The chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR2 have been found to be expressed on microglia in many neurodegenerative diseases, such as multiple sclerosis and Alzheimer's disease. There is emerging evidence that chemokines, besides chemoattraction, might directly modulate reactive profiles of microglia. To address this hypothesis we have investigated the effects of CCL2, CCL3, CCL5, and CXCL1 on cytokine and growth factor production, NO synthesis, and phagocytosis in non-stimulated and lipopolysaccharide-stimulated primary rat microglia. The respective receptors CCR1, CCR5, and CXCR2 were shown to be functionally expressed on microglia. All tested chemokines stimulated chemotaxis whereas only CCL5 increased NO secretion and attenuated IL-10 as well as IGF-1 production in activated microglia. Based on these findings we propose that besides its chemoattractant function CCL5 has a modulatory effect on activated microglia.

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.

OBJECTIVE

Interleukin-13 (IL-13) is a pleiotropic cytokine that can affect vessel formation, an important component of the rheumatoid arthritis (RA) synovial tissue pannus. The purpose of this study was to use a gene therapy approach to investigate the role of IL-13 in angiogenesis in vivo, using a rat adjuvant-induced arthritis model of RA.

METHODS

Ankle joints of female rats were injected preventatively with an adenovirus vector containing human IL-13 (AxCAIL-13), a control vector with no insert (AxCANI), or phosphate buffered saline (PBS). Joints were harvested at the peak of arthritis, and histologic and biochemical features were evaluated.

RESULTS

AxCAIL-13-treated joint homogenates had lower hemoglobin levels, suggesting reduced joint vascularity, and both endothelial cell migration and tube formation were significantly inhibited (P < 0.05). Similarly, AxCAIL-13 inhibited capillary sprouting in the rat aortic ring assay and vessel growth in the Matrigel plug in vivo assay. IL-13 gene delivery resulted in up-regulation and association of phosphorylated ERK-1/2 and protein kinase Calpha/betaII, suggesting a novel pathway in IL-13-mediated angiostasis. The angiostatic effect of AxCAIL-13 was associated with down-regulation of proangiogenic cytokines (IL-18, cytokine-induced neutrophil chemoattractant 1/CXCL1, lipopolysaccharide-induced CXC chemokine/CXCL5) and up-regulation of the angiogenesis inhibitor endostatin. The expression and activity of matrix metalloproteinases 2 and 9, which participate in angiogenesis, was impaired in response to IL-13 as compared with AxCANI and PBS treatment.

CONCLUSIONS

Our findings support a role for IL-13 as an in vivo antiangiogenic factor and provide a rationale for its use in RA to control pathologic neovascularization.

OBJECTIVE

Using the murine sponge model, we investigated the temporal relationship between angiogenesis, leukocyte accumulation and endogenous generation of the pro-inflammatory chemokines CXCL1-3/KC and CCL2/JE. Furthermore, the effects of exogenous administration of these chemokines were studied.

METHODS

Angiogenesis in the implants was assessed by measuring the hemoglobin content (vascular index) and leukocyte accumulation quantified by evaluating MPO and NAG enzyme activities.

RESULTS

A progressive increase in hemoglobin content and in enzymatic activities was observed during the whole period. The levels of CXCL1-3/KC and CCL2/JE in the implants peaked at days 7 and 1, respectively. Exogenous administration of CXCL1-3/KC (100 ng/day intra-implant) applied at days 1-3 resulted in increased neovascularization and macrophage accumulation. Intra-implant injections of CCL2/JE (100 ng/day) also resulted in increased angiogenesis and macrophage accumulation.

CONCLUSIONS

These results demonstrated that the chemokines, CXCL1-3/KC and CCL2/JE, are generated within the sponge compartment and that neovascularization and inflammatory cells influx can be modulated by exogenous administration of the chemokines.

