GSDMD contributes to host defence against Staphylococcus aureus skin infection by suppressing the Cxcl1-Cxcr2 axis.

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1ß 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-1ß production, the critical role of IL-1ß was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1ß 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.

Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms.

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Macrophage migration inhibitory factor inhibits neutrophil apoptosis by inducing cytokine release from mononuclear cells.

The chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) is a pivotal driver of acute and chronic inflammatory conditions, cardiovascular disease, autoimmunity, and cancer. MIF modulates the early inflammatory response through various mechanisms, including regulation of neutrophil recruitment and fate, but the mechanisms and the role of the more recently described MIF homolog MIF-2 (D-dopachrome tautomerase; D-DT) are incompletely understood. Here, we show that both MIF and MIF-2/D-DT inhibit neutrophil apoptosis. This is not a direct effect, but involves the activation of mononuclear cells, which secrete CXCL8 and other prosurvival mediators to promote neutrophil survival. Individually, CXCL8 and MIF (or MIF-2) did not significantly inhibit neutrophil apoptosis, but in combination they elicited a synergistic response, promoting neutrophil survival even in the absence of mononuclear cells. The use of receptor-specific inhibitors provided evidence for a causal role of the noncognate MIF receptor CXCR2 expressed on both monocytes and neutrophils in MIF-mediated neutrophil survival. We suggest that the ability to inhibit neutrophil apoptosis contributes to the proinflammatory role ascribed to MIF, and propose that blocking the interaction between MIF and CXCR2 could be an important anti-inflammatory strategy in the early inflammatory response.

C-X-C chemokine receptor 2 (Cxcr2) promotes hepatocellular carcinoma immune evasion via regulating programmed death-ligand 1 (PD-L1).

Strategies to sensitize hepatocellular carcinomas (HCC) to programmed death-1 (PD1)/programmed death-ligand 1 (PD-L1) inhibitor therapies are important in improving the survival of HCC patients. The aim of the study was to characterize C-X-C chemokine receptor 2 (Cxcr2) as a therapeutic target in HCC and evaluate the effects of Cxcr2 suppression in sensitizing HCC to PD1/PD-L1 inhibitor therapies. To this end, we constructed a Cxcr2-knockout HCC cell line (Hepa1-6 KO) using the CRISPR-Cas9 approach and assessed the tumor growth rate and survival of mice after subcutaneously inoculating Hepa1-6 KO cells in mice. We show that Cxcr2 knockdown does not dramatically inhibit tumor growth and improve mouse survival. In tumor xenografts, the proportion of T cells is not affected but the ratio of M1/M2 macrophage is greatly increased. Cxcr2 knockdown does not alter cell viability but macrophages co-cultured with Hepa1-6 KO cells are shifted to M1 phenotypes compared to WT cells. Hepa-1-6 KO cells exhibit lower levels of PD-L1 expression. c-Myc is suppressed in Hepa1-6 KO cells, which contributes to PD-L1 downregulation. Knockdown of Cxcr2 decreases PD-L1 levels and consequently promotes the shift of macrophages to the M1 phenotype, which is mediated by downregulating c-Myc. In summary, Cxcr2 is a potential target for suppressing immune escape in HCC.

