MCP-1 Gene Research Articles

Abstract 70: Cellular senescence as a driver of preeclampsia

Background: Preeclampsia (PE) is a severe hypertensive disorder of pregnancy of unknown pathophysiology. We previously have reported the presence of senescence in kidney and fat tissue of PE women, as well as accelerated epigenetic aging, which prompted us to postulate that cellular senescence may drive development of PE. Methods: We prospectively collected sera from pregnant women throughout pregnancy, and using multiplex Luminex analysis, measured 50 markers of the senescence-associated secretory phenotype (SASP). Comparisons were made between women who developed PE (n=10) vs. normotensive pregnancies (n=10). We then injected human PE serum into the IL-10KO mouse to induce PE and assess whether PE is associated with development of senescence. We measured plasma p16, a circulating indicator of cellular senescence, before and after inducing PE. We also compared plasma p16 and expressions of senescence-associated genes in the hearts and aortas of regular pregnant (PG, no PE serum injection) and PE mice (n=4). Results: There was a significant increase in several SASP markers in sera of women who developed PE, beginning in the first trimester, through delivery. Notably, eotaxin, MCP1, GROb, MIF, MMP2, TNFRII, IGFBP2 were significantly increased in PE women compared to their normotensive peers throughout all trimesters. Mice injected with PE serum exhibited all features of PE, as reported earlier. When measuring plasma p16 before and after PE serum injection, animals had higher values upon inducing PE. Plasma samples of PE mice demonstrated higher p16 concentrations compared to the PG group. The aortas of PE mice exhibited increased expressions of senescence associated genes p16, il6, and mcp1, whereas in the hearts, p16, Il1a and il8 were upregulated. Conclusion: The SASP is present very early in human pregnancy, even before PE has developed. Injection of SASP-enriched PE serum into IL-10KO mice induces cellular senescence in different tissues resulting in a PE phenotype. These results suggest that senescence may be an underlying mechanism of PE.

The adipocyte apolipoprotein M is negatively associated with inflammation

Obesity is associated with the development of local adipose tissue (AT) and systemic inflammation. Most adipokines are upregulated with obesity and have pro-inflammatory properties. Few are downregulated and possess beneficial anti-inflammatory effects. The apolipoprotein M (APOM) is an adipokine whose expression is low during obesity and associated with a metabolically healthy AT. Here, the role of adipose-derived APOM on obesity-associated AT inflammation was investigated by measuring the expression of pro-inflammatory genes in human and mouse models. In 300 individuals with obesity, AT APOM mRNA level was negatively associated with plasma hs-CRP. The inflammatory profile was assessed in Apom−/− and WT mice fed a normal chow diet (NCD), or a high-fat diet (HFD) to induce AT inflammation. After HFD, mice had a higher inflammatory profile in AT and liver, and a 50% lower Apom gene expression compared with NCD-fed mice. Apom deficiency was associated with a higher inflammatory signature in AT compared with WT mice but not in the liver. Adeno-associated viruses encoding human APOM were used to induce APOM overexpression: in vivo, in WT mice AT prior to HFD; in vitro, in human adipocytes which conditioned media was applied to ThP-1 macrophages. The murine AT overexpressing APOM gene had a reduced inflammatory profile. The macrophages treated with APOM-enriched media from adipocytes exhibited lower IL6 and MCP1 gene expression compared with macrophages treated with control media, independently of S1P. Our study highlights the protective role of adipocyte APOM against obesity-induced AT inflammation.

Abstract We140: Age-Dependent miR-34a and AGTRAP Cross-Modulation: a Novel Mechanism of Vascular Aging

