A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis.

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

Osteocytes support bone metastasis of melanoma cells by CXCL5.

Bone metastasis is a common complication of certain cancers such as melanoma. The spreading of cancer cells into the bone is supported by changes in the bone marrow environment. The specific role of osteocytes in this process is yet to be defined. By RNA-seq and chemokines screening we show that osteocytes release the chemokine CXCL5 when they are exposed to melanoma cells. Osteocytes-mediated CXCL5 secretion enhanced the migratory and invasive behaviour of melanoma cells. When the expression of the CXCL5 receptor, CXCR2, was down-regulated in melanoma cells in vitro, we observed a significant decrease in melanoma cell migration in response to osteocytes. Furthermore, melanoma cells with down-regulated CXCR2 expression showed less bone metastasis and less bone loss in the bone metastasis model in vivo. Furthermore, when simultaneously down-regulating CXCL5 in osteocytes and CXCR2 in melanoma cells, melanoma progression was abrogated in vivo. In summary, these data suggest a significant role of osteocytes in bone metastasis of melanoma, which is mediated through the CXCL5-CXCR2 pathway.

Exercise training inhibits macrophage-derived IL-17A-CXCL5-CXCR2 inflammatory axis to attenuate pulmonary fibrosis in mice exposed to silica.

Exposure to crystalline silica leads to health effects beyond occupational silicosis. Exercise training's potential benefits on pulmonary diseases yield inconsistent outcomes. In this study, we utilized experimental silicotic mice subjected to exercise training and pharmacological interventions, including interleukin-17A (IL-17A) neutralizing antibody or clodronate liposome for macrophage depletion. Findings reveal exercise training's ability to mitigate silicosis progression in mice by suppressing scavenger receptor B (SRB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Toll-like receptor 4 (TLR4) pathways. Macrophage-derived IL-17A emerges as primary source and trigger for silica-induced pulmonary inflammation and fibrosis. Exercise training effectively inhibits IL-17A-CXC motif chemokine ligand 5 (CXCL5)-Chemokine (C-X-C motif) Receptor 2 (CXCR2) axis in silicotic mice. Our study evidences exercise training's potential to reduce collagen deposition, preserve elastic fibers, slow pulmonary fibrosis advancement, and enhance pulmonary function post silica exposure by impeding macrophage-derived IL-17A-CXCL5-CXCR2 axis.

CXCL5 promotes tumorigenesis and angiogenesis of glioblastoma via JAK-STAT/NF-κb signaling pathways.

BACKGROUND: The tumor microenvironment contains chemokines that play a crucial role in various processes, such as tumorigenesis, inflammation, and therapy resistance, in different types of cancer. CXCL5 is a significant chemokine that has been shown to promote tumor proliferation, invasion, angiogenesis, and therapy resistance when overexpressed in various types of cancer. This research aims to investigate the impact of CXCL5 on the biological functions of glioblastoma (GBM). METHODS: The TCGA GBM and GEO databases were utilized to perform transcriptome microarray analysis and oncogenic signaling pathway analysis of CXCL5 in GBM. Validation of CXCL5 expression was performed using RT-qPCR and Western Blot. The impact of CXCL5 on cell proliferation, tumorigenesis, and angiogenesis in GBM was assessed through various methods, including cell proliferation assay, cloning assay, intracranial xenograft tumor models, and tube formation assay. Clinical prognosis was evaluated in 59 samples of gliomas with varying degrees of malignancy (grades 2, 3, and 4) and the TCGA GBM database, based on CXCL5 expression levels. The activities of the JAK-STAT and NF-κB signaling pathways were detected using Western Blot. RESULTS: The expression of CXCL5 was highly enriched in GBM. Moreover, the inhibition of CXCL5 showed a significant efficacy in suppressing cellular proliferation and angiogenesis, resulting in extended survival rates in xenograft mouse models in comparison to the control group. Notably, pretreatment with dapsone exhibited a reversal of the impact of CXCL5 on the formation of colonies and tubes in GBM cells. Elevated expression of CXCL5 was correlated with poor outcomes in GBM patients. Furthermore, the overexpression of CXCL5 has been associated with the activation of JAK-STAT and NF-κB signaling pathways. CONCLUSIONS: CXCL5 plays an important role in tumorigenesis and angiogenesis, indicating the potential for novel therapies targeting CXCL5 in GBM.

CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus.

