Inhibition on CXCL5 reduces aortic matrix metalloproteinase 9 expression and protects against acute aortic dissection.

Acute aortic dissection (AAD) is an acute inflammatory vascular condition associated with significant morbidity and mortality. Depletion of neutrophils can attenuate the development of AAD. The CXC-motif chemokine 5 (CXCL5) can attract and activate neutrophils. This study aimed to investigate whether direct inhibition of CXCL5 could protect against AAD formation. A set of AAD animal models was designed using an angiotensin II infusion for 3 days after treatment with the lysyl oxidase inhibitor beta-aminopropionitrile for 4 weeks in 4-week-old male BALB/c mice. While AAD developed successfully in all the animals, approximately 31% of the mice died before sacrifice. The morphological changes at different time points during the experimental period indicated that angiotensin II could trigger AAD formation in this model. CXCL5 protein expression in the aorta tissue was increased after treatment with angiotensin II. Moreover, the ex vivo and in vitro study showed that vascular smooth muscle cells and monocytes isolated from the animals could generate CXCL5. CXCL5 inhibition by a specific monoclonal antibody significantly decreased the severity of AAD evaluated by ultrasound, aorta wet weight, and en face assay. The immunohistochemical analysis showed that the aortic tissues from AAD mice had higher expressions of matrix metalloproteinase (MMP) 9 and neutrophil-positive areas in the medial layer compared to control mice. Treatment with a CXCL5 antibody reduced MMP9 and neutrophil expressions as well as neutrophil and CXCL5 double-positive areas compared to untreated AAD mice. In conclusion, direct inhibition on CXCL5 reduced aortic MMP9 expression as well as neutrophil infiltration and attenuated the development of AAD, suggesting the mechanistic role of CXCL5 in neutrophil-triggered AAD. CXCL5 may be a potential therapeutic target for AAD.

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats.

BACKGROUND: Prenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure. METHODS: Pregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro. RESULTS: Both male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment. CONCLUSION: CRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.

CXCL5/CXCR2 axis in tumor microenvironment as potential diagnostic biomarker and therapeutic target.

The components of the tumor microenvironment (TME) in solid tumors, especially chemokines, are currently attracting much attention from scientists. C-X-C motif chemokine ligand 5 (CXCL5) is one of the important chemokines in TME. Overexpression of CXCL5 is closely related to the survival time, recurrence and metastasis of cancer patients. In TME, CXCL5 binds to its receptors, such as C-X-C motif chemokine receptor 2 (CXCR2), to participate in the recruitment of immune cells and promote angiogenesis, tumor growth, and metastasis. The CXCL5/CXCR2 axis can act as a bridge between tumor cells and host cells in TME. Blocking the transmission of CXCL5/CXCR2 signals can increase the sensitivity and effectiveness of immunotherapy and slow down tumor progression. CXCL5 and CXCR2 are also regarded as biomarkers for predicting prognosis and molecular targets for customizing the treatment. In this review, we summarized the current literature regarding the biological functions and clinical significance of CXCL5/CXCR2 axis in TME. The possibility to use CXCL5 and CXCR2 as potential prognostic biomarkers and therapeutic targets in cancer is also discussed.

The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis.

Bone is one of the most common sites for metastasis across cancers. Cancer cells that travel through the vasculature and invade new tissues can remain in a non-proliferative dormant state for years before colonizing the metastatic site. Switching from dormancy to colonization is the rate-limiting step of bone metastasis. Here we develop an ex vivo co-culture method to grow cancer cells in mouse bones to assess cancer cell proliferation using healthy or cancer-primed bones. Profiling soluble factors from conditioned media identifies the chemokine CXCL5 as a candidate to induce metastatic colonization. Additional studies using CXCL5 recombinant protein suggest that CXCL5 is sufficient to promote breast cancer cell proliferation and colonization in bone, while inhibition of its receptor CXCR2 with an antagonist blocks proliferation of metastatic cancer cells. This study suggests that CXCL5 and CXCR2 inhibitors may have efficacy in treating metastatic bone tumors dependent on the CXCL5/CXCR2 axis.

Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common inflammatory autoimmune disease, characterized by pathological osteogenesis. Mesenchymal stem cells (MSCs), as the main source of osteoblasts, participate in bone remodeling not only through differentiation into osteoblasts but also through indirect regulation of osteoclastogenesis. Our previous study indicated that the stronger osteogenic differentiation of MSCs from AS patients (ASMSCs) involved in pathological osteogenesis. However, whether there is any abnormality in the regulation of osteoclastogenesis by ASMSCs remains unclear. In this study, ASMSCs or MSCs from healthy donors (HDMSCs) were co-cultured with CD14 + monocytes in osteoclast induction medium. Our results demonstrated that ASMSCs exhibited a stronger capacity to inhibit osteoclastogenesis than HDMSCs. To explore underlying mechanisms, cytokine array assays were performed, showing that ASMSCs secreted more CXCL5 than HDMSCs, which was confirmed by enzyme-linked immunosorbent assays. Moreover, inhibition of osteoclastogenesis by ASMSCs was recovered by decreasing CXCL5. Besides, the inhibitory effect of CXCL5 on osteoclastogenesis was confirmed by exogenous addition. Bioinformatics analysis was applied to find the interaction between miR-4284 and CXCL5, which was verified by luciferase reporter assays. Furthermore, we used miR-4284 inhibitors or mimics to prove that the expression of CXCL5 was regulated by miR-4284. Further analysis showed that downregulation of miR-4284 in MSCs resulted in increase of CXCL5, markedly inhibiting osteoclastogenesis, whereas upregulation of miR-4284 in MSCs had the opposite effect. Our findings indicate that ASMSCs exhibit a stronger capacity to inhibit osteoclastogenesis than HDMSCs through the miR-4284/CXCL5 axis, which provide a new perspective on the mechanism of pathologic osteogenesis in AS.

ONZIN Upregulation by Mutant p53 Contributes to Osteosarcoma Metastasis Through the CXCL5-MAPK Signaling Pathway.

BACKGROUND/AIMS: Gain-of-function of mutant p53 is associated with a high rate of lung metastasis in osteosarcoma. To investigate the mechanism of mutant p53-induced osteosarcoma metastasis, expression array analysis was performed, comparing non-metastatic osteosarcomas from p53+/- mice with metastatic osteosarcomas from p53R172H/+ mice. Onzin (Plac8) was identified as one of the genes upregulated in p53R172H/+ mouse metastatic osteosarcomas. Accordingly, we investigated the role of ONZIN in human osteosarcoma metastasis. METHODS: ONZIN function and its downstream targets were examined in osteosarcoma cell lines. Assays related to tumorigenesis and metastasis, including cell migration, invasion, clonogenic survival, and soft agar colony formation, were performed in osteosarcoma cells. Additionally, mouse xenograft models were used to examine the role of ONZIN overpression in tumorigenesis in vivo. Lastly, 87 osteosarcoma patients were recruited to investigate the clinical relevance of ONZIN overexpression in metastasis and prognosis. RESULTS: ONZIN overexpression enhanced osteosarcoma cell proliferation, clonogenic survival, migration, and invasion independent of p53 status. Furthermore, ONZIN overexpression induced CXCL5 upregulation and resulted in increased ERK phosphorylation, which contributed to more aggressive osteosarcoma metastatic phenotypes. More importantly, overexpression of ONZIN in human osteosarcoma patients was closely associated with lung metastasis, poor prognoses, and survival. CONCLUSIONS: Overexpression of ONZIN promotes osteosarcoma progression and metastasis, and can serve as a clinical biomarker for osteosarcoma metastasis and prognosis.

Spinal CXCL5 contributes to nerve injury-induced neuropathic pain via modulating GSK-3ß phosphorylation and activity in rats.

