Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/CXCR2 results in antitumor immunity and durable response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered non-immunogenic, with trials showing its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and to identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and antitumor immunity, characterized by modulating T cell subsets with antitumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the expression of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and the availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

Synovial Fluid Neutrophils From Patients With Juvenile Idiopathic Arthritis Display a Hyperactivated Phenotype.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood. The predominant subtypes, oligoarticular and polyarticular JIA, are traditionally considered to be autoimmune diseases with a central role for T cells and autoantibodies. Mounting evidence suggests an important role for neutrophils in JIA pathogenesis. We undertook this study to investigate the phenotypic features of neutrophils present in the blood and inflamed joints of patients. METHODS: JIA synovial fluid (SF) and parallel blood samples from JIA patients and healthy children were collected. SF-treated neutrophils from healthy donors and pleural neutrophils from patients with pleural effusion were investigated as controls for SF exposure and extravasation. Multicolor flow cytometry panels allowed for in-depth phenotypic analysis of neutrophils, focusing on the expression of adhesion molecules, activation, and maturation markers and chemoattractant receptors. Multiplex technology was used to quantify cytokines in plasma and SF. RESULTS: SF neutrophils displayed an activated, hypersegmented phenotype with decreased CD62L expression, up-regulation of adhesion molecules CD66b, CD11b, and CD15, and down-regulation of CXCR1/2. An elevated percentage of CXCR4-positive neutrophils was detected in SF from patients. Pleural neutrophils showed less pronounced maturation differences. Strikingly, significant percentages of SF neutrophils showed a profound up-regulation of atypical neutrophil markers, including CXCR3, intercellular adhesion molecule 1, and HLA-DR. CONCLUSION: Our data show that neutrophils in inflamed joints of JIA patients have an activated phenotype. This detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives JIA.

Upregulation of interleukin (IL)-31, a cytokine producing CXCR1 peripheral immune cells, contributes to the immune abnormalities of autism spectrum disorder.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by communication deficits, impaired social interactions, and restricted stereotypical behaviors. Several immune cells are associated with immune dysfunction in ASD; however, IL-31 has not been explored in ASD. This study aims to investigate the role of inflammatory cytokines and transcription factors of the CXCR1 cells in children with ASD. In the current study, we investigated the cytokines and transcription factors produced by CXCR1+ cells (IL-31, IL-9, IL-21R, IL-21, NF-κB p65, RORγT, STAT1, and FoxP3) in peripheral blood mononuclear cells (PBMCs), from children with ASD and typically developing (TD) control children, using flow cytometric analysis. In addition, we measured mRNA and protein expression levels of IL-31 using quantitative real-time PCR and western blot analyses in PBMCs. In our study, children with ASD had increased CXCR1+IL-31+, CXCR1+IL-9+, CXCR1+IL-21R+, CXCR1+IL-21+, CXCR1+NF-κB+ p65, CXCR1+RORγT+, and CXCR1+STAT1+, and decreased CXCR1+FoxP3+ cells as compared with cells from the TD control samples. Similarly, children with ASD showed increased IL-31 mRNA and protein expression levels as compared to those of TD control samples. Our results suggest that upregulated production of inflammatory cytokines and transcription factors in CXCR1+ cells cause immunological imbalance in children with ASD. Therefore, attenuation of inflammatory cytokines/mediators and transcription factors could have a therapeutic potential in the treatment of ASD.

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.

Truncation of CXCL8 to CXCL8(9-77) enhances actin polymerization and in vivo migration of neutrophils.

CXCL8 is the principal human neutrophil-attracting chemokine and a major mediator of inflammation. The chemokine exerts its neutrophil-chemotactic and neutrophil-activating activities via interaction with glycosaminoglycans (GAGs) and activation of the G protein-coupled receptors (GPCRs) CXCR1 and CXCR2. Natural CXCL8 displays an exceptional degree of amino (NH2 )-terminal heterogeneity. Most CXCL8 forms result from proteolytic processing of authentic CXCL8(1-77). Here, we compared the potencies to activate and recruit neutrophils of the 3 most abundant natural CXCL8 forms: full-length 77 amino acid CXCL8 and the 2 major natural truncated forms lacking 5 or 8 NH2 -terminal amino acids. NH2 -terminal truncation hardly affected the capacity of CXCL8 to induce shedding of CD62L or to up-regulate the expression of the adhesion molecules CD11a, CD11b, or CD15 on human neutrophils. In addition, the potency of CXCL8 to induce neutrophil degranulation and its effect on phagocytosis remained unaltered upon removal of 5 or 8 NH2 -terminal residues. However, NH2 -terminal truncation strongly potentiated CXCL8-induced actin polymerization. CXCL8(6-77) and CXCL8(9-77) showed a comparable capacity to induce Ca2+ signaling in human neutrophils and to direct in vitro neutrophil migration. Strikingly, the ability of CXCL8(9-77) to recruit neutrophils into the peritoneal cavity of mice was significantly enhanced compared to CXCL8(6-77). These results suggest that NH2 -terminal truncation influences specific biological activities of CXCL8 and indicate that CXCL8(9-77) may be the most potent neutrophil-attracting CXCL8 form in vivo.

