In normal physiologic responses to injury and infection, inflammatory cells enter tissue and sites of inflammation through a chemotactic process regulated by several families of proteins, including inflammatory chemokines, a family of small inducible cytokines. In neutrophils, chemokines chemokine (CXC motif) ligand 1 (CXCL1) and CXCL8 are potent chemoattractants and activate G protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXCR2. Several small-molecule antagonists of CXCR2 have been developed to inhibit the inflammatory responses mediated by this receptor. Here, we present the data describing the pharmacology of AZD5069 [N-(2-(2,3-difluorobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)[2,4,5,6-(13)C4, 1,3-(15)N2]pyrimidin-4-yl)azetidine-1-sulfonamide,[(15)N2,(13)C4]N-(2-(2,3-difluoro-6-[3H]-benzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide], a novel antagonist of CXCR2. AZD5069 was shown to inhibit binding of radiolabeled CXCL8 to human CXCR2 with a pIC50 value of 9.1. Furthermore, AZD5069 inhibited neutrophil chemotaxis, with a pA2 of approximately 9.6, and adhesion molecule expression, with a pA2 of 6.9, in response to CXCL1. AZD5069 was a slowly reversible antagonist of CXCR2 with effects of time and temperature evident on the pharmacology and binding kinetics. With short incubation times, AZD5069 appeared to have an antagonist profile with insurmountable antagonism of calcium response curves. This behavior was also observed in vivo in an acute lipopolysaccharide-induced lung inflammation model. Altogether, the data presented here show that AZD5069 represents a novel, potent, and selective CXCR2 antagonist with potential as a therapeutic agent in inflammatory conditions.

Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.

OBJECTIVE

The effects of high-refined carbohydrate-containing diet (HC) on inflammatory parameters and metabolic disarrangement of adipose tissue are poorly understood. The aim of this study was to evaluate the timing and progression of metabolic and inflammatory dysfunction induced by HC diet in mice.

METHODS

BALB/c mice were fed chow or HC diet for 1 and 3 days, 1, 2, 4, 6, 8, 10, and 12 weeks.

RESULTS

Animals given HC diet exhibited acute and sustained increase in visceral adiposity, glucose intolerance, low insulin sensitivity, hyperlipemia, acute increase in mRNA expression of ACC, LPL, PPARγ, SREBP-1, and ChREBP and altered circulating levels of adiponectin, resistin, and leptin. There was leucocyte rolling and adhesion on adipose tissue microvessels already at 3 days and until 8 weeks of HC diet. Adipose tissue of mice had increased number of macrophages (M1 and M2), lymphocytes (CD8+ and CD4+ Foxp3+), and neutrophils (GR1+) already at 3 days after initiation of HC diet. Overall, concentration of cytokines and chemokines, TNF-α, IL-6, IL-10, TGF-β1, CCL2, and CXCL1, in adipose tissue was elevated throughout the experimental period. Levels of IL-10 and TGF-β1 tended to reach baseline levels at 12 weeks of HC diet.

CONCLUSIONS

We describe a novel murine model of fat pad expansion induced by HC diet that is characterized by early onset and sustained adipose tissue inflammation and metabolic disarrangement. The acute inflammatory response in adipose tissue occurs very early and is sustained, suggesting that adipose tissue inflammation is a homeostatic mechanism to regulate nutrient overload and adipose expansion.

The excellent clinical efficacy of anti-interleukin 17A (IL-17A) biologics on psoriasis indicates a crucial pathogenic role of IL-17A in this autoinflammatory skin disease. IL-17A accelerates the proliferation of epidermal keratinocytes. Keratinocytes produce a myriad of antimicrobial peptides and chemokines, such as CXCL1, CXCL2, CXCL8, and CCL20. Antimicrobial peptides enhance skin inflammation. IL-17A is capable of upregulating the production of these chemokines and antimicrobial peptides in keratinocytes. CXCL1, CXCL2, and CXCL8 recruit neutrophils and CCL20 chemoattracts IL-17A-producing CCR6⁺ immune cells, which further contributes to forming an IL-17A-rich milieu. This feed-forward pathogenic process results in characteristic histopathological features, such as epidermal hyperproliferation, intraepidermal neutrophilic microabscess, and dermal CCR6⁺ cell infiltration. In this review, we focus on IL-17A and keratinocyte interaction regarding psoriasis pathogenesis.

Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

Epidermal growth factor receptor (EGFR) signaling is a known mediator of colorectal carcinogenesis. Studies have focused on the role of EGFR signaling in epithelial cells, although the exact nature of the role of EGFR in colorectal carcinogenesis remains a topic of debate. Here, we present evidence that EGFR signaling in myeloid cells, specifically macrophages, is critical for colon tumorigenesis in the azoxymethane-dextran sodium sulfate (AOM-DSS) model of colitis-associated carcinogenesis (CAC). In a human tissue microarray, colonic macrophages demonstrated robust EGFR activation in the pre-cancerous stages of colitis and dysplasia. Utilizing the AOM-DSS model, mice with a myeloid-specific deletion of Egfr had significantly decreased tumor multiplicity and burden, protection from high-grade dysplasia and significantly reduced colitis. Intriguingly, mice with gastrointestinal epithelial cell-specific Egfr deletion demonstrated no differences in tumorigenesis in the AOM-DSS model. The alterations in tumorigenesis in myeloid-specific Egfr knockout mice were accompanied by decreased macrophage, neutrophil and T-cell infiltration. Pro-tumorigenic M2 macrophage activation was diminished in myeloid-specific Egfr-deficient mice, as marked by decreased Arg1 and Il10 mRNA expression and decreased interleukin (IL)-4, IL10 and IL-13 protein levels. Surprisingly, diminished M1 macrophage activation was also detectable, as marked by significantly reduced Nos2 and Il1b mRNA levels and decreased interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-1β protein levels. The alterations in M1 and M2 macrophage activation were confirmed in bone marrow-derived macrophages from mice with the myeloid-specific Egfr knockout. The combined effect of restrained M1 and M2 macrophage activation resulted in decreased production of pro-angiogenic factors, CXCL1 and vascular endothelial growth factor (VEGF), and reduced CD31+ blood vessels, which likely contributed to protection from tumorigenesis. These data reveal that EGFR signaling in macrophages, but not in colonic epithelial cells, has a significant role in CAC. EGFR signaling in macrophages may prove to be an effective biomarker of CAC or target for chemoprevention in patients with inflammatory bowel disease.

OBJECTIVE

Rheumatoid arthritis is a chronic destructive autoimmune disease, but the course is unpredictable in individual patients. An attractive treatment would provide a disease-regulated therapy that offers personalised drug delivery. Therefore, we expressed the anti-inflammatory interleukin-10 (IL-10) gene under the control of inflammation-dependent promoters in a mouse model of arthritis.

METHODS

Proximal promoters of S100a8, Cxcl1, Mmp13, Saa3, IL-1b and Tsg6 were selected by whole-genome expression analysis of inflamed synovial tissues from arthritic mice. Mice were injected intraarticularly in knee joints with lentiviral vectors expressing a luciferase reporter or the therapeutic protein IL-10 under control of the Saa3 or Mmp13 promoter. After 4 days, arthritis was induced by intraarticular injection of streptococcal cell walls (SCW). At different time points after arthritis induction, in vivo bioluminescent imaging was performed and knee joints were dissected for histological and RNA analysis.

RESULTS

The disease-regulated promoter-luciferase reporter constructs showed different activation profiles during the course of the disease. The Saa3 and Mmp13 promoters were significantly induced at day 1 or day 4 after arthritis induction respectively and selected for further research. Overexpression of IL-10 using these two disease-inducible promoters resulted in less synovitis and markedly diminished cartilage proteoglycan depletion and in upregulation of IL-1Ra and SOCS3 gene expression.

CONCLUSIONS

Our study shows that promoters of genes that are expressed locally during arthritis can be candidates for disease-regulated overexpression of biologics into arthritic joints, as shown for IL-10 in SCW arthritis. The disease-inducible approach might be promising for future tailor-made local gene therapy in arthritis.

BACKGROUND

Diabetes is a significant risk factor for developing West Nile virus (WNV)-associated encephalitis (WNVE) in humans, the leading cause of arboviral encephalitis in the United States. Using a diabetic mouse model (db/db), we recently demonstrated that diabetes enhanced WNV replication and the susceptibility of mice to WNVE. Herein, we have examined immunological events in the brain of wild type (WT) and db/db mice after WNV infection. We hypothesized that WNV-induced migration of protective leukocytes into the brain is attenuated in the presence of diabetes, leading to a high viral load in the brain and severe disease in diabetic mice.

