OBJECTIVE

Subarachnoid hemorrhage (SAH) results in the expression of inflammatory and extracellular matrix (ECM)-related genes and various G protein-coupled receptors. In the present study, the authors evaluated the time course and sequence of the transduction pathways, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase-1 and 2 (ERK1/2), and associated transcription factor activation as well as gene regulation and associated protein levels.

METHODS

Subarachnoid hemorrhage was induced in rats by injecting 250 microl of blood into the suprachiasmatic cistern, and gene regulation in the cerebral arteries was examined at various points in time following SAH by using quantitative polymerase chain reaction (PCR) and immunohistochemistry.

RESULTS

Immunohistochemical findings demonstrated that SAH phosphorylates and activates p38 and ERK1/2 as well as the downstream transcription factors Elk-1 and activating transcription factor-2. The pattern of activation consists of a rapid phase within the first few hours and a late phase that occurs from 24 to 48 hours. Activation is followed by an increase in the transcription of the inflammatory and ECM-related genes (IL6, TNFalpha, IL1beta, CXCL1, CXCL2, CCL20, MMP8, MMP9, MMP13, and iNOS), as demonstrated using real-time PCR. For MMP13 and iNOS, the changes in transcription were translated into functional proteins, as revealed on immunohistochemistry.

CONCLUSIONS

Activation of the p38 and ERK1/2 signaling pathways and their downstream transcription factors can explain the increase in the transcription of the genes studied. This increase and the subsequent augmentation in protein levels suggest that the inflammatory response may in part explain the remodeling that occurs in cerebral arteries following SAH.

BACKGROUND

Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen-associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by Toll-like receptor (TLR) 4, and neutrophil chemotaxis via integrins.

METHODS

Here we investigated the role of lumican in the development of colitis mediated by intrarectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum(+/+) and Lum(-/-) mice.

RESULTS

The TNBS treated Lum(+/+) mouse colons showed marked increases in CXCL1, tumor necrosis factor alpha (TNF-α), and neutrophil infiltration, whereas these responses were significantly dampened in the Lum(-/-) mice. The nuclear factor kappa B (NF-κB) transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum(+/+) but not in Lum(-/-) colons. Also, nuclear translocation of NF-κB was delayed in LPS stimulated Lum(-/-) primary peritoneal macrophages.

CONCLUSIONS

The Lum(-/-) mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4(-/-) and MyD88(-/-) mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

1. It was proposed previously that oxidative stress is a main component of the inflammatory process in chronic obstructive pulmonary disease (COPD). Thus, in the present study, we investigated the inflammatory response in mice deficient for the p47(phox) subunit of NADPH oxidase (p47 KO) exposed to cigarette smoke (CS). 2. Exposure of mice to CS elicited an increase in the number of macrophages and neutrophils and levels of interleukin (IL)-6, keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein-1 (MCP1/CCL2) in bronchoalveolar lavage fluid (BALF), which were lower in p47 KO mice compared with control mice. In contrast, 24 h after lipopolysaccharide (LPS) exposure, the number of macrophages and neutrophils, as well as KC/CXCL1 levels, in BALF was significantly greater in p47 KO mice compared with control mice. 3. The present study has shown that airway inflammation is decreased in p47 KO mice after exposure to CS, but not LPS, suggesting that oxidative stress is involved in the pathogenesis of airway inflammation associated with COPD.

Expression of Bcl-x(L) correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-x(L) prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.

