temperature requirement serine protease A 2 (HtrA2) is known to be involved in growth, unfolded protein response to stress, apoptosis, and autophagy. However, whether HtrA2 controls inflammation and immune response remains elusive.MethodsExpression of HtrA2 in the synovial tissue of patients was examined using immunohistochemistry and immunofluorescence staining. Enzyme-linked immunosorbent assay was used to determine the concentrations of HtrA2, interleukin-6 (IL-6), interleukin-8 (IL-8), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor α (TNFα). Synoviocyte survival was assessed by MTT assay. For the downregulation of HtrA2 transcripts, cells were transfected with HtrA2 siRNA.ResultsWe found that the concentration of HtrA2 was elevated in rheumatoid arthritis (RA) synovial fluid (SF) than in osteoarthritis (OA) SF, and its concentrations were correlated with the number of immune cells in the RA SF. Interestingly, HtrA2 levels in the SF of RA patients were elevated in proportion to synovitis severity and correlated with the expression of proinflammation cytokines and chemokines, such as IL-6, IL-8, and CCL2. In addition, HtrA2 was highly expressed in RA synovium and primary synoviocytes. RA synoviocytes released HtrA2 when stimulated with ER stress inducers. Knockdown of HtrA2 inhibited the IL1β-, TNFα-, and LPS-induced release of proinflammatory cytokines and chemokines by RA synoviocytes.ConclusionHtrA2 is a novel inflammatory mediator and a potential target for the development of an anti-inflammation therapy for RA.

Potential utility of urinary chemokine CCL2 to creatinine ratio in prognosis of 5-year graft failure and mortality post 1-year protocol biopsy in kidney transplant recipients.

Chemokines (chemotactic cytokines) are small proteins which are engaged in many pathophysiological processes, including inflammation and homeostasis. In recent years, application of chemokines in transplant medicine was intensively studied. The aim of this study was to determine the utility of urinary chemokines CCL2 (C-C motif ligand 2) and CXCL10 (C-X-C motif chemokine ligand 10) in prognosis of 5-year graft failure and mortality post 1-year protocol biopsy in renal transplant recipients. Forty patients who had a protocol biopsy 1 year after renal transplantation were included. Concentrations of CCL2 and CXCL10 in urine with reference to urine creatinine were measured. All patients were under the supervision of one transplant center. Long-term outcomes within 5 years after 1-year posttransplant biopsy were analyzed. Urinary CCL2:Cr at the time of biopsy was significantly increased in patients who died or had graft failure. CCL2:Cr was proven to be a significant predictor of 5-year graft failure and mortality (odds ratio [OR]:1.09, 95% confidence interval [CI]: 1.02-1.19, p = .02; OR: 1.08, 95% CI: 1.02-1.16, p = .04; respectively). Chemokines are easily detected by current methods. In the era of personalized medicine, urinary CCL2:Cr can be considered as a factor providing complementary information regarding risk of graft failure or increased mortality.

Aberrant inflammatory responses in intoxicated burn-injured patients parallel impaired cognitive function.

Burn-injured patients with alcohol use disorder (AUD) have increased morbidity and mortality compared to alcohol-abstaining individuals with similar injuries. It is hypothesized that this is due, in part, to alcohol-induced dysregulation of the systemic inflammatory response, leading to worsened clinical outcomes, including increased susceptibility to infection, and heightened cognitive impairment. To examine the effects of alcohol on inflammatory markers after burn injury, we used multiplex assays to measure a panel of 48 cytokines, chemokines, and growth factors in the plasma of burn patents within 24h of admission to the University of Colorado Burn Center. Thirty patients were enrolled between July 2018 to February 2020 and were stratified based on presence of AUD and total body surface area (TBSA) burn of ≥20% into four groups: [AUD-, TBSA <20%, N=12], [AUD+, TBSA <20%, N=3], [AUD-, TBSA ≥20%, N=8], [AUD+, TBSA ≥20%, N=7]. In addition, Confusion Assessment Method (CAM) scores were collected to evaluate patient delirium during the course of hospitalization. Multivariate statistical analysis demonstrated a number of cytokines and other factors that were significantly different between the groups. For example, the anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1ra) was dampened in the AUD+, TBSA ≥20% cohort with a 75.2% decrease compared to AUD-, TBSA ≥20%, and an 83.9% decrease compared to AUD-, TBSA <20% (p=0.008). Additionally, plasma levels of the pro-inflammatory mediator CXCL12 (C-X-C motif chemokine ligand 12, also known as stromal cell-derived factor 1, SDF-1) was higher in the AUD+groups (p=0.03) and similarly, IL-18 levels were greater in AUD+, TBSA ≥20% (p=0.009). Eotaxin (also known as cytokine CC motif ligand 11, CCL11) was markedly elevated in the AUD+, TBSA ≥20% cohort with a 2.4-fold increase over the AUD-, TBSA ≥20%, and a 1.7-fold rise compared to the AUD-, TBSA <20% cohorts (p=0.04). Interestingly, there was also a marked rise in CAM+delirium scores (85.7%) among the AUD+patients with TBSA ≥20% (p=0.02). Not surprisingly, we found that hospital stays increased with AUD+ and larger burns (p=0.0009). Our findings reveal that burn patients who misuse alcohol have aberrant inflammatory responses that may lead to greater immune dysregulation and worse clinical outcomes.

