CCR5 mRNA Expression Research Articles

The Long COVID: Further Advances in Our Understanding about the Role of Specific Chemokines (CCR5, CCR6, CCR9 and CCL3) in Pediatrics

Introduction: Long COVID, also known as post-COVID-19 syndrome (PCS), is a condition where individuals continue to experience symptoms for weeks or months after recovering from COVID-19. The aim of this study was to evaluate the long-term effects of severe COVID-19 on promotor methylation and expression genes including CCR5, CCR6, CCR9 and CCL3 in children. Material and Methods: Clinical data and blood samples from 94 long COVID patients and 25 healthy subjects were collected. The control group was age-matched. Promotor methylation and mRNA expression of CCR5, CCR6, CCR9 and CCL3 genes in these patients and control group were assessed through methylation-specific PCR and Real-time PCR assay. Results: Our result indicated that promotor of CCR5 (p = 0.01) and CCL3 (p = 0.006) in long COVID children were hyper-methylated compared to healthy control group. Subsequently CCR5 and CCL3 transcript was decreased compared to control group (p = 0.01). In addition, CCL3 transcript in children with long COVID was decreased compared to control group (p = 0.008). On the other hand, we did not observe any significant modification in the transcript levels of CCR6 (p = 0.7) and CCR9 (p = 0.46) in children with long COVID in comparison to all control groups. Conclusion: The CCR5 and CCL3 promoter region DNA methylation and the subsequent decrease in the expression of these genes were possibly correlated with long COVID occurrence in children. Our study revealed additional data on the SARS-CoV-2 mediated inflammatory response. conclusion: The CCR5 and CCL3 promoter region DNA methylation and the subsequent decrease in the expression of these genes were possibly correlated with long COVID occurrence in children. Our study revealed additional data on the SARS-CoV-2 mediated inflammatory response.

Female genital tract host factors and tenofovir and lamivudine active metabolites.

We previously reported the effect of contraception on cervical tenofovir concentrations in Ugandan women living with HIV. Here we explored the role of cervicovaginal cytokines and drug metabolizing enzymes and transporters (DMETs) to elucidate FGT drug disposition in a Ugandan cohort. Cervicovaginal fluid and cervical biopsies were collected from Ugandan women living with HIV receiving tenofovir/lamivudine-based therapy and intramuscular depot medroxyprogesterone acetate (DMPA-IM; n=25), copper IUD (cuIUD; n=12), or condoms (n=13) as contraception. Cytokines were measured in cervicovaginal fluid (CVF). Ectocervical tenofovir diphosphate (TFVdp) and lamivudine triphosphate (3TCtp), dATP/dCTP concentrations, and immune marker/DMETs gene expression were measured in cervical biopsies. Cervical 3TCtp was not correlated with any CVF cytokines. Cervical TFVdp was correlated with IL-10, IL-7, and IL-17 in CVF. CCR5 mRNA expression in cervical biopsies was higher in cuIUD-users versus condoms-users. Using multivariable linear regression, CVF IL-17, tissue dATP, plasma estradiol, and plasma tenofovir were all significant predictors of cervical TFVdp. Tissue dCTP and plasma lamivudine were significant predictors of cervical 3TCtp. TFVdp concentrations in cervix appear to be influenced by local inflammation. In contrast, 3TCtp FGT exposure was not affected by genital inflammation or DMETS. CuIUD users have more immune cells present, which may in turn influence local TFVdp disposition.

Cytokines and chemokines skin gene expression in correlation with immune cells in blood and severity in equine insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses. In the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success. By qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood. Our data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1β, and CCL26 mRNA expression in skin punch biopsies of IBH horses. In summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses.

Mechanism of Lian Hua Qing Wen capsules regulates the inflammatory response caused by M1 macrophage based on cellular experiments and computer simulations

