MIP-1beta Research Articles

A glucose-rich heteropolysaccharide from Marsdenia tenacissima (Roxb.) Wight et Arn. and its zinc-modified complex enhance immunoregulation by regulating TLR4-Myd88-NF-κB pathway

A previously unreported immunological polysaccharide (MTP70–1) was obtained from Marsdenia tenacissima (Roxb.) Wight et Arn. MTP70–1 (2738 Da) is a heteropolysaccharide that mainly consists of (1 → 5)-linked-L-Araf, t-D-Glcp, (1 → 3,5)-linked-L-Araf, (1 → 4)-linked-D-Galp, (1 → 6)-linked-D-Glcp, and (1 → 3,6)-linked-D-Manp. In vitro cell assays revealed that MTP70–1 exhibits moderate immunomodulatory effects at the cellular level, and MTP70–1 was further modified with zinc to improve these effects. These modifications enhanced the immunomodulatory effects of MTP70–1, as phagocytosis was enhanced, the secretion of cytokines (TNF-α, IL-6, IL-1β, and IL-18) was increased, and the generation of chemokines (NO and ROS) in macrophages was enhanced. The intracellular mechanism by which MTP70–1 and MTP70-Zn activate macrophages was further revealed to be closely related to the TLR4-Myd88-NF-κB signaling pathway. In addition, a microscale thermophoresis binding (MST) assay confirmed that Zn modification can effectively enhance the binding affinity of MTP70–1 for TLR4. Ultimately, better immune-enhancing activity was attained with MTP70-Zn than MTP70–1. The immune-enhancing activity of MTP70-Zn was further demonstrated through zebrafish assays, which revealed that MTP70-Zn can effectively enhance the proliferation of macrophages and neutrophils.

Protective role of Metrnl on macrophage recruitment during the acute inflammatory phase in a mouse model of myocardial infarction

Abstract Introduction Meteorin-like protein (Metrnl) is a cytokine involved in the attenuation of inflammation. Previously, a cardioprotective role of Metrnl against cardiac hypertrophy and ischemia/reperfusion has been described. After myocardial infarction (MI), macrophages recruited from the spleen, and their capacity to shift from inflammatory (M1) to reparative (M2) phases determines the fate of cardiac repair. Purpose The aim of this study was to analyse the function of Metrnl in a mouse model of permanent MI during the acute inflammatory phase. Methods Wild-type (WT), Metrnl Knock-out (Metrnl-/-) and Metrnl-reconstituted Metrnl-/- mice (AAV9-Metrnl, with myocardial-specific tropism) were subjected to MI by coronary artery ligation. After 4 days, cardiac function was assessed using echocardiography at baseline and 4 days post-MI. Monocyte populations were analysed in spleen and blood by flow cytometry, and macrophages infiltrating the infarcted myocardium by gene expression analyses. Results Echocardiographic analysis demonstrated that Metrnl deficiency led to cardiac dysfunction (FAC p=0.02) accompanied by myocardial dilation 4 days post-MI (EDV p=0.02; ESV p=0.01). On the contrary, the overexpression of Metrnl in the myocardium of AVV9-Metrnl mice restored cardiac function compared to Metrnl-/- group (FAC p=0.01, EDV p=0.04, ESV p=0.09). Furthermore, we observed a reduction in splenic monocytes (CD11b+F4/80+) in Metrnl-/- mice compared to the WT group at 4 days post-MI (p=0.06). In particular, a significant decrease in pro-inflammatory Ly6CHigh and CD86+ monocytes was noted in the spleens of Metrnl-/- mice compared to the WT group (p=0.03; p=0.04 respectively), suggesting their mobilization out of the spleen, but not in the AVV9-Metrnl group. Although the total number of Ly6CHigh and Ly6CLow monocytes in the bloodstream was similar in all groups, there was a trend to different expression in the CCR2 and CCR5 chemokine receptors (p=0.07; p=0.06 respectively). At the same time, M1 macrophage recruitment chemokines (CCL2) and inflammatory markers (TNFα) were significantly induced in the ischemic area of the infarcted myocardium compared to the remote zone in the WT and Metrnl-/- groups (CCL2 p=0.03; p=0.01 respectively; TNFα p=0.008;p=0.04 respectively). In contrast, TNFα and CCL2 genes were not induced in the ischemic area of AVV9-Metrnl mice. Regarding M2 macrophages, the Arg1 marker was significantly reduced in the ischemic zone of Metrnl-/- mice compared to the WT group (p= 0.01), while in the AVV9-Metrnl group was recovered (p= 0.04). Conclusions Our results demonstrate that the absence of Metrnl promotes the recruitment of inflammatory macrophages to the ischemic myocardium and is associated to cardiac dysfunction.

