Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts.

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Send it, receive it, quick erase it: A mouse model to decipher chemokine communication.

A method to precisely determine which cells respond to chemokines in vivo is currently lacking. A novel class of dual fluorescence reporter mice could help identify cells that produce and/or sense a given chemokine in vitro and in vivo (Rodrigo et al. 2024. J. Exp. Med.https://doi.org/10.1084/jem.20231814).

Chemokines and lymphocyte homing in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that typically presents with lymphocyte, dendritic cell, and macrophage infiltration of exocrine gland ducts and the formation of ectopic germinal centers. The interactions of lymphocyte homing receptors and addressins and chemokines and their receptors, such as α4ß7/MAdCAM-1, LFA-1/ICAM-1, CXCL13/CXCR5, CCL25/CCR9, CX3CL1/CX3CR1, play important roles in the migration of inflammatory cells to the focal glands and the promotion of ectopic germinal center formation in SS. A variety of molecules have been shown to be involved in lymphocyte homing, including tumor necrosis factor-α, interferon (IFN)-α, IFN-ß, and B cell activating factor. This process mainly involves the Janus kinase-signal transducer and activator of transcription signaling pathway, lymphotoxin-ß receptor pathway, and nuclear factor-κB signaling pathway. These findings have led to the development of antibodies to cell adhesion molecules, antagonists of chemokines and their receptors, compounds interfering with chemokine receptor signaling, and gene therapies targeting chemokines and their receptors, providing new targets for the treatment of SS in humans. The aim of this study was to explore the relationship between lymphocyte homing and the pathogenesis of SS, and to provide a review of recent studies addressing lymphocyte homing in targeted therapy for SS.

Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model.

The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen-glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Bronchom assuages airway hyperresponsiveness in house dust mite-induced mouse model of allergic asthma and moderates goblet cell metaplasia, sub-epithelial fibrosis along with changes in Th2 cytokines and chemokines.

Background: Asthma is a common obstructive airway disease with an inflammatory etiology. The main unmet need in the management of asthma is inadequate adherence to pharmacotherapy, leading to a poorly-controlled disease state, necessitating the development of novel therapies. Bronchom is a calcio-herbal formulation, which is purported to treat chronic asthma. The objective of the current study was to examine the in-vivo efficacy of Bronchom in mouse model of allergic asthma. Methods: Ultra high performance liquid chromatography was utilized to analyze the phytocompounds in Bronchom. Further, the in-vivo efficacy of Bronchom was evaluated in House dust mite (HDM)-induced allergic asthma in mice. Mice were challenged with aerosolized methacholine to assess airway hyperresponsiveness. Subsequently, inflammatory cell influx was evaluated in bronchoalveolar lavage fluid (BALF) followed by lung histology, wherein airway remodeling features were studied. Simultaneously, the levels of Th2 cytokines and chemokines in the BALF was also evaluated. Additionally, the mRNA expression of pro-inflammatory and Th2 cytokines was also assessed in the lung along with the oxidative stress markers. Results: Phytocompounds present in Bronchom included, gallic acid, protocatechuic acid, methyl gallate, rosmarinic acid, glycyrrhizin, eugenol, 6-gingerol and piperine. Bronchom effectively suppressed HDM-induced airway hyperresponsiveness along with the influx of leukocytes in the BALF. Additionally, Bronchom reduced the infiltration of inflammatory cells in the lung and it also ameliorated goblet cell metaplasia, sub-epithelial fibrosis and increase in α-smooth muscle actin. Bronchom decreased Th2 cytokines (IL-4 and IL-5) and chemokines (Eotaxin and IP-10) in the BALF. Likewise, it could also suppress the mRNA expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-33), and IL-13. Moreover, Bronchom restored the HDM-induced diminution of endogenous anti-oxidants (GSH and SOD) and the increase in pro-oxidants (GSSG and MDA). Furthermore, Bronchom could also decrease the nitrosative stress by lowering the observed increase in nitrite levels. Conclusion: Taken together, the results of the present study data convincingly demonstrate that Bronchom exhibits pharmacological effects in an animal model of allergic asthma. Bronchom mitigated airway hyperresponsiveness, inflammation and airway remodeling evoked by a clinically relevant allergen and accordingly it possesses therapeutic potential for the treatment of asthma.