BACKGROUND: Higher chemokine C-X-C motif ligand 5 (CXCL5) level was observed in type 2 diabetes mellitus (DM) patients; however, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis and wound healing in DM. METHODS: Endothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and Leprdb/JNarl mice were used as type 1 and type 2 DM models. Moreover, CXCL5 knockout mice were used to generate diabetic mice. Hindlimb ischemia surgery, aortic ring assays, matrigel plug assay, and wound healing assay were conducted. RESULTS: CXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 neutralizing antibody upregulated vascular endothelial growth factor (VEGF)/stromal cell-derived factor-1 (SDF-1) and promoted cell function in EPCs from type 2 DM patients and high glucose-treated EPCs from non-DM subjects as well as HAECs. CXCL5 directly up-regulated interleukin (IL)-1ß/IL-6/tumor necrosis factor-α and down-regulated VEGF/SDF-1 via ERK/p65 activation through chemokine C-X-C motif receptor 2 (CXCR2). CXCL5 neutralizing antibody recovered the blood flow after hindlimb ischemia, increased circulating EPC number, and enhanced VEGF and SDF-1 expression in ischemic muscle. CXCL5 suppression promoted neovascularization and wound healing in different diabetic animal models. The above observation could also be seen in streptozotocin-induced CXCL5 knockout diabetic mice. CONCLUSIONS: CXCL5 suppression could improve neovascularization and wound healing through CXCR2 in DM. CXCL5 may be regarded as a potential therapeutic target for vascular complications of DM.

Renal clear cell carcinoma-derived CXCL5 drives tumor-associated fibroblast formation and facilitates cancer progression.

BACKGROUND: Kidney renal clear cell carcinoma (KIRC, ccRCC) is one of the most common and aggressive subtypes of urinary system cancer. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) exacerbate the malignant phenotype of KIRC. It is necessary to explore further how KIRC induces normal fibroblasts (NFs) into CAFs. METHODS: The transcriptome data of KIRC was obtained from The Cancer Genome Atlas (TCGA), and the hub-genes and their corresponding functions in the co-expression module were obtained through differential analysis, enrichment analysis, and weighted correlation network analysis (WGCNA) analysis. RT-PCR, western-blot, and Elisa assays were used to detect the expression of CXCL5 (C-X-C Motif Chemokine Ligand 5) in KIRC cells and medium. Western-blot and immunofluorescence were used to demonstrate the transformation of NFs to CAF-like cells and relevant pathways. Human umbilical vein endothelial cells (huvec) were seeded within collagen gel to represent the neo-vascular network. Transwell, scrape, colony formation, and CCK-8 assays were performed to reveal the feedback effect of KIRC cells. RESULTS: Bioinformatics analysis showed that CXCL5 was a core gene in differential expression genes (DEGs) and was associated with extracellular matrix (ECM), which was associated with CAFs. KIRC-derived CXCL5 promoted the conversion of NFs to CAF-like cells. It included morphological and corresponding molecular marker changes. Activation of the JAK/STAT3 pathway was involved in this process. Corresponding, CAFs cells could secrete vascular endothelial growth factor (VEGF), which induced angiogenesis. CXCL5 promoted KIRC invasion and proliferation. CONCLUSIONS: Our research suggested that KIRC-derived CXCL5 could induce NFs to become CAFs-like cells that promote angiogenesis in the TME. The positive feedback of CXCL5 promoted its own invasive growth. The intercellular communication with CXCL5 as the core might be the critical node in the occurrence and development of KIRC.

Targeting CXCL5 in Pancreatic Cancer Cells Inhibits Cancer Xenograft Growth by Reducing Proliferation and Inhibiting EMT Progression.

BACKGROUND: Pancreatic cancer (PC) is the most lethal malignant tumor, with average survival period of about 10 months. C-X-C ligand 5 (CXCL5), an important chemokine for immune cell accumulation in tumor tissues, has been reported to be involved in a variety of human cancers. However, the exact role of CXCL5 in PC progression has not been well defined. METHODS: The expression of CXCL5 in PC was analyzed based on online databases and clinical specimens immunohistochemical staining, and Western blotting of CXCL5 in PC cell lines and patient samples. The correlation between CXCL5 expression and prognosis in PC was explored. The role of CXCL5 in PC was investigated through in vitro and in vivo experiments. RESULTS: The expression of CXCL5 was significantly increased in PC tissues compared with that in pancreas tissues, and CXCL5 high expression predicts poor prognosis in PC patients. Further analyses demonstrated that overexpression of CXCL5 in PC cells was positively related to higher proliferation rate, higher migration ability, and higher EMT markers including SNAI2 and TWIST1 of tumor cells in vitro. Consistently, the knockdown of CXCL5 in PC cells harmed the proliferation rate, migration ability, and expression of EMT indexes of tumor cells in vitro. Importantly, knockdown of CXCL5 inhibited the growth of xenograft tumors in vivo. CONCLUSION: CXCL5 high expression predicts poor prognosis in PC patients. CXCL5 promotes PC cell growth and EMT process. Inhibition of CXCL5 may be a potential therapeutic approach for PC.

CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality.

BACKGROUND: Metastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30. METHODS: PC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings. RESULTS: Human membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30's ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test, p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30NegSOCS3PosPC, when compared to patients with IL30PosSOCS3NegPC. CONCLUSIONS: Membrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.

ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

The tumor microenvironment represents one of the main obstacles in breast cancer treatment owing to the presence of heterogeneous stromal cells, such as adipose-derived stem cells (ADSCs), that may interact with breast cancer cells and promote cancer development. Resistin is an adipocytokine associated with adverse breast cancer progression; however, its underlying mechanisms in the context of the breast tumor microenvironment remain largely unidentified. Here, we utilized a transwell co-culture model containing patient-derived ADSCs and breast cancer cell lines to investigate their potential interaction, and observed that breast cancer cells co-cultured with resistin-treated ADSCs (R-ADSCs) showed enhanced cancer cell growth and metastatic ability. Screening by proteome arrays revealed that C-X-C motif chemokine ligand 5 (CXCL5) was released in the conditioned medium of the co-culture system, and phosphorylated ERK was increased in breast cancer cells after co-culture with R-ADSCs. Breast cancer cells treated with the recombinant proteins of CXCL5 showed similarly enhanced cell migration and invasion ability as occurred in the co-culture model, whereas application of neutralizing antibodies against CXCL5 reversed these phenomena. The orthotopic xenograft in mice by breast cancer cells after co-culture with R-ADSCs had a larger tumor growth and more CXCL5 expression than control. In addition, clinical analysis revealed a positive correlation between the expression of resistin and CXCL5 in both tumor tissues and serum specimens of breast cancer patients. The current study suggests that resistin-stimulated ADSCs may interact with breast cancer cells in the tumor microenvironment via CXCL5 secretion, leading to breast cancer cell malignancy.

miR-145-3p Hampers the Malignant Progression of Esophageal Carcinoma via CXCL5 Downregulation.

Esophageal carcinoma (EC) is the most prevalent malignant tumor that occurs frequently worldwide. The early diagnostic biomarkers are crucial for EC treatment. miRNA can regulate EC progression, with diagnostic and prognostic value. Herein, differentially expressed miRNAs and mRNAs (DEmRNAs) in EC were predicted based on TCGA database. The target mRNAs of miRNA were predicted through databases, which were then intersected with DEmRNAs. Next, the correlation between miRNA and candidate mRNAs was analyzed. qRT-PCR was introduced to analyze expression of miR-145-3p and CXCL5 mRNA in EC cell lines, and western blot was performed to assess protein expression of CXCL5. Cell proliferation, migration, invasion, and apoptosis in EC were examined through CCK-8, wound healing, Transwell invasion, and flow cytometry assays. Moreover, targeting relationship between miR-145-3p and CXCL5 was verified through luciferase reporter gene analysis. The experimental results revealed a decreased miR-145-3p expression and an increased CXCL5 expression in EC. Enforced expression of miR-145-3p hindered proliferation, migration, invasion, and stimulated apoptosis of EC cells by repressing CXCL5. This study manifested that miR-145-3p may be a tumor suppressor in EC, and miR-145-3p/CXCL5 axis restrained the malignant progression of EC. These results supply an underlying target for prognosis and treatment of EC patients.

[CXCL5 inhibits tumor immune of lung cancer via modulating PD1/PD-L1 signaling].

