ABSTRACT
Interleukin-8 (IL-8 or C-X-C motif chemokine ligand 8, CXCL8) is a cytokine secreted by numerous cell types and is best known for its functional roles in inflammatory response by binding to specific receptors (the interleukin-8 receptors, IL-8Rs). From the transcriptomic data of largemouth bass (Micropterus salmoides), we identified an IL-8R that is highly homologous to the functionally validated teleost IL-8Rs. The M. salmoides IL-8 receptor (MsCXCR2) was further compared with the C-X-C motif chemokine receptor 2 subfamily by phylogenetic analysis. Briefly, the full-length CDS sequence of MsCXCR2 was cloned into the pEGFP-N1 plasmid, and the membrane localization of fusion expressing MsCXCR2-EGFP was revealed in HEK293 cells. To determine the functional interaction between IL-8 and MsCXCR2, secretory expressed Larimichthys crocea IL-8 (LcIL-8) was used to stimulate MsCXCR2 expressing cells. MsCXCR2 was demonstrated to be activated by LcIL-8, leading to receptor internalization, which was further revealed by the detection of extracellular regulated protein kinase (ERK) phosphorylation. Quantitative real-time PCR was used to evaluate the expressional distribution and variation of MsCXCR2 in healthy and Nocardia seriolae infected fish. Based on our findings, MsCXCR2 was constitutively expressed in all examined tissues, despite at different levels. Furthermore, gene expression was found to be significantly upregulated in the liver and head kidney of diseased fish. Collectively, our findings reveal the molecular activity of MsCXCR2 and indicate the functional involvement of this IL-8R in the immune response induced by N. seriolae in M. salmoides.
Subject(s)
Bass , Fish Diseases , Nocardia Infections , Receptors, Interleukin-8B/genetics , Animals , Bass/genetics , Bass/immunology , Fish Diseases/immunology , Fish Diseases/microbiology , Fish Proteins/genetics , Fish Proteins/immunology , HEK293 Cells , Humans , Interleukin-8/pharmacology , Nocardia , Nocardia Infections/immunology , Nocardia Infections/veterinary , Phylogeny , Receptors, Interleukin-8B/immunologyABSTRACT
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.
Subject(s)
Chemokine CXCL1/genetics , Intracellular Signaling Peptides and Proteins/genetics , Phosphate-Binding Proteins/genetics , Receptors, Interleukin-8B/genetics , Skin Diseases/veterinary , Staphylococcal Infections/veterinary , Staphylococcus aureus/physiology , Animals , Chemokine CXCL1/immunology , Female , Intracellular Signaling Peptides and Proteins/immunology , Male , Mice , Mice, Inbred C57BL , Phosphate-Binding Proteins/immunology , Receptors, Interleukin-8B/immunology , Skin Diseases/genetics , Skin Diseases/immunology , Staphylococcal Infections/genetics , Staphylococcal Infections/immunologyABSTRACT
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.
Subject(s)
Antibodies/pharmacology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Neutrophil Activation/drug effects , Receptors, Interleukin-8B/metabolism , Animals , Binding Sites , Chemokines , Chemotaxis , Encephalomyelitis, Autoimmune, Experimental/immunology , Endocytosis , Epitopes , Humans , Immunoglobulin G , Interleukin-8/metabolism , Ligands , Mice , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , Signal Transduction , Tumor Necrosis Factor-alphaABSTRACT
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.
Subject(s)
Aging/immunology , Biological Transport/immunology , Inflammation/immunology , Neutrophils/immunology , Animals , Chemokine CXCL1/immunology , Endothelial Cells/immunology , Endothelium, Vascular/immunology , Female , Intercellular Junctions/immunology , Lung/immunology , Male , Mice , Mice, Inbred C57BL , Receptors, Interleukin-8B/immunology , Venules/immunologyABSTRACT
COVID-19 is characterized by respiratory symptoms of various severities, ranging from mild upper respiratory signs to acute respiratory failure/acute respiratory distress syndrome associated with a high mortality rate. However, the pathophysiology of the disease is largely unknown. Shotgun metagenomics from nasopharyngeal swabs were used to characterize the genomic, metagenomic and transcriptomic features of patients from the first pandemic wave with various forms of COVID-19, including outpatients, patients hospitalized not requiring intensive care, and patients in the intensive care unit, to identify viral and/or host factors associated with the most severe forms of the disease. Neither the genetic characteristics of SARS-CoV-2, nor the detection of bacteria, viruses, fungi or parasites were associated with the severity of pulmonary disease. Severe pneumonia was associated with overexpression of cytokine transcripts activating the CXCR2 pathway, whereas patients with benign disease presented with a T helper "Th1-Th17" profile. The latter profile was associated with female gender and a lower mortality rate. Our findings indicate that the most severe cases of COVID-19 are characterized by the presence of overactive immune cells resulting in neutrophil pulmonary infiltration which, in turn, could enhance the inflammatory response and prolong tissue damage. These findings make CXCR2 antagonists, in particular IL-8 antagonists, promising candidates for the treatment of patients with severe COVID-19.