PurposeThe polarization of macrophages with the resulting inflammatory response play a crucial part in tissue and organ damage due to inflammatory. Study has proved Lian Hua Qing Wen capsules (LHQW) can reduce activation of inflammatory response and damage of tissue derived from the inflammatory reactions. However, the mechanism of LHQW regulates the macrophage-induced inflammatory response is unclear. Therefore, we investigated the mechanism of LHQW regulated the inflammatory response of M1 macrophages by cellular experiments and computer simulations. MethodsThis study has analysed the targets and mechanisms of macrophage regulating inflammatory response at gene and protein levels through bioinformatics. The monomeric components of LHQW were analyzed by High Performance Liquid Chromatography (HPLC). We established the in vitro cell model by M1 macrophages (Induction of THP-1 cells into M1 macrophages). RT-qPCR and immunofluorescence were used to detect changes in gene and protein levels of key targets after LHQW treatment. Computer simulations were utilized to verify the binding stability of monomeric components and protein targets. ResultsMacrophages had 140,690 gene targets, inflammatory response had 12,192 gene targets, intersection gene targets were 11,772. Key monomeric components (including: Pinocembrin, Fargesone-A, Nodakenin and Bowdichione) of LHQW were screened by HPLC. The results of cellular experiments indicated that LHQW could significantly reduce the mRNA expression of CCR5, CSF2, IFNG and TNF, thereby alleviating the inflammatory response caused by M1 macrophage. The computer simulations further validated the binding stability and conformation of key monomeric components and key protein targets, and IFNG/Nodakenin was able to form the most stable binding conformation for its action. ConclusionIn this study, the mechanism of LHQW inhibits the polarization of macrophages and the resulting inflammatory response was investigated by computer simulations and cellular experiments. We found that LHQW may not only reduce cell damage and death by acting on TNF and CCR5, but also inhibit the immune recognition process and inflammatory response by regulating CSF2 and IFNG to prevent polarization of macrophages. Therefore, these results suggested that LHQW may act through multiple targets to inhibit the polarization of macrophages and the resulting inflammatory response.

Abstract TMP113: RNA Sequencing Reveals Novel Pericytic MiR-15a/16-1 Targets in Post-Stroke Cerebral Angiogenesis

Introduction: Angiogenesis actively contributes to post-stroke functional recovery and improves long-term survival in stroke patients. Previously, we demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis by enhancing classic pro-angiogenic factors and their receptors, and genetic deletion of miR-15a/16-1 in pericytes likewise promotes post-stroke functional recovery by stimulating cerebral angiogenesis. Here, we further investigate the underlying mechanisms and downstream targets of pericytic miR-15a/16-1 in cerebral angiogenesis after ischemic stroke. Methods: Pericyte-miR15a/16-1 conditional knockout mice (Pericyte-miR-15a/16-1 cKO) and wild-type (WT) controls were subjected to 1h MCAO followed by 7d reperfusion. Cerebral microvessels were extracted and subjected to RNA-seq to identify the significant differentially expressed genes (DEGs) that related to miR-15a/16-1 and angiogenesis among experimental groups. TargetScan and miRDB were used to predict potential miR-15a/16-1 downstream targets. Primary mouse brain vascular pericytes (mBVPs) were treated with lentivirus to achieve loss- or gain-of-miR-15a/16-1 function, and subjected to OGD 8h and reperfusion 24h. Pro-angiogenic factors were further verified by dual-luciferase reporter assay, qPCR, and western blotting. Results: RNA-seq analysis showed 289 upregulated genes (P < 0.05 & at least one CPM > 1) and 399 upregulated genes from 100 positive enriched gene sets (GESA) in cerebral microvessels of pericyte-miR-15a/16-1 cKO mice compared to WT controls 7d after MCAO. By comparing to miR-15a/16-1 potential target database from TargetScan and miRDB, 9 angiogenic genes were identified. Further biochemical analysis revealed that Pappa2, Fgf9, Islr, and Ccr2 mRNA and protein expression are significantly increased in cerebral microvessels of cKO mice and mBVPs with loss-of-miR-15a/16-1 function. Furthermore, dual-luciferase assay results verified that loss- or gain-of-miR-15a/16-1 function significantly increased or decreased luciferase reporter activity of these four genes, respectively. Conclusion: Our findings reveal novel pericytic miR-15a/16-1 mediated angiogenic targets in ischemic stroke.