METHODS

Nine-week old C57BL/6 WT and db/db mice were infected with WNV. Leukocyte infiltration, expression of cell adhesion molecules (CAM), neuroinflammatory responses, activation of astrocytes, and neuronal death were analyzed using immunohistochemistry, qRT-PCR, flow cytometry, and western blot.

RESULTS

We demonstrate that infiltration of CD45+ leukocytes and CD8+T cells was significantly reduced in the brains of db/db mice, which was correlated with attenuated expression of CAM such as E-selectin and ICAM-1. WNV infection in db/db mice was associated with an enhanced inflammatory response in the brain. mRNA and protein levels of key chemokines such as CXCL1CXCL1, CCL2, CCL5, CCL3, and G-CSF, and cytokines such as IL-1β, TNF, IL-6, IFNγ, and IL-1α were significantly elevated in the brains of db/db mice compared to WT mice. Elevated levels of cytokines also correlated with increased astrocytes activation and neuronal damage in the brains of db/db mice.

CONCLUSIONS

These data suggest that reduced leukocytes recruitment, in part, due to lower levels of CAM results in failure to clear WNV infection from the brain leading to increased production of inflammatory molecules, which mediates increased neuronal death and mortality in db/db mice. This is the first study to elucidate the expression of CAM and their correlation with the migration of leukocytes, specifically cytotoxic CD8+ T cells, in increasing disease severity in the diabetic mouse model.

Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.

Acute myelogenous leukemia (AML) is a bone marrow disease in which the leukemic cells show constitutive release of a wide range of CCL and CXCL chemokines and express several chemokine receptors. The AML cell release of various chemokines is often correlated and three release clusters have been identified: CCL2-4/<em>CXCL1</em>/8, CCL5/CXCL9-11, and CCL13/17/22/24/CXCL5. CXCL8 is the chemokine usually released at highest levels. Based on their overall constitutive release profile, patients can be classified into distinct subsets that differ in their T cell chemotaxis towards the leukemic cells. The release profile is modified by hypoxia, differentiation status, pharmacological interventions, and T cell cytokine responses. The best investigated single chemokine in AML is <em>CXCL1</em>2 that binds to CXCR4. <em>CXCL1</em>2/CXCR4 is important in leukemogenesis through regulation of AML cell migration, and CXCR4 expression is an adverse prognostic factor for patient survival after chemotherapy. Even though AML cells usually release high levels of several chemokines, there is no general increase of serum chemokine levels in these patients and the levels are also influenced by patient age, disease status, chemotherapy regimen, and complicating infections. However, serum CXCL8 levels seem to partly reflect the leukemic cell burden in AML. Specific chemokine inhibitors are currently being developed, although redundancy and pleiotropy of the chemokine system are obstacles in drug development.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) cell signaling pathway is important in inflammation and cell survival. Inflammation and cell death in the kidney are features of cisplatin-induced AKI. While it is known that cisplatin induces NF-κB signaling in the kidney, the NF-κB responsive genes and the effect of direct NF-κB transcriptional inhibition in cisplatin-induced AKI is not known. Mice injected with cisplatin, 25mg/kg, developed AKI, acute tubular necrosis (ATN) and apoptosis on day 3. Mice were treated with JSH-23 (20 or 40 mg/kg) which directly affects NF-κB transcriptional activity. Kidney function, tubular injury (ATN, serum neutrophil gelatinase-associated lipocalin [NGAL], but not apoptosis) and myeloperoxidase (MPO) activity were significantly improved by JSH-23 (40 mg/kg). Sixty one NF-κB responsive genes were increased by cisplatin of which 21 genes were decreased by JSH-23. Genes that were decreased by JSH-23 that are known to play a role in cisplatin-induced AKI were IL-10, IFN-γ, chemokine [C-C motif] ligand 2 (CCL2) and caspase-1. Another gene, caspase recruitment domain family, member 11 (CARD11), not previously known to play a role in AKI, was increased more than 20-fold and completely inhibited by JSH-23. CXCL1 and TNF-α, known mediators of cisplatin-induced AKI, were decreased by JSH-23. RIPK1 and 3, receptor-interacting serine/threonine-protein kinases, that play an important role in necroptosis, were decreased by JSH-23. In mouse proximal tubule cells in culture, JSH-23 resulted in an increase in apoptosis suggesting that the mechanism of protection against AKI by JSH-23 is not due to a direct effect on proximal tubules. In conclusion, NF-κB transcriptional inhibition in cisplatin-induced AKI ameliorates kidney function and ATN without a significant effect on apoptosis and is associated with a decrease pro-inflammatory mediators and CARD11.