Porcine CXCR1/2 antagonist CXCL8(3-72)G31P inhibits lung inflammation in LPS-challenged mice.

Swine pneumonia is a great threat for pig industry around the world, which is usually accompanied with neutrophils infiltration in the airway. Although interleukin-8 (CXCL8) and its receptors, CXC chemokine receptor 1 and 2 (CXCR1/2) in human have been well documented, the expression and function of CXCR1/2 is still unknown in swine. To explore the feasibility to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, we detected CXCR1/2 expression in swine pneumonia through Real-Time PCR and immunohistochemistry for the first time. Two porcine CXCR1/2 antagonists, CXCL8(3-72)N11R/G31P (pN11R) and CXCL8(3-72)G31P (pG31P) were prepared and their anti-inflammatory effects were evaluated using cell chemotaxis assays and animal experiments. Our data showed that CXCR1/2 expression, which was closely related to neutrophil infiltration in the lung, was significantly up-regulated in swine pneumonia. The pN11R and pG31P could effectively inhibit the directional migration of neutrophils in vitro. In vivo data also indicated that both pN11R and pG31P significantly relieved LPS-induced pneumonia in mice through decreasing the expression of TNF-α, CXCL8, and IL-1ß, and inhibiting neutrophil influx into the lung. pG31P was more efficient. Our study suggested that it is possible to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, and pG31P is a promising candidate.

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.

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.

Functional identification and expressional responses of large yellow croaker (Larimichthys crocea) interleukin-8 and its receptor.

Interleukin-8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by multiple cell types. It promotes chemotaxis and phagocytosis via interaction with chemokine receptors CXCR1 and CXCR2. Using published data, IL-8 gene (LcIL-8) of the large yellow croaker (Larimichthys crocea) was cloned into the pcDNA3.1 plasmid, and an interleukin-8 receptor (LcCXCR2) was cloned into the pEGFP-N1 plasmid. Secratory expression of LcIL-8 in HEK293T cells was carried out, and product in culture medium was collected for LcCXCR2 stimulation in HEK293 cells. Following receptor internalization observation and intracellular signaling detection, the functional interaction of LcIL-8 and LcCXCR2 was further determined and the ERK phosphorylation signal activation mediated by LcCXCR2 was demonstrated. Quantitative real-time PCR analysis was used to analyze transcription level regulation of LcIL-8 and LcCXCR2 in various tissues of large yellow croaker. Expression of LcIL-8 and LcCXCR2 was elevated in the spleen, head kidney, and liver after Vibrio parahemolyticus challenge. Results illustrated the functional interaction between LcIL-8 and LcCXCR2 in mediating intracellular ERK1/2 phosphorylation signaling and suggested that the LcIL-8 and LcCXCR2 system is part of the immune response induced by V. Parahemolyticus in L. crocea.

Blastemal progenitors modulate immune signaling during early limb regeneration.

Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic il-8 expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the il-8 receptor cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.

Intravenous immunoglobulin replacement treatment reduces in vivo elastase secretion in patients with common variable immune disorders.

BACKGROUND: Intravenous immunoglobulin (IVIg) treatment partially replaces antibody defects and modulates innate and adaptive immune cells in patients with primary antibody deficiencies. MATERIALS AND METHODS: This study was focused on the evaluation of the effects of in vivo IVIg administration on neutrophils from patients with common variable immune disorders (CVID). We examined polymorphonuclear neutrophil (PMN) phagocytosis, PMN oxidative burst, release of neutrophil elastase, serum level of interleukin-8 and PMN expression of CXCR1, CD11c and CD66b. RESULTS: CVID patients on chronic IVIg treatment had reduced elastase release, but normal expression of CXCR1, CD66b and CD11c receptors on PMN, normal phagocytic ability and normal secretion of interleukin-8. We found that IVIg infusions rapidly reduced the serum level of interleukin-8, the expression of its receptor, CXCR1, and the release of neutrophil elastase, suggesting that IVIg exert a dampening effect on neutrophil activity. In contrast, IVIg infusions did not alter neutrophil phagocytosis or the expression of the other receptors analysed. DISCUSSION: These findings add further information regarding the anti-inflammatory role of immunoglobulins and suggest additional benefits in keeping with recent attempts to use new therapies targeting neutrophil inflammation.

CXCR2 is critical for bacterial control and development of joint damage and pain in Staphylococcus aureus-induced septic arthritis in mouse.

Staphylococcus aureus is the main pathogen associated with septic arthritis. Upon infection, neutrophils are quickly recruited to the joint by different chemoattractants, especially CXCR1/2 binding chemokines. Although their excessive accumulation is associated with intense pain and permanent articular damage, neutrophils have an important function in controlling bacterial burden. This work aimed to study the role of CXCR2 in the control of infection, hypernociception and tissue damage in S. aureus-induced septic arthritis in mice. The kinetics of neutrophil recruitment correlated with the bacterial load recovered from inflamed joint after intra-articular injection of S. aureus. Treatment of mice from the start of infection with the non-competitive antagonist of CXCR1/2, DF2156A, reduced neutrophil accumulation, cytokine production in the tissue, joint hypernociception and articular damage. However, early DF2156A treatment increased the bacterial load locally. CXCR2 was important for neutrophil activation and clearance of bacteria in vitro and in vivo. Start of treatment with DF2156A 3 days after infection prevented increase in bacterial load and reduced the hypernociception in the following days, but did not improve tissue damage. In conclusion, treatment with DF2156A seems be effective in controlling tissue inflammation and dysfunction but its effects are highly dependent on the timing of the treatment start.