Chemokines as Prognostic Factor in Colorectal Cancer Patients: A Systematic Review and Meta-Analysis.

Chemokines orchestrate many aspects of tumorigenic processes such as angiogenesis, apoptosis and metastatic spread, and related receptors are expressed on tumor cells as well as on inflammatory cells (e.g., tumor-infiltrating T cells, TILs) in the tumor microenvironment. Expressional changes of chemokines and their receptors in solid cancers are common and well known, especially in affecting colorectal cancer patient outcomes. Therefore, the aim of this current systematic review and meta-analysis was to classify chemokines as a prognostic biomarker in colorectal cancer patients. A systematic literature search was conducted in PubMed, CENTRAL and Web of Science. Information on the chemokine expression of 25 chemokines in colorectal cancer tissue and survival data of the patients were investigated. The hazard ratio of overall survival and disease-free survival with chemokine expression was examined. The risk of bias was analyzed using Quality in Prognosis Studies. Random effects meta-analysis was performed to determine the impact on overall respectively disease survival. For this purpose, the pooled hazard ratios (HR) and their 95% confidence intervals (CI) were used for calculation. Twenty-five chemokines were included, and the search revealed 5556 publications. A total of thirty-one publications were included in this systematic review and meta-analysis. Overexpression of chemokine receptor CXCR4 was associated with both a significantly reduced overall survival (HR = 2.70, 95%-CI: 1.57 to 4.66, p = 0.0003) as well as disease-free survival (HR = 2.68, 95%-CI: 1.41 to 5.08, p = 0.0026). All other chemokines showed either heterogeneous results or few studies were available. The overall risk of bias for CXCR4 was rated low. At the current level of evidence, this study demonstrates that CXCR4 overexpression in patients with colorectal cancer is associated with a significantly diminished overall as well as disease-free survival. Summed up, this systematic review and meta-analysis reveals CXCR4 as a promising prognostic biomarker. Nevertheless, more evidence is needed to evaluate CXCR4 and its antagonists serving as new therapeutic targets.

Analyzing the Functional Roles and Immunological Features of Chemokines in COAD.

Chemokines are key proteins that regulate cell migration and immune responses and are essential for modulating the tumor microenvironment. Despite their close association with colon cancer, the expression patterns, prognosis, immunity, and specific roles of chemokines in colon cancer are still not fully understood. In this study, we investigated the mutational features, differential expression, and immunological characteristics of chemokines in colon cancer (COAD) by analyzing the Tumor Genome Atlas (TCGA) database. We clarified the biological functions of these chemokines using Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. By univariate and multivariate COX regression analyses, we developed chemokine-based prognostic risk models. In addition, using Gene Set Enrichment Analysis (GSEA) and Gene Set Variant Analysis (GSVA), we analyzed the differences in immune responses and signaling pathways among different risk groups. The results showed that the mutation rate of chemokines was low in COAD, but 25 chemokines were significantly differentially expressed. These chemokines function in several immune-related biological processes and play key roles in signaling pathways including cytokine-cytokine receptor interactions, NF-kappa B, and IL-17. Prognostic risk models based on CCL22, CXCL1, CXCL8, CXCL9, and CXCL11 performed well. GSEA and GSVA analyses showed significant differences in immune responses and signaling pathways across risk groups. In conclusion, this study reveals the potential molecular mechanisms of chemokines in COAD and proposes a new prognostic risk model based on these insights.

Lapagyl mitigates UV-induced inflammation and immunosuppression via Foxp3+ Tregs and CCL pathway: A single-cell transcriptomics study.

