Cellular Inflammation in Models of Acute Gout

<p>Gout is a common form of inflammatory arthritis that is caused by the precipitation of monosodium urate crystals (MSU) in the joints. The acute form of gout is associated with sudden painful inflammatory episodes characterised by a large infiltration of neutrophils. The involvement of monocytes and macrophages is also recognised to be important, although the exact roles of monocytes and macrophages in gout need to be clarified. The overall objective of this thesis was to investigate the contributions of neutrophils, monocytes and macrophages to acute gouty inflammation. To determine whether the presence of other cells may be affecting neutrophil activation in gout inflammation, human blood neutrophils were stimulated with MSU as a purified population and in a mixed white blood cell population. The half-life of neutrophils in culture increased from 10h in purified neutrophil cultures to >24h in mixed cell cultures. The increase in viability was associated with large increases in cytokine production (TNF[alpha], IL-1[Beta], IL-6, IL-8) in mixed cell cultures. Exposure of neutrophils to media conditioned by MSU-stimulated mononuclear cells improved both neutrophil viability and stimulated the IL-8 production from neutrophils to a greater extent than direct contact with MSU. Exposure to conditioned media also primed MSU-stimulated neutrophil superoxide responses. High superoxide production was also observed when serum was lowered to <10%. These results indicate that neutrophil activation in gout largely occurs via soluble factors present in the pro-inflammatory environment, rather than by direct contact with MSU alone. Polygodial, a compound derived from the plant, Horopito (Pseudowintera colorata), and a number of structurally related sesquiterpene dialdehydes were tested for their potential to suppress neutrophil activation in gout. Polygodial inhibited MSU- and PMAstimulated neutrophil superoxide production in vitro with IC50 values of 0.78 [micron] and 0.16 [micron] respectively. This activity was largely dependent on the dialdehyde functionality but was enhanced by the presence of ring hydroxyl groups. The removal of hydroxyl groups was also associated with an increased cytoxicity. Polygodial and two other sesquiterpene dialdehyde compounds inhibited neutrophil superoxide production in an MSU model of acute gout, while Polygodial and a second compound also inhibited neutrophil recruitment. These studies confirm Polygodial and two structurally related sesquiterpene dialdehyde as potential anti-inflammatory compounds for potential use in gout. To confirm whether monocytes and/or macrophages were important in the onset of gout, resident macrophages and MSU-recruited monocytes were studied in a peritoneal model of MSU-induced inflammation. Gr-1+,7/4- monocytes were recruited to MSU-induced inflammation within 4h, however high cytokine production (IL-1[beta], TNF[alpha], IL-6, MCP-1) in the peritoneum preceded peak monocyte infiltration. Infiltrating monocytes did not produce high amounts of IL-6 and TNF[alpha], nor were they able to produce proinflammatory cytokines when re-stimulated with MSU ex vivo. However, resident macrophages exhibited production of IL-1[beta], TNF[alpha] and IL-6 following exposure to MSU ex vivo. Depletion of macrophages in vivo by clodronate liposomes led to a reduced recruitment of neutrophils and a lowered production of IL-1[beta] and IL-6 without affecting the recruitment of monocytes. These data identify macrophages, rather than monocytes, as key cells in initiationing and driving of inflammation in acute gout. The lack of responses from MSU-stimulated monocytes led to the question of what function, if any, was being exhibited by monocytes during MSU-induced inflammation. Therefore, the phenotype and corresponding function of monocytes was profiled over the course of MSU-induced inflammation. Newly recruited monocytes were small in size and expressed high levels of Gr-1 and 7/4, low levels of F4/80 and CD80. Over 48h the expression of Gr-1 and 7/4 was lost, while the expression of CD80 increased. These changes were associated with an increase in cell size, phagocytic capacity, and the production of pro-inflammatory cytokines in response to MSU. Changes in phenotype indicated differentiation into immature and then resident-like macrophages, and this was confirmed by PKH26-labelling infiltrating monocytes, which acquired an F4/80hi, resident macrophage phenotype after 3 to 5 days. Differentiation into macrophages was associated with an increased uptake of apoptotic neutrophils both in vivo and following ex vivo incubation. The phagocytosis of apoptotic neutrophils was associated with a reduced production of IL-1[beta] following stimulation with MSU ex vivo. These results indicate that MSU-recruited monocytes are not pro-inflammatory cells in gout, rather, they differentiate into resident macrophages over 3 to 5 days, and may over time aid in the resolution of gouty inflammation through the clearance of apoptotic neutrophils, a process that also suppresses further inflammatory responses. Together, the results of this thesis indicate that macrophages are key pro-inflammatory cells in gout contributing to neutrophil recruitment and cytokine production; that recruited neutrophils are activated by soluble mediators produced by MSU-activated cells, as well as by low serum environments; and that recruited monocytes are capable of both mediating the resolution of MSU-induced inflammation and replenishing the local resident macrophage population. These results provide a more clear model of the cellular events that occur during acute gout in humans that to date have been limited by a lack of in vivo-based studies.</p>

Cellular Inflammation in Models of Acute Gout

<p>Gout is a common form of inflammatory arthritis that is caused by the precipitation of monosodium urate crystals (MSU) in the joints. The acute form of gout is associated with sudden painful inflammatory episodes characterised by a large infiltration of neutrophils. The involvement of monocytes and macrophages is also recognised to be important, although the exact roles of monocytes and macrophages in gout need to be clarified. The overall objective of this thesis was to investigate the contributions of neutrophils, monocytes and macrophages to acute gouty inflammation. To determine whether the presence of other cells may be affecting neutrophil activation in gout inflammation, human blood neutrophils were stimulated with MSU as a purified population and in a mixed white blood cell population. The half-life of neutrophils in culture increased from 10h in purified neutrophil cultures to >24h in mixed cell cultures. The increase in viability was associated with large increases in cytokine production (TNF[alpha], IL-1[Beta], IL-6, IL-8) in mixed cell cultures. Exposure of neutrophils to media conditioned by MSU-stimulated mononuclear cells improved both neutrophil viability and stimulated the IL-8 production from neutrophils to a greater extent than direct contact with MSU. Exposure to conditioned media also primed MSU-stimulated neutrophil superoxide responses. High superoxide production was also observed when serum was lowered to <10%. These results indicate that neutrophil activation in gout largely occurs via soluble factors present in the pro-inflammatory environment, rather than by direct contact with MSU alone. Polygodial, a compound derived from the plant, Horopito (Pseudowintera colorata), and a number of structurally related sesquiterpene dialdehydes were tested for their potential to suppress neutrophil activation in gout. Polygodial inhibited MSU- and PMAstimulated neutrophil superoxide production in vitro with IC50 values of 0.78 [micron] and 0.16 [micron] respectively. This activity was largely dependent on the dialdehyde functionality but was enhanced by the presence of ring hydroxyl groups. The removal of hydroxyl groups was also associated with an increased cytoxicity. Polygodial and two other sesquiterpene dialdehyde compounds inhibited neutrophil superoxide production in an MSU model of acute gout, while Polygodial and a second compound also inhibited neutrophil recruitment. These studies confirm Polygodial and two structurally related sesquiterpene dialdehyde as potential anti-inflammatory compounds for potential use in gout. To confirm whether monocytes and/or macrophages were important in the onset of gout, resident macrophages and MSU-recruited monocytes were studied in a peritoneal model of MSU-induced inflammation. Gr-1+,7/4- monocytes were recruited to MSU-induced inflammation within 4h, however high cytokine production (IL-1[beta], TNF[alpha], IL-6, MCP-1) in the peritoneum preceded peak monocyte infiltration. Infiltrating monocytes did not produce high amounts of IL-6 and TNF[alpha], nor were they able to produce proinflammatory cytokines when re-stimulated with MSU ex vivo. However, resident macrophages exhibited production of IL-1[beta], TNF[alpha] and IL-6 following exposure to MSU ex vivo. Depletion of macrophages in vivo by clodronate liposomes led to a reduced recruitment of neutrophils and a lowered production of IL-1[beta] and IL-6 without affecting the recruitment of monocytes. These data identify macrophages, rather than monocytes, as key cells in initiationing and driving of inflammation in acute gout. The lack of responses from MSU-stimulated monocytes led to the question of what function, if any, was being exhibited by monocytes during MSU-induced inflammation. Therefore, the phenotype and corresponding function of monocytes was profiled over the course of MSU-induced inflammation. Newly recruited monocytes were small in size and expressed high levels of Gr-1 and 7/4, low levels of F4/80 and CD80. Over 48h the expression of Gr-1 and 7/4 was lost, while the expression of CD80 increased. These changes were associated with an increase in cell size, phagocytic capacity, and the production of pro-inflammatory cytokines in response to MSU. Changes in phenotype indicated differentiation into immature and then resident-like macrophages, and this was confirmed by PKH26-labelling infiltrating monocytes, which acquired an F4/80hi, resident macrophage phenotype after 3 to 5 days. Differentiation into macrophages was associated with an increased uptake of apoptotic neutrophils both in vivo and following ex vivo incubation. The phagocytosis of apoptotic neutrophils was associated with a reduced production of IL-1[beta] following stimulation with MSU ex vivo. These results indicate that MSU-recruited monocytes are not pro-inflammatory cells in gout, rather, they differentiate into resident macrophages over 3 to 5 days, and may over time aid in the resolution of gouty inflammation through the clearance of apoptotic neutrophils, a process that also suppresses further inflammatory responses. Together, the results of this thesis indicate that macrophages are key pro-inflammatory cells in gout contributing to neutrophil recruitment and cytokine production; that recruited neutrophils are activated by soluble mediators produced by MSU-activated cells, as well as by low serum environments; and that recruited monocytes are capable of both mediating the resolution of MSU-induced inflammation and replenishing the local resident macrophage population. These results provide a more clear model of the cellular events that occur during acute gout in humans that to date have been limited by a lack of in vivo-based studies.</p>

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF

Background and AimsPatients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients.Material and MethodsBlood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions.ResultsSeveral features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion.ConclusionsGenomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies.

The effect of pamidronate delivery in bisphosphonate-naïve patients on neutrophil chemotaxis and oxidative burst

The pathogenesis of medication-related osteonecrosis of the jaw (MRONJ), a morbid condition associated with bisphosphonate administration, has not been fully elucidated. Recent research utilizing a murine model has revealed that the neutrophil becomes dysfunctional following exposure to bisphosphonates. Accordingly, the impairment of neutrophil function could play an important role in the pathogenesis of MRONJ via an infectious mechanism mediated by the suppression of the innate immune system. Currently, the existing human data are insufficient to substantiate this theory. To investigate, we isolated neutrophils from blood and oral rinse samples from bisphosphonate-naïve patients who were recently diagnosed with multiple myeloma both prior to and one month following their initial infusion of pamidronate, an intravenous bisphosphonate agent. Stimulated blood and oral neutrophil superoxide production and chemotactic capabilities were found to be impaired relative to baseline values. These results suggest that impaired neutrophil function may partially contribute to the aetiology underlying the pathophysiological processes linked to the development of MRONJ. Further, as the functional status of circulating neutrophils was reflected in the oral cavity where sampling can be accomplished in a non-invasive fashion, it is conceivable that neutrophil function could serve as a potential biomarker for MRONJ prognostication.

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2-complex

Two formyl peptide receptors (FPR1 and FPR2), abundantly expressed by neutrophils, regulate both pro-inflammatory tissue recruitment of neutrophils and resolution of inflammatory reactions. This dual functionality of the FPRs, opens for a possibility to develop receptor selective therapeutics as mechanism for novel anti-inflammatory treatments. In line with this, high throughput screening studies have identified numerous FPR ligands belonging to different structural classes, but a potent FPR1 agonist with defined biased signaling and functional selectivity has not yet been reported. In this study, we used an FPR1 selective small compound agonist (RE) that represents a chemical entity developed from NOX2 activators identified from our earlier screening studies (WO2012127214). This FPR1 agonist potently activates neutrophils to produce reactive oxygen species (ROS, EC50 ~1 nM), whereas it is a weaker chemoattractant than the prototype FPR1 agonist fMLF. At the signaling level, RE has a strong bias towards the PLC-PIP2-Ca2+ pathway and ERK1/2 activation but away from β-arrestin recruitment and the ability to recruit neutrophils chemotactically. In addition, FPR1 when activated by RE could cross-regulate other receptor-mediated neutrophil functions. In comparison to the peptide agonist fMLF, RE is more resistant to oxidization-induced inactivation by the MPO-H2O2-halide system. In summary, this study describes as a novel FPR1 agonist displaying a biased signaling and functional selectivity when activating FPR1 in human blood neutrophils. RE could possibly be a useful tool compound not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1specific drug therapeutics.

Eugenol prevents fMLF-induced superoxide anion production in human neutrophils by inhibiting ERK1/2 signaling pathway and p47phox phosphorylation

Eugenol is a polyphenol extracted from Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL–20 µg/mL) of eugenol for 30 min, inhibited significantly (p < 0.001) superoxide anion generation induced by the chemotactic peptide formyl-Met-Leu-Phe (fMLF) with an IC50 of 5 µg/mL. Phorbolmyristate acetate (PMA)-stimulated O2− production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.

Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.