Inflammatory Chemokines Research Articles

Platelet Activation and Mechanisms of Thromboembolism Formation in Patients with Severe COVID-19. Alternative Mechanisms of Hemostasis System Activity

The review highlights the mechanism of development of hypercoagulation and thrombosis in severe forms of COVID-19. The introduction of the SARS-CoV-2 virus into the host organism is carried out by the interaction of the spike protein S with the angiotensin-converting enzyme ACE-2, which is located in type 2 alveocytes, vascular endothelium, kidneys, liver and other organs. In the event of a serious condition in patients with COVID-19, both nonspecific and adaptive immunity are activated. Stimulation of the complement system with the appearance of C3a, C3b, C5a fragments and the membrane attack complex (MAC) creates conditions for the development of hypercoagulability. The involvement of the renin-angiotensin-aldosterone system in this process and the appearance of angiotensin 2 (Ang-2) further increase the intensity of hypercoagulability. When the SARS-CoV-2 virus enters cells, the protective reaction of the adaptive immune system can turn into a pathological one (a cytokine storm develops), characterized by a high level of pro-inflammatory cytokines IL-1α, IL-6, Il-8, TNF-α, IL-17, etc.) and chemokines (CCL-2, CCL-11, etc.), which ultimately leads to the development of thromboangiopathy or otherwise immunothrombosis in seriously ill patients with COVID-19. Patients with more severe lesions may develop a condition similar to DIC. At the same time, patients with COVID-19 have mild thrombocytopenia, elevated levels of fibrinogen, D-dimer, fibrinogen degradation products (FDP), which indicates intense thrombus formation, as well as short PT and APTT, due to a largely increased level of FVIII. In COVID-19, along with the classical one, an alternative pathway (bypassing thrombin) of regulation of the hemostasis system and thrombus formation appears, mainly associated with the influence of the spike protein S (PS, PROS1) of the SARS-CoV-2 virus and papain-like protease (PROS1). Protein S directly affects the conversion of fibrinogen to fibrin, as well as the activation of individual plasma coagulation factors. The alternative pathway of blood coagulation is also due to the activation of the complement system via the lectin pathway with the inclusion of metalloproteinases MASP-1, 2 and 3. In addition, the S protein activates tPA, which may be accompanied by hyperfibrinolysis. In seriously ill patients with COVID-19, platelets play an important role in the occurrence of thromboembolic complications. During the release reaction, platelets are released from the cytoplasm into the blood α and dense granules containing inflammatory cytokines and chemokines, which enhances the cytokine storm and, consequently, thrombus formation. By acting on the spike protein S, platelets enhance an alternative way of regulating the hemostasis system and thrombus formation.

CCL2 promotes osteogenesis by facilitating macrophage migration during acute inflammation.

Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1day) but not sustained (7days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.

Specialized Pro-Resolving Lipid Mediators: Endogenous Roles and Pharmacological Activities in Infections

During an infection, inflammation mobilizes immune cells to eliminate the pathogen and protect the host. However, inflammation can be detrimental when exacerbated and/or chronic. The resolution phase of the inflammatory process is actively orchestrated by the specialized pro-resolving lipid mediators (SPMs), generated from omega-3 and -6 polyunsaturated fatty acids (PUFAs) that bind to different G-protein coupled receptors to exert their activity. As immunoresolvents, SPMs regulate the influx of leukocytes to the inflammatory site, reduce cytokine and chemokine levels, promote bacterial clearance, inhibit the export of viral transcripts, enhance efferocytosis, stimulate tissue healing, and lower antibiotic requirements. Metabolomic studies have evaluated SPM levels in patients and animals during infection, and temporal regulation of SPMs seems to be essential to properly coordinate a response against the microorganism. In this review, we summarize the current knowledge on SPM biosynthesis and classifications, endogenous production profiles and their effects in animal models of bacterial, viral and parasitic infections.

