The Role of Pro-Inflammatory Chemokines CCL-1, 2, 4, and 5 in the Etiopathogenesis of Type 2 Diabetes Mellitus in Subjects from the Asir Region of Saudi Arabia: Correlation with Different Degrees of Obesity.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is becoming a major global health concern, especially in developing nations. The high prevalence of obesity and related diabetes cases are attributed to rapid economic progress, physical inactivity, the consumption of high-calorie foods, and changing lifestyles. OBJECTIVES: We investigated the roles of pro-inflammatory chemokines CCL1, 2, 4, and 5 in T2DM with varying levels of obesity in the Asir region of Saudi Arabia. MATERIALS AND METHODS: In total, 170 confirmed T2DM subjects and a normal control group were enrolled. Demographic data, serum levels of CCL-1, 2, 4, and 5, and biochemical indices were assessed in the subjects and control groups by standard procedures. RESULTS: T2DM subjects were divided into four groups: A (normal body weight), B (overweight), C (obese), and D (highly obese). We observed that male and female control subjects had similar mean serum concentrations of pro-inflammatory chemokines CCL-1, 2, 4, and 5. T2DM subjects in all the four groups showed significantly higher levels of all the four chemokines compared to the controls, regardless of gender. In T2DM subjects with obesity and severe obesity, the rise was most significant. There was a progressive rise in the concentrations of CCL-1, 2, and 4 in T2DM subjects with increasing BMI. Serum CCL5 levels increased significantly in all T2DM subject groups. The increase in CCL5 was more predominant in normal-weight people, compared to overweight and obese T2DM subjects. CONCLUSIONS: Male and female control subjects had similar serum levels of pro-inflammatory chemokines CCL-1, 2, 4, and 5. The progressive rise in blood concentrations of three pro-inflammatory chemokines CCL-1, 2, and 4 in T2DM subjects with increasing BMI supports the idea that dyslipidemia and obesity contribute to chronic inflammation and insulin resistance. Serum CCL5 levels increased significantly in all T2DM subject groups. The selective and more pronounced increase in CCL5 in the T2DM group with normal BMI, compared to subjects with varying degrees of obesity, was rather surprising. Further research is needed to determine if CCL5 underexpression in overweight and obese T2DM subjects is due to some unexplained counterbalancing processes.

Reduced chemokine C-C motif ligand 1 expression may negatively regulate colorectal cancer progression at liver metastatic sites.

Colorectal cancer (CRC) liver metastasis, albeit a stage-IV disease, is completely curable by surgical resection in selected patients. In addressing the molecular basics of this phenomenon, differentially expressed genes at primary and liver metastatic sites were screened by RNA sequencing with the use of paraffin-embedded surgical specimens. Chemokine C-C motif ligand 1 (CCL1), a chemotactic factor for a ligand of the chemokine C-C motif receptor 8 (CCR8), was isolated as one of the differentially expressed genes. Histological analysis revealed that the number of CCL1-positive cells, mainly tumour associated macrophages (TAMs) located in the stroma of CRC, decreased significantly at liver metastatic sites, while the expression level of CCR8 on CRC remained unchanged. To explore the biological significance of the CCL1-CCR8 axis in CRC, CCR8-positive CRC cell line Colo320DM was used to assess the effect of the CCL1-CCR8 axis on major signalling pathways, epithelial mesenchymal transition induction and cell motility. Upon stimulation of recombinant CCL1 (rCCL1), phosphorylation of AKT was observed in Colo320DM cells; on the other hand, the corresponding significant increase in MMP-2 levels demonstrated by RT-qPCR was nullified by siRNA (siCCR8). In the scratch test, rCCL1 treatment significantly increased the motility of Colo320DM cells, which was similarly nullified by siCCR8. Thus, the activation of the CCL1-CCR8 axis is a positive regulator of CRC tumour progression. Reduced CCL1 expression of TAMs at liver metastatic sites may partly explain the unique slow tumour progression of CRC, thus providing for a grace period for radical resection of metastatic lesions.

