OBJECTIVE

The central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA).

METHODS

The expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA).

RESULTS

Protein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels.

CONCLUSIONS

The alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

The pathogenesis of myocarditis during Trypanosoma cruzi infection is poorly understood. We investigated the role played by chemokine receptor 5 (CCR5) in the influx of T cells to the cardiac tissue of T. cruzi-infected mice. mRNA and protein for the CCR5 ligands CCL3, CCL4, and CCL5 were detected in the hearts of infected mice in association with CD4+ and CD8+ T cells. There was a high level of CCR5 expression on CD8+ T cells in the hearts of infected mice. Moreover, CCR5 expression on CD8+ T cells was positively modulated by T. cruzi infection. CCR5-deficient mice infected with T. cruzi experienced a dramatically inhibited migration of T cells to the heart and were also more susceptible to infection. These results suggest that CCR5 and its ligands play a central role in the control of T cell influx in T. cruzi-infected mice. Knowledge of the mechanisms that trigger and control the migration of cells to the heart in patients with Chagas disease may help in the design of drugs that prevent myocarditis and protect against the development of severe disease.

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G(0) phase and abolishes the production of inflammatory cytokines such as TNF-alpha, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Statins exert favorable effects on lipoprotein metabolism but may also possess anti-inflammatory effects. Here, we explored the effects of atorvastatin in a model of adjuvant-induced arthritis in rat. Oral treatment with atorvastatin (1-10 mg/kg) from days 10 to 15 after arthritis induction caused inhibition of the increase in paw volume. Maximal inhibition occurred at a dose of 10 mg/kg. At this dose, atorvastatin markedly ameliorated the histopathological findings of joints obtained from day 16 of arthritic animals. This was mirrored by an effective blockade of neutrophil influx, as assessed by the tissue myeloperoxidase levels. The concentrations of the cytokines interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha and the chemokines CCL5 and CCL2 were significantly decreased in arthritic rats treated with atorvastatin. In contrast, the levels of interleukin-10 were enhanced by the drug treatment. The drug also prevented the hypernociception observed in the inflamed joints. These data clearly illustrate the therapeutic potential of a statin-sensitive pathway in inflammatory arthritis.

Macrophages have a central role in innate-immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram-positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP-3, CCL19/MIP-3beta, and CCL20/MIP-3alpha and CXC chemokine ligands CXCL8/interleukin (IL)-8, CXCL9/monokine induced by interferon-gamma (IFN-gamma), and CXCL10/IFN-inducible protein 10. Bacteria-induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria-induced IFN-alpha/beta production. CCL19 and CCL20 mRNA expression was up-regulated by IL-1beta or tumor necrosis factor alpha (TNF-alpha), and in addition, IFN-alpha together with TNF-alpha further enhanced CCL19 gene expression. Synergy between IFN-alpha and TNF-alpha was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria-stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus-induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.

Undoubtedly, platelets are key elements in the regulation of thrombosis and haemostasis. Along with their primary task to prevent blood loss from injured vessels, platelets have emerged as regulators of a variety of processes in the vasculature. Multiple challenges, from the contact and adhesion to subendothelial matrix after injury of the vessel wall, to interactions with blood cells in inflammatory conditions, result in platelet activation with concomitant shape change and release of numerous substances. Among these, chemokines have been found to modulate several processes in the vasculature, such as atherosclerosis and angiogenesis. In particular, the chemokines connective tissue activating protein III (CTAP-III) and its precursors, or truncation products (CXCL7), platelet factor 4, (PF4, CXCL4) and its variant PF4alt (CXCL4L1) or regulated upon activation and normal T cell expressed and secreted (RANTES, CCL5), have been investigated thoroughly. Defined common properties as their aptitude to bind glycosaminoglycans or their predisposition to associate and form homooligomers are pre-requisites for their role in the vasculature and function in vivo. The current review summarizes the development of these single chemokines, and their cooperative effects that may in part be dependent on their physical interactions.

