CXCL8 Secretion Research Articles

Pro-pigmentary action of 5-fluorouracil through the stimulated secretion of CXCL12 by dermal fibroblasts.

Background:There is growing evidence that 5-fluorouracil (5-FU) combined with therapeutic trauma can effectively induce skin repigmentation in vitiligo patients who are unresponsive to conventional treatments. Previous studies have mainly focused on identifying the antimitotic activity of 5-FU for the treatment of skin cancer, but few studies have investigated its extra-genotoxic actions favoring melanocyte recruitment.Methods:We utilized the full thickness excisional skin wound model in Dct-LacZ transgenic mice to dynamically assess the migration of melanocytes in the margins of wounds treated with or without 5-FU. The in-situ expression of CXCL12 was examined in the wound beds using immunofluorescence staining. Quantitative real-time polymerase chain reaction and Western blotting analyses were performed to detect the expression levels of CXCL12 mRNA and protein in primary mouse dermal fibroblasts treated with or without 5-FU. Transwell assays and fluorescein isothiocyanate (FITC)-phalloidin staining were used to observe cell migration and filamentous actin (F-actin) changes of melan-a murine melanocytes.Results:Whole mount and cryosection X-gal staining showed that the cell numbers of LacZ-positive melanocytes were much higher in the margins of dorsal and tail skin wounds treated with 5-FU compared with the controls. Meanwhile, CXCL12 immunostaining was significantly increased in the dermal compartment of wounds treated with 5-FU (control vs. 5-FU, 22.47 ± 8.85 vs. 44.69 ± 5.97, P < 0.05). Moreover, 5-FU significantly upregulated the expression levels of CXCL12 mRNA (control vs. 5-FU, 1.00 ± 0.08 vs. 1.54 ± 0.06, P < 0.05) and protein (control vs. 5-FU, 1.00 ± 0.06 vs. 2.93 ± 0.10, P < 0.05) in cultured fibroblasts. Inhibition of the CXCL12/CXCR4 axis suppressed melanocyte migration in vitro using a CXCL12 small interfering RNA (siRNA) or a CXCR4 antagonist (AMD3100).Conclusion:5-FU possesses a pro-pigmentary activity through activation of the CXCL12/CXCR4 axis to drive the chemotactic migration of melanocytes.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

The indole-hydantoin derivative exhibits anti-inflammatory activity by preventing the transactivation of NF-κB through the inhibition of NF-κB p65 phosphorylation at Ser276

Indole- and hydantoin-based derivatives both exhibit anti-inflammatory activity, suggesting that the structures of indole and hydantoin are functional for this activity. In the present study, we synthesized two types of indole-hydantoin derivatives, IH-1 (5-(1H-indole-3-ylmethylene) imidazolidine-2,4-dione) and IH-2 (5-(1H-indole-3-ylmethyl) imidazolidine-2,4-dione) and examined their effects on LPS-induced inflammatory responses in murine macrophage-like RAW264.7 cells. LPS-induced inflammatory responses were not affected by indole, hydantoin, or IH-2. In contrast, IH-1 significantly inhibited the LPS-induced production of nitric oxide (NO) and secretion of CCL2 and CXCL1 by suppressing the mRNA expression of inducible NO synthase (iNOS), CCL2, and CXCL1. IH-1 markedly inhibited the LPS-induced activation of NF-κB without affecting the degradation of IκBα or nuclear translocation of NF-κB. IH-1 markedly attenuated the transcriptional activity of NF-κB by suppressing the LPS-induced phosphorylation of the NF-κB p65 subunit at Ser276. Furthermore, IH-1 prevented the LPS-induced interaction of NF-κB p65 subunit with a transcriptional coactivator, cAMP response element-binding protein (CBP). Collectively, these results revealed the potential of the novel indole-hydantoin derivative, IH-1 as an anti-inflammatory drug.

