Cellular Distribution of C-C Motif Chemokine Ligand 2 Like Immunoreactivities in Frontal Cortex and Corpus Callosum of Normal and Lipopolysaccharide Treated Animal

Background: C-C motif chemokine ligand 2 (CCL2) is reported to be involved in the pathogenesis of various neurological and/or psychiatric diseases. Tissue or cellular expression of CCL2, in normal or pathological condition, may play an essential role in recruiting of monocytes or macrophages into the targeted organs, and be involved in a certain pathogenic mechanism. However, only a few studies focused on tissue and cellular distribution of the CCL2 peptide in the brain’s grey and white matters (GM, WM), and the changes of the GM and WM cellular CCL2 level in septic or endotoxic encephalopathy was not explored. Hence, the CCL2 cellular distribution in the front brain cortex and the corpus callosum (CC) WM was investigated in the present work by using immunofluorescent staining. Results: 1) Normally, CCL2 like immunoreactivity (CCL2-ir) in the CC is significantly higher than the cortex, especially when the measurement includes ependymal layer attached to the CC. 2) Structures surrounding the vasculatures contribute major CCL2-ir positive profiles in both GM and WM, but significantly more in the CC WM, in which they are bilaterally distributed and predominantly located in the lateral CC between the cingulate cortex and the lateral ventricles. 3) Following systemic lipopolysaccharide (LPS), the number of neuron-like CCL2-ir positive cells are increased significantly in the cortex, but not in the CC. 4) More CCL2-ir positive elements are accumulated inside microvasculature like structures in the CC WM, compared to those found in the cortex following systemic LPS. 5) Few macrophage/microglia marker-Iba-1 labeled structures exhibit CCL2-ir in normal cortex and CC, but the co-localization is significantly increased following systemic LPS. 6) Following saline or LPS injection, CCL2-ir and GFAP or Iba-1 double labeled structures are observed within the ependymal layer between the lateral ventricles and the CC. No accumulation of neutrophils was detected.Conclusion: there exist differences in the cellular distribution of the CCL2 peptide in the front brain cortex GM and the subcortical WM - the CC, in both the physiological condition and experimental endotoxemia. Which might cause different pathological change in the GM and WM.

IL-17A and CCL2 in blood serum and circulating neutrophils in patients with ovarian tumors.

e17536 Background: During the ovarian carcinogenesis, circulating neutrophils (Nph) phenotype switches to the pro-tumor, with an increase in the Nph C-C motif ligand 2 (CCL2) expression. Proinflammatory interleukin-17A (IL-17A) is able to induce the CCL2 expression in Nph. The study aimed to evaluate the IL-17A and CCL2 levels in serum and circulating Nph in patients with ovarian tumors. Methods: The study included 97 ovarian cancer (OC) patients, 30 patients with benign ovarian tumors (BT) and the control (n=22). The levels of IL-17A (LLC "Cytokine", Russia) and CCL2 (Vector-Best-Volga, Russia) in serum and Nph lysate were assessed by ELISA. The systemic inflammation was assessed by neutrophil to lymphocyte ratio (NLR). Informed voluntary consent was obtained from all women. Statistical processing was performed using one-way ANOVA, Spearman correlations (Statistica 13.0 (TIBCO, USA)). Results: We found an increase in NLR in BT (4.3 ± 1.0) compared with the control (2.2 ± 0.1) (p = 0.020), a decrease in stage I-II OC (2.4 ± 0.2) compared with BT (p = 0.053), and an increase in stage III (4.5 ± 0.9) and IV (4.6 ± 0.5) compared with stage I-II OC (p = 0.054 and p = 0.046, respectively). The cytokines levels are presented in Table. The serum level of IL-17A in BT was higher than in the control (p = 0.010). In stage I-II OC, the serum IL-17A level was decreased in comparison with the BT (p = 0.004). In stage IV OC, the serum IL-17A was higher than in stage I-II (p = 0.015) and the control (p = 0.017). The level of IL-17A in Nph in BT did not differ from the control. In all stages of the OC, the IL-17A Nph level was lower than in the control (p1, 2, 3 = 0.0001). In stage IV OC, the expression of IL-17A in Nph was lower compared with stage III (p = 0.031). The serum and Nph levels of IL-17A were correlated only in stage I-II OC (r = 0.679, p = 0.011). The CCL2 serum levels in BT (p = 0.0002) and OC stage I-II, III, IV were higher compared with the control (p1 = 0.031, p2 = 0.001, p3 = 0.0005, respectively). The Nph CCL2 level was higher in BT than in the control (p = 0.001). In stage I-II OC, the expression of CCL2 in Nph was higher than in the control (p = 0.034), but lower than in the BT (p = 0.003). In OC stages III and IV, the Nph CCL2 level was higher than in the control, but did not differ from that in BT and stages I-II. In stage III OC, the CCL2 level in Nph negatively correlated with the serum IL-17A level (r = -0.405, p = 0.049). Conclusions: The pro-tumor polarization of circulating Nph is not IL-17A-dependent in patients with ovarian tumors. The patterns of the systemic immune response differ in benign ovarian tumors, early, and advanced ovarian cancer.[Table: see text]

