Atopic dermatitis (AD) is the most common inflammatory skin disease, but treatment options for moderate-to-severe disease are limited. Ustekinumab is an IL-12/IL-23p40 antagonist that suppresses Th1, Th17 and Th22 activation, commonly used for psoriasis patients. We sought to assess efficacy and safety of ustekinumab in patients with moderate-to-severe AD. In this phase II, double-blind, placebo-controlled study, 33 patients with moderate-to-severe AD were randomly assigned to either ustekinumab (n=16) or placebo (n=17), with subsequent crossover at 16 weeks, and last dose at 32 weeks. Background therapy with mild topical steroids was allowed to promote retention. Study endpoints included clinical (SCORAD50) and biopsy-based measures of tissue structure and inflammation, using protein and gene expression studies. The ustekinumab group achieved higher SCORAD50 responses at 12, 16 (the primary endpoint) and 20 weeks compared to placebo, but the difference between groups was not significant. The AD molecular profile/transcriptome showed early robust gene modulation, with sustained further improvements until 32 weeks in the initial ustekinumab group. Distinct and more robust modulation of Th1, Th17 and Th22 but also Th2-related AD genes was seen after 4 weeks of ustekinumab treatment (i.e. MMP12, IL-22, IL-13, IFN-γ, elafin/PI3, CXCL1 and CCL17; P<.05). Epidermal responses (K16, terminal differentiation) showed faster (4 weeks) and long-term regulation (32 weeks) from baseline in the ustekinumab group. No severe adverse events were observed. Ustekinumab had clear clinical and molecular effects, but clinical outcomes might have been obscured by a profound "placebo" effect, most likely due to background topical glucocorticosteroids and possibly insufficient dosing for AD.

The crucial role of chemokines in the initiation and progression of atherosclerosis has been widely recognized. Through essential functions in leukocyte recruitment, chemokines govern the infiltration with mononuclear cells and macrophage accumulation in atherosclerotic lesions. Beyond recruitment, chemokines also provide homeostatic functions supporting cell survival and mediating the mobilization and homing of progenitor cells. As a new regulatory layer, several microRNAs (miRNAs) have been found to modulate the function of endothelial cells (ECs), smooth muscle cells and macrophages by controlling the expression levels of chemokines and thereby affecting different stages in the progression of atherosclerosis. For instance, the expression of <em>CXCL1</em> can be down-regulated by miR-181b, which inhibits nuclear factor-κB activation in atherosclerotic endothelium, thus attenuating the adhesive properties of ECs and exerting early atheroprotective effects. Conversely, <em>CXCL1</em>2 expression can be induced by miR-126 in ECs through an auto-amplifying feedback loop to facilitate endothelial regeneration, thus limiting atherosclerosis and mediating plaque stabilization. In contrast, miR-155 plays a pro-atherogenic role by promoting the expression of CCL2 in M1-type macrophages, thereby enhancing vascular inflammation. Herein, we will review novel aspects of chemokines and their regulation by miRNAs during atherogenesis. Understanding the complex cross-talk of miRNAs controlling chemokine expression may open novel therapeutic options to treat atherosclerosis.

The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between type II and type III Toxoplasma strains and quantitative trait locus (QTL) mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside the parasites and is found within the parasitophorous vacuole. In vitro experiments with a type II Δgra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 than those infected with the wild-type or complemented control parasites. In vivo experiments showed that mice infected with a type II Δgra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites and that complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the type II and type III versions of GRA25 differ in endogenous expression levels; however, both are able to promote parasite expansion in vivo when expressed in a type II Δgra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.

BACKGROUND

Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma.

RESULTS

We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFkappaB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFkappaB dependent manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFkappaB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression.

