Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.

Accumulating evidence suggest that platelets play an important role in regulating neutrophil recruitment in septic lung injury. Herein, we hypothesized that platelet-derived CCL5 might facilitate sepsis-induced neutrophil accumulation in the lung. Abdominal sepsis was induced by CLP in C57BL/6 mice. CLP increased plasma levels of CCL5. Platelet depletion and treatment with the Rac1 inhibitor NSC23766 markedly reduced CCL5 in the plasma of septic mice. Moreover, Rac1 inhibition completely inhibited proteasePAR4-induced secretion of CCL5 in isolated platelets. Immunoneutralization of CCL5 decreased CLP-induced neutrophil infiltration, edema formation, and tissue injury in the lung. However, inhibition of CCL5 function had no effect on CLP-induced expression of Mac-1 on neutrophils. The blocking of CCL5 decreased plasma and lung levels of CXCL1 and CXCL2 in septic animals. CCL5 had no effect on neutrophil chemotaxis in vitro, suggesting an indirect effect of CCL5 on neutrophil recruitment. Intratracheal challenge with CCL5 increased accumulation of neutrophils and formation of CXCL2 in the lung. Administration of the CXCR2 antagonist SB225002 abolished CCL5-induced pulmonary recruitment of neutrophils. Isolated alveolar macrophages expressed significant levels of the CCL5 receptors CCR1 and CCR5. In addition, CCL5 triggered significant secretion of CXCL2 from isolated alveolar macrophages. Notably, intratracheal administration of clodronate not only depleted mice of alveolar macrophages but also abolished CCL5-induced formation of CXCL2 in the lung. Taken together, our findings suggest that Rac1 regulates platelet secretion of CCL5 and that CCL5 is a potent inducer of neutrophil recruitment in septic lung injury via formation of CXCL2 in alveolar macrophages.

OBJECTIVE

Platelets are potent regulators of neutrophil accumulation in septic lung damage. We hypothesized that platelet-derived CXCL4 might support pulmonary neutrophilia in a murine model of abdominal sepsis.

METHODS

Polymicrobial sepsis was triggered by coecal ligation and puncture (CLP) in C57BL/6 mice. Platelet secretion of CXCL4 was studied by using confocal microscopy. Plasma and lung levels of CXCL4, CXCL1 and CXCL2 were determined by elisa. Flow cytometry was used to examine surface expression of Mac-1 on neutrophils.

RESULTS

CLP increased CXCL4 levels in plasma, and platelet depletion reduced plasma levels of CXCL4 in septic animals. Rac1 inhibitor NSC23766 decreased the CLP-enhanced CXCL4 in plasma by 77%. NSC23766 also abolished PAR4 agonist-induced secretion of CXCL4 from isolated platelets. Inhibition of CXCL4 reduced CLP-evoked neutrophil recruitment, oedema formation and tissue damage in the lung. However, immunoneutralization of CXCL4 had no effect on CLP-induced expression of Mac-1 on neutrophils. Targeting CXCL4 attenuated plasma and lung levels of CXCL1 and CXCL2 in septic mice. CXCL4 had no effect on neutrophil chemotaxis in vitro, indicating it has an indirect effect on pulmonary neutrophilia. Intratracheal CXCL4 enhanced infiltration of neutrophils and formation of CXCL2 in the lung. CXCR2 antagonist SB225002 markedly reduced CXCL4-provoked neutrophil accumulation in the lung. CXCL4 caused secretion of CXCL2 from isolated alveolar macrophages.

CONCLUSIONS

Rac1 controls platelet secretion of CXCL4 and CXCL4 is a potent stimulator of neutrophil accumulation in septic lungs via generation of CXCL2 in alveolar macrophages. Platelet-derived CXCL4 plays an important role in lung inflammation and tissue damage in polymicrobial sepsis.