Single-dose thioacetamide (TAA) administration induces inflammation and acute liver damage. The mechanism of inflammatory cell recruitment in the liver is still unclear. The aim of this study was to examine the sequence and recruitment of inflammatory cells in different liver regions in relation to CXC- and CC-chemokine and cytokine expression during acute liver injury. Single-dose TAA was administered to rats intraperitoneally, and animals were killed at different time points thereafter. Serum and liver tissue were taken and frozen immediately. Tissue was used for immunostaining cryostat sections, RNA, and protein extraction. RT-PCR and western blotting were performed for RNA and protein analysis, respectively. An early increase (3 h) in CXCL8/IL-8 levels was measured followed by a marked release in MCP1/CCL2 (24 h) serum levels after TAA administration compared with controls. Similarly, an early increase in specific RNA of hepatic chemokines CXCL1/KC and CXCL8/IL-8 was found at 3 h, followed by an upregulation of CXCL5/LIX (6 h), CXCL2/MIP-2 (12 h), and MCP1/CCL2 gene expression at 24-48 h. Further, an induction of pro-inflammatory cytokines IFN-γ and IL-1β followed by IL-6 and TNF-α was observed with a maximum at 12 h. The magnitude of increase in gene expression of TNF-α and MCP1/CCL2 was the highest among all cytokines and chemokines, respectively. By means of immunohistochemistry, an early (12-24 h) increase in the number of only neutrophil granulocytes (NGs) attached to and around portal vessel walls was observed, followed by increased numbers of mononuclear phagocytes (24-48 h) along the sinusoids. Treatment of the human monocytic cell line U-937 with TNF-α increased the gene expression of CXCL1/KC, CXCL8/IL-8, and MCP1/CCL2. Conversely, adding of infliximab (IFX) to the culture medium inhibited this upregulation significantly. In conclusion, single-dose TAA administration induces a sequence of events with a defined upregulation of gene expression of inflammatory chemokines and cytokines and a transient accumulation of NGs within the portal area and macrophages along the sinusoids throughout the liver. Periportal inflammation seems to precede hepatocellular damage.

OBJECTIVE

Inflammation at the level of the sensory dorsal root ganglia (DRGs) leads to robust mechanical pain behavior and the local inflammation has direct excitatory effects on sensory neurons including small, primarily nociceptive, neurons. These neurons express the transient receptor potential vanilloid-1 (TRPV1) channel, which integrates multiple signals of pain and inflammation. The aim of this study was to characterize the regulation of the TRPV1 channel by local DRG inflammation and by growth-related oncogene (GRO/KC, systemic name: CXCL1), a cytokine known to be upregulated in inflamed DRGs.

METHODS

Activation of the TRPV1 receptor with capsaicin was studied with patch clamp methods in acutely isolated small-diameter rat sensory neurons in primary culture. In vivo, behavioral effects of TRPV1 and GRO/KC were examined by paw injections.

RESULTS

Neurons isolated from lumbar DRGs 3 days after local inflammation showed enhanced TRPV1 function: tachyphylaxis (the decline in response to repeated applications of capsaicin) was significantly reduced. A similar effect on tachyphylaxis was observed in neurons pre-treated for 4 h in vitro with GRO/KC. This effect was blocked by H-89, a protein kinase A inhibitor. Consistent with the in vitro results, in vivo behavioral responses to paw injection of capsaicin were enhanced and prolonged by pre-injecting the paw with GRO/KC 4 h before the capsaicin injection. GRO/KC paw injections alone did not elicit pain behaviors.

CONCLUSIONS

Function of the TRPV1 channel is enhanced by DRG inflammation and these effects are preserved in vitro during short-term culture. The effects (decreased tachyphylaxis) are mimicked by incubation with GRO/KC, which has previously been found to be strongly upregulated in this and other pain models.

Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women, worldwide. In the early stages of the disease, biomarkers predicting early relapse would improve survival rates. In metastatic patients, the use of predictive biomarkers could potentially result in more personalized treatments and better outcomes. The CXC family of chemokines (CXCL1 to 17) are small (8 to 10 kDa) secreted proteins that attract neutrophils and lymphocytes. These chemokines signal through chemokine receptors (CXCR) 1 to 8. Several studies have reported that these chemokines and receptors have a role in either the promotion or inhibition of cancer, depending on their capacity to suppress or stimulate the action of the immune system, respectively. In general terms, activation of the CXCR1/CXCR2 pathway or the CXCR4/CXCR7 pathway is associated with tumor aggressiveness and poor prognosis; therefore, the specific inhibition of these receptors is a possible therapeutic strategy. On the other hand, the lesser known CXCR3 and CXCR5 axes are generally considered to be tumor suppressor signaling pathways, and their stimulation has been suggested as a way to fight cancer. These pathways have been studied in tumor tissues (using immunohistochemistry or measuring mRNA levels) or serum [using enzyme-linked immuno sorbent assay (ELISA) or multiplexing techniques], among other sample types. Common variants in genes encoding for the CXC chemokines have also been investigated as possible biomarkers of the disease. This review summarizes the most recent findings on the role of CXC chemokines and their receptors in CRC and discusses their possible value as prognostic or predictive biomarkers as well as the possibility of targeting them as a therapeutic strategy.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-β, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

BACKGROUND

Chronic rhinosinusitis (CRS) is characterized by persistent mucosal inflammation and frequent exacerbations.

OBJECTIVE

To determine whether innate epithelial responses to cigarette smoke or bacterial or viral pathogens may be abnormal in CRS leading to an inappropriate inflammatory response.

METHODS

Primary nasal epithelial cells (PNECs) were grown from middle turbinate biopsies of 9 healthy controls and 11 patients with CRS. After reaching 80% to 90% confluence, PNECs were exposed to medium or cigarette smoke extract (CSE) 5% (vol/vol) for 1 hour, washed, then stimulated with staphylococcal lipoteichoic acid, LPS, or double-stranded RNA (dsRNA). After 24 hours, gene expression was quantified by QRT-PCR.

RESULTS

At baseline, PNECs revealed elevated TNF-alpha and growth-related oncogene-alpha (a C-X-C chemokine)/CXCL1 (GRO-alpha) (4-fold increase, P = .02; and 16-fold increase, P = .004, respectively) in subjects with CRS compared with controls with normal levels of IL-1beta, IL-6, IL-8/CXCL8, human beta-defensin-2, monocyte chemoattractant protein 2/CCL8, monocyte chemoattractant protein 3/CCL7, and regulated upon activation, normal T-cell expressed and secreted (RANTES)/CCL5. Immunostaining of nasal biopsies, however, revealed comparable epithelial staining for TNF-alpha, GRO-alpha, and RANTES. There were no differences in mRNA induction by CSE, TNF-alpha, lipoteichoic acid, LPS, or dsRNA alone. The combination of CSE+dsRNA induced exaggerated RANTES (12,115-fold vs 1500-fold; P = .03) and human beta-defensin-2 (1120-fold vs 12.5-fold; P = .05) in subjects with CRS. No other genes were differentially induced. Furthermore, CSE+dsRNA induced normal levels of IFN-beta, IFN-lambda1, and IFN-lambda2/3 mRNA in subjects with CRS.

CONCLUSIONS

Cigarette smoke extract plus dsRNA induces exaggerated epithelial RANTES expression in patients with CRS. We propose that an analogous response to cigarette smoke plus viral infection may contribute to acute exacerbations and eosinophilic mucosal inflammation in CRS.

Infection with respiratory syncytial virus (RSV) in neonatal mice leads to exacerbated disease if mice are reinfected with the same virus as adults. Both T cells and the host major histocompatibility complex genotype contribute to this phenomenon, but the part played by innate immunity has not been defined. Since macrophages and natural killer (NK) cells play key roles in regulating inflammation during RSV infection of adult mice, we studied the role of these cells in exacerbated inflammation following neonatal RSV sensitization/adult reinfection. Compared to mice undergoing primary infection as adults, neonatally sensitized mice showed enhanced airway fluid levels of interleukin-6 (IL-6), alpha interferon (IFN-α), CXCL1 (keratinocyte chemoattractant/KC), and tumor necrosis factor alpha (TNF-α) at 12 to 24 h after reinfection and IL-4, IL-5, IFN-γ, and CCL11 (eotaxin) at day 4 after reinfection. Weight loss during reinfection was accompanied by an initial influx of NK cells and granulocytes into the airways and lungs, followed by T cells. NK cell depletion during reinfection attenuated weight loss but did not alter T cell responses. Depletion of alveolar macrophages with inhaled clodronate liposomes reduced both NK and T cell numbers and attenuated weight loss. These findings indicate a hitherto unappreciated role for the innate immune response in governing the pathogenic recall responses to RSV infection.

