Higher Levels Of CCL2 Research Articles

CCL3 predicts exceptional response to TGFβ inhibition in basal-like pancreatic cancer enriched in LIF-producing macrophages

The TGFβ receptor inhibitor galunisertib showed promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the phase 2 H9H-MC-JBAJ study. Identifying biomarkers for this treatment remains essential. Baseline plasma levels of chemokine CCL3 were integrated with clinical outcomes in PDAC patients treated with galunisertib plus gemcitabine (n = 104) or placebo plus gemcitabine (n = 52). High CCL3 was a poor prognostic factor in the placebo group (mOS 3.6 vs. 10.1 months; p < 0.01) but a positive predictor for galunisertib (mOS 9.2 vs. 3.6 months; p < 0.01). Mechanistically, tumor-derived CCL3 activates Tgfβ signaling in macrophages, inducing their M2 phenotype and Lif secretion, sustaining a mesenchymal/basal-like ecotype. TGFβ inhibition redirects macrophage polarization to M1, reducing Lif and shifting PDAC cells to a more epithelial/classical phenotype, improving gemcitabine sensitivity. This study supports exploring TGFβ-targeting agents in PDAC with a mesenchymal/basal-like ecotype driven by high CCL3 levels.

Expression of immune-related genes and breast cancer recurrence in women with ductal carcinoma in situ

Background and aimDuctal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer with highly variable clinical behavior, but risk stratification is still challenging. We sought to identify immune-related gene expression signatures of pure DCIS associated with different risks of breast cancer recurrence. MethodsA retrospective nested case-control study of 143 pure DCIS was performed including 70 women with subsequent ipsilateral breast event (IBE, in situ or invasive; cases) and 73 DCIS women with no IBE and matched for age, tumor size, treatment, hormone receptors/HER2 status, and follow-up time (controls). RNA was extracted from DCIS samples and subjected to next-generation sequencing gene expression analysis of 395 immune-related genes. Correlations between DCIS immune-related gene expression and IBE were analyzed using weighted Cox regression for nested case-control data. ResultsEight immune-related genes were differentially expressed between cases and controls. MAGEA10 expression (present vs. absent) and high expression levels of IFNA17 and CBLB (Q4 vs. Q1) were observed more frequently in DCIS of women with subsequent IBE, mainly invasive (p-valueFDR < 0.05). Conversely, expression of IL3RA1, TAGAP, TNFAIP8, and high expression levels of CCL2 and LRP1 were associated with a lower risk of IBE (p-valueFDR < 0.05). ConclusionThis exploratory analysis of pure DCIS showed significant differences in immune-related gene expression profiles between women with and with no subsequent IBE, particularly as invasive IBE. These results, after additional validation, could improve risk stratification and management of DCIS patients.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

Evaluation of plasma CCL3 levels in individuals with newly diagnosed chronic lymphocytic leukemia and it’s correlation with clinical and laboratory parameters

Abstract: BACKGROUND: The most prevalent type of leukemia in adults is called chronic lymphocytic leukemia (CLL). is a lymphoproliferative condition is defined by the mature clone’s expansion in blood, secondary lymphoid tissues, spleen and the bone marrow. CCL3 is a cytokine that is involved in development, repair, and the acute inflammatory state. CCL3, therefore, should become useful for risk assessment in patients with CLL. OBJECTIVES: The aims of the study were to estimate the level of plasma CCL3 in newly diagnosed patients with CLL matched to healthy controls and correlate it with hematological parameters and clinical parameters and prognostic markers such as CD38 and LAIR-1. PATIENTS, MATERIALS AND METHODS: This study was cross-sectional in nature. Patient group included 55 patients with newly established diagnosis of CLL proven by morphology and immunophenotyping and control group included sex and age matched of 30 healthy individuals. The plasma CCL3 levels were measured using an enzyme-linked immunosorbent assay. Microsoft Office Excel 2019 and SPSS version 26 were used for the statistical analysis. The mean and standard deviation of normally distributed numerical values whereas the terms “median” and “range” were used to describe numerical values that are not regularly distributed. P &lt;0.05 was used to determine the degree of significance. RESULTS: There were statistically significant relationships between CCL3 of patient (median level about 766.42 ng/ml) and control (median level about 453.35 ng/ml) groups (P &lt; 0.001). There was a strong correlation between CCL3 and white blood cell, hemoglobin, and absolute lymphocyte count (P = 0.013, P &lt; 0.001, and P = 0.011, respectively). There was a strong relationship between CCL3 expression with Binet stage and with hepatomegaly (P &lt; 0.001 and P = 0.020, respectively), but no significant relation between CCL3 expression and CD38 and LAIR-1. CONCLUSIONS: Patients with CLL had high levels of CCL3, which increased with advancing Binet stages. Therefore, our research clarified the useful and simplicity of measurement of CCL3 plasma levels for follow-up in CLL patients and risk assessment.

