Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2-/- as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-alpha, IL-1beta, IL-6, IL-12, CCL7, CCL8, CCL9, <em>CXCL1</em>, CXCL2, CXCL4, and CXCL5. CXCL9 and <em>CXCL1</em>0 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2-/- mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.

BACKGROUND

A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.

RESULTS

We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, <em>CXCL1</em>, <em>CXCL1</em>3, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53).

CONCLUSIONS

Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

Host response to viral infection involves distinct effectors of innate and adaptive immunity, whose mobilization needs to be coordinated to ensure protection. Here we show that influenza virus triggers, in human blood dendritic-cell (DC) subsets (ie, plasmacytoid and myeloid DCs), a coordinated chemokine (CK) secretion program with 3 successive waves. The first one, occurring at early time points (2 to 4 hours), includes CKs potentially attracting effector cells such as neutrophils, cytotoxic T cells, and natural killer (NK) cells (<em>CXCL1</em>6, <em>CXCL1</em>, CXCL2, and CXCL3). The second one occurs within 8 to 12 hours and includes CKs attracting effector memory T cells (CXCL8, CCL3, CCL4, CCL5, CXCL9, <em>CXCL1</em>0, and <em>CXCL1</em>1). The third wave, which occurs after 24 to 48 hours, when DCs have reached the lymphoid organs, includes CCL19, CCL22, and <em>CXCL1</em>3, which attract naive T and B lymphocytes. Thus, human blood DC subsets carry a common program of CK production, which allows for a coordinated attraction of the different immune effectors in response to viral infection.

OBJECTIVE

The identification of the cellular and molecular pathways that mediate the development of non-alcoholic steatohepatitis is of crucial importance. Cytokines produced by liver-resident and infiltrating inflammatory cells, play a pivotal role in liver inflammation. The role of the proinflammatory cytokines IL-1α and IL-1β in steatohepatitis remains elusive.

METHODS

We employed IL-1α and IL-1β-deficient mice and transplanted marrow cells to study the role of liver-resident and bone marrow-derived IL-1 in steatosis and its progression to steatohepatitis.

RESULTS

Atherogenic diet-induced steatohepatitis in wild-type mice was associated with 16 and 4.6 fold-elevations in mRNA levels of hepatic IL-1α and IL-1β, respectively. In mice deficient in either IL-1α or IL-1β the transformation of steatosis to steatohepatitis and liver fibrosis was markedly reduced. This protective effect in IL-1α-deficient mice was noted despite increased liver cholesterol levels. Deficiency of IL-1α markedly reduced plasma serum amyloid A and steady-state levels of mRNA coding for inflammatory genes (P-selectin, CXCL1, IL-6, and TNFα) as well as pro-fibrotic genes (MMP-9 and Collagen) and particularly a 50% decrease in TGFβ levels (p = 0.004). IL-1α mRNA levels were two-folds lower in IL-1β-deficient mice, and IL-1β transcripts were three-folds lower in IL-1α-deficient compared to wild-type mice. Hepatic cell derived IL-1α rather than from recruited bone marrow-derived cells was required for steatohepatitis development.

CONCLUSIONS

These data demonstrate the critical role of IL-1α and IL-1β in the transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. Therefore, the potential of neutralizing IL-1α and/or IL-1β to inhibit the development of steatohepatitis should be explored.

Many diseases have an inflammatory component, where neutrophil interactions with the vascular endothelium lead to barrier dysfunction and increased permeability. Neutrophils increase permeability through secreted products such as the chemokines CXCL1, 2, 3, and 8, through adhesion-dependent processes involving beta(2) integrins interacting with endothelial ICAM-1, and through combinations where beta(2) integrin engagement leads to degranulation and secretion of heparin-binding protein. Some neutrophil products, such as arachidonic acid or the leukotriene LTA4, are further processed by endothelial enzymes via transcellular metabolism before the resulting products thromboxane A2 or LTC4 can activate their cognate receptors. Neutrophils also generate reactive oxygen species that induce vascular leakage. This review focuses on the mechanisms of neutrophil-mediated leakage.