BACKGROUND

A heightened proinflammatory state has been hypothesized to enhance human immunodeficiency virus type 1 (HIV-1) transmission - both susceptibility of HIV-1-exposed persons and infectiousness of HIV-1-infected persons.

METHODS

Using prospective data from heterosexual African couples with HIV-1 serodiscordance, we conducted a nested case-control analysis to assess the relationship between cytokine concentrations and the risk of HIV-1 acquisition. Case couples (n = 120) were initially serodiscordant couples in which HIV-1 was transmitted to the seronegative partner during the study; control couples (n = 321) were serodiscordant couples in which HIV-1 was not transmitted to the seronegative partner. Differences in a panel of 30 cytokines were measured using plasma specimens from both HIV-1-susceptible and HIV-1-infected partners. Plasma was collected before seroconversion for cases.

RESULTS

For both HIV-1-infected and HIV-1-susceptible partners, cases and controls had significantly different mean responses in cytokine panels (P < .001, by the Hotelling T(2) test), suggesting a broadly different pattern of immune activation for couples in which HIV-1 was transmitted, compared with couples without transmission. Individually, log10 mean concentrations of interleukin 10 (IL-10) and <em>CXCL1</em>0 were significantly higher for both HIV-1-susceptible and HIV-1-infected case partners, compared with HIV-1-susceptible and HIV-1-infected control partners (P < .01 for all comparisons). In multivariate analysis, HIV-1 transmission was significantly associated with elevated <em>CXCL1</em>0 concentrations in HIV-1-susceptible partners (P = .001) and with elevated IL-10 concentrations in HIV-1-infected partners (P = .02).

CONCLUSIONS

Immune activation, as measured by levels of cytokine markers, particularly elevated levels of IL-10 and CXCL1, are associated with increased HIV-1 susceptibility and infectiousness.

Acute lung injury (ALI) is characterized by hypoxemia, enhanced permeability of the air-blood barrier, and pulmonary edema. Particularly in the elderly, ALI is associated with increased morbidity and mortality. The reasons for this, however, are poorly understood. We hypothesized that age-related changes in pulmonary structure, function, and inflammation lead to a worse prognosis in ALI. ALI was induced in young (10 wk old) and old (18 mo old) male C57BL/6 mice by intranasal application of 2.5 mg lipopolysaccharide (LPS)/kg body wt or saline (control mice). After 24 h, lung function was assessed, and lungs were either processed for stereological or inflammatory analysis, such as bronchoalveolar lavage fluid (BALF) cytometry and qPCR. Both young and old mice developed severe signs of ALI, including alveolar and septal edema and enhanced inflammatory BALF cells. However, the pathology of ALI was more pronounced in old compared with young mice with nearly sixfold higher BALF protein concentration, twice the number of neutrophils, and significantly higher expression of neutrophil chemokine Cxcl1, adhesion molecule Icam-1, and metalloprotease-9, whereas the expression of tight junction protein occludin significantly decreased. The old LPS mice had thicker alveolar septa attributable to higher volumes of interstitial cells and extracellular matrix. Tissue resistance and elastance reflected observed changes at the ultrastructural level in the lung parenchyma in ALI of young and old mice. In summary, the pathology of ALI with advanced age in mice is characterized by a greater neutrophilic inflammation, leakier air-blood barrier, and altered lung function, which is in line with findings in elderly patients.