Dissecting the cytomegalovirus CC chemokine: Chemokine activity and gHgLchemokine-dependent cell tropism are independent players in CMV infection.

Like all herpesviruses, cytomegaloviruses (CMVs) code for many immunomodulatory proteins including chemokines. The human cytomegalovirus (HCMV) CC chemokine pUL128 has a dual role in the infection cycle. On one hand, it forms the pentameric receptor-binding complex gHgLpUL(128,130,131A), which is crucial for the broad cell tropism of HCMV. On the other hand, it is an active chemokine that attracts leukocytes and shapes their activation. All animal CMVs studied so far have functionally homologous CC chemokines. In murine cytomegalovirus (MCMV), the CC chemokine is encoded by the m131/m129 reading frames. The MCMV CC chemokine is called MCK2 and forms a trimeric gHgLMCK2 entry complex. Here, we have generated MCK2 mutant viruses either unable to form gHgLMCK2 complexes, lacking the chemokine function or lacking both functions. By using these viruses, we could demonstrate that gHgLMCK2-dependent entry and MCK2 chemokine activity are independent functions of MCK2 in vitro and in vivo. The gHgLMCK2 complex promotes the tropism for leukocytes like macrophages and dendritic cells and secures high titers in salivary glands in MCMV-infected mice independent of the chemokine activity of MCK2. In contrast, reduced early antiviral T cell responses in MCMV-infected mice are dependent on MCK2 being an active chemokine and do not require the formation of gHgLMCK2 complexes. High levels of CCL2 and IFN-γ in spleens of infected mice and MCMV virulence depend on both, the formation of gHgLMCK2 complexes and the MCK2 chemokine activity. Thus, independent and concerted functions of MCK2 serving as chemokine and part of a gHgL entry complex shape antiviral immunity and virus dissemination.

Cytokines profile in pure neural leprosy.

Pure Neural Leprosy (PNL) is a form of this long time known disease that affects only the peripheral nervous system. Since it is a rare form of the disease, its pathophisiology is still poorly understood. Describe the cytokines profile in patients with PNL. 30 Patients diagnosed with PNL in the Souza Araujo Outpatient Clinic and with cytokines evaluated were selected. They were evaluated by neurologists and diagnosed after a nerve biopsy. Serum levels of IL-1 β, IL-6, IL-10, IL-17, TNF, CCL-2/MCP-1, IFN-ϒ, CXCL-10/IP-10 and TGF-β were evaluates at the moment of the diagnosis. Neural thickening was a common clinical finding in this groups of patients. Small and medium sensitive fibers signs and symptoms were present in 92% of the patients and motor involvement in 53%. 43% of patients presented neuropathic pain and no one had neuritis TGF-beta, IL-17, CCl-2 and IP-10. CCL-2 levels were associated with demyelinating patters and IP-10 and IL-1o were associated with axonal patterns at NCS. PNL patients' cytokine profile appears to be different of other clinical forms of leprosy, with the presence of cytokines described in both tuberculoid and lepromatous leprosy. High levels of CCl-2 may be related to the presence of silent neuritis as well as the presence of IL-10. PNL is unique a form of leprosy, therefore, understanding its immunological profiles essential to better understand the disease itself.

Characterizing the lavage and serum cytokine profiles of interstitial pneumonia with autoimmune features and their implications for progressive fibrosis.