MiR-184-5p represses neuropathic pain by regulating CCL1/CCR8 signaling interplay in the spinal cord in diabetic mice.

BACKGROUND: Diabetic neuropathic pain (DNP) is a serious complication for diabetic patients involving nervous system. MicroRNAs (miRNAs) are small-noncoding RNAs which are dysregulated in neuropathic pain, and might be critical molecules for pain treatment. Our previous study has shown miR-184-5p was significantly downregulated in DNP. Therefore, the mechanism of miR-184-5p in DNP was investigated in this study. METHODS: A DNP model was established through streptozotocin (STZ). The pharmacological tools were injected intrathecally, and pain behavior was evaluated by paw withdrawal mechanical thresholds (PWMTs). Bioinformatics analysis, Dual-luciferase reporter assay and fluorescence-in-situ-hybridization (FISH) were used to seek and confirm the potential target genes of miR-184-5p. The expression of relative genes and proteins was analyzed by quantitative reverse transcriptase real-time PCR (qPCR) and western blotting. RESULTS: MiR-184-5p expression was down-regulated in spinal dorsal on days 7 and 14 after STZ, while intrathecal administration of miR-184-5p agomir attenuates neuropathic pain induced by DNP and intrathecal miR-184-5p antagomir induces pain behaviors in naïve mice. Chemokine CC motif ligand 1 (CCL1) was found to be a potential target of miR-184-5p and the protein expression of CCL1 and the mRNA expression of CCR8 were up-regulated in spinal dorsal on days 7 and 14 after STZ. The luciferase reporter assay and FISH demonstrated that CCL1 is a direct target of miR-184-5p. MiR-184-5p overexpression attenuated the expression of CCL1/CCR8 in DNP; intrathecal miR-184-5p antagomir increased the expression of CCL1/CCR8 in spinal dorsal of naïve mice. CONCLUSION: This research illustrates that miR-184-5p alleviates DNP through the inhibition of CCL1/CCR8 signaling expression.

The chemokine CCL1 facilitates pulmonary fibrosis by promoting macrophage migration and M2 polarization.

Macrophage M2 polarization has been identified in the pathogenesis of pulmonary fibrosis (PF), but the mediators that drive the macrophage M2 program in PF need to be clarified. We showed that the expression of AMFR and CCR8, two known receptors of CCL1, was increased in macrophages from lungs of mice with bleomycin (BLM)-induced PF. Deficiency in either AMFR or CCR8 in macrophages protected mice from BLM-induced PF. In vitro experiments revealed that CCL1 recruited macrophages by binding to its classical receptor CCR8 and drove the macrophage M2 phenotype via its interaction with the recently identified receptor AMFR. Mechanistic studies revealed that the CCL1-AMFR interaction enhanced CREB/C/EBPß signaling to promote the macrophage M2 program. Together, our findings reveal that CCL1 acts as a mediator of macrophage M2 polarization and could be a therapeutic target in PF.

Suppressive Effects of 4-(Phenylsulfanyl) Butan-2-One on CCL-1 Production via Histone Acetylation in Monocytes.

The 4-(phenylsulfanyl) butan-2-one (4-PSB-2), a marine-derived compound from soft coral, was proven to have multiple biological activities including neuroprotection and potent anti-inflammatory effects. CC chemokine ligand (CCL)-1 belongs to T helper (Th)2-related chemokines that are involved in the recruitment of Th2 inflammatory cells. Histone acetylation has been recognized as a critical mechanism underlying the regulated cytokine and chemokine production. Our study tried to investigate the anti-inflammatory effect of 4-PSB-2 on CCL-1 production in human monocytes and explore possible underlying intracellular processes, including epigenetic regulation. To confirm our hypothesis, human monocyte THP-1 cell line and primary CD14+ cells were pretreated with various concentrations of 4-PSB-2 and then were stimulated with lipopolysaccharide (LPS). The CCL-1 concentration was measured by enzyme-linked immunosorbent assays, and the intracellular signaling pathways and epigenetic regulation of 4-PSB-2 were investigated by using Western blotting and chromatin immunoprecipitation analysis. In this study, we found that 4-PSB-2 had a suppressive effect on LPS-induced CCL-1 production. Moreover, this suppressive effect of 4-PSB-2 was mediated via intracellular signaling such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In addition, 4-PSB-2 could suppress CCL-1 production by epigenetic regulation through downregulating histone H3 and H4 acetylation. In short, our study demonstrated that 4-PSB-2 may have a potential role in the treatment of allergic inflammation.

Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1+ fibroblast and CCR8+ Treg cell interactions.

BACKGROUND: Since the lung is one of the most common sites for cancer metastasis, it could provide a suitable microenvironment for pre-metastatic niche (PMN) formation to facilitate tumor cell colonization. Regulatory T cells (Tregs) are an immunosuppressive cell type found ubiquitously in tumors and may play a crucial role in PNM formation. In this study, we investigated tumor-derived exosome (TDE)-induced Treg differentiation in the lung PMN as well as the underlying mechanisms. METHODS: TDEs were isolated from the Lewis lung carcinoma cell line (LLC-exo) and their effects on mouse pulmonary fibroblasts was investigated in vitro as well as on lung tumor formation and metastasis in a pre-injected mouse model. Immune cell populations in the lung were analyzed by flow cytometry. Expression of CCL1 and CCR8 was evaluated by immunofluorescence staining, qRT-PCR and Western blot analyses. Cytokine expression was measured using mouse cytokine arrays and ELISA. RESULTS: The number of CD4+ FoxP3+ Tregs was significantly increased in lungs in a LLC-exo pre-injected mouse model. Lung fibroblasts secreted increased amounts of CCL1 after co-culture with LLC-exo, which induced Treg differentiation by activating its specific receptor CCR8, ultimately contributing to the establishment of an immunologically tolerant PMN. Moreover, inhibiting the release of LLC-exo by GW4869, or blocking the CCL1-CCR8 axis using AZ084, suppressed Tregs differentiation and tumor metastasis in the lung. CONCLUSIONS: Collectively, our study provides a novel mechanism by which Tregs are activated to form an immunologically tolerant PMN and demonstrates a critical link among lung fibroblasts, Tregs and metastatic tumor cells.

Effect of naturally derived surgical hemostatic materials on the proliferation of A549 human lung adenocarcinoma cells.

Hemostatic materials are generally applied in surgical operations for cancer, but their effects on the growth and recurrence of tumors are unclear. Herein, three commonly used naturally derived hemostatic materials, gelatin sponge, Surgicel (oxidized regenerated cellulose), and biopaper (mixture of sodium hyaluronate and carboxymethyl chitosan), were cocultured with A549 human lung adenocarcinoma cells in vitro. Furthermore, the performance of hemostatic materials and the tumorigenicity of the materials with A549 â€‹cells were observed after subcutaneous implantation into BALB/c mice. The in vitro results showed that biopaper was dissolved quickly, with the highest cell numbers at 2 and 4 days of culture. Gelatin sponges retained their structure and elicited the least cell infiltration during the 2- to 10-day culture. Surgicel partially dissolved and supported cell growth over time. The in vivo results showed that biopaper degraded rapidly and elicited an acute Th1 lymphocyte reaction at 3 days after implantation, which was decreased at 7 days after implantation. The gelatin sponge resisted degradation and evoked a hybrid M1/M2 macrophage reaction at 7-21 days after implantation, and a protumor M2d subset was confirmed. Surgicel resisted early degradation and caused obvious antitumor M2a macrophage reactions. Mice subjected to subcutaneous implantation of A549 â€‹cells and hemostatic materials in the gelatin sponge group had the largest tumor volumes and the shortest overall survival (OS), while the Surgicel and the biopaper group had the smallest volumes and the longest OS. Therefore, although gelatin sponges exhibited cytotoxicity to A549 â€‹cells in vitro, they promoted the growth of A549 â€‹cells in vivo, which was related to chronic M2d macrophage reaction. Surgicel and biopaper inhibited A549 â€‹cell growth in vivo, which is associated with chronic M2a macrophage reaction or acute Th1 lymphocyte reaction.

Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells.

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Chemokines in the Landscape of Cancer Immunotherapy: How They and Their Receptors Can Be Used to Turn Cold Tumors into Hot Ones?

Over the last decade, monoclonal antibodies to immune checkpoint inhibitors (ICI), also known as immune checkpoint blockers (ICB), have been the most successful approach for cancer therapy. Starting with mAb to cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors in metastatic melanoma and continuing with blockers of the interactions between program cell death 1 (PD-1) and its ligand program cell death ligand 1 (PDL-1) or program cell death ligand 2 (PDL-2), that have been approved for about 20 different indications. Yet for many cancers, ICI shows limited success. Several lines of evidence imply that the limited success in cancer immunotherapy is associated with attempts to treat patients with "cold tumors" that either lack effector T cells, or in which these cells are markedly suppressed by regulatory T cells (Tregs). Chemokines are a well-defined group of proteins that were so named due to their chemotactic properties. The current review focuses on key chemokines that not only attract leukocytes but also shape their biological properties. CXCR3 is a chemokine receptor with 3 ligands. We suggest using Ig-based fusion proteins of two of them: CXL9 and CXCL10, to enhance anti-tumor immunity and perhaps transform cold tumors into hot tumors. Potential differences between CXCL9 and CXCL10 regarding ICI are discussed. We also discuss the possibility of targeting the function or deleting a key subset of Tregs that are CCR8+ by monoclonal antibodies to CCR8. These cells are preferentially abundant in several tumors and are likely to be the key drivers in suppressing anti-cancer immune reactivity.

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells.

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein-protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

The Role of Chemokines in Cervical Cancers.

Both clinical-pathological and experimental studies have shown that chemokines play a key role in activating the immune checkpoint modulator in cervical cancer progression and are associated with prognosis in tumor cell proliferation, invasion, angiogenesis, chemoresistance, and immunosuppression. Therefore, a clear understanding of chemokines and immune checkpoint modulators is essential for the treatment of this disease. This review discusses the origins and categories of chemokines and the mechanisms that are responsible for activating immune checkpoints in cervical dysplasia and cancer, chemokines as biomarkers, and therapy development that targets immune checkpoints in cervical cancer research.

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis.

Introduction: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. Materials and Methods: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. Results: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. Conclusion: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.

Splenectomy increases blood pressure and abolishes sex differences in renal T-regulatory cells in spontaneously hypertensive rats.

Over the past decade there has been increasing support for a role of the immune system in the development of hypertension. Our lab has previously reported that female spontaneously hypertensive rats (SHRs) have a blood pressure (BP)-dependent increase in anti-inflammatory renal regulatory T cells (Tregs), corresponding to lower BP compared with males. However, little is known regarding the mechanism for greater renal Tregs in females. The current study was designed to test the hypothesis that the greater relative abundance of renal Tregs in female SHR is due to greater Treg production. To test this hypothesis, T cell profiles were measured in the spleen by flow cytometry in male and female SHR at 5 and 14 weeks of age. Splenic Tregs did not differ between males and females, suggesting sex differences in renal Tregs is not due to differences in production. To assess the role of the spleen in sex differences in renal Tregs and BP control, rats were randomized to receive sham surgery (CON) or splenectomy (SPLNX) at 12 weeks of age and implanted with telemeters to measure BP. After 2 weeks, kidneys were harvested for flow cytometric analysis of T cells. Splenectomy increased BP in both sexes after 2 weeks. Renal Tregs decreased in both sexes after splenectomy, abolishing the sex differences in renal Tregs. In conclusion, splenic Tregs were comparable in male and female SHRs, suggesting that sex differences in renal Tregs is due to differences in renal Treg recruitment, not Treg production.

The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis.

Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.

CCL1 and IL-2Ra differentiate Tuberculosis disease from latent infection Irrespective of HIV infection in low TB burden countries.

