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Neuropathic pain resulting from damage or dysfunction of the nervous system is a highly debilitating chronic pain state and is often resistant to currently available treatments. It has become clear that neuroinflammation, mainly mediated by proinflammatory cytokines and chemokines, plays an important role in the establishment and maintenance of neuropathic pain. Chemokines were originally identified as regulators of peripheral immune cell trafficking and were also expressed in neurons and glial cells in the central nervous system. In recent years, accumulating studies have revealed the expression, distribution and function of chemokines in the spinal cord under chronic pain conditions. In this review, we provide evidence showing that several chemokines are upregulated after peripheral nerve injury and contribute to the pathogenesis of neuropathic pain via different forms of neuron–glia interaction in the spinal cord. First, chemokine CX3CL1 is expressed in primary afferents and spinal neurons and induces microglial activation via its microglial receptor CX3CR1 (neuron-to-microglia signaling). Second, CCL2 and CXCL1 are expressed in spinal astrocytes and act on CCR2 and CXCR2 in spinal neurons to increase excitatory synaptic transmission (astrocyte-to-neuron signaling). Third, we recently identified that CXCL13 is highly upregulated in spinal neurons after spinal nerve ligation and induces spinal astrocyte activation via receptor CXCR5 (neuron-to-astrocyte signaling). Strategies that target chemokine-mediated neuron-glia interactions may lead to novel therapies for the treatment of neuropathic pain.

ObjectiveThe role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME.DesignClinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. YTHDF1 function was determined in syngeneic tumours, intestine-specific Ythdf1 knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated YTHDF1-siRNA was used for YTHDF1 silencing in vivo.Results YTHDF1 expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8+ T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of Ythdf1 augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while Ythdf1 knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or ApcMin/+ models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in Ythdf1 knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8+ T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-siYTHDF1 boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC.ConclusionYTHDF1 impairs antitumour immunity via an m6A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.

ObjectiveHepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy.DesignNeutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry.ResultsNeutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs.ConclusionCXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

ObjectiveOur previous studies have identified CXCL8 as the crucial chemokine responsible for gastric cancer metastasis mediated by loss of RACK1. However, the regulatory effect of CXCL8 on immune surveillance in gastric cancer remains obscure.DesignFlow cytometry analyses were performed to examine major source of CXCL8 and phenotypes of immune cells in fresh tumour tissues from 76 patients with gastric cancer. Real-time PCR was performed to analyse CXCL8 mRNA level in gastric cancer tissues. For immunohistochemical analyses, a total of 420 patients with gastric cancer undergoing curative resection were enrolled. In vitro culture of fresh tumour tissue was performed to evaluate the potential therapeutic effect of blocking CXCL8 pathway in gastric cancer.ResultsIncreased level of CXCL8 indicates poor clinical outcome and tumour progression in patients with gastric cancer. In gastric cancer tissues, CXCL8 is predominantly secreted by macrophages and colony stimulating factor 2 (CSF-2) facilitates macrophage-derived CXCL8 secretion. High level of CXCL8 is associated with decreased CD8+ T cells infiltration and Ki67+ CD8+ T cells proportion. Moreover, CXCL8 also inhibits CD8+ T cells function by inducing the expression of PD-L1 on macrophages. Finally, we show that a small-molecule CXCR2 inhibitor, reparixin, drives the decreased programmed death-ligand 1 (PD-L1+) macrophages and promotes antitumour immunity. Accordingly, high levels of CXCL8+ macrophages are positively correlated with poor prognosis in patients with gastric cancer.ConclusionsCXCL8 is predominantly secreted by macrophages and contributes to the immunosuppressive microenvironment by inducing PD-L1+ macrophages in gastric cancer. CXCL8 inhibitors may drive antitumour response, providing potential therapeutic effects for patients with gastric cancer.