BACKGROUND

Respiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study.

METHODS

A549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection.

CONCLUSIONS

RSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, <em>CXCL1</em>2 and stem cell growth factor beta (SCGF-β). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1β, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, <em>CXCL1</em>, IP-10/<em>CXCL1</em>0, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1β, IL-5, <em>CXCL1</em>2, MIF, LIF, <em>CXCL1</em>2 and IFN-γ. Silencing of retinoic acid-inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment.

CONCLUSIONS

RSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.

A variety of chemokines has been shown to recruit human bone marrow-derived mesenchymal stem cells (MSC) and may be potential candidates for chemokine-based tissue regeneration approaches. The aim of our study was to determine whether the chemokine CXCL7 stimulates migration of human bone marrow-derived MSC and to analyze the effect of CXCL7 on the recruitment of MSC on the broad molecular level. Chemotaxis assays documented that high doses of CXCL7 significantly recruited MSC. Gene expression profiling using oligonucleotide microarrays showed that MSC treated with CXCL7 differentially expressed genes related to cell migration, cell adhesion and extracellular matrix remodeling. Pathway analysis showed that CXCL7 induced the expression of all chemokines binding the interleukin (IL) receptors A and B, CXCR1 and CXCR2, as well as the IL6 signal transducer (gp130) and its ligands IL6 and leukemia inhibitory factor (LIF). Induction of differentially expressed chemokines CXCL1-3, CXCL5, and CXCL6 as well as LIF and gp130 in MSC by CXCL7 was verified by real-time polymerase chain reaction. Immunoassay of cell culture supernatants confirmed elevated levels of the interleukins 6 and 8 in MSC upon treatment with CXCL7. Chemotaxis assays showed that interleukin 6 did not recruit MSC. In conclusion, CXCL7 significantly stimulates the migration of human MSC in vitro. Pathway analysis suggests that recruitment of human MSC by CXCL7 is supported by the induction of ligands of the interleukin 8 receptors, synergistically activating the respective signaling pathways.

Systemic inflammation shifts the brain microenvironment towards a proinflammatory state. However, how peripheral inflammation mediates changes in the brain remains to be clarified. We aimed to identify hippocampal cells and cytokines that respond to endotoxemia. Mice were intraperitoneally injected with lipopolysaccharide (LPS) or saline, and examined 1, 4, and 24 h after injection. Tissue cytokine concentrations in the spleens and hippocampi were determined by multiplex assays. Another group of mice were studied immunohistologically. Fourteen cytokines showed an increased concentration in the spleen, and 10 showed an increase in the hippocampus after LPS injection. Cytokines increased at 4 h (CCL2, <em>CXCL1</em>, CXCL2, and interleukin-6) were expressed by leptomeningeal stromal cells, choroid plexus stromal cells, choroid plexus epithelial cells, and hippocampal vascular endothelial cells, all of which were located at the brain-immune interface. Receptors for these cytokines were expressed by astrocytic endfeet. Cytokines increased at 24 h (CCL11, <em>CXCL1</em>0, and granulocyte-colony stimulating factor) were expressed by astrocytes. Cells of the brain-immune interface therefore respond to endotoxemia with cytokine signals earlier than hippocampal parenchymal cells. In the parenchyma, astrocytes play a key role in responding to signals by using endfeet located in close apposition to the interface cells via cytokine receptors.

