The coadministration of trehalose dibehenate and monosodium urate crystals promotes an antitumor phenotype in human-derived myeloid cells.

Trehalose dibehenate (TDB), a ligand for the macrophage-inducible C-type lectin, has shown promise as an adjuvant for preventative vaccines and also as an anticancer agent in murine assays. The potential for TDB to affect the antitumor immune response of human myeloid cells, however, has not been studied. We investigated the effect of the adjuvants TDB and monosodium urate (MSU) crystals on the protumor or antitumor immune phenotype of human monocytes, macrophages and monocyte-derived dendritic cells (Mo-DCs). TDB treatment alone led to an inflammatory response in all three cell types, which was most pronounced when using human monocytes, with MSU augmenting this response. TDB also decreased cell surface markers associated with a protumorigenic phenotype, with MSU showing some ability to augment this response. Notably, a significant reduction in CD115 was observed for all antigen-presenting cells upon TDB or MSU + TDB treatment. The potential to increase the antigen-presenting capabilities of the myeloid cells was also observed upon treatment with TDB and MSU + TDB, as indicated by the upregulation of cell surface markers such as CD86 for all three cell types and a favorable ratio of interleukin (IL)-12p40 to IL-10 for monocytes stimulated with MSU + TDB. There was no significant production of IL-12p40 by Mo-DC; however, in a mixed lymphocyte assay, MSU + TDB costimulation of Mo-DC led to a significant increase in CD4+ T-cell numbers and in the IL-12p40-to-IL-10 ratio. Taken together, these findings show for the first time the potential of MSU + TDB costimulation to favor a tumor-suppressive phenotype in human-derived myeloid cells.

The effects of trehalose glycolipid presentation on cytokine production by GM-CSF macrophages.

Trehalose glycolipids (TGLs) are promising vaccine adjuvants, however effects of glycolipid presentation in the in vitro evaluation, and ultimate selection, of lead vaccine adjuvants are often overlooked. To this end, we synthesised a variety of TGLs and determined how the physicochemical presentation of these lipids influenced the cytokine response by bone marrow derived macrophages (BMMs). The TGLs were presented to wild-type and Mincle-/- BMMs as micellar solutions, coated on plates, coated on beads or surfactant solubilised. Medium to long-chain TGLs, either coated on plates or surfactant solubilised, resulted in the highest BMM activation. Stimulation of BMMs with TGLs coated on beads led to a decreased cytokine response, as compared to TGLs alone. All the TGL responses were Mincle dependent, however the mode of presentation did not have the same effect for each individual TGL. This was most apparent for the C22 trehalose monoester, which showed reduced activity compared to its diester counterpart when presented on a plate, but similar activity to the diester when presented as micelles or on beads. Taken together, our findings support the use of several in vitro assays for selecting lead vaccine adjuvants, particularly if structural differences between the adjuvants are pronounced. Graphical abstract The mode of glycolipid presentation, such as micellar solutions, coated on plates, coated on beads or surfactant solubilised, influences the immune response to trehalose glycolipids.

A Phenomic Scan of the Norfolk Island Genetic Isolate Identifies a Major Pleiotropic Effect Locus Associated with Metabolic and Renal Disorder Markers.