Cognitive decline in Alzheimer's disease (AD) occurs as a result of the buildup of pathological proteins and downstream events including an elevated and altered inflammatory response. Inflammation has previously been linked to increased abnormal phosphorylation of tau protein. To determine if endogenous amyloid-beta (Abeta)-induced neuroinflammation drives tau phosphorylation in vivo, we treated 8-month-old 3xTg-AD with minocycline, an anti-inflammatory agent, to assess how it influenced cognitive decline and development of pathology. 4 months of treatment restored cognition to non-transgenic performance. Inflammatory profiling revealed a marked decrease in GFAP, TNFalpha, and IL6 and an increase in the CXCL1 chemokines KC and MIP1a. Minocycline also reduced levels of insoluble Abeta and soluble fibrils. Despite reducing levels of the tau kinase cdk5 coactivator p25, minocycline did not have wide effects on tau pathology with only one phospho-epitope showing reduction with treatment (S212/S214). The sum of these findings shows that reduction of the inflammatory events in an AD mouse model prevents cognitive deficits associated with pathology, but that endogenous Abeta-derived neuroinflammation does not contribute significantly to the development of tau pathology.

White adipose tissue (WAT) overgrowth in obesity is linked with increased aggressiveness of certain cancers. Adipose stromal cells (ASCs) can become mobilized from WAT, recruited by tumours and promote cancer progression. Mechanisms underlying ASC trafficking are unclear. Here we demonstrate that chemokines CXCL1 and CXCL8 chemoattract ASC by signalling through their receptors, CXCR1 and CXCR2, in cell culture models. We further show that obese patients with prostate cancer have increased epithelial CXCL1 expression. Concomitantly, we observe that cells with ASC phenotype are mobilized and infiltrate tumours in obese patients. Using mouse models, we show that the CXCL1 chemokine gradient is required for the obesity-dependent tumour ASC recruitment, vascularization and tumour growth promotion. We demonstrate that αSMA expression in ASCs is induced by chemokine signalling and mediates the stimulatory effects of ASCs on endothelial cells. Our data suggest that ASC recruitment to tumours, driven by CXCL1 and CXCL8, promotes prostate cancer progression.

Ocular infection with HSV causes corneal neovascularization (CV), an essential step in the pathogenesis of the blinding immunoinflammatory lesion stromal keratitis. The infection results in IL-17A production, which contributes to CV in ways that together serve to shift the balance between corneal concentrations of vascular endothelial growth factor A (VEGF-A) and the soluble vascular endothelial growth factor receptor 1 molecule, which binds to VEGF-A and blocks its function (a so-called VEGF trap). Accordingly, animals lacking responses to IL-17A signaling, either because of IL-17 receptor A knockout or wild-type animals that received neutralizing mAb to IL-17A, had diminished CV, compared with controls. The procedures reduced VEGF-A protein levels but had no effect on the levels of soluble vascular endothelial growth factor receptor 1. Hence the VEGF trap was strengthened. IL-17A also caused increased CXCL1/KC synthesis, which attracts neutrophils to the inflammatory site. Neutrophils further influenced the extent of CV by acting as an additional source of VEGF-A, as did metalloproteinase enzymes that degrade the soluble receptor, inhibiting its VEGF-blocking activity. Our results indicate that suppressing the expression of IL-17A, or increasing the activity of the VEGF trap, represents a useful approach to inhibiting CV and the control of an ocular lesion that is an important cause of human blindness.

Dual-specificity phosphatase (DUSP) 1 dephosphorylates and inactivates members of the MAPK superfamily, in particular, JNKs, p38α, and p38β MAPKs. It functions as an essential negative regulator of innate immune responses, hence disruption of the Dusp1 gene renders mice extremely sensitive to a wide variety of experimental inflammatory challenges. The principal mechanisms behind the overexpression of inflammatory mediators by Dusp1(-/-) cells are not known. In this study, we use a genetic approach to identify an important mechanism of action of DUSP1, involving the modulation of the activity of the mRNA-destabilizing protein tristetraprolin. This mechanism is key to the control of essential early mediators of inflammation, TNF, CXCL1, and CXCL2, as well as the anti-inflammatory cytokine IL-10. The same mechanism also contributes to the regulation of a large number of transcripts induced by treatment of macrophages with LPS. These findings demonstrate that modulation of the phosphorylation status of tristetraprolin is an important physiological mechanism by which innate immune responses can be controlled.

Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract.

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL1CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFβ2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.

The chemokine (C-X-C motif) ligand 1 (CXCL1) regulates tumor-stromal interactions and tumor invasion. However, the precise role of CXCL1 on gastric tumor growth and patient survival remains unclear. In the current study, protein expressions of CXCL1, vascular endothelial growth factor (VEGF) and phospho-signal transducer and activator of transcription 3 (p-STAT3) in primary tumor tissues from 98 gastric cancer patients were measured by immunohistochemistry (IHC). CXCL1 overexpressed cell lines were constructed using Lipofectamine 2000 reagent or lentiviral vectors. Effects of CXCL1 on VEGF expression and local tumor growth were evaluated in vitro and in vivo. CXCL1 was positively expressed in 41.4% of patients and correlated with VEGF and p-STAT3 expression. Higher CXCL1 expression was associated with advanced tumor stage and poorer prognosis. In vitro studies in AGS and SGC-7901 cells revealed that CXCL1 increased cell migration but had little effect on cell proliferation. CXCL1 activated VEGF signaling in gastric cancer (GC) cells, which was inhibited by STAT3 or chemokine (C-X-C motif) receptor 2 (CXCR2) blockade. CXCL1 also increased p-STAT3 expression in GC cells. In vivo, CXCL1 increased xenograft local tumor growth, phospho-Janus kinase 2 (p-JAK2), p-STAT3 levels, VEGF expression and microvessel density. These results suggested that CXCL1 increased local tumor growth through activation of VEGF signaling which may have mechanistic implications for the observed inferior GC survival. The CXCL1/CXCR2 pathway might be potent to improve anti-angiogenic therapy for gastric cancer.

BACKGROUND

In-hospital outcomes are generally acceptable in patients with type B dissection; however, some patients present with undesirable complications, such as aortic expansion and rupture. Excessive inflammation is an independent predictor of adverse clinical outcomes.

OBJECTIVE

We have investigated the underlying mechanisms of catastrophic complications after acute aortic dissection (AAD) in mice.

RESULTS

When angiotensin II was administered in lysyl oxidase inhibitor-preconditioned mice, AAD emerged within 24 hours. The dissection was initiated at the proximal site of the descending thoracic aorta and propagated distally into an abdominal site. Dissection of the aorta caused dilatation, and ≈70% of the mice died of aortic rupture. AAD triggered CXCL1 and granulocyte-colony stimulating factor expression in the tunica adventitia of the dissected aorta, leading to elevation of circulating CXCL1/granulocyte-colony stimulating factor levels. Bone marrow CXCL1CXCL1 function using an anti-CXCR2 antibody reduced neutrophil accumulation and limited aortic rupture post AAD. The tunica adventitia of the expanded dissected aorta demonstrated high levels of interleukin-6 (IL-6) expression. Neutrophils were the major sources of IL-6, and CXCR2 neutralization significantly reduced local and systemic levels of IL-6. Furthermore, disruption of IL-6 effectively suppressed dilatation and rupture of the dissected aorta without any influence on the incidence of AAD and neutrophil mobilization.

CONCLUSIONS

Adventitial CXCL1/granulocyte-colony stimulating factor expression in response to AAD triggers local neutrophil recruitment and activation. This leads to adventitial inflammation via IL-6 and results in aortic expansion and rupture.

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)(-/-) mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS(-/-) mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage.

BACKGROUND

During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite.

RESULTS

We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of <em>CXCL1</em>, CCL2, CCL4 and TNF-alpha expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased <em>CXCL1</em>0 expression and increased IL-10 expression.

CONCLUSIONS

These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.