ObjectiveInvestigating the effect of ferroptosis in the tumour microenvironment to identify combinatory therapy for liver cancer treatment.DesignGlutathione peroxidase 4 (GPx4), which is considered the master regulator of ferroptosis, was genetically altered in murine models for hepatocellular carcinoma (HCC) and colorectal cancer (CRC) to analyse the effect of ferroptosis on tumour cells and the immune tumour microenvironment. The findings served as foundation for the identification of additional targets for combine therapy with ferroptotic inducer in the treatment of HCC and liver metastasis.ResultsSurprisingly, hepatocyte-restricted GPx4 loss does not suppress hepatocellular tumourigenesis. Instead, GPx4-associated ferroptotic hepatocyte death causes a tumour suppressive immune response characterised by a CXCL10-dependent infiltration of cytotoxic CD8+ T cells that is counterbalanced by PD-L1 upregulation on tumour cells as well as by a marked HMGB1-mediated myeloid derived suppressor cell (MDSC) infiltration. Blocking PD-1 or HMGB1 unleashes T cell activation and prolongs survival of mice with Gpx4-deficient liver tumours. A triple combination of the ferroptosis inducing natural compound withaferin A, the CXCR2 inhibitor SB225002 and α-PD-1 greatly improves survival of wild-type mice with liver tumours. In contrast, the same combination does not affect tumour growth of subcutaneously grown CRC organoids, while it decreases their metastatic growth in liver.ConclusionOur data highlight a context-specific ferroptosis-induced immune response that could be therapeutically exploited for the treatment of primary liver tumours and liver metastases.

Background Drug-resistance and severe side effects of chemotherapeutic agents result in unsatisfied survival of patients with lung cancer. CXCLs/CXCR2 axis plays an important role in progression of cancer including lung cancer. However, the specific anti-cancer mechanism of targeting CXCR2 remains unclear. Methods Immunohistochemical analysis of CXCR2 was performed on the microarray of tumor tissues of clinical lung adenocarcinoma and lung squamous cell carcinoma patients. CCK8 test, TUNEL immunofluorescence staining, PI-Annexin V staining, β-galactosidase staining, and Western blot were used to verify the role of CXCR2 in vitro. Animal models of tail vein and subcutaneous injection were applied to investigate the therapeutic role of targeting CXCR2. Flow cytometry, qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry analysis were performed for further mechanistic investigation. Results The expression of CXCR2 was elevated in both human lung cancer stroma and tumor cells, which was associated with patients’ prognosis. Inhibition of CXCR2 promoted apoptosis, senescence, epithelial-to-mesenchymal transition (EMT), and anti-proliferation of lung cancer cells. In vivo study showed that tumor-associated neutrophils (TANs) were significantly infiltrate into tumor tissues of mouse model, with up-regulated CXCLs/CXCR2 signaling and suppressive molecules, including Arg-1 and TGF-β. SB225002, a selective inhibitor of CXCR2 showed promising therapeutic effect, and significantly reduced infiltration of neutrophils and enhanced anti-tumor T cell activity via promoting CD8 + T cell activation. Meanwhile, blockade of CXCR2 could enhance therapeutic effect of cisplatin via regulation of neutrophils infiltration. Conclusions Our finds verify the therapeutic effects of targeting CXCR2 in lung cancer and uncover the potential mechanism for the increased sensitivity to chemotherapeutic agents by antagonists of CXCR2.

Background Major depressive disorder (MDD) is a highly prevalent psychiatric disorder, and inflammation has been considered crucial components of the pathogenesis of depression. NLRP1 inflammasome-driven inflammatory response is believed to participate in many neurological disorders. However, it is unclear whether NLRP1 inflammasome is implicated in the development of depression. Methods Animal models of depression were established by four different chronic stress stimuli including chronic unpredictable mild stress (CUMS), chronic restrain stress (CRS), chronic social defeat stress (CSDS), and repeat social defeat stress (RSDS). Depressive-like behaviors were determined by sucrose preference test (SPT), forced swim test (FST), tail-suspension test (TST), open-field test (OFT), social interaction test (SIT), and light-dark test (LDT). The expression of NLRP1 inflammasome complexes, BDNF, and CXCL1/CXCR2 were tested by western blot and quantitative real-time PCR. The levels of inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. Results Chronic stress stimuli activated hippocampal NLRP1 inflammasome and promoted the release of pro-inflammatory cytokines IL-1β, IL-18, IL-6, and TNF-α in mice. Hippocampal Nlrp1a knockdown prevented NLRP1 inflammasome-driven inflammatory response and ameliorated stress-induced depressive-like behaviors. Also, chronic stress stimuli caused the increase in hippocampal CXCL1/CXCR2 expression and low BDNF levels in mice. Interestingly, Nlrp1a knockdown inhibited the up-regulation of CXCL1/CXCR2 expression and restored BDNF levels in the hippocampus. Conclusions NLRP1 inflammasome-driven inflammatory response contributes to chronic stress induced depressive-like behaviors and the mechanism may be related to CXCL1/CXCR2/BDNF signaling pathway. Thus, NLRP1 inflammasome could become a potential antidepressant target.