Multiphenotype genome-wide association studies (GWAS) may reveal pleiotropic genes, which would remain undetected using single phenotype analyses. Analysis of large pedigrees offers the added advantage of more accurately assessing trait heritability, which can help prioritise genetically influenced phenotypes for GWAS analysis. In this study we performed a principal component analysis (PCA), heritability (h2) estimation and pedigree-based GWAS of 37 cardiovascular disease -related phenotypes in 330 related individuals forming a large pedigree from the Norfolk Island genetic isolate. PCA revealed 13 components explaining >75% of the total variance. Nine components yielded statistically significant h2 values ranging from 0.22 to 0.54 (P<0.05). The most heritable component was loaded with 7 phenotypic measures reflecting metabolic and renal dysfunction. A GWAS of this composite phenotype revealed statistically significant associations for 3 adjacent SNPs on chromosome 1p22.2 (P<1x10-8). These SNPs form a 42kb haplotype block and explain 11% of the genetic variance for this renal function phenotype. Replication analysis of the tagging SNP (rs1396315) in an independent US cohort supports the association (P = 0.000011). Blood transcript analysis showed 35 genes were associated with rs1396315 (P<0.05). Gene set enrichment analysis of these genes revealed the most enriched pathway was purine metabolism (P = 0.0015). Overall, our findings provide convincing evidence for a major pleiotropic effect locus on chromosome 1p22.2 influencing risk of renal dysfunction via purine metabolism pathways in the Norfolk Island population. Further studies are now warranted to interrogate the functional relevance of this locus in terms of renal pathology and cardiovascular disease risk.

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1ß production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo.

Boysenberry ingestion supports fibrolytic macrophages with the capacity to ameliorate chronic lung remodeling.

Lung fibrosis negatively impacts on lung function in chronic asthma and is linked to the development of profibrotic macrophage phenotypes. Epidemiological studies have found that lung function benefits from increased consumption of fruit high in polyphenols. We investigated the effect of boysenberry consumption, in both therapeutic and prophylactic treatment strategies in a mouse model of chronic antigen-induced airway inflammation. Boysenberry consumption reduced collagen deposition and ameliorated tissue remodeling alongside an increase in the presence of CD68+CD206+arginase+ alternatively activated macrophages in the lung tissue. The decrease in tissue remodeling was associated with increased expression of profibrolytic matrix metalloproteinase-9 protein in total lung tissue. We identified alternatively activated macrophages in the mice that consumed boysenberry as a source of the matrix metalloproteinase-9. Oral boysenberry treatment may moderate chronic tissue remodeling by supporting the development of profibrolytic alternatively activated macrophages expressing matrix metalloproteinase-9. Regular boysenberry consumption therefore has the potential to moderate chronic lung remodeling and fibrosis in asthma and other chronic pulmonary diseases.

Mapping eQTLs in the Norfolk Island genetic isolate identifies candidate genes for CVD risk traits.

Cardiovascular disease (CVD) affects millions of people worldwide and is influenced by numerous factors, including lifestyle and genetics. Expression quantitative trait loci (eQTLs) influence gene expression and are good candidates for CVD risk. Founder-effect pedigrees can provide additional power to map genes associated with disease risk. Therefore, we identified eQTLs in the genetic isolate of Norfolk Island (NI) and tested for associations between these and CVD risk factors. We measured genome-wide transcript levels of blood lymphocytes in 330 individuals and used pedigree-based heritability analysis to identify heritable transcripts. eQTLs were identified by genome-wide association testing of these transcripts. Testing for association between CVD risk factors (i.e., blood lipids, blood pressure, and body fat indices) and eQTLs revealed 1,712 heritable transcripts (p < 0.05) with heritability values ranging from 0.18 to 0.84. From these, we identified 200 cis-acting and 70 trans-acting eQTLs (p < 1.84 × 10(-7)) An eQTL-centric analysis of CVD risk traits revealed multiple associations, including 12 previously associated with CVD-related traits. Trait versus eQTL regression modeling identified four CVD risk candidates (NAAA, PAPSS1, NME1, and PRDX1), all of which have known biological roles in disease. In addition, we implicated several genes previously associated with CVD risk traits, including MTHFR and FN3KRP. We have successfully identified a panel of eQTLs in the NI pedigree and used this to implicate several genes in CVD risk. Future studies are required for further assessing the functional importance of these eQTLs and whether the findings here also relate to outbred populations.

Delayed liver regeneration in C3H/HeJ mice: possible involvement of haemodynamic and structural changes in the hepatic microcirculation.

