M2 macrophages promote PD-L1 expression in triple-negative breast cancer via secreting CXCL1.

BACKGROUND: M2 macrophages are known to play a significant role in the progression of triple-negative breast cancer (TNBC) by creating an immunosuppressive microenvironment. The aim of this study is to investigate the impact of M2 macrophages on TNBC and their correlation with programmed death-ligand 1 (PD-L1) expression. METHODS: We employed a co-culture system to analyze the role of the mutual regulation of M2 macrophages and TNBC cells. Employing a multifaceted approach, including bioinformatics analysis, Western blotting, flow cytometry analysis, ELISA, qRT-PCR, lentivirus infection, mouse models, and IHC, we aimed to elucidate the influence and mechanism of M2 macrophages on PD-L1 expression. RESULTS: The results showed a substantial infiltration of M2 macrophages in TNBC tissue, which demonstrated a positive correlation with PD-L1 expression. CXCL1 exhibited abnormally high expression in M2 macrophages and enhanced the expression of PD-L1 in TNBC cells. Notably, silencing CXCL1 or its receptor CXCR2 inhibited M2 macrophages-induced expression of PD-L1. Mechanistically, CXCL1 derived from M2 macrophages binding to CXCR2 activated the PI3K/AKT/NF-κB signaling pathway, resulting in increased PD-L1 expression in TNBC. CONCLUSION: Broadly speaking, these results provide evidence for the immunosuppressive role of M2 macrophages and CXCL1 in TNBC cells, indicating their potential as therapeutic biomarkers.

Crosstalk between cancer cells and macrophages promotes OSCC cell migration and invasion through a CXCL1/EGF positive feedback loop.

BACKGROUND: C-X-C motif chemokine ligand 1 (CXCL1) and epithelial growth factor (EGF) are highly secreted by oral squamous cell carcinoma (OSCC) cells and tumor-associated macrophages, respectively. Recent studies have shown that there is intricate "cross-talk" between OSCC cells and macrophages. However, the underlying mechanisms are still poorly elucidated. METHODS: The expression of CXCL1 was detected by immunohistochemistry in OSCC clinical samples. CXCL1 levels were evaluated by RT‒PCR and ELISA in an OSCC cell line and a normal epithelial cell line. The expression of EGF was determined by RT‒PCR and ELISA. The effect of EGF on the proliferation of OSCC cells was evaluated by CCK-8 and colony formation assays. The effect of EGF on the migration and invasion ability and epithelial-mesenchymal transition (EMT) of OSCC cells was determined by wound healing, Transwell, RT‒PCR, Western blot and immunofluorescence assays. The polarization of macrophages was evaluated by RT‒PCR and flow cytometry. Western blotting was used to study the molecular mechanism in OSCC. RESULTS: The expression of C-X-C motif chemokine ligand 1 (CXCL1) was higher in the OSCC cell line (Cal27) than in immortalized human keratinocytes (Hacat cells). CXCL1 derived from Cal27 cells upregulates the expression of epithelial growth factor (EGF) in macrophages. Paracrine stimulation mediated by EGF further facilitates the epithelial-mesenchymal transition (EMT) of Cal27 cells and initiates the upregulation of CXCL1 in a positive feedback-manner. Mechanistically, EGF signaling-induced OSCC cell invasion and migration can be ascribed to the activation of NF-κB signaling mediated by the epithelial growth factor receptor (EGFR), as determined by western blotting. CONCLUSIONS: OSCC cell-derived CXCL1 can stimulate the M2 polarization of macrophages and the secretion of EGF. Moreover, EGF significantly activates NF-κB signaling and promotes the migration and invasion of OSCC cells in a paracrine manner. A positive feedback loop between OSCC cells and macrophages was formed, contributing to the promotion of OSCC progression.

Depressive-like Behavior Is Accompanied by Prefrontal Cortical Innate Immune Fatigue and Dendritic Spine Losses after HIV-1 Tat and Morphine Exposure.