Background: Renin-Angiotensin Aldosterone System (RAAS) activation plays a central role in vascular aging, leading to structural and functional alterations including vascular smooth muscle cell (VSMC) proliferation, migration and secretion of extracellular matrix and inflammatory molecules. The age-associated increase in Angiotensin II (Ang II) expression enhances AT1-receptor activity and determinates a pro-fibrotic and pro-inflammatory phenotype. Further, miR-34a expression is modulated by aging and increases in different tissues, including the mouse aorta, but no direct miR-34a target has been identified in the RAAS pathway that sustains age-dependent increase of pro-inflammatory signaling. Aims: The aim of our study is to evaluate the potential interaction between miR-34a and RAAS signaling in VSMCs. Methods and Results: miR-34a expression exhibited an age-dependent increase in Rhesus Monkey (8- to 26-year-old) and rat central arteries (8- and 30-month-old). In silico analysis indicated that AT1R-associated protein (AGTRAP), a negative modulator of AT1R signaling, is a potential miR-34a target suggesting that the age-dependent increase in miR-34a in the arterial vascular wall may decrease AGTRAP expression in arterial VSMC. Ang II enhanced miR-34a expression in 30-month-old rat and in human aorta SMCs (HASMCs), whereas AGTRAP and Sirtuin-1 (SIRT1) mRNA and protein level decreased. Immunohistochemistry analysis showed a decrease of AGTRAP in 18-month-old miR-34a knock-out mice compared to 2-month-old mice. By luciferase assay, we demonstrated that AGTRAP is a direct miR-34a target. Moreover, AGTRAP and SIRT1 decreased in HASMCs infected with miR-34a-encoding lentivirus and this effect was partially rescued in AGTRAP/miR-34a co-infected cells. Finally, pro-inflammatory genes IL-6, COX-2, MCP-1 and MFG-E8 expression was upregulated in Ang II-treated and miR-34a overexpressing HASMCs, and this effect was abolished in AGTRAP/miR-34a co-transfected cells. Conclusions: Our results show cross-modulation between miR-34a and AGTRAP. MiR-34a targeting AGTRAP increases the expression of pro-inflammatory molecules in VSMCs and represents a novel mechanism that modulates RAAS signaling in vascular aging. Statement: This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.

Laminin 332 functionalized surface improve implant roughness and oral keratinocyte bioactivity

ObjectiveThe biological seal (BS) at the implant-tissue interface is essential for the success of dental implants (DIs), and the absence of a proper BS can lead to peri-implantitis. The basement membrane (BM) and junctional epithelium are critical for sealing the peri-implant mucosa, and laminin 332 is an important protein in binding the epithelium to the implant surface. The aim of this study was to evaluate the response of oral keratinocytes to titanium dental implant surfaces biofunctionalized with laminin 332. DesignThe dental implant surface was treated with a piranha solution to create hydroxyl (OH) groups, facilitating biofunctionalization with laminin 332. The modified surface underwent scanning electron microscopy, surface roughness evaluation, and chemical composition analysis. Human keratinocytes from the Cal-27 line were then cultured on the modified implants for 24 and 48 h to assess viability, morphology, cytokine secretion, and mRNA expression of tissue repair-associated genes. ResultsThe results showed that laminin 332 biofunctionalization of the implant surface resulted in lower values of Ra, Rq and positive surface roughness parameters Rsk, Rku and Rv. The elemental composition showed an increase in nitrogen and carbon content corresponding to protein binding. The biofunctionalized surfaces did not affect cell viability and promoted cytokine secretion (IL-1a and IL-8) and a significant increase (p < 0.05) in MCP-1, EGF, FGF, TGF and VEGF gene expression compared to the control. ConclusionIn conclusion, laminin 332 coating Ti implants was shown to be effective in promoting keratinocyte adhesion, spreading, and viability. This approach could be an alternative way to improve biocompatibility.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model

BackgroundExposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis.Methods and resultsEight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation.ConclusionsCTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Optimization of Naringenin-loaded nanoparticles for targeting of Vanin-1, iNOS, and MCP-1 signaling pathway in HFD-induced obesity