Sex-Specific Variations in the mRNA Levels of Candidate Genes in Peripheral Blood Mononuclear Cells from Patients with Diabetes: A Multistep Study

ABSTRACT Background Type 2 diabetes (T2D) is one of the most prevalent diseases that also show sexual dimorphism in many different aspects. Objectives This study aimed to distinguish the mRNA expression of genes in peripheral blood mononuclear cells (PBMCs) in men or women with T2D using a multistep analysis. Methods A total of 95 patients with T2D were compared based on their sex in terms of clinical variables and mRNA expression in their PBMCs. Results Men with T2D had lower LDLC, HDLC, and HbA1c values in their blood, but greater creatinine levels. In men with T2D, TLR4, CCR2, NOX2, and p67phox mRNA expression was greater, but IL6 and NF-κB mRNA expression was lesser in PBMCs. There was a link between fasting plasma glucose (FPG), triglycerides, and hs-CRP, as well as COX1 mRNA in men with T2D. In women with T2D, FPG was associated with the mRNA expression of THBS1 and p67phox, as well as triglycerides and HDLC levels. We found the exclusive effect of FPG on HDLC, HbA1c, as well as p67phox mRNA in PBMCs of women with T2D. Analysis revealed the exclusive effect of FPG on hs-CRP and PAFR mRNA in PBMCs of men with T2D. FPG was shown to be associated with body mass index, hs-CRP, triglycerides, and COX1 mRNA in men with T2D, and with serum triglycerides, THSB1, and p67phox mRNA in women with T2D, according to network analysis. HbA1c was linked with NF-κB mRNA in women with T2D. Conclusions Using a multistep analysis, it was shown that network analysis outperformed traditional analytic techniques in identifying sex-specific alterations in mRNA gene expression in PBMCs of T2D patients. The development of sex-specific therapeutic approaches may result from an understanding of these disparities.

Veronica persica Ethanol Extract Ameliorates Dinitrochlorobenzene-Induced Atopic Dermatitis-like Skin Inflammation in Mice, Likely by Inducing Nrf2/HO-1 Signaling.

Atopic dermatitis (AD) is chronic allergic contact dermatitis with immune dysregulation. Veronica persica has pharmacological activity that prevents asthmatic inflammation by ameliorating inflammatory cell activation. However, the potential effects of the ethanol extract of V. persica (EEVP) on AD remain elusive. This study evaluated the activity and underlying molecular pathway of EEVP in two AD models: dinitrochlorobenzene (DNCB)-induced mice and interferon (IFN)-γ/tumor necrosis factor (TNF)-α-stimulated human HaCaT keratinocytes. EEVP attenuated the DNCB-induced increase in serum immunoglobulin E and histamine levels, mast cell counts in toluidine-blue-stained dorsal skin, inflammatory cytokine (IFN-γ, interleukin [IL]-4, IL-5, and IL-13) levels in cultured splenocytes, and the mRNA expression of IL6, IL13, IL31 receptor, CCR-3, and TNFα in dorsal tissue. Additionally, EEVP inhibited the IFN-γ/TNF-α-induced mRNA expression of IL6, IL13, and CXCL10 in HaCaT cells. Furthermore, EEVP restored the IFN-γ/TNF-α-induced downregulation of heme oxygenase (HO)-1 in HaCaT cells by inducing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. A molecular docking analysis demonstrated that EEVP components have a strong affinity to the Kelch-like ECH-associated protein 1 Kelch domain. In summary, EEVP inhibits inflammatory AD by attenuating immune cell activation and inducing the Nrf2/HO-1 signaling pathway in skin keratinocytes.

Benefits of bone marrow-derived mesenchymal stem cells primed with estradiol in alleviating collagen-induced arthritis.

To investigate the effects of the oestradiol (ES) pulsed bone marrow-derived mesenchymal stem cells (BM-MSC) to treat adjuvant-induced arthritis in Wistar rats. BM-MSCs were pulsed with ES (0, 10,100, and 1000 nM) for 24 hr. RA was induced by collagen and Freund's Complete Adjuvant into the base of the tail of Wistar rats. The least effective concentration of ES that can promote potent anti-inflammatory properties in the MSC population is 100 nM. At this concentration, ES increases the inhibition of the polyclonal T lymphocyte proliferation, production of IDO, IL-10, Nitric oxide, and TGF-β, and expression of CXCR4 and CCR2 mRNA in the MSC population. Accordingly, the RA rats were treated with 2×106 MSCs or ES-pulsed MSCs (100 nM) on day 10 when all animals had developed signs of RA. ES-pulsed BM-MSCs reduced the severity of RA more profoundly than treatment with BM-MScs alone. The ability of ES-pulsed BM-MSCs to reduce symptoms and RA markers like CRP, RF, and nitric oxide was comparable to that of prednisolone. Prednisolone was more successful in reducing inflammatory cytokines than treatment with ES-pulsed BM-MSCs. ES-pulsed BM-MSCs were more successful in increasing anti-inflammatory cytokines than treatment with Prednisolone. The ability of ES-pulsed BM-MSCs to decrease the level of nitric oxide was comparable to that of prednisolone. ES-pulsed BM-MSCs may be a helpful strategy in RA control.

Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice.

Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

Shensong Yangxin Capsule Reduces the Susceptibility of Arrhythmia in db/db Mice via Inhibiting the Inflammatory Response Induced by Endothelium Dysfunction.

The aim of our study was to investigate the mechanism by which the Chinese compound Shensong Yangxin Capsule (SSYX) reduces susceptibility to arrhythmia in db/db mice. The db/db mice without drug treatment served as the model group. Other-treated db/db mice were administered SSYX for 8 weeks. Electrocardiogram (ECG), electrical mapping, pathological changes, immunofluorescence staining, real-time quantitative PCR, and Western blot analyses were then conducted. SSYX decreased arrhythmia susceptibility and shortened the abnormal ECG parameters of db/db mice. Meanwhile, SSYX restored irregular conduction direction and shortened the conduction time of the isolated heart. HE and Masson staining showed that SSYX alleviated inflammatory infiltration and collagen fiber deposition. Western blot showed that SSYX decreased the protein expression of ICAM-1, VCAM-1, and MCP-1 and increased the protein expression of occludin, ZO-1, eNOS, and Cx43. SSYX also increased the content of NO, decreased ET-1, TNF-α, IL-1β, IL-6, MCP-1, and CCR-2 mRNA expression, and increased Kv 4.2, Kv 4.3, Cav 1.2, and Nav 1.5 mRNA expression. Furthermore, SSYX decreased the fluorescence intensity of F4/80 and iNOS, increased the fluorescence intensity of CD31 and eNOS, and improved the Cx43 and α-actinin connection structure in cardiac tissues. The above therapeutic effects of SSYX were inhibited by L-NAME. SSYX reduced the susceptibility of db/db mice to arrhythmia by inhibiting the inflammatory response and macrophage polarization, and this effect of SSYX occurred through protection of endothelial cell function.

CCR5 contributes to adverse outcomes during malaria in pregnancy.

CCR5 is a chemokine receptor that mediates cell recruitment to sites of inflammation. It has been previously reported that the expression of CCR5 is increased in the placentas of women with malaria, a disease characterized by causing deliveries with low birth weight among other complications. CCR5 has been associated with pathology of protozoan infections during pregnancy but its role during malaria in pregnancy has not been elucidated. In the present work, we assessed the pregnancy outcome, placental structure, and levels of inflammatory markers of pregnant C57BL/6 and CCR5-/- mice infected or not with Plasmodium berghei NK65, with the purpose of determine the role of CCR5 in pregnancy associated malaria complications. We demonstrated that the expression of CCR5 mRNA increases in late pregnancy placentas of C57BL/6 when compared to uninfected controls. Infected pregnant C57BL/6 mice showed preterm birth, decreased fetal weight, placental inefficiency, and reduced placental vascular space. On the other hand, CCR5 deficiency led to increased levels of maternal parasitemia, reduced fetal weight and placental inefficiency compared to C57BL/6 mice. However, the infection did not cause additional changes in these parameters or in the incidence of preterm delivery in infected CCR5-/- mice in relation to C57BL/6 mice, showing that CCR5 may contribute to the adverse effects caused by infection during pregnancy. This improvement in pregnancy outcome, observed in infected CCR5-/- mice, was accompanied by lower placental levels of the inflammatory markers, such as TNF and NAG. Furthermore, it was observed that the placentas of CCR5-/- animals showed structural differences in relation to C57BL/6 mice, which could improve the efficiency of maternal-fetal exchanges, reflecting on fetal weight. Taken together, these results indicate that CCR5 expression contributes to the adverse outcomes caused by malaria in late pregnancy.

Pregnancy preparation: redistribution of CCR7-positive cells in the rat uterus.