To explore whether cytokines could be potential biomarkers to predict the occurrence of the progressive fibrosis (PF) phenotype among patients with interstitial pneumonia with autoimmune features (IPAF). This study prospectively collected 51 IPAF and 15 idiopathic pulmonary fibrosis (IPF) patients who were diagnosed at the First Affiliated Hospital of Guangzhou Medical University from July 2020 to June 2021. All IPAF patients were followed up for 1 year to assess the development of PF phenotype. Paired bronchoalveolar lavage fluid (BALF) and serum samples were collected at enrolment and analysed for differences in 39 cytokines expression. Principal component analysis (PCA) and cluster analysis were conducted to identify a subgroup of IPAF patients at high risk for developing the PF phenotype. Finally, cytokine differences were compared between subgroups to identify potential biomarkers for PF-IPAF occurrence. According to the PCA analysis, 81.25% of PF-IPAF patients share overlapped BALF cytokine profiles with IPF. Cluster analysis indicated that IPAF patients in subtype 2 had a higher risk of developing the PF phenotype within 1 year (P = 0.048), characterized by higher levels of CCL2 and CXCL12, and lower lymphocyte proportion (LYM%) in BALF. Elevated levels of BALF CCL2 (>299.16 pg/ml) or CXCL12 (>660.115 pg/ml)were associated with a significantly higher risk of developing PF phenotype within the 1-year follow-up period (P = 0.009, 0.001, respectively). PF-IPAF phenotype exhibits similar inflammatory cytokine profiles to IPF. Cytokine CCL2 and CXCL12, and LYM% in BALF serveas potential biomarkers for predicting the PF phenotype in IPAF patients. Register: Qian Han, Website: http://www.chictr.org.cn/showproj.aspx?proj=61619, Registration number: ChiCTR2000040998.

Suppression of microglial Ccl2 reduces neuropathic pain associated with chronic spinal compression.

Chronic spinal compression is a common complication of spinal cord injury (SCI), which can lead to spinal stenosis or herniated discs. The ensuing neuropathic pain is often associated with the activation of microglia. In this investigation, our objective was to explore whether modifying the levels of chemokine (C-C motif) ligand 2 (Ccl2) in microglia could alleviate neuropathic pain resulting from chronic spinal compression. We used a public database to look for major altered gene associated in a SCI model established in rats. We then employed adeno-associated virus (AAV) vectors, expressing siRNA for the identified significantly altered gene under a microglia-specific TMEM119 promoter. We also tested the impact of this treatment in microglia in vivo on the severity of chronic spinal compression and associated pain using a ttw mouse model for progressive spinal compression. We identified chemokine (C-C motif) ligand 2 (Ccl2) as the primary gene altered in microglia within a rat SCI model, utilizing a public database. Microglial Ccl2 levels were then found to be significantly elevated in disc specimens from SCI patients diagnosed with chronic spinal compression and strongly correlated with the Thompson classification of the degeneration level and pain score. Depletion of Ccl2 in microglia-specific TMEM119 promoter were developed to transfect mouse microglia in vitro, resulting in a proinflammatory to anti-inflammatory phenotypic adaption. In vivo depletion of Ccl2 in microglia mitigated the severity of chronic spinal compression and related pain in ttw mice, likely due to significant changes in pain-associated cytokines and factors. Disc microglia expressing high levels of Ccl2 may contribute to chronic spinal compression and SCI-associated pain. Therapeutically targeting Ccl2 in microglia could offer a potential avenue for treating chronic spinal compression and SCI-associated pain.

A Lasso Cox score consisting of CTLA4+ regulatory T cells (Treg), monocytic myeloid derived suppressor cells (M-MDSC), and CXCR3+ CD8+ T cells: Association with adjuvant ipilimumab (ipi) survival outcomes in patients (pts) with high-risk melanoma.