Epigenetic BET inhibitor apabetalone counters inflammatory and fibrotic processes in activated cardiac fibroblasts providing insight into reduced hospitalizations for heart failure in BETonMACE trial

Abstract Background After myocardial infarction (MI), sustained crosstalk between monocytes and cardiac fibroblasts (CFs) promotes chronic inflammation and interstitial fibrosis that can contribute to heart failure (HF). Resident and myocardium-infiltrating immune cells produce proinflammatory cytokines IL-1β and TNFα that stimulate CFs to produce monocyte chemoattractants. Monocytes secrete TGF-β1 that transforms CFs into contractile myofibroblasts overproducing the extracellular matrix (ECM) responsible for cardiac fibrosis. Epigenetic BET inhibitors (BETi) reduce CF transdifferentiation to prevent cardiac fibrosis and HF in animal models. In patients with cardiovascular disease, type 2 diabetes and recent MI, administration of the BETi apabetalone (APA) resulted in less hospitalization due to HF vs. placebo (BETonMACE phase 3 trial; hazard ratio 0.59, p=0.03). Purpose To examine APA’s in vitro effects on inflammatory and profibrotic processes in CFs associated with cardiac fibrosis and HF. Methods Immortalized and primary human CFs were stimulated with cytokines (10ng/mL IL-1β, TNFα, TGF-β1) or with conditioned media from 10ng/mL IFNγ and 100ng/mL lipopolysaccharide-treated THP-1 macrophages ± 5μM APA on plastic or in 3D collagen gels. Gene expression was analyzed by PCR, protein levels by FACS or ELISA, collagen deposition with picrosirius red, THP-1 monocyte migration in Boyden chambers, and CF contraction with 3D collagen gels. Results IL-1β or TNFα stimulation of CFs upregulated monocyte chemoattractant 1 (CCL2) and vascular cell adhesion protein 1 (VCAM1) expression, promoting THP-1 cell migration and adhesion to CFs. APA treatment reduced cytokine-induced CCL2 (>20%) and VCAM1 (>70%) gene expression as well as THP-1 cell migration and adhesion to stimulated CFs (>60%). TGF-β1 treatment induced CF transdifferentiation into myofibroblasts as shown by increased expression of α smooth muscle actin (α-SMA) protein, and secretion of ECM proteins fibronectin and periostin. APA treatment reduced myofibroblast mRNA and protein expression of α-SMA (~30%), fibronectin (~30%) and periostin (97%). Myofibroblast-mediated collagen deposition was also reduced by APA by ~20%. α-SMA-dependent CF contraction can be visualized in attached 3D collagen gels. TGF-β1, IL-1β, TNFα or macrophage-conditioned media promoted CF-mediated gel contraction by 2.5, 1.4, 2.1, or 1.8-fold, respectively. APA treatment countered gel contraction by 30-60%, thereby reducing the myofibroblast-like behaviour in organotypic cultures. Conclusions In vitro, APA treatment reduced proinflammatory crosstalk between monocytes and CFs, resulting in less monocyte migration, monocyte - CF adhesion and CF contraction. APA also reduced signaling by TGF-β1, thus reducing profibrotic and contractile behaviour of CFs responsible for cardiac fibrosis. Since fibrosis contributes to HF, this data provides insight into the observed reduction in hospitalization due to HF in the BETonMACE trial.

Clopidogrel ameliorates high-fat diet-induced hepatic steatosis in mice through activation of the AMPK signaling pathway and beyond.