Pain is one of the hallmarks of inflammation. Opioid receptors mediate antipain responses in both the peripheral nervous system and CNS. In the present study, pretreatment of CCR1: mu-opioid receptor/HEK293 cells with CCL3 (MIP-1alpha) induced internalization of mu-opioid receptors and severely impaired the mu-opioid receptor-mediated inhibition of cAMP accumulation. Immunohistochemical staining showed that CCR1 and mu-opioid receptors were coexpressed on small to medium diameter neurons in rat dorsal root ganglion. Analysis of ligand-induced calcium flux showed that both types of receptors were functional. Pretreatment of neurons with CCL3 exhibited an impaired [D-Ala(2),N-MePhe(4),Gly-o15]enkephalin-elicited calcium response, indicative of the heterologous desensitization of mu-opioid receptors. Other chemokines, such as CCL2, CCL5, and CXCL8, exhibited similar inhibitory effects. Our data indicate that proinflammatory chemokines are capable of desensitizing mu-opioid receptors on peripheral sensory neurons, providing a novel potential mechanism for peripheral inflammation-induced hyperalgesia.

Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1alpha], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1alpha]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.

Human colorectal cancer (CRC), the second largest cause of tumor-related death in Western countries, represents a paradigm for the now well-established connections between inflammation and cancer. In this study, we investigated which inflammatory mediators are mostly expressed in the microenvironment of human CRC. The RNA profile of a large panel of inflammatory genes, in particular chemokines and chemokine receptors, was analyzed in eight surgical tumor samples and in paired normal tissues from CRC patients. We employed an "inflammatory gene card" (TaqMan Low Density Array by Applied Biosystem), designed by our group, containing probes for 24 chemokines and 17 chemokine receptors. Several chemokines were strongly upregulated in the tumor microenvironment, most frequently CCL4 and CCL5, chemotactic for monocytes/macrophages and T cells, and the corresponding receptors CCR1 and CCR5; the angiogenic chemokines CXCL1 and CXCL8, and the receptor CXCR2. The antiangiogenic chemokines CXCL9 and CXCL10 were also expressed, but in the absence of the receptor CXCR3. Selected results have been confirmed in a larger number of samples. The levels of mRNA CXCL8 were significantly associated with the levels of osteopontin, a matrix-associated protein that shares with chemokines important functions such as induction of cell migration and survival, and modulation of the neoangiogenesis. Overall these results could be helpful to identify the most relevant inflammatory pathways present in CRC tumors and to build a solid rationale for future therapeutic interventions based on anti-inflammatory strategies.

Leukocyte migration into the central nervous system (CNS) is mediated by chemokines expressed on CNS endothelial cell surfaces. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBECs) in vitro and in situ. Four chemokines (CCL2, CCL5, CXCL8, and CXCL10) were demonstrated by immunohistochemistry in endothelial cells in brain samples from patients with multiple sclerosis. CXCL8 and CCL2 were constitutively released and increased by primary HBECs and the brain endothelial cell line hCEMC/D3 in response to tumor necrosis factor and/or interferon gamma. CXCL10 and CCL5 were undetectable in resting endothelial cells but were secreted in response to these proinflammatory cytokines. Tumor necrosis factor strongly increased the production of CCL2, CCL5, and CXCL8; interferon gamma upregulated CXCL10 exclusively. CCL3 was not secreted by HBECs and seemed to be confined to astrocytes in situ. The chemokine receptors CXCR1 and CXCR3 were expressed by HBECs both in vitro and in situ; CXCR3 was upregulated in response to cytokine stimulation in vitro. In contrast, CXCR3 expression was reduced in noninflammatory (silent) multiple sclerosis lesions. The particularly high levels of CXCL10 and CXCL8 expressed by brain endothelium may contribute to the predominant TH1-type inflammatory response observed in chronic inflammatory conditions such as multiple sclerosis.

Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC>> TARC>> RANTES>> MIP1 beta>> MCP1>> MIP1 alpha>> FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.

Breastfeeding represents the continued exposure of the infant to the maternal immune environment.Uterine, perinatal, and postnatal exposure to immune factors may contribute to an infant’s risk of developing immune-mediated disorders, including allergies. A PubMed search was conducted to review studies in humans and analyze concentrations of immune markers (TGF-beta, IFN-gamma, eotaxin, CCL5, CXCL10, TNF-alpha, MCP-1, IL-1beta, IL-4, IL-5, IL-6,IL-8, IL-10, IL-12, IL-13, sCD14, sIgA, IgG4, IgM) found in maternal serum, amniotic fluid, cord serum, colostrum, transition and mature milk. Concentrations of immune markers showed large variations across samples and studies. Reports documented conflicting results. Small sample sizes, differences in population characteristics, inconsistent sample collection times, and various sample collection and measurement methods may have led to wide variations in the concentrations of immune markers. Studies analyzing the associations between immune markers in maternal fluids and infant allergies remain inconclusive because of gaps in knowledge and a lack of standardized methods.

The objective of this study is to test whether the activation of toll-like receptors (TLRs) 2 and 3 (innate immune receptors for gram-positive and viral pathogens, respectively) can induce preterm delivery. One uterine horn of preterm pregnant CD-1 mice at approximately 75% of gestation was injected with TLR-2 ligands (lipoteichoic acid [LTA] or peptidoglycan [PGN]) or the TLR-3 ligand polyinosinic:cytidylic acid (poly[I:C]). Preterm delivery was recorded. In a separate group of mice, tissue mRNAs were quantified by reverse transcriptase polymerase chain reaction 5 hours after treatment with PGN or poly(I:C). Intrauterine PGN and LTA induced preterm delivery, reaching 100% at maximal doses. Intraperitoneal PGN also induced preterm delivery but at lower rates (maximum = 55%). Intrauterine poly(I:C) induced preterm birth in up to 31% of mice. Poly(I:C) induced uterine interferon beta and chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) but not interleukin 1beta, tumor necrosis factor, or lipopolysaccharide-induced CXC chemokine. PGN did not alter these mRNAs when compared with saline. Neither treatment induced gene expression in fetal membranes. Activation of either TLR-2 or -3 can induce preterm delivery in the mouse. Activation of TLR-3 with poly(I:C) induces interferon beta and the chemokine CCL5 in uterine tissues but not in fetal membranes.

BACKGROUND

Adipose tissue is a primary site of obesity-induced inflammation, which is emerging as an important contributor to obesity-related diseases such as type 2 diabetes. Dietary fibre consumption appears to be protective. Short-chain fatty acids, e.g. propionic acid, are the principal products of the colonic fermentation of dietary fibre and may have beneficial effects on adipose tissue inflammation.

METHODS

Human omental adipose tissue explants were obtained from overweight (mean BMI 28·8) gynaecological patients who underwent surgery. Explants were incubated for 24 h with propionic acid. Human THP-1 monocytic cells were differentiated to macrophages and incubated with LPS in the presence and absence of propionic acid. Cytokine and chemokine production were determined by multiplex-ELISA, and mRNA expression of metabolic and macrophages genes was determined by RT-PCR.

RESULTS

Treatment of adipose tissue explants with propionic acid results in a significant down-regulation of several inflammatory cytokines and chemokines such as TNF-α and CCL5. In addition, expression of lipoprotein lipase and GLUT4, associated with lipogenesis and glucose uptake, respectively, increased. Similar effects on cytokine and chemokine production by macrophages were observed.

CONCLUSIONS

We show that propionic acid, normally produced in the colon, may have a direct beneficial effect on visceral adipose tissue, reducing obesity-associated inflammation and increasing lipogenesis and glucose uptake. Effects on adipose tissue as a whole are at least partially explained by effects on macrophages but likely also adipocytes are involved. This suggests that, in vivo, propionic acid and dietary fibres may have potential in preventing obesity-related inflammation and associated diseases.