Although intestinal epithelial cells are known to up-regulate the expression of several chemokine genes in response to the stimulation with B. fragilis enterotoxin (BFT), there has been little understanding on the cellular mechanisms of BFT-induced mucosal inflammation. To test whether nuclear transcriptional factor-kappa B (NF-kappaB) is involved in the process, we stimulated intestinal epithelial cells with BFT, and evaluated the signalling NF-kappaB pathways. BFT increased signals of NF-kappaB in HT-29 and T84 epithelial cell lines as well as primary human colon epithelial cells. NF-kappaB molecules activated by BFT stimulation were composed of p65 and p50 heterodimers. In contrast, BFT decreased the signals of IkappaBalpha and IkappaB epsilon, as assessed by immunoblot. Super-repressors of IkappaBalpha, IkappaB kinase (IKK)beta, and NF-kappaB inducing kinase (NIK) inhibited an up-regulated transcription of downstream target gene (CXCL8) of NF-kappaB. Moreover, blocking the activation of NF-kappaB by MG-132 or antisense p50 oligonucleotide transfection resulted in down-regulated expression of chemokines such as CXCL1, CXCL8, and CCL2 in BFT-stimulated HT-29 cells. In addition, NF-kappaB inhibition suppressed the BFT-induced neutrophil transepithelial migration in T84 cells. These results indicate that NF-kappaB can be a central regulator of chemokine gene expression in BFT-stimulated intestinal epithelial cells and may be an important regulator of neutrophil migration.

Ozone exposure produces acute inflammation and neutrophil influx in the distal lung. Alveolar epithelial cells cover a large surface area, secrete chemokines, and may initiate or modify the inflammatory response. The effect of ozone on chemokine production by these cells has not been defined. Isolated rat type II cells were cultured in different conditions to express the morphologic appearance and biochemical markers for the type I and the type II cell phenotypes. These cells were exposed to ozone at an air/liquid interface. The type I-like cells were more susceptible to injury than the type II cells and showed signs of injury at exposure levels of 100 ppb ozone for 60 min. Both phenotypes showed evidence of lipid peroxidation after ozone exposure as measured by 8-isoprostane production, but neither phenotype secreted increased amounts of MIP-2 (CXCL3), CINC-1 (CXCL1), or MCP-1 (CCL2) in response to ozone. Both cell phenotypes secreted MIP-2 and MCP-1 in response to IL-1beta or lipopolysaccharide, but there was no priming or synergy with ozone. It is likely that the inflammatory response to ozone in the alveolar compartment is not due to the direct effect of ozone on epithelial cells.

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.

SpyCEP is a Streptococcus pyogenes protease that cleaves CXCL8/IL-8 and its activity is associated with human invasive disease severity. We investigated the role of SpyCEP in S. pyogenes necrotizing fasciitis and respiratory tract infection in mice using isogenic strains differing only in SpyCEP expression. SpyCEP cleaved human CXCL1, 2, 6 and 8 plus murine CXCL1 and 2 at a structurally conserved site. Mice were infected in thigh muscle with a strain of S. pyogenes that expresses a high level of SpyCEP, or with an isogenic non-SpyCEP expressing strain. SpyCEP expression by S. pyogenes hindered bacterial clearance from muscle, and enhanced bacterial spread, associated with cleavage of murine chemoattractant CXCL1. Mice were then infected with Lactococcus lactis strains that differed only in SpyCEP expression. In contrast to the parent L. lactis strain (lacks SpyCEP), which was avirulent when administered intramuscularly, infection with a strain that expressed SpyCEP heterologously led to dramatic systemic illness within 24 h, failure to clear bacteria from muscle and marked dissemination to other organs. In the upper airways, SpyCEP expression was required for survival of L. lactis but not S. pyogenes. However, dissemination of S. pyogenes to the lung was SpyCEP-dependent and was associated with evidence of chemokine cleavage. Taken together, the studies provide clear evidence that SpyCEP is necessary and sufficient for systemic bacterial dissemination from a soft tissue focus in this model and also underlies dissemination in the respiratory tract.