Subject(s)
COVID-19 , Genome, Viral , Metagenomics , SARS-CoV-2 , Th1 Cells/immunology , Th17 Cells/immunology , Transcriptome , Adult , Aged , Aged, 80 and over , COVID-19/genetics , COVID-19/immunology , Female , Humans , Male , Middle Aged , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunologyABSTRACT
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.
Subject(s)
B7-H1 Antigen/metabolism , Carcinoma, Hepatocellular/immunology , Liver Neoplasms/immunology , Receptors, Interleukin-8B/immunology , Animals , B7-H1 Antigen/genetics , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Cell Survival , Humans , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/immunology , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Interleukin-8B/deficiency , Receptors, Interleukin-8B/genetics , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Apoptosis/immunology , Intramolecular Oxidoreductases/immunology , Leukocytes, Mononuclear/immunology , Macrophage Migration-Inhibitory Factors/immunology , Neutrophils/immunology , Cytokines/immunology , Humans , Inflammation/immunology , Receptors, Interleukin-8B/immunologyABSTRACT
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.
Subject(s)
Neoplasm Metastasis/pathology , Pancreatic Neoplasms/pathology , Receptors, Interleukin-8B/immunology , Animals , Cell Line, Tumor , Cell Proliferation/physiology , Endothelial Cells/immunology , Endothelial Cells/pathology , Mice , Neoplasm Metastasis/immunology , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/pathology , Neutrophils/immunology , Pancreatic Neoplasms/immunology , Tumor Microenvironment/immunology , Pancreatic NeoplasmsABSTRACT
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.
Subject(s)
Arthritis, Rheumatoid/genetics , Fibroblasts/metabolism , Neutrophils/metabolism , Synovial Membrane/metabolism , Anti-Citrullinated Protein Antibodies/immunology , Arthritis, Rheumatoid/immunology , Bayes Theorem , Databases, Genetic , Fibroblasts/immunology , Gene Expression Profiling , Histone Deacetylases/genetics , Histone Deacetylases/immunology , Humans , Janus Kinase 3/genetics , Janus Kinase 3/immunology , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/immunology , Neutrophils/immunology , Osteogenesis/genetics , Osteogenesis/immunology , Phenotype , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/immunology , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/immunology , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , Synovial Membrane/immunology , Systems Analysis , Transcriptome , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunology , src-Family Kinases/genetics , src-Family Kinases/immunologyABSTRACT
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.
Subject(s)
Arthritis, Juvenile/immunology , Neutrophil Activation/immunology , Neutrophils/immunology , Receptors, CXCR3/immunology , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/immunology , Synovial Fluid/cytology , Adaptive Immunity/immunology , Adolescent , Antigens, CD/metabolism , Arthritis, Juvenile/metabolism , CD11b Antigen/metabolism , Cell Adhesion , Cell Adhesion Molecules/metabolism , Child , Child, Preschool , Down-Regulation , Female , Flow Cytometry , GPI-Linked Proteins/metabolism , HLA-DR Antigens/immunology , Humans , Immunity, Innate/immunology , Immunophenotyping , Intercellular Adhesion Molecule-1/metabolism , L-Selectin/immunology , Lewis X Antigen/metabolism , Male , Neutrophils/metabolism , Pleural Effusion , Up-RegulationABSTRACT
Rhinovirus (RV) infections in asthmatic patients are often associated with asthma exacerbation, characterized by worsened airways hyperreactivity and increased immune cell infiltration to the airways. The C-X-C chemokines, CXCL3 and CXCL5, regulate neutrophil trafficking to the lung via CXCR2, and their expression in the asthmatic lung is associated with steroid-insensitive type 2 inflammatory signatures. Currently, the role of CXCL3 and CXCL5 in regulating neutrophilic and type 2 responses in viral-induced asthma exacerbation is unknown. Inhibition of CXCL3 or CXCL5 with silencing RNAs in a mouse model of RV-induced exacerbation of asthma attenuated the accumulation of CXCR2+ neutrophils, eosinophils, and innate lymphoid cells in the lung and decreased production of type 2 regulatory factors IL-25, IL-33, IL-5, IL-13, CCL11, and CCL24. Suppression of inflammation was associated with decreased airways hyperreactivity, mucus hypersecretion, and collagen deposition. Similar results were obtained by employing RC-3095, which has been shown to bind to CXCR2, or by depletion of neutrophils. Our data demonstrate that CXCL3 and CXCL5 may be critical in the perpetuation of RV-induced exacerbation of asthma through the recruitment of CXCR2-positive neutrophils and by promoting type 2 inflammation. Targeting the CXCL3/CXCL5/CXCR2 axis may provide a new therapeutic approach to attenuating RV-induced exacerbations of asthma.