Resolvins of the D series are generated from docosahexaenoic acid, which are enriched in fish oils and are believed to exert beneficial roles on diverse inflammatory disorders, including inflammatory bowel disease (IBD). In this study, we investigated the anti-inflammatory effects of the aspirin-triggered resolvin D1 (AT-RvD1), its precursor (17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) and resolvin D2 (RvD2) in dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Our results showed that the systemic treatment with AT-RvD1, RvD2, or 17R-HDHA in a nanogram range greatly improved disease activity index, body weight loss, colonic damage, and polymorphonuclear infiltration in both colitis experimental models. Moreover, these treatments reduced colonic cytokine levels for TNF-α, IL-1β, MIP-2, and CXCL1/KC, as well as mRNA expression of NF-κB and the adhesion molecules VCAM-1, ICAM-1, and LFA-1. Furthermore, AT-RvD1, but not RvD2 or 17R-HDHA, depended on lipoxin A4 receptor (ALX) activation to inhibit IL-6, MCP-1, IFN-γ, and TNF-α levels in bone marrow-derived macrophages stimulated with LPS. Similarly, ALX blockade reversed the beneficial effects of AT-RvD1 in DSS-induced colitis. To our knowledge, our findings showed for the first time the anti-inflammatory effects of resolvins of the D series and precursor 17R-HDHA in preventing experimental colitis. We also demonstrated the relevant role exerted by ALX activation on proresolving action of AT-RvD1. Moreover, AT-RvD1 showed a higher potency than 17R-HDHA and RvD2 in preventing DSS-induced colitis. The results suggest that these lipid mediators possess a greater efficacy when compared with other currently used IBD therapies, such as monoclonal anti-TNF, and have the potential to be used for treating IBD.

Through the activity of macrophage-specific matrix metalloproteinase-12 (MMP-12), we found that macrophages dampen the lipopolysaccharide (LPS)-induced influx of polymorphonuclear leukocytes (PMNs)-thus providing a new mechanism for the termination of PMN recruitment in acute inflammation. MMP-12 specifically cleaves human ELR(+) CXC chemokines (CXCL1, -2, -3, -5, and -8) at E-LR, the critical receptor-binding motif or, for CXCL6, carboxyl-terminal to it. Murine (m) MMP-12 also cleaves mCXCL1, -2, and -3 at E-LR. MMP-12-cleaved mCXCL2 (macrophage-inflammatory protein-2 [MIP-2]) and mCXCL3 (dendritic cell inflammatory protein-1 [DCIP-1]) lost chemotactic activity. Furthermore, MMP-12 processed and inactivated monocyte chemotactic proteins CCL2, -7, -8, and -13 at position 4-5 generating CCR antagonists. Indeed, PMNs and macrophages in bronchoalveolar lavage fluid were significantly increased 72 hours after intranasal instillation of LPS in Mmp12(-/-) mice compared with wild type. Specificity occurred at 2 levels. Macrophage MMP-1 and MMP-9 did not cleave in the ELR motif. Second, unlike human ELR(+)CXC chemokines, mCXCL5 (LPS-induced CXC chemokine [LIX]) was not inactivated. Rather, mMMP-12 cleavage at Ser4-Val5 activated the chemokine, promoting enhanced PMN early infiltration in wild-type mice compared with Mmp12(-/-) mice 8 hours after LPS challenge in air pouches. We propose that the macrophage, specifically through MMP-12, assists in orchestrating the regulation of acute inflammatory responses by precise proteolysis of ELR(+)CXC and CC chemokines.

BACKGROUND

The diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood.

OBJECTIVE

Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties.

RESULTS

In primary hepatocytes from healthy animals, metformin and the IKKβ (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α-dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1β, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/β activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02-0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22-2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11).

CONCLUSIONS

We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups.

BACKGROUND

Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.

Systemic small vessel vasculitis is associated with antineutrophil cytoplasm antibodies (ANCAs). While there is mounting in vitro evidence to suggest that ANCAs are capable of enhancing leukocyte-endothelial interactions, no in vivo evidence for this has been provided. In this study a novel rat model of ANCA-associated experimental autoimmune vasculitis (EAV), induced by immunization with human myeloperoxidase (MPO), was used to analyze directly the potential effect of ANCAs on leukocyte-venular wall interactions in vivo as observed by intravital microscopy. These rats developed anti-MPO antibodies directed against rat leukocytes, showed pathologic evidence of small vessel vasculitis, and had enhanced leukocyte adhesion and transmigration in response to the chemokine Groalpha (CXCL1 [CXC ligand 1]). Passive transfer of immunoglobulin from rats with EAV to naive rats conferred enhanced adhesion and transmigration responses in the recipients. Furthermore, rats with EAV and recipients of ANCA-positive immunoglobulin developed extensive microvascular injury, as manifested by mesenteric hemorrhage, in response to CXCL1. This study provides the first direct in vivo evidence for the ability of ANCAs to enhance leukocyte-endothelial interactions and cause microvascular hemorrhage, thereby providing a mechanism by which ANCAs could exert pathogenic effects in systemic vasculitis.

