BACKGROUND

The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).

RESULTS

Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.

CONCLUSIONS

AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG-I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG-I (ΔRIG-I), MAVS, TRIF, IKKε, and TBK1-induced activation of IFN-β promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose-dependent manner inhibited IKKε and especially TBK1-induced IRF3 phosphorylation. GST pull-down experiments with GST-NS2 and in vitro-translated and cell-expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3-5D) activated readily IFN-β promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses.

The gamma amino butyric acid B (GABA(B)) receptor is a G protein-coupled receptor (GPCR) involved in synaptic transmission. Recent data indicate it to be also expressed on immune cells, along with chemokine receptors, which are also GPCRs. As GPCRs can undergo heterologous desensitization, we have examined the ability of baclofen, a GABA(B) receptor selective agonist, to interfere with the function of pro-inflammatory chemokine receptors known to be upregulated in cutaneous inflammation. In vitro, baclofen reduces chemotaxis of human peripheral blood mononuclear cells towards CCL2, CCL5, CXCL10, CXCL2 and CX3CL1 in a dose-dependant manner. Protein kinase C inhibitors calphostin C and G0 6976 could reverse this effect, pointing towards the involvement of both calcium-dependent and -independent protein kinase C in baclofen-induced inhibition of chemokine receptors. In an in vivo model of contact hypersensitivity in C57BL/6 mice, intraperitoneal injection of baclofen markedly alleviated signs of inflammation as well as recruitment of neutrophils, monocytes and lymphocytes into the skin. This study demonstrates a new role for the GABA(B) receptor in inflammation, making it a potential new therapeutic target to treat inflammatory skin diseases.

In traditional Chinese medicine (TCM), diagnosis of pathology and choice of treatment prescriptions are based on a method of differentiation of signs and symptoms known as syndrome differentiation or ZHENG. The cornerstone of TCM, ZHENG, relies on the gathering of clinical information through inspection, auscultation and olfaction, inquiry, and palpation. However, the biomolecular basis of the ZHENG remains unclear. In this study, we established mouse xenograft pancreatic cancer models with Shi-Re (Dampness-Heat), Pi-Xu (Spleen-Deficiency), or Xue-Yu (Blood-Stasis) ZHENG, which are regarded as the three major ZHENGs in pancreatic cancer. We found that tumors of the different ZHENG models exhibited significantly altered cancer-associated fibroblast (CAF) proliferative activity and tumor-associated macrophage (TAM) infiltration, which led to altered levels of CAF- and TAM-derived secreted cytokines such as SDF-1 and CCL5. The ZHENG model type also significantly influenced tumor growth, and administration of herbal medicine to the ZHENG model modified the tumor microenvironment. Therefore, this study partially unveiled the molecular basis of TCM ZHENG in pancreatic cancer.

West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine ex vivo spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses.

OBJECTIVE

There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

Chemokines play a pivotal role in the regulation of inflammatory cell infiltration in glomerular immune injury. To characterize mechanisms relevant for the regulation of chemokine expression in vivo, the LPS-mediated model of renal inflammation in rats was used in which we have previously demonstrated that the chemokine RANTES/CCL5 is expressed and secreted in glomeruli. Glomerular RANTES/CCL5 expression in this model correlated with an increased glomerular binding activity of the transcription factors AP-1, C/EBP, and NF-kappaB. To gain further insight into the functional roles of these transcription factors in the regulation of glomerular RANTES/CCL5 expression, we cloned the rat RANTES/CCL5 promoter and established the model of in vivo LPS tolerance. In tolerant rats, LPS-induced glomerular RANTES/CCL5 expression and activation of the transcription factors AP-1 and C/EBP were significantly reduced using both consensus and rat RANTES/CCL5-specific oligonucleotides. Reduced glomerular NF-kappaB binding activity after LPS injection could be demonstrated in tolerant rats only when using rat RANTES/CCL5-specific oligonucleotides. Reduced binding activity to this RANTES/CCL5-specific NF-kappaB binding site in the context of broad NF-kappaB activation might be due to changes in transcription factor interactions or chromatin remodeling processes.

OBJECTIVE

Evaluation of the efficacy of green tea extract (GTE) in regulating chemokine production and chemokine receptor expression in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA).