e21557 Background: High-risk melanoma pts treated with ipi had a more favorable outcome than those treated with interferon-α2b (IFN) in phase III adjuvant trial E1609. We hypothesized that further profiling of circulating immune cells and cytokines would inform underlying mechanisms associated with immune response vs. resistance to CTLA4 blockade. Methods: Multiparameter flow cytometry was used to compare surface protein expression on PBMC collected at baseline, focusing on Treg, MDSC and effector T cells (N = 210 ipi, N = 119 IFN). Multiplex Luminex assays were used to test baseline serum for concentrations of candidate cytokines, chemokines and growth factors. A survival analysis tool, DRPPM-PATH-SURVEIOR, was then used to perform a univariate screening of these analytes using a Cox proportional hazards (PH) model. A median cut-point was derived for each marker, and pts were split into above or below median cohorts. The dichotomized scores were then processed through a Cox regression analysis. Features were ranked based on p-values calculated by a likelihood test for overall survival (OS) and recurrence-free survival (RFS). A Lasso Cox regression model was further developed using Glmnet by optimizing lambda at minimum mean cross-validated error. Pts were randomly and evenly split into training and testing datasets. Results: Initial univariate Cox PH regression model screening identified 6 cellular and 2 serum protein biomarkers associated with OS and RFS. Enriched populations of CTLA4+ Treg (CD3+/CD4+/CD25hi+/CD152+) and monocytic (M)-MDSC (Lin-CD33+/HLA-DRlo+/CD14+/CD15+) were identified as being associated with poor outcome, while enriched populations of CXCR3+/CD4+ T cells, CXCR3+/CD8+ T cells, CTLA4+/INFg+/CD8+ T cells and CD39+ Treg (CD3+/CD4+/CD25hi+/CD39+) were associated with better outcome. Higher levels of CCL3 and CXCL11 chemokines in serum were associated with improved OS and RFS. To develop a prognostic model predictive of ipi treatment benefits, we focused on top ranked biomarkers and generated and tested a Lasso Cox regression model based on CTLA4+ Treg, M-MDSC and CXCR3+/CD8+ T cell populations using a training subset (N = 105 ipi pts). This model was predictive of pt OS and RFS in the testing subset (N = 105) with C-index = 0.67. To examine broader applicability of our Lasso Cox model, we analyzed an additional 119 pts treated on the IFN control arm. By incorporating treatment arm as an interaction term with our 3 biomarker Lasso Cox score, our model was determined to be predictive of risk in pts treated with ipi but not IFN. Conclusions: Our analysis confirms the predictive value of a 3 biomarker Lasso Cox score predicated on CTLA4+ Treg, M-MDSC and CXCR3+/CD8+ T cell content in patient blood at baseline as it relates to adjuvant ipi treatment outcomes.

Abstract 284: Pro-Inflammatory Adventitial Fibroblast Contribute To Aortic Aneurysms In Loeys Dietz Syndrome

Introduction: Vascular smooth muscle cell (SMC) phenotype modulation is implicated in thoracic aneurysm formation, however, the role of adventitial fibroblasts (AdvFib) in aneurysm formation remains poorly characterized. Loeys-Dietz Syndrome (LDS), caused by a spectrum of mutations in the TGF-β signaling cascade, leads to aggressive thoracic aneurysms. Given the ubiquitous nature of TGF-β signaling machinery, the exact cell type(s) responsible for initiation of LDS pathogenesis is poorly understood. Methods: We performed single cell transcriptomic characterization of aortic root and ascending aortic samples from LDS patients ((n=6 LDS (harboring TGFB2 , TGFBR1 , TGFBR2 , or SMAD3 mutation); n=2 Donor control)) as well as root/ascending aorta from a murine model of LDS ( Tgfbr2 G357W/+ ) to understand cell-state transitions and transcriptomic alterations in LDS. Results: Single cell RNA sequencing (scRNAseq) of human and mice LDS samples (in total &gt;40,000 cells) revealed a secondary pattern of cellular activation in LDS as compared to Marfan Syndrome. Instead of SMC phenotypic modulation as seen in MFS, LDS appears to preferentially modulate adventitial fibroblasts (AdvFib) phenotype in the Tgfbr2 G357W/+ mouse model. Human LDS patient’s AdvFib turn on high levels of macrophage-recruiting pro-inflammatory cytokines CCL2 and HLA-B, with a corresponding 1.8-fold increase in LDS aortic wall macrophages compared to Donor. A more extreme pattern was observed in the Tgfbr2 G357W/+ LDS mouse model. While Tgfbr2 G357W/+ SMC are transcriptomically similar to wild type counterparts, AdvFib were activated into a pro-inflammatory state associated with increased aortic macrophage recruitment ( Ccl2 , Il6 , Vcam1, and Cxcl2 ) and fibrotic response ( Col1a1 , Col1a2 , Col3a1, and Fn1 ). A 6-fold increase in the number of aortic wall macrophages was identified in Tgfbr2 G357W/+ compared to WT. Conclusions: LDS mouse and human AdvFib undergo phenotypic alterations into a pro-inflammatory state evidenced by increased expression of numerous cytokines, macrophage recruitment, and ECM deposition. AdvFib, in addition to SMCs, are potentially another important pathological cell population in LDS aneurysm formation.