Host Cxcr2-Dependent Regulation of Pancreatic Cancer Growth, Angiogenesis, and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) manifests aggressive tumor growth and early metastasis. Crucial steps in tumor growth and metastasis are survival, angiogenesis, invasion, and immunosuppression. Our prior research showed that chemokine CXC- receptor-2 (CXCR2) is expressed on endothelial cells, innate immune cells, and fibroblasts, and regulates angiogenesis and immune responses. Here, we examined whether tumor angiogenesis, growth, and metastasis of CXCR2 ligands expressing PDAC cells are regulated in vivo by a host CXCR2-dependent mechanism. C57BL6 Cxcr2-/- mice were generated following crosses between Cxcr2-/+ female and Cxcr2-/- male. Cxcr2 ligands expressing Kirsten rat sarcoma (KRAS-PDAC) cells were orthotopically implanted in the pancreas of wild-type or Cxcr2-/- C57BL6 mice. No significant difference in PDAC tumor growth was observed. Host Cxcr2 loss led to an inhibition in microvessel density in PDAC tumors. Interestingly, an enhanced spontaneous and experimental liver metastasis was observed in Cxcr2-/- mice compared with wild-type mice. Increased metastasis in Cxcr2-/- mice was associated with an increase in extramedullary hematopoiesis and expansion of neutrophils and immature myeloid precursor cells in the spleen of tumor-bearing mice. These data suggest a dynamic role of host CXCR2 axis in regulating tumor immune suppression, tumor growth, and metastasis.

Deep phenotyping of synovial molecular signatures by integrative systems analysis in rheumatoid arthritis.

OBJECTIVE: RA encompasses a complex, heterogeneous and dynamic group of diseases arising from molecular and cellular perturbations of synovial tissues. The aim of this study was to decipher this complexity using an integrative systems approach and provide novel insights for designing stratified treatments. METHODS: An RNA sequencing dataset of synovial tissues from 152 RA patients and 28 normal controls was imported and subjected to filtration of differentially expressed genes, functional enrichment and network analysis, non-negative matrix factorization, and key driver analysis. A naïve Bayes classifier was applied to the independent datasets to investigate the factors associated with treatment outcome. RESULTS: A matrix of 1241 upregulated differentially expressed genes from RA samples was classified into three subtypes (C1-C3) with distinct molecular and cellular signatures. C3 with prominent immune cells and proinflammatory signatures had a stronger association with the presence of ACPA and showed a better therapeutic response than C1 and C2, which were enriched with neutrophil and fibroblast signatures, respectively. C2 was more occupied by synovial fibroblasts of destructive phenotype and carried highly expressed key effector molecules of invasion and osteoclastogenesis. CXCR2, JAK3, FYN and LYN were identified as key driver genes in C1 and C3. HDAC, JUN, NFKB1, TNF and TP53 were key regulators modulating fibroblast aggressiveness in C2. CONCLUSIONS: Deep phenotyping of synovial heterogeneity captured comprehensive and discrete pathophysiological attributes of RA regarding clinical features and treatment response. This result could serve as a template for future studies to design stratified approaches for RA patients.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

CXCR2-modified CAR-T cells have enhanced trafficking ability that improves treatment of hepatocellular carcinoma.

Unlike hematological malignancies, solid tumors have proved to be less susceptible to chimeric antigen receptor (CAR)-T cell therapy, which is partially caused by reduced accumulation of therapeutic T cells in tumor site. Since efficient trafficking is the precondition and pivotal step for infused CAR-T cells to exhibit their anti-tumor function, strategies are highly needed to improve the trafficking ability of CAR-T cells for solid tumor treatment. Here, based on natural lymphocyte chemotaxis theory and characteristics of solid tumor microenvironments, we explored the possibility of enhancing CAR-T cell trafficking by using chemokine receptors. Our study found that compared with other chemokines, several CXCR2 ligands showed relatively high expression level in human hepatocellular carcinoma tumor tissues and cell lines. However, both human peripheral T cells and hepatocellular carcinoma tumor infiltrating T cells lacked expression of CXCR2. CXCR2-expressing CAR-T cells exhibited identical cytotoxicity but displayed significantly increased migration ability in vitro. In a xenograft tumor model, we found that expressing CXCR2 in CAR-T cells could significantly accelerate in vivo trafficking and tumor-specific accumulation, and improve anti-tumor effect of these cells.

Targeting CXCR1/2: The medicinal potential as cancer immunotherapy agents, antagonists research highlights and challenges ahead.