METHODS

Fibroblasts isolated from human RA synovium were used in the study. Regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, growth-regulated oncogene (GRO)alpha/CXCL1 and IL-8/CXCL8 production was measured by ELISA. Western blotting was used to study the phosphorylation of protein kinase C (PKC)delta and c-Jun N-terminal kinases (JNK). Chemokine and chemokine receptor expression was determined by quantitative RT-PCR. The benefit of GTE administration in rat AIA was determined.

RESULTS

GTE (2.5-40 microg/ml) inhibited IL-1beta-induced MCP-1/CCL2 (10 ng/ml), RANTES/CCL5, GROalpha/CXCL1 and IL-8/CXCL8 production in human RA synovial fibroblasts (P < 0.05). However, GTE inhibited MCP-1/CCL2 and GROalpha/CXCL1 mRNA synthesis in RA synovial fibroblasts. Furthermore, GTE also inhibited IL-1beta-induced phosphorylation of PKCdelta, the signalling pathway mediating IL-1beta-induced chemokine production. Interestingly, GTE preincubation enhanced constitutive and IL-1beta-induced CCR1, CCR2b, CCR5, CXCR1 and CXCR2 receptor expression. GTE administration (200 mg/kg/day p.o.) modestly ameliorated rat AIA, which was accompanied by a decrease in MCP-1/CCL2 and GROalpha/CXCL1 levels and enhanced CCR-1, -2, -5 and CXCR1 receptor expression in the joints of GTE administered rats.

CONCLUSIONS

Chemokine receptor overexpression with reduced chemokine production by GTE may be one potential mechanism to limit the overall inflammation and joint destruction in RA.

BACKGROUND

Chemokines produced by resident renal cells promote the infiltration of leukocyte subsets. We have analysed the chemotactic responses of CD3+ peripheral blood lymphocytes (PBLs) to factors secreted by proximal tubular epithelial cells (PTEC), assessing the role of chemokines and chemokine receptors in this process.

METHODS

By FACS we analysed expression of the chemokine receptors CCR5, CXCR3, CX3CR1, CCR2, CXCR1 and CXCR2 on both freshly isolated and activated PBLs. Using Boyden chambers we studied the chemotactic activity of supernatant from resting and cytokine-stimulated (TNF-alpha and IFN-gamma) PTEC towards PBLs. Soluble recombinant chemokines and blocking antibodies were used to study the role of individual chemokine receptors. Chemokine secretion by PTEC was analysed by ELISA.

RESULTS

Only a small proportion of freshly isolated cells expressed the chemokine receptors and there was low grade chemotaxis of these cells towards cytokine-stimulated PTEC supernatant compared with unstimulated PTEC supernatant. After activation, 84% of PBLs expressed CCR5, 90% expressed CXCR3 and 19% expressed CX3CR1. There remained low expression levels of CXCR1, CXCR2 and CCR2. Activated PBLs showed strong chemotactic responses to supernatant from cytokine-stimulated PTEC compared with unstimulated PTEC (P<0.001). Chemotaxis of these cells was inhibited by blocking CCR5, CXCR3 and CX3CR1 by 69%, 71% and 29% respectively, with complete inhibition following combined blockade. ELISA showed high levels of the chemokine RANTES/CCL5 (for CCR5) and IP-10/CXCL10 (for CXCR3) in cytokine-stimulated PTEC supernatant.

CONCLUSIONS

Chemokines produced by cytokine activated PTEC promote the selective recruitment of activated T cells via the receptors, CCR5, CXCR3 and CX3CR1. These receptors may be amenable to therapeutic manipulation in renal inflammation.