Exploring the Profile of Cell Populations and Soluble Immunological Mediators in Bothrops atrox Envenomations.

Bothrops atrox envenomations are common in the Brazilian Amazon. The venom of B. atrox is highly inflammatory, which results in severe local complications, including the formation of blisters. Moreover, there is little information on the immune mechanisms associated with this condition. Thus, a longitudinal study was carried out to characterize the profile of the cell populations and soluble immunological mediators in the peripheral blood and blisters in B. atrox patients s according to their clinical manifestations (mild and severe). A similar response in both B. atrox patient groups (MILD and SEV) was observed, with an increase in inflammatory monocytes, NKT, and T and B cells, as well as CCL2, CCL5, CXCL9, CXCL10, IL-1β and IL-10, when compared with the group of healthy blood donors. After the administration of antivenom, the participation of patrolling monocytes and IL-10 in the MILD group was observed. In the SEV group, the participation of B cells was observed, with high levels of CCL2 and IL-6. In the blister exudate, a hyperinflammatory profile was observed. In conclusion, we revealed the involvement of cell populations and soluble mediators in the immune response to B. atrox envenomation at the local and peripheral level, which is related to the onset and extent of the inflammation/clinical manifestation.

Expression Profiles of Hepatic Immune Response Genes in HEV Infection.

Hepatitis E is a liver inflammation caused by infection with the hepatitis E virus (HEV). Every year, there are an estimated 20 million HEV infections worldwide, leading to an estimated 3.3 million symptomatic cases of hepatitis E. HEV viral load has been studied about the disease progression; however, hepatic the host gene expression against HEV infection remains unknown. Methods: We identified the expression profiles of hepatic immune response genes in HEV infections. Fresh blood samples were collected from all the study subjects (130 patients and 124 controls) in 3ml EDTA vacutainers. HEV viral load was determined by a real-time PCR. The total RNA was isolated from the blood using the TRIZOL method. The expression of theCCL2, CCL5, CXCL10, CXCL16, TNF, IFNGR1, and SAMSN1 genes was studied in the blood of 130 HEV patients and 124 controls using a real-time PCR. Results: Gene expression profiles indicate high levels of CCL2, CCL5, CXCL10, CXCL16, TNF, IFNGR1, and SAMSN1 genes that might lead to the recruitment of leukocytes and infected cell apoptosis. Conclusion: Our study demonstrated distinct differences in the expression profiles of host immune response-related genes of HEV infections and provided valuable insight into the potential impact of these genes on disease progression.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Abstract 11486: Hormone-Independent Sex Dimorphisms in Cardiac Reparative Functions of Bone Marrow Stem Cells: Underlying Epigenetic Insights