CONCLUSIONS

Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

Regulatory molecules produced by stromal cells are often membrane bound until cleaved by matrix metalloproteinases (MMPs); cleavage can either activate or inactivate regulatory functions. We report here that marrow stromal cells induce the expression of MMP-9 in monocytes. Induction was contact independent and could be reproduced with recombinant MCP-1/CCL2, whereas IL-6, M-CSF, G-CSF, GM-CSF, IL-8/CXCL8, SDF-1/CXCL1CXCL1 did not have this effect. Stroma-induced levels of MMP-9 in the monocyte population from healthy donors were relatively consistent, whereas induced levels varied significantly (P < .001) in the CD14+ population from 27 patients with myelodysplastic syndrome (MDS). In patients with a clonal chromosomal marker, the level of inducible MMP-9 expression in the monocyte population was inversely correlated with the percentage of marker-positive cells (n = 11, P = .01), suggesting that the ability to induce MMP-9 may be compromised in clonally derived monocytes. The inducible levels of MMP-9 were also inversely correlated with marrow cellularity observed in biopsies from MDS patients (P < .001). We conclude that monocytes can express MMP-9 in response to stromal factors and that this response may be significantly decreased in MDS-derived monocytes.

OBJECTIVE

The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium.

METHODS

To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents.

RESULTS

EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment.

CONCLUSIONS

This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.

BACKGROUND

The regulation of gene expression in nociceptive pathways contributes to the induction and maintenance of pain sensitization. Histone acetylation is a key epigenetic mechanism controlling chromatin structure and gene expression. Chemokine CC motif receptor 2 (CXCR2) is a proinflammatory receptor implicated in neuropathic and inflammatory pain and is known to be regulated by histone acetylation in some settings. The authors sought to investigate the role of histone acetylation on spinal CXCR2 signaling after incision.

METHODS

Groups of 5-8 mice underwent hind paw incision. Suberoylanilide hydroxamic acid and anacardic acid were used to inhibit histone deacetylase and histone acetyltransferase, respectively. Behavioral measures of thermal and mechanical sensitization as well as hyperalgesic priming were used. Both message RNA quantification and chromatin immunoprecipitation analysis were used to study the regulation of CXCR2 and ligand expression. Finally, the selective CXCR2 antagonist SB225002 was administered intrathecally to reveal the function of spinal CXCR2 receptors after hind paw incision.

RESULTS

Suberoylanilide hydroxamic acid significantly exacerbated mechanical sensitization after incision. Conversely, anacardic acid reduced incisional sensitization and also attenuated incision-induced hyperalgesic priming. Overall, acetylated histone H3 at lysine 9 was increased in spinal cord tissues after incision, and enhanced association of acetylated histone H3 at lysine 9 with the promoter regions of CXCR2 and keratinocyte-derived chemokine (CXCL1) was observed as well. Blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision.

CONCLUSIONS

Histone modification is an important epigenetic mechanism regulating incision-induced nociceptive sensitization. The spinal CXCR2 signaling pathway is one epigenetically regulated pathway controlling early and latent sensitization after incision.

BACKGROUND

Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors.

METHODS

We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses.

RESULTS

In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients.

CONCLUSIONS

Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.

Klebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores, low-molecular-weight, high-affinity iron chelators, for bacterial replication and full virulence. The specific combination of siderophores secreted by K. pneumoniae during infection can impact tissue localization, systemic dissemination, and host survival. However, the effect of these potent iron chelators on the host during infection is unknown. In vitro, siderophores deplete epithelial cell iron, induce cytokine secretion, and activate the master transcription factor hypoxia inducible factor-1α (HIF-1α) protein that controls vascular permeability and inflammatory gene expression. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to inflammation and bacterial dissemination during pneumonia. To examine the effects of siderophore secretion independently of bacterial growth, we performed infections with tonB mutants that persist in vivo but are deficient in siderophore import. Using a murine model of pneumonia, we found that siderophore secretion by K. pneumoniae induces the secretion of interleukin-6 (IL-6), CXCL1, and CXCL2, as well as bacterial dissemination to the spleen, compared to siderophore-negative mutants at an equivalent bacterial number. Furthermore, we determined that siderophore-secreting K. pneumoniae stabilized HIF-1α in vivo and that bacterial dissemination to the spleen required alveolar epithelial HIF-1α. Our results indicate that siderophores act directly on the host to induce inflammatory cytokines and bacterial dissemination and that HIF-1α is a susceptibility factor for bacterial invasion during pneumonia.