OBJECTIVES: To evaluate the performance of selected host immunological biomarkers in differentiating tuberculosis (TB) disease from latent TB infection (LTBI) in HIV uninfected and infected individuals enrolled in TB low-burden countries. DESIGN: Participants with TB disease (N = 85) and LTBI (N = 150) were recruited from prospective cohorts at hospitals in Norway and Denmark. Plasma concentrations of 54 host markers were assessed by Luminex multiplex immunoassays. Using receiver operator characteristic curves and general discriminant analysis, we determined the abilities of individual and combined biomarkers to discriminate between TB disease and LTBI including when patients were stratified according to HIV infection status. RESULTS: Regardless of the groups compared, CCL1 and IL-2Ra were the most accurate single biomarkers in differentiating TB disease from LTBI. Regardless of HIV status, a 4-marker signature (CCL1+RANTES+CRP+MIP-1α) derived from a training set (n = 155) differentiated TB disease from LTBI in the test set (n = 67) with a sensitivity of 56.0% (95% CI, 34.9-75.6) and a specificity of 85.7% (95% CI, 71.5-94.6). A 5-marker signature derived from the HIV uninfected group (CCL1+RANTES+MIP-1α+procalcitonin+IP-10) performed in HIV-infected individuals with a sensitivity of 75.0% and a specificity of 96.7% after leave-one-out cross validation. A 2-marker signature (CCL1+TNF-α) identified in HIV-infected persons performed in HIV-uninfected with a sensitivity and specificity of 66.7% and 100% respectively in the test set. CONCLUSIONS: Plasma CCL1 and IL-2Ra have potential as biomarkers for differentiating TB disease from LTBI in low TB burden settings unaffected by HIV infection. Combinations between these and other biomarkers in bio-signatures for global use warrant further exploration.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

BACKGROUND: The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. OBJECTIVES: Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. METHODS: Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. RESULTS: The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. CONCLUSIONS: Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

Human Adipose-Derived Mesenchymal Stem Cells-Derived Exosomal microRNA-19b Promotes the Healing of Skin Wounds Through Modulation of the CCL1/TGF-ß Signaling Axis.

INTRODUCTION: Human adipose-derived mesenchymal stem cells (ADMSCs) with their secretory factors are able to induce collagen synthesis and fibroblast migration in the wound healing process. This study is launched to figure out the effect of human ADMSCs-derived exosomes on skin wound healing. METHODS: ADMSCs were extracted and ADMSCs-derived exosomes were identified. Skin damage models were established by treating HaCaT cells and human skin fibroblasts with H2O2. Next, the roles of ADMSCs and their derived exosomes were investigated. The exosomal miRNA then was analyzed, and the function of miRNA on the H2O2-induced cells was studied by miRNA suppression. Bioinformatics analysis, luciferase activity and RIP assays were implemented to find the target genes ofthe miRNA and the modulated pathways. A mouse skin damage model was induced to elucidate the effects of exosomes in vivo by injecting exosomes. RESULTS: H2O2 treatment significantly reduced the viability of HaCaT cells and increased their apoptosis rate. Co-culture with ADMSCs or their derived exosomes could improve the cell damage caused by H2O2. Meanwhile, H2O2 treatment promoted the internalization of exosomes. ADMSCs and their derived exosomes significantly increased miR-19b expression in the recipient cells, while inhibiting miR-19b resulted in a reduction in the therapeutic effect of ADMSCs-derived exosomes. Besides, miR-19b regulated the TGF-ß pathway by targeting CCL1. The therapeutic effect of exosomes was further confirmed by a mouse model of skin damage. CONCLUSION: Our study indicates that exosomal miR-19b derived from ADMSCs regulates the TGF-ß pathway by targeting CCL1, thereby promoting the healing of skin wounds.

The Role of IgG4 in the Fine Tuning of Tolerance in IgE-Mediated Allergy and Cancer.