Naringenin, a natural dihydrochalcone flavonoid, exhibits diverse pharmacological properties. This study investigates the hypolipidemic effects of Nar-NPs on obese mice. The characteristics of Nar-NPs, including morphology, particle size, zeta potential, UV–vis, and FT-IR spectra, were examined. The anti-obesity properties of Nar-NPs were evaluated in obese rats, considering LD50, 1/20 LD50, and 1/50 LD50 for treatment preparation. Results indicated that synthesized Nar-NPs were uniform, spherical, and well-dispersed, with a size of 130.06 ± 1.98 nm and with a zeta potential of −25.6 ± 0.8 mV. Nar-NPs exhibited enhancement in the cumulative release of naringenin (56.87 ± 2.45 %) as compared to pure naringenin suspension 87.83 ± 1.84 % in 24 h of the study. The LD50 of Nar-NPs was determined as 412.5 mg/kg.b.w. HFD induced elevated glycemic, oxidative stress, and inflammatory biomarkers while reducing HDL-C, GSH, and superoxide dismutase (SOD) levels. Administration of Nar-NPs significantly mitigated body weight, glucose, insulin, leptin, TC, TG, SREBP1c, pAMPK, PPAR-α, as well as vanin-1, MCP-1, and iNOS mRNA gene expression. Histological investigations supported the biochemical and PCR findings. In a nutshell, the study suggests that the Nar-NPs could serve as a promising and viable pharmacological strategy for the treatment of obesity-related disorders.

Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy.

Atherosclerotic plaques are sites of chronic inflammation with diverse cell contents and complex immune signaling. Plaque progression and destabilization are driven by the infiltration of immune cells and the cytokines that mediate their interactions. Here, we attempted to compare the systemic cytokine profiles in the blood plasma of patients with atherosclerosis and the local cytokine production, using ex vivo plaque explants from the same patients. The developed method of 41-plex xMAP data normalization allowed us to differentiate twenty-two cytokines produced by the plaque that were not readily detectable in free circulation and six cytokines elevated in blood plasma that may have other sources than atherosclerotic plaque. To verify the xMAP data on the putative atherogenesis-driving chemokines MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and fractalkine (CX3CL1), qPCR was performed. The MIP1A (CCL3), MIP1B (CCL4), FKN (CX3CL1), and MCP1 (CCL2) genes were expressed at high levels in the plaques, whereas RANTES (CCL5) was almost absent. The expression patterns of the chemokines were restricted to the plaque cell types: the MCP1 (CCL2) gene was predominantly expressed in endothelial cells and monocytes/macrophages, MIP1A (CCL3) in monocytes/macrophages, and MIP1B (CCL4) in monocytes/macrophages and T cells. RANTES (CCL5) was restricted to T cells, while FKN (CX3CL1) was not differentially expressed. Taken together, our data indicate a plaque-specific cytokine production profile that may be a useful tool in atherosclerosis studies.

Comparing Methods for Induction of Insulin Resistance in Mouse 3T3-L1 Cells.

Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders. 1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR). In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel. We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected. In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.

Novel Peritoneal Sclerosis Rat Model Developed by Administration of Bleomycin and Lansoprazole.

In our preliminary experiment, peritoneal sclerosis likely induced by peritoneal dialysis was unexpectedly observed in the livers of rats given bleomycin and lansoprazole. We examined whether this peritoneal thickening around the liver was time-dependently induced by administration of both drugs. Male Wistar rats were injected with bleomycin and/or lansoprazole for 2 or 4 weeks. The 3YB-1 cell line derived from rat fibroblasts was treated by bleomycin and/or lansoprazole for 24 h. The administration of both drugs together, but not individually, thickened the peritoneal tissue around the liver. There was accumulation of collagen fibers, macrophages, and eosinophils under mesothelial cells. Expressions of Col1a1, Mcp1 and Mcp3 genes were increased in the peritoneal tissue around the liver and in 3YB-1 cells by the administration of both drugs together, and Opn genes had increased expressions in this tissue and 3YB-1 cells. Mesothelial cells indicated immunoreactivity against both cytokeratin, a mesothelial cell marker, and αSMA, a fibroblast marker, around the livers of rats given both drugs. Administration of both drugs induced the migration of macrophages and eosinophils and induced fibrosis associated with the possible activation of fibroblasts and the possible promotion of the mesothelial-mesenchymal transition. This might become a novel model of peritoneal sclerosis for peritoneal dialysis.