Opioid use disorder (OUD) and HIV are comorbid epidemics that can increase depression. HIV and the viral protein Tat can directly induce neuronal injury within reward and emotionality brain circuitry, including the prefrontal cortex (PFC). Such damage involves both excitotoxic mechanisms and more indirect pathways through neuroinflammation, both of which can be worsened by opioid co-exposure. To assess whether excitotoxicity and/or neuroinflammation might drive depressive behaviors in persons infected with HIV (PWH) and those who use opioids, male mice were exposed to HIV-1 Tat for eight weeks, given escalating doses of morphine during the last two weeks, and assessed for depressive-like behavior. Tat expression decreased sucrose consumption and adaptability, whereas morphine administration increased chow consumption and exacerbated Tat-induced decreases in nesting and burrowing-activities associated with well-being. Across all treatment groups, depressive-like behavior correlated with increased proinflammatory cytokines in the PFC. Nevertheless, supporting the theory that innate immune responses adapt to chronic Tat exposure, most proinflammatory cytokines were unaffected by Tat or morphine. Further, Tat increased PFC levels of the anti-inflammatory cytokine IL-10, which were exacerbated by morphine administration. Tat, but not morphine, decreased dendritic spine density on layer V pyramidal neurons in the anterior cingulate. Together, our findings suggest that HIV-1 Tat and morphine differentially induce depressive-like behaviors associated with increased neuroinflammation, synaptic losses, and immune fatigue within the PFC.

Nobiletin enhances plasma Interleukin-6 and C-X-C motif chemokine ligand 1 levels that are increased by treadmill running.

Exercise increases the muscular secretion of Interleukin-6 (IL-6), which is partially regulated by ß2-adrenergic receptor signaling. Nobiletin is a polymethoxyflavone (PMF) found in citrus fruits that induces the secretion of IL-6 from C2C12 myotubes, but it remains unclear whether nobiletin promotes IL-6 secretion during exercise. The aim of this study was to clarify the effects of nobiletin on IL-6 secretion during exercise. Nobiletin and epinephrine were found to synergistically increase IL-6 secretion from differentiated C2C12 cells, which was suppressed by the inhibition of adenylyl cyclase (AC) or protein kinase A (PKA). Treadmill running for 60 min increased plasma levels of IL-6, epinephrine, and norepinephrine in rats. Nobiletin (5 mg/kg) orally administered 30 min before running increased plasma IL-6 levels further, although it did not increase plasma epinephrine and norepinephrine. In a similar manner to IL-6, nobiletin and epinephrine synergistically increased the secretion of C-X-C motif chemokine ligand 1 (CXCL-1) from C2C12 cells, or the increase in plasma CXCL-1 was enhanced by nobiletin after treadmill running of rats. Our results suggest that nobiletin promotes IL-6 and CXCL-1 secretion from skeletal muscle by synergistic enhancement of the PKA pathway in ß2-adrenergic receptor signaling.

Sensory nerves: A driver of the vicious cycle in bone metastasis?

Bone is one of the preferential target organs of cancer metastasis. Bone metastasis is associated with various complications, of which bone pain is most common and debilitating. The cancer-associated bone pain (CABP) is induced as a consequence of increased neurogenesis, reprogramming and axonogenesis of sensory nerves (SNs) in harmony with sensitization and excitation of SNs in response to the tumor microenvironment created in bone. Importantly, CABP is associated with increased mortality, of which precise cellular and molecular mechanism remains poorly understood. Bone is densely innervated by autonomic nerves (ANs) (sympathetic and parasympathetic nerves) and SNs. Recent studies have shown that the nerves innervating the tumor microenvironment establish intimate communications with tumors, producing various stimuli for tumors to progress and disseminate. In this review, our current understanding of the role of SNs innervating bone in the pathophysiology of CABP will be overviewed. Then the hypothesis that SNs facilitate cancer progression in bone will be discussed in conjunction with our recent findings that SNs play an important role not only in the induction of CABP but also the progression of bone metastasis using a preclinical model of CABP. It is suggested that SNs are a critical component of the bone microenvironment that drives the vicious cycle between bone and cancer to progress bone metastasis. Suppression of the activity of bone-innervating SNs may have potential therapeutic effects on the progression of bone metastasis and induction of CABP.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Homeobox B5 promotes metastasis and poor prognosis in Hepatocellular Carcinoma, via FGFR4 and CXCL1 upregulation.