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

Lung cancer is the leading cause of cancer death in the world and metastasis is an essential aspect of lung cancer progression. ITGB8 has been implicated in metastasis of human tumors. However, the molecular mechanism by which ITGB8 is involved in tumor metastasis is still unclear. In this study, we compared the gene expression profiles of human lung cancer cell lines A549 and PC9 by ITGB8 gene silencing with that of parent cells and negative control cells to comprehensively investigate ITGB8-mediated changes with respect to the metastatic potential and gene expression of human lung cancer cell lines. Our results showed that ITGB8 silencing cells exhibited significant cell cycle arrest and less adhesion and invasion abilities. We confirmed by Western blot, ELISA, and real-time PCR that the expression of metastasis-related genes CXCL1, CXCL2, CXCL5, MMP-2, and MMP-9 were significantly decreased while that of E-Cadherin and cystatin B were dramatically increased in A549- and PC9-ITGB8 silencing cells. Furthermore, silencing of ITGB8 caused Snail and NF-κB transcriptional activation, and MEK and Akt phosphorylation level changes in lung cancer cell lines. Our results indicated that ITGB8 may play an important role in metastasis of human lung cancer cells. The ITGB8 silencing may change the lung cancer cells to a less invasive phenotype through alteration in the expression of metastasis-related genes.

Osteoclast (OC) progenitors (OCP) have been defined in the bone marrow (BM) as CD3(-)CD45R(B220)(-)GR1(-)CD11b(lo/)(-)CD115(+) (dOCP) and more recently in the peripheral blood (PB) as Lym(-)Ly6G(-)CD11b(+)Ly6C(+). These progenitors respond to stimuli, including LPS from periopathogenic Aggregatibacter actinomycetemcomitans, activating MAPK signaling, resulting in cytokine/chemokine-mediated osteoclastogenesis. Intracellular negative signaling pathways, including MAPK phosphatase-1 (MKP-1, gene Dusp1) deactivate MAPK pathways (p-p38 and p-JNK) and reduce inflammatory cytokines/chemokines.

OBJECTIVE

To delineate the role of MKP-1 in chemokine-mediated OC formation using defined OC progenitor populations. Given its role in innate immune inflammatory signaling, we hypothesize that MKP-1 regulates LPS-induced OC formation from BM OCP through deregulated chemokines.

METHODS

BM and PB from WT and Dusp1(-/-) female mice (8-12weeks) was obtained and sorted into defined progenitor populations. BM sorted dOCP were primed with MCSF and RANKL (48h), blocked with vehicle or chemokine blocking antibodies and stimulated with LPS (48-96h). TRAP assay and OC activity were measured for OC formation and activity following treatments. NanoString Array and qPCR were utilized for gene expression analysis.

RESULTS

Dusp1(-/-) dOCPs formed more and larger osteoclasts from CD11b(hi) and dOCP compared to matched WT (P<0.05 each). PB-derived dOCP produced larger and more functional osteoclasts from Dusp1(-/-) mice compared to WT controls. NanoString array data revealed significant deregulation in chemokine expression from Dusp1(-/-) versus WT cells. qPCR validation of target genes revealed that Dusp1 deficient CD11b(+) populations display 1.5-3.5-fold greater expression of CXCL1 and 2-3-fold greater expression of CXCL2 compared to WT in CD11b(hi) and dOCP (P<0.05 each). Antibody blocking studies using anti-CXCL1 and CXCL2 antibodies blunted osteoclastogenesis in Dusp1(-/-) cells.

CONCLUSIONS

MKP-1 negatively regulates chemokine-driven OC formation and subsequent bone resorption in response to LPS stimulation. Collectively, these data provide useful insight into mechanisms potentially leading to the development of therapeutic treatment of periodontal disease.

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

The granulocyte-specific microRNA-223 (miR-223) has recently emerged as a negative regulator of NOD-like receptor 3 (NLRP3) expression, a central key player in chronic hepatic injuries such as fibrotic nonalcoholic steatohepatitis (NASH), as well as in other liver conditions including acute hepatitis. In this study, we evaluated the therapeutic effect of the synthetic miR-223 analog miR-223 3p in a murine model of lipopolysaccharide (LPS)/D-GalN-induced endotoxin acute hepatitis (EAH) or fibrotic NASH resultant of long-term feeding with a high-fat, fructose, and cholesterol (FFC) diet. miR-223 3p ameliorated the infiltration of monocytes, neutrophils, and early activated macrophages and downregulated the transcriptional expression of the pro-inflammatory cytokines Il6 and Il12 and the chemokines Ccl2, Ccl3, Cxcl1, and Cxcl2 in EAH. In fibrotic NASH, treatment with miR-223 3p led to a remarkable mitigation of fibrosis development and activation of hepatic stellate cells (HSCs). miR-223 3p disrupted the activation of the NLRP3 inflammasome by impairing the synthesis of cleaved interleukin-1β (IL-1β), mature IL-1β, and NLRP3, and the activation of caspase-1 p10 in both EAH and fibrotic NASH. Our data enlightens miR-223 3p as a post-transcriptional approach to treat acute and chronic hepatitis by silencing the activation of the NLRP3 inflammasome.