Negative chemotaxis of Ligilactobacillus agilis BKN88 against gut-derived substances

Ligilactobacillus agilis is a motile lactic acid bacterium found in the gastrointestinal tracts of animals. The findings of our previous study suggest that the motility of L. agilis BKN88 enables gut colonization in murine models. However, the chemotactic abilities of motile lactobacilli remain unknown. This study aimed to identify the gut-derived chemoeffectors and their corresponding chemoreceptors in L. agilis BKN88. Chemotaxis assays with chemotactic and non-chemotactic (ΔcheA) L. agilis strains revealed that low pH, organic acids, and bile salts served as repellents. L. agilis BKN88 was more sensitive to bile and acid than the gut-derived non-motile lactobacilli, implying that L. agilis might utilize motility and chemotaxis instead of exhibiting stress tolerance/resistance. L. agilis BKN88 contains five putative chemoreceptor genes (mcp1–mcp5). Chemotaxis assays using a series of chemoreceptor mutants revealed that each of the five chemoreceptors could sense multiple chemoeffectors and that these chemoreceptors were functionally redundant. Mcp2 and Mcp3 sensed all tested chemoeffectors. This study provides further insights into the interactions between chemoreceptors and ligands of motile lactobacilli and the unique ecological and evolutionary features of motile lactobacilli, which may be distinct from those of non-motile lactobacilli.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

Oxidative stress and inflammation are mediated via aryl hydrocarbon receptor signalling in idiopathic membranous nephropathy

Membranous nephropathy (MN) patients are diagnosed by the presence of phospholipase A2 receptor (PLA2R) before they progress to renal failure. However, the subepithelium-like immunocomplex deposit-mediated downstream molecular pathways are poorly understood. The aryl hydrocarbon receptor (AHR), NF-ƙB and Nrf2 pathways play central roles in the pathogenesis and progression of chronic kidney disease. However, their mutual effects on MN require further examination. Thus, we investigated the effect of AHR signalling on the NF-ƙB and Nrf2 pathways in IMN patients, cationic bovine serum albumin (CBSA)-injected rats and zymosan activation serum (ZAS)-treated podocytes. IMN patients show significantly decreased serum total protein and albumin levels, increased urine protein levels and intrarenal IgG4 and PLA2R protein expression in glomeruli compared with controls. IMN patients exhibited increased mRNA expression of intrarenal AHR and its target genes, including CYP1A1, CYP1A2, CYP1B1 and COX-2. This increase was accompanied by significantly upregulated protein expression of CD3, NF-ƙB p65 and COX-2 and significantly downregulated Nrf2 and HO-1 expression. Similarly, CBSA-induced rats showed severe proteinuria and activated intrarenal AHR signalling. This was accompanied by significantly upregulated protein expression of intrarenal p-IκBα, NF-κB p65 and its gene products, including COX-2, MCP-1, iNOS, 12-LOX, p47phox and p67phox, and significantly downregulated protein expression of Nrf2 and its gene products, including HO-1, catalase, GCLC, GCLM, MnSOD and NQO1. These results were further verified in ZAS-induced podocytes. Treatment with the AHR antagonist CH223191 and AHRsiRNA significantly preserved podocyte-specific protein expression and improved the NF-ƙB and Nrf2 pathways in ZAS-induced podocytes. In contrast, similar results were obtained in ZAS-induced podocytes treated with the NF-ƙB inhibitor BAY 11–7082 and NF-κBp65 siRNA. However, neither method had a significant effect on AHR signalling. Collectively, these results indicate that the NF-ƙB pathway is a downstream target of AHR signalling. Our findings suggest that blocking AHR signalling inhibits oxidative stress and inflammation, thereby improving proteinuria and renal injury.