Aiduqing formula inhibits breast cancer metastasis by suppressing TAM/CXCL1-induced Treg differentiation and infiltration

BackgroundMetastasis represents the leading cause of death in patients with breast cancer. Traditional Chinese medicine is particularly appreciated for metastatic diseases in Asian countries due to its benefits for survival period prolongation and immune balance modulation. However, the underlying molecular mechanisms remain largely unknown. This study aimed to explore the antimetastatic effect and immunomodulatory function of a clinical formula Aiduqing (ADQ).MethodsNaive CD4+ T cells, regulatory T cells (Tregs), and CD8+ T cells were sorted by flow cytometry. Then, breast cancer cells and these immune cells were co-cultured in vitro or co-injected into mice in vivo to simulate their coexistence. Flow cytometry, ELISA, qPCR, double luciferase reporter gene assay, and chromatin immunoprecipitation assay were conducted to investigate the immunomodulatory and antimetastatic mechanisms of ADQ.ResultsADQ treatment by oral gavage significantly suppressed 4T1-Luc xenograft growth and lung metastasis in the orthotopic breast cancer mouse model, without noticeable hepatotoxicity, nephrotoxicity, or hematotoxicity. Meanwhile, ADQ remodeled the immunosuppressive tumor microenvironment (TME) by increasing the infiltration of tumor-infiltrating lymphocytes (TILs) and cytotoxic CD8+ T cells, and decreasing the infiltration of Tregs, naive CD4+ T cells, and tumor-associated macrophages (TAMs). Molecular mechanism studies revealed that ADQ remarkably inhibited CXCL1 expression and secretion from TAMs and thus suppressed the chemotaxis and differentiation of naive CD4+ T cells into Tregs, leading to the enhanced cytotoxic effects of CD8+ T cells. Mechanistically, TAM-derived CXCL1 promoted the differentiation of naive CD4+ T cells into Tregs by transcriptionally activating the NF-κB/FOXP3 signaling. Lastly, mouse 4T1-Luc xenograft experiments validated that ADQ formula inhibited breast cancer immune escape and lung metastasis by suppressing the TAM/CXCL1/Treg pathway.ConclusionsThis study not only provides preclinical evidence supporting the application of ADQ in inhibiting breast cancer metastasis but also sheds novel insights into TAM/CXCL1/NF-κB/FOXP3 signaling as a promising therapeutic target for Treg modulation and breast cancer immunotherapy.5spcww6AwskguE5dGnjCJQVideo

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6′-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.The therapeutic effects of CP-25 on experimental Sjögren's syndrome has been shown to be associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. The study provides an experimental foundation for CP-25 as a potential drug in the treatment of human autoimmune disorder, Sjögren's syndrome.

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1β and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

LAMC1 upregulation via TGFβ induces inflammatory cancer-associated fibroblasts in esophageal squamous cell carcinoma via NF-κB-CXCL1-STAT3.

Cancer‐associated fibroblasts (CAF) are a heterogeneous cell population within the tumor microenvironment,and play an important role in tumor development. By regulating the heterogeneity of CAF, transforming growth factor β (TGFβ) influences tumor development. Here, we explored oncogenes regulated by TGFβ1 that are also involved in signaling pathways and interactions within the tumor microenvironment. We analyzed sequencing data of The Cancer Genome Atlas (TCGA) and our own previously established RNA microarray data (GSE53625), as well as esophageal squamous cell carcinoma (ESCC) cell lines with or without TGFβ1 stimulation. We then focused on laminin subunit gamma 1 (LAMC1), which was overexpressed in ESCC cells, affecting patient prognosis, which could be upregulated by TGFβ1 through the synergistic activation of SMAD family member 4 (SMAD4) and SP1. LAMC1 directly promoted the proliferation and migration of tumor cells, mainly via Akt–NFκB–MMP9/14 signaling. Additionally, LAMC1 promoted CXCL1 secretion, which stimulated the formation of inflammatory CAF (iCAF) through CXCR2–pSTAT3. Inflammatory CAF promoted tumor progression. In summary, we identified the dual mechanism by which the upregulation of LAMC1 by TGFβ in tumor cells not only promotes ESCC proliferation and migration, but also indirectly induces carcinogenesis by stimulating CXCL1 secretion to promote the formation of iCAF. This finding suggests that LAMC1 could be a potential therapeutic target and prognostic marker for ESCC.

LRG1 facilitates corneal fibrotic response by inducing neutrophil chemotaxis via Stat3 signaling in alkali-burned mouse corneas.