Immune suppression in the tumor microenvironment (TME) is an intractable issue in anti-cancer immunotherapy. The chemokine receptors CXCR1 and CXCR2 recruit immune suppressive cells such as the myeloid derived suppressor cells (MDSCs) to the TME. Therefore, CXCR1/2 antagonists have aroused pharmaceutical interest in recent years. In this review, the medicinal chemistry of CXCR1/2 antagonists and their relevance in cancer immunotherapy have been summarized. The development of the drug candidates, along with their design rationale, clinical status and current challenges have also been discussed.

AhR Activation Leads to Massive Mobilization of Myeloid-Derived Suppressor Cells with Immunosuppressive Activity through Regulation of CXCR2 and MicroRNA miR-150-5p and miR-543-3p That Target Anti-Inflammatory Genes.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, is a potent ligand for aryl hydrocarbon receptor (AhR). In the current study, we made an exciting observation that naive C57BL/6 mice that were exposed i.p. to TCDD showed massive mobilization of myeloid-derived suppressor cells (MDSCs) in the peritoneal cavity. These MDSCs were highly immunosuppressive and attenuated Con A-induced hepatitis upon adoptive transfer. TCDD administration in naive mice also led to induction of several chemokines and cytokines in the peritoneal cavity and serum (CCL2, CCL3, CCL4, CCL11, CXCL1, CXCL2, CXCL5, CXCL9, G-CSF, GM-CSF, VEGF, and M-CSF) and chemokine receptors on MDSCs (CCR1, CCR5, and CXCR2). Treatment with CXCR2 or AhR antagonist in mice led to marked reduction in TCDD-induced MDSCs. TCDD-induced MDSCs had high mitochondrial respiration and glycolytic rate and exhibited differential microRNA (miRNA) expression profile. Specifically, there was significant downregulation of miR-150-5p and miR-543-3p. These two miRNAs targeted and enhanced anti-inflammatory and MDSC-regulatory genes, including IL-10, PIM1, ARG2, STAT3, CCL11 and its receptors CCR3 and CCR5 as well as CXCR2. The role of miRs in MDSC activation was confirmed by transfection studies. Together, the current study demonstrates that activation of AhR in naive mice triggers robust mobilization of MDSCs through induction of chemokines and their receptors and MDSC activation through regulation of miRNA expression. AhR ligands include diverse compounds from environmental toxicants, such as TCDD, that are carcinogenic to dietary indoles that are anti-inflammatory. Our studies provide new insights on how such ligands may regulate health and disease through induction of MDSCs.

Distinct Spatio-Temporal Dynamics of Tumor-Associated Neutrophils in Small Tumor Lesions.

Across a majority of cancer types tumor-associated neutrophils (TAN) are linked with poor prognosis. However, the underlying mechanisms, especially the intratumoral behavior of TAN, are largely unknown. Using intravital multiphoton imaging on a mouse model with neutrophil-specific fluorescence, we measured the migration of TAN in distinct compartments of solid tumor cell lesions in vivo. By longitudinally quantifying the infiltration and persistence of TAN into growing tumors in the same animals, we observed cells that either populated the peripheral stromal zone of the tumor (peritumoral TAN) or infiltrated into the tumor core (intratumoral TAN). Intratumoral TAN showed prolonged tumor-associated persistence and reduced motility compared to peritumoral TAN, whose velocity increased with tumor progression. Selective pharmacological blockade of CXCR2 receptors using AZD5069 profoundly inhibited recruitment of TAN into peritumoral regions, while intratumoral infiltration was only transiently attenuated and rebounded at later time points. Our findings unravel distinct spatial dynamics of TAN that are partially and differentially regulated via the CXCR2 signaling pathway.

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein.