Therapeutic targeting of adipose tissue macrophages ameliorates liver fibrosis in non-alcoholic fatty liver disease

Background & Aims: The accumulation of adipose tissue macrophages (ATMs) in obesity has been associated with hepatic injury. However, the contribution of ATMs to hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) remains to be elucidated. Herein, we investigate the relationship between ATMs and liver fibrosis in patients with patients with NAFLD and evaluate the impact of modulation of ATMs over hepatic fibrosis in an experimental non-alcoholic steatohepatitis (NASH) model. MethodsAdipose tissue and liver biopsies from 42 patients with NAFLD with different fibrosis stages were collected. ATMs were characterised by immunohistochemistry and flow cytometry and the correlation between ATMs and liver fibrosis stages was assessed. Selective modulation of the ATM phenotype was achieved by i.p. administration of dextran coupled with dexamethasone in diet-induced obesity and NASH murine models. Chronic administration effects were evaluated by histology and gene expression analysis in adipose tissue and liver samples. In vitro crosstalk between human ATMs and hepatic stellate cells (HSCs) and liver spheroids was performed. ResultsPatients with NAFLD presented an increased accumulation of pro-inflammatory ATMs that correlated with hepatic fibrosis. Long-term modulation of ATMs significantly reduced pro-inflammatory phenotype and ameliorated adipose tissue inflammation. Moreover, ATMs modulation was associated with an improvement in steatosis and hepatic inflammation and significantly reduced fibrosis progression in an experimental NASH model. In vitro, the reduction of the pro-inflammatory phenotype of human ATMs with dextran–dexamethasone treatment reduced the secretion of inflammatory chemokines and directly attenuated the pro-fibrogenic response in HSCs and liver spheroids. ConclusionsPro-inflammatory ATMs increase in parallel with fibrosis degree in patients with NAFLD and their modulation in an experimental NASH model improves liver fibrosis, uncovering the potential of ATMs as a therapeutic target to mitigate liver fibrosis in NAFLD. Impact and implicationsWe report that human adipose tissue pro-inflammatory macrophages correlate with hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD). Furthermore, the modulation of adipose tissue macrophages (ATMs) by dextran-nanocarrier conjugated with dexamethasone shifts the pro-inflammatory phenotype of ATMs to an anti-inflammatory phenotype in an experimental murine model of non-alcoholic steatohepatitis. This shift ameliorates adipose tissue inflammation, hepatic inflammation, and fibrosis. Our results highlight the relevance of adipose tissue in NAFLD pathophysiology and unveil ATMs as a potential target for NAFLD.

Targeting Cellular Senescence in Organ Transplantation.

Targeting Cellular Senescence in Organ Transplantation.

The Effect of Radiotherapy on Cell Survival and Inflammatory Cytokine and Chemokine Secretion in a Co-Culture Model of Head and Neck Squamous Cell Carcinoma and Normal Cells.

Radiotherapy (RT) is one of the main treatments for head and neck squamous cell carcinomas (HNSCCs). Unfortunately, radioresistance is observed in many cases of HNSCCs. The effectiveness of RT depends on both the direct effect inducing cell death and the indirect effect of changing the tumor microenvironment (TME). Knowledge of interactions between TME components after RT may help to design a new combined treatment with RT. In the study, we investigated the effect of RT on cell survival and cell secretion in a co-culture model of HNSCCs in vitro. We examined changes in cell proliferation, colony formation, cell cycle phases, type of cell death, cell migration and secretion after irradiation. The obtained results suggest that the presence of fibroblasts and endothelial cells in co-culture with HNSCCs inhibits the function of cell cycle checkpoints G1/S and G2/M and allows cells to enter the next phase of the cell cycle. We showed an anti-apoptotic effect in co-culture of HNSCCs with fibroblasts or endothelial cells in relation to the execution phase of apoptosis, although we initially observed increased activation of the early phase of apoptosis in the co-cultures after irradiation. We hypothesize that the anti-apoptotic effect depends on increased secretion of IL-6 and MCP-1.

Inflammatory cytokines and chemokines in obese adolescents with antibody against to adenovirus 36

