The MAPK phosphatase DUSP1 is an essential negative regulator of TLR-triggered innate immune activation. Here, we have investigated the impact of DUSP1 on inflammatory and antimicrobial host responses to the intracellular pathogen Chlamydophila pneumoniae. Following nasal infection, DUSP1-deficient mice mounted an enhanced pulmonary cytokine (IL-1beta, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), leading to increased leukocyte infiltration. Of interest, the increased inflammatory response, in the absence of DUSP1, was associated with higher bacterial numbers in the lungs, although the expression of IFN-gamma and critical antichlamydial effector molecules, such as iNOS, was intact. Blockade of IL-6 trans-signaling by injection of a soluble gp130-Fc fusion protein corrected the overshooting chemokine production as well as the increased chlamydial load in Dusp1(-/-) mice. Furthermore, IL-6 enhanced the replication of C. pneumoniae in embryonic fibroblasts in vitro. These data show that DUSP1 is required to achieve a balanced response to chlamydial infection and identify IL-6 as critical for amplifying inflammation and benefiting chlamydial growth through direct effects on infected cells.

Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-ras(G12D); Pdx-1cre) showed early expression of Ncoa3 during pre-neoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels.

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.

Lung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.

C57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.

Lungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.

These results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

Galectins (Gals), a family of mammalian lectins, play diverse roles under physiological and pathological conditions. Here, we analyzed the tandem-repeat Gal-8 synthesis, secretion and effects on the endothelium physiology. Gal-8M and Gal-8L isoforms were secreted under basal conditions by human microvascular endothelial cells (HMEC-1). However, expression and secretion of the Gal-8M isoform, but not Gal-8L, were increased in response to bacterial lipopolysaccharide (LPS) stimulus and returned to control values after LPS removal. Similarly, cell surface Gal-8 exposure was increased after stimulation with LPS. To evaluate Gal-8 effects on the endothelium physiology, HMEC-1 cells were incubated in the presence of recombinant Gal-8M. Pretreated HMEC-1 cells became proadhesive to human normal platelets, indicating that Gal-8 actually activates endothelial cells. This effect was specific for lectin activity as it was prevented by the simultaneous addition of lactose, but not by sucrose. Endothelial cells also increased their exposition of von Willebrand factor after Gal-8 treatment, which constitutes another feature of cell activation that could be, in turn, responsible for the observed platelet adhesion. Several pro-inflammatory molecules were abundantly produced by Gal-8 stimulated endothelial cells: CXCL1 (GRO-α), GM-CSF, IL-6 and CCL5 (RANTES), and in a lower degree CCL2 (MCP-1), CXCL3 (GRO-γ) and CXCL8 (IL-8). In agreement, Gal-8M induced nuclear factor kappa B phosphorylation. Altogether, these results not only confirm the pro-inflammatory role we have already proposed for Gal-8 in other cellular systems but also suggest that this lectin is orchestrating the interaction between leukocytes, platelets and endothelial cells.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory arthropathy associated with articular damage and attendant comorbidities. Even although RA treatment has advanced remarkably over the last decade, a significant proportion of patients still do not achieve sustained remission. The cause of RA is not yet known despite the many potential mechanisms proposed. It has been confirmed that RA is associated with dysregulated immune system and persistent inflammation. Therefore, management of inflammation is always the target of therapy. Sinomenine (SIN) is the prescription drug approved by the Chinese government for RA treatment. A previous study found that SIN was a robust anti-inflammation drug. In this study, we screened the different secretory cytokines using inflammation antibody arrays and qRT-PCR in both LPS-induced and SIN-treated RAW264.7 cells followed by evaluation of the ability of SIN to modulate cytokine secretion in a cell model, collagen-induced arthritis (CIA) mouse model, and RA patients. Several clinical indexes affecting the 28-joint disease activity score (DAS28) were determined before and after SIN treatment. Clinical indexes, inflammatory cytokine secretion, and DAS28 were compared among RA patients treated with either SIN or methotrexate (MTX). To explore the mechanism of SIN anti-inflammatory function, RA-associated monocyte/macrophage subsets were determined using flow cytometry in CIA mouse model and RA patients, both treated with SIN. The results demonstrated that SIN regulated IL-6, GM-CSF, IL-12 p40, IL-1α, TNF-α, IL-1β, KC (CXCL1), Eotaxin-2, IL-10, M-CSF, RANTES, and MCP-1 secretion in vivo and in vitro and reduced RA activity and DAS28 in a clinical setting. Furthermore, SIN attenuated CD11b+F4/80+CD64+ resident macrophages in the synovial tissue, CD11b+Ly6C+CD43+ macrophages in the spleen and draining lymph nodes of CIA mice. The percentage of CD14+CD16+ peripheral blood mononuclear cells was reduced by SIN in RA patients. These data indicated that SIN regulates the secretion of multiple inflammatory cytokines and monocyte/macrophage subsets, thereby suppressing RA progression. Therefore, along with MTX, SIN could be an alternative cost-effective anti-inflammatory agent for treating RA.