Membrane antigens (mAgs) are important targets for the development of antibody (Ab) drugs. However, native mAgs are not easily prepared, causing difficulties in acquiring functional Abs. In this study, we present a platform in which human mAgs were expressed in native form on cell adjuvants made with membrane-bound cytokines that were then used immunize syngeneic mice directly. The membrane-bound cytokines were used as immune stimulators to enhance specific Ab responses against the desired mAgs. Then, mAgs-expressing xenogeneic cells were used for Ab characterization to reduce non-specific binding. We established cell adjuvants by expressing membrane-bound cytokines (mIL-2, mIL-18, or mGM-CSF) on BALB/3T3 cells, which were effective in stimulating splenocyte proliferation in vitro. We then transiently expressed ecotropic viral integration site 2B (EVI2B) on the adjuvants and used them to directly immunize BALB/c mice. We found that 3T3/mGM-CSF cells stimulated higher specific anti-EVI2B Ab response in the immunized mice than the other cell adjuvants. A G-protein coupled receptor (GPCR), CXCR2, was then transiently expressed on 3T3/mGM-CSF cell adjuvant to immunize mice. The immune serum exhibited relatively higher binding to xenogeneic 293 A/CXCR2 cells than 293 A cells (~3.5-fold). Several hybridoma clones also exhibited selective binding to 293 A/CXCR2 cells. Therefore, the cell adjuvant could preserve the native conformation of mAgs and exhibit anti-mAg Ab stimulatory ability, providing a more convenient and effective method to generate functional Abs, thus possibly accelerating Ab drug development.

The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo.

Human cytomegalovirus (HCMV) is a betaherpesvirus that is a significant pathogen within newborn and immunocompromised populations. Morbidity associated with HCMV infection is the consequence of viral dissemination. HCMV has evolved to manipulate the host immune system to enhance viral dissemination and ensure long-term survival within the host. The immunomodulatory protein vCXCL-1, a viral chemokine functioning primarily through the CXCR2 chemokine receptor, is hypothesized to attract CXCR2+ neutrophils to infection sites, aiding viral dissemination. Neutrophils harbor HCMV in vivo; however, the interaction between vCXCL-1 and the neutrophil has not been evaluated in vivo Using the mouse model and mouse cytomegalovirus (MCMV) infection, we show that murine neutrophils harbor and transfer infectious MCMV and that virus replication initiates within this cell type. Utilizing recombinant MCMVs expressing vCXCL-1 from the HCMV strain (Toledo), we demonstrated that vCXCL-1 significantly enhances MCMV dissemination kinetics. Through cellular depletion experiments, we observe that neutrophils impact dissemination but that overall dissemination is largely neutrophil independent. This work adds neutrophils to the list of innate cells (i.e., dendritic and macrophages/monocytes) that contribute to MCMV dissemination but refutes the hypothesis that neutrophils are the primary cell responding to vCXCL-1.IMPORTANCE An adequate in vivo analysis of HCMV's viral chemokine vCXCL-1 has been lacking. Here we generate recombinant MCMVs expressing vCXCL-1 to study vCXCL-1 function in vivo using MCMV as a surrogate. We demonstrate that vCXCL-1 increases MCMV dissemination kinetics for both primary and secondary dissemination. Additionally, we provide evidence, that the murine neutrophil is largely a bystander in the mouse's response to vCXCL-1. We confirm the hypothesis that vCXCL-1 is a HCMV virulence factor. Infection of severely immunocompromised mice with MCMVs expressing vCXCL-1 was lethal in more than 50% of infected animals, while all animals infected with parental virus survived during a 12-day period. This work provides needed insights into vCXCL-1 function in vivo.

The YIN and YANG of lipoproteins in developing and preventing infectious arthritis by Staphylococcus aureus.