BACKGROUND

Chemokines and chemokine receptors cooperate to promote immune cell recruitment to the central nervous system (CNS). In this study, we investigated the roles of CXCR2 and CXCL1 in leukocyte recruitment to the CNS using a murine model of neuroinflammation.

METHODS

Wild-type (WT), CXCL1(-/-), and CXCR2(-/-) mice each received an intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). Esterase staining and intravital microscopy were performed to examine neutrophil recruitment to the brain. To assess endothelial activation in these mice, the expression of adhesion molecules was measured via quantitative real-time polymerase chain reaction (PCR) and Western blotting. To identify the cellular source of functional CXCR2, chimeric mice were generated by transferring bone marrow cells between the WT and CXCR2(-/-) mice.

RESULTS

Expression levels of the chemokines CXCL1, CXCL2, and CXCL5 were significantly increased in the brain following the i.c.v. injection of LPS. CXCR2 or CXCL1 deficiency blocked neutrophil infiltration and leukocyte recruitment in the cerebral microvessels. In the CXCR2(-/-) and CXCL1(-/-) mice, the cerebral endothelial expression of adhesion molecules such as P-selectin and VCAM-1 was dramatically reduced. Furthermore, the bone marrow transfer experiments demonstrated that CXCR2 expression on CNS-residing cells is essential for cerebral endothelial activation and leukocyte recruitment. Compared with microglia, cultured astrocytes secreted a much higher level of CXCL1 in vitro. Astrocyte culture conditioned medium significantly increased the expression of VCAM-1 and ICAM-1 in cerebral endothelial cells in a CXCR2-dependent manner. Additionally, CXCR2 messenger RNA (mRNA) expression in cerebral endothelial cells but not in microglia or astrocytes was increased following tumor necrosis factor-α (TNF-α) stimulation. The intravenous injection of the CXCR2 antagonist SB225002 significantly inhibited endothelial activation and leukocyte recruitment to cerebral microvessels.

CONCLUSIONS

CXCL1 secreted by astrocytes and endothelial CXCR2 play essential roles in cerebral endothelial activation and subsequent leukocyte recruitment during neuroinflammation.

Recruitment of neutrophils into alveolar air spaces is an early event in the pathogenesis of pneumonia due to Streptococcus pneumoniae. This results from chemokines released by activated endothelial and epithelial cells and alveolar macrophages. Culture supernatants of 6 wild-type strains of S. pneumoniae, shown to contain choline-binding protein A (CbpA; clades A and B), induced release of chemokine CXCL8 from the human alveolar epithelial cell line A549, whereas a CbpA deletion mutant elicited significantly reduced CXCL8 release, compared with that of its isogenic parent (P<.01). Recombinant CbpA up-regulated expression of messenger RNA of CXCL8 and CCL2 but not of XCL1, CXCL1CXCL1, and CXCL5 in a dose- and time-dependent manner. CbpA also increased the expression of intercellular adhesion molecule 1 (CD54) by A549 cells. Thus, CbpA of S. pneumoniae induces the transcription and release of proinflammatory molecules by human alveolar epithelial cells.

The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai(-/-) and Apoe(-/-) mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.

Purpose: Tumor development has been closely linked to tumor microenvironment, particularly in terms of myeloid-derived suppressive cells (MDSCs), a heterogeneous population of immature myeloid cells that protect tumors from elimination by immune cells. Approaches aimed at blocking MDSC accumulation could improve cancer clinical outcome. Experimental Design: We investigated that metformin suppressed MDSC migration to inhibit cancer progression. Primary tumor tissues were incubated with metformin, and proinflammatory chemokine production was measured. To study MDSC chemotaxis in vivo, BALB/C nude mice were injected subcutaneously with TE7 cells and treated with metformin. Migration of adoptively transferred MDSCs was analyzed using flow cytometry and immunohistochemistry. Results: The frequency of tumor-infiltrated polymorphonuclear (PMN)-MDSCs was increased compared to their circulating counterparts. There was a significant correlation between PMN-MDSCs accumulation in tumors and ESCC prognosis. Moreover, PMN-MDSCs displayed immunosuppressive activity in vitro. Treatment with metformin reduced MDSC migration in patients. Metformin inhibited CXCL1 secretion in ESCC cells and tumor xenografts by enhancing AMPK phosphorylation and inducing DACH1 expression, leading to NF-κB inhibition and reducing MDSC migration. Knockdown of AMPK and DACH1 expression blocked the effect of metformin on MDSC chemotaxis. Conclusions: A novel anti-tumor effect of metformin, which is mediated by reducing PMN-MDSC accumulation in the tumor microenvironment via AMPK/DACH1/CXCL1 axis.

Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C-X-C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high-fat diet (HFD)-fed mice. Here we demonstrate that overexpression of Cxcl1 in the liver alone promotes steatosis-to-NASH progression in HFD-fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as ASK1 and p38MAPK) activation. Myeloid cell-specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47^phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1-induced NASH and stress kinase activation in HFD-fed mice. Treatment with interleukin (IL)-22, a cytokine with multiple targets, ameliorated CXCL1/HFD-induced NASH or methionine-choline deficient diet-induced NASH in mice. Mechanistically, IL-22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL-22 treatment attenuated the inflammatory functions of hepatocyte-derived, mitochondrial DNA-enriched extracellular vesicles, thereby suppressing liver inflammation in NASH. CONCLUSION: Hepatic overexpression of CXCL1 is sufficient to drive steatosis-to-NASH progression in HFD-fed mice through neutrophil-derived reactive oxygen species and activation of stress kinases, which can be reversed by IL-22 treatment via the induction of metallothionein.

Gut dysbiosis is commonly observed in patients with cirrhosis and chronic gastrointestinal disorders, however, its effect on anti-tumor immunity in the liver is largely unknown. Here we studied how the gut microbiome affects anti-tumor immunity in cholangiocarcinoma. Primary sclerosing cholangitis (PSC) or colitis, two known risk factors for cholangiocarcinoma, which promote tumor development in mice caused an accumulation of CXCR2+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). A decrease in gut barrier function observed in mice with PSC and colitis allowed gut derived bacteria and lipopolysaccharide (LPS) to appear in the liver and induced CXCL1 expression in hepatocytes through a TLR4-dependent mechanism and an accumulation of CXCR2+ PMN-MDSC. On the contrary, neomycin treatment blocked CXCL1 expression, PMN-MDSC accumulation and inhibited tumor growth even in the absence of liver disease or colitis. Our study demonstrates that the gut microbiome controls hepatocytes to form an immunosuppressive environment by increasing PMN-MDSC to promote liver cancer.

Gout manifests as recurrent episodes of acute joint inflammation and pain due to the deposition of monosodium urate (MSU) crystals within the affected tissue in a process dependent on NLRP3 inflammasome activation. The synthesis, activation, and release of IL-1β are crucial for MSU-induced inflammation. The current study evaluated the mechanism by which TNF-α contributed to MSU-induced inflammation. Male C57BL/6J or transgenic mice were used in this study and inflammation was induced by the injection of MSU crystals into the joint. TNF-α was markedly increased in the joint after the injection of MSU. There was inhibition in the infiltration of neutrophils, production of CXCL1 and IL-1β, and decreased hypernociception in mice deficient for TNF-α or its receptors. Pharmacological blockade of TNF-α with Etanercept or pentoxyfylline produced similar results. Mechanistically, TNF-α blockade resulted in lower amounts of IL-1β protein and pro-IL-1β mRNA transcripts in joints. Gene-modified mice that express only transmembrane TNF-α had an inflammatory response similar to that of WT mice and blockade of soluble TNF-α (XPro™1595) did not decrease MSU-induced inflammation. In conclusion, TNF-α drives expression of pro-IL-1β mRNA and IL-1β protein in experimental gout and that its transmembrane form is sufficient to trigger MSU-induced inflammation in mice.