Changes in the endometrium prior to implantation may be critical in predicting pregnancy outcomes. This study shows that the endocrine system directs positional changes in CCR7+ cells before implantation, which may be critical for developing maternal tolerance. Suppression of the maternal immune system is vital for the implantation of the semi-allogeneic embryo. Although progress in understanding the dialogue between mother and embryo has been made, key interactions between maternal immune cells, hormones, and chemokines remain elusive. Uterine expression of the C-C chemokine receptor type 7 (CCR7) could recruit T regulatory cells and facilitate localized immune suppression. To test this concept, Ccr7 mRNA and protein were assessed in uterine tissue. Ccr7 mRNA expression peaked at day 4 in pregnant rat uteri and then declined at days 5 and 6. CCR7 protein showed similar quantitative changes. To test if female sex steroids affected the spatial distribution of CCR7-expressing cells, uteri from ovariectomized rats, progesterone-pretreated rats (2 mg daily), and progesterone-pretreated rats injected with estradiol (0.2 µg) were analyzed. Progesterone increased CCR7-positive (+) cells in the antimesometrial stroma. Progesterone and estradiol increased CCR7+ cells in the mesometrial stroma. Estradiol increased the density of cluster of differentiation 4 (CD4) positive cells in the mesometrial stromal region over progesterone alone. The density of cells expressing the T regulatory cell marker, forkhead box protein 3 (FOXP3), increased in the antimesometrial stroma in response to progesterone alone. Progesterone and estradiol increased FOXP3+ cells in the antimesometrial region of the stroma. Co-localization of CCR7, CD4, and FOXP3 in the stroma suggests CCR7+ cells are T regulatory cells. Polarization of CCR7+ cells in the endometrial stroma was an intrinsic response regulated by sex steroids and did not require the presence of an embryo.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 -CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5- Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

The Significance of the Alter miR let-7a and miR-335 Expression Level Regulating the CCR7/CCL19 Axis as Potential Biomarkers of Tumor Progression in NSCLC.

The chemokine receptor 7/C-C ligand 19 chemokine (CCR7/CCL19) has been implicated in the development and progression of NSCLC. Its expression is regulated by various epigenetic factors including miRNAs. The aim of this study was to assess the expression of CCR7/CCL19 in cancer tissue in relation to that of miRNAs (miR-let-7a, miR-335) as transcriptional regulators. The expression of the tested miRNAs was also evaluated in serum exosomes. Sixty patients (n = 60) were enrolled in the study. The total expression of the studied mRNA and miRNAs were evaluated using qPCR. Tumor tissue fragments, macroscopically unchanged adjacent tissue, and serum were used as controls. Higher CCR7 and CCL19 mRNA expression levels were observed in tumor tissue compared to control. According to stages of the disease (AJCC tumor staging), the greatest expression level of the studied genes’ mRNA was observed in patients with stage III. In NSCLC patients, lower miR let-7a expression level was observed in tumor tissue compared to serum; however, miR-335 expression level was higher (p < 0.05). The expression level of miR-335 positively correlated with tumor size (T features according to pTNM staging) and AJCC tumor staging, while miR let-7a had a negative correlation (p > 0.05) with liquid biopsy. Significantly greater miR-335 expression level and lower miR let-7a expression level in serum were observed in patients with metastases to lymph nodes. Our findings reveal a significant correlation between the expression levels of the mRNA of the studied genes and miRNAs. Changes in miR-335 and miR let-7a expression levels in the serum exosomes of NSCLC patients in relation to lymph node metastases and tumor stage may serve as a non-invasive molecular biomarker of tumor progression.

Reduced CCR5 Expression and Immune Quiescence in Black South African HIV-1 Controllers.