Klebsiella pneumoniae causes a wide range of bacterial diseases, including pneumonia, urinary tract infections, and sepsis. To cause infection, K. pneumoniae steals iron from its host by secreting siderophores, small iron-chelating molecules. Classically, siderophores are thought to worsen infections by promoting bacterial growth. In this study, we determined that siderophore-secreting K. pneumoniae causes lung inflammation and bacterial dissemination to the bloodstream independently of bacterial growth. Furthermore, we determined that siderophore-secreting K. pneumoniae activates a host protein, hypoxia inducible factor (HIF)-1α, and requires it for siderophore-dependent bacterial dissemination. Although HIF-1α can protect against some infections, it appears to worsen infection with K. pneumoniae Together, these results indicate that bacterial siderophores directly alter the host response to pneumonia in addition to providing iron for bacterial growth. Therapies that disrupt production of siderophores could provide a two-pronged attack against K. pneumoniae infection by preventing bacterial growth and preventing bacterial dissemination to the blood.

The role of serum amyloid A (SAA) proteins, which are ligands for toll-like receptors, was analyzed in human bone marrow-derived mesenchymal stem cells (hMSCs) and their osteogenic offspring with a focus on senescence, differentiation and mineralization. In vitro aged hMSC developed a senescence-associated secretory phenotype (SASP), resulting in enhanced SAA1/2, TLR2/4 and proinflammatory cytokine (IL6, IL8, IL1β, CXCL1, CXCL2) expression before entering replicative senescence. Recombinant human SAA1 (rhSAA1) induced SASP-related genes and proteins in MSC, which could be abolished by cotreatment with the TLR4-inhibitor CLI-095. The same pattern of SASP-resembling genes was stimulated upon induction of osteogenic differentiation, which is accompanied by autocrine SAA1/2 expression. In this context additional rhSAA1 enhanced the SASP-like phenotype, accelerated the proinflammatory phase of osteogenic differentiation and enhanced mineralization. Autocrine/paracrine and rhSAA1 via TLR4 stimulate a proinflammatory phenotype that is both part of the early phase of osteogenic differentiation and the development of senescence. This signaling cascade is tightly involved in bone formation and mineralization, but may also propagate pathological extraosseous calcification conditions such as calcifying inflammation and atherosclerosis.

It is not currently possible to predict the probability of whether a woman with a chlamydial genital infection will develop pelvic inflammatory disease (PID). To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (C. muridarum Var001 [CmVar001] and CmVar004) that differ in their abilities to elicit upper genital tract pathology in a mouse model. CmVar004 has a lower growth rate in vitro and induces pathology in only 20% of C57BL/6 mouse oviducts versus 83.3% of oviducts in CmVar001-infected mice. To determine if chemokine and cytokine production within 24 h of infection is associated with the outcome of pathology, levels of 15 chemokines and cytokines were measured. CmVar004 infection induced significantly lower levels of CXCL1, CXCL2, tumor necrosis factor alpha (TNF-α), and CCL2 in comparison to CmVar001 infection with similar rRNA (rs16) levels for Chlamydiae. A combination of microRNA (miRNA) sequencing and quantitative real-time PCR (qRT-PCR) analysis of 134 inflammation-related miRNAs was performed 24 h postinfection to determine if the chemokine/cytokine responses would also be reflected in miRNA expression profiles. Interestingly, 12 miRNAs (miR-135a-5p, miR298-5p, miR142-3p, miR223-3p, miR299a-3p, miR147-3p, miR105, miR325-3p, miR132-3p, miR142-5p, miR155-5p, and miR-410-3p) were overexpressed during CmVar004 infection compared to CmVar001 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge, this is the first report demonstrating that early biomarkers elicited in the host can differentiate between two pathological variants of chlamydiae and be predictive of upper tract disease.

OBJECTIVE

It is apparent that an infecting chlamydial population consists of multiple genetic variants with differing capabilities of eliciting a pathological response; thus, it may be possible to identify biomarkers specific for a given virulence pathotype. miRNAs are known to regulate genes that in turn regulate signaling pathways involved in disease pathogenesis. Importantly, miRNAs are stable and can reflect a tissue response and therefore have the potential to be biomarkers of disease severity. Currently, with respect to chlamydial infections, there is no way to predict whether an infected patient is more or less likely to develop PID. However, data presented in this study indicate that the expression of a specific miRNA profile associated with a virulent variant early in the infection course may be predictive of an increased risk of pelvic inflammatory disease, allowing more aggressive treatment before significant pathology develops.