Podocyte loss has been reported to relate to disease severity and progression in IgA nephropathy (IgAN). However, the underlying mechanism for its role in IgAN remain unclear. Recent evidence has shown that IgA1 complexes from patients with IgAN could activate mesangial cells to induce soluble mediator excretion, and further injure podocytes through mesangial-podocytic cross-talk. In the present study, we explored the underlying mechanism of mesangial cell-induced podocyte loss in IgAN. We found that IgA1 complexes from IgAN patients significantly up-regulated the expression of CXCL1 and TGF-β1 in mesangial cells compared with healthy controls. Significantly higher urinary levels of CXCL1 and TGF-β1 were also observed in patients with IgAN compared to healthy controls. Moreover, IgAN patients with higher urinary CXCL1 and TGF-β1 presented with severe clinical and pathological manifestations, including higher 24-hour urine protein excretion, lower eGFR and higher cresentic glomeruli proportion. Further in vitro experiments showed that increased podocyte death and reduced podocyte adhesion were induced by mesangial cell conditional medium from IgAN (IgAN-HMCM), as well as rhCXCL1 together with rhTGF-β1. In addition, the over-expression of CXCR2, the receptor for CXCL1, by podocytes was induced by IgAN-HMCM and rhTGF-β1, but not by rhCXCL1. Furthermore, the effect of increased podocyte death and reduced podocyte adhesion induced by IgAN-HMCM and rhCXCL1 and rhTGF-β1 was rescued partially by a blocking antibody against CXCR2. Moreover, we observed the expression of CXCR2 in urine exfoliated podocytes in IgAN patients. Our present study implied that IgA1 complexes from IgAN patients could up-regulate the secretion of CXCL1 and TGF-β1 in mesangial cells. Additionally, the synergistic effect of CXCL1 and TGF-β1 further induced podocyte death and adhesion dysfunction in podocytes via CXCR2. This might be a potential mechanism for podocyte loss observed in IgAN.

Cytotoxic chemotherapeutic drugs, especially when used in combination, are widely employed to treat a variety of cancers in patients but often lead to serious symptoms that negatively affect physical functioning and quality of life. There is compelling evidence that implicates cytotoxic chemotherapy-induced inflammation in the etiology of these symptoms. Because IL-1β plays a central role as an initiator cytokine in immune responses, we compared doxorubicin, a drug known to induce IL-1β production, with ten other commonly prescribed chemotherapeutic drugs in their ability to lead to processing and secretion of IL-1β by primary mouse macrophages. Seven of them (melphalan, cisplatin, vincristine, etoposide, paclitaxel, methotrexate, and cytarabine) caused the production of IL-1β in cells pretreated with lipopolysaccharide. When delivered in combination with doxorubicin, one of the drugs, vincristine, was also capable of synergistically activating the NLRP3-dependent inflammasome and increasing expression of IL-1β, IL-6, and CXCL1. The absence of TNF-α and IL-1 signaling caused a partial reduction in the production of mature IL-1β. Three small-molecule inhibitors known to suppress activity of kinases situated upstream of mitogen-activated kinases (MAPKs) inhibited the expression of IL-1β, IL-6, and CXCL1 when doxorubicin and vincristine were used singly or together, so specific kinase inhibitors may be useful in reducing inflammation in patients receiving chemotherapy.

Determining the molecular events induced in the spleen during schistosome infection is an essential step in better understanding the immunopathogenesis of schistosomiasis and the mechanisms by which schistosomes modulate the host immune response. The present study defines the transcriptional and cellular events occurring in the murine spleen during the progression of Schistosoma japonicum infection. Additionally, we compared and contrasted these results with those we have previously reported for the liver. Microarray analysis combined with flow cytometry and histochemistry demonstrated that transcriptional changes occurring in the spleen were closely related to changes in cellular composition. Additionally, the presence of alternatively activated macrophages, as indicated by up-regulation of Chi3l3 and Chi3l4 and expansion of F4/80(+) macrophages, together with enhanced expression of the immunoregulatory genes ANXA1 and CAMP suggests the spleen may be an important site for the control of S. japonicum-induced immune responses. The most striking difference between the transcriptional profiles of the infected liver and spleen was the contrasting expression of chemokines and cell adhesion molecules. Lymphocyte chemokines, including the homeostatic chemokines <em>CXCL1</em>3, CCL19 and CCL21, were significantly down-regulated in the spleen but up-regulated in the liver. Eosinophil (CCL11, CCL24), neutrophil (<em>CXCL1</em>) and monocyte (<em>CXCL1</em>4, CCL12) chemokines and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver but unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Co-ordinated expression of these genes probably contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver, thereby facilitating the development of hepatic granulomas and fibrosis. Together these data provide, for the first time, a comprehensive overview of the molecular events occurring in the spleen during schistosomiasis and will substantially further our understanding of the local and systemic mechanisms driving the immunopathogenesis of this disease.

Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, β3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

The idiopathic inflammatory myopathies (IM) are subdivided into dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (IBM). These autoimmune muscle diseases represent different immunopathological entities. DM is a humoral endotheliopathy initiated by complement deposition in intramuscular blood vessels, and characterized by perimysial inflammation and muscle fiber atrophy in perifascicular regions. In PM and IBM, nonnecrotic muscle fibers are actively invaded by autoaggressive macrophages and cytotoxic T cells. Chemokines are key mediators of inflammatory disease as they regulate leukocyte recruitment to tissue target sites. We studied a large selection of alpha/beta-chemokines and receptors in normal controls and in the IM using immunohistochemistry, immunofluorescence, in situ hybridization, and Western blotting. We showed that the chemokine array of normal myocytes was limited, while the blood vessels in normal skeletal muscle tissue displayed a broad chemokine profile. The IM were characterized by a general increase of specific chemokines and chemokine receptors, while chemokine distribution reflected the two different immune responses represented within these muscle diseases. In DM, endothelial expression of CCL2 and <em>CXCL1</em>2beta was highly increased. In PM and IBM, macrophages and cytotoxic T cells actively invading nonnecrotic muscle fibers expressed highest levels of <em>CXCL1</em>0 and CCL2. Some chemokines were selectively expressed by different IM infiltrates: CCL4 was present only in the perimysial inflammatory foci of a subset of DM biopsies, while <em>CXCL1</em>, CXCL2, CXCL3, and CCL7-positive cells were exclusively detected in endomysial infiltrates of a number of PM and IBM samples. The chemokine receptor profile of the IM indicated the predominance of Th1-mediated immune responses in all three IM. Our studies identified three ligand-receptor pairs, namely <em>CXCL1</em>0/CXCR3, <em>CXCL1</em>2/CXCR4, and CCL2/CCR2, as potential targets for chemokine-based therapy in IM.

OBJECTIVE

Stomach cancer has a high mortality rate in East Asia, and is strongly associated with Helicobacter pylori (H. pylori) infection. H. pylori is known to express chemokine genes in the gastric mucosa, chemokines that are important host immune factors facilitating inflammation and tumor growth. To investigate the mechanism of carcinogenesis in the stomach, it is essential to determine which molecule of H. pylori is involved in induction of chemokines, but this has remained unclear. We previously reported that a tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) secreted from H. pylori acts as a tumor promoter in stomach cancer development, and thus started to investigate whether Tipalpha is involved in induction of chemokine genes.

METHODS

Comprehensive gene expression analysis was conducted using DNA microarray and KeyMolnet analyses. The gene expression was quantitatively analyzed by real-time RT-PCR.

RESULTS

Comprehensive and quantitative gene expression analyses revealed that Tipalpha induces gene expression of the chemokines Ccl2, Ccl7, Ccl20, <em>Cxcl1</em>, Cxcl2, Cxcl5 and <em>Cxcl1</em>0 extensively and simultaneously in mouse stomach cancer cells, MGT-40. Tipalpha induced high levels of chemokine gene expression, whereas inactive deleted Tipalpha, del-Tipalpha, showed only marginal expression, suggesting a correlation between tumor promotion and chemokine gene expression by Tipalpha. MG-132, a proteasome inhibitor which represses NF-kappaB-activation, inhibited chemokine gene expressions.

CONCLUSIONS

We report here that Tipalpha of H. pylori gene product is a strong inducer of chemokine gene expressions, providing a new model for stomach cancer development.

Improved biomarkers are needed to facilitate clinical decision-making and as surrogate endpoints in clinical trials in multiple sclerosis (MS). We assessed whether neurodegenerative and neuroinflammatory markers in cerebrospinal fluid (CSF) at initial sampling could predict disease activity during 2 years of follow-up in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS.

Using multiplex bead array and enzyme-linked immunosorbent assay, <em>CXCL1</em>, CXCL8, <em>CXCL1</em>0, <em>CXCL1</em>3, CCL20, CCL22, neurofilament light chain (NFL), neurofilament heavy chain, glial fibrillary acidic protein, chitinase-3-like-1, matrix metalloproteinase-9 and osteopontin were analysed in CSF from 41 patients with CIS or relapsing-remitting MS and 22 healthy controls. Disease activity (relapses, magnetic resonance imaging activity or disability worsening) in patients was recorded during 2 years of follow-up in this prospective longitudinal cohort study.

