Neutrophil extracellular trap formation is associated with IL-1β and autophagy-related signaling in gout

Background

Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.

Methodology/Principal Findings

Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.

Conclusions/Significance

These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.     

MicroRNA-155 as a proinflammatory regulator via SHIP-1 down-regulation in acute gouty arthritis

Introduction

Gout is characterized by episodes of intense joint inflammation in response to intra-articular monosodium urate monohydrate (MSU) crystals. miR-155 is crucial for the proinflammatory activation of human myeloid cells and antigen-driven inflammatory arthritis. The functional role of miR-155 in acute gouty arthritis has not been defined. Therefore, the aim of this study was to examine the role of miR-155 in pathogenesis of acute gouty arthritis.

Methods

Samples from 14 patients with acute gouty arthritis and 10 healthy controls (HCs) were obtained. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were cultured in vitro with MSU crystals, and gene expression (human miR-155 and SHIP-1) were assessed by real-time PCR. THP-1 cells were stimulated by MSU crystals and/or miR-155 transfection and then subjected to Western blot analysis. Levels of human tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β in cell culture supernatants were measured by Luminex. Immunohistochemistry was performed on formalin-fixed gout tissues with anti–SHIP-1 antibody. A C57BL/6 J male mouse model of gout was used to analyze the expressions of miR-155, SHIP-1, and inflammatory cytokines.

Results

The samples from gouty arthritis were highly enriched in miR-155, with levels of expression being higher than those found in PBMC from HC. Treatment of the cells with MSU crystals strongly induced miR-155. In addition, overexpression of miR-155 in the cells decreased levels of SHIP-1 and promoted production of MSU-induced proinflammatory cytokines, such as TNF-α and IL-1β. Consistent with in vitro observations, miR-155 expression was elevated in the mouse model of gout. The production of inflammatory cytokines was markedly increased in MSU crystal induced peritonitis mice.

Conclusions

Overexpression of miR-155 in the gouty SFMC leads to suppress SHIP-1 levels and enhance proinflammatory cytokines.     

Decreased CXCR1 and CXCR2 expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration

Introduction

In anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.

Methods

Membrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.

Results

Expression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.

Conclusions

Expression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.     

Involvement of Neutrophil Hyporesponse and the Role of Toll-Like Receptors in Human Immunodeficiency Virus 1 Protection

Objectives

Neutrophils contribute to pathogen clearance through pattern recognition receptors (PRRs) activation. However, the role of PRRs in neutrophils in both HIV-1-infected [HIV-1(+)] and HIV-1-exposed seronegative individuals (HESN) is unknown. Here, a study was carried out to evaluate the level of PRR mRNAs and cytokines produced after activation of neutrophils from HIV-1(+), HESN and healthy donors.

Methods

The neutrophils were stimulated with specific agonists for TLR2, TLR4 and TLR9 in the presence of HIV-1 particles. Pro-inflammatory cytokine production, expression of neutrophil activation markers and reactive oxygen species (ROS) production were analyzed in neutrophils from HESN, HIV-1(+) and healthy donors (controls).

Results

We found that neutrophils from HESN presented reduced expression of PRR mRNAs (TLR4, TLR9, NOD1, NOD2, NLRC4 and RIG-I) and reduced expression of cytokine mRNAs (IL-1β, IL-6, IL-18, TNF-α and TGF-β). Moreover, neutrophils from HESN were less sensitive to stimulation through TLR4. Furthermore, neutrophils from HESN challenged with HIV-1 and stimulated with TLR2 and TLR4 agonists, produced significantly lower levels of reactive oxygen species, versus HIV-1(+).

Conclusions

A differential pattern of PRR expression and release of innate immune factors in neutrophils from HESN is evident. Our results suggest that lower neutrophil activation can be involved in protection against HIV-1 infection.     

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.     

Siglec-E Promotes β2-Integrin-dependent NADPH Oxidase Activation to Suppress Neutrophil Recruitment to the Lung

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.     

Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.     

The relationship of apolipoprotein B and very low density lipoprotein triglyceride with hyperuricemia and gout

Introduction

Gout results from an innate immune response to monosodium urate (MSU) crystals deposited in joints. Increased very low-density lipoprotein (VLDL) has been associated with gout. The apolipoprotein B (apo B), which is present on VLDL, regulates neutrophil response to MSU crystals and has been positively associated with gout. Furthermore, the gene (A1CF) encoding the complementation factor for the APOB mRNA-editing enzyme is associated with urate levels. However, the relationship of apo B and VLDL with gout and hyperuricaemia (HU) is still unclear. Therefore, we tested the association of VLDL and apo B with HU and with gout compared to HU.

Methods

New Zealand European (n = 90) and Māori and Pacific Island (Polynesian) (n = 90) male gout case and control sample sets were divided into normouricaemia (NU), asymptomatic HU and gout groups. Size exclusion chromatography and enzyme-linked immunosorbant assay was used to measure VLDL and apo B. Multivariate logistic regression was used to assess the risk of gout and HU per unit change in VLDL and apo B.