The GM-CSF/CCL17 pathway in obesity-associated osteoarthritic pain and disease in mice

We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r=-0.458and p=0.0096), with elevated circulating CCL17 levels (r=0.2108and p=0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

Effect of Simvastatin Treatment on Mitochondrial Function and Inflammatory Status of Human White Adipose Tissue.

Statin therapy has shown pleiotropic effects affecting both mitochondrial function and inflammatory status. However, few studies have investigated the concurrent effects of statin exposure on mitochondrial function and inflammatory status in human subcutaneous white adipose tissue. In a cross-sectional study, we investigated the effects of simvastatin on mitochondrial function and inflammatory status in subcutaneous white adipose tissue of 55 human participants: 38 patients (19 females/19 males) in primary prevention with simvastatin (> 40 mg/d, > 3 mo) and 17 controls (9 females/8 males) with elevated plasma cholesterol. The 2 groups were matched on age, body mass index, and maximal oxygen consumption. Anthropometrics and fasting biochemical characteristics were measured. Mitochondrial respiratory capacity was assessed in white adipose tissue by high-resolution respirometry. Subcutaneous white adipose tissue expression of the inflammatory markers IL-6, chemokine (C-C motif) ligand 2 (CCL2), CCL-5, tumor necrosis factor-α, IL-10, and IL-4 was analyzed by quantitative PCR. Simvastatin-treated patients showed lower plasma cholesterol (P < .0001), low-density lipoprotein (P < .0001), and triglyceride levels (P = .0116) than controls. Simvastatin-treated patients had a lower oxidative phosphorylation capacity of mitochondrial complex II (P = .0001 when normalized to wet weight, P < .0001 when normalized to citrate synthase activity [intrinsic]), and a lower intrinsic mitochondrial electron transport system capacity (P = .0004). Simvastatin-treated patients showed higher IL-6 expression than controls (P = .0202). Simvastatin treatment was linked to mitochondrial respiratory capacity in human subcutaneous white adipose tissue, but no clear link was found between statin exposure, respiratory changes, and inflammatory status of adipose tissue.

A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a β-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Persistent alveolar inflammatory response in critically ill patients with COVID-19 is associated with mortality

IntroductionPatients with COVID-19-related acute respiratory distress syndrome (ARDS) show limited systemic hyperinflammation, but immunomodulatory treatments are effective. Little is known about the inflammatory response in the lungs and if this...

Effects of chemokine receptor CCR7 in the pathophysiology and clinical features of the immuno-inflammatory response in primary pterygium.

Chemokine receptor 7 (CCR7) has been considered a critical biomarker in inflammation and the immune response; however, little is known about CCR7 in pterygia. This study aimed to investigate whether CCR7 participates in the pathogenesis of primary pterygia and how CCR7 affects the progression of pterygia. This was an experimental study. Slip-lamp photographs of 85 pterygium patients were used to measure the width, extent, and area of pterygia with computer software. Pterygium blood vessels and general ocular redness were quantitatively analyzed with a specific algorithm. The expression of CCR7 and its ligands C-C motif ligand 19 (CCL19) and C-C motif ligand 21 (CCL21) in control conjunctivae and excised pterygia collected during surgery were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The phenotype of CCR7-expressing cells was identified by costaining for major histocompatibility complex II (MHC II), CD11b or CD11c. The CCR7 level was significantly increased by 9.6-fold in pterygia compared with control conjunctivae (p=0.008). The higher the expression of CCR7 was, the more blood vessels appeared in pterygia (r=0.437, p=0.002) and the more general ocular redness was (r=0.51, p<0.001) in pterygium patients. CCR7 was significantly associated with pterygium extent (r=0.286, p=0.048). In addition, we found that CCR7 colocalized with CD11b, CD11c or MHC II in dendritic cells, and immunofluorescence staining showed that CCR7-CCL21 is a potential chemokine axis in pterygium. This work verified that CCR7 impacts the extent of primary pterygia invading the cornea and inflammation at the ocular surface, which may provide a possibility for a further in-depth understanding of the immunological mechanism in pterygia.