Objective: To investigate the role of CXCL5 in tumor immune of lung cancer and to explore the potential molecular mechanisms. Methods: A total of 62 cases of patients with lung cancer admitted in the First Affiliated Hospital of Henan University from May 2018 to December 2019 were recruited as study object. Another 20 cases of patients with pulmonary infectious diseases and 20 cases of healthy control were selected as control. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of CXCL5 in patients with lung cancer, pulmonary infectious diseases and healthy control. Immunohistochemical staining (IHC) was used to detect the expressions of CXCL5 and PD-1/PD-L1 in tumor and paracarcinoma tissues of patients with lung cancer. Pearson correlation analysis was used to evaluate the correlation between CXCL5 and PD-1 in tumor and paracarcinoma tissues of patients with lung cancer. Lewis cells either expressing CXCL5 or vector plasmids were used to establish C57BL/6J mice model of lung cancer, and all mice were then divided into vehicle and PD-1 antibody treatment groups, 10 mice for each group. The mice survival and tumor growth curves were recorded. IHC was used to evaluate the expressions of CXCL5, PD-1 as well as the proportions of CD8(+) T and Treg cells in xenograft tumor tissues. Results: In patients with lung cancer, the serum level of CXCL5 [(351.7±51.5) ng/L] was significant higher than that in patients with pulmonary infectious diseases and healthy control [(124.7±23.4) ng/L, P<0.001]. The expression levels of CXCL5 (0.136±0.034), CXCR2 (0.255±0.050), PD-1 (0.054±0.012) and PD-L1 (0.350±0.084) in tumor were significant higher than those in paracarcinoma normal tissues [(0.074±0.022), (0.112±0.023), (0.041±0.007) and (0.270±0.043) respectively, P<0.001]. CXCL5 was significant positively correlated with PD-1 in tumor tissues of lung cancer (r=0.643, P<0.001), but not correlated with PD-1 in paracarcinoma tissues(r=0.088, P=0.496). The vector control group, CXCL5 overexpression group, vector control + anti-PD-1 antibody treatment group and CXCL5 overexpression + anti-PD-1 antibody treatment group all successfully formed tumors in mice, while CXCL5 overexpression increased the tumor growth significantly (P<0.01), which was abrogated by the treatment of anti-PD-1 antibody. CXCL5 overexpression decreased the mice survival time significantly (P<0.01), this effect was also abrogated by the treatment of anti-PD-1 antibody. The proportion of CD8(+) T cells in CXCL5 overexpression group [(10.40±2.00)%] was significant lower than that in vector control group [(21.20±3.30)%, P=0.002]. The proportion of CD4(+) Foxp3(+) Treg cells in CXCL5 overexpression group [(38.40±3.70)%] was significant higher than that in vector control group [(23.30±2.25)%, P<0.001]. After the treatment of anti-PD-1 antibody, no significant difference were observed for the proportion of CD8(+) T cells [(34.10±5.00)% and (33.40±4.00)% respectively] and Treg cells [(14.70±3.50)% and (14.50±3.30)% respectively] in xenograft tumor tissues between CXCL5 overexpression+ anti-PD-1 antibody treatment group and vector control + anti-PD-1 antibody treatment group (P>0.05). Conclusion: The expressions of CXCL5 and PD-1/PD-L1 are all increased significantly in the tumor tissues of patients with lung cancer, CXCL5 may inhibit tumor immune of lung cancer via modulating PD-1/PD-L1 signaling.

KIF4A promotes tumor progression of bladder cancer via CXCL5 dependent myeloid-derived suppressor cells recruitment.

Although KIF4A has been found to play an important role in a variety of tumors and is closely associated with the activation of immunocytes, its role in bladder cancer (BC) remains unclear. Here, we report increased expression of KIF4A in both lymph node-positive and high grade BC tissues. High expression of KIF4A has been significantly correlated with fewer CD8+ tumor-infiltrating lymphocytes (TILs) and a much worse prognosis in patients with BC. With respect to promoting tumor growth, the expression of KIF4A in promoting tumor growth was more pronounced in immune-competent mice (C57BL/6) than in immunodeficient mice (BALB/C). In addition, the more increased accumulation of myeloid-derived suppressor cells (MDSCs) was observed in tumor-bearing mice with KIF4A overexpression than in the control group. Transwell chemotaxis assays revealed that KIF4A overexpression in T24 cells increased MDSC recruitment. Furthermore, according to ELISA results, CXCL5 was the most noticeably increased cytokine in the KIF4A-transduced BC cells. Additional studies in vitro and in vivo showed that the capability of KIF4A to promote BC cells to recruit MDSCs could be significantly inhibited by anti-CXCL5 antibody. Therefore, our results demonstrated that KIF4A-mediated BC production of CXCL5 led to an increase in MDSC recruitment, which contributed to tumor progression.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

EMT-mediated regulation of CXCL1/5 for resistance to anti-EGFR therapy in colorectal cancer.