Metabolic dysfunction-associated steatotic liver disease (MASLD) frequently confers an increased risk of vascular thrombosis; however, the marketed antiplatelet drugs are investigated for the prevention and treatment of MASLD in patients with these coexisting diseases. To determine whether clopidogrel could ameliorate high-fat diet (HFD)-induced hepatic steatosis in mice and how it works, mice were fed on normal diet or HFD alone or in combination with or without clopidogrel for 14weeks, and primary mouse hepatocytes were treated with palmitate/oleate alone or in combination with the compounds examined for 24h. Body weight, liver weight, insulin resistance, triglyceride and total cholesterol content in serum and liver, histological morphology, transcriptomic analysis of mouse liver, and multiple key MASLD-associated genes and proteins were measured, respectively. Clopidogrel mitigated HFD-induced hepatic steatosis (as measured with oil red O staining and triglyceride kit assay) and reduced elevations in serum aminotransferases, liver weight, and the ratio of liver to body weight. Clopidogrel downregulated the expression of multiple critical lipogenic (Acaca/Acacb, Fasn, Scd1, Elovl6, Mogat1, Pparg, Cd36, and Fabp4), profibrotic (Col1a1, Col1a2, Col3a1, Col4a1, Acta2, and Mmp2), and proinflammatory (Ccl2, Cxcl2, Cxcl10, Il1a, Tlr4, and Nlrp3) genes, and enhanced phosphorylation of AMPK and ACC. However, compound C (an AMPK inhibitor) reversed enhanced phosphorylation of AMPK and ACC in clopidogrel-treated primary mouse hepatocytes and alleviated accumulation of intracellular lipids. We concluded that clopidogrel may prevent and/or reverse HFD-induced hepatic steatosis in mice, suggesting that clopidogrel could be repurposed to fight fatty liver in patients.

Nppb contributes to Sepsis-Induced myocardial injury by regulating Senescence-Related genes

BackgroundSepsis, a systemic inflammatory condition, is a leading cause of mortality due to cardiovascular injury. Sepsis and cellular senescence are closely related, yet the specific mechanisms are still unclear. This study aims to identify a novel therapeutic target for mitigating sepsis-induced myocardial injury. MethodsWe initially assessed serum inflammatory markers and myocardial injury indicators in septic mice. This involved observing inflammatory cell infiltration in ventricular muscle tissue, assessing cardiac function, and recording electrocardiograms. We examined the expression of connexin 40 (Cx40) and connexin 43 (Cx43) and analyzed mitochondrial structures in ventricular tissues. A conditional heart-specific Nppb knockout mouse model was then developed in mice to evaluate these changes. Ventricular tissues were analyzed via mRNA sequencing to identify differentially expressed genes (DEGs), which were cross-referenced with senescence-related genes to identify key hub genes. The expression and distribution of hub genes were subsequently analyzed by single-cell analysis. Finally, the expression of the senescence-related gene CCL2, along with cardiac structure and function, was validated in a conditional heart-specific Nppb knockout sepsis mouse model. ResultsMyocardial injury severity positively correlated with serum brain natriuretic peptide (BNP) in septic mice. Conditional heart-specific Nppb knockout improved myocardial injury outcomes in mice with sepsis. The DEGs identified in the conditional heart-specific Nppb knockout model overlapped with senescence-related genes, identifying seven upregulated genes associated with inflammation, cardiac contraction and apoptotic pathways. Single-cell analysis confirmed high CCL2 levels and an increased macrophage presence correlating with sepsis progression. Conditional heart-specific Nppb knockout reduced CCL2 levels, which were associated with improved cardiac structure and function. ConclusionThis study confirms that conditional heart-specific Nppb knockout reduces CCL2 expression, thereby ameliorating myocardial injury in septic mice. Targeting Nppb to regulate the senescence-related gene CCL2 may represent an effective strategy for treating myocardial injury in septic patients.

Osteopontin Promotes Cholangiocyte Secretion of Chemokines to Support Macrophage Recruitment and Fibrosis in MASH.

Osteopontin (OPN) promotes the ductular reaction and is a major driver of chronic liver disease (CLD) progression. Although CLD is characterised by the accumulation of inflammatory cells including macrophages around the peri-portal regions, the influence of OPN on recruitment is unclear. We investigated the role of OPN in cholangiocyte chemokine production and macrophage recruitment by combining invivo, invitro, and in silico approaches. The effects of OPN on cholangiocyte chemokine production and macrophage migration were assessed in culture, alongside RNA-sequencing to identify genes and pathways affected by OPN depletion. Murine liver injury models were used to assess liver chemokine expression and liver macrophage/monocyte recruitment. OPN and chemokine expression were analysed in liver tissue and plasma from biopsy-proven metabolic dysfunction-associated alcoholic steatohepatitis (MASH) patients. OPN-knockdown in cholangiocytes reduced chemokine secretion. RNA-sequencing showed OPN-related effects clustered around immunity, chemotaxis and chemokine production. Macrophage exposure to cholangiocyte-conditioned media showed OPN-supported migration via chemokines chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand (CXCL)1. These effects were related to NF-κB signalling. Murine liver fibrosis was accompanied by upregulated liver OPN, CCL2, CCL5 and CXCL1 mRNA, and accumulation of liver cluster of differentiation (CD)11b/F4/80+CC chemokine receptors (CCR2)high macrophages but treatment with OPN-specific neutralising aptamers reduced fibrosis, chemokine mRNAs and accumulation of liver CD11b/F4/80+CCR2high/lymphocyte antigen 6 complexhigh inflammatory monocytes. In human MASH, liver OPN correlated with chemokines CCL2 and IL8 in association with portal injury and fibrosis. Plasma OPN, serum CCL2 and IL8 also increased with fibrosis stage. OPN promotes cholangiocyte chemokine secretion and the accumulation of pro-inflammatory monocytes. These data support neutralisation of OPN as an anti-inflammatory and anti-fibrotic strategy.