Recent studies have demonstrated that human dental pulp cells sense pathogens and elicit innate and/or adaptive immunity. Particular attention has been paid to odontoblasts that are situated at the pulp-dentin interface and constitute the first line of defense to cariogenic bacteria entering dentin after enamel disruption. In this review, recent in vitro and in vivo data suggesting that odontoblasts initiate immune/inflammatory events within the dental pulp in response to cariogenic bacteria are discussed. These data include sensing of pathogens by Toll-like receptors (TLRs), production of chemokines upon cell stimulation with microbial by-products and induction of dendritic cell migration. Additional results presented here reveal that all TLR genes are expressed in the healthy human dental pulp that is thus well equipped to combat pathogens entering the tissue. Seventeen chemokine genes including CXCL1CXCL1CXCL1CXCL1 are upregulated in odontoblasts both under caries lesions and upon stimulation with pathogen by-products. These molecules thus appear as preferential targets for the design of therapeutic agents able to reduce the immune/inflammatory response to cariogenic bacteria and favor pulp healing.

The nuclear receptor PPARγ acts as a key modulator of lipid metabolism, inflammation and pathogenesis in BCG-infected macrophages. However, the molecular mechanisms involved in PPARγ expression and functions during infection are not completely understood. Here, we investigate signaling pathways triggered by TLR2, the involvement of co-receptors and lipid rafts in the mechanism of PPARγ expression, lipid body formation and cytokine synthesis in macrophages during BCG infection. BCG induces NF-κB activation and increased PPARγ expression in a TLR2-dependent manner. Furthermore, BCG-triggered increase of lipid body biogenesis was inhibited by the PPARγ antagonist GW9662, but not by the NF-κB inhibitor JSH-23. In contrast, KC/CXCL1 production was largely dependent on NF-κB but not on PPARγ. BCG infection induced increased expression of CD36 in macrophages in vitro. Moreover, CD36 co-immunoprecipitates with TLR2 in BCG-infected macrophages, suggesting its interaction with TLR2 in BCG signaling. Pretreatment with CD36 neutralizing antibodies significantly inhibited PPARγ expression, lipid body formation and PGE2 production induced by BCG. Involvement of CD36 in lipid body formation was further confirmed by decreased BCG-induced lipid body formation in CD36 deficient macrophages. Similarly, CD14 and CD11b/CD18 blockage also inhibited BCG-induced lipid body formation, whereas TNF-α synthesis was not affected. Disruption of rafts recapitulates the latter result, inhibiting lipid body formation, but not TNF-α synthesis in BCG-infected macrophages. In conclusion, our results suggest that CD36-TLR2 cooperation and signaling compartmentalization within rafts, divert host response signaling through PPARγ-dependent and NF-κB-independent pathways, leading to increased macrophage lipid accumulation and down-modulation of macrophage response.

Neutrophil attraction and adhesion to endothelial cells occurs via well defined mechanisms, yet the ability of other cell types to express neutrophil-binding adhesion molecules is not well studied. Cardiac fibroblasts (CF) are a key cell type involved in repair of the infarcted myocardium, a scenario in which neutrophil recruitment is perceived to be detrimental. Here we determined the effects of proinflammatory cytokines on expression of neutrophil-binding adhesion molecules and neutrophil-attracting chemokines in CF cultured from multiple patients, and explored the underlying regulatory mechanisms. An adhesion molecule focused RT-PCR array identified 5 transcripts that were increased markedly in human CF treated with the proinflammatory cytokine interleukin-1 (IL-1, 10 ng/ml, 6 h); including intercellular cell adhesion molecule (ICAM-1) and E-selectin. Real-time RT-PCR verified the array data and immunoblotting confirmed cytokine-induced ICAM-1 and E-selectin protein expression. Treatment with a panel of pharmacological inhibitors identified the NF-κB pathway as mediating IL-1-induced ICAM-1 and E-selectin mRNA and protein expression. Additionally, E-selectin expression in human CF was markedly potentiated by the JNK inhibitor SP600125, but this was not observed when a more selective inhibitor ((L)-JNKI-1) was used, or in human vascular endothelial cells. IL-1 also stimulated CF to secrete the neutrophil chemoattractant CXCL8 via a p38- and NF-κB-dependent mechanism, as well as inducing CXCL1, CXCL2 and CXCL5 mRNA expression. In conclusion, human CF express neutrophil-binding adhesion molecules and neutrophil chemoattractants in response to proinflammatory cytokines suggesting that, in addition to EC, CF may play an important role in regulating neutrophil recruitment into the infarcted myocardium.