Polymorphonuclear cells (PMN) are recruited in early inflammation and are believed to contribute to inflammatory pain. However, studies demonstrating a hyperalgesic role of PMN did not examine selective PMN recruitment or did not document effective PMN recruitment. We hypothesized that hyperalgesia does not develop after chemokine-induced PMN selective recruitment and is independent of PMN infiltration in complete Freund's adjuvant (CFA)-induced, local inflammation. PMN were recruited by intraplantar injection of CXC chemokine ligand 1 (CXCL1; keratinocyte-derived chemokine), CXCL2/3 (macrophage inflammatory protein-2), or CFA, with or without preceding systemic PMN depletion. Chemokine inoculation resulted in dose (0-30 microg)- and time (0-12 h)-dependent, selective recruitment of PMN as quantified by flow cytometry. CXCL2/3, but not CXCL1, was less effective at high doses, probably as a result of significant down-regulation of CXC chemokine receptor 2 expression on blood PMN. Neither chemokine caused mechanical or thermal hyperalgesia as determined by the Randall-Selitto and Hargreaves test, respectively, despite comparable expression of activation markers (i.e., CD11b, CD18, and L-selectin) on infiltrating PMN. In contrast, CFA injection induced hyperalgesia, independent of PMN recruitment. c-Fos mRNA and immunoreactivity in the spinal cord were increased significantly after inoculation of CFA-independent of PMN-migration but not of CXCL2/3. Measurement of potential hyperalgesic mediators showed that hyperalgesia correlated with local prostaglandin E2 (PGE2) but not with interleukin-1beta production. In summary, hyperalgesia, local PGE2 production, and spinal c-Fos expression occur after CFA-induced inflammation but not after CXCL1- or CXCL2/3-induced, selective PMN recruitment. Thus, PMN seem to be less important in inflammatory hyperalgesia than previously thought.

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.

BACKGROUND

Chronic pain models are commonly defined as either nerve-injury or inflammation models, but recent work suggests inflammatory processes are important in nerve injury-induced pain.

METHODS

In the rat spinal nerve ligation model, the authors examined effects of systemic corticosteroid triamcinolone acetonide (TA) on the cytokine protein profile and sympathetic sprouting in the axotomized sensory ganglia, excitability of sensory neurons, and mechanical sensitivity.

RESULTS

By postoperative day 3, marked increases (5- to 16-fold) in monocyte chemoattractant protein-1, growth-related oncogene (GRO/KC or CXCL1), and interleukin (IL)-6 were observed, whereas IL-4 and IL-2 levels fell more than fourfold. The increased cytokines and number of sympathetic basket formations in the sensory ganglia were reduced toward normal values by TA given starting at the time of injury. Interleukin-4 and IL-2 levels were not restored by TA. Systemic TA also reduced the firing rate and incidence of bursting activity, but not the overall incidence of spontaneous activity, in large- and medium-sized neurons. Mechanical hypersensitivity on postoperative day 3 was reduced by TA, and some effect could still be observed 4 days after cessation of TA. However, starting TA at day 7 was ineffective.

CONCLUSIONS

Several components of the spinal nerve injury model are responsive to corticosteroid, suggesting inflammatory processes are important in the development of neuropathic pain. The observation that TA was effective when given starting at the time of injury suggests that steroid treatment might alter the development of chronic pain after surgical procedures that involve nerve injury, such as amputation or hernia repair.

BACKGROUND

Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq).

OBJECTIVE

We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort.

METHODS

RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, ≥ 2.0; false discovery rate ≤ 0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation.

RESULTS

Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory (S100A8/A9/A12, CXCL1, and 2'-5'-oligoadenylate synthetase-like [OASL]) and barrier (MKi67, keratin 16 [K16], and claudin 8 [CLDN8]) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation.

CONCLUSIONS

This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.