PAR1 plays a central role in mediating the interplay between coagulation and inflammation, but its role in regulating acute neutrophilic inflammation is unknown. We report that antagonism of PAR1 was highly effective at reducing acute neutrophil accumulation in a mouse model of LPS-induced lung inflammation. PAR1 antagonism also reduced alveolar-capillary barrier disruption in these mice. This protection was associated with a reduction in the expression of the chemokines, CCL2 and CCL7, but not the proinflammatory cytokines, TNF and IL-6, or the classic neutrophil chemoattractants, CXCL1 and CXCL2. Antibody neutralization of CCL2 and CCL7 significantly reduced LPS-induced total leukocyte and neutrophil accumulation, recovered from the bronchoalveolar lavage fluid of challenged mice. Immunohistochemical analysis revealed that CCL2 predominantly localized to alveolar macrophages and pulmonary epithelial cells, whereas CCL7 was restricted to the pulmonary epithelium. In keeping with these observations, the intranasal administration of recombinant CCL2 (rCCL2) and rCCL7 led to the accumulation of neutrophils within the lung airspaces of naive mice in the absence of any underlying inflammation. Flow cytometry analysis further demonstrated an increase in Ly6G(hi) neutrophils expressing the chemokine receptors, CCR1 and CCR2, isolated from mouse lungs compared with circulating neutrophils. Conversely, the expression of CXCR2 decreased on neutrophils isolated from the lung compared with circulating neutrophils. Furthermore, this switch in chemokine receptor expression was accentuated after acute LPS-induced lung inflammation. Collectively, these findings reveal a novel role for PAR1 and the chemokines, CCL2 and CCL7, during the early events of acute neutrophilic inflammation.

Nuclear Factor kappa B (NF-κB) signaling is frequently deregulated in a variety of cancers and is constitutively active in estrogen receptor negative (ER-) breast cancer subtypes. These molecular subtypes of breast cancer are associated with poor overall survival. We focused on mechanisms of NF-κB regulation by microRNAs (miRNAs), which regulate eukaryotic gene expression at the post-transcriptional level. In a previous genome-wide miRNA screen, we had identified miR-30c-2-3p as one of the strongest negative regulators of NF-κB signaling. Here we have uncovered the underlying molecular mechanisms and its consequences in breast cancer. In vitro results show that miR-30c-2-3p directly targets both TNFRSF1A-associated via death domain (TRADD), an adaptor protein of the TNFR/NF-κB signaling pathway, and the cell cycle protein Cyclin E1 (CCNE1). Ectopic expression of miR-30c-2-3p downregulated essential cytokines IL8, IL6, CXCL1, and reduced cell proliferation as well as invasion in MDA-MB-231 breast cancer cells. RNA interference (RNAi) induced silencing of TRADD phenocopied the effects on invasion and cytokine expression caused by miR-30c-2-3p, while inhibition of CCNE1 phenocopied the effects on cell proliferation. We further confirmed the tumor suppressive role of this miRNA using a dataset of 781 breast tumors, where higher expression was associated with better survival in breast cancer patients. In summary we have elucidated the mechanism by which miR-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression in breast cancer.

Obesity is recognized as a low-grade chronic inflammatory state which involves a chemokine network contributing to a variety of diseases. As a first step toward understanding the roles of the obesity-driven chemokine network, we used a 3T3-L1 cell differentiation model to identify the chemokine profiles elicited during adipogenesis and how this profile is modified by epidermal growth factor (EGF) and tumor necrosis factor-α (TNF) as a growth and proinflammatory factor, respectively. The chemokine network was monitored using PCR arrays and qRT-PCR while main signaling pathways of EGF and TNF were measured using immunoblotting. The dominant chemokines in preadipocytes were CCL5, CCL8, CXCL1, and CXCL1CXCL1CXCL1CXCL1CXCL1 and CXCL5 in adipocytes, indicating the potentiation of CXCR2-mediated pathway in adipocytes. TNF induced CCL2, CCL7, and CXCL1 in preadipocytes but had no response in adipocytes. EGFR downstream activation was dominant in adipocytes whereas NFκB activation was dominant in preadipocytes. Taken together, the adipocyte-driven chemokine network in the 3T3-L1 cell differentiation model involves CXCR2-mediated signaling which appears more potentiated to growth factors like EGF than proinflammatory factors like TNF.