Among the four immunoglobulin G (IgG) subclasses, IgG4 is the least represented in serum of a healthy human and it is considered an "odd" antibody. The IgG4 antibody has unique structural features that affect its biological function. These include the ability to undergo antigen-binding fragment (Fab)-arm exchange, to create fragment crystallizable (Fc) - Fc binding with other IgG4 and other IgG subclass antibodies, have a unique affinity profile for Fc gamma receptors (FcγRs) and no binding to complement component C1q. Altogether, these characteristics support anti-inflammatory roles of IgG4 leading to immune tolerance. Under conditions of chronic antigenic stimulation and Th2-type inflammation, both tissue and serum IgG4 levels are increased. This review seeks to highlight how in allergen immunotherapy IgG4 can confer a protective role as a "blocking" antibody and safeguard from subsequent allergen exposure, while IgG4 can confer immunomodulatory functions to support malignancy. While Th2 conditions drive polarization of macrophages to the M2a subtype, chronic antigen stimulation drives B cell class switching to IgG4 to further support phenotypical macrophage changes towards an M2b-like state. M2b-like macrophages can secrete chemokine (C-C motif) ligand 1 (CCL1) and interleukin-10 (IL-10) to support regulatory cell recruitment and to further shape a tolerogenic microenvironment. Thereby, IgG4 have a Janus-faced role, favorable in allergy but detrimental in cancer.

Ectosomal PKM2 Promotes HCC by Inducing Macrophage Differentiation and Remodeling the Tumor Microenvironment.

Tumor-derived extracellular vesicles are important mediators of cell-to-cell communication during tumorigenesis. Here, we demonstrated that hepatocellular carcinoma (HCC)-derived ectosomes remodel the tumor microenvironment to facilitate HCC progression in an ectosomal PKM2-dependent manner. HCC-derived ectosomal PKM2 induced not only metabolic reprogramming in monocytes but also STAT3 phosphorylation in the nucleus to upregulate differentiation-associated transcription factors, leading to monocyte-to-macrophage differentiation and tumor microenvironment remodeling. In HCC cells, sumoylation of PKM2 induced its plasma membrane targeting and subsequent ectosomal excretion via interactions with ARRDC1. The PKM2-ARRDC1 association in HCC was reinforced by macrophage-secreted cytokines/chemokines in a CCL1-CCR8 axis-dependent manner, further facilitating PKM2 excretion from HCC cells to form a feedforward regulatory loop for tumorigenesis. In the clinic, ectosomal PKM2 was clearly detected in the plasma of HCC patients. This study highlights a mechanism by which ectosomal PKM2 remodels the tumor microenvironment and reveals ectosomal PKM2 as a potential diagnostic marker for HCC.

Spinal CCL1/CCR8 regulates phosphorylation of GluA1-containing AMPA receptor in postoperative pain after tibial fracture and orthopedic surgery in mice.

Chronic postoperative pain might be a pivotal component hindering recovery and regains the function after bone fracture and orthopedic surgery. However, the underlying mechanisms remain largely unclear. AMPA receptor of excitatory synapses is considered due to its critical role in pathologic pain. Chemokine CCL1 related neuroinflammation plays a role in excitatory synaptic transmission and nociceptive transduction. This study examined whether spinal CCL1 is associated with fracture-associated postoperative pain via AMPA receptor. We herein discovered that the tibial fracture with orthopedic surgery initiated and maintained chronic postoperative pain along with spinal up-regulation of CCL1/CCR8 expression and phosphorylation of GluA1-containing AMPA receptor. Central CCL1/CCR8 inhibition impaired mechanical and cold allodynia, and phosphorylated GluA1-containing AMPA receptor in the spinal dorsal horn. Intrathecal injection of GluA1-containing AMPA receptor antagonist NASPM alleviated fracture-related postoperative pain. Also, exogenous CCL1 delivery facilitated acute pain behaviors and spinal phosphorylation of GluA1-containing AMPA receptor in naïve mice, reversing by co-application of NASPM. Our current results indicated that spinal CCL1/CCR8-mediated GluA1-containing AMPA receptor activation is vital in the pathogenesis of fracture associated postoperative pain in mice.