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel profibrotic factor that modulates transforming growth factor-β (TGF-β) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophil infiltration. LRG1 promoted neutrophil chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, whereas the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophil-induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophil infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage

BackgroundPulmonary fibrosis (PF) is a growing clinical problem with limited therapeutic options. Human umbilical cord mesenchymal stromal cell (hucMSC) therapy is being investigated in clinical trials for the treatment of PF patients. However, little is known about the underlying molecular and cellular mechanisms of hucMSC therapy on PF. In this study, the molecular and cellular behavior of hucMSC was investigated in a bleomycin-induced mouse PF model.MethodsThe effect of hucMSCs on mouse lung regeneration was determined by detecting Ki67 expression and EdU incorporation in alveolar type 2 (AT2) and lung fibroblast cells. hucMSCs were transfected to express the membrane localized GFP before transplant into the mouse lung. The cellular behavior of hucMSCs in mouse lung was tracked by GFP staining. Single cell RNA sequencing was performed to investigate the effects of hucMSCs on gene expression profiles of macrophages after bleomycin treatment.ResultshucMSCs could alleviate collagen accumulation in lung and decrease the mortality of mouse induced by bleomycin. hucMSC transplantation promoted AT2 cell proliferation and inhibited lung fibroblast cell proliferation. By using single cell RNA sequencing, a subcluster of interferon-sensitive macrophages (IFNSMs) were identified after hucMSC infusion. These IFNSMs elevate the secretion of CXCL9 and CXCL10 following hucMSC infusion and recruit more Treg cells to the injured lung.ConclusionsOur study establishes a link between hucMSCs, macrophage, Treg, and PF. It provides new insights into how hucMSCs interact with macrophage during the repair process of bleomycin-induced PF and play its immunoregulation function.

Abstract 1637: A fully human anti-vista antibody as a promising therapy against poorly immunogenic tumors

Abstract V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is an immune checkpoint regulator of the B7 family. VISTA can be found on the cell surface of some tumor types, however for the majority of cancers, VISTA is highly expressed in the immunological myeloid cell compartment in the tumor microenvironment (TME). VISTA has been shown, in vitro and in vivo, to inhibit T cell activation and prevent T cell recruitment into tumors. In patients, high VISTA expression is associated with poor prognosis and is also a potential mediator of resistance to anti-CTLA-4 and anti-PD-(L)1 therapies. Therefore, VISTA is a very attractive new target for cancer immunotherapy. Kineta has selected a lead candidate anti-VISTA monoclonal antibody after a deep screen of 107 fully human and highly diverse antibodies directed against the VISTA extracellular domain. The candidate exhibits high potency in the subnanomolar range and is characterized by a long constant of dissociation evaluated by ELISA and Octet binding. It targets human and cynomolgus monkey VISTA on a unique epitope. Cross reactivity against other B7 family members has also been evaluated, and the lead candidate demonstrates high specificity against VISTA. The candidate antibody also efficiently induces T cell activation, proliferation and IFNg secretion on a Staphylococcal EnterotoxinB assay, as well as in a coculture experiment with a cell line overexpressing VISTA. The candidate promotes maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion in a monocyte activation assay. The mechanism of action is mediated in part by NK cells. This anti-VISTA antibody also prevents the immunosuppressive function of differentiated MDSCs in vitro against T cells. In Knock-In-human VISTA mice, anti-VISTA antibody treatment mediates single-agent antitumor activity in vivo in multiple syngeneic tumor models and shows enhanced efficacy in combination with either anti-PD-(L)1 or anti-CTLA-4 treatment. Finally, anti-VISTA antibody treatment was well-tolerated in exploratory toxicology studies in cynomolgus monkey and has a half-life consistent with other monoclonal antibodies. Our results strongly support the continued development of our anti-VISTA antibody for the treatment of colder, less immunogenic tumors. Citation Format: Thierry Guillaudeux, Eric Tarcha, Robert Bader, Benjamin Dutzar, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig, Yulia Ovechkina, David Peckham, Jeff Posakony, Shaarwari Sridhar, Mei Xu, Shawn Iadonato. A fully human anti-vista antibody as a promising therapy against poorly immunogenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1637.