Early cutaneous inflammatory response at different degree of burn and its significance for clinical diagnosis and management

A complete characterization of the burn wound based on cutaneous architectural changes and inflammatory response is extremely important to provide evidence for progressive changes in the burn wound. Burn wounds are highly susceptible to conversion into deeper wounds, which need special care and attention; thereby, the complete characterization of burn wound type and their subsequent inflammatory status in the cutaneous system at the earliest is of paramount importance. Inflammatory markers at different degrees will help clinicians devise better and more specific treatment strategies for each burn type.The present study is carried out to profile pro-inflammatory gene expression along with immune cell quantification, vascular perfusion, and histopathological assessment in the cutaneous system of murine models. The study revealed that burn injury caused an immediate increase in vascular perfusion in superficial and partial-thickness burns, whereas there was a decrease in vascular perfusion in full-thickness burns. An influx of lymphocytes at the edges of burn wounds in each type of burn injury was well-orchestrated with the event of vascular perfusion. Further, pro-inflammatory gene expression profiling revealed significant upregulation vis-à-vis upregulation of TNF-α and MCP-1 genes, with an increase in the number of neutrophils following 72 h of injury that evidently cemented the conversion of superficial burn into partial-thickness burn. The molecular findings were profoundly supported by the histopathological changes.Thus, our foundational studies show distinct characteristic cutaneous changes correlated with the expression of key pro-inflammatory genes in three different types of burn injuries. Characterization of these cutaneous inflammatory responses provides a promising future for medical interventions involved with different degrees of burn injury, and it will also help in the pre-clinical testing of therapies for burn injury.

#5108 SUSTAINED IL-6 SECRETION CAN LEAD TO AN AMPLIFIED INFLAMMATORY RESPONSE, MEDIATED BY THE ACTIVATION OF STAT3 AND NFKB VIA THE IL-6 AMPLIFIER LOOP

Abstract Background and Aims Organ transplants are the preferred treatment for end-stage organ failure. Potent and specific immunosuppressive agents have significantly decreased acute allograft rejection following renal transplantation. A significant barrier to long-term kidney allograft outcomes is chronic antibody-mediated rejection (CABMR). Chronic inflammation is a major cause of late graft loss that is mediated by soluble mediators released by immune and non-immune cells. IL-6 is a crucial cytokine that plays a central role in developing chronic inflammation. Non-immune cells, such as fibroblasts, have recently been identified as mediating chronic allograft rejection by activating the IL-6 amplifier loop (IL-6+IL-17). Our Aim of the study: -to study the effect of IL-6+IL-17 on secretion of IL-6 in the culture supernatant of fibroblasts derived from renal biopsy of chronic antibody mediated rejection (CABMR) patients; -to see the effect of anti-IL-6 (Tocilizumab)and anti-IL-17 on the secretion of IL-6 from the fibroblasts derived from kidney tissue of chronic antibody mediated rejection (CABMR) patients; and -to elucidate the pro-inflammatory pathway leading to increased IL-6 secretion by induction of Amplifier loop. Method Fibroblasts from grafted kidney from CABMR patients (n = 6) were cultured and stimulated with IL-6 (20ng/ µl), IL-17(50ng/ µl), IL-6 plus IL-17 for 24 hours. Levels of IL-6, MCP-1 and CCL20 were estimated in culture supernatants by ELISA as marker of IL-6 amplifier loop activation. mRNA expression of IL-6, MCP1, CCL20, and SOCS3 genes were measured in the stimulated fibroblasts. Stimulated Renal fibroblast cells from CABMR patients were lysed with Lysis buffer and subjected to SDS–PAGE and western blotting with anti-phospho-STAT3 and anti-phospho-NFκB p65. Additionally, IL-6, MCP1, and CCL20 levels were measured in Healthy control (n = 10), CABMR (n = 20), and non-CABMR (n = 30) patients. Results IL-6 and IL-17 synergistically induced more IL-6, CCL-20 &amp; MCP-1 production from fibroblasts in culture supernatant. Gene expression analysis of IL-6, MCP1, and CCL20 was significantly higher with synergistic activation of IL-6 and IL-17 as compared to either IL-6 or IL-17 alone, while SOCS3 gene expression was downregulated. Our results also suggested that IL-6 Amplifier loop activation induces the NFκB and STAT3 signalling pathway activation in the non-immune cells like fibroblast derived from CABMR patients. Additionally, concentrations of IL-6, CCL-20 &amp; MCP-1 in sera were significantly higher in CABMR patients compared to non-CABMR patients (p&amp;lt;0.001). There was a significant reduction in IL-6 concentration in culture supernatant with IL-6 and IL-17 inhibitor together and mRNA expression of IL-6, CCL20 and MCP-1 was significantly reduced while SOCS3 gene expression was upregulated. Conclusion In humans after kidney transplantation, IL-6 amplifier activation plays an active role in chronic rejection responses. Inhibition of IL-6 with Anti-IL-6 (Tocilizumab) and inhibition of IL-17 with Anti-IL-17 together reduces markers of tissue injury (IL-6, MCP1, CCL20) and rejection of allografts. so, IL-6 amplifier may be a therapeutic target for Chronic transplant rejection.