Potential Role of Macrophage Phenotypes and CCL2 in the Pathogenesis of Takayasu Arteritis

ObjectivesTo investigate vascular macrophage phenotype as well as vascular and peripheral chemokine (C-C motif) ligand 2 (CCL2) expression during different stages of disease progression in patients with Takayasu Arteritis (TA).MethodsIn this study, 74 patients with TA and 50 controls were recruited. TA disease activity was evaluated with Kerr scores. Macrophage phenotype and CCL2 expression were examined by immunohistochemistry in vascular specimens from 8 untreated and 7 treated TA patients, along with 4 healthy controls. Serum CCL2 were quantified by enzyme-linked immune-absorbent assay from TA patients at baseline (n=59), at 6-months (n=38), and from 46 healthy volunteers. Vascular macrophage phenotype, vascular CCL2 expression and serum CCL2 levels during different stages, as well as the relationship between serum CCL2 and disease activity or other inflammatory parameters (erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and interleukin 6 (IL-6)) were investigated.ResultsIn untreated patients, vascular M1 macrophages and CCL2 showed increased expression, mainly in the adventitia. In contrast, in treated patients, vascular adventitial M1 and CCL2 expression were decreased, while vascular medial M2 macrophages and CCL2 levels were increased. Distribution of macrophages and CCL2 was consistent within the TA vascular lesions regardless of the disease stage. Furthermore, peripheral CCL2 was elevated in patients with TA (TA: 160.30 ± 120.05 vs. Control: 65.58 ± 54.56 pg/ml, P < 0.001). CCL2 levels were found to correlate with ESR, CRP, and IL-6 (all R values between 0.55 and 0.6, all P < 0.001). Receiver operating curve analysis demonstrated that CCL2 (at the cut-off value of 100.36 pg/ml) was able to predict disease activity (area under the curve = 0.74, P = 0.03). Decrease in CCL2 level was observed in patients with clinical remission (CR), but not in patients without CR, after 6 months of treatment (CR patients: baseline 220.18 ± 222.69 vs. post-treatment 88.71 ± 55.89 pg/ml, P = 0.04; non-CR patients: baseline 142.45 ± 104.76 vs. post-treatment 279.49 ± 229.46 pg/ml, P = 0.02).ConclusionsMacrophages contribute to vascular pathological changes in TA by undergoing phenotype transformation. CCL2 is an important factor for recruiting macrophages and a potential biomarker for disease activity.

MicroRNA-874-3p/ADAM (A Disintegrin and Metalloprotease) 19 Mediates Macrophage Activation and Renal Fibrosis After Acute Kidney Injury

Inflammation and maladaptive repair play a crucial role in the development of chronic kidney disease and hypertension after acute kidney injury. To study the mechanisms involved in acute kidney injury-to-chronic kidney disease transition, we established a chronic renal fibrosis mouse model that was triggered by an initial ischemia/reperfusion–induced acute kidney injury (acute-chronic model). Downregulation of microRNA-874-3p during renal fibrosis was identified by a genome-wide RNA-sequencing and was further confirmed in cell-based assays, mouse models, and human samples. Overexpression of microRNA-874-3p in the kidneys markedly alleviated renal fibrosis, accompanied with decreased infiltrated macrophages and expression of α-smooth muscle actin, type I collagen, fibronectin, CCL (C-C motif chemokine ligand) 2, and ADAM (A Disintegrin and Metalloprotease) 19. ADAM19 is a target gene of microRNA-874-3p as shown by luciferase reporter assays and was upregulated in the acute-chronic model. Overexpression of ADAM19 directly induced the expression of fibrotic genes, CCL2, and macrophage infiltration in vivo. Depletion of macrophages using clodronate liposomes ameliorated the fibrogenic effects of ADAM19. Overexpression of ADAM19 also induced accumulation of the Notch1 intracellular domain, an upstream regulator of CCL2 expression, whereas Notch1 pathway antagonist N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester reduced CCL2 level in ADAM19-overexpressed cells. Collectively, microRNA-874-3p/ADAM19 mediates renal fibrosis after acute kidney injury by increasing macrophage infiltration via the Notch1/CCL2 pathway.