Obesity in adolescents has reached epidemic proportions and is associated with the inflammatory response and viral infections. The aim of this study was to understand the profile of inflammatory cytokines and chemokines associated with the inflammatory response and metabolic syndrome (MetS) in obese adolescents with positive serology for adenovirus 36 (ADV36). Thirty-six overweight, 36 obese, and 25 severe obesity adolescents aged 10 to 16 years were included in the study. The following variables were analyzed: sex, age, body mass index (BMI), blood pressure, total cholesterol and fractions, triglycerides, glucose, serum cytokine concentrations, and ADV36 antibodies. Cytokines and chemokines were quantified by cytometry and ADV36 serology was determined by enzyme-linked immunosorbent assay (ELISA). The results showed higher levels of the cytokines interleukin-1beta (IL-1β), IL-6, IL-10 and of the chemokine interferon-gamma-inducible protein 10 (IP-10) in severe obesity adolescents compared to the obese and overweight groups, as well as in the group with MetS compared to the group without this syndrome. The frequency of ADV36-positive individuals did not differ between groups. The findings revealed differences in BMI between the obese and severe obesity groups versus the overweight group in the presence of positivity for ADV36, suggesting an association with weight gain and possibly MetS installation.

Agomir miRNA-150-5p alleviates pristane-induced lupus by suppressing myeloid dendritic cells activation and inflammation via TREM-1 axis.

Triggering receptors expressed on myeloid cells-1 (TREM-1) has been shown to participate in inflammatory autoimmune diseases. Nevertheless, the detailed underlying mechanisms and therapeutic benefits by targeting TREM-1 remain elusive, especially in myeloid dendritic cells (mDCs) and systemic lupus erythematosus (SLE). Disorders of epigenetic processes including non-coding RNAs give rise to SLE, resulting in complicated syndromes. Here, we aim to address this issue and explore the miRNA to inhibit the activation of mDCs and alleviate the progress of SLE by targeting TREM-1 signal axis. Bioinformatics methods were used to analyze the differentially expressed genes (DEGs) between patients with SLE and healthy individuals by four mRNA microarray datasets from Gene Expression Omnibus (GEO). Then we identified the expression of TREM-1 and its soluble form (sTREM-1) in clinical samples by ELISA, quantitative real-time PCR and Western blot. Phenotypic and functional changes of mDCs elicited by TREM-1 agonist were determined. Three databases of miRNAs target prediction and a dual-luciferase reporter assay were used to screen and verify miRNAs that can directly inhibit TREM-1 expression in vitro. Moreover, pristane-induced lupus mice were injected with miR-150-5p agomir to evaluate the effects of miR-150-5p on mDCs in lymphatic organs and disease activity in vivo. We screened TREM-1 as one of the hub genes closely correlated with the progression of SLE and identified sTREM-1 in serum as a valuable diagnostic biomarker for SLE. Moreover, activation of TREM-1 by its agonist promoted activation and chemotaxis of mDCs and increased the production of inflammatory cytokines and chemokines, showing higher expression of IL-6, TNF-α, and MCP-1. We showed that lupus mice displayed a unique miRNA signature in spleen, among which miR-150 was the most significantly expressed miRNA that targeting TREM-1 compared with wild type group. Transfection of miRNA-150-5p mimics directly suppressed the expression of TREM-1 by binding to its 3' UTR. Our in vivo experiments first indicated that administration of miR-150-5p agomir effectively ameliorated lupus symptoms. Intriguingly, miR-150 inhibited the over activation of mDCs through TREM-1 signal pathway in lymphatic organs and renal tissues. TREM-1 represents a potentially novel therapeutic target and we identify miR-150-5p as one of the mechanisms to alleviate lupus disease, which is attributable for inhibiting mDCs activation through TREM-1 signaling pathway.

Important functional role of the protein osteopontin in the progression of malignant pleural mesothelioma.

Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components. Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model. In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene (Spp1) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo. These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo. These findings have translational potential to improve the therapeutic response of human MPM.

#3261 SAR131675, A SELECTIVE VEGFR3 INHIBITOR, AMELIORATES RENAL INFLAMMATION AND LYMPHANGIOGENESIS IN THE MURINE LUPUS NEPHRITIS MODEL