Unique Individuals who exhibit either suppressive HIV-1 control, or the ability to maintain low viral load set-points and preserve their CD4+ T cell counts for extended time periods in the absence of antiretroviral therapy, are broadly termed HIV-1 controllers. We assessed the extent to which black South African controllers (n=9), differ from uninfected healthy controls (HCs, n=22) in terms of lymphocyte and monocyte CCR5 expression (density and frequency of CCR5-expressing cells), immune activation as well as peripheral blood mononuclear cell (PBMC) mitogen-induced chemokine/cytokine production. In addition, relative CD4+ T cell CCR5 mRNA expression was assessed in a larger group of controllers (n=20) compared to HCs (n=10) and HIV-1 progressors (n=12). Despite controllers having significantly higher frequencies of activated CD4+ and CD8+ T cells (HLA-DR+) compared to HCs, CCR5 density was significantly lower in these T cell populations (P=0.039 and P=0.064, respectively). This lower CCR5 density was largely attributable to controllers with higher VLs (>400 RNA copies/ml). Significantly lower CD4+ T cell CCR5 density in controllers was maintained (P=0.036) when HCs (n=12) and controllers (n=9) were matched for age. CD4+ T cell CCR5 mRNA expression was significantly less in controllers compared to HCs (P=0.007) and progressors (P=0.002), whereas HCs and progressors were similar (P=0.223). The levels of soluble CD14 in plasma did not differ between controllers and HCs, suggesting no demonstrable monocyte activation. While controllers had lower monocyte CCR5 density compared to the HCs (P=0.02), significance was lost when groups were age-matched (P=0.804). However, when groups were matched for both CCR5 promoter haplotype and age (n=6 for both) reduced CCR5 density on monocytes in controllers relative to HCs was highly significant (P=0.009). Phytohemagglutinin-stimulated PBMCs from the controllers produced significantly less CCL3 (P=0.029), CCL4 (P=0.008) and IL-10 (P=0.028) compared to the HCs, which was largely attributable to the controllers with lower VLs (<400 RNA copies/ml). Our findings support a hypothesis of an inherent (genetic) predisposition to lower CCR5 expression in individuals who naturally control HIV-1, as has been suggested for Caucasian controllers, and thus, likely involves a mechanism shared between ethnically divergent population groups.

Rs7853346 Polymorphism in lncRNA-PTENP1 and rs1799864 Polymorphism in CCR2 are Associated with Radiotherapy-Induced Cognitive Impairment in Subjects with Glioma Via Regulating PTENP1/miR-19b/CCR2 Signaling Pathway.

LncRNA-PTENP1 was reported to promote multiple myeloma cancer stem cell proliferation, and the G allele of rs7853346 polymorphism in lncRNA-PTENP1 was demonstrated to enhance the effect of lncRNA-PTENP1. In this study, we aimed to study the potential effect of lncRNA-PTENP1 and CCR2 mRNA polymorphisms on cognitive impairment in glioma patients. In this study, 279 glioma patients were recruited and grouped according to their genotypes of rs7853346 in PTENP1 and rs1799864 in CCR1. Pathogenic parameters were collected from patients before radiotherapy (month 0) or at month 1 and month 3 after radiotherapy to study the effect of rs7853346 and rs1799864 on cognitive impairment. Sequence analysis, luciferase assay, real-time PCR, and Western blot were performed to study the regulatory relationships between lncRNA-PTENP1, miR-18b, and CCR2. The glioma patient groups exhibited no significant differences concerning basic characteristics. However, the CG&GG/GG genotype alleviated radiotherapy-induced cognitive impairment by exhibiting the highest MMSE among the four groups. On the contrary, parameters including the severity of depression, bladder control, global health status, itchy skin, and weakness of legs all showed no difference among different patient groups at month 0, month 1, and month 3. Also, a long-term positive effect of CG&GG/GG genotype on role functioning and social functioning was also observed after radiotherapy. Compared with patients carrying the CC genotype of rs7853346, the expression of lncRNA-PTENP1 was reduced while the miR-19b level was elevated in patients carrying the CG&GG genotypes of rs7853346. Moreover, the expression of CCR2 mRNA was the highest in the CC/GA&AA group and the lowest in the CG&GG/GG group. Subsequent sequence analysis and luciferase assay indicated that miR-19b could bind to lncRNA-PTENP1 and 3'UTR of CCR2 mRNA, and the knockdown of lncRNA-PTENP1 led to evident up-regulation of miR-19b and down-regulation of CCR2 mRNA/protein in a cellular model, thus verifying the presence of the lncRNA-PTENP1/miR-19b/CCR2 mRNA signaling pathway. In conclusion, by studying the changes in the key parameters of glioma patients who were subjected to radiotherapy, we concluded that the rs7853346 polymorphism in lncRNA-PTENP1 and the rs1799864 polymorphism in CCR2 could independently affect cognitive impairment, while a more significant combined effect on cognitive impairment was exerted in glioma patients via the signaling pathway of PTENP1/miR-19b/CCR2.

BCG-trained innate immunity leads to fetal growth restriction by altering immune cell profile in the mouse developing placenta.