Chemokines are an indispensable component of our immune system through the regulation of directional migration and activation of leukocytes. CXCL8 is the most potent human neutrophil-attracting chemokine and plays crucial roles in the response to infection and tissue injury. CXCL8 activity inherently depends on interaction with the human CXC chemokine receptors CXCR1 and CXCR2, the atypical chemokine receptor ACKR1, and glycosaminoglycans. Furthermore, (hetero)dimerization and tight regulation of transcription and translation, as well as post-translational modifications further fine-tune the spatial and temporal activity of CXCL8 in the context of inflammatory diseases and cancer. The CXCL8 interaction with receptors and glycosaminoglycans is therefore a promising target for therapy, as illustrated by multiple ongoing clinical trials. CXCL8-mediated neutrophil mobilization to blood is directly opposed by CXCL12, which retains leukocytes in bone marrow. CXCL12 is primarily a homeostatic chemokine that induces migration and activation of hematopoietic progenitor cells, endothelial cells, and several leukocytes through interaction with CXCR4, ACKR1, and ACKR3. Thereby, it is an essential player in the regulation of embryogenesis, hematopoiesis, and angiogenesis. However, CXCL12 can also exert inflammatory functions, as illustrated by its pivotal role in a growing list of pathologies and its synergy with CXCL8 and other chemokines to induce leukocyte chemotaxis. Here, we review the plethora of information on the CXCL8 structure, interaction with receptors and glycosaminoglycans, different levels of activity regulation, role in homeostasis and disease, and therapeutic prospects. Finally, we discuss recent research on CXCL12 biochemistry and biology and its role in pathology and pharmacology.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and to provide a new paradigm for PDAC therapy.DesignTwo unselected cohorts of 154 and 20 patients with PDAC were subjected to correlation between interleukin (IL)-33 and CXCL3 levels and survivals. Unbiased expression profiling, and genetic and pharmacological gain-of-function and loss-of-function approaches were employed to identify molecular signalling in tumour-associated macrophages (TAMs) and myofibroblastic cancer-associated fibroblasts (myoCAFs). The role of the IL-33–ST2–CXCL3–CXCR2 axis in PDAC metastasis was evaluated in three clinically relevant mouse PDAC models.ResultsIL-33 was specifically elevated in human PDACs and positively correlated with tumour inflammation in human patients with PDAC. CXCL3 was highly upregulated in IL-33-stimulated macrophages that were the primary source of CXCL3. CXCL3 was correlated with poor survival in human patients with PDAC. Mechanistically, activation of the IL-33–ST2–MYC pathway attributed to high CXCL3 production. The highest level of CXCL3 was found in PDAC relative to other cancer types and its receptor CXCR2 was almost exclusively expressed in CAFs. Activation of CXCR2 by CXCL3 induced a CAF-to-myoCAF transition and α-smooth muscle actin (α-SMA) was uniquely upregulated by the CXCL3–CXCR2 signalling. Type III collagen was identified as the CXCL3–CXCR2-targeted adhesive molecule responsible for myoCAF-driven PDAC metastasis.ConclusionsOur work provides novel mechanistic insights into understanding PDAC metastasis by the TAM-CAF interaction and targeting each of these signalling components would provide an attractive and new paradigm for treating pancreatic cancer.