Recent evidence indicates that cancer cells express chemokine (CK) receptors and that their signaling is crucial for tumor proliferation, migration, and angiogenesis. The profiles of expression of CXC CK receptors (CXCR1-5) and their main ligands (growth-related oncogene, GRO1-2-3/<em>CXCL1</em>-2-3; interleukin 8, IL-8/CXCL8; monokine-induced gamma-interferon MIG/CXCL9; gamma-interferon-inducible-protein-10, IP-10/<em>CXCL1</em>0; stromal cell-derived factor-1, SDF1/<em>CXCL1</em>2; B-cell activating CK-1, BCA-1/<em>CXCL1</em>3) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) in surgical samples of human meningiomas. All the five receptors displayed high percentages of positive cases: 92% CXCR1, 89% CXCR2, 83% CXCR3, 78% CXCR4, and 94% CXCR5. Conversely, their ligands showed a lower pattern of expression: 40% IL-8, 42% GRO1-3, 42% IP-10, 28% MIG, 53% SDF1, and 3% BCA-1. SDF1/CXCR4 interaction plays a pivotal role in cancer proliferation. Thus, the signaling mechanisms activated by the exclusive binding between SDF1 and CXCR4 was investigated in 12 primary cultures from meningioma tissues. CXCR4 was functionally coupled as demonstrated by the significant increase of DNA synthesis in meningioma cells in response to SDF1, measured by [3H]-thymidine uptake. In three primary cultures, the SDF1-dependent mitogenic activity was associated with a marked phosphorylation of extracellular signal-regulated kinase (ERK1/2) as evaluated by Western blots. PD98059 (a MEK inhibitor) significantly reduced ERK1/2 activation, thus linking the SDF1/CXCR4 pathway to meningioma cell proliferation via ERK1/2 signal transduction. We demonstrate, for the first time in human meningiomas, the simultaneous expression of CXCR1-5 and their CKs and the mitogenic activity of SDF1/CXCR4, suggesting a pivotal role of these receptor-ligand pairs in meningeal tumors.

The Borrelia burgdorferi surface lipoprotein OspC is a critical virulence factor, but its precise role in the establishment of B. burgdorferi infection remains unclear. To determine whether OspC affects the host response at the site of inoculation of the bacterium, the recruitment of macrophages and neutrophils and the production of cytokines were examined at the site of infection by wild-type, ospC mutant, and complemented mutant B. burgdorferi strains. Of the 21 cytokines tested, monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived chemokine (KC, CXCL1), and vascular endothelial growth factor (VEGF) were found at increased levels at the site of inoculation of B. burgdorferi, and the levels varied with the production of OspC at one or more time points over the 1-week course of infection. The kinetics of expression and the dependence on OspC production by B. burgdorferi varied among the cytokines. The production of KC and MCP-1, and the appearance of monocytic infiltrates, correlated with the presence of the bacteria rather than with OspC specifically. In contrast, VEGF production was not correlated simply to the presence of the bacteria and is influenced by the presence of OspC. In in vitro assays, OspC and B. burgdorferi expressing OspC stimulated the growth of endothelial cells more than did the controls. These data suggest the possibility of a novel role for OspC in the life of B. burgdorferi at the interface of its mammalian and tick hosts.