BACKGROUND: As the largest organ of the body, the skin is constantly subjected to ultraviolet radiation (UVR), leading to inflammations and changes that mirror those seen in chronological aging. Although various small molecule drugs have been explored for treating skin photoaging, they typically suffer from low stability and a high incidence of adverse reactions. Consequently, the continued investigation of photoaging treatments, particularly those utilizing herbal products, remains a critical clinical endeavor. One such herbal product, Lapagyl, is derived from the bark of the lapacho tree and possesses antioxidant efficacies that could be beneficial in combating skin photoaging. PURPOSE: This research aimed to evaluate the efficacy of the herbal product Lapagyl in combating UVR-induced skin photoaging. Additionally, it sought to unravel the mechanisms by which Lapagyl promotes the regeneration of the skin extracellular matrix. METHODS: To investigate whether Lapagyl can alleviate skin aging and damage, a UVR radiation model was established using SKH-1 hairless mice. The dorsal skins of these mice were evaluated for wrinkle formation, texture, moisture, transepidermal water loss (TEWL), and elasticity. Pathological assessments were conducted to determine Lapagyl's efficacy. Additionally, single-cell sequencing and spectrum analysis were employed to elucidate the working mechanisms and primary components of Lapagyl in addressing UVR-induced skin aging and injury. RESULTS: Lapagyl markedly reduced UVR-induced wrinkles, moisture loss, and elasticity decrease in SKH-1 mice. Single-cell sequencing demonstrated that Lapagyl corrected the imbalance in cell proportions caused by UVR, decreased UVR-induced ROS expression, and protected basal and spinous cells from skin damage. Additionally, Lapagyl effectively prevented the entry of inflammatory cells into the skin by reducing CCL8 expression and curtailed the UVR-induced formation of Foxp3+ regulatory T cells (Tregs) in the skin. Both pathological assessments and ex vivo skin model results demonstrated that Lapagyl effectively reduced UVR-induced damage to collagen and elastin. Spectrum analysis identified Salidroside as the primary compound remaining in the skin following Lapagyl treatment. Taken together, our study elucidated the skin protection mechanism of the herbal product Lapagyl against UVR damage at the cellular level, revealing its immunomodulatory effects, with salidroside identified as the primary active compound for skin. CONCLUSION: Our study provided a thorough evaluation of Lapagyl's protective effects on skin against UVR damage, delving into the mechanisms at the cellular level. We discovered that Lapagyl mitigates skin inflammation and immunosuppression by regulating Foxp3+ Tregs and the CCL pathway. These insights indicate that Lapagyl has potential as a novel therapeutic option for addressing skin photoaging.

Structural basis of chemokine recognition by the class A3 tick evasin EVA-ACA1001.

Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.

Chemokine­ and chemokine receptor-based subtypes predict prognosis, immunotherapy and chemotherapy response in colorectal cancer patients.

BACKGROUND: The clinical significance and comprehensive characteristics of chemokines and chemokine receptors in colorectal cancer (CRC) have not been previously reported. Our study aims to investigate the expression profiles of chemokines and chemokine receptors, as well as establish subtypes in CRC. METHODS: 1009 CRC samples were enrolled in our study. Consensus unsupervised clustering analysis was conducted to establish subtypes, and a risk score model was developed using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses. 36 pairs of tissue specimens of CRC patients and two CRC cell lines were used to validate the subtypes and risk score in vitro. Quantitative real-time PCR and western blotting were employed to validate mRNA and protein expression levels, respectively. Flow cytometry was utilized for analyzing cell apoptosis, while cell viability assay and EdU assay were conducted to assess cell proliferation ability. RESULTS: The Cluster B group shares similarities with the low-risk group in terms of exhibiting a higher level of immune cell infiltration and belonging to hot tumor. Patients CRC in the Cluster B group demonstrate a more favorable prognosis and exhibit better response to immunotherapy and chemotherapy. On the other hand, the Cluster A group resembles the high-risk group as it displays lower levels of immune cell infiltration, indicating a cold tumor phenotype. CRC patients in the Cluster A group have poorer prognoses and show less therapeutic efficacy towards immunotherapy and chemotherapy. Furthermore, we utilized a total of 36 pairs of tissue samples obtained from patients with CRC, along with two CRC cell lines for validation in vitro. This comprehensive approach further enhances the scientific validity and reliability of the identified subtypes and risk score in their ability to predict prognosis, response to immunotherapy, and response to chemotherapy among CRC patients. CONCLUSION: We first established robust prognostic subtypes based on chemokines and chemokine receptors, which could potentially serve as a novel biomarker for guiding individualized treatment in patients with CRC undergoing immunotherapy and chemotherapy.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

A Lacticaseibacillus rhamnosus secretome induces immunoregulatory transcriptional, functional and immunometabolic signatures in human THP-1 monocytes.

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX3CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells.