Abstract Background and Aims Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune-complex deposits and inflammatory cell infiltrations in multiple organs, and approximately half of lupus patients have nephritis. Lymphangiogenesis is the proliferation of pre-existing lymphatic vessels (LVs), which regulate tissue fluid homeostasis and immune cell trafficking, responding to the tissue environment. In this study, we evaluated the therapeutic effect of SAR131672, a selective VEGFR3 inhibitor, on the murine lupus nephritis model by regulating inflammation and lymphangiogenesis. Method First, we reviewed medical records for the biopsy-proven lupus nephritis and performed an immunohistochemistry study for D2-40. For animal experiments, seven to eight-week-old male BALB/c mice were used. The back area's skin was shaved and treated topically three times per week, with 100 μg of resiquimod in 100 μl of acetone for eight weeks and concomitantly treatment of VEGFR3 inhibitor, SAR 131672 by oral gavage. We evaluated renal histology and immunofluorescent study for inflammatory cells and lymphatic vessels. We also evaluated inflammatory cytokines and chemokines, lymphangiogenic factors by qRT-PCR. Results In a human study, we found that the higher lupus activity index, the more D2-40(+) lymphatic vessels are expressed in the tubulointerstitial areas. In an animal study, eight weeks of topical treatment of resiquimod to Balb/c mice induces lupus-like symptoms such as weight loss, splenomegaly, and glomerular immune complexes deposits such as IgG, IgM, and C3 in immunofluorescent staining. Histologically, glomerular mesangial cell proliferation and increased inflammatory cells in tubulointerstitial areas were noted in the H&amp;E stain. Inhibition of VEGFR3 by oral SAR131672 treatment decreases glomerular and tubulointerstitial inflammation and decreases LYVE-1(+) lymphatic vessels. The pro-inflammatory cytokines and chemokines such as ICAM-1, VCAM-1, MCP-1, CCL19, CCL21, CCR7, CXCL13, and BAFF mRNA levels were increased compared with the vehicle-treated group. Treatment SAR131672 decreases pro-inflammatory cytokines and chemokine. Conclusion Inhibition of VEGFR3 by SAR131672 decreases the resiquimod-induced lupus nephritis model by regulating inflammation and lymphangiogenesis.

#4321 ACTION3 PHASE 3 STUDY: EVALUATING A NOVEL THERAPEUTIC STRATEGY OF A CCR2 INHIBITOR (DMX-200) WITH ANGIOTENSIN RECEPTOR BLOCKADE IN FSGS

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a disease of podocytes. Complications include nephrotic syndrome and progressive kidney failure. FSGS is a condition with high unmet need and there is no approved treatment. The angiotensin II receptor type 1 (AT1R) and chemokine receptor 2 (CCR2) are G protein coupled receptors that form functional heteromers. Simultaneous antagonism of these receptors suggested synergistic renoprotective effects in preclinical and early phase clinical studies of proteinuric kidney disease. DMX-200 (repagermanium) is a C-C chemokine receptor type 2 (CCR2) inhibitor that, when administered concurrently with an angiotensin II receptor blocker (ARB), is designed to inhibit recruitment of monocytes implicated in the inflammatory chemokine environment of chronic disease. In a Phase 2a open-label study of 27 patients with proteinuric chronic kidney disease, 25% of patients achieved &amp;gt; 50% reduction in uPCR with combined use of DMX-200 and irbesartan. A Phase 2a placebo-controlled cross-over study in 8 patients with primary FSGS receiving stable dose of irbesartan demonstrated evidence of promising efficacy with clinically relevant reduction in uPCR of 17% with DMX-200 compared with placebo. These encouraging data suggest that treatment with DMX-200 may result in clinically meaningful reduction in proteinuria when added to ARB in patients with glomerular diseases such as FSGS. Proteinuria reduction is a positive prognostic sign for preserving kidney function. The observations from earlier trials have led to the initiation of an international randomised double-blind, placebo-controlled Phase 3 clinical trial (ACTION3) to further evaluate the efficacy of DMX-200 in patients with FSGS receiving an ARB. Method Eligible patients are adults (18-80 years) with biopsy-proven primary FSGS, genetic FSGS or FSGS of undetermined cause (FSGS-UC), and uPCR of &amp;gt;1.5g/g based on 24hr urine collection and eGFR of ≥25 ml/min/1.73 m2. Blood pressure ≤160/100 mmHg and BMI ≤40kg/m2. Patients with secondary FSGS or those treated with strong immunomodulatory agents (other than corticosteroids) are excluded. All patients are required to receive concomitant ARB treatment at a dose of at least 50% of the maximum approved dose according to the relevant product label. Patients receiving ACE-inhibitors are required to switch to an ARB. Other background therapies such as SGLT-2 inhibitors and mineralocorticoid receptor antagonists are permitted if receiving a stable dose in the 3 months prior to screening. Patients are randomized in a 1:1 ratio to DMX-200 (120 mg twice daily) or placebo. The primary objective is to evaluate the efficacy of DMX-200 on reduction in PCR after 35 weeks of treatment and eGFR slope after 104 weeks of treatment. Secondary objectives are safety and tolerability of DMX-200 and to evaluate the effect of DMX-200 on response criteria and composite endpoint of worsening kidney function. Exploratory objectives include assessment of changes in inflammatory biomarkers such as MCP-1 and the pharmacokinetic profile of DMX-200. Two interim analyses (IA) are planned, first planned IA for futility will be performed by an Independent Data Monitoring Committee after the first ​72 randomized patients (36 patients per group) complete 35 weeks ​of treatment. A second IA will be performed once approximately​ 144 enrolled patients complete 35 weeks ​of treatment.​ The final analysis will be performed once approximately 286 patients complete 104 weeks of treatment. The study is currently open in 11 countries at approximately 75 investigational sites and will be expanded to include more countries and sites subject to a successful first interim analysis. Results ACTION3 is a trial in progress and is expected to be completed in 2026. Conclusion ACTION3 is a Phase 3 randomized, placebo-controlled efficacy and safety trial which evaluates the novel approach of combining an investigational CCR2 inhibitor, DMX-200, with angiotensin receptor blockade in patients with FSGS.