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis in susceptible humans. Here, we infected Diversity Outbred (DO) mice with ∼100 bacilli by aerosol to model responses in a highly heterogeneous population. Following infection, 'supersusceptible', 'susceptible' and 'resistant' phenotypes emerged. TB disease (reduced survival, weight loss, high bacterial load) correlated strongly with neutrophils, neutrophil chemokines, tumor necrosis factor (TNF) and cell death. By contrast, immune cytokines were weak correlates of disease. We next applied statistical and machine learning approaches to our dataset of cytokines and chemokines from lungs and blood. Six molecules from the lung: TNF, CXCL1, CXCL2, CXCL5, interferon-γ (IFN-γ), interleukin 12 (IL-12); and two molecules from blood - IL-2 and TNF - were identified as being important by applying both statistical and machine learning methods. Using molecular features to generate tree classifiers, CXCL1, CXCL2 and CXCL5 distinguished four classes (supersusceptible, susceptible, resistant and non-infected) from each other with approximately 77% accuracy using completely independent experimental data. By contrast, models based on other molecules were less accurate. Low to no IFN-γ, IL-12, IL-2 and IL-10 successfully discriminated non-infected mice from infected mice but failed to discriminate disease status amongst supersusceptible, susceptible and resistant M.-tuberculosis-infected DO mice. Additional analyses identified CXCL1 as a promising peripheral biomarker of disease and of CXCL1 production in the lungs. From these results, we conclude that: (1) DO mice respond variably to M. tuberculosis infection and will be useful to identify pathways involving necrosis and neutrophils; (2) data from DO mice is suited for machine learning methods to build, validate and test models with independent data based solely on molecular biomarkers; (3) low levels of immunological cytokines best indicate a lack of exposure to M. tuberculosis but cannot distinguish infection from disease.

OBJECTIVE

Pancreatic cancer is characterized by perineural invasion, early lymph node and liver metastases, and an extremely dismal prognosis. In the present study we aimed at investigating the expression profile of pro-inflammatory and angiogenic CXC chemokines as potential factors contributing to the aggressive biology of this gastrointestinal malignancy.

METHODS

Protein expression profiles of the CXC chemokines growth-related oncogene alpha (GRO-alpha/CXCL1), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78/CXCL5), granulocyte chemoattractant protein-2 (GCP-2/CXCL6), neutrophil-activating protein-2 (NAP-2/CXCL7), and interleukin-8 (IL-8/CXCL8) were assessed by enzyme-linked immunosorbent assay in pancreatic carcinoma, cancer of the papilla of Vater, pancreatic cystadenoma, and chronic pancreatitis specimens.

RESULTS

IL-8 and ENA-78 protein expression was most pronounced in pancreatic carcinoma specimens, showing an 11-fold and 17-fold overexpression in comparison with non-affected neighbouring tissues, a 66-fold and 24-fold upregulation compared to pancreatic cystadenoma, and a 6-fold and 9-fold overexpression with respect to chronic pancreatitis, respectively (p < 0.05 between all groups). In addition, a close correlation between IL-8 and ENA-78 protein expression and advanced pancreatic carcinomas in relation to the T category was evident (p < 0.05).

CONCLUSIONS

Our results demonstrate that ELR+ CXC chemokines are differentially expressed in malignant and non-malignant human pancreatic specimens, suggesting a potential contribution of these chemokines to the pathogenesis of pancreatic carcinoma.

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (<em>CXCL1</em>, CXCL2, <em>CXCL1</em>2, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, <em>CXCL1</em>, and CXCL2 that increased with additional days of social defeat. While <em>CXCL1</em>2 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner.