Rapid bone destruction often leads to permanent joint dysfunction in patients with septic arthritis, which is mainly caused by Staphylococcus aureus (S. aureus). Staphylococcal cell wall components are known to induce joint inflammation and bone destruction. Here, we show that a single intra-articular injection of S. aureus lipoproteins (Lpps) into mouse knee joints induced chronic destructive macroscopic arthritis through TLR2. Arthritis was characterized by rapid infiltration of neutrophils and monocytes. The arthritogenic effect was mediated mainly by macrophages/monocytes and partially via TNF-α but not by neutrophils. Surprisingly, a S. aureus mutant lacking Lpp diacylglyceryl transferase (lgt) caused more severe joint inflammation, which coincided with higher bacterial loads of the lgt mutant in local joints than those of its parental strain. Coinjection of pathogenic S. aureus LS-1 with staphylococcal Lpps into mouse knee joints caused improved bacterial elimination and diminished bone erosion. The protective effect of the Lpps was mediated by their lipid moiety and was fully dependent on TLR2 and neutrophils. The blocking of CXCR2 on neutrophils resulted in total abrogation of the protective effect of the Lpps. Our data demonstrate that S. aureus Lpps elicit innate immune responses, resulting in a double-edged effect. On the one hand, staphylococcal Lpps boost septic arthritis. On the other hand, Lpps act as adjuvants and activate innate immunity, which could be useful for combating infections with multiple drug-resistant strains.

Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy.

Recruitment of myeloid-derived suppressor cells (MDSCs) into tumors induces local immunosuppression in carcinomas. Here, we assessed whether SX-682, an orally bioavailable small-molecule inhibitor of CXCR1 and CXCR2, could block tumor MDSC recruitment and enhance T cell activation and antitumor immunity following multiple forms of immunotherapy. CXCR2+ neutrophilic MDSCs (PMN-MDSCs) were the most abundant myeloid cell subset within oral and lung syngeneic carcinomas. PMN-MDSCs demonstrated greater suppression of tumor-infiltrating lymphocyte killing of targets compared with macrophages. SX-682 significantly inhibited trafficking of PMN-MDSCs without altering CXCR2 ligand expression. Trafficking of CXCR1+ macrophages was unaltered, possibly due to coexpression of CSF1R. Reduced PMN-MDSC tumor infiltration correlated with enhanced accumulation of endogenous or adoptively transferred T cells. Accordingly, tumor growth inhibition or the rate of established tumor rejection following programed death-axis (PD-axis) immune checkpoint blockade or adoptive cell transfer of engineered T cells was enhanced in combination with SX-682. Despite CXCR1/2 expression on tumor cells, SX-682 appeared to have little direct antitumor effect on these carcinoma models. These data suggest that tumor-infiltrating CXCR2+ PMN-MDSCs may prevent optimal responses following both PD-axis immune checkpoint blockade and adoptive T cell transfer therapy. Abrogation of PMN-MDSC trafficking with SX-682 enhances T cell-based immunotherapeutic efficacy and may be of benefit to patients with MDSC-infiltrated cancers.

Crosstalk between NFκB-dependent astrocytic CXCL1 and neuron CXCR2 plays a role in descending pain facilitation.

BACKGROUND: Despite accumulating evidence on the role of glial cells and their associated chemicals in mechanisms of pain, few studies have addressed the potential role of chemokines in the descending facilitation of chronic pain. We aimed to study the hypothesis that CXCL1/CXCR2 axis in the periaqueductal gray (PAG), a co-restructure of the descending nociceptive system, is involved in descending pain facilitation. METHODS: Intramedullary injection of Walker 256 mammary gland carcinoma cells of adult female Sprague Dawley rats was used to establish a bone cancer pain (BCP) model. RT-PCR, Western blot, and immunohistochemistry were performed to detect pNfkb, Cxcl1, and Cxcr2 and their protein expression in the ventrolateral PAG (vlPAG). Immunohistochemical co-staining with NeuN, GFAP, and CD11 were used to examine the cellular location of pNFκB, CXCL1, and CXCR2. The effects of NFκB and CXCR2 antagonists and CXCL1 neutralizing antibody on pain hypersensitivity were evaluated by behavioral testing. RESULTS: BCP induced cortical bone damage and persistent mechanical allodynia and increased the expression of pNFκB, CXCL1, and CXCR2 in vlPAG. The induced phosphorylation of NFκB was co-localized with GFAP and NeuN, but not with CD11. Micro-injection of BAY11-7082 attenuated BCP and reduced CXCL1 increase in the spinal cord. The expression level of CXCL1 in vlPAG showed co-localization with GFAP, but not with CD11 and NeuN. Micro-administration of CXCL1 neutralizing antibody from 6 to 9 days after inoculation attenuated mechanical allodynia. Furthermore, vlPAG application of CXCL1 elicited pain hypersensitivity in normal rats. Interestingly, CXCR2 was upregulated in vlPAG neurons (not with CD11 and GFAP) after BCP. CXCR2 antagonist SB225002 completely blocked the CXCL1-induced mechanical allodynia and attenuated BCP-induced pain hypersensitivity. CONCLUSION: The NFκB-dependent CXCL1-CXCR2 signaling cascade played a role in glial-neuron interactions and in descending facilitation of BCP.