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis. Conversely, cancer cells often disrupt apoptosis to survive. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle--its cognate receptor--was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

BACKGROUND

IBC (Inflammatory Breast cancer) is a rare form of breast cancer with a particular phenotype. New molecular targets are needed to improve the treatment of this rapidly fatal disease. Given the role of NF-kappaB-related genes in cell proliferation, invasiveness, angiogenesis and inflammation, we postulated that they might be deregulated in IBC.

METHODS

We measured the mRNA expression levels of 60 NF-kappaB-related genes by using real-time quantitative RT-PCR in a well-defined series of 35 IBCs, by comparison with 22 stage IIB and III non inflammatory breast cancers. Twenty-four distant metastases of breast cancer served as "poor prognosis" breast tumor controls.

RESULTS

Thirty-five (58%) of the 60 NF-kappaB-related genes were significantly upregulated in IBC compared with non IBC. The upregulated genes were NF-kappaB genes (NFKB1, RELA, IKBKG, NFKBIB, NFKB2, REL, CHUK), apoptosis genes (MCL1L, TNFAIP3/A20, GADD45B, FASLG, MCL1S, IER3L, TNFRSF10B/TRAILR2), immune response genes (CD40, CD48, TNFSF11/RANKL, TNFRSF11A/RANK, CCL2/MCP-1, CD40LG, IL15, GBP1), proliferation genes (CCND2, CCND3, CSF1R, CSF1, SOD2), tumor-promoting genes (CXCL1CXCL1/GRO1). Only two of these 35 genes (PTGS2/COX2 and CXCL1/GRO1)were also upregulated in breast cancer metastases. We identified a five-gene molecular signature that matched patient outcomes, consisting of IL8 and VEGF plus three NF-kappaB-unrelated genes that we had previously identified as prognostic markers in the same series of IBC.

CONCLUSIONS

The NF-kappaB pathway appears to play a major role in IBC, possibly contributing to the unusual phenotype and aggressiveness of this form of breast cancer. Some upregulated NF-kappaB-related genes might serve as novel therapeutic targets in IBC.

Recent advances have facilitated the use of blood-derived RNA to conduct genomic analyses of human diseases. This emerging technology represents a rigorous and convenient alternative to traditional tissue biopsy-derived RNA, as it allows for larger sample sizes, better standardization of technical procedures, and the ability to non-invasively profile human subjects. In the present pilot study, we have collected RNA from blood of patients diagnosed with schizophrenia or bipolar disorder (BPD), as well as normal control subjects. Using microarray analysis, we found that each disease state exhibited a unique expressed genome signature, allowing us to discriminate between the schizophrenia, BPD, and control groups. In addition, we validated changes in several potential biomarker genes for schizophrenia and BPD by RT-PCR, and some of these were found to code to chromosomal loci previously linked to schizophrenia. Linear and non-linear combinations of eight putative biomarker genes (APOBEC3B, ADSS, ATM, CLC, CTBP1, DATF1, CXCL1, and S100A9) were able to discriminate between schizophrenia, BPD, and control samples, with an overall accuracy of 95%-97% as indicated by receiver operating characteristic (ROC) curve analysis. We therefore propose that blood cell-derived RNA may have significant value for performing diagnostic functions and identifying disease biomarkers in schizophrenia and BPD.

Chemokines are small proteins that control several tissue functions, including cell recruitment and activation under homeostatic and inflammatory conditions. CXCL8 (interleukin-8) is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 are also ELR+ chemokine members that bind to these receptors, especially CXCR2. The majority of studies on the biology of CXCL8 and their receptors have been performed in polymorphonuclear leukocytes. However, many other cells express CXCR1/CXCR2, including epithelial, endothelial, fibroblasts and neurons, contributing to the biological effects of CXCL8. There is substantial amount of experimental data suggesting that CXCL8 and receptors contribute to elimination of pathogens, but may also contribute significantly to disease-associated processes, including tissue injury, fibrosis, angiogenesis and tumorigenesis. Here, we discuss the biology of CXCL8 family and the potential therapeutic use of antagonists or blockers of these molecules in the context of organ-specific diseases.

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (<em>CXCL1</em>), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (<em>CXCL1</em>0), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). We previously identified a TIC population derived from hepatocellular carcinoma (HCC) that is characterized by membrane expression of CD133. Here, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133(+) liver TICs obtained from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133(+) cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133(+) liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis, and initiate tumors. In further support of these observations, IL-8 repression in CD133(+) liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be preferentially expressed in CD133(+) liver TICs. Addition of exogenous NTS resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, both key components of the mitogen-activated protein kinase (MAPK) pathway. Enhanced IL-8 secretion by CD133(+) liver TICs can in turn activate an IL-8-dependent feedback loop that signals through the MAPK pathway. Further, in its role as a liver TIC marker CD133 also plays a functional part in regulating tumorigenesis of liver TICs by way of regulating NTS, IL-8, CXCL1, and MAPK signaling.