Exploring the Role of C-C Motif Chemokine Ligand-2 Single Nucleotide Polymorphism in Pulmonary Tuberculosis: A Genetic Association Study from North India

The C-C motif chemokine ligand-2 (CCL2) was evidenced to be associated with tuberculosis susceptibility in some ethnic groups. In the present study, effort was made to find out the association of CCL2-2518 A>G and -362 G>C variants with susceptibility to TB in a population from North India. The genotyping was carried out in 373 participants with pulmonary TB (PTB) and 248 healthy controls (HCs) for CCL2-2518 A>G and -362 G>C polymorphisms by PCR-RFLP and by melting curve analysis using fluorescence-labeled hybridization fluorescent resonance energy transfer (FRET) probes, respectively, followed by DNA sequencing in a few representative samples. Genotype and allele frequencies were compared by the chi-squared test and crude and Mantel-Haenszel (M-H) odds ratio (OR). OR was calculated using STATA/MP16.1 software. Further, CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-β levels were measured in serum samples of these participants using commercially available kits. Our analysis indicated that the homozygous mutant in both -2518 GG ( OR = 2.07 , p = 0.02 ) and -362 CC ( OR = 1.92 , p = 0.03 ) genotypes was associated with susceptibility to pulmonary TB. Further, heterozygous genotypes -2518AG ( OR = 0.60 , p = 0.003 ) and -362GC ( OR = 0.64 , p = 0.013 ) provide resistance from PTB disease. Haplotype analysis revealed AC haplotype ( p = 0.006 ) to be a risk factor associated with PTB susceptibility. The serum CCL2 level was significantly elevated among participants with -2518 AA genotype compared to -2518 GG genotype. CCL2 level was observed to be positively correlated with IL12p70, IFN-γ and TNF-α, thus suggesting the immunological regulatory role of CCL2 against pulmonary tuberculosis. CCL2-2518 GG and -362 CC genotypes were found to be associated with susceptibility to pulmonary tuberculosis and CCL2-2518AG and CCL2-362GC with resistance from PTB. AC haplotype was found to be a risk factor for PTB in the present study. It may be hypothesized from the findings that -2518G allele could be responsible for lower production of CCL2 which leads to defective Th1 response and makes a host susceptible for pulmonary tuberculosis.

FRI0012 THERAPEUTIC VALUE OF CURCUMIN ON INITIATION AND DEVELOPMENT OF INFLAMMATION IN TAKAYASU’S ARTERITIS CAUSED BY HSP65-MEDIATED CCL2 OVEREXPRESSION