Neutrophil Extracellular Traps Induced by IL8 Promote Diffuse Large B-cell Lymphoma Progression via the TLR9 Signaling.

PURPOSE: More than 30% of patients with diffuse large B-cell lymphoma (DLBCL) experience treatment failure after first-line therapy. Neutrophil extracellular traps (NETs), a pathogen-trapping structure in tumor microenvironment, can promote the transition of autoimmunity to lymphomagenesis. Here, we investigate whether NETs play a novel role in DLBCL progression and its underlying mechanism.Experimental Design: NETs in DLBCL tumor samples and plasma were detected by immunofluorescence and ELISA, respectively. The correlation between NETs and clinical features were analyzed. The effects of NETs on cellular proliferation and migration and mechanisms were explored, and the mechanism of NET formation was also studied by a series of in vitro and in vivo assays. RESULTS: Higher levels of NETs in plasma and tumor tissues were associated with dismal outcome in patients with DLBCL. Furthermore, we identified NETs increased cell proliferation and migration in vitro and tumor growth and lymph node dissemination in vivo. Mechanistically, DLBCL-derived IL8 interacted with its receptor (CXCR2) on neutrophils, resulting in the formation of NETs via Src, p38, and ERK signaling. Newly formed NETs directly upregulated the Toll-like receptor 9 (TLR9) pathways in DLBCL and subsequently activated NFκB, STAT3, and p38 pathways to promote tumor progression. More importantly, disruption of NETs, blocking IL8-CXCR2 axis or inhibiting TLR9 could retard tumor progression in preclinical models. CONCLUSIONS: Our data reveal a tumor-NETs aggressive interaction in DLBCL and indicate that NETs is a useful prognostic biomarker and targeting this novel cross-talk represents a new therapeutic opportunity in this challenging disease.

The role of CXCR2 in acute inflammatory responses and its antagonists as anti-inflammatory therapeutics.

PURPOSE OF REVIEW: CXCR2 is key stimulant of immune cell migration and recruitment, especially of neutrophils. Alleviating excessive neutrophil accumulation and infiltration could prevent prolonged tissue damage in inflammatory disorders. This review focuses on recent advances in our understanding of the role of CXCR2 in regulating neutrophil migration and the use of CXCR2 antagonists for therapeutic benefit in inflammatory disorders. RECENT FINDINGS: Recent studies have provided new insights into how CXCR2 signaling regulates hematopoietic cell mobilization and function in both health and disease. We also summarize several CXCR2 regulatory mechanisms during infection and inflammation such as via Wip1, T-bet, P-selectin glycoprotein ligand-1, granulocyte-colony-stimulating factor, and microbiome. Moreover, we provide an update of studies investigating CXCR2 blockade in the laboratory and in clinical trials. SUMMARY: Neutrophil homeostasis, migration, and recruitment must be precisely regulated. The CXCR2 signaling pathway is a potential target for modifying neutrophil dynamics in inflammatory disorders. We discuss the recent clinical use of CXCR2 antagonists for controlling inflammation.