Highlights d Tumor-secreted CXCR1 and CXCR2 ligands induce extrusion of NETs d NETs protect tumor cells from CTL and NK cytotoxicity in 3D cultures d Inhibition of NETosis sensitizes tumors to PD-1+CTLA-4 dual checkpoint blockade d NETs impair contact of immune cytotoxic cells with tumor cells in living mice Authors

The chemokine CXC ligand 8 (CXCL8)͞IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors. L eukocyte trafficking into tissue sites of inflammation is directed by chemokines. Chemokines are grouped into four families based on a cysteine motif in the amino terminus of the protein (1, 2). Human CXC ligand 8 (CXCL8)͞IL-8 and related molecules are polymorphonuclear cells (PMN) chemoattractants. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Only one corresponding receptor has been identified in the mouse, and this is recognized by ligands that act as neutrophil attractant, although a mouse orthologue of CXCL8 has not been identified. By recruiting and activating PMN, CXCL8 and related rodent molecules have been implicated in a wide range of disease states characterized by PMN infiltration in organs, including reperfusion injury (RI) (3).G protein-coupled receptors (GPCR) are a prime target for the development of new strategies to control diverse pathologies (4-6). Antichemokine strategies include antibodies, N-terminal modified chemokines, and small-molecule antagonists (7-9). Here we describe a class of GPCR inhibitors that specifically block the inflammatory CXCL8 chemokine receptors CXCR1 and CXCR2 by means of an allosteric noncompetitive mode of interaction and protection against RI. Materials and MethodsReagents. Repertaxin (R)(Ϫ)-2-(4-isobutylphenyl)propionyl methansulfonamide) salified with L-lysine was dissolved in saline. Chemokines were from PeproTech (London). Chemicals, cell culture reagents, and protease inhibitors were from Sigma.Migration. Cell migration of human PMN and monocytes and rodent peritoneal PMN were evaluated in a 48-well microchemotaxis chamber with or without Repertaxin. Agonists (1 nM CXCL8, 10 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 10 nM CXCL1, 2.5 nM CCL2, 1 nM C5a, 5 nM rat and mouse CXCL1, and 2.5 nM rat and mouse CXCL2) were seeded in the lower compartment. The chemotaxis chamber was incubated for 45 min (human PMN), 1 h (rodent PMN), or 2 h (monocytes). L1.2 migration was evaluated by using 5-m pore-size Transwell filters (Costar) (10). Mutation Analysis of CXCR1 and Signaling. The human CXCR1 ORF was PCR amplified from a CXCR1͞pCEP4 plasmid (kindly provided by P. M. Murphy, National Institutes of Health, Bethesda). Receptor mutants and chimeric re...

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes.Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n ¼ 10) and advanced cancer patients (n ¼ 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures.Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset.Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control.

Topical rhNGF is safe and more effective than vehicle in promoting healing of moderate-to-severe NK.

To evaluate the efficacy and safety of topical cenegermin (recombinant human nerve growth factor) in patients with neurotrophic keratopathy.Design: Multicenter, randomized, double-masked, vehicle-controlled trial.Participants: Patients with neurotrophic persistent epithelial defect with or without stromal thinning. Methods: The NGF0214 trial, conducted among 11 sites in the United States, randomized 48 patients 1:1 to cenegermin 20 mg/ml or vehicle eye drops, 6 drops daily for 8 weeks of masked treatment. Follow-up was 24 weeks. Safety was assessed in all patients who received study drug. Efficacy was assessed by intention to treat. Main Outcome Measures: The primary end point was healing of the neurotrophic lesion (persistent epithelial defect or corneal ulcer) after 8 weeks of masked treatment. Masked central readers measured neurotrophic lesions in randomized clinical pictures, then assessed healing status conventionally (<0.5 mm of fluorescein staining in the greatest dimension of the lesion area) and conservatively (0-mm lesion staining and no other residual staining). Secondary variables included corneal healing at 4 weeks of masked treatment (key secondary end point), overall changes in lesion size, rates of disease progression, and changes in visual acuity and corneal sensitivity from baseline to week 8.Results: Conventional assessment of corneal healing showed statistically significant differences at week 8: compared to 7 of 24 vehicle-treated patients (29.2%), 16 of 23 cenegermin-treated patients (69.6%) achieved less than 0.5 mm of lesion staining (þ40.4%; 95% confidence interval [CI], 14.2%e66.6%; P ¼ 0.006). Conservative assessment of corneal healing also reached statistical significance at week 8: compared to 4 of 24 vehicle-treated patients (16.7%), 15 of 23 cenegermin-treated patients (65.2%) achieved 0 mm of lesion staining and no other residual staining (þ48.6%; 95% CI, 24.0%e73.1%; P < 0.001). Moreover, the conservative measure of corneal healing showed statistical significance at week 4 (key secondary end point). Compared to vehicle, cenegermintreated patients showed statistically significant reductions in lesion size and disease progression rates during masked treatment. Cenegermin was well tolerated; adverse effects were mostly local, mild, and transient.Conclusions: Cenegermin treatment showed higher rates of corneal healing than vehicle in neurotrophic keratopathy associated with nonhealing corneal defects.

No abstract

No abstract

Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation. IntroductionAchieving long-lasting insulin independence after portal vein islet transplantation has improved, but remains challenging. Nonspecific immune activation (1-6), along with preexisting and transplant-induced auto-and allospecific immune responses (7-9), are components affecting outcome; these are not fully suppressed by ongoing protocols of generalized immunosuppression. Increasing general immunosuppression potency is not ideal because of side effects. Consequently, the development of novel protocols that specifically target proinflammatory immune cell compartments that impede islet function and survival is compelling.