Adipose tissue is an active endocrine gland, synthesizing and secreting multiple signaling molecules termed adipokines. Following the detection of adipokines and their receptors in the mammary tissue of various species, it is indicated that adipokines play a role in the development of the mammary gland. The aim of the present study was to determine the concentration-dependent influence of three adipokines, leptin, adiponectin, and chemerin, on the viability, apoptosis, and secretory activity of BME-UV1 bovine mammary epithelial cells. The study confirmed that BME-UV1 cells contain the leptin receptor (Ob-R) protein, and express transcripts of adiponectin (ADIPOR1 and ADIPOR2) and chemerin (CMLKR1 and GPR1) receptors. Regardless of the administered dose, none of the three tested adipokines had an effect on the viability of BME-UV1 cells, and the number of apoptotic cells remained unchanged. However, chemerin (100 ng/mL) stimulated BME-UV1 cells to synthesize and secrete αS1-casein, the major protein component of milk. These results indicate that chemerin may be a potent regulator of the bovine mammary epithelial cells' functional differentiation, contributing, along with the major systemic hormones and local growth factors, to the development of the bovine mammary gland.

Effect of Regulation of Chemerin/Chemokine-like Receptor 1/Stimulator of Interferon Genes Pathway on Astrocyte Recruitment to Aß Plaques.

Recruitment and accumulation of reactive astrocytes around senile plaques are common pathological features of Alzheimer's disease (AD), with unclear mechanisms. Chemerin, an adipokine implicated in neuroinflammation, acts through its receptor, chemokine-like receptor 1 (CMKLR1), which also functions as a receptor for amyloid ß (Aß). The impact of the chemerin/CMKLR1 axis on astrocyte migration towards Aß plaques is unknown. Here we investigated the effect of CMKLR1 on astrocyte migration around Aß deposition in APP/PS1 mice with Cmklr1 knockout (APP/PS1-Cmklr1-/-). CMKLR1-expressed astrocytes were upregulated in the cortices and hippocampi of 9-month-old APP/PS1 mice. Chemerin mainly co-localized with neurons, and its expression was reduced in the brains of APP/PS1 mice, compared to WT mice. CMKLR1 deficiency decreased astrocyte colocalization with Aß plaques in APP/PS1-Cmklr1-/- mice, compared to APP/PS1 mice. Activation of the chemerin/CMKLR1 axis promoted the migration of primary cultured astrocytes and U251 cells, and reduced astrocyte clustering induced by Aß42. Mechanistic studies revealed that chemerin/CMKLR1 activation induced STING phosphorylation. Deletion of STING attenuated the promotion of the chemerin/CMKLR1 axis relative to astrocyte migration and abolished the inhibitory effect of chemerin on Aß42-induced astrocyte clustering. These findings suggest the involvement of the chemerin/CMKLR1/STING pathway in the regulation of astrocyte migration and recruitment to Aß plaques/Aß42.

Exosomes derived from vMIP-II-Lamp2b gene-modified M2 cells provide neuroprotection by targeting the injured spinal cord, inhibiting chemokine signals and modulating microglia/macrophage polarization in mice.

Inflammation is one of the key injury factors for spinal cord injury (SCI). Exosomes (Exos) derived from M2 macrophages have been shown to inhibit inflammation and be beneficial in SCI animal models. However, lacking targetability restricts their application prospects. Considering that chemokine receptors increase dramatically after SCI, viral macrophage inflammatory protein II (vMIP-II) is a broad-spectrum chemokine receptor binding peptide, and lysosomal associated membrane protein 2b (Lamp2b) is the key membrane component of Exos, we speculated that vMIP-II-Lamp2b gene-modified M2 macrophage-derived Exos (vMIP-II-Lamp2b-M2-Exo) not only have anti-inflammatory properties, but also can target the injured area by vMIP-II. In this study, using a murine contusive SCI model, we revealed that vMIP-II-Lamp2b-M2-Exo could target the chemokine receptors which highly expressed in the injured spinal cords, inhibit some key chemokine receptor signaling pathways (such as MAPK and Akt), further inhibit proinflammatory factors (such as IL-1ß, IL-6, IL-17, IL-18, TNF-α, and iNOS), and promote anti-inflammatory factors (such as IL-4 and Arg1) productions, and the transformation of microglia/macrophages from M1 into M2. Moreover, the improved histological and functional recoveries were also found. Collectively, our results suggest that vMIP-II-Lamp2b-M2-Exo may provide neuroprotection by targeting the injured spinal cord, inhibiting some chemokine signals, reducing proinflammatory factor production and modulating microglia/macrophage polarization.