Background:Takayasu’s arteritis (TA) is a chronic inflammatory disease characterized with macrophages infiltration. During active stage, aorta adventitial fibroblasts (AAFs) proliferate excessively and produce numerous pro-inflammatory factors in the adventitia, which is the main target of TA therapy. Monocyte chemokine CCL2 may contribute to the infiltration of macrophages in TA arteries[1]but whether with relationship with HSP65, an antigen of Mycobacterium tuberculosis (M. TB) which might involve in the pathogenesis of TA[2]and activate AAFs to produce inflammatory factors, has not been reported. The treatment of TA is full of difficulties and contradictions[3]. Curcumin is a traditional Chinese medicine with anti-inflammatory effect[4], whether it is effective on TA and the underlying mechanism remains unclear.Objectives:To explore the mechanism of TA inflammation triggered by M. TB associated antigen HSP65 activating AAFs, as well as the therapeutic value of curcumin in the initiation and development of TA.Methods:We first verified high HSP65 expression in aortic adventitia of TA patients by IHC. mRNA-seq was used to profile DEGs between AAFs stimulated by HSP65 with or without pretreated with curcumin, and AAFs without any treatment. Then the key chemokine CCL2 screened by mRNA-seq was detected in the adventitia of TA aorta, and its correlation with HSP65 expression was analyzed by double-labelled IF. Subsequently, we explored how HSP65 affected the production of inflammatory factors by AAFs at cellular level and its related signal pathway. Simultaneously, we explored whether curcumin could hinder this process. and verified the effect of curcumin on serum CCL2 level in patients with TA. Finally, serum CCL2 and other inflammation indicators of TA patients at baseline and after 3 months treatment by curcumin were determined.Results:HSP65 was highly expressed in the adventitia of TA arteries. DEGs analysis showed a key role of CCL2. The expression of CCL2 in adventitia of TA arteries was significantly higher than healthy subjects, and was correlated with HSP65. HSP65 facilitated the production of CCL2, IL-6 and IL-1β by AAFs via activating TLR4-JAK2/AKT/STAT3 pathway, among which the change of CCL2 was the most remarkable. Curcumin reversed the upregulation of CCL2 induced by HSP65 in vitro, which was more obvious than that of MTX and tofacitinib. Finally, curcumin significantly downregulated the level of serum CCL2 of TA patients.Conclusion:HSP65 initiates and promotes inflammation of TA by upregulated CCL2 in AAFs through JAK/AKT/STAT3 pathway, while curcumin can reverse this process and slow down the initiation and development of TA.References:[1]L, A., J, H., A, M., G, G. & Z, A. Pathogenesis of Takayasu’s arteritis: a 2011 update.Autoimmunity reviews11, 61-67 (2011).[2]Y, S., et al.Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu’s arteritis.The Journal of clinical investigation93, 750-758 (1994).[3]L, B., G, Y. & C, P. Non-glucocorticoid drugs for the treatment of Takayasu’s arteritis: A systematic review and meta-analysis. Autoimmunity reviews 17, 683-693 (2018).[4]T, E., et al.Curcumin--from molecule to biological function.Angewandte Chemie (International ed. In English)51, 5308-5332 (2012).Figure A.High expression of HSP65 and CCL2 in aortic adventitia of TA patients (n=8) than that of healthy controls (n=6).Figure B.HSP65 increased production of CCL2 in AAFs through TLR4/JAK2-STAT3 pathway.Figure C.Curcumin reversed inflammatory response initiated by HSP65 via inhibiting JAK2/AKT/STAT3 signal pathway in AAFs and significantly reduced serum CCL2 concentration of TA patients.Acknowledgments:We thank Ningli Li for her technical support in this studyDisclosure of Interests:None declared

Study on plasma CC chemokine ligand 2 level and its promoter region 2518A/G polymorphism in MS patients

The increase of CC chemokine ligand 2 (CCL2) is associated with multiple sclerosis (MS), but the relationship between gene promoter region 2518A/G and the pathogenesis of MS is still not obvious. Collected 54 cases of relapsing-remitting MS patients and 54 healthy controls. By detecting the CCL2-2518A/G polymorphism of MS patients and analyzing the plasma CCL2 level. High levels of A/A genotype and A allele frequency in serum CCL2 and PBMC were found in MS patients. The serum CCL2 of MS patients with A/A genotype is higher than other genotypes. Lipopolysaccharide stimulated PBMC, CCL2 levels in the supernatant of all genotypes were higher, and the A/A genotype levels of MS patients were the highest. Finally, CCL2-2518A/G polymorphism is related to the pathogenesis of MS.

Potential Role of Carvedilol in the Cardiac Immune Response Induced by Experimental Infection with Trypanosoma cruzi

Trypanosoma cruzi causes a cardiac infection characterized by an inflammatory imbalance that could become the inciting factor of the illness. To this end, we evaluated the role of carvedilol, a beta-blocker with potential immunomodulatory properties, on the immune response in C57BL/6 mice infected with VL-10 strain of T. cruzi in the acute phase. Animals (n=40) were grouped: (i) not infected, (ii) infected, (iii) infected + carvedilol, and (iv) not infected + carvedilol. We analyzed parameters related to parasitemia, plasma levels of TNF, IL-10, and CCL2, and cardiac histopathology after the administration of carvedilol for 30 days. We did not observe differences in the maximum peaks of parasitemia in the day of their detection among the groups. The plasma TNF was elevated at 60 days of infection in mice treated or not with carvedilol. However, we observed a decreased CCL2 level and increased IL-10 levels in those infected animals treated with carvedilol, which impacted the reduction of the inflammatory infiltration in cardiac tissue. For this experimental model, carvedilol therapy was not able to alter the levels of circulating parasites but modulates the pattern of CCL2 and IL-10 mediators when the VL10 strain of T. cruzi was used in C57BL6 mice.