Impact of the MCP-1-2518A>G polymorphism on COVID-19 severity in the Iranian population: A case-control study.

As a result of SARS-CoV-2 infection, the host's immune system is disrupted, and chemokines and cytokines are intensified to eliminate the virus, resulting in cytokine storm syndrome and acute respiratory distress syndrome (ARDS). Patients with COVID-19 have been observed to have elevated levels of MCP-1, a chemokine associated with the severity of the disease. In some diseases, polymorphisms in the regulatory region of the MCP-1 gene correspond to serum levels and disease severity. An attempt was made in this study to assess the relationship between MCP-1 G-2518A and serum MCP-1 levels in Iranian COVID-19 patients and the severity of the disease. In this study, patients were randomly sampled from outpatients on the first day of diagnosis and from inpatients on the first day of their hospitalization. Patients were classified into the outpatient (without symptoms or with mild symptoms) and inpatient (with moderate, severe, and critical symptoms) groups. The serum level of MCP-1 was measured by ELISA and the frequency of MCP-1 G-2518A gene polymorphism genotypes in COVID-19 patients was checked by the RFLP-PCR method. Participants with COVID-19 infection had a higher rate of underlying diseases, such as diabetes, high blood pressure, kidney disease, and cardiovascular disease than the control group (P-value<0.001). Also, the frequency of these factors in inpatients was significantly higher compared to outpatients (P-value<0.001). Additionally, the level of MCP-1 in serum was significantly different with an average of 11.90 in comparison to 2.98 in the control group (P-value, 0.05), which is attributed to elevated serum levels among patients in hospitals with an average of 11.72 in comparison to 2.98 in the control group. Compared with outpatients, inpatients had a higher frequency of the G allele of the MCP-1-2518 polymorphism (P-value<0.05), while a notable difference was observed in the serum level of MCP-1 in COVID-19 patients with the MCP-1-2518 AA genotype in the whole group in comparison to the control group (P-value: 0.024). Totally, the results showed that a high frequency of the G allele is related to hospitalization and poor outcome in COVID-19 cases.

Significant association of MCP1 rs1024611 and CCR2 rs1799864 polymorphisms with colorectal cancer and liver metastases susceptibility and aggressiveness: A case-control study

BackgroundThe MCP-1/CCR2 axis is one of the major chemokine signaling pathways that play a crucial role in the tumor microenvironment and has been involved in triggering various tumor progression mechanisms, such as increasing the immunosuppressive cells recruitment and promoting tumor cell proliferation and invasiveness. AimThe current study investigated the association of MCP1 (rs1024611) and CCR2 (rs1799864) genes variants with the risk as well as prognosis of colorectal cancer (CRC) and colorectal liver metastases (CRLM). Subjects and methodsA retrospective cohort study involved 408 patients (284 CRC and 124 CRLM), and 284 healthy control was conducted. Genotyping of selected polymorphisms was performed by PCR-RFLP assays and confirmed by microchip and capillary electrophoresis. ResultsThe results highlighted a positive association between MCP1 rs1024611 (non-AA) and CCR2 rs1799864 (GA) genotypes with increased CRC and CRLM risk. Correlation between SNPs and clinicopathological characteristics revealed a positive association between MCP1 rs1024611 and CCR2 rs1799864 (dominant model) and CRC poor prognosis features. Kaplan-Meier survival analysis revealed a significant association between MCP1 rs1024611 non-AA carriers and decreased survival rate. Neoadjuvant treatment showed an improvement in CRC and CRLM survival rates among carriers of MCP1 and CCR2 wild-type genotype. FOLFIRI chemotherapy exhibits reduced survival rates for patients who carried mutated genotypes of MCP1 and CCR2 polymorphisms. ConclusionConsidering our results, we suggest That both MCP1 and CCR2 polymorphisms may constitute independent factors for CRC and CRLM occurrence and can be helpful targets for an efficient therapeutic approach.