Trained immunity is a new concept illustrating that innate immune cells are able to undergo a long-term metabolic and epigenetic reprogramming after infection or vaccination, thus displaying either a pro- or an anti-inflammatory phenotype during a sequential unrelated challenge. Innate immune cells such as natural killer (NK) cells and macrophages constitute a large part of the decidual leukocyte population at the maternal-fetal interface, playing an important role in placental development and as such in fetal growth and development. In this study, we hypothesized that training the innate immune cells before pregnancy could have an impact on pregnancy. To test this hypothesis, we used CBA/J x DBA/2 mouse model to investigate pregnancy outcomes and leukocyte population at the maternal-fetal interface. Although we were not able to show a beneficial effect of LPS-tolerogenic training on fetal resorption, Bacillus Calmette-Guérin (BCG) training, known to prime innate immune cells to be proinflammatory, led to fetal growth restriction, without aggravating the fetal resorption rate. We also found that BCG training led to less NK cells and macrophages at the maternal-fetal interface at the early stage of placentation (E9.5), associated with a down-regulation of Ccr3 and Lif mRNA expression. This induced altered leucocyte population profile can be an explanation for the subsequent fetal growth restriction. These data suggest that preconceptional infections-induced trained immunity could influence pregnancy outcomes.

Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells

BackgroundThis study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. MethodsPlasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-β, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. ResultsTransduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-β levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. ConclusionsSimultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.

Basal renal phenotype and response to induction of aristolochic acid nephropathy in mice lacking the neutral amino acid transporter B0AT1 (SLC6A19)

Background B0AT1 (SLC6A19) mediates the reabsorption of neutral amino acids, including branched-chain amino acids (BCAA), in the intestine and kidney (expressed in early and mid-proximal tubule - S1/S2). Dietary restriction of BCAA has been proposed to improve metabolic health by inhibiting mTOR and inducing autophagy. Here we characterized basal kidney function and determined whether B0AT1 KO protects the kidney from aristolochic acid-induced nephropathy (AAN), a model of proximal tubular injury. Methods In male B0AT1 KO and littermate wild-type mice (WT) mice, we measured fractional renal amino acid excretion and performed renal FITC-sinistrin clearance studies under terminal anesthesia to determine renal creatinine handling. A set of female mice was injected with aristolochic acid (3 mg/kg i.p.) or vehicle every 3 days for 3 weeks, and kidneys were harvested 3 weeks later for RTqPCR analyses (n=7-16 mice per group). Results RTqPCR and Western blotting confirmed absence of renal B0AT1 mRNA in KO, associated with increased fractional renal excretion of neutral amino acids, including BCAA (to 20-40%), whereas plasma levels of BCAA were not changed. In vehicle-treated mice, plasma creatinine was slightly higher in KO vs WT (0.080±0.002 vs 0.074±0.002 mg/dl; *P<0.05). Clearance studies revealed a trend for lesser fractional excretion of creatinine in KO vs WT (P=0.057), suggesting lesser creatinine secretion contributed to higher plasma levels. Urinary albumin/creatinine ratio (UACR) and urinary glucose/creatinine ratio (UGCR) were tripled and doubled in female KO vs WT (180±18 vs 62±5 ng/μg*; 1.35±0.26 vs 0.70±0.06 mg/mg*), respectively, suggesting a modest impairment in proximal tubular albumin and glucose reabsorption. The renal expression of the alternative neutral amino acid transporter B0AT3 (SLC6A18), expressed in S1-S3, was reduced in KO (to 32±5% of WT*). This may be due to KO model generation, as B0AT1 and B0AT3 are co-expressed side-by-side on the same chromosome. Whole kidney CCR2 and KIM-1 mRNA expression were modestly higher in KO vs WT, which may reflect enhanced amino acid and glucose transport in the vulnerable S3 segment in the outer medulla. In the absence of robust effects on plasma creatinine, AAN significantly increased UACR vs vehicle in WT (+164±39 vs +52±14 ng/μg*) but not in KO (-18±30 vs +57±49 ng/μg) when measured after completion of injections. Moreover, increases in CCR2 and TIMP1 expression in response to AAN vs vehicle observed in WT (+206±28%; +203±45%) were attenuated in KO (+54±20%*; +81±39%*). Conclusions Genetic deletion of B0AT1 in male and female mice induces distinct effects on kidney transport beyond amino acids. Follow up studies with higher doses of AA are needed to more fully evaluate the nephroprotective potential of B0AT1 inhibition.