Abstract 1692: A novel immunoregulatory role of tumor treating fields (TTFields) on macrophage polarization

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. In this study we investigated the potential of TTFields for regulating macrophage phenotype skewing, with or without lipopolysaccharide (LPS) and interferon-γ (IFN-γ) or IL-4 activation. Methods: Bone marrow cells were flushed from the femurs and tibias of 5-8 week old Balb\C mice to generate bone marrow-derived macrophages (BMDMs). To study individual macrophage responses to TTFields, we profiled macrophages that were stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization), followed by treatment with TTFields (150 kHz) for 24 hours. Flow cytometry was used to assess surface expression of F4/80 (a macrophage biomarker) and the activation markers that included major histocompatibility complex class II (MHC II), CD80, and inducible nitric oxide synthase (iNOS). A multiplexed secretion assay was used to measure the heterogeneity of the stimulated macrophages. We captured the secretion of 13 different proteins including CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, free active TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC) and IL-1β. Results: TTFields treatment polarized IL-4 stimulated M2 macrophages to the M1 phenotype, as indicated by a significant increase in the percentage of CD80+MHC IIhigh cells. Treatment with TTFields of BMDMs that were either stimulated with IFN-γ and LPS (M1 phenotype) or had remained unstimulated increased the percentage of CD80+MHC IIhigh cells to a lesser degree. Moreover, treatment with TTFields significantly increased intracellular iNOS levels in M1 polarized BMDMs. In unstimulated BMDMs, application of TTFields resulted in significant increases in secretion of the pro-inflammatory cytokines CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. Also, a similar pattern was observed in cell supernatants of M1 and M2 stimulated BMDMs after TTFields application. Conclusions: These results elucidate a novel immunoregulatory role of TTFields on macrophage polarization. Future studies will aim to focus on the mechanism governing phenotypic skewing of macrophages treated with TTFields and to explore the effect of TTFields on macrophage in the tumor microenvironment. Citation Format: Boris Brant, Tali Voloshin, Alexandra Volodin, Lilach Koren, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Moshe Giladi, Uri Weinberg, Yoram Palti. A novel immunoregulatory role of tumor treating fields (TTFields) on macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1692.

A Protective Role of FAM13A in Human Airway Epithelial Cells Upon Exposure to Cigarette Smoke Extract.

BackgroundChronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease characterized by chronic inflammation upon inhalation of noxious particles, e.g., cigarette smoke. FAM13A is one of the genes often found to be associated with COPD, however its function in the pathophysiology of COPD is incompletely understood. We studied its role in airway epithelial barrier integrity and cigarette smoke-induced epithelial responses.Materials and MethodsProtein level and localization of FAM13A was assessed with immunohistochemistry in lung tissue from COPD patients and non-COPD controls. In vitro, FAM13A expression was determined in the absence or presence of cigarette smoke extract (CSE) in primary airway epithelial cells (AECs) from COPD patients and controls by western blotting. FAM13A was overexpressed in cell line 16HBE14o- and its effect on barrier function was monitored real-time by electrical resistance. Expression of junctional protein E-cadherin and β-catenin was assessed by western blotting. The secretion of neutrophil attractant CXCL8 upon CSE exposure was measured by ELISA.ResultsFAM13A was strongly expressed in airway epithelium, but significantly weaker in airways of COPD patients compared to non-COPD controls. In COPD-derived AECs, but not those of controls, FAM13A was significantly downregulated by CSE. 16HBE14o- cells overexpressing FAM13A built up epithelial resistance significantly more rapidly, which was accompanied by higher E-cadherin expression and reduced CSE-induced CXCL8 levels.ConclusionOur data indicate that the expression of FAM13A is lower in airway epithelium of COPD patients compared to non-COPD controls. In addition, cigarette smoking selectively downregulates airway epithelial expression of FAM13A in COPD patients. This may have important consequences for the pathophysiology of COPD, as the more rapid build-up of epithelial resistance upon FAM13A overexpression suggests improved (re)constitution of barrier function. The reduced epithelial secretion of CXCL8 upon CSE-induced damage suggests that lower FAM13A expression upon cigarette smoking may facilitate epithelial-driven neutrophilia.