OBJECTIVE

The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor α (TNFα)-driven inflammatory program in human FLS.

METHODS

FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-κB signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays.

RESULTS

In FLS, TNFα induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-κB activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages.

CONCLUSIONS

TNFα signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNFα contribute to the persistence of synovial inflammation in RA.

OBJECTIVE

The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines.

METHODS

Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting.

RESULTS

Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic.

CONCLUSIONS

Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1β). The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are underinvestigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumour-secreted factors, express CCL5 that is not expressed either in normal LECs or in cancer cells, and direct tumour dissemination into these tissues. Moreover, tumour-conditioned LECs promote angiogenesis in these organs, allowing tumour extravasation and colonization. Mechanistically, tumour cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.

OBJECTIVE

FoxO4 is a member of the forkhead box transcription factor O (FoxO) subfamily. FoxO proteins are involved in diverse biological processes. In this study, we examine the role of FoxO4 in intestinal mucosal immunity and inflammatory bowel disease (IBD).

METHODS

Foxo4-null mice were subjected to trinitrobenzene sulfonic acid (TNBS) treatment. Microarray analysis and quantitative reverse transcription polymerase chain reaction were used to identify the cytokine transcripts that were altered by Foxo4 deletion. The effects of Foxo4 deficiency on the intestinal epithelial permeability and levels of tight junction proteins were examined by permeable fluorescent dye and Western blot. The molecular and cellular mechanisms by which FoxO4 regulates the mucosal immunity were explored through immunologic and biochemical analyses. The expression level of FoxO4 in intestinal epithelial cells of patients with IBD was examined with immunohistochemistry.

RESULTS

Foxo4-null mice were more susceptible to TNBS injury-induced colitis. The chemokine CCL5 is significantly up-regulated in the colonic epithelial cells of Foxo4-null mice, with increased recruitment of CD4(+) intraepithelial T cells and up-regulation of cytokines interferon-gamma and tumor necrosis factor-alpha in the colon. Foxo4 deficiency also resulted in an increase in intestinal epithelial permeability and down-regulation of the tight junction proteins ZO-1 and claudin-1. Mechanistically, FoxO4 inhibited the transcriptional activity of nuclear factor-kappaB (NF-kappaB), and Foxo4 deficiency is associated with increased NF-kappaB activity in vivo. FoxO4 transcription is transiently repressed in response to TNBS treatment and in patients with IBD.

CONCLUSIONS

These results indicate that FoxO4 is an endogenous inhibitor of NF-kappaB and identify a novel function of FoxO4 in the regulation of NF-kappaB-mediated mucosal immunity.

Recent studies have revealed that sex hormones manifest a variety of biological and immunological effects in the skin. Pregnancy, menstruation and the menopause modulate the natural course of psoriasis, indicating a female hormone-induced regulation of skin inflammation. Estrogen in vitro down-regulates the production of the neutrophil, type 1 T cell and macrophage-attracting chemokines, CXCL8, CXCL10, CCL5, by keratinocytes, and suppresses IL-12 production and antigen-presenting capacity while enhancing anti-inflammatory IL-10 production by dendritic cells. These data indicate that estrogen may attenuate inflammation in psoriatic lesions. Estrogen, alone or together with progesterone, prevents or reverses skin atrophy, dryness and wrinkles associated with chronological or photo-aging. Estrogen and progesterone stimulate proliferation of keratinocytes while estrogen suppresses apoptosis and thus prevents epidermal atrophy. Estrogen also enhances collagen synthesis, and estrogen and progesterone suppress collagenolysis by reducing matrix metalloproteinase activity in fibroblasts, thereby maintaining skin thickness. Estrogen maintains skin moisture by increasing acid mucopolysaccharide or hyaluronic acid levels in the dermis. Progesterone increases sebum secretion. Estrogen accelerates cutaneous wound healing stimulating NGF production in macrophages, GM-CSF production in keratinocytes and bFGF and TGF-beta1 production in fibroblasts, leading to the enhancement of wound re-innervation, re-epithelialization and granulation tissue formation. In contrast, androgens prolong inflammation, reduce deposition of extracellular matrix in wounds, and reduce the rate of wound healing. Estrogen enhances VEGF production in macrophages, an effect that is antagonized by androgens and which may be related to the development of granuloma pyogenicum during pregnancy. These regulatory effects of sex steroids may be manipulated as therapeutic or prophylactic measures in psoriasis, aging, chronic wounds or granuloma pyogenicum.