Extracellular vesicles derived from the intestinal microbiota of patients with CGD-associated inflammatory bowel disease have pro-inflammatory effects

BackgroundChronic granulomatous disease (CGD) is an inborn error of immunity characterized by mutations in any one of the 6 subunits forming the nicotinamide-adenine dinucleotide phosphate oxidase complex 2 leading to defective phagocyte-derived ROS production. As a result, patients with CGD have recurrent infections and increased inflammation and 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). Although we have previously demonstrated that patients with CGD-IBD have intestinal dysbiosis, its functional impact is poorly understood. Interestingly, intestinal microbes can produce gut microbiota-derived extracellular vesicles (GMEVs) which can cross the intestinal barrier and have systemic and/or end-organ immunomodulatory effects. Yet, the role of GMEVs in patients with CGD has never been evaluated. ObjectiveTo investigate the impact of GMEVs isolated from patients with CGD, with or without CGD-IBD, on intestinal permeability and inflammatory responses using wild type and NOX2-deficient (CYBB-/-) models. MethodsGMEVs were isolated and characterized from fecal samples obtained from 11 patients without CGD-IBD and 11 patients with active CGD-IBD recruited at the National Institutes of Health. Stimulation with GMEVs was performed on HT29, THP1 and CYBB-/- THP1 cell lines, HT29/THP1 co-culture models, and induced pluripotent stem cell-derived human intestinal organoids. TNF-α, IL-6, IL-8, IL-1β, IP-10, GM-CSF and MCP1 gene expression and production were assessed by RT-qPCR and ELISA, respectively. ResultsOur data showed that stimulation with GMEVs from patients with active CGD-IBD increased intestinal permeability compared to cells stimulated with GMEVs from patients without CGD-IBD. In HT29 cells, increased intestinal permeability was associated with increased gene expression and production of IL-8 and IL-1β. IL-8 was similarly increased in intestinal organoids. In THP1 cells, stimulation led to increased gene expression and production of TNF-α, IL-6 and IP-10, while stimulation in CYBB-/- THP1 cells increased TNF-α and MCP1 gene expression. Finally, stimulation of HT29/THP1 cell co-cultures resulted in increased gene expression of TNF-α, IL-6 and GM-CSF. ConclusionsOur results suggest that GMEVs isolated from patients with CGD-IBD may induce pro-inflammatory immune responses in patients with CGD, thereby contributing to their underlying inflammatory pathology. These findings bring forward GMEVs as a possible new therapeutic target for CGD-IBD. [Display omitted]

Dihydrotestosterone, and Not Testosterone, Enhances the LPS-Induced Inflammatory Cytokine Gene Expression in Human Adipocytes

The development of obesity-related complications lies in the low-grade inflammatory state consequent to adipocyte dysfunction. The direct involvement of sex hormones in adipose tissue inflammation has been previously suggested, but the evidence is scarce. In this study, we evaluated the effects of sex steroids on the in-vitroexpression of inflammatory mediators in human-derived adipocytes before and after lipopolysaccharide (LPS) exposure. Human adipocytes were differentiated from the vascular stromal fraction of adipose tissue samples of subjects undergoing abdominoplasty. We evaluated MCP-1, IL-1β, IL-6, and TNF-α gene expression in the presence of the main sex steroids, testosterone (T), and 17β-estradiol (E). Furthermore, we analyzed the effects of adipocytes exposure to the non-aromatizable androgen dihydrotestosterone (DHT), together with the effects of adipocytes pre-incubation with the aromatase inhibitor anastrozole alone (A), and in combination with T (A/T) before incubation with LPS. DHT, but not T, significantly enhanced the LPSinduction of MCP-1, IL-1β, IL-6, and TNF-α. Intriguingly, the exposure of adipocytes with A/T dramatically increased the LPS-induced expression of all considered inflammatory cytokines, even more than a hundred-fold. DHT and A/T dramatically enhance LPS-induced inflammatory cytokine expression in human-derived adipocytes. These results confirm the involvement of sex hormones in adipose tissue inflammation, suggesting a specific role for non-aromatizable androgens as the amplificatory sex hormones of the inflammatory response.