In this second review on chemokines, we focus on the polymorphisms and alternative splicings and on their consequences in disease. Because chemokines are key mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic disorders, a large number of studies attempting to relate particular polymorphisms of chemokines to given diseases have already been conducted, sometimes with contradictory results. Reviewing the published data, it becomes evident that some chemokine genes that are polymorphic have alleles that are found repeatedly, associated with disease of different aetiologies but sharing some aspects of pathogenesis. Among CXC chemokines, single nucleotide polymorphisms (SNPs) in the CXCL8 and CXCL12 genes stand out, as they have alleles associated with many diseases such as asthma and human immunodeficiency virus (HIV), respectively. Of CC chemokines, the stronger associations occur among alleles from SNPs in CCL2 and CCL5 genes and a number of inflammatory conditions. To understand how chemokines contribute to disease it is also necessary to take into account all the isoforms resulting from differential splicing. The first part of this review deals with polymorphisms and the second with the diversity of molecular species derived from each chemokine gene due to alternative splicing phenomena. The number of molecular species and the level of expression of each of them for every chemokine and for each functionally related group of chemokines reaches a complexity that requires new modelling algorithms akin to those proposed in systems biology approaches.

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

BACKGROUND

Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins.

RESULTS

We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1:1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent pi-pi interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine.

CONCLUSIONS

However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the "address" whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.

Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44(+) NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

T cells migrate into inflamed sites through the extracellular matrix (ECM) in response to chemotactic areas and are then simultaneously or sequentially exposed to multiple chemotactic ligands. We examined the responses of human peripheral blood T cells, present in an ECM-like context, to combinatorial signaling transduced by SDF-1alpha (CXCL12), and two CCR5 ligands, RANTES (CCL5) and MIP-1beta (CCL4). Separately, these chemokines, at G protein-coupled receptor (GPCR)-stimulating concentrations, induced T cell adhesion to fibronectin (FN) and T cell chemotaxis. However, the pro-adhesive and pro-migratory capacities of SDF-1alpha and RANTES or MIP-1beta were mutually suppressed by the simultaneous or sequential exposure of the cells to these CCR5 or CXCR4 ligands. This cross-talk did not involve the internalization of the SDF-1alpha receptor, CXCR4, but rather, a decrease in phosphorylation of ERK and Pyk-2, as well as inhibition of Ca(2+) mobilization. Strikingly, early CXCR4 signaling of phosphatidylinositol-3-kinase, detected by SDF-1alpha-induced AKT phosphorylation, was insensitive to RANTES-CCR5 signals. Accordingly, early chemotaxis to SDF-1alpha was not susceptible to CCR5 occupancy, whereas late stages of T cell chemotaxis were markedly down-regulated. This is an example of a specialized functional desensitization of heterologous chemokine receptors that induces GPCR interference with T cell adhesion to ECM ligands and chemotaxis within chemokine-rich extravascular contexts.

Human immunodeficiency virus type 1 (HIV-1) entry into CD4(+) cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, DeltaCXCR1, produced greater signals upon activation (Ca(2+) mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since DeltaCXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.

Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered "mixing vessels" that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2'5'-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics.

OBJECTIVE

The transcription factor nuclear factor-kappa-B (NFkappaB) is implicated in inflammatory responses, obesity and the metabolic syndrome, while immune cells appear to play a central role in mediating insulin resistance and can be used as a model to study inflammation and its relationship with insulin resistance. In peripheral blood mononuclear cells of overweight participants with the metabolic syndrome, we evaluated (1) the effect of diet-induced weight loss on the expression of genes involved in NFkappaB activation and (2) their association with insulin sensitivity. The genes studied were: TNF receptors TNFRSF1A and TNFRSF1B, and IL1R1, TLR4, TLR2, ICAM1, CCL5 and IKBKB.

METHODS

We analysed data from 34 overweight participants with abnormal glucose metabolism and the metabolic syndrome, who were randomised to a weight-reduction (n = 24) or control group (n = 10) for 33 weeks. The mRNA expression was measured using real-time PCR. Measures of insulin and glucose homeostasis were assessed by IVGTT and OGTT.

RESULTS

In general, the genes studied were downregulated after weight loss intervention. The changes in TLR4, TLR2, CCL5 and TNFRSF1A mRNA expression were associated with an increase in insulin sensitivity index independently of the change in waist circumference (p < 0.05). The change in IKBKB expression correlated with most of the changes in gene expression in the weight-reduction group.

CONCLUSIONS

These results suggest that proteins encoded by CCL5, TLR2 and TLR4, and TNFRSF1A might contribute to insulin-resistant states that characterise obesity and the metabolic syndrome.

BACKGROUND

ClinicalTrials.gov NCT 00621205.

Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Prostate cancer is the most common nonskin neoplasm and second leading cause of death in men. 6-Shogaol (6-SHO), a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been shown to possess anti-inflammatory and anticancer activity. In the present study, the effect of 6-SHO on the growth of prostate cancer cells was investigated. 6-SHO effectively reduced survival and induced apoptosis of cultured human (LNCaP, DU145, and PC3) and mouse (HMVP2) prostate cancer cells. Mechanistic studies revealed that 6-SHO reduced constitutive and interleukin (IL)-6-induced STAT3 activation and inhibited both constitutive and TNF-α-induced NF-κB activity in these cells. In addition, 6-SHO decreased the level of several STAT3 and NF-κB-regulated target genes at the protein level, including cyclin D1, survivin, and cMyc and modulated mRNA levels of chemokine, cytokine, cell cycle, and apoptosis regulatory genes (IL-7, CCL5, BAX, BCL2, p21, and p27). 6-SHO was more effective than two other compounds found in ginger, 6-gingerol, and 6-paradol at reducing survival of prostate cancer cells and reducing STAT3 and NF-κB signaling. 6-SHO also showed significant tumor growth inhibitory activity in an allograft model using HMVP2 cells. Overall, the current results suggest that 6-SHO may have potential as a chemopreventive and/or therapeutic agent for prostate cancer and that further study of this compound is warranted.

The mouse model of genital herpes relies on medoxyprogesterone treatment of female mice to render the vaginal lumen susceptible to inoculation with herpes simplex virus 2 (HSV-2). In the present study, we report that mice deficient in the A1 chain of the type I interferon receptor (CD118(-/-)) are susceptible to HSV-2 in the absence of medroxyprogesterone preconditioning. In the absence of hormone pretreatment, 2,000 PFU of a clinical isolate of HSV-2 was sufficient to establish a productive infection in the vagina of 75% ± 17% and in the spinal cord of 71% ± 14% of CD118(-/-) mice, whereas the same dose of HSV-2 replicated to detectable levels in only 13% ± 13% of vaginal samples and 0% of spinal cord samples from wild-type mice, as determined at day 5 postinfection. The susceptibility to HSV-2 infection in the CD118(-/-) mice was associated with a significant reduction in the infiltration of HSV-specific cytotoxic T lymphocytes into the vaginal tissue, the local production of gamma interferon (IFN-γ), and the expression of T cell-recruiting chemokines CCL5, CXCL9, and CXCL10. Collectively, the results underscore the significant contribution of type I IFNs in resistance to genital HSV-2 infection.

Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

BACKGROUND

Chemokines play a crucial role in breast cancer tumorigenesis and progression. Recently, the chemokine (C-C motif) ligand 5 (CCL5), which can be secreted either by tumor cells or by mesenchymal stromal cells recruited to the tumor, has been identified as a key node in the bidirectional communication between breast cancer and normal cells.

METHODS

In this review, the authors discuss the role of CCL5/chemokine receptor 5 (CCR5) axis in promoting breast cancer onset and progression. Interrogation of large clinical databases has demonstrated increased expression of the CCL5/CCR5 axis in specific subtypes of breast cancer. The activation of the receptor CCR5 in breast cancer cells controls their invasiveness serving as a driver for metastasis. Furthermore, the CCL5/CCR5 axis participates in the recruitment of specific immune cells into tumors, inducing local immunosuppression and favoring tumor progression.

CONCLUSIONS

The role of CCR5 in HIV infection led to the development of specific and potent CCR5 antagonists. The data reviewed here includes basic and translational studies that support the use of such CCR5 antagonists in breast cancer patients as adjuvant therapy to block the metastasis.

We review evidence that Stem Cell Factor (SCF) plays an important role in the pathophysiology of asthma. SCF is produced by a wide variety of cells present in asthmatic lung, including mast cells and eosinophils. Its receptor, c-kit, is broadly expressed on mature mast cells and eosinophils. SCF promotes recruitment of mast cell progenitors into tissues, as well as their local maturation and activation. It also promotes eosinophil survival, maturation and functional activation. SCF enhances IgE-dependent release of mediators from mast cells, including histamine, leukotrienes, cytokines (TNF-alpha, IL-5, GM-CSF) and chemokines (