Results

Increased levels of VLDL triglycerides (Tg) were observed in the gout sample set compared to NU and HU in Europeans (P = 1.8 × 10^-6 and 1 × 10^-3, respectively), but only compared to NU in Polynesians (P = 0.023). This increase was driven by increased number of VLDL particles in the European participants and by the Tg-enrichment of existing VLDL particles in the Polynesian participants. Each mmol/L increase in VLDL Tg was significantly associated with gout in the presence of HU in Europeans, with a similar trend in Polynesians (OR = 7.61, P = 0.011 and 2.84, P = 0.069, respectively). Each μmol/L increase in total apo B trended towards decreased risk of HU (OR = 0.47; P = 0.062) and, conversely, with increased risk of gout compared to HU (OR = 5.60; P = 0.004).

Conclusions

Increased VLDL Tg is associated with the risk of gout compared to HU. A genetic approach should be taken to investigate the possibility for causality of VLDL in gout. Apolipoprotein B may have pleiotropic effects in determining HU and gout.     

Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis

Introduction

It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.

Methods

RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.

Results

We found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.

Conclusions

Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.     

Baeckein E suppressed NLRP3 inflammasome activation through inhibiting both the priming and assembly procedure: Implications for gout therapy

BackgroundBaeckein E (BF-2) was isolated from the aerial parts of Baeckea frutescens L., which has a long history of use in traditional medicine in Southeast Asia to treat inflammatory disease.PurposeBF-2 was identified to have inhibitory activity on nucleotide oligomerization domain (NOD)-like receptor protein-3 inflammasome (NLRP3) activation. This study aimed to investigate the related signaling cascade of BF-2 in both lipopolysaccharides (LPS)/ATP induced pyroptosis in J774A.1 macrophages and its application in a mouse model of gout induced by monosodium urate crystal (MSU).MethodsThe effect of BF-2 on NLRP3 inflammasome activation and gouty arthritis was studied in J774A.1 macrophages and male C57BL/6 mice. The J774A.1 macrophages were primed with LPS and stained by propidium iodide (PI) for cell pyroptosis detection. A gout mouse model was established by subcutaneous injection of MSU crystals into the hind paw of C57BL/6 mice. Mice were then randomly divided into different groups. The concentrations of IL-1β and IL-18 in both J774A.1 macrophage and gout mouse model were analyzed by ELISA. The NLRP3 inflammasome related protein expression was detected by western blot analysis. The inhibitory effects of BF-2 on NLRP3 inflammasome assembly were analyzed by immunoprecipitation assay. The roles of BF-2 in mitochondrial damage were imaged by Mito Tracker Green and Mito Tracker Red probes. The inhibitory effects of BF-2 on ROS production were imaged by DCF (2′,7′-dichlorofluorescein diacetate) probe.ResultsThe results demonstrated BF-2 could significantly suppress the cell pyroptosis and IL-1β secretion in macrophages. Furthermore, BF-2 significantly inhibited NLRP3 inflammasome activation and reduced ankle swelling in the gout mouse model. In detail, it alleviated mitochondrial damage mediated oxidative stress and inhibited the assembly of NLRP3 inflammasome by affecting the binding of pro-Caspase 1 and ASC. Moreover, BF-2 blocked NLRP3 activation by inhibiting the MAPK/NF-κB signaling pathways.ConclusionsResults demonstrated BF-2 inhibited NLRP3 inflammasome activation in both LPS primed macrophages and mouse model of gout through blocking MAPK/NF-κB signaling pathway and mitochondrial damage mediated oxidative stress. This study strongly suggests BF-2 could be a promising drug candidate against inflammatory diseases associated with NLRP3 inflammasome activation.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

The interaction between C5a and sphingosine-1-phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation

Introduction

C5a plays an crucial role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the interaction between C5a and sphingosine-1-phosphate (S1P) in neutrophils for ANCA-mediated activation.

Methods

The plasma levels of S1P from 29 patients with ANCA-associated vasculitis (AAV) in active stage and in remission were tested by enzyme-linked immunosorbent assay (ELISA). The generation of S1P was tested in C5a-triggered neutrophils. The effect S1P receptor antagonist was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA.

Results

The plasma level of circulating S1P was significantly higher in patients with AAV with active disease compared with patients in remission (2034.2 ± 438.5 versus 1489.3 ± 547.4 nmol/L, P < 0.001). S1P can prime neutrophils for ANCA-induced respiratory burst and degranulation. Compared with non-triggered neutrophils, the mean fluorescence intensity (MFI) value for CD88 expression was up-regulated significantly in S1P-triggered neutrophils. S1P receptor antagonist decreased oxygen radical production in C5a primed neutrophils induced by ANCA-positive IgG from patients. Blocking S1P inhibited C5a-primed neutrophil migration.

Conclusions

S1P triggered by C5a-primed neutrophils could further activate neutrophils. Blocking S1P could attenuate C5a-induced activation of neutrophils by ANCA. The interaction between S1P and C5a plays an important role in neutrophils for ANCA-mediated activation.     