Subject(s)
Asthma/immunology , Chemokine CXCL5/immunology , Chemokines, CXC/immunology , Chemotaxis, Leukocyte/immunology , Neutrophils/immunology , Receptors, Interleukin-8B/immunology , Rhinovirus/immunology , Animals , Bronchial Hyperreactivity/immunology , Eosinophils/immunology , Immunity, Innate/immunology , Inflammation/immunology , Lung/immunology , Lymphocytes/immunology , Male , Mice , Mice, Inbred BALB CABSTRACT
Sepsis-associated encephalopathy (SAE) is a devastating neurological complication of sepsis with intolerable high motility. SAE is accompanied with brain vascular injury, endothelial hyperpermeability, and neutrophil infiltration into the brain tissue, key inflammatory processes leading to further brain edema and neuronal cell apoptosis. Recent studies from us and others suggest that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is crucial for neutrophil recruitment during SAE. Here we use CXCR2 antagonist SB225002 to characterize the role of CXCR2 in brain infiltration of neutrophil in a murine model of SAE. Systemic administration of high-dose LPS (10 mg/kg) induced evident neutrophil infiltration into the cerebral cortex in wild-type mice. However, CXCR2 antagonist SB225002 markedly attenuated neutrophil infiltration into brain. The CXCR2 expression on neutrophils in the peripheral circulation was dramatically downregulated in response to this LPS dose, and endothelial CXCR2 was significantly upregulated, suggesting endothelial but not neutrophil CXCR2 plays a more important role in neutrophil infiltration into brain. Strikingly, although these CXCR2 antagonist SB225002 treated mice displayed reduced neutrophil infiltration, no change in neutrophil rolling and adhesion was observed. Furthermore, we confirmed that CXCR2 agonist CXCL1 induced a marked increase in actin stress fiber synthesis and paracellular gap formation in cultured cerebral endothelial cells, which is attenuated by SB225002. Thus, these results demonstrate a selective role for endothelial CXCR2 to regulate cerebral vascular permeability and neutrophil transmigration in high-dose LPS induced neuroinflammation, and also suggest a therapeutic potential of CXCR2 antagonist SB225002 in SAE.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Phenylurea Compounds/therapeutic use , Receptors, Interleukin-8B/antagonists & inhibitors , Sepsis-Associated Encephalopathy/drug therapy , Animals , Brain/drug effects , Brain/immunology , Brain Edema , Cell Line , Disease Models, Animal , Lipopolysaccharides/immunology , Male , Mice, Inbred C57BL , Neutrophils/immunology , Receptors, Interleukin-8B/immunology , Sepsis-Associated Encephalopathy/immunologyABSTRACT
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.
Subject(s)
Host-Pathogen Interactions/immunology , Myeloid Differentiation Factor 88/immunology , Papillomaviridae/immunology , Papillomavirus Infections/immunology , RNA, Messenger/immunology , RNA, Viral/immunology , Receptors, Interleukin-1 Type I/immunology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Alternative Splicing , Animals , Caspase 1/deficiency , Caspase 1/genetics , Caspase 1/immunology , Cervix Uteri/immunology , Cervix Uteri/virology , Female , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Humans , Immunity, Innate , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Papillomaviridae/growth & development , Papillomaviridae/metabolism , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , RNA, Messenger/genetics , RNA, Viral/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/deficiency , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/immunology , Receptors, CCR6/deficiency , Receptors, CCR6/genetics , Receptors, CCR6/immunology , Receptors, CXCR3/deficiency , Receptors, CXCR3/genetics , Receptors, CXCR3/immunology , Receptors, Interleukin-1 Type I/deficiency , Receptors, Interleukin-1 Type I/genetics , Receptors, Interleukin-8B/deficiency , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , STAT1 Transcription Factor/deficiency , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , Signal Transduction , Vagina/immunology , Vagina/virologyABSTRACT
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.