SLC7A2 deficiency promotes hepatocellular carcinoma progression by enhancing recruitment of myeloid-derived suppressors cells

The main reason for poor prognosis in hepatocellular carcinoma (HCC) patients is high metastasis and recurrence. Cancer progression depends on a tumor-supportive microenvironment. Therefore, illustrating the mechanisms of tumor immunity in underlying HCC metastasis is essential. Here, we report a novel role of solute carrier family 7 member 2 (SLC7A2), a member of the solute carrier family, in HCC metastasis. The reduction of SLC7A2 was an independent and significant risk factor for the survival of HCC patients. Upregulation of SLC7A2 decreased HCC invasion and metastasis, whereas downregulation of SLC7A2 promoted HCC invasion and metastasis. We further found that deficient SLC7A2 medicated the upregulation of CXCL1 through PI3K/Akt/NF-kκB pathway to recruit myeloid-derived suppressor cells (MDSCs), exerting tumor immunosuppressive effect. Moreover, we found that G9a-mediated di-methylation of H3K9 (H3K9me2) silenced the expression of SLC7A2 to suppress HCC metastasis and immune escape. In conclusion, G9a-mediated silencing of SLC7A2 exerts unexpected functions in cancer metastasis by fostering a tumor-supportive microenvironment through CXCL1 secretion and MDSCs recruitment. Thus, SLC7A2 may provide new mechanistic insight into the cancer-promoting property of MDSCs.

Tumor-derived lactate inhibit the efficacy of lenvatinib through regulating PD-L1 expression on neutrophil in hepatocellular carcinoma

BackgroundNeutrophils play a controversial role in tumor development. The function of programmed cell death-1 ligand (PD-L1+) neutrophils, however, may inhibit the cytotoxicity of anti-tumor immunity. In this study, we elucidate...

Hsa-miR-605 regulates the proinflammatory chemokine CXCL5 in complex regional pain syndrome

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.

Mitochondrial DNA leakage induces odontoblast inflammation via the cGAS-STING pathway

BackgroundMitochondrial DNA (mtDNA) is a vital driver of inflammation when it leaks from damaged mitochondria into the cytosol. mtDNA stress may contribute to cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway activation in infectious diseases. Odontoblasts are the first cells challenged by cariogenic bacteria and involved in maintenance of the pulp immune and inflammatory responses to dentine-invading pathogens. In this study, we investigated that mtDNA as an important inflammatory driver participated in defending against bacterial invasion via cGAS-STING pathway in odontoblasts.MethodsThe normal tissues, caries tissues and pulpitis tissues were measured by western blotting and immunohistochemical staining. Pulpitis model was built in vitro to evaluated the effect of the cGAS-STING pathway in odontoblast-like cell line (mDPC6T) under inflammation. Western blot and real-time PCR were performed to detect the expression of cGAS-STING pathway and pro-inflammatory cytokines. The mitochondrial function was evaluated reactive oxygen species (ROS) generated by mitochondria using MitoSOX Red dye staining. Cytosolic DNA was assessed by immunofluorescent staining and real-time PCR in mDPC6T cells after LPS stimulation. Furthermore, mDPC6T cells were treated with ethidium bromide (EtBr) to deplete mtDNA or transfected with isolated mtDNA. The expression of cGAS-STING pathway and pro-inflammatory cytokines were measured.ResultsThe high expression of cGAS and STING in caries and pulpitis tissues in patients, which was associated with inflammatory progression. The cGAS-STING pathway was activated in inflamed mDPC6T. STING knockdown inhibited the nuclear import of p65 and IRF3 and restricted the secretion of the inflammatory cytokines CXCL10 and IL-6 induced by LPS. LPS caused mitochondrial damage in mDPC6T, which promoted mtDNA leakage into the cytosol. Depletion of mtDNA inhibited the cGAS-STING pathway and nuclear translocation of p65 and IRF3. Moreover, repletion of mtDNA rescued the inflammatory response, which was inhibited by STING knockdown.ConclusionOur study systematically identified a novel mechanism of LPS-induced odontoblast inflammation, which involved mtDNA leakage from damaged mitochondria into the cytosol stimulating the cGAS-STING pathway and the inflammatory cytokines IL-6 and CXCL10 secretion. The mtDNA-cGAS-STING axis could be a potent therapeutic target to prevent severe bacterial inflammation in pulpitis.11Tk2-r6oLFg_Eti7nH7V6Video Graphic abstract