Promoting metastasis: neutrophils and T cells join forces

The role neutrophils play in cancer is a matter of debate as both pro- and anti-tumor functions have been documented. In a recent publication in Nature, Coffelt et al. identify a new mechanism where neutrophils and T cells cooperate to generate metastasis-supporting immune suppression.Neutrophils are an important component of the innate immune system and play a critical role in fighting infections and inflammation. However, they are also propagated and mobilized by tumors and accumulate at the tumor bed and in pre-metastatic and metastatic tissues where they exert important effects. There are conflicting reports regarding the role that neutrophils play in cancer. Neutrophils have been shown to have tumoricidal properties and can kill tumor cells either by direct cytotoxicity or via antibody-dependent cell-mediated cytotoxicity (ADCC). A number of reports have also shown that neutrophils can recruit and activate T cells, inducing acquired anti-tumor immune responses. On the other hand, neutrophils were shown to possess bona fide pro-tumor properties including the promotion of tumor angiogenesis, supply of pro-tumor cytokines, stimulation of growth by neutrophil elastase, and promotion of immune evasion by immunosuppression of T cells¹. Confusion about the role of neutrophils in cancer might be explained by the realization that neutrophil precursors have some plasticity² and that multiple neutrophil subsets with differing properties likely exist within tumors, which are driven by factors in the tumor microenvironment, such as TGFβ³. It appears that early on in tumor growth, both murine and human neutrophils tend to inhibit the primary cancer growth and actually recruit and activate CD8⁺ T cells^4,5 (Figure 1), however, as tumors become larger and the microenvironment changes, the neutrophils (along with other tumor-associated cell types, such as macrophages) begin to become immunosuppressive and inhibit T cell activity^2,6,7.Open in a separate windowFigure 1The consequences of the interplay between neutrophils and lymphocytes on tumor growth and metastatic progression. Neutrophils were previously shown to possess both tumor-promoting and tumor-limiting properties. Neutrophils have the capacity to propagate cytotoxic CD8⁺ cells, through secretion of factors such as TNFα, cathepsin G and neutrophil elastase, thereby limiting primary tumor growth. Coffelt et al.¹⁰ identified a tumor-promoting cascade where tumor-secreted IL-1β stimulates the secretion of IL-17α from γδ T cells. Consequently, neutrophils acquire a suppressive phenotype and inhibit the propagation of cytotoxic CD8⁺ cells, ultimately enhancing metastatic seeding in the pre-metastatic lung.In addition to their role in influencing primary tumor growth, interesting new observations have been made about the role of neutrophils in cancer metastasis. In recent years, it has become apparent that while tumor cell-autonomous traits play a key role in the metastatic process, the normal stromal cells that surround and interact with tumor cells also play a critical part in the metastatic cascade. Again, the role of neutrophils in metastasis is unclear. We, and others, recently reported that tumor-stimulated neutrophils possess anti-metastatic activity and actively limit metastatic seeding by direct elimination of tumor cells at the pre-metastatic site^8,9.In contrast to these studies, Coffelt et al.¹⁰ recently presented data to show that neutrophils could enhance metastasis in the highly aggressive KEP mouse model of metastatic breast cancer. They elegantly show that depletion of neutrophils in this model leads to a dramatic reduction in spontaneous lung metastases. They further show that the combined depletion of both neutrophils and CD8⁺ cells results in reversal of the metastatic phenotype, implicating CD8⁺ cells and neutrophils as partners in crime. Looking for the mechanism through which tumors induce this metastasis-enhancing process, the authors found that several cytokines capable of inducing IL-17α release from γδ T cells are significantly increased, and showed that IL-17α was indeed required for upregulation of G-CSF, which in turn, regulated both neutrophil mobilization and activation of the immunosuppressive neutrophil phenotype (Figure 1). Finally, the authors demonstrated that it is tumor-secreted IL-1β that stimulates the release of IL-17α, inducing the unique immune suppressive phenotype in neutrophils which acquire the ability to suppress CD8⁺ cytotoxic T cells and directly support metastatic spread. This complex mechanism may thus be perturbed by eliminating γδ T cells, IL-17α or neutrophils, firmly supporting the author''s conclusions.Interestingly, while this novel mechanism involving the interplay between tumor-stimulated neutrophils and two distinct T cell subsets has profound implications for metastatic spread, it apparently has no significant implications for primary tumor growth.This study raises a number of interesting issues. Are IL-1β, γδ T cells or IL-17α important in other tumors? Are these results generalizable to other mouse models and to human tumors? It is unclear why the results of this paper are so different than other reports showing that neutrophils prevent metastasis^8,9. Tumor type, location, size, and the timing of interventions are all likely to be important. Regardless, this paper is a sophisticated demonstration of how tumor cells, innate immune cells and adaptive immune cells have the potential to interact in a specific tumor model. This study thus provides an interesting paradigm that should be examined in other systems.     

IL-32γ Delays Spontaneous Apoptosis of Human Neutrophils through MCL-1, Regulated Primarily by the p38 MAPK Pathway

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.     

Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis

During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1β and G-CSF as well as reduced levels of anti-inflammatory TGF-β. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.