The underlying mechanisms by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect host resistance to Pseudomonas aeruginosa are unclear. The aim of this study was to determine their role on the kinetic of pro- and antiinflammatory response in lung infection. Mice fed either a control diet or a diet enriched with EPA and DHA were infected intratracheally and we studied lung expression of proinflammatory markers [CXCL1, interleukin (IL)-6, tumor necrosis factor-alpha], antiinflammatory markers (IL-10, A20, and IkappaB alpha), and PPARalpha and PPARgamma. The inflammatory response was assessed using recruitment of neutrophils and macrophages into bronchoalveolar lavage fluid, bacterial clearance from the lung, pulmonary injury, and 7-d survival rate. Compared with the control group, EPA and DHA delayed the expression of proinflammatory markers during the first 2 h (P < 0.05), upregulated proinflammatory marker expression (P < 0.05), and induced overexpression of antiinflammatory markers at 8 h (P < 0.05), enhanced recruitment of neutrophils at 16 h (P < 0.05), and induced PPARalpha and PPARgamma overexpression at 4 and 8 h (P < 0.01), respectively. Pulmonary bacterial load decreased and pulmonary injury and mortality were reduced during the first 24 h (P < 0.05). In conclusion, EPA and DHA modulate the balance between pro- and antiinflammatory cytokines, alter the early response of the host to P. aeruginosa infection, and affect the early outcome of infection.

Bone cancer pain (BCP) is still an intractable problem currently because the analgesic pharmacological intervention remains insufficient. Thus, the development of novel therapeutic target is critical for the treatment of BCP. Emerging evidence demonstrated that some chemokines and their receptors contribute to the induction and maintenance of BCP. In this article, we reviewed the current evidence for the role of different chemokines and their receptors (e.g. <em>CXCL1</em>2/CXCR4, <em>CXCL1</em>/CXCR2, CCL2/CCR2, CCL5/CCR5, CX3CL1/CX3CR1 and <em>CXCL1</em>0/CXCR3) in mediating BCP. By extensively understanding the involvement of chemokines and their receptors in BCP, novel therapeutic targets may be revealed for the treatment of BCP.

Fibroblasts have been reported to play an important role in hepatocellular carcinoma (HCC). However, the role of fibroblasts have not been fully understood. Conditioned medium collected from human peri-tumor tissue-derived fibroblasts (CM-pTAFs) showed high metastasis ability than human HCC tissues-derived fibroblasts (CM-TAFs). To determine what component was secreted from fibroblasts, we used Bio-Plex analysis system and compared the factors secreted from CM-pTAFs and CM-TAFs, found a series of up-regulated cytokines in the CM-pTAFs, including IL-6, CCL2, CXCL1, CXCL8, SCGF-β, HGF and VEGF. Pretreatment of IL-6 inhibitor Tocilizumab could inhibit metastasis the HCC cell treated with CM-pTAFs in vitro and in vivo. The expression of CCR2 and CXCR1 were up-regulated after CM-pTAFs treatment in HCC cell line SMMC-7721. Flow cytometric analysis experiment showed that most CCR2 or CXCR1 positive cells were also EpCAM positive. In vitro studies also showed that CM-pTAFs could increase stemness of SMMC-7721. In addition, neutralization of SCGF-β and HGF could significantly reduce metastasis and viability of cancer stem cells treated with CM-pTAFs. Taken together, these results indicated that the peri-tumor tissues derived fibroblasts may promote development of HCC by recruiting cancer stem cells and maintaining their stemness characteristic.