GSDMD contributes to host defence against Staphylococcus aureus skin infection by suppressing the Cxcl1\u2013Cxcr2 axis

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1β secretion. To date, the role and regulatory mechanism of GSDMD during cutaneous microbial infection remain unclear. Here, we showed that GSDMD protected against Staphylococcus aureus skin infection by suppressing Cxcl1–Cxcr2 signalling. GSDMD deficiency resulted in larger abscesses, more bacterial colonization, exacerbated skin damage, and increased inflammatory cell infiltration. Although GSDMD deficiency resulted in defective IL-1β production, the critical role of IL-1β was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1β production but did not aggravate disease severity during S. aureus skin infection. Interestingly, GSDMD-deficient mice had increased Cxcl1 secretion accompanied by increased recruitment of neutrophils, whereas Caspase-1/11-deficient mice presented similar levels of Cxcl1 and neutrophils as wild-type mice. Moreover, the absence of GSDMD promoted Cxcl1 secretion in bone marrow-derived macrophages induced by live, dead, or different strains of S. aureus. Corresponding to higher transcription and secretion of Cxcl1, enhanced NF-κB activation was shown in vitro and in vivo in the absence of GSDMD. Importantly, inhibiting the Cxcl1–Cxcr2 axis with a Cxcr2 inhibitor or anti-Cxcl1 blocking antibody rescued host defence defects in the GSDMD-deficient mice. Hence, these results revealed an important role of GSDMD in suppressing the Cxcl1–Cxcr2 axis to facilitate pathogen control and prevent tissue damage during cutaneous S. aureus infection.

Tumor-Associated Macrophage Promotes the Survival of Cancer Cells upon Docetaxel Chemotherapy via the CSF1/CSF1R-CXCL12/CXCR4 Axis in Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is an advanced stage of prostate cancer that can progress rapidly even in patients treated with castration. Previously, we found that tumor-associated macrophages (TAM) can be recruited by CSF-1 secreted by docetaxel-treated prostate cancer cells and promote the survival of cancer cells in response to chemotherapy. The inhibition of CSF-1R can impede this effect and significantly prolong survival in xenograft mice. However, the actual mechanism of how TAM improves cancer cell survival still remains elusive and controversial. Here, for the first time, we found that the enhanced survival of cancer cells achieved by TAM was mainly mediated by CXCR4 activation from the increased secretion of CXCL12 from CSF-1 activated TAM. This finding helps to clarify the mechanism of chemoresistance for second-line chemotherapy using docetaxel, facilitating the development of novel drugs to overcome immune tolerance in castration-resistant prostate cancer.

Exosomes From Subjects With Multiple Sclerosis Express EBV-Derived Proteins and Activate Monocyte-Derived Macrophages.

ObjectiveTo investigate in a cross-sectional study the effect of serum-derived exosomes on primary human blood monocyte-derived macrophages (MDMs) comparing exosomes from healthy donors vs patients with relapsing-remitting multiple sclerosis in remission and in relapse and to assess whether the response correlates with exosomal Epstein-Barr virus (EBV) protein expression.MethodsA total of 45 serum-derived exosome preparations were isolated from patients and healthy controls and verified for the expression of exosomal and EBV markers. MDMs were differentiated from monocytes for 7 days and incubated for 24 hours with exosomes, and then, cell supernatants were collected for cytokine measurement by cytometric bead array. Cells were immunophenotyped before and after differentiation.ResultsSerum-derived exosomes of patients with multiple sclerosis (MS) expressed higher levels of EBV proteins than healthy controls. Of interest, expression of EBV nuclear antigen EBNA1 and latent membrane proteins LMP1 and 2A was higher on exosomes derived from patients with active RRMS compared with healthy controls and stable patients. After data normalization, we observed that incubation with EBV(+) exosomes induced CXCL10 and CCL2 secretion by MDMs. MDMs differentiated from patients with active disease were better secretors of CXCL10 and other interferon-γ–inducible chemokines, including CCL2 and CXCL9, than MDMs from healthy and stable MS groups. MDMs from active patients had a higher frequency of a CD14(++) subset that correlated with the secreted CXCL10.ConclusionExosomes expressing EBV proteins correlate with disease activity and induce an inflammatory response in MDMs that is compounded by the origin of the responder cells.