NEW FINDINGS: What is the central question of this study? The liver regenerative process is complex and involves a sequence of signalling events, but the possible involvement of structural and haemodynamic changes in vivo during this process has never been explored. What is the main finding and its importance? Normal sinusoidal blood flow and velocity are crucial for a normal regenerative response, and delays in these haemodynamic events resulted in impaired liver regeneration in lipopolysaccharide-insensitive, C3H/HeJ mice. Toll-like receptor 4 signalling is required for restoration of normal liver architecture during the liver regenerative process. Liver regeneration is delayed in mice with a defective Toll-like receptor 4 (TLR4; C3H/HeJ mice) but is normal in TLR4 knockouts (TLR4-/- ). Here, we investigated the possible involvement of structural and haemodynamic changes in vivo in the underlying mechanism. In lipopolysaccharide-sensitive (C3H/HeN and C57BL/6) and lipopolysaccharide-insensitive (C3H/HeJ and TLR4-/- ) mice, a 70% partial hepatectomy (PH) was performed under inhalational anaesthesia. At days 3 and 7 after PH, the hepatic microcirculation was interrogated using intravital microscopy. Delayed liver regeneration was confirmed in C3H/HeJ, but not in C3H/HeN, C57BL/6 (WT) or TLR4-/- mice by liver weight-to-body-weight ratio, the percentage of proliferating cell nuclear antigen (PCNA)-positive cells and mitotic index data. At day 3 after PH, sinusoidal red blood cell velocity increased by 100% in C3H/HeN mice, but by only 40% in C3H/HeJ mice. Estimated sinusoidal blood flow was significantly higher at day 7 after PH in C3H/HeN than in C3H/HeJ mice. The hepatic cord width was significantly larger in C3H/HeN than in C3H/HeJ mice at day 3 and it was significantly larger in TLR4-/- than in C57BL/6 WT mice at day 7 after PH. Hepatocyte nucleus density and functional sinusoidal density was significantly reduced at days 3 and 7 after PH in all mouse strains compared with their zero-time controls. Functional sinusoidal density was significantly lower in C3H/HeJ compared with C3H/HeN mice at day 7 after PH. The present study indicates that altered sinusoidal blood flow and velocity in C3H/HeJ mice may contribute to the observed delay in the regenerative response in these mice. In addition, restoration of normal liver architecture may be delayed in TLR4-/- mice.

Absence of airway inflammation in a large proportion of adolescents with asthma.

BACKGROUND AND OBJECTIVE: Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. METHODS: Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12-17 years). Asthma was identified on the basis of wheeze and asthma history. RESULTS: The proportion of NEA (sputum eosinophils <2.5%) was 54%. In this group, atopy, sputum neutrophil, eosinophil, eosinophil cationic protein (ECP), endotoxin, neutrophil elastase and IL-8 levels were not different from those without asthma. In contrast, eosinophilic asthma (EA) was associated with atopy and sputum ECP and IL-8. The majority of NEA had no evidence of inflammation; only 14% had neutrophilia (≥61% neutrophils), compared with 11% of EA, and 15% of those without asthma. Small differences in FEV1 (NS) were found between EA and NEA, but symptom prevalence and severity was not different (63% of EA and 52% of NEA were classified moderate to severe). CONCLUSION: NEA is common in adolescent asthma and has similar clinical characteristics as EA. Neutrophils do not appear to play a role in NEA in adolescents, and underlying mechanisms may not involve airway inflammation.

The uptake of trehalose glycolipids by macrophages is independent of Mincle.

Trehalose glycolipids play an important role in the pathogenesis of Mycobacterium tuberculosis and are used as adjuvants for vaccines; however, much still remains unanswered about the mechanisms through which these glycolipids exert their immunomodulatory potential. Recently, the macrophage-inducible C-type lectin Mincle was determined to be the receptor for trehalose glycolipids, yet the role played by Mincle in glycolipid uptake is unknown. Accordingly, we developed several fluorescent trehalose glycolipid reporter systems that can be used to study the uptake of soluble trehalose glycolipids and glycolipid-coated particles by macrophages. Our studies revealed that, although Mincle is essential for the activation of macrophages by trehalose glycolipids, the receptor does not play a role in the uptake of these glycolipids or of glycolipid-coated particles.