Introduction: This work was designed to identify the hormone-independent mechanisms that govern the superiority of female endothelial progenitor cells (F-EPC) for cardiac repair post myocardial infarction (MI). A direct comparison of therapeutic efficacy of gender-specific stem cells or estrogen-independent mechanisms of gender-specific dimorphism in the reparative properties of EPCs has not been established. Hypothesis: We hypothesized that epigenetic mechanisms contribute to the sex-specific functional dimorphisms of Sca-1+/CD31+ EPCs in regulating cardiac reparative functions post-MI. Methods: Male (M), female (F), and ovariectomized (OVX) mice derived Sca-1+/CD31+ EPCs were subjected through a series of cytokine quantifications and epigenetic CUT&amp;Tag screening, followed by assessment of their therapeutic function in vitro and in vivo . Results: F-EPC and OVX EPC secrete high levels of proangiogenic factors and low amounts of pro-inflammatory cytokines compared to M-EPC. Further evaluation of the inflammasome showed that the M-EPC secreted the most elevated amounts of CCL3, and other inflammatory chemokines compared to F-EPC and OVX EPC. The secretome of F-EPC and OVX EPC was found to be substantially proangiogenic in vitro than the male EPCs. Post-MI injection of F-EPC and OVX EPC resulted in remarkable preservation of cardiac structure and functions. In contrast, post-MI injection of M-EPC resulted in an inflamed myocardium. Epigenetic sequencing of the sex differential EPCs for the H3K9me3 mark showed high methylation in M-EPC compared to F-EPC and OVX EPC. The inhibition of histone methyltransferase, G9a using BIX-01294 upregulated the secretion of inflammatory factors in all EPCs. The conditioned medium from all EPCs with high levels of CCL3 inhibited angiogenesis in vitro . Neutralizing CCL3 in the same medium restored in vitro angiogenesis. Conclusion: EPCs from both fertile and OVX female mice retain better cardiac reparative activity than their male counterparts irrespective of sex hormone influence. Mechanistically, we show that this gender-based functional dimorphism of EPCs is mediated, in part, by differential epigenetic regulation of chemokine CCL3.

Long-term effect of childhood trauma: Role of inflammation and white matter in mood disorders

Bipolar disorder (BD) and major depressive disorder (MDD) are severe psychiatric illnesses that share among their environmental risk factors the exposure to adverse childhood experiences (ACE). Exposure to ACE has been associated with long-term changes in brain structure and the immune response. In the lasts decades, brain abnormalities including alterations of white matter (WM) microstructure and higher levels of peripheral immune/inflammatory markers have been reported in BD and MDD and an association between inflammation and WM microstructure has been shown. However, differences in these measures have been reported by comparing the two diagnostic groups. The aim of the present study was to investigate the interplay between ACE, inflammation, and WM in BD and MDD. We hypothesize that inflammation will mediate the association between ACE and WM and that this will be different in the two groups. A sample of 200 patients (100 BD, 100 MDD) underwent 3T MRI scan and ACE assessment through Childhood Trauma Questionnaire. A subgroup of 130 patients (75 MDD and 55 BD) underwent blood sampling for the assessment of immune/inflammatory markers. We observed that ACE associated with higher peripheral levels of IL-2, IL-17, bFGF, IFN-γ, TNF-α, CCL3, CCL4, CCL5, and PDGF-BB only in the BD group. Further, higher levels of CCL3 and IL-2 associated with lower FA in BD. ACE were found to differently affect WM microstructure in the two diagnostic groups and to be negatively associated with FA and AD in BD patients. Mediation analyses showed a significant indirect effect of ACE on WM microstructure mediated by IL-2. Our findings suggest that inflammation may mediate the detrimental effect of early experiences on brain structure and different mechanisms underlying brain alterations in BD and MDD.

Microglia-derived CCL2 has a prime role in neocortex neuroinflammation

BackgroundIn myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), several areas of demyelination are detectable in mouse cerebral cortex, where neuroinflammation events are associated with scarce inflammatory infiltrates and blood–brain barrier (BBB) impairment. In this condition, the administration of mesenchymal stem cells (MSCs) controls neuroinflammation, attenuating astrogliosis and promoting the acquisition of stem cell traits by astrocytes. To contribute to the understanding of the mechanisms involved in the pathogenesis of EAE in gray matter and in the reverting effects of MSC treatment, the neocortex of EAE-affected mice was investigated by analyzing the cellular source(s) of chemokine CCL2, a molecule involved in immune cell recruitment and BBB-microvessel leakage.MethodsThe study was carried out by immunohistochemistry (IHC) and dual RNAscope IHC/in situ hybridization methods, using astrocyte, NG2-glia, macrophage/microglia, and microglia elective markers combined with CCL2.ResultsThe results showed that in EAE-affected mice, hypertrophic microglia are the primary source of CCL2, surround the cortex neurons and the damaged BBB microvessels. In EAE-affected mice treated with MSCs, microgliosis appeared diminished very soon (6 h) after treatment, an observation that was long-lasting (tested after 10 days). This was associated with a reduced CCL2 expression and with apparently preserved/restored BBB features. In conclusion, the hallmark of EAE in the mouse neocortex is a condition of microgliosis characterized by high levels of CCL2 expression.ConclusionsThis finding supports relevant pathogenetic and clinical aspects of the human disease, while the demonstrated early control of neuroinflammation and BBB permeability exerted by treatment with MSCs may have important therapeutic implications.