The emergence of RAS/RAF mutant clone is the main feature of EGFR inhibitor resistance in KRAS wild-type colon cancer. However, its molecular mechanism is thought to be multifactorial, mainly due to cellular heterogeneity. In order to better understand the resistance mechanism in a single clone level, we successfully isolated nine cells with cetuximab-resistant (CR) clonality from in vitro system. All CR cells harbored either KRAS or BRAF mutations. Characteristically, these cells showed a higher EMT (Epithelial to mesenchymal transition) signature, showing increased EMT markers such as SNAI2. Moreover, the expression level of CXCL1/5, a secreted protein, was significantly higher in CR cells compared to the parental cells. In these CR cells, CXCL1/5 expression was coordinately regulated by SNAI2/NFKB and transactivated EGFR through CXCR/MMPI/EGF axis via autocrine singling. We also observed that combined cetuximab/MEK inhibitor not only showed growth inhibition but also reduced the secreted amounts of CXCL1/5. We further found that serum CXCL1/5 level was positively correlated with the presence of RAS/RAF mutation in colon cancer patients during cetuximab therapy, suggesting its role as a biomarker. These data indicated that the application of serum CXCL1/5 could be a potential serologic biomarker for predicting resistance to EGFR therapy in colorectal cancer.

Sox9/CXCL5 axis facilitates tumour cell growth and invasion in hepatocellular carcinoma.

High rates of metastasis and postsurgical recurrence contribute to the higher mortality of hepatocellular carcinoma (HCC), partly due to cancer stem cell (CSC)-dependent tumorigenesis and metastasis. Sex-determining region Y-box 9 (Sox9) has been previously characterized as a candidate CSC marker of HCC. Here, we observed that the increase of Sox9 significantly promoted HCC cell growth and invasion in cell cultures, whereas knockdown of Sox9 showed the opposite effects, suggesting that Sox9 may regulate the proliferation and invasion of hepatoma cells in an autocrine manner. RNA sequencing, together with functional assays and clinical analyses, identified CXCL5 as a key mediator downstream of Sox9 in HCC cells. Mechanistic studies revealed that Sox9 induced CXCL5 expression by directly binding to a promoter region. Using gain- and loss-of-function approaches, we demonstrated that the intrinsic effective role of Sox9 in hepatoma cell growth and invasion depended on CXCL5, and that blockade of CXCL5/CXCR2 signalling abolished Sox9-triggered HCC cell proliferation and migration. Furthermore, the Sox9/CXCL5 axis activated PI3K-AKT and ERK1/2 signalling which are implicated in regulating HCC cell proliferation and invasion. Finally, the Sox9/CXCL5 axis contributed to the infiltration of neutrophils and macrophages in both tumour and peritumoral tissues from the orthotopic xenograft model. In summary, our data identify the Sox9/CXCL5 axis as an endogenous factor in controlling HCC cell growth and invasion, thereby raising the possibility of pharmacologic intervention with CXCL5/CXCR2 pathway inhibitors in therapy for HCC patients with higher Sox9 expression.

Postoperative proliferative vitreoretinopathy development is linked to vitreal CXCL5 concentrations.

The specific changes linked to de novo development of postoperative PVR have remained elusive and were the object of the underlying study. Vitreous fluid (VF) was obtained at the beginning of vitrectomy from 65 eyes that underwent vitrectomy for primary rhegmatogenous retinal detachment (RRD) without preoperative PVR. Eyes developing postoperative PVR within 6 months after re-attachment surgery were compared to those which did not regarding the preoperative concentrations of 43 cytokines and chemokines in the VF, using multiplex beads analysis. For all comparisons Holm's correction was applied in order to control for multiple comparisons. Twelve out of 65 eyes (18.5%) developed PVR postoperatively. While 12 of the chemokines and cytokines presented concentration differences on a statistical level of p < 0.05 (CXCL5, CCL11, CCL24, CCL26, GM-CSF, IFN-γ, CCL8, CCL7, MIF, MIG/CXCL9, CCL19, and CCL25), CXCL5 was the only cytokine with sufficiently robust difference in its VF concentrations to achieve significance in eyes developing postoperative PVR compared to eyes without PVR. CXCL5 may represent a potent biomarker for the de novo development of postoperative PVR. In line with its pathophysiological role in the development of PVR, it might serve as a basis for the development of urgently needed preventive options.

Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection.

Respirovirus such as influenza virus infection induces pulmonary anti-viral immune response, orchestration of innate and adaptive immunity restrain viral infection, otherwise causes severe diseases such as pneumonia. Chemokines regulate leukocyte recruitment to the inflammation site. One chemokine CXCL5, plays a scavenging role to regulate pulmonary host defense against bacterial infection, but its role in pulmonary influenza virus infection is underdetermined. Here, using an influenza (H1N1) infected CXCL5-/- mouse model, we found that CXCL5 not only responds to neutrophil infiltration into infected lungs at the innate immunity stage, but also affects B lymphocyte accumulation in the lungs by regulating the expression of the B cell chemokine CXCL13. Inhibition of CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs, and in vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge. CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to an enhanced B cell immune response and facilitating induced bronchus-associated lymphoid tissue formation in the infected lungs during the late infection and recovery stages. These data highlight multiple regulatory roles of CXCL5 in leukocyte chemotaxis during pulmonary influenza infection.

Tumor Lymphatic Interactions Induce CXCR2-CXCL5 Axis and Alter Cellular Metabolism and Lymphangiogenic Pathways to Promote Cholangiocarcinoma.

Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.

CXCL5 Downregulation in Villous Tissue Is Correlated With Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) affects 5% of childbearing-age women worldwide. Inadequate trophoblast invasion is one of the main reasons for the development of RSA; however, the underlying molecular mechanisms for RSA have not been fully understood, and further explanation is urgently needed. C-X-C motif chemokine ligand 5 (CXCL5) is reported to contribute to the invasion of cancer cells, and its aberrant expression is associated with the cellular process of tumor pathology. Considering the high behavioral similarity between trophoblast cells and cancer cells, we hypothesized that CXCL5 may influence trophoblast invasion, and its expression levels in villous tissue may be correlated with RSA. In this study, we firstly investigated the CXCL5 expression in placental villous tissues of 15 RSA patients and 13 control patients, and the results showed that CXCL5 levels were significantly lower in villous tissue from RSA patients than those of the controls. Further in vitro experiments presented that recombinant human CXCL5 can enhance trophoblast migration, invasion, and epithelial-to-mesenchymal transition (EMT) process. We also demonstrated that CXCL5 exerted these effects on trophoblast cells through PI3K/AKT/ERK1/2 signaling pathway. In conclusion, these data indicate that CXCL5 downregulation in human villous tissue is correlated with RSA. Additionally, we found that estrogen, progesterone, human chorionic gonadotropin, and decidual stromal cells can regulate CXCL5 and chemokine receptor 2 (CXCR2) expression of trophoblast in a cell manner.

CXCL5 secreted from macrophages during cold exposure mediates white adipose tissue browning.

Adipose tissue affects metabolic-related diseases because it consists of various cell types involved in fat metabolism and adipokine release. CXC ligand 5 (CXCL5) is a member of the CXC chemokine family and is highly expressed by macrophages in white adipose tissue (WAT). In this study, we generated and investigated the function of CXCL5 in knockout (KO) mice using CRISPR/Cas9. The male KO mice did not show significant phenotype differences in normal conditions. However, proteomic analysis revealed that many proteins involved in fatty acid beta-oxidation and mitochondrial localization were enriched in the inguinal WAT (iWAT) of Cxcl5 KO mice. Cxcl5 KO mice also showed decreased protein and transcript expression of genes associated with thermogenesis, including uncoupling protein 1 (UCP1), a well-known thermogenic gene, and increased expression of genes associated with inflammation. The increase in UCP1 expression in cold conditions was significantly retarded in Cxcl5 KO mice. Finally, we found that CXCL5 treatment increased the expression of transcription factors that mediate Ucp1 expression and Ucp1 itself. Collectively, our data show that Ucp1 expression is induced in adipocytes by CXCL5, which is secreted upon ß-adrenergic stimulation by cold stimulation in M1 macrophages. Our data indicate that CXCL5 plays a crucial role in regulating energy metabolism, particularly upon cold exposure. These results strongly suggest that targeting CXCL5 could be a potential therapeutic strategy for people suffering from disorders affecting energy metabolism.