Subject(s)
Actins/genetics , Base Sequence , Interleukin-8/genetics , Neutrophils/metabolism , Protein Processing, Post-Translational/immunology , Sequence Deletion , Actins/immunology , Animals , CD11a Antigen/genetics , CD11a Antigen/immunology , CD11b Antigen/genetics , CD11b Antigen/immunology , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Movement/drug effects , Cell Movement/immunology , Chemotaxis, Leukocyte , Female , Gene Expression Regulation , Glycosaminoglycans , Humans , Interleukin-8/immunology , Interleukin-8/pharmacology , Lewis X Antigen/genetics , Lewis X Antigen/immunology , Mice , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/immunology , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/immunology , Polymerization , Primary Cell Culture , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
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.
Subject(s)
Extracellular Traps/metabolism , Neoplasms, Experimental/therapy , Receptors, Chemokine/agonists , Receptors, Interleukin-8A/agonists , Receptors, Interleukin-8B/agonists , Animals , Cell Line, Tumor , Cytotoxicity, Immunologic/immunology , HT29 Cells , Humans , Intravital Microscopy/methods , Killer Cells, Natural/immunology , Ligands , Mice , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Receptors, Chemokine/immunology , Receptors, Chemokine/metabolism , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/immunology , Receptors, Interleukin-8B/metabolism , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
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.
Subject(s)
Interleukin-8/pharmacology , Interleukin-8/therapeutic use , Peptide Fragments/pharmacology , Peptide Fragments/therapeutic use , Pneumonia/drug therapy , Pneumonia/veterinary , Receptors, Interleukin-8A/antagonists & inhibitors , Receptors, Interleukin-8B/antagonists & inhibitors , Swine Diseases/drug therapy , Animals , Cell Movement/drug effects , Disease Models, Animal , Drug Discovery/methods , Female , Immunohistochemistry , Interleukin-8/metabolism , Lipopolysaccharides/adverse effects , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Neutrophils/metabolism , Pneumonia/chemically induced , Pneumonia/pathology , Real-Time Polymerase Chain Reaction , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8A/isolation & purification , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/immunology , Receptors, Interleukin-8B/isolation & purification , Signal Transduction/drug effects , SwineABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular/therapy , Immunotherapy, Adoptive/methods , Liver Neoplasms/therapy , Receptors, Chimeric Antigen/genetics , Receptors, Interleukin-8B/genetics , T-Lymphocytes, Cytotoxic/immunology , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement , Chemokine CCL2/genetics , Chemokine CCL2/immunology , Chemokine CXCL5/genetics , Chemokine CXCL5/immunology , Cytotoxicity, Immunologic , Gene Expression , Humans , Interleukin-8/genetics , Interleukin-8/immunology , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Mice , Receptors, Chimeric Antigen/immunology , Receptors, Interleukin-8B/immunology , T-Lymphocytes, Cytotoxic/cytology , Tumor Burden , Tumor Microenvironment/immunology , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Immunologic Factors/pharmacology , Immunotherapy , Neoplasms/therapy , Receptors, Interleukin-8A/agonists , Receptors, Interleukin-8B/agonists , Animals , Dose-Response Relationship, Drug , Humans , Immunologic Factors/chemistry , Molecular Structure , Neoplasms/immunology , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/immunology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/immunology , Gene Expression Regulation/immunology , Immune Tolerance , Myeloid-Derived Suppressor Cells/immunology , Receptors, Aryl Hydrocarbon/immunology , Receptors, Interleukin-8B/immunology , Animals , Basic Helix-Loop-Helix Transcription Factors/agonists , Chemokines/immunology , Female , Gene Expression Regulation/drug effects , Inflammation/chemically induced , Inflammation/immunology , Inflammation/pathology , Mice , MicroRNAs , Myeloid-Derived Suppressor Cells/pathology , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/agonistsABSTRACT
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.