Abstract P3093: Gender-specific Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells: Estrogen Independent Epigenetic Mechanisms

Introduction: Several studies, including our labs, have previously determined the role of estrogen in augmenting EPC-based cardiac repair; however, a direct comparison of therapeutic efficacy of gender-specific stem cells or estrogen-independent mechanisms of gender-specific dimorphism in the reparative properties of BM-EPCs, has not been established. Hypothesis: We hypothesized that epigenetic mechanisms contribute to the sex-specific functional dimorphism of Sca-1+/CD31+ BM-EPCs in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to the enhanced reparative function of female EPCs. Methods: To evaluate our hypothesis, we sorted the male, female and ovariectomized (OVX) mice derived Sca-1+/CD31+ BM-EPCs using MACS multi-sort method. We then subjected the BM-EPCs through a series of cytokine quantifications and epigenetic screening followed by assessment of their therapeutic function in vitro and in vivo . Results: Female and ovariectomized (OVX) female BM-EPCs secrete high levels of pro-angiogenic factors and low levels of pro-inflammatory cytokines compared to male BM-EPCs. Further evaluation of the secretome showed that the male EPCs secreted high levels of interleukins and chemokines compared to female and OVX EPCs. We found that male EPCs exclusively secreted CCL3/Mip-1α. Functional in vitro angiogenic evaluation of the EPC secretome showed higher propensity of female and OVX EPCs than the male EPCs. Post-MI injection of BM-EPCs resulted in remarkable preservation of cardiac structure and functions in both female BM-EPC groups compared to the male EPCs. Male EPC injection resulted in high inflammation in the heart tissue. Epigenetic sequencing of the BM-EPCs for the H3K9me3 mark showed high methylation in male EPCs compared to female and OVX EPCs. The inhibition of histone methyltransferase, Ehmt2/G9a using BIX-01294 upregulated the secretion of inflammatory factors in all EPCs. The conditioned medium from all EPCs with high levels of CCL3 inhibited angiogenesis in vitro . Neutralizing CCL3 in the same medium restored in vitro angiogenesis. Conclusion: Estrogen-independent epigenetic mechanisms govern the enhanced cardiac reparative properties of female BM-EPC.

Mesenchymal stem cells pretreated with proinflammatory cytokines accelerate skin wound healing by promoting macrophages migration and M2 polarization.

IntroductionNumerous studies have shown that mesenchymal stem cells (MSCs) promote cutaneous wound healing via paracrine signaling. Our previous study found that the secretome of MSCs was significantly amplified by treatment with IFN-γ and TNF-α (IT). It has been known that macrophages are involved in the initiation and termination of inflammation, secretion of growth factors, phagocytosis, cell proliferation and collagen deposition in wound, which is the key factor during wound healing. In the present study, we used a unique supernatant of MSCs from human umbilical cord-derived MSCs (UC-MSCs) pretreated with IT, designated S-IT MSCs, to explore whether S-IT MSCs have a better effect on improving wound healing by improving the biological function of macrophages than the control supernatant of MSCs (S-MSCs).MethodsIn the present study, we used a unique supernatant of MSCs pretreated with IT subcutaneously injected into a mice total skin excision. We evaluated the effect of S-IT MSCs on wound healing and the quality of wound repair via promoting macrophages migration and M2 polarization in vivo. In addition, the effect of S-IT MSCs on macrophages migration, converting toward M2 phenotype and phagocytosis were also investigated in vitro.ResultsIndeed, S-IT MSCs were found to be more potent in promoting macrophage migration, M2 polarization, phagocytosis, and promoting wound closure than S-MSCs during the wound repair. High levels of CCL2 and IL-6 were found in S-IT MSCs, which indicated that the optimization of macrophage function by S-IT MSCs may be achieved through their high expression of CCL2 and IL-6.ConclusionsOur results suggest that the beneficial paracrine effect of MSCs on wound healing can be amplified by pretreatment with IT, which may represent a new strategy for optimizing the therapeutic effect of MSCs on wound healing.