In a logistic regression analysis model, NFL in CSF at baseline emerged as the best predictive marker, correctly classifying 93% of patients who showed evidence of disease activity during 2 years of follow-up and 67% of patients who did not, with an overall proportion of 85% (33 of 39 patients) correctly classified. Combining NFL with either neurofilament heavy chain or osteopontin resulted in 87% overall correctly classified patients, whereas combining NFL with a chemokine did not improve results.

This study demonstrates the potential prognostic value of NFL in baseline CSF in CIS and relapsing-remitting MS and supports its use as a predictive biomarker of disease activity.

Maternal bacterial infections adversely affect lung development by crossing the placental barrier and infecting the developing fetus. The underlying mechanism negatively affecting pulmonary development remains unknown. Herein, we investigated whether a systemic maternal infection affects postnatal inflammation and alveolar development. Pregnant rats were injected with 2.5 mg/kg LPS on day 20 and 21 (term = 22 days). Postnatal (PN0-21) mRNA and protein expression of cytokines (IL-1beta, IL-6, IL-10, CXCL1/2, TNFalpha) and genes implicated in alveologenesis [tropoelastin, lysyl oxidase (LOX), lysyl oxidase-like (LOXL)1, tenascin-C (TNC), fibulin 5, vascular endothelial growth factor (VEGF-A), VEGF receptor (VEGFR)2, VEGFR1, platelet-derived growth factor (PDGF)A, PDGFB, and PDGFRalpha] were quantified by real-time PCR and beadlyte technology. Lung transcript and protein levels of IL-1beta, IL-6, and CXCL1/2 were significantly greater in LPS-exposed pups than those of control pups at PN0, 2, 6, 10, and 14. Bronchoalveolar lavage fluid (BALF) of LPS-exposed animals contained significantly more macrophages at PN2 and 14 than BALF of control pups. Morphometric analysis revealed that LPS-exposed animals had fewer and larger alveoli, fewer secondary septa, and decreased peripheral vessel density when compared with control pups. This morphological delay in alveolar development disappeared after PN14. Tropoelastin, LOXL1, VEGF, VEGFR2, and PDGFRalpha mRNA expression of LPS-exposed animals was significantly greater than those of control animals in PN2-14 lungs. TNC, LOX, fibulin 5, VEGFR1, PDGFA, and PDGFB expression was not affected by maternal LPS exposure. Together, the data demonstrate that maternal exposure to endotoxin results in a prolonged pulmonary inflammation postnatally, altered gene expression of molecules implicated in alveologenesis, and delayed morphological maturation of the lung.

The murine chemokine CXCL1/KC is known as a chemoattractant for neutrophil infiltration and as a promoter of tumor growth. To determine its relevance in tumorigenesis, we first asked whether okadaic acid (OKA), a natural tumor promoter and a potent protein phosphatase 1 and 2A inhibitor, stimulates KC expression and if it does, through what pathway, in a promotable mouse epidermal-like JB6 cell line commonly used for studying molecules related to tumor promotion. We found that OKA stimulated the de novo synthesis of KC mRNA and protein in a dose- and time-dependent manner. To determine the mechanism by which OKA stimulated the expression of KC at the transcriptional level, transient transfection assays using serially deleted sections of KC promoter fused to luciferase reporter gene were performed. These studies showed that transactivation of KC promoter by OKA specifically involved the region between -104 and -59 containing the two nuclear factor-kappaB (NF-kappaB) response elements (kappaB1 and kappaB2). Further analyses using the mutated NF-kappaB response elements kappaB1 and kappaB2 indicated that both regions were required for optimum transactivation of KC by OKA with the former NF-kappaB response element playing a more significant role in regulating KC expression. Gel-shift and supershift analyses demonstrated the involvement of three NF-kappaB subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-kappaB dimer complex. Additionally, immunohistochemistry and western blot analyses confirmed the presence of p65 in the nucleus with its transactivation domain phosphorylated at serine 536. In summary, this is the first report to show that the tumor promoter OKA can stimulate the de novo synthesis and secretion of KC, and that this stimulation is mediated through the NF-kappaB pathway in JB6 cells.