reparixin and Disease-Models--Animal

Neutrophil extracellular traps (NETs) facilitate bacterial clearance but also promote thrombosis and organ injury in sepsis. We quantified ex vivo NET induction in septic humans and murine models of sepsis to identify signalling pathways that may be modulated to improve outcome in human sepsis.. NET formation in human donor neutrophils was quantified after incubation with plasma obtained from patients with sepsis or systemic inflammation (double-blinded assessment of extracellular DNA using immunofluorescence microscopy). NET formation (% neutrophils forming NETs) was correlated with plasma cytokine levels (MultiPlex assay). Experimental sepsis (caecal ligation and puncture or intraperitoneal injection of Escherichia coli) was assessed in C57/BL6 male mice. The effect of pharmacological inhibition of CXCR1/2 signalling (reparixin) on NET formation, organ injury (hepatic, renal, and cardiac biomarkers), and survival in septic mice was examined.. NET formation was higher after incubation with plasma from septic patients (median NETs=25% [10.5-46.5%]), compared with plasma obtained from patients with systemic inflammation (14% [4.0-23.3%]; P=0.02). Similar results were observed after incubation of plasma from mice with neutrophils from septic non-septic mice. Circulating CXCR1/2 ligands correlated with NETosis in patients (interleukin-8; r=0.643) and mice (macrophage inflammatory protein-2; r=0.902). In experimental sepsis, NETs were primarily observed in the lungs, correlating with fibrin deposition (r=0.702) and lung injury (r=0.692). Inhibition of CXCR1/2 using reparixin in septic mice reduced NET formation, multi-organ injury, and mortality, without impairing bacterial clearance.. CXCR1/2 signalling-induced NET formation is a therapeutic target in sepsis, which may be guided by ex vivo NET assays.

Topics: Animals; Cytokines; Disease Models, Animal; Extracellular Traps; Humans; Inflammation; Lung Injury; Male; Mice; Mice, Inbred C57BL; Neutrophils; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Retrospective Studies; Sepsis; Sulfonamides; Thrombosis

Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study.. Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array.. IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice.. These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.

Topics: Adult; Animals; Cytokines; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Interleukin-8; Intervertebral Disc Degeneration; Low Back Pain; Magnetic Resonance Imaging; Male; Mice; Mice, Knockout; Middle Aged; Osteonectin; Signal Transduction; Sulfonamides

Expression of IL-8 and its receptors CXCR1 and CXCR2 is a common occurrence in human epithelial thyroid cancer (TC). In human TC samples, IL-8 expression is associated with tumor progression. IL-8 enhances proliferation, survival, motility, and leads to the maintenance of stemness features and tumor-initiating ability of TC cells. Here, we studied the effects of Reparixin (formerly Repertaxin), a small molecular weight CXCR1 and CXCR2 inhibitor, on the malignant phenotype of various TC cell lines.. Reparixin impaired the viability of epithelial thyroid cancerous cells, but not that of the non-malignant counterpart. Reparixin treatment significantly decreased TC cell survival, proliferation, Epithelial-to-Mesenchymal Transition (EMT) and stemness. CXCR1 and CXCR2 silencing abolished these effects. Reparixin sensitized TC cells to Docetaxel and Doxorubicin in culture. Used as single agent, Reparixin significantly inhibited TC cell tumorigenicity in immunodeficient mice. Finally, Reparixin potentiated the effects of Docetaxel on TC cell xenotransplants in mice.. We assessed the effects of Reparixin on TC cell viability (by growth curves, BrdU incorporation, TUNEL assay), EMT (by RT-PCR, Flow Cytometry, Migration assays), stemness (by RT-PCR, Flow Cytometry, sphere-formation and self-renewal), and tumorigenicity (by xenotransplantation in nude mice).. The present study suggests that Reparixin, both alone and in combination with classic chemotherapics, represents a novel potential therapeutic strategy for aggressive forms of TC.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Disease Models, Animal; DNA Replication; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Mice; Neoplastic Stem Cells; Rats; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Sulfonamides; Thyroid Neoplasms; Xenograft Model Antitumor Assays