Role of miR-146a in regulation of the acute inflammatory response to monosodium urate crystals.

OBJECTIVES: MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional repressors of gene expression. We hypothesised that miRNA regulate gene expression of proinflammatory cytokines in response to monosodium urate (MSU) crystals. METHODS: We stimulated human monocytic THP-1 cells with MSU crystals and examined miRNA and proinflammatory cytokine gene expression. The effects of miR-146a overexpression were examined by transfecting THP-1 cells with miR-146a precursor. miR-146a expression was examined in the urate peritonitis model, in peripheral blood mononuclear cells from people with gout and control participants, and in gouty tophus samples. RESULTS: MSU crystals increased miR-146a expression in THP-1 cells, but not other miRNA implicated in interleukin (IL)-1ß regulation. Overexpression of miR-146a expression reduced MSU crystal-induced IL-1ß, tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and IL-8 gene expression. In the urate peritonitis model, reduced miR-146a expression was observed during the acute inflammatory response to MSU crystal injection. In people with intercritical gout, peripheral blood mononuclear cells expressed significantly higher levels of miR-146a, compared with normouricaemic and hyperuricaemic control participants and those with acute gout flares. Expression of miR-146a was also observed in all tophus samples. CONCLUSIONS: Collectively, these data suggest that miR-146a is a transcriptional brake that is lost during the acute inflammatory response to MSU crystals.

Increased numbers of monocyte-derived dendritic cells during successful tumor immunotherapy with immune-activating agents.

Local treatment with selected TLR ligands or bacteria such as bacillus Calmette-Guérin increases antitumor immune responses and delays tumor growth. It is thought that these treatments may act by activating tumor-associated dendritic cells (DCs), thereby supporting the induction of antitumor immune responses. However, common parameters of successful immune activation have not been identified. We used mouse models to compare treatments with different immune-activating agents for the ability to delay tumor growth, improve priming of tumor-specific T cells, and induce early cytokine production and DC activation. Treatment with polyinosinic-polycytidylic acid or a combination of monosodium urate crystals and Mycobacterium smegmatis was effective at delaying the growth of s.c. B16 melanomas, orthotopic 4T1 mammary carcinomas, and reducing 4T1 lung metastases. In contrast, LPS, monosodium urate crystals, or M. smegmatis alone had no activity. Effective treatments required both NK1.1(+) and CD8(+) cells, and resulted in increased T cell priming and the infiltration of NK cells and CD8(+) T cells in tumors. Unexpectedly, both effective and ineffective treatments increased DC numbers and the expression of costimulatory molecules in the tumor-draining lymph node. However, only effective treatments induced the rapid appearance of a population of monocyte-derived DCs in the draining lymph node, early release of IL-12p70 and IFN-γ, and low IL-10 in the serum. These results suggest that the activation of existing DC subsets is not sufficient for the induction of antitumor immune responses, whereas early induction of Th1 cytokines and monocyte-derived DCs are features of successful activation of antitumor immunity.

Structure-Activity Relationships of the Bioactive Thiazinoquinone Marine Natural Products Thiaplidiaquinones A and B.