GPR84 is a member of the metabolic G protein-coupled receptor family, and its expression has been described predominantly in immune cells. GPR84 activation is involved in the inflammatory response, but the mechanisms by which it modulates inflammation have been incompletely described. In this study, we investigated GPR84 expression, activation, and function in macrophages to establish the role of the receptor during the inflammatory response. We observed that GPR84 expression in murine tissues is increased by endotoxemia, hyperglycemia, and hypercholesterolemia. Ex vivo studies revealed that GPR84 mRNA expression is increased by LPS and other pro-inflammatory molecules in different murine and human macrophage populations. Likewise, high glucose concentrations and the presence of oxidized LDL increased GPR84 expression in macrophages. Activation of the GPR84 receptor with a selective agonist, 6-(octylamino) pyrimidine-2,4(1H,3H)-dione (6-n-octylaminouracil, 6-OAU), enhanced the expression of phosphorylated Akt, p-ERK, and p65 nuclear translocation under inflammatory conditions and elevated the expression levels of the inflammatory mediators TNFα, IL-6, IL-12B, CCL2, CCL5, and CXCL1. In addition, GPR84 activation triggered increased bacterial adhesion and phagocytosis in macrophages. The enhanced inflammatory response mediated by 6-OAU was not observed in GPR84-/- cells nor in macrophages treated with a selective GPR84 antagonist. Collectively, our results reveal that GPR84 functions as an enhancer of inflammatory signaling in macrophages once inflammation is established. Therefore, molecules that antagonize the GPR84 receptor may be potential therapeutic tools in inflammatory and metabolic diseases.

OBJECTIVE

Searching for useful diagnostic tools to discriminate between asymptomatic bacteriuria (ASB) and acute cystitis, this study compared urinary levels of cytokines/chemokines and leukocyte esterase in three groups of elderly subjects; those with acute cystitis, those with ASB, and those without bacteriuria.

METHODS

Comparative laboratory.

METHODS

Primary care.

METHODS

A total of 16 patients with acute cystitis, 24 subjects with ASB, and 20 controls without bacteriuria, all of whom were aged 80 or over.

METHODS

Urinary levels of IL-1 beta, TNF-alpha, IL-12, IL-18, CXCL1 (GRO-alpha), CXCL8 (IL-8), CCL2 (MCP-1), IL-6, IL-10, and leukocyte esterase.

RESULTS

Urinary levels of CXCL1, CXCL8, and IL-6 were significantly higher in acute cystitis patients than in the ASB group. The sensitivities and specificities for CXCL8, IL-6, and leukocyte esterase to discriminate between acute cystitis and ASB were 63% (95% CI 36-84) and 96% (95% CI 77-100) (cut-off>> 285 pg/mg creatinine), 81% (95% CI 54-95) and 96% (95% CI 77-100) (cut-off>> 30 pg/mg creatinine), and 88% (95% CI 60-98) and 79% (95% CI 57-92) (cut-off>> 2, on a scale of 0-4), respectively.

CONCLUSIONS

The results indicate that measurement of urinary cytokines, and also leukocyte esterase, when using a cut-off value>> 2, could be useful in clinical practice to discriminate between symptomatic and asymptomatic urinary tract infections in the elderly. A combination of IL-6 and leukocyte esterase could be even more useful. This needs to be evaluated in prospective studies on the diagnosis and treatment of urinary tract infections in an elderly population.

Neutrophil migration to the site of bacterial infection is a critical step in host defense. Exclusively produced in the bone marrow, neutrophil release into the blood is tightly controlled. Although the chemokine CXCL1 induces neutrophil influx during bacterial infections, its role in regulating neutrophil recruitment, granulopoiesis, and neutrophil mobilization in response to lung infection-induced sepsis is unclear. Here, we used a murine model of intrapulmonary Streptococcus pneumoniae infection to investigate the role of CXCL1 in host defense, granulopoiesis, and neutrophil mobilization. Our results demonstrate that CXCL1 augments neutrophil influx to control bacterial growth in the lungs, as well as bacterial dissemination, resulting in improved host survival. This was shown in Cxcl1^-/- mice, which exhibited defective amplification of early neutrophil precursors in granulocytic compartments, and CD62L- and CD49d-dependent neutrophil release from the marrow. Administration of recombinant CXCL2 and CXCL5 after infection rescues the impairments in neutrophil-dependent host defense in Cxcl1^-/- mice. Taken together, these findings identify CXCL1 as a central player in host defense, granulopoiesis, and mobilization of neutrophils during Gram-positive bacterial pneumonia-induced sepsis.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and β-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca2+ channels and phospholipase C. β-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and β-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. β-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/β-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/β-arrestin axis and neutrophil function.

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.