Background: Since metastasis remains the main reason for HCC-associated death, a better understanding of molecular mechanism underlying HCC metastasis is urgently needed. Here, we elucidated the role of Homeobox B5 (HOXB5), a member of the HOX transcriptional factor family, in promoting HCC metastasis. Method: The expression of HOXB5 and its functional targets fibroblast growth factor receptor 4 (FGFR4) and C-X-C motif chemokine ligand 1 (CXCL1) were detected by immunohistochemistry. Luciferase reporter and chromatin immunoprecipitation assays were performed to measure the transcriptional regulation of target genes by HOXB5. The effects of FGFR4 and CXCL1 on HOXB5-mediated metastasis were analyzed by an orthotopic metastasis model. Results: Elevated expression of HOXB5 had a positive correlation with poor tumour differentiation, higher TNM stage, and indicated unfavorable prognosis. Overexpression of HOXB5 promoted HCC metastasis through transactivating FGFR4 and CXCL1 expression, whereas knockdown of FGFR4 and CXCL1 decreased HOXB5-enhanced HCC metastasis. Moreover, HOXB5 overexpression in HCC cells promoted myeloid derived suppressor cells (MDSCs) infiltration through CXCL1/CXCR2 axis. Either depletion of MDSCs by anti-Gr1 or blocking CXCL1-CXCR2 axis by CXCR2 inhibitor impaired HOXB5-mediated HCC metastasis. In addition, fibroblast growth factor 19 (FGF19) contributed to the HOXB5 upregulation through PI3K/AKT/HIF1α pathway. Overexpression of FGF15 (an analog of FGF19 in mouse) promoted HCC metastasis, whereas knockdown of HOXB5 significantly inhibited FGF15-enhanced HCC metastasis in immunocompetent mice. HOXB5 expression was positively associated with CXCL1 expression and intratumoral MDSCs accumulation in human HCC tissues. Patients who co-expressed HOXB5/CXCL1 or HOXB5/CD11b exhibited the worst prognosis. Furthermore, the combination of FGFR4 inhibitor BLU-554 and CXCR2 inhibitor SB265610 dramatically decreased HOXB5-mediated HCC metastasis. Conclusion: HOXB5 was a potential prognostic biomarker in HCC patients and targeting this loop may provide a promising treatment strategy for the inhibition of HOXB5-mediated HCC metastasis.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.

Calcium-activated chloride channel regulator 1 (CLCA1): More than a regulator of chloride transport and mucus production.

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl- transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases.

XIAOPI Formula Inhibits Breast Cancer Stem Cells via Suppressing Tumor-Associated Macrophages/C-X-C Motif Chemokine Ligand 1 Pathway.

Macrophages are the most abundant stromal cells associated with the host immune system in multiple malignancies including breast cancer. With proven clinical efficacy and no noticeable adverse effects, XIAOPI formula (XPS) has been approved for breast hyperplasia treatment by the State Food and Drug Administration of China (SFDA) in 2018. The existing knowledge about the anti-breast cancer activities and mechanisms of XPS has been very limited. The present study aimed to investigate whether XPS could exert an anti-breast cancer effect by regulating tumor-associated macrophages (TAMs) in tumor microenvironment. Herein, breast cancer cells and TAMs were co-cultured using the transwell co-culture system to simulate the coexistence of them. XPS could significantly inhibit the proliferation, colony formation, breast cancer stem cells (CSCs) subpopulation, mammosphere formation abilities as well as stemness-related genes expression in both human and mouse breast cancer cells in the co-culture system. Additionally, XPS could suppress M2 phenotype polarization as well as C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of TAMs. Notably, further mechanistic explorations verified TAMs/CXCL1 as the critical target of XPS in inhibiting breast CSCs self-renewal in the co-culture system as the exogenous CXCL1 administration could abrogate the inhibitory effect of XPS on breast CSCs self-renewal. More importantly, XPS significantly inhibited mammary tumor growth, breast CSCs subpopulation, and TAMs/CXCL1 activity in mouse 4T1-Luc xenografts in vivo without any detectable side effects. Taken together, this study not only uncovers the immunomodulatory mechanism of XPS in treating breast cancer but also sheds novel insights into TAMs/CXCL1 as a potential molecular target for breast CSCs elimination.