Acute kidney injury associated to COVID-19 leads to a strong unbalance of circulant immune mediators

BackgroundSevere cases of coronavirus disease 2019 (COVID-19) have increased risk for acute kidney injury (AKI). The exacerbation of the immune response seems to contribute to AKI development, but the immunopathological process is not completely understood. ObjectivesTo analyze levels of circulant immune mediators in COVID-19 patients evolving with or without AKI. We have also investigated possible associations of these mediators with viral load and clinical outcomes. MethodsThis is a longitudinal study performed with hospitalized patients with moderate to severe COVID-19. Serum levels of 27 immune mediators were measured by a multiplex immunoassay. Data were analyzed at two timepoints during the follow-up: within the first 13 days of the disease onset (early sample) and from the 14th day to death or hospital discharge (follow-up sample). ResultsWe studied 82 COVID-19 patients (59.5 ± 17.5 years, 54.9% male). Of these, 34 (41.5%) developed AKI. These patients presented higher SARS-CoV-2 viral load (P = 0.03), higher frequency of diabetes (P = 0.01) and death (P = 0.0004). Overall, AKI patients presented significantly higher and sustained levels (P < 0.05) of CCL-2, CCL-3, CCL-4, CXCL-8, CXCL-10, IFN-γ, IL-2, IL-6, TNF-α, IL-1Ra, IL-10 and VEGF. Importantly, higher levels of CCL-2, CXCL-10, IL-2, TNF-α, IL-10, FGFb, and VEGF were observed in AKI patients independently of death. ROC curves demonstrated that early alterations in CCL-2, CXCL-8, CXCL-10, IFN-γ, IL-6, IL-1Ra and IL-10 show a good predictive value regarding AKI development. Lastly, immune mediators were significantly associated with each other and with SARS-CoV-2 viral load in AKI patients. ConclusionsCOVID-19 associated AKI is accompanied by substantial alterations in circulant levels of immune mediators, which could significantly contribute to the establishment of kidney injury.

EPEN-26. Chemokine receptor blockade reverses CCL2 mediated immunosuppression and restores CAR-T cell function in posterior fossa ependymoma

Trastuzumab-based HER2 CAR-T constructs have demonstrated preclinical efficacy in medulloblastoma and are being evaluated for use in children and young adults with recurrent or refractory CNS tumors. Preliminary results demonstrate immune activation but no objective tumor response in three patients, including two patients with posterior fossa (PF)-EPN. A key finding in the serum and CSF of all three patients was very high levels of the inflammatory chemokine CCL2 following treatment with CAR-T cells. Preclinical studies suggest that high levels of CCL2 may impede T cell mediated anti-tumor activity in CNS tumors. The role of CCL2 to enhance or diminish CAR-T cell efficacy for CNS tumors is unknown. We evaluated a second generation trastuzumab-based HER2 CAR construct with a 4-1BB co-stimulatory domain in two ultra-high-risk patient-derived xenograft (PDX) models that faithfully recapitulate PFA-EPN. In contrast to preclinical studies in other cancers, treatment with trastuzumab-based HER2 CAR-T cell alone causes only partial regression of tumors and robust infiltration of immunosuppressive monocytes in PFA-EPN PDX mouse models. We studied constitutive NF-kB activation because it is a hallmark of PFA-EPN that drives dysregulation of inflammatory genes and forms an immunosuppressive tumor microenvironment. Upon tumor recognition, CAR-T cells produce high amounts of the cytokine tumor necrosis factor-alpha, which is an extracellular stimulus that propagates NF-kB activation in PFA-EPN. We show that HER2 CAR-T cell treatment causes increased nuclear translocation of the RELA NF-kB subunit, which induces CCL2 gene transcription and chemokine release. This results in CCL2-CCR2 ligand/receptor mediated influx of inflammatory monocytes and regulatory T cells, impairing CAR-T cell effector function. Inhibition of CCR2 restores anti-tumor CAR-T cytotoxicity against bulky orthotopic tumors by decreasing the infiltration of inflammatory monocytes and regulatory T cells. Combinatorial strategies addressing tumor mediated immunosuppression should be evaluated in upcoming CAR-T cell trials for patients with high-risk CNS tumors.