Prophylactic simvastatin increased survival during endotoxemia and inhibited granulocyte trafficking in a cell-intrinsic manner

Cross sectional studies have shown that statin-users have improved odds of surviving severe sepsis. Meanwhile controlled clinical trials failed to demonstrate improved sepsis survival with acute statin administration following hospitalization. Here, a lethal murine peritoneal lipopolysaccharide (LPS) endotoxemia model was used to assess the efficacy of chronic versus acute simvastatin on survival. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival. At a pre-mortality time point in LPS-treated mice, chronic simvastatin suppressed granulocyte trafficking in to the lungs and peritoneum without otherwise suppressing emergency myelopoiesis, myeloid cells in circulation, or inflammatory cytokines. Chronic simvastatin treatment significantly downregulated inflammatory chemokine gene signature in the lungs of LPS-treated mice. Thus, it was unclear if simvastatin was inhibiting granulocyte chemotaxis in a cell intrinsic or extrinsic manner. Adoptive transfer of fluorescently labeled granulocytes from statin and vehicle treated mice into LPS-treated mice showed that simvastatin inhibited lung-granulocyte trafficking in a cell intrinsic manner. Congruent with this, chemotaxis experiments using in vitro macrophages and ex vivo granulocytes demonstrated that simvastatin inhibited chemotaxis in a cell-intrinsic manner. Collectively, chronic but not acute simvastatin treatment improved survival in murine endotoxemia, and this was associated with cell-intrinsic inhibition of granulocyte chemotaxis.

Thymoquinone Inhibition of Chemokines in TNF-α-Induced Inflammatory and Metastatic Effects in Triple-Negative Breast Cancer Cells.

The lack of identifiable molecular targets or biomarkers hinders the development of treatment options in triple-negative breast cancer (TNBC). However, natural products offer a promising alternative by targeting inflammatory chemokines in the tumor microenvironment (TME). Chemokines are crucial in promoting breast cancer growth and metastasis and correlate to the altered inflammatory process. In the present study, we evaluated the anti-inflammatory and antimetastatic effects of the natural product thymoquinone (TQ) on TNF-α-stimulated TNBC cells (MDA-MB-231 and MDA-MB-468) to study the cytotoxic, antiproliferative, anticolony, antimigratory, and antichemokine effects using enzyme-linked immunosorbent assays, quantitative real-time reverse transcription-polymerase chain reactions, and Western blots were used in sequence to validate the microarray results further. Four downregulated inflammatory cytokines were identified, CCL2 and CCL20 in MDA-MB-468 cells and CCL3 and CCL4 in MDA-MB-231 cells. Furthermore, when TNF-α-stimulated MDA-MB-231 cells were compared with MDA-MB-468 cells, the two cells were sensitive to TQ's antichemokine and antimetastatic effect in preventing cell migration. It was concluded from this investigation that genetically different cell lines may respond to TQ differently, as TQ targets CCL3 and CCL4 in MDA-MB-231 cells and CCL2 and CCL20 in MDA-MB-468 cells. Therefore, the results indicate that TQ may be recommended as a component of the therapeutic strategy for TNBC treatment. These outcomes stem from the compound's capacity to suppress the chemokine. Even though these findings support the usage of TQ as part of a therapy strategy for TNBC associated with the identified chemokine dysregulations, additional in vivo studies are needed to confirm these in vitro results.