Bacterial meningitis causes high mortality and brain damage. The host immune response is associated with brain injury. Chemokine (C-X-C motif) (CXC) chemokines are neutrophil chemoattractants. This study focused on the beneficial effects of intracerebroventricular administration of reparixin, an inhibitor of chemokine (C-X-C motif) receptor (CXCR)1/2, to rats at 2 h following experimental Klebsiella pneumoniae meningoencephalitis.. We used a previously established meningoencephalitis animal model in which Sprague-Dawley rats were infected by K. pneumoniae. Sham and infected animals were treated with vehicle or reparixin and sacrificed at various time points. Leukocyte infiltration into cerebrospinal fluid (CSF) and brain as well as gene and protein expression of chemokines and receptors, and neuronal apoptosis were examined. Primary cultures of neuron/glia were infected with K. pneumoniae as an in vitro model of meningoencephalitis.. Levels of chemokine (C-X-C motif) ligand (CXCL)2 in CSF time-dependently increased markedly as early as 2 h, and peaked at 8 h following infection and were much higher than those in serum collected simultaneously. Reparixin significantly reduced leukocyte infiltration into CSF and brain tissues, clinical illness, and brain cell apoptosis at 24 h. Reparixin reduced the elevated CSF concentrations of chemokines [CXCL1, CXCL2, chemokine (C-C motif) ligand (CCL)2 and CCL5] and proinflammatory cytokines. Reparixin also reduced the expression of mRNA of various chemokines, chemokine receptors and proinflammatory cytokines in infected brain tissues. Using primary cultures that are devoid of leukocytes, we further observed that reparixin attenuated the neuronal, but not microglial cell death after infection.. Reparixin not only reduces amplified inflammation, but also provides direct neuroprotective effects in K. pneumoniae meningoencephalitis.

Topics: Animals; Apoptosis; Brain; Chemokine CXCL2; Disease Models, Animal; Inflammation Mediators; Klebsiella Infections; Male; Meningoencephalitis; Neuroprotective Agents; Neutrophil Infiltration; Rats, Sprague-Dawley; RNA, Messenger; Sulfonamides

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.. MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 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.. 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.. IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924-36. ©2016 AACR.

Topics: Animals; Biomarkers; Cell Line, Tumor; Chemotaxis, Leukocyte; Disease Models, Animal; Extracellular Traps; Humans; Interleukin-8; Mice; Mice, Knockout; Myeloid-Derived Suppressor Cells; Neoplasms; Neutrophils; Sulfonamides; T-Lymphocyte Subsets

We previously demonstrated that a synthetic retinoic acid receptor agonist, Am80, attenuated intracerebral hemorrhage (ICH)-induced neuropathological changes and neurological dysfunction. Because inflammatory events are among the prominent features of ICH pathology that are affected by Am80, this study investigated the potential involvement of proinflammatory cytokines/chemokines in the effect of Am80 on ICH. ICH induced by collagenase injection into mouse striatum caused prominent upregulation of mRNAs for interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, CXCL1, CXCL2, and CCL3. We found that dexamethasone (DEX) and Am80 differently modulated the increase in expression of these cytokines/chemokines; TNF-α expression was attenuated only by DEX, whereas CXCL2 expression was attenuated only by Am80. Expression of IL-1β and IL-6 was inhibited both by DEX and Am80. Neurological assessments revealed that Am80, but not DEX, significantly alleviated motor dysfunction of mice after ICH. From these results, we suspected that CXCL2 might be critically involved in determining the extent of motor dysfunction. Indeed, magnetic resonance imaging-based classification of ICH in individual mice revealed that invasion of hematoma into the internal capsule, which has been shown to cause severe neurological disabilities, was associated with higher levels of CXCL2 expression than ICH without internal capsule invasion. Moreover, a CXCR1/2 antagonist reparixin ameliorated neurological deficits after ICH. Overall, suppression of CXCL2 expression may contribute to the beneficial effect of Am80 as a therapeutic agent for ICH, and interruption of CXCL2 signaling may provide a promising target for ICH therapy.

Topics: Animals; Benzoates; Cerebral Hemorrhage; Chemokine CXCL2; Cytokines; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Sulfonamides; Tetrahydronaphthalenes; Up-Regulation