BACKGROUND: Neuroinflammation is identified to be crucial in the development of neuropathic pain, whereas definite molecular mechanisms remain obscure. Recently, chemokine CXCL5 is manifested to participate in the inflammatory process of central nervous system, however, little is known about the potential effect of spinal CXCL5 on pathogenesis of pain. This study investigated whether and how CXCL5 and its receptor CXCR2 regulated neuropathic pain in a rat model of chronic constriction injury (CCI) of the sciatic nerves. METHODS: Recombinant CXCL5, a neutralizing antibody against CXCL5, selective CXCR2 antagonist SB225002 and GSK-3ß inhibitor TDZD-8 were injected intrathecally. PWT and PWL were documented to assess mechanical allodynia and thermal hyperalgesia. Simultaneously, levels of CXCL5 and CXCR2 in spinal dorsal horn were measured by RT-qPCR after nociceptive testing. Western blot was utilized to evaluate spinal GSK-3ß expression and phosphorylation. RESULTS: We found that CCI engendered rapid and long-lasting mechanical allodynia and thermal hyperalgesia, which was accompanied by dramatical rise of spinal CXCL5 and CXCR2 expression. CCI also caused an increase of pGSK-3ß (Tyr216) and a decrease of pGSK-3ß (Ser9) without affecting total protein level of GSK-3ß. Moreover, spinal blockage of CXCL5/CXCR2 pathway attenuated neuropathic pain and inhibited the enhancement of GSK-3ß activity. Also, intrathecal delivery of exogenous CXCL5 dose-dependently induced nociceptive hypersensitivity in naïve rats, which was prevented by the supplemental addition of TDZD-8. CONCLUSION: These present findings indicate that up-regulation of spinal CXCL5 and CXCR2 is involved in neuropathic pain after nerve injury, through regulating GSK-3ß activity in rats.

DACH1 inhibits lung adenocarcinoma invasion and tumor growth by repressing CXCL5 signaling.

Whole-genome and transcriptome sequencing of non-small cell lung cancer (NSCLC) identified that DACH1, is a human homolog of drosophila gene dac, is involved in NSCLC. Here we showed that expression of DACH1 was significantly decreased in human NSCLC tissues and DACH1 abundance was inversely correlated with tumor stages and grades. Restoration of DACH1 expression in NSCLC cells significantly reduced cellular proliferation, clone formation, migration and invasion in vitro, as well as tumor growth in vivo. Unbiased screen and functional study suggested that DACH1 mediated effects were dependent in part on suppression of CXCL5. There was an inverse correlation between DACH1 mRNA levels and CXCL5 in both lung cancer cell lines and human NSCLC tissues. Kaplan-Mier analysis of human NSCLC samples demonstrated that high DACH1 mRNA levels predicted favorable prognosis for relapse-free and overall survival. In agreement, high CXCL5 expression predicted a worse prognosis for survival.

CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration.

CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.

Anti-CXCL5 therapy ameliorates IL-17-induced arthritis by decreasing joint vascularization.

IL-17-induced joint inflammation is associated with increased angiogenesis. However, the mechanism by which IL-17 mediates angiogenesis is undefined. Therefore, the pathologic role of CXCL1 and CXCL5 was investigated in arthritis mediated by local expression of IL-17, employing a neutralizing antibody to each chemokine. Next, endothelial chemotaxis was utilized to examine whether endothelial migration was differentially mediated by CXCL1 and CXCL5. Our results demonstrate that IL-17-mediated disease activity was not affected by anti-CXCL1 treatment alone. In contrast, mice receiving anti-CXCL5 demonstrated significantly reduced clinical signs of arthritis, compared to the mice treated with IgG control. Consistently, while inflammation, synovial lining thickness, bone erosion and vascularization were markedly reduced in both the anti-CXCL5 and combination anti-CXCL1 and 5 treatment groups, mice receiving anti-CXCL1 antibody had clinical scores similar to the control group. In contrast to joint FGF2 and VEGF levels, TNF-α was significantly reduced in mice receiving anti-CXCL5 or combination of anti-CXCL1 and 5 therapies compared to the control group. We found that, like IL-17, CXCL1-induced endothelial migration is mediated through activation of PI3K. In contrast, activation of NF-κB pathway was essential for endothelial chemotaxis induced by CXCL5. Although CXCL1 and CXCL5 can differentially mediate endothelial trafficking, blockade of CXCR2 can inhibit endothelial chemotaxis mediated by either of these chemokines. These results suggest that blockade of CXCL5 can modulate IL-17-induced inflammation in part by reducing joint blood vessel formation through a non-overlapping IL-17 mechanism.