Investigation of parasite genetic variation and systemic immune responses in patients presenting with different clinical presentations of cutaneous leishmaniasis caused by Leishmania aethiopica

BackgroundCutaneous leishmaniasis (CL) is a neglected tropical skin disease, caused by the protozoan parasite Leishmania. In Ethiopia, CL is mainly caused by Leishmania aethiopica and can present in different clinical forms. The aim of this study was to assess whether these different forms are associated with differences in parasite genetic and host systemic immune signatures.MethodsHere we analysed the whole genome sequence data for 48 clinical parasite isolates and the systemic immune signature from a cohort of CL patients, who were recruited in Nefas Mewcha, Northern Ethiopia, from January 2019 to January 2022.ResultsOur results show that parasites from CL cases with different presentations in a single Ethiopian setting are from the same genetic population based on a permutation test of genome-wide similarity. Furthermore, a logistic regression test for genome wide association did not identify any individual genetic variants significantly associated with disease presentation. We also measured plasma chemokine and cytokine levels of 129 CL patients presenting with different forms of CL. None of the chemokine [eotaxin, eotaxin-3, interleukin (IL)-8, interferon (IFN)-γ-induced protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-4, macrophage-derived chemokines (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β and thymus- and activation-regulated chemokine (TARC)] or cytokine (IFN-γ, IL-1β, interleukin-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, tumor necrosis factor-α) levels measured were significantly different between the different clinical presentations of CL, as measured by Kruskal–Wallis test. We also compared those with healthy nonendemic controls: our results show a chemokine (IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β and TARC) but not a cytokine immune signature in patients with CL as compared to healthy nonendemic controls, as measured by Mann-Whitney test.ConclusionsThe results of our study did not identify a systemic immune signature or parasite genetic factors associated with different clinical presentation of CL.Graphical abstract

Resveratrol and Its Natural Analogs Mitigate Immune Dysregulation and Oxidative Imbalance in the Endometriosis Niche Simulated in a Co-Culture System of Endometriotic Cells and Macrophages.

Background: Inflammation and immune cell dysfunction are critical facilitators of endometriosis pathophysiology. Macrophages are renowned for stimulating lesion growth, vascularization, innervation, and pain generation. By combining macrophages and endometriotic cells, we determined if resveratrol and its natural analogs can target the immune dysregulation and oxidative imbalance in endometriosis. Methods: After treatment with compounds (5, 10, 25 µM), we evaluated the expression of key inflammatory and oxidative stress markers, cytokines release, and ROS production by applying q-PCR, ELISA, Cytometric Beads Array, and multiplexed fluorogenic staining and flow cytometry analysis with bioimaging. Results: The results showed that endometriosis-related macrophages treated with stilbenes have impaired expression of pro-inflammatory markers (IL6, IL8, IL1B, TNF, CCL2, CXCL10, PTGS2). The effect of resveratrol, pterostilbene, and piceatannol was observed, especially in reducing IL1B, CCL2, and CXCL10 genes up to 3.5-, 5-, and 7.7-fold at 25 µM, respectively. Also, with piceatannol or polydatin exposure, the IL-6 decrease was noticeable. This study reported an antioxidant effect by reducing ROS-positive cells from 96% to 48% by pterostilbene. Results from flow cytometry correlated with the transcript activation of detoxification enzymes (SOD, GPX). Conclusions: Prospects for potential therapy based on regulating the immune microenvironment and reducing the accumulation of free radicals with stilbenes application were described in the article.

SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

SATB1, a key regulator of T cell development, governs lineage-specific transcriptional programs upon T cell activation. The absence of SATB1 has been linked to the initiation and progression of autoimmunity. However, its precise roles in this process remain incompletely understood. Here we show that conditional knockout of Satb1 in CD4+ T cells in mice led to T cell hyperactivation and inflammatory cell infiltration across multiple organs. Transcriptional profiling on activated T cells revealed that the loss of SATB1 led to aberrant upregulation of CC chemokines. Treating Satb1 conditional knockout mice with CC chemokine receptor inhibitor alleviated inflammatory cell infiltration. Intriguingly, SATB1’s transcriptional regulation of chemokine genes could not be attributed to its direct binding to chemokine promoters. Instead, SATB1 exerted its regulatory effects by controlling higher-order chromatin organization at a CC chemokine locus. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9 genes and a distal enhancer, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing their upregulation. Collectively, these results demonstrate that SATB1 protects organs from immune cell infiltration by regulating chemokine expression, providing valuable insights into the development of autoimmunity-related phenotypes.

Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage.

The skin microenvironment created by keratinocytes (KC) influences the stress responses of melanocytes (MC) to UVB insults. This study employed RNA sequencing analysis as well as in vitro and in vivo models to elucidate the underlying mechanisms. Our RNA-Seq analysis revealed a statistically significant upregulation of GCSF and CCL20 genes in UVB-irradiated KC, correlating with the protective effects of KC on MC responses to UVB exposure. Recombinant GCSF and CCL20 exhibited the most pronounced modulation of UVB-induced MC responses. These effects included the attenuation of apoptosis and reduction of ROS formation, along with the upregulation of tyrosinase and tyrosinase-related protein-1, which are involved in the melanogenic pathway. ELISA was also used to confirm that UVB could induce the secretion of GCSF and CCL20 from KC. A similar correlation between GCSF and CCL20 expression in KC and tyrosinase levels in MC was observed in UVB-irradiated mouse skin. Our study provides novel insights into the protective role of GCSF and CCL20 in the paracrine effects of KC on UVB-induced MC damage through the modulation of stress response pathways, the MITF-tyrosinase axis, and the regulation of p53. These findings have implications for the development of pharmacological strategies targeting KC-derived paracrine factors for the prevention of skin photodamage.

SGLT2i and GLP1-RA exert additive cardiorenal protection with a RAS blocker in uninephrectomized db/db mice.

Diabetic Kidney Disease (DKD) is the main cause of end-stage renal disease in the developed world. The current treatment of the DKD with renin-angiotensin system (RAS) blockade does not totally halt the progression to end stage kidney disease. Currently, several drugs have shown to delay DKD progression such as sodium-glucose co-transporter-2 inhibitors (SGLT2i) and glucagon-like-1 receptor agonists (GLP-1RA). We hypothesized that by combining several drugs that prevent DKD progression on top of RAS blockade a synergistic effect would be achieved in terms of cardiorenal protection. In the present study, we analysed if the combination of a RAS blocker (ramipril) with a SGLT2i (empagliflozin) and/or GLP-1RA (semaglutide) in a type 2 diabetic mouse model could have add-on effects in kidney and heart protection. Male and female uninephrectomized type 2 diabetic db/db mice were treated with empagliflozin and/or semaglutide on top of ramipril during 8weeks. During the study body weight, water and food intake were weekly monitored, glycaemia biweekly and albuminuria and glomerular filtration rate (GFR) before and after the treatment. At the end of the experiment, kidney and heart were isolated for histological and gene expression studies as well as for intrarenal RAS state assessment. Semaglutide combined with ramipril and/or empagliflozin significantly decreased albuminuria but only when combined with both compounds, semaglutide further decreased blood glucose, glomerular hyperfiltration in male mice and glomerular mesangial matrix expansion. In kidney, only the triple treatment with empagliflozin, semaglutide and ramipril reduced the expression of the proinflammatory and profibrotic genes ccl2 and TGFß1. In addition, the combination of empagliflozin and semaglutide on top of RAS blockade was superior in decreasing cardiomyocyte hypertrophy and heart fibrosis in db/db mice. Our results suggest that the combination of SGLT2i with GLP-1RA is superior in cardiorenal protection in DKD than the drugs administered alone on top of RAS blockade.