Tumor-related inflammation does influence the biological behavior of neoplastic cells and ultimately the patient's outcome. With specific regard to thyroid cancer, the issue of tumor-associated inflammation has been extensively studied and recently reviewed. However, the role of chemokines, which play a crucial role in determining the immuno-phenotype of tumor-related inflammation, was not addressed in previous reviews on the topic. Experimental evidence shows that thyroid cancer cells actively secrete a wide spectrum of chemokines and, at least for some of them, solid scientific data support a role for these immune-active molecules in the aggressive behavior of the tumor. Our proposal for a review article on chemokines and thyroid cancer stems from the notion that chemokines, besides having the ability to attract and maintain immune cells at the tumor site, also produce several pro-tumorigenic actions, which include proangiogenetic, cytoproliferative, and pro-metastatic effects. Studies taking into account the role of CCL15, C-X-C motif ligand 12, CXCL1CXCL1, CCL20, and CCL2 in the context of thyroid cancer will be reviewed with particular emphasis on CXCL8. The reason for focusing on CXCL8 is that this chemokine is the most studied one in human malignancies, displaying multifaceted pro-tumorigenic effects. These include enhancement of tumor cells growth, metastatization, and angiogenesis overall contributing to the progression of several cancers including thyroid cancer. We aim at reviewing current knowledge on the (i) ability of both normal and tumor thyroid cells to secrete CXCL8; (ii) direct/indirect pro-tumorigenic effects of CXCL8 demonstrated by in vitro and in vivo studies specifically performed on thyroid cancer cells; and (iii) pharmacologic strategies proven to be effective for lowering CXCL8 secretion and/or its effects on thyroid cancer cells.

OBJECTIVE

To investigate whether di-(2-ethylhexyl) phthalate (DEHP) affects the production of inflammatory cytokines by human macrophages.

METHODS

Differentiated macrophage-like THP-1 cells were exposed to 200 μM DEHP for 3 h, followed by incubation in the presence or absence of opsonized zymosan A, and the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the culture media were determined by ELISA. DNA microarray and quantitative real-time RT-PCR analyses were performed to identify genes that showed changes in expression in response to DEHP.

RESULTS

DEHP treatment increased the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the media, regardless of whether the cells phagocytosed zymosan. DNA microarray analysis showed that DEHP increased the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP3, MMP10, MMP14, and CSF2 mRNA, and real-time RT-PCR showed that DEHP significantly enhanced the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP10, CSF2, TNF-α, IL-1β, and IL-6 mRNA in THP-1 cells. DEHP significantly induced translocation of p65 NF-κB into the nucleus.

CONCLUSIONS

DEHP enhances the production of inflammatory cytokines and chemokines by macrophages, and exacerbates their inflammatory response.

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/HeJ mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-α, IL-1β and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-α, IL-1β and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis.

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60min, followed by reperfusion. RvD1 (5, 10, and 15μg/kg, i.p.) was administered to the mice 1h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80(+)Gr-1(+) cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.

The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.

BACKGROUND

Rhinoviruses (RVs) are the major triggers of asthma exacerbations. We have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental RV-induced asthma exacerbations.

OBJECTIVE

We hypothesized that neutrophil-related CXC chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in RV-induced exacerbations of asthma.

METHODS

Protein levels of antimicrobial peptides (SLPI, HNP 1-3, elafin, and LL-37) and neutrophil chemokines (CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, and CXCL8/IL-8) were determined in bronchoalveolar lavage (BAL) fluid of 10 asthmatics and 15 normal controls taken before, at day four during and 6 weeks post-experimental infection.

RESULTS

BAL HNP 1-3 and Elafin were higher, CXCL7/NAP-2 was lower in asthmatics compared with controls at day 4 (P = 0.035, P = 0.048, and P = 0.025, respectively). BAL HNP 1-3 and CXCL8/IL-8 were increased during infection (P = 0.003 and P = 0.011, respectively). There was a trend to increased BAL neutrophils at day 4 compared with baseline (P = 0.076). BAL HNP 1-3 was positively correlated with BAL neutrophil numbers at day 4. There were no correlations between clinical parameters and HNP1-3 or IL-8 levels.

CONCLUSIONS

We propose that RV infection in asthma leads to increased release of CXCL8/IL-8, attracting neutrophils into the airways where they release HNP 1-3, which further enhances airway neutrophilia. Strategies to inhibit CXCL8/IL-8 may be useful in treatment of virus-induced asthma exacerbations.