BSHXF-medicated serum combined with ADSCs regulates the TGF-β1/Smad pathway to repair oxidatively damaged NPCs and its component analysis

Ethnopharmacological relevanceLower back pain (LBP) is a common and frequent clinical condition, and intervertebral disc degeneration (IDD) is recognized as the leading cause of LBP, typically manifested by increased nucleus pulposus cell (NPC) senescence and death. In recent years, the treatment of IDD with stem cell injections has had great potential compared to surgical treatment. Combining the two may achieve better results, as BuShenHuoXueFang (BSHXF) is an herbal formula that improves the survival rate of transplanted stem cells and enhances their efficacy. Aim of the studyWe aimed to qualitatively and quantitatively analyze BSHXF-medicated serum and investigate the molecular mechanism of BSHXF-mediated serum in promoting the differentiation of adipose mesenchymal stem cells (ADSCs) into NPCs and delaying the senescence of NPCs by regulating the TGF-β1/Smad pathway. Materials and methodsIn this study, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS) was used to establish a method for the analysis of rat serum samples to track the active components in vivo; the oxidative damage model of NPCs was induced by T-BHP, and a Transwell chamber was used to construct a coculture system of ADSCs and NPCs. Flow cytometry was used to determine the cell cycle; SA-β-Gal staining was used to assess cell senescence; ELISA was used to detect IL-1β, IL-6 inflammatory factors, CXCL-1, CXCL-3, CXCL-10 chemokines, and TGF-β1 in the supernatants of ADSCs and NPCs. WB was used to detect COL2A1, COL1A1, and Aggrecan in ADSCs to assess the manifestation of NP differentiation in ADSCs, and the WB method was used to detect COL2A1, COL1A1, Aggrecan, p16, p21, p53, and p-p53 protein expression in NPCs to reflect the cellular senescence status and to detect TGF-β1, Smad2, Smad3, p- Smad2, and p- Smad3 protein expression in NPCs to reflect the pathway condition. ResultsWe finally identified 70 blood components and their metabolites, including 38 prototypes, from the BSHXF-medicated serum. Compared with that in the nonmedicated serum group, the TGF-β1/Smad pathway was activated in the medicated serum group, ADSCs moved toward NPC characteristics, the number of NPCs in the S/G2M phase increased, the number of senescent NPCs decreased, IL-1β and IL-6 inflammatory factors in the Transwell decreased, CXCL-1, CXCL-3, and CXCL-10 chemokines decreased, and the expression of p16, p21, p53 and p-p53 proteins in NPCs was inhibited. ConclusionBy regulating the TGF-β1/Smad pathway, BSHXF-medicated serum promoted ADSCs to NPCs, effectively alleviated the cycle blockage of NPCs after oxidative damage, encouraged the growth and proliferation of NPCs, delayed the aging of NPCs, improved the deteriorating microenvironment around NPCs, and repaired oxidatively damaged NPCs. The combination of BSHXF or its compounds with ADSCs has great potential for the treatment of IDD in the future.

USP14 inhibition promotes recovery by protecting BBB integrity and attenuating neuroinflammation in MCAO mice.

Blood-brain barrier (BBB) dysfunction is one of the hallmarks of ischemic stroke. USP14 has been reported to play a detrimental role in ischemic brain injury. However, the role of USP14 in BBB dysfunction after ischemic stroke is unclear. In this study, we tested the role of USP14 in disrupting BBB integrity after ischemic stroke. The USP14-specific inhibitor IU1 was injected into middle cerebral artery occlusion (MCAO) mice once a day. The Evans blue (EB) assay and IgG staining were used to assess BBB leakage 3 days after MCAO. FITC-detran test was slected to examine the BBB leakage in vitro. Behavior tests were conducted to evaluate recovery from ischemic stroke. Middle cerebral artery occlusion increased endothelial cell USP14 expression in the brain. Furthermore, the EB assay and IgG staining showed that USP14 inhibition through IU1 injection protected against BBB leakage after MCAO. Analysis of protein expression revealed a reduction in the inflammatory response and chemokine release after IU1 treatment. In addition, IU1 treatment was found to rescue neuronal loss resulting from ischemic stroke. Behavior tests showed a positive effect of IU1 in attenuating brain injury and improving motor function recovery. In vitro study showed that IU1 treatment could alleviate endothelial cell leakage induced by OGD in cultured bend.3 cells through modulating ZO-1 expression. Our results demonstrate a role for USP14 in disrupting the integrity of the BBB and promoting neuroinflammation after MCAO.