Deubiquitination of RIPK2 by OTUB2 augments NOD2 signalling and protective effects in intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, but the molecular mechanisms underlying IBD are incompletely understood. In this study, we explored the role and regulating mechanism of otubain 2 (OTUB2), a deubiquitinating enzyme, in IBD. To study the function of OTUB2 in IBD, we generated Otub2-/- mice and treated them with dextran sulfate sodium (DSS) to induce experimental colitis. Bone marrow transplantation was performed to identify the cell populations that were affected by OTUB2 in colitis. The molecular mechanism of OTUB2 in signal transduction was studied by various biochemical methods. OTUB2 was highly expressed in colon-infiltrating macrophages in both humans with IBD and mice with DSS-induced experimental colitis. Colitis was significantly aggravated in Otub2-/- mice and bone marrow chimeric mice receiving Otub2-/- bone marrow. OTUB2-deficiency impaired the production of cytokines and chemokines in macrophages in response to the NOD2 agonist muramyl dipeptide (MDP). Upon MDP stimulation, OTUB2 promoted NOD2 signaling by stabilizing RIPK2. Mechanistically, OTUB2 inhibited the proteasomal degradation of RIPK2 by removing K48-linked polyubiquitination on RIPK2, which was mediated by the active C51 residue in OTUB2. In mice, OTUB2 ablation abolished the protective effects of MDP administration in colitis. This study identified OTUB2 as a novel regulator of intestinal inflammation.

Dual targeting macrophages and microglia is a therapeutic vulnerability in models of PTEN-deficient glioblastoma.

Tumor-associated macrophages and microglia (TAMs) are critical for tumor progression and therapy resistance in glioblastoma (GBM), a type of incurable brain cancer. We previously identified lysyl oxidase (LOX) and olfactomedin like-3 (OLFML3) as essential macrophage and microglia chemokines, respectively, in GBM. Here, single-cell transcriptomics and multiplex sequential immunofluorescence followed by functional studies demonstrate that macrophages negatively correlate with microglia in the GBM tumor microenvironment. LOX inhibition in PTEN-deficient GBM cells upregulates OLFML3 expression via the NF-κB-PATZ1 signaling pathway, inducing a compensatory increase of microglia infiltration. Dual targeting macrophages and microglia via inhibition of LOX and the CLOCK-OLFML3 axis generates potent anti-tumor effects and offers a complete tumor regression in more than 60% of animals when combined with anti-PD1 therapy in PTEN-deficient GBM mouse models. Thus, our findings provide a translational triple therapeutic strategy for this lethal disease.

Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 (MOP3) attenuates inflammation and improves survival in hepatic ischemia/reperfusion injury

IntroductionHepatic ischemia/reperfusion injury is a severe clinical condition leading to high mortality as the result of excessive inflammation, partially triggered by released damage-associated molecular patterns. Extracellular cold-inducible RNA-binding protein is a new damage-associated molecular pattern. Current clinical management of hepatic ischemia/reperfusion injury is limited to supportive therapy, necessitating the development of novel and effective treatment strategies. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 is a newly invented oligopeptide originating from milk fat globule-epidermal growth factor-VIII. This peptide acts as an opsonic compound that specifically binds to extracellular cold-inducible RNA-binding protein to facilitate its clearance by phagocytes, thereby attenuating inflammation. In this study, we hypothesized that milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 attenuated hepatic ischemia/reperfusion injury by inhibiting extracellular cold-inducible RNA-binding protein–induced inflammation in Kupffer cells. MethodsWe treated Kupffer cells isolated from male C57BL/6 mice with extracellular cold-inducible RNA-binding protein and various doses of milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 for 4 hours, then measured cytokines in the culture supernatants. In addition, mice underwent 70% hepatic ischemia for 60 minutes immediately followed by the intravenous administration of either vehicle or milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3. Blood and ischemic liver tissues were collected 24 hours later, and inflammatory markers including cytokines, liver enzymes, chemokines, myeloperoxidase activity, and Z-DNA-binding protein 1 were measured. Hepatic tissue damage and cell death were evaluated histologically. Survival rates were monitored for 10 days posthepatic ischemia/reperfusion. ResultsThe release of interleukin-6 and tumor necrosis factor-α from extracellular cold-inducible RNA-binding protein–challenged Kupffer cells was significantly reduced by milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 in a dose-dependent manner. In hepatic ischemia/reperfusion mice, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly decreased serum levels of extracellular cold-inducible RNA-binding protein, interleukin-6, tumor necrosis factor-α, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment also significantly reduced mRNA levels of interleukin-6, tumor necrosis factor-α, interleukin-1β, Z-DNA-binding protein 1, and chemokine macrophage inflammatory protein-2, as well as myeloperoxidase activity in hepatic tissues. Histologic evaluation demonstrated that treatment with milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated tissue damage and cell death in the liver of hepatic ischemia/reperfusion mice. Milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 treatment significantly improved the survival rate of hepatic ischemia/reperfusion mice. ConclusionMilk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 significantly attenuated inflammation and liver tissue damage and improved survival after hepatic ischemia/reperfusion. Thus, milk fat globule-epidermal growth factor-VIII–derived oligopeptide 3 holds promise as a potential future therapeutic strategy for hepatic ischemia/reperfusion injury.