In an effort to more accurately define the mechanism of cell death and to establish structure-activity relationship requirements for the marine meroterpenoid alkaloids thiaplidiaquinones A and B, we have evaluated not only the natural products but also dioxothiazine regioisomers and two precursor quinones in a range of bioassays. While the natural products were found to be weak inducers of ROS in Jurkat cells, the dioxothiazine regioisomer of thiaplidiaquinone A and a synthetic precursor to thiaplidiaquinone B were found to be moderately potent inducers. Intriguingly, and in contrast to previous reports, the mechanism of Jurkat cell death (necrosis vs. apoptosis) was found to be dependent upon the positioning of one of the geranyl sidechains in the compounds with thiaplidiaquinone A and its dioxothiazine regioisomer causing death dominantly by necrosis, while thiaplidiaquinone B and its dioxothiazine isomer caused cell death via apoptosis. The dioxothiazine regioisomer of thiaplidiaquinone A exhibited more potent in vitro antiproliferative activity against human tumor cells, with NCI sub-panel selectivity towards melanoma cell lines. The non-natural dioxothiazine regioisomers were also more active in antiplasmodial and anti-farnesyltransferase assays than their natural product counterparts. The results highlight the important role that natural product total synthesis can play in not only helping understand the structural basis of biological activity of natural products, but also the discovery of new bioactive scaffolds.

The effect of diet-induced obesity on the inflammatory phenotype of non-adipose-resident macrophages in an in vivo model of gout.

OBJECTIVE: Gout is strongly associated with obesity. The aim of this study was to determine if obesity altered the inflammatory phenotype of non-adipose tissue-resident macrophages in response to the gout-causing agent monosodium urate (MSU) crystals. METHODS: C57BL/6J mice were fed a high-fat diet for 12 weeks. Resident peritoneal macrophages were stimulated ex vivo with MSU crystals (200 µg/ml for 18 h) and the supernatants were collected. Mice were challenged with MSU crystals in vivo (3 mg, intraperitonal) and the peritoneal lavage fluid was collected (8 and 16 h). Cytokine and chemokine levels were analysed by multiplex bead array and peritoneal cell populations were analysed by flow cytometry. RESULTS: Peritoneal macrophages from obese mice produced elevated background levels of IL-6, monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived cytokine (KC) that decreased following MSU crystal stimulation ex vivo. MSU-induced IL-1ß production was higher for macrophages from obese mice compared with controls. High background levels of IL-6, MCP-1, KC and GM-CSF, but not IL-1ß, were measured in the peritoneal fluid of unchallenged obese mice. MSU crystal challenge in vivo raised IL-1ß levels equally in both control and obese mice, whereas elevated background levels of IL-6, MCP-1, KC and GM-CSF levels dropped in obese mice. There was a consistent trend towards lower numbers of naive peritoneal resident macrophages and MSU-recruited monocytes and neutrophils in obese mice. CONCLUSION: Obesity induces a background pro-inflammatory environment orchestrated by non-adipose tissue-resident macrophages. However, this may not automatically translate into exacerbation of MSU crystal-induced inflammation in gout.

Discovery of lipids from B. longum subsp. infantis using whole cell MALDI analysis.

Bifidobacteria are dominant members of the microbial community in the intestinal tract of infants, and studies have shown that glycolipids extracted from the cell surface of these bacteria elicit beneficial immune responses. Accordingly, the identification and structural characterization of glycolipids from the cell wall of bifidobacteria is the first step in correlating glycolipid structure with biological activity. Using whole cell MALDI as a screening tool, we herein present for the first time the identification and structural elucidation of the major polar lipids from Bifidobacterium longum subs. infantis. The lipids identified include an unprecedented plasmenyl cyclophosphatidic acid and a mixed acetal glycolipid, with the latter subsequently being isolated and found to suppress the innate immune response.

Mechanisms of spontaneous resolution of acute gouty inflammation.

Acute gout is an auto-inflammatory disease characterized by self-limiting inflammation in response to the deposition of monosodium urate (MSU) crystals in the joints or tissues. Recognition of MSU triggers activation of the NLRP3 inflammasome, release of active interleukin (IL)-1ß, and amplification of the inflammatory response by the surrounding tissue followed by recruitment and activation of inflammatory leukocytes. The shutdown of this inflammatory response is linked to a number of regulatory events ranging from crystal coating and apoptotic cell clearance through to pro-inflammatory cytokine regulation and transforming growth factor ß1 (TGFß1) production. This review will highlight mechanisms that limit acute inflammation triggered by MSU crystals and suggests areas for further research.