MiR-10a-3p modulates adiposity and suppresses adipose inflammation through TGF-β1/Smad3 signaling pathway.

Obesity is a multifactorial disease characterized by an enhanced amount of fat and energy storage in adipose tissue (AT). Obesity appears to promote and maintain low-grade chronic inflammation by activating a subset of inflammatory T cells, macrophages, and other immune cells that infiltrate the AT. Maintenance of AT inflammation during obesity involves regulation by microRNAs (miRs), which also regulate the expression of genes implicated in adipocyte differentiation. This study aims to use ex vivo and in vitro approaches to evaluate the role and mechanism of miR-10a-3p in adipose inflammation and adipogenesis. Wild-type BL/6 mice were placed on normal (ND) and high-fat diet (HFD) for 12 weeks and their obesity phenotype, inflammatory genes, and miRs expression were examined in the AT. We also used differentiated 3T3-L1 adipocytes for mechanistic in vitro studies. Microarray analysis allowed us to identify an altered set of miRs in the AT immune cells and Ingenuity pathway analysis (IPA) prediction demonstrated that miR-10a-3p expression was downregulated in AT immune cells in the HFD group as compared to ND. A molecular mimic of miR-10a-3p reduced expression of inflammatory M1 macrophages, cytokines, and chemokines, including transforming growth factor-beta 1 (TGF-β1), transcription factor Krüppel-like factor 4 (KLF4), and interleukin 17F (IL-17F) and induced expression of forkhead box P3 (FoxP3) in the immune cells isolated from AT of HFD-fed mice as compared to ND. In differentiated 3T3-L1 adipocytes, the miR-10a-3p mimics also reduced expression of proinflammatory genes and lipid accumulation, which plays a role in the dysregulation of AT function. In these cells, overexpression of miR-10a-3p reduced the expression of TGF-β1, Smad3, CHOP-10, and fatty acid synthase (FASN), relative to the control scramble miRs. Our findings suggest that miR-10a-3p mimic mediates the TGF-β1/Smad3 signaling to improve metabolic markers and adipose inflammation. This study provides a new opportunity for the development of miR-10a-3p as a novel therapeutic for adipose inflammation, and its associated metabolic disorders.

Black carbon nanoparticles activate the crosstalk mechanism between necroptosis and macrophage extracellular traps to change macrophages fate

PM2.5 still poses a threat to public health even at very low levels. Black carbon (BC) is a key component of PM2.5. Macrophage extracellular traps (METs) are a means by which macrophages capture and destroy invading pathogens. Necroptosis is an inflammatory programmed cell death. However, there is no research on the crosstalk mechanism between necroptosis and METs after BC exposure. In our study, fluorescence labeling, fluorescent probes, qPCR, and immunofluorescence were applied. Our research found that under normal physiological conditions, when macrophages receive external stimuli (in our experiment, phorbol 12-myristate 13-acetate (PMA)), they will form METs, thus exhibiting innate immune function. However, exposure to BC can cause necroptosis in macrophages accompanied by increased levels of ROS and cytosolic calcium ions as well as altered expression of inflammatory factors and chemokines that prevent the formation of METs, and weakening innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibits the formation of METs. Our experiment will enrich the understanding of the mechanism of macrophage injury caused by BC exposure, provide a new direction for studying harmful atmospheric particle toxicity, and propose new therapeutic insights for diseases caused by atmospheric particulate matter. This study is the first to explore the crosstalk mechanism between necroptosis and METs after BC exposure.