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.

Biomimetic siRNA nanogels for regulating macrophage polarization and promoting osteogenesis

BackgroundBone fracture regeneration poses significant clinical challenges due to complications such as delayed healing, nonunion, and the limitations of current treatments. ObjectiveThis study introduces a novel therapeutic approach utilizing biomimetic nanogels to silence the Ccl4 gene, aiming to promote bone repair by regulating macrophage polarization. MethodsThe nanogels, composed of tannic acid (TA) and small interfering RNA (siRNA), were designed for targeted gene delivery. ResultsIn vitro findings indicate that siRNA-mediated Ccl4 reduction significantly improves M2 macrophage polarization, which, in turn, promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Increased expression of osteogenic markers and enhanced mineral deposition were observed. The nanogels demonstrated optimal particle size, stability, and cellular uptake, and biocompatibility assays confirmed their non-toxicity. ConclusionThis study underscores the potential of targeted siRNA delivery in modulating immune responses to enhance bone regeneration, offering promising treatment options for complex bone healing scenarios.

Network pharmacology study and in vitro experimental validation of Xiaojianzhong decoction against gastric cancer.

Cancer is one of the most serious threats to human health worldwide. Conventional treatments such as surgery and chemotherapy are associated with some drawbacks. In recent years, traditional Chinese medicine treatment has been increasingly advocated by patients and attracted attention from clinicians, and has become an indispensable part of the comprehensive treatment for gastric cancer. To investigate the mechanism of Xiaojianzhong decoction (XJZ) in the treatment of gastric cancer (GC) by utilizing network pharmacology and experimental validation, so as to provide a theoretical basis for later experimental research. We analyzed the mechanism and targets of XJZ in the treatment of GC through network pharmacology and bioinformatics. Subsequently, we verified the impact of XJZ treatment on the proliferative ability of GC cells through CCK-8, apoptosis, cell cycle, and clone formation assays. Additionally, we performed Western blot analysis and real-time quantitative PCR to assess the protein and mRNA expression of the core proteins. XJZ mainly regulates IL6, PTGS2, CCL2, MMP9, MMP2, HMOX1, and other target genes and pathways in cancer to treat GC. The inhibition of cell viability, the increase of apoptosis, the blockage of the cell cycle at the G0/G1 phase, and the inhibition of the ability of cell clone formation were observed in AGS and HGC-27 cells after XJZ treatment. In addition, XJZ induced a decrease in the mRNA expression of IL6, PTGS2, MMP9, MMP2, and CCL2, and an increase in the mRNA expression of HOMX1. XJZ significantly inhibited the expression of IL6, PTGS2, MMP9, MMP2, and CCL2 proteins and promoted the expression of the heme oxygenase-1 protein. XJZ exerts therapeutic effects against GC through multiple components, multiple targets, and multiple pathways. Our findings provide a new idea and scientific basis for further research on the molecular mechanisms underlying the therapeutic effects of XJZ in the treatment of GC.

MIP3α-Rel Mtb intranasal DNA vaccination induces reactive T-cell infiltration into the lungs in mice and macaques.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of mortality due to a single infectious organism. While generally curable, TB requires a lengthy and complex antibiotic regimen, due in large part to bacteria that can shift to a persistent state in the presence of antibiotic pressure. Rel Mtb is the primary enzyme regulating the stringent response, which contributes to the metabolic shift of Mtb to a persistent state. Targeting Rel Mtb with a vaccine to eliminate persistent bacteria through the induction of Rel Mtb -specific T-cell immunity in combination with antibiotics to kill dividing bacteria has shown promise in model systems. In a mouse model of Mtb infection, a vaccine created by genetically fusing rel Mtb to the chemokine macrophage inflammatory protein 3α ( MIP3 α), a ligand for the CC chemokine receptor type 6 (CCR6) present on immature dendritic cells, has been shown to enhance T-cell responses and accelerate eradication of infection in mouse models compared to a vaccine lacking the chemokine component. In this study, immunogenicity studies in the mouse and rhesus macaque models provide evidence that intranasal administrations of the DNA form of the MipRel vaccine led to enhanced lung infiltration of T cells after a series of immunizations. Furthermore, despite similar T-cell immunity seen in PBMCs between MipRel and Rel vaccinations, lung and bronchoalveolar lavage cell samples are more enriched for cytokine-secreting T cells in MipRel groups compared to Rel groups. We conclude that intranasal immunization with a MIP-3α fusion vaccine represents a novel strategy for use of a simple DNA vaccine formulation to elicit T-cell immune responses within the respiratory tract. That this formulation is immunogenic in a non-human primate model historically viewed as poorly responsive to DNA vaccines indicates the potential for clinical application in the treatment of Mtb infection, with possible application to other respiratory pathogens. Future studies will further characterize the protective effect of this vaccination platform.