Divergent roles for Ly6C+CCR2+CX3CR1+ inflammatory monocytes during primary or secondary infection of the skin with the intra-phagosomal pathogen Leishmania major.

Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

IL-8 Alterations in HIV-1 Infected Children With Disease Progression.

Disease progression in HIV-1 infected children is faster than in adults. Less than 5% of the infected children maintain stable CD4 counts beyond 7 years of infection and are termed long-term nonprogressors (LTNPs). Delineating the host immune response in antiretroviral naïve (ART) and treated HIV-1 infected children at different disease stages will help in understanding the immunopathogenesis of the disease.A total of 79 asymptomatic, perinatally HIV-1 infected children (50 ART naïve and 29 ART treated) and 8 seronegative donors were recruited in this study. T- and B-cell activation PCR arrays were performed from the cDNA, using total RNA extracted from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1 infected children at different stages of the disease. The differentially expressed genes were identified. Quantitative RT-PCR was performed for the (interleukin-8) IL-8 gene and its transcriptional mediators, that is, SHP2, GRB2, and IL-8R (IL-8 receptor/CXCR1). Plasma levels of IL-8 were measured by flow cytometry.Gene array data revealed a higher expression of IL-8 in the ART naïve HIV-1 infected progressors and in ART nonresponders than LTNPs and ART responders, respectively. Quantitative RT-PCR analysis demonstrated a significant higher expression of IL-8 (P < 0.001), its receptor CXCR1 (P = 0.03) and the upstream signaling molecule SHP2 (P = 0.04) in the progressors versus LTNPs. Plasma levels of IL-8 were significantly higher in progressors versus LTNPs (P < 0.001), and ART nonresponders versus ART responders (P < 0.001). A significant negative correlation of plasma levels of IL-8 with CD4 counts (cells/µL) was observed in HIV-1 infected ART naïve subjects (r = -0.488; P < 0.001), while the IL-8 levels positively correlated with viral load in the ART treated children (r = 0.5494; P < 0.001). ART naïve progressors on follow up demonstrated a significant reduction in the mRNA expression (P = 0.05) and plasma levels of IL-8 (P = 0.05) post 6 months of ART initiation suggesting the beneficial role of ART therapy in reducing inflammation in infected children.Our data suggest that IL-8 may serve as a potential prognostic marker in adjunct with CD4 counts to monitor disease progression in the HIV-1 infected children and the efficacy of ART.

Interleukin-8 and Its Receptors in Human Milk from Mothers of Full-Term and Premature Infants.

In addition to its nutritional benefits, human milk also has bioactive elements. Limited immunological functions of newborns are supported and altered by the immunological elements of mother milk. Chemokines are of importance among these immune factors. Interleukin-8 (IL-8) has been demonstrated in mother's milk, and its receptors, CXC chemokine receptors (CXCR)-1 and CXCR-2, were detected on cells, responsible for immunological reactions and mammary glandular cells. The soluble forms of these receptors are yet to be described in human milk. In this study, it was aimed to assess the IL-8 levels and the concentrations of its receptors in colostrum and mature mother's milk in regard to preterm and term delivery. The results of this study indicated a decline in IL-8 levels with the lactation stage, but no difference was observed between term and preterm mother's milk. Regarding the CXCR-1 and CXCR-2, the concentrations of these receptors were similar in both colostrum and mature milk. Furthermore, there was not any significant difference between term and preterm mother's milk. In conclusion, this is the first study to investigate the concentrations of CXCR-1 and CXCR-2 with the levels of IL-8 in colostrum and mature human milk of term and preterm newborns. The alterations in IL-8 levels were similar in some of the studies reported. CXCR-1 and CXCR-2 levels did not demonstrate any significant difference. Further studies are required to investigate the soluble forms of these receptors and their relation to IL-8 with larger cohort.

CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense.

Pseudomonas aeruginosa is a key opportunistic pathogen causing disease in cystic fibrosis (CF) and other lung diseases such as chronic obstructive pulmonary disease (COPD). However, the pulmonary host defense mechanisms regulating anti-P. aeruginosa immunity remain incompletely understood. Here we demonstrate, by studying an airway P. aeruginosa infection model, in vivo bioluminescence imaging, neutrophil effector responses and human airway samples, that the chemokine receptor CXCR1 regulates pulmonary host defense against P. aeruginosa. Mechanistically, CXCR1 regulates anti-Pseudomonas neutrophil responses through modulation of reactive oxygen species and interference with Toll-like receptor 5 expression. These studies define CXCR1 as a novel, noncanonical chemokine receptor that regulates pulmonary anti-Pseudomonas host defense with broad implications for CF, COPD and other infectious lung diseases.