Associations of IL17A G-197A single nucleotide polymorphism with immunological parameters and structural changes of the brain in schizophrenia

Schizophrenia is a chronic mental disorder that is caused by a complex palette of genetic, epigenetic and environmental factors. Some of the important components of its pathogenesis are systemic inflammation and the dysfunction of immunity, which lead to neuroinflammation, contributing to development of structural brain changes. Earlier we have shown that increase in interleukin-17A levels is associated with morphometric changes and immune dysregulation in schizophrenia. IL17A G-197A (rs2275913) genetic polymorphism is involved in determining interleukin-17A secretion. The goal of this work was to investigate the associations between rs2275913 polymorphism, immune disorders and structural neurovisualization findings in schizophrenia to provide new insights into the immunopathogenesis of this disease. 60 patients aged 18 to 42 years diagnosed with schizophrenia were enrolled. 85 healthy volunteers were included into the control group. Multiplex assay was used to determine cytokine and chemokine serum levels. Rs2275913 polymorphism was assessed by polymerase chain reaction with electrophoretic detection of amplification products. A number of relationships between rs2275913 polymorphism and the immune parameters in schizophrenia were revealed. Carriers of G allele showed significant increase in IFNY, a key cytokine of Th1-link of adaptive immunity, and IL-8, an inflammatory chemokine. Also, increased levels of CXCL16 were observed in patients carrying the G allele. CXCL16 activates secretion of other proinflammatory chemokines and is involved in activation of Th1 adaptive immunity. Associations of heterozygous GA genotype with reduced cortical thickness in a number of areas of the frontal cortex in schizophrenia were found. Changes in cortical thickness in some of these areas, including middle frontal gyrus and orbitofrontal cortex, can be relevant to the pathogenesis of schizophrenia. The results highlight the importance of immunogenetic factors in the pathogenesis of schizophrenia and indicate that the rs2275913 polymorphism requires further studies as a potential biomarker of immune dysregulation and morphometric brain changes in schizophrenia.

THE SYNERGISTIC EFFECTS OF PROINFLAMMATORY CYTOKINES ON CARDIOVASCULAR FUNCTION AND SYMPATHETIC OUTFLOW IN RAT

Objective: Inflammation has been implicated in the pathophysiology of cardiovascular dysfunction and neurohumoral activation in cardiovascular diseases. Our previous work has demonstrated that proinflammatory cytokines (PICs) in cardiovascular/autonomic brain regions contribute significantly to the sympathetic excitation and cardiac dysfunction in heart failure and hypertension. Notably, the chronic inflammatory conditions in these disease settings are caused by numerous inflammatory cytokines and chemokines. Given that the elevated levels of a single effector cytokine in the pathophysiological condition are insufficient to cause a significant change in hemodynamic and sympathetic responses, how PIC-driven neuroinflammation promotes sympathetic activation remains unclear. This work sought to determine whether PICs in the brain have a synergistic action to exaggerate their inflammatory effects on hemodynamic and sympathetic responses in rats. Design and method: Blood pressure (BP, mmHg), heart rate (HR, beats/min) and renal sympathetic nerve activity (RSNA, % change) were continuously recorded for 4-5 hours in urethane anesthetized male Sprague Dawley rats. The inflammatory cytokines tumor necrosis factor-↑ (TNF-↑), interleukin (IL)-1↓, IL-6 and vehicle were injected intracerebroventricularly (ICV). Results: We found that while ICV injection of TNF-↑, IL-1↓ or IL-6 alone at a low dose (10 ng, respectively) did not induce significant changes in BP, HR and RSNA, the combined injection of all three cytokines at the same dose (10 ng) elicited a substantial (*p&lt;0.05) increase in BP (18.2 ± 2.1*), HR (62 ± 7*) and RSNA (94.5 ± 5.2 %*), that began within 15-20 mins. These responses peaked at 2-3 hours after ICV injection and remained elevated for the rest of the recording period. Additionally, ICV injection of TNF-↑, IL-1↓ or IL-6 alone at a high dose (50 ng, respectively), produced a dramatic and long-lasting increase in BP, HR and RSNA, that closely resembles the time course and response pattern induced by ICV injection of all three cytokines concurrently at the low dose (10 ng). Conclusions: These data suggest a synergistically excitatory action of PICs in the brain on sympathetic outflow. Interventions that simultaneously target multiple cytokines may have an extended anti-inflammatory effect of reducing sympathetic excitation in hypertension and heart failure.