Airway and/or lung remodeling, involving exaggerated extracellular matrix (ECM) protein deposition, is a critical feature common to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Fibulin-1 (Fbln1), an important ECM protein involved in matrix organization, may be involved in the pathogenesis of these diseases. We found that Fbln1 was increased in COPD patients and in cigarette smoke-induced (CS-induced) experimental COPD in mice. Genetic or therapeutic inhibition of Fbln1c protected against CS-induced airway fibrosis and emphysema-like alveolar enlargement. In experimental COPD, this occurred through disrupted collagen organization and interactions with fibronectin, periostin, and tenascin-c. Genetic inhibition of Fbln1c also reduced levels of pulmonary inflammatory cells and proinflammatory cytokines/chemokines (TNF-α, IL-33, and CXCL1) in experimental COPD. Fbln1c-/- mice also had reduced airway remodeling in experimental chronic asthma and pulmonary fibrosis. Our data show that Fbln1c may be a therapeutic target in chronic respiratory diseases.

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20-30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including <em>CXCL1</em>, CXCL8/IL8, <em>CXCL1</em>0/IP-10, and CCL20/MIP3a, and IL-36 (10-100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, <em>CXCL1</em>, <em>CXCL1</em>0/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound.

Staphylococcus aureus is the leading cause of skin infections. In a mouse model of S. aureus skin infection, we found that lesion size did not correlate with bacterial burden. Athymic nude mice had smaller skin lesions that contained lower levels of myeloperoxidase, IL-17A, and CXCL1, compared with wild type mice, although there was no difference in bacterial burden. T cell deficiency did not explain the difference in lesion size, because TCR βδ (-/-) mice did not have smaller lesions, and adoptive transfer of congenic T cells into athymic nude mice prior to infection did not alter lesion size. The differences observed were specific to the skin, because mortality in a pneumonia model was not different between wild type and athymic nude mice. Thus, the clinical severity of S. aureus skin infection is driven by the inflammatory response to the bacteria, rather than bacterial burden, in a T cell independent manner.

MicroRNAs regulate the maladaptation of endothelial cells (ECs) to naturally occurring disturbed blood flow at arterial bifurcations resulting in arterial inflammation and atherosclerosis in response to hyperlipidemic stress. Here, we show that reduced endothelial expression of the RNAse Dicer, which generates almost all mature miRNAs, decreases monocyte adhesion, endothelial C-X-C motif chemokine 1 (CXCL1) expression, atherosclerosis and the lesional macrophage content in apolipoprotein E knockout mice (Apoe(-/-)) after exposure to a high-fat diet. Endothelial Dicer deficiency reduces the expression of unstable miRNAs, such as miR-103, and promotes Krüppel-like factor 4 (KLF4)-dependent gene expression in murine atherosclerotic arteries. MiR-103 mediated suppression of KLF4 increases monocyte adhesion to ECs by enhancing nuclear factor-κB-dependent CXCL1 expression. Inhibiting the interaction between miR-103 and KLF4 reduces atherosclerosis, lesional macrophage accumulation and endothelial CXCL1 expression. Overall, our study suggests that Dicer promotes endothelial maladaptation and atherosclerosis in part by miR-103-mediated suppression of KLF4.

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF-alpha and IL-1beta) and chemokines (KC/CXCL-1) in the genesis of mechanical hypernociception during antigen-induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anesthesiometer) adapted with a 0.5 mm(2) polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham-immunized (SI) mice, induced mechanical hypernociception in a dose-dependent manner. Hypernociception induced by antigen was reduced in animals pretreated with IL-1ra and reparixin (a non-competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1-/-) mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1beta and KC/CXCL-1 in IM, but not in SI, mice. The increase in TNF-alpha levels preceded the increase in IL-1beta and KC/CXCL1. Antigen-induced release of IL-1beta and KC/CXCL1 was reduced in TNFR1-/- mice, and TNF-alpha-induced hypernociception was inhibited by IL-1ra and reparixin. Hypernociception induced by IL-1beta in immunized mice was inhibited by indomethacin, whereas KC/CXCL1-induced hypernociception was inhibited by indomethacin and guanethidine. Antigen-induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF-alpha, which triggers the subsequent release of IL-1beta and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct-acting final mediators, prostanoids and sympathetic amines.