Albuminuria is both a hallmark and a risk factor for progressive glomerular disease, and results in increased exposure of podocytes to serum albumin with its associated factors. Here in vivo and in vitro models of serum albumin-overload were used to test the hypothesis that albumin-induced proteinuria and podocyte injury directly correlate with COX-2 induction. Albumin induced COX-2, MCP-1, CXCL1, and the stress protein HSP25 in both rat glomeruli and cultured podocytes, whereas B7-1 and HSP70i were also induced in podocytes. Podocyte exposure to albumin induced both mRNA and protein and enhanced the mRNA stability of COX-2, a key regulator of renal hemodynamics and inflammation, which renders podocytes susceptible to injury. Podocyte exposure to albumin also stimulated several kinases (p38 MAPK, MK2, JNK/SAPK, and ERK1/2), inhibitors of which (except JNK/SAPK) downregulated albumin-induced COX-2. Inhibition of AMPK, PKC, and NFκB also downregulated albumin-induced COX-2. Critically, albumin-induced COX-2 was also inhibited by glucocorticoids and thiazolidinediones, both of which directly protect podocytes against injury. Furthermore, specific albumin-associated fatty acids were identified as important contributors to COX-2 induction, podocyte injury, and proteinuria. Thus, COX-2 is associated with podocyte injury during albuminuria, as well as with the known podocyte protection imparted by glucocorticoids and thiazolidinediones. Moreover, COX-2 induction, podocyte damage, and albuminuria appear mediated largely by serum albumin-associated fatty acids.

Celecoxib is a specific inhibitor of cyclooxygenase 2 (COX2). While it has been used for the treatment of chronic inflammatory conditions, including rheumatoid arthritis, its detailed anti-inflammatory mechanism has not been clarified. Here, we found that Celecoxib potently inhibited TNFalpha-induced transcriptional activity and DNA binding activity of NF-kappaB; however, Celecoxib had no effect on TNFalpha-induced IKK activation and degradation of IkappaBalpha and IkappaBbeta, suggesting that it inhibited NF-kappaB activation via suppressing downstream of IKK activation and IkappaBs degradation. Interestingly, it was also found that Celecoxib abrogated TNFalpha-induced nuclear accumulation of the NF-kappaB p65 subunit. As a result, TNFalpha-induced expression of inflammatory cytokines, CXCL1/KC and CCL2/MCP-1, was clearly inhibited by Celecoxib. On the other hand, Celecoxib had no effect on the TNFalpha-induced nuclear translocation of c-jun and activation of ERK, JNK, p38 and Akt. Taken together, these data indicate that Celecoxib specifically inhibits TNFalpha-induced NF-kappaB activation at the level of its nuclear translocation. This negative regulation of NF-kappaB activation by Celecoxib might be an important mechanism leading to its anti-inflammatory activity.

BACKGROUND

The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action.

RESULTS

Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1β, CXCL1/KC, MCP-1, MIP-2, TNF-α and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-κB (NF-κB). In addition, euphol decreased LPS-induced MCP-1, TNF-α, IL-6 and IFN-γ, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue.

CONCLUSIONS

Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant-derived compound might be a potential molecule in the management of inflammatory bowel diseases.

Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin-1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)-induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c-deficient (Fbln1c-/- ) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild-type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c-/- mice with AAD also had reduced numbers of α-smooth muscle actin-positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)-5, IL-13, IL-33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL-5, IL-33 and TNF levels in lung-draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3-positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL-5 and IL-13 from co-cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium-treated controls. Our data show that Fbln1c may be a therapeutic target in chronic asthma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a 'cast' carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14⁺CD16⁻, intermediate monocytes (intM) CD14⁺ CD16⁺ and nonclassical monocytes (ncM) CD14⁺ CD16⁺ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1β after LPS stimulation. Based on IL-1β secretion after LPS/ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNγ increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes.

Solid tumour and leukemic cells expressing chemokine receptors, metastasize to chemokine-secreting organs. Chemokines indirectly affect tumour development by attracting immunocompetent cells with pro- or anti-tumoral activities. Various membrane-associated and soluble proteases selectively cleave specific chemokines. Precursor plasma chemokines (CXCL7, CCL14) need to be proteolytically processed to obtain receptor affinity. Angiogenic CXC chemokines (<em>CXCL1</em>, CXCL8) have increased CXCR1/CXCR2 affinity after limited NH2-terminal processing, whereas truncated angiostatic chemokines (<em>CXCL1</em>0) show lower CXCR3 affinity without loss of angiostatic potential. NH2-terminally cleaved monocyte chemotactic proteins (CCL2, CCL7, CCL8) have impaired capacity to attract tumour-associated macrophages and function as receptor antagonists for intact CC chemokines. Migration of Th1/CCR5+ and Th2/CCR4+ effector lymphocytes toward CCR5 (CCL5, CCL3L1) and CCR4 (CCL22) ligands is affected by cleavage. Although proteolytical processing of chemokines is well studied in vitro, the direct or indirect effects on tumour invasion and metastasis are only poorly evaluated.

BACKGROUND

Alcohol-induced intestinal dysbiosis disrupts homeostatic gut-liver axis function and is essential in the development of alcoholic liver disease. Here, we investigate changes in enteric microbiome composition in a model of early alcoholic steatohepatitis and dissect the pathogenic role of intestinal microbes in alcohol-induced liver pathology.

METHODS

Wild type mice received a 10-day diet that was either 5% alcohol-containing or an isocaloric control diet plus a single binge. 16S rDNA sequencing defined the bacterial communities in the cecum of alcohol- and pair-fed animals. Some mice were treated with an antibiotic cocktail prior to and throughout alcohol feeding. Liver neutrophils, cytokines and steatosis were evaluated.

RESULTS

Acute-on-chronic alcohol administration induced shifts in various bacterial phyla in the cecum, including increased Actinobacteria and a reduction in Verrucomicrobia driven entirely by a reduction in the genus Akkermansia. Antibiotic treatment reduced the gut bacterial load and circulating bacterial wall component lipopolysaccharide (LPS). We found that bacterial load suppression prevented alcohol-related increases in the number of myeloperoxidase- (MPO) positive infiltrating neutrophils in the liver. Expression of liver mRNA tumor necrosis factor alpha (Tnfα), C-X-C motif chemokine ligand 1 (Cxcl1) and circulating protein monocyte chemoattractant protein-1 (MCP-1) were also reduced in antibiotic-treated alcohol-fed mice. Alcohol-induced hepatic steatosis measured by Oil-Red O staining was significantly reduced in antibiotic treated mice. Genes regulating lipid production and storage were also altered by alcohol and antibiotic treatment. Interestingly, antibiotic treatment did not protect from alcohol-induced increases in serum aminotransferases (ALT/AST).

CONCLUSIONS

Our data indicate that acute-on-chronic alcohol feeding alters the microflora at multiple taxonomic levels and identifies loss of Akkermansia as an early marker of alcohol-induced gut dysbiosis. We conclude that gut microbes influence liver inflammation, neutrophil infiltration and liver steatosis following alcohol consumption and these data further emphasize the role of the gut-liver axis in early alcoholic liver disease.