The systemic deletion of interleukin-1α reduces myocardial inflammation and attenuates ventricular remodeling in murine myocardial infarction

Myocardial inflammation following myocardial infarction (MI) is crucial for proper myocardial healing, yet, dysregulated inflammation may promote adverse ventricular remodeling and heart failure. IL-1 signaling contributes to these processes, as shown by dampened inflammation by inhibition of IL-1β or the IL-1 receptor. In contrast, the potential role of IL-1α in these mechanisms has received much less attention. Previously described as a myocardial-derived alarmin, IL-1α may also act as a systemically released inflammatory cytokine. We therefore investigated the effect of IL-1α deficiency on post-MI inflammation and ventricular remodeling in a murine model of permanent coronary occlusion. In the first week post-MI, global IL-1α deficiency (IL-1α KO mice) led to decreased myocardial expression of IL-6, MCP-1, VCAM-1, hypertrophic and pro-fibrotic genes, and reduced infiltration with inflammatory monocytes. These early changes were associated with an attenuation of delayed left ventricle (LV) remodeling and systolic dysfunction after extensive MI. In contrast to systemic Il1a-KO, conditional cardiomyocyte deletion of Il1a (CmIl1a-KO) did not reduce delayed LV remodeling and systolic dysfunction. In conclusion, systemic Il1a-KO, but not Cml1a-KO, protects against adverse cardiac remodeling after MI due to permanent coronary occlusion. Hence, anti-IL-1α therapies could be useful to attenuate the detrimental consequences of post-MI myocardial inflammation.

Antifungal bio-coating of endotracheal tube built by overexpressing the MCP1 gene of Saccharomyces boulardii and employing hydrogel as a “house” to antagonize Candida albicans

BackgroundFor some ICU patients, an artificial airway must be established with an endotracheal tube, but Candida albicans can easily adhere to the tube surface and form a biofilm, leading to potentially life threatening fungal infections. Therefore, it is urgent to prevent and reduce C. albicans infections introduced by the endotracheal tube. However, there are few antifungal drugs effective against C. albicans, and each of these drugs may have adverse effects on human cells. Saccharomyces boulardii is regarded as an alternative strategy to inhibit the adhesion of C. albicans, but it is affected by environmental stress. We hypothesized that it is feasible to strengthen the antagonistic ability of S. boulardii via encapsulating and genetically modification.MethodsIn this study, a bioactive material carrying the overexpressed MCP1 gene of Saccharomyces boulardii was constructed based on one-step photo-crosslinking. This material achieved spatial growth control of S. boulardii by encapsulating each S. boulardii cell within a hydrogel pore. The bioactive material was coated on an endotracheal tube and tested for its ability to inhibit the adhesion of C. albicans. Additionally, the material’s antagonistic activity towards C. albicans was evaluated by detecting intracellular Adenosine-triphosphate content, reactive oxygen species level and the activity of antioxidative enzymes. Tissue invasion experiment was executed to further evaluate the anti-adhesion ability of S. boulardii bio-coating.ResultsEncapsulating the overexpression of MCP1 by S. boulardii in hydrogel pores enhanced the viability of probiotics in the presence of high salt and oxidation stress. When used as the coating of an endotracheal tube, the S. boulardii bioactive material efficiently inhibited the adhesion of C. albicans by impairing the activities of superoxide dismutase and catalase and disturbing mitochondrial functions. In vivo, the S. boulardii bioactive material coating displayed good biocompatibility and reduced the host tissue invasion and virulence of C. albicans.ConclusionsThe integration of genetic modification and immobilization model breaks the bottleneck of previous application of microorganisms, and provides a new way to prevent fungal infections introduced by endotracheal tubes.Graphical