CONCLUSIONS

CD133(+) liver TICs promote angiogenesis, tumorigenesis, and self-renewal through NTS-induced activation of the IL-8 signaling cascade.

OBJECTIVE

Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis.

RESULTS

Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor alpha (TNFalpha) and IL-1beta synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFalpha antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFalpha responded to IL-33 in chemotaxis.

CONCLUSIONS

These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFalpha therapy of inflammation.

Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with>>/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant,>>/=threefold, upregulation of the expression of >100 transcripts while 73 were>>/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1beta and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that beta-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1(-/-) corneas have impaired IL-1beta and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high beta-glucan. In contrast to Dectin 1(-/-) mice, cellular infiltration into infected TLR2(-/-), TLR4(-/-), and MD-2(-/-) mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4(-/-) mice, but not TLR2(-/-) or MD-2(-/-) mice. We also found that TRIF(-/-) and TIRAP(-/-) mice exhibited no fungal-killing defects, but that MyD88(-/-) and IL-1R1(-/-) mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which beta-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1beta, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE(2) levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE(2) and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-alpha, IL-1beta, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1beta produced PGE(2), and IL-1beta-induced PGE(2) production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE(2). Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1β in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.

CNS injury stimulates the expression of several proinflammatory cytokines and chemokines, some of which including MCP-1 (also known as CCL2), KC (CXCL1), and MIP-2 (CXCL2) act to recruit Gr-1(+) leukocytes at lesion sites. While earlier studies have reported that neutrophils and monocytes/macrophages contribute to secondary tissue loss after spinal cord injury (SCI), recent work has shown that depletion of Gr-1(+) leukocytes compromised tissue healing and worsened functional recovery. Here, we demonstrate that astrocytes distributed throughout the spinal cord initially contribute to early neuroinflammation by rapidly synthesizing MCP-1, KC, and MIP-2, from 3 up to 12h post-SCI. Chemokine expression by astrocytes was followed by the infiltration of blood-derived immune cells, such as type I "inflammatory" monocytes and neutrophils, into the lesion site and nearby damaged areas. Interestingly, astrocytes from mice deficient in MyD88 signaling produced significantly less MCP-1 and MIP-2 and were unable to synthesize KC. Analysis of the contribution of MyD88-dependent receptors revealed that the astrocytic expression of MCP-1, KC, and MIP-2 was mediated by the IL-1 receptor (IL-1R1), and not by TLR2 or TLR4. Flow cytometry analysis of cells recovered from the spinal cord of MyD88- and IL-1R1-knockout mice confirmed the presence of significantly fewer type I "inflammatory" monocytes and the almost complete absence of neutrophils at 12h and 4days post-SCI. Together, these results indicate that MyD88/IL-1R1 signals regulate the entry of neutrophils and, to a lesser extent, type I "inflammatory" monocytes at sites of SCI.

Circulating endothelial progenitor cells (EPCs) may contribute to endothelial regeneration; however, the exact mechanisms of their arterial homing remain elusive. We examined the role of the angiogenic chemokine receptor CXCR2 in the homing of human EPCs. Isolated EPCs expressed CXCR2 together with kinase insert domain-containing receptor, CD31, vascular endothelial cadherin, and CXCR4. Adhesion assays under flow conditions showed that EPCs preferentially adhered to beta(2)-integrin ligands, that firm arrest on fibronectin or fibrinogen was enhanced by the CXCR2 ligands CXCL1 or CXCL7, and that blockade of CXCR2 significantly reduced EPC adhesion on platelet-coated endothelial matrix. This was corroborated by the involvement of CXCR2 in EPC recruitment to denuded areas of murine carotid arteries ex vivo and in vivo. Notably, blocking CXCR2 inhibited the incorporation of human EPCs expressing CXCR2 at sites of arterial injury in athymic nude mice. Immunoreactivity for the beta-thromboglobulin isoform CXCL7 was observed in murine platelets and denuded smooth muscle cells (SMCs) early after wire injury, and transcripts for CXCL7 and CXCL1 were detected in isolated human arterial SMCs. Human KDR(+)CXCR2(+) cells showed better in situ adhesion to injured murine carotid arteries than KDR(+)CXCR2(-) cells, were predominantly CD14(+), and improved CXCR2-dependent endothelial recovery after injury in nude mice. In conclusion, our data clearly demonstrate the importance of CXCR2 for the homing of circulating EPCs to sites of arterial injury and for endothelial recovery in vivo.