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.

Pneumococcal disease continues to account for significant morbidity and mortality worldwide. For the development of novel prophylactic and therapeutic strategies against the disease spectrum, a complete understanding of pneumococcal behavior in vivo is necessary. We evaluated the expression patterns of the proven and putative virulence factor genes adcR, cbpA, cbpD, cbpG, cpsA, nanA, pcpA, piaA, ply, psaA, pspA, and spxB after intranasal infection of CD1 mice with serotype 2, 4, and 6A pneumococci by real-time reverse transcription-PCR. Simultaneous gene expression patterns of selected host immunomodulatory molecules, CCL2, CCL5, CD54, CXCL2, interleukin-6, and tomor necrosis factor alpha, were also investigated. We show that pneumococcal virulence genes are differentially expressed in vivo, with some genes demonstrating niche- and serotype-specific differential expression. The in vivo expression patterns could not be attributed to in vitro differences in expression of the genes in transparent and opaque variants of the three strains. The host molecules were significantly upregulated, especially in the lungs, blood, and brains of mice. The pneumococcal-gene expression patterns support their ascribed roles in pathogenesis, providing insight into which protein combinations might be more appropriate as vaccine antigens against invasive disease. This is the first simultaneous comparison of bacterial- and host gene expression in the same animal during pathogenesis. The strategy provides a platform for prospective evaluation of interaction kinetics between invading pneumococci and human patients in culture-positive cases and should be feasible in other infection models.

Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors leads to cross-phosphorylation and cross-internalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 formed constitutive homo- as well as heterooligomeric complexes together with C5aR but not with the unrelated AT(1a)R in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-DeltaNT mutant, resulted in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylated CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/C5aR cells, whereas AT(1a)R stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-DeltaNT together with a phosphorylation-deficient CCR5 mutant that neither binds beta-arrestin nor undergoes internalization partially restored the CCL5-induced association of beta-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of (125)I-CCL5. Co-expression of C5aR, but not of AT(1a)R, promoted CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 was also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28(CT) mutant led to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and beta-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes.

We have previously shown that mice challenged with a lethal dose of A/Puerto Rico/8/34-OVA(I) are protected by injection of 4-8 × 10(6) in vitro-generated Tc1 or Tc17 CD8(+) effectors. Viral load, lung damage, and loss of lung function are all reduced after transfer. Weight loss is reduced and survival increased. We sought in this study to define the mechanism of this protection. CD8(+) effectors exhibit multiple effector activities, perforin-, Fas ligand-, and TRAIL-mediated cytotoxicity, and secretion of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, IL-17, IL-21, IL-22, IFN-γ, and TNF) and chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10). Transfer of CD8(+) effectors into recipients, before infection, elicits enhanced recruitment of host neutrophils, NK cells, macrophages, and B cells. All of these events have the potential to protect against viral infections. Removal of any one, however, of these potential mechanisms was without effect on protection. Even the simultaneous removal of host T cells, host B cells, and host neutrophils combined with the elimination of perforin-mediated lytic mechanisms in the donor cells failed to reduce their ability to protect. We conclude that CD8(+) effector T cells can protect against the lethal effects of viral infection by means of a large number of redundant mechanisms.