Neutrophils clear viruses, but excessive neutrophil responses induce tissue injury and worsen disease. Aging increases mortality to influenza infection; however, whether this is due to impaired viral clearance or a pathological host immune response is unknown. Here we show that aged mice have higher levels of lung neutrophils than younger mice after influenza viral infection. Depleting neutrophils after, but not before, infection substantially improves the survival of aged mice without altering viral clearance. Aged alveolar epithelial cells (AECs) have a higher frequency of senescence and secrete higher levels of the neutrophil-attracting chemokines CXCL1 and CXCL2 during influenza infection. These chemokines are required for age-enhanced neutrophil chemotaxis in vitro. Our work suggests that aging increases mortality from influenza in part because senescent AECs secrete more chemokines, leading to excessive neutrophil recruitment. Therapies that mitigate this pathological immune response in the elderly might improve outcomes of influenza and other respiratory infections.

We recently demonstrated that immunization with polyester poly(lactide-co-glycolide acid) (PLGA) nanoparticles loaded with the 11-kDa Leishmania vaccine candidate kinetoplastid membrane protein 11 (KMP-11) significantly reduced parasite load in vivo. Presently, we explored the ability of the recombinant PLGA nanoparticles to stimulate innate responses in macrophages and the outcome of infection with Leishmania braziliensis in vitro. Incubation of macrophages with KMP-11-loaded PLGA nanoparticles significantly decreased parasite load. In parallel, we observed the augmented production of nitric oxide, superoxide, TNF-α and IL-6. An increased release of CCL2/MCP-1 and CXCL1/KC was also observed, resulting in macrophage and neutrophil recruitment in vitro. Lastly, the incubation of macrophages with KMP-11-loaded PLGA nanoparticles triggered the activation of caspase-1 and the secretion of IL-1β and IL-18, suggesting inflammasome participation. Inhibition of caspase-1 significantly increased the parasite load. We conclude that KMP-11-loaded PLGA nanoparticles promote the killing of intracellular Leishmania parasites through the induction of potent innate responses.

UNASSIGNED

In this novel study, KMP-11-loaded PLGA nanoparticles are demonstrated to promote the killing of intracellular Leishmania parasites through enhanced innate immune responses by multiple mechanisms. Future clinical applications would have a major effect on our efforts to address parasitic infections.

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.

BACKGROUND

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.

METHODS

The mRNA expression levels of cumulus cells were detected by qRT- PCR.

RESULTS

Consistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

CONCLUSIONS

Several cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

Chemokine signalling is required for the homing of leukocytes during retinal inflammation, and is associated with pathogenesis of diseases such as age-related macular degeneration (AMD). Here, we explore the role of interleukin-1β (IL-1β) in modulating AMD-associated chemokines Ccl2, <em>Cxcl1</em>, and <em>Cxcl1</em>0 during photo-oxidative retinal damage, and the effect on both the accumulation of outer-retinal macrophages, and death of photoreceptors.

Inhibition of retinal IL-1β expression was performed using either siRNA or antibody neutralisation, which was intravitreally injected in SD rats prior to photo-oxidative damage. Changes in the expression and localisation of Il-1β, Ccl2, <em>Cxcl1</em> and <em>Cxcl1</em>0 genes were assessed using qPCR and in situ hybridisation, while the recruitment of retinal macrophages was detected using immunohistochemistry for IBA1. Levels of photoreceptor cell death were determined using TUNEL.

Photo-oxidative damage elevated the expression of Il-1β and inflammasome-related genes, and IL-1β protein was detected in microglia infiltrating the outer retina. This was associated with increased expression of Ccl2, <em>Cxcl1</em>, and <em>Cxcl1</em>0. Intravitreal IL-1β inhibitors suppressed chemokine expression following damage and reduced macrophage accumulation and photoreceptor death. Moreover, in Müller and RPE cell cultures, and in vivo, Ccl2, <em>Cxcl1</em> and <em>Cxcl1</em>0 were variously upregulated when stimulated with IL-1β, with increased macrophage accumulation detected in vivo.

IL-1β is produced by retinal microglia and macrophages and promotes chemokine expression by Müller cells and RPE in retinal degeneration. Targeting IL-1β may prove efficacious in broadly suppressing chemokine-mediated inflammation in retinal dystrophies such as AMD.