Comparison of serum cytokines and chemokines levels and clinical significance in patients with pemphigus vulgaris-A retrospective study.

In this study, we aimed to examine the relationship between the serum cytokine levels of patients with pemphigus vulgaris (PV) and the Pemphigus Disease Area Index (PDAI), along with the presence of anti-desmoglein (Dsg) 1 antibody, anti-Dsg3 antibody and co-infection among patients with pemphigus vulgaris. This retrospective study included 62 PV patients and 59 healthy individuals who attended the Second Affiliated Hospital of Kunming Medical University from November 2014 to November 2022. The serum concentrations of cytokines and chemokines were assessed using the Luminex 200 System (a high-throughput cytokine detection method). Additionally, anti-Dsg1 and anti-Dsg3 antibodies were determined through enzyme-linked immunosorbent assay, while disease severity was evaluated using the PDAI scoring system. The PV group exhibited elevated levels of Th1 cytokines (such as interleukin (IL)-1RA, IL-1β, IL-2, IL-12p70, GM-CSF, TNF-α, IL-18, IFN-γ), Th2 cytokines (IL-5, IL-10, IL-13) and Th17/Th22-related cytokines (IL-17A, IL-22) compared to the healthy control group (p < 0.05). Conversely, the levels of chemokines (macrophage inflammatory protein-1 alpha (MIP-1α), stromal cell-derived factor-1 alpha (SDF-1α), interferon-inducible protein-10 (IP-10), Regulated on Activation in Normal T-Cell Expressed And Secreted (RANTES), growth-regulated on-gene-alpha (GRO-α), MIP-1β) and Th2 (IL-31) were lower in the PV group compared to the healthy control group (p < 0.05). No significant differences were observed in other cytokines and chemokines (p > 0.05). Additionally, IL-7, IFN-γ, IL-18 and GRO-α showed positive correlations with PDAI, IL-6 correlated positively with anti-Dsg3 antibody levels, and IL-12p70, IL-18, and IFN-γ correlated positively with anti-Dsg1 antibody levels. Furthermore, IL-15 exhibited a positive association with skin infections. PV patients have elevated levels of various cytokines and chemokines, and there are different degrees of elevation in cytokines and chemokines associated with the activation of various T cell subsets. PDAI and the Dsg1 antibody levels are mainly related to the Th1-related cytokines.

Gene expression analysis identifies hub genes and pathways distinguishing fatal from survivor outcomes of Ebola virus disease.

The Ebola virus poses a severe public health threat, yet understanding factors influencing disease outcomes remains incomplete. Our study aimed to identify critical pathways and hub genes associated with fatal and survivor Ebola disease outcomes. We analyzed differentially expressed hub genes (DEGs) between groups with fatal and survival outcomes, as well as a healthy control group. We conducted additional analysis to determine the functions and pathways associated with these DEGs. We found 13,198 DEGs in the fatal and 12,039 DEGs in the survival group compared to healthy controls, and 1873 DEGs in the acute fatal and survivor groups comparison. Upregulated DEGs in the comparison between the acute fatal and survivor groups were linked to ECM receptor interaction, complement and coagulation cascades, and PI3K-Akt signaling. Upregulated hub genes identified from the acute fatal and survivor comparison (FGB, C1QA, SERPINF2, PLAT, C9, SERPINE1, F3, VWF) were enriched in complement and coagulation cascades; the downregulated hub genes (IL1B, 1L17RE, XCL1, CXCL6, CCL4, CD8A, CD8B, CD3D) were associated with immune cell processes. Hub genes CCL2 and F2 were unique to fatal outcomes, while CXCL1, HIST1H4F, and IL1A were upregulated hub genes unique to survival outcomes compared to healthy controls. Our results demonstrate for the first time the association of EVD outcomes to specific hub genes and their associated pathways and biological processes. The identified hub genes and pathways could help better elucidate Ebola disease pathogenesis and contribute to the development of targeted interventions and personalized treatment for distinct EVD outcomes.