Female Wistar rats (225 g) underwent spinal cord injury (SCI) at the T4 segment and were assigned to one of the three groups treated with: (1) saline; (2) 7.5 mg kg(-1) Reparixin; or (3) 15 mg kg(-1) Reparixin. Reparixin is a small molecule, allosteric noncompetitive inhibitor of CXCR1 and CXCR2 chemokine receptors involved in inflammation.. Spinal cord homogenates at 12 and 72 h post-SCI were assayed for tumor necrosis factor α (TNF-α) and cytokine-induced neutrophil chemoattractant (CINC)-1 using enzyme-linked immunosorbant assay (ELISA). Myeloperoxidase activity and western blots for CD68, Fas and p75 content were used to assess inflammation and death receptor ligands, respectively. Histopathology and neurological outcomes were assessed by immunohistochemistry, locomotion scoring and cardiovascular measurement of autonomic dysreflexia 4 weeks post-SCI.. Both 7.5 and 15 mg kg(-1) doses of Reparixin reduced levels of TNF-α and CINC-1 72 h post-SCI and decreased macrophage (CD68) content in the spinal cord lesion. Only 15 mg kg(-1) Reparixin reduced both Fas and p75 levels in the spinal cord compared with untreated SCI. We observed a reduced lesion area and increased neuron number in the gray matter of Reparixin-treated rats. Hindlimb motor scores at 7 and 28 days post-SCI were improved by 15 mg kg(-1) Reparixin treatment. Both 7.5 and 15 mg kg(-1) Reparixin reduced development of autonomic dysreflexia 4 weeks post-SCI. The change in mean arterial pressure, induced by cutaneous or visceral stimulation, was reduced by 40-50%.. Acute treatment with 15 mg kg(-1) Reparixin reduces acute inflammation and is associated with minor improvements in motor function and a significant reduction in the severity of autonomic dysreflexia.

Topics: Acute Disease; Animals; Autonomic Dysreflexia; Disease Models, Animal; Female; Inflammation Mediators; Nerve Degeneration; Rats; Rats, Wistar; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Spinal Cord Injuries; Sulfonamides

Autologous nucleus pulposus obtained from coccygeal intervertebral discs was grafted on the proximal of L5 dorsal root ganglion. Pain behavior, mRNA expression of Interleukin-8 (IL-8), and immunohistochemical changes were assessed.. The purpose of this study is to investigate temporal changes of IL-8 expression in the spinal cord and dorsal root ganglion and the pain-related behaviors with time course and to elucidate whether repertaxin (IL-8 receptor inhibitor) attenuates pain-related behaviors in a rat model of lumbar disc herniation.. Inflammatory mediators like cytokines and chemokines have been implicated in radicular pain because of disc herniation. IL-8, known as CXCL8, is a chemokine, which has been reported to be associated with painful degenerative disc disorders and chronic inflammatory pain states.. Lumbar disc herniated rat model was made by implantation of the autologous nucleus pulposus, harvested from the coccygeal vertebra of each tail, on the left L5 nerve root just proximal to the dorsal root ganglion. Rats were tested for mechanical allodynia and thermal hyperalgesia at 2 days before surgery, and on days 1, 5, 10, 20, 30, 40, 50, and 60 postoperatively. Experimental group was intrathecally injected with the IL-8 receptor inhibitor at L5 level on postoperative day 10. Mechanical allodynia of the plantar surface of both hindpaw was tested on 30 minutes, 1, 3 hours, 1, 3, 5, and 10 days after administration. For the staining of astrocytes and microglia, immunohistochemical study was done 20 days after surgery.. Mechanical allodynia in ipsilateral hindpaw developed 1 day after surgery and lasted until 60 days and thermal withdrawal latency decreased significantly on the ipsilateral side 10 days after surgery and gradually increased through day 60. The IL-8 receptor inhibitor attenuated the mechanical allodynia caused by nucleus pulposus when it was administered on postoperative day 10 and reduced microglial activation and phosphorylated form of mitogen-activated protein kinase (pERK) expression in the spinal dorsal horn.. IL-8 might be a potential therapeutic target in chronic radicular neuropathic pain because of disc herniation, CXCL8 inhibitor could be one of its promising therapeutic agents.

Topics: Animals; Chemokine CXCL1; Disease Models, Animal; Ganglia, Spinal; Gene Expression; Hyperalgesia; Immunohistochemistry; Interleukin-8; Intervertebral Disc Displacement; Lumbar Vertebrae; Male; Pain; Pain Measurement; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-8A; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Sulfonamides; Time Factors

Acute lung injury (ALI) remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability, but no small molecule CXCR2 inhibitor has been demonstrated to be effective in ALI or animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its effects in two models of ALI, induced by either lipopolysaccharide (LPS) inhalation or acid instillation.. In two ALI models in mice, we measured vascular permeability by Evans blue and evaluated neutrophil recruitment into the lung vasculature, interstitium and airspace by flow cytometry.. Pharmacological inhibition of CXCR2 by Reparixin reduced CXCL1-induced leukocyte arrest in the microcirculation of the cremaster muscle, but did not influence arrest in response to leukotriene B4 (LTB4) demonstrating specificity. Reparixin (15 microg g(-1)) reduced neutrophil recruitment in the lung by approximately 50% in a model of LPS-induced ALI. A higher dose did not provide additional reduction of neutrophil recruitment. This dose also reduced accumulation of neutrophils in the interstitial compartment and vascular permeability in LPS-induced ALI. Furthermore, both prophylactic and therapeutic application of Reparixin improved gas exchange, and reduced neutrophil recruitment and vascular permeability in a clinically relevant model of acid-induced ALI.. Reparixin, a non-competitive allosteric CXCR2 inhibitor attenuates ALI by reducing neutrophil recruitment and vascular permeability.

Topics: Animals; Capillary Permeability; Cell Movement; Disease Models, Animal; Dose-Response Relationship, Drug; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neutrophils; Pulmonary Gas Exchange; Receptors, Interleukin-8B; Respiratory Distress Syndrome; Sulfonamides

The chemokine receptors CXCR1 and CXCR2 play a role in mediating neutrophil recruitment and neutrophil-dependent injury in several models of inflammation. We undertook this study to investigate the role of these receptors in mediating neutrophil adhesion, subsequent migration, and neutrophil-dependent hypernociception in a murine model of monarticular antigen-induced arthritis (AIA).. AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic pressure meter. Neutrophil accumulation in the tissue was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity. Levels of tumor necrosis factor alpha (TNFalpha) and the chemokines CXCL1 and CXCL2 were quantified by enzyme-linked immunosorbent assay. Histologic analysis was performed to evaluate the severity of arthritis and leukocyte infiltration.. Antigen challenge in immunized mice induced production of TNFalpha, CXCL1, and CXCL2 and also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment with reparixin or DF2162 (allosteric inhibitors of CXCR1/CXCR2) decreased neutrophil recruitment, an effect that was associated with marked inhibition of neutrophil adhesion. Drug treatment also inhibited TNFalpha production, hypernociception, and the overall severity of the disease in the tissue.. Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the adhesion of neutrophils to synovial microvessels. As a consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies suggest a potential therapeutic role for the modulation of CXCR1/CXCR2 receptor signaling in the treatment of arthritis.

Topics: Adjuvants, Immunologic; Animals; Arthritis, Experimental; Benzeneacetamides; Cell Adhesion; Cell Movement; Disease Models, Animal; Endothelium, Vascular; Male; Mesylates; Mice; Mice, Inbred C57BL; Neutrophils; Peroxidase; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Sulfonamides; Synovial Membrane; Tumor Necrosis Factor-alpha

Alzheimer's disease (AD) is a progressive chronic disorder that leads to cognitive decline. Several studies have associated up-regulation of some of the chemokines and/or their receptors with altered APP processing leading to increased production of beta-amyloid protein (Abeta) and AD pathological changes. However, there is no direct evidence to date to determine whether the altered processing of APP results in up-regulation of these receptors or whether the up-regulation of the chemokine receptors causes modulated processing of APP. In the current study, we demonstrate that treatment of the chemokine receptor CXCR2 with agonists leads to enhancement of Abeta production and treatment with antagonists or immunodepletion of CXCR2's endogenous agonists leads to Abeta inhibition. Further, we found that the inhibitory effect of the antagonist of CXCR2 on Abeta40 and Abeta42 is mediated via gamma-secretase, specifically through reduction in expression of presenilin (PS), one of the gamma-secretase components. Also, in vivo chronic treatment with a CXCR2 antagonist blocked Abeta40 and Abeta42 production. Using small interfering RNAs for CXCR2, we further showed that knockdown of CXCR2 in vitro accumulates gamma-secretase substrates C99 and C83 with reduced production of both Abeta40 and Abeta42. Taken together, these findings strongly suggest for the first time that up-regulation of the CXCR2 receptor can be the driving force in increased production of Abeta. Our findings unravel new mechanisms involving the CXCR2 receptor in the pathogenesis of AD and pose it as a potential target for developing novel therapeutics for intervention in this disease. Also, we propose here a new chemical series of interest that can serve as a prototype for drug development.

Topics: Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Cells, Cultured; Chemokine CXCL1; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; gamma-Aminobutyric Acid; Humans; Interleukin-8; Mice; Mice, Transgenic; Phenylurea Compounds; Presenilins; Receptors, Interleukin-8B; Recombinant Proteins; RNA, Small Interfering; Sulfonamides; Triglycerides