Neutrophil cannibalism triggers transforming growth factor ß1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal-induced inflammation in mice.

OBJECTIVE: To identify macrophage-independent sources of transforming growth factor ß1 (TGFß1) production during monosodium urate monohydrate (MSU) crystal-induced inflammation and to determine how TGFß1 alters MSU crystal-recruited neutrophil functions. METHODS: C57BL/6J mice were injected intraperitoneally with MSU crystals with or without TGFß1-neutralizing antibody. MSU crystal-recruited peritoneal and blood neutrophils were purified and cultured ex vivo. Peritoneal neutrophils were treated with the caspase inhibitor Q-VD-OPh, anti-TGFß1 antibody, or fluorochrome-labeled apoptotic neutrophils. Neutrophils were analyzed for expression of annexin V, caspase 3, and TGFß1 by flow cytometry or fluorescence microscopy, for superoxide production using the redox-sensitive dye water-soluble tetrazolium 1, and for TGFß1 and interleukin-1ß (IL-1ß) production by enzyme-linked immunosorbent assay. RESULTS: Eighteen hours after MSU crystal administration in vivo, TGFß1 levels were elevated in peritoneal lavage fluids, and a significant number of peritoneal neutrophils were TGFß1+. Purified blood or peritoneal neutrophils cultured ex vivo showed TGFß1+ neutrophils coexpressing the apoptosis marker caspase 3 and increased TGFß1 production, both of which dropped following inhibition of apoptosis. Live neutrophils that had phagocytosed apoptotic neutrophils showed greatest TGFß1 expression. Superoxide production by purified MSU crystal-recruited neutrophils ex vivo was enhanced by anti-TGFß1 antibody treatment. Neutrophils purified from the peritoneum of MSU crystal-challenged mice treated with anti-TGFß1 antibody produced elevated levels of superoxide, but neutrophil IL-1ß production was unaffected. CONCLUSION: Neutrophil cannibalism and TGFß1 production have the potential to make a significant contribution to the controlled resolution of neutrophil-driven inflammatory diseases such as gout.

Hyperuricaemia elevates circulating CCL2 levels and primes monocyte trafficking in subjects with inter-critical gout.

OBJECTIVE: To investigate the effect of hyperuricaemia on serum chemokine (C-C motif) ligand 2 (CCL2) levels and blood monocytes in people with gout. METHODS: Whole blood was collected from subjects with a history of acute or chronic gout but not currently experiencing an attack of gout, subjects with asymptomatic hyperuricaemia and healthy individuals with normouricaemia. Serum concentrations of CCL2 were measured by bead array and levels of CD14(+)/CD11b(+) blood monocytes determined by flow cytometry. RESULTS: Subjects with gout and asymptomatic hyperuricaemia had higher serum levels of CCL2 and showed an increase in the percentage of circulating CD14(+) monocytes compared with subjects with normouricaemia. CONCLUSION: Hyperuricaemia causes elevated serum CCL2 levels and increased monocyte recruitment that may be driven by soluble uric acid-induced CCL2 production. Hyperuricaemia may initiate subclinical priming of circulating blood monocytes for adhesion and trafficking during a gout attack.

Induction of T cell responses and recruitment of an inflammatory dendritic cell subset following tumor immunotherapy with Mycobacterium smegmatis.

Mycobacteria and their cell wall components have been used with varying degrees of success to treat tumors, and Mycobacterium bovis BCG remains in use as a standard treatment for superficial bladder cancer. Mycobacterial immunotherapy is very effective in eliciting local immune responses against solid tumors when administered topically; however, its effectiveness in eliciting adaptive immune responses has been variable. Using a subcutaneous mouse thymoma model, we investigated whether immunotherapy with Mycobacterium smegmatis, a fast-growing mycobacterium of low pathogenicity, induces a systemic adaptive immune response. We found that M. smegmatis delivered adjacent to the tumor site elicited a systemic anti-tumor immune response that was primarily mediated by CD8(+) T cells. Of note, we identified a CD11c(+)CD40(int)CD11b(hi)Gr-1(+) inflammatory DC population in the tumor-draining lymph nodes that was found only in mice treated with M. smegmatis. Our data suggest that, rather than rescuing the function of the DC already present in the tumor and/or tumor-draining lymph node, M. smegmatis treatment may promote anti-tumor immune responses by inducing the involvement of a new population of inflammatory cells with intact function.

Monosodium urate monohydrate crystal-recruited noninflammatory monocytes differentiate into M1-like proinflammatory macrophages in a peritoneal murine model of gout.

OBJECTIVE: To profile monosodium urate monohydrate (MSU) crystal-recruited monocyte inflammatory function during the course of in vivo differentiation, in a murine model of peritoneal MSU crystal-induced inflammation. METHODS: C57BL/6J mice were injected intraperitoneally with MSU crystals, and the peritoneal cells were harvested at different time points. The MSU crystal-recruited monocyte/macrophage population was analyzed for the expression of differentiation and activation markers, cytokine production following MSU crystal restimulation ex vivo and in vivo, expression of NLRP3-associated proteins (ASC, caspase 1) and pro-interleukin-1ß (proIL-1ß), and phagocytic capacity. RESULTS: Monocytes recruited 8 hours after MSU crystal stimulation (F4/80(low) Gr-1(int) 7/4+) exhibited poor phagocytic capacity, expressed low levels of proIL-1ß, and failed to produce proinflammatory cytokines in response to MSU crystal restimulation. In the absence of MSU crystal restimulation, differentiating monocytes produced low levels of transforming growth factor ß1 ex vivo, and this was abrogated following MSU crystal restimulation. Over time these cells developed a proinflammatory phenotype in vivo, characterized by the production of IL-1ß, tumor necrosis factor α, IL-6, CCL2 (monocyte chemotactic protein 1), and CXCL1 (cytokine-induced neutrophil chemoattractant) following ex vivo MSU crystal restimulation, and leading to IL-1ß production and cell infiltration following MSU crystal rechallenge in vivo. Proinflammatory function was associated with differentiation toward a macrophage phenotype (F4/80(high) Gr-1-7/4-), an increase in phagocytic capacity, and an increase in the expression of proIL-1ß. CONCLUSION: MSU crystal-recruited monocytes differentiate into proinflammatory M1-like macrophages in vivo. This proinflammatory macrophage phenotype is likely to play a key role in perpetuating inflammation in gouty arthritis in the presence of ongoing deposition of fresh MSU crystals.

Bradykinin receptor 2 extends inflammatory cell recruitment in a model of acute gouty arthritis.

The aim of this study was determine the effect of bradykinin receptor antagonism on MSU crystal-induced chemokine production and leukocyte recruitment. Mice were injected intraperitoneally with monosodium urate (MSU) crystals ± bradykinin B1- or B2 receptor antagonists, Des-Arg-HOE-140 and HOE-140, respectively. MSU crystal-induced chemokine production and leukocyte recruitment in the peritoneum were measured over 24h and B1 and B2 receptor expression on leukocytes and peritoneal membrane was determined by flow cytometry and fluorescence microscopy. Data analysis showed that only B2 receptor antagonism decreased monocyte and neutrophil infiltration 24 h post MSU crystal administration. Decreased leukocyte infiltration was associated with reduced monocyte (CCL2) chemokine levels. MSU crystal-induced damage to the surrounding visceral membrane was also attenuated in the presence of B2 receptor antagonism. Together, these data show that bradykinin receptor 2 plays a role in maintaining MSU crystal-induced leukocyte infiltration and membrane permeability and identify the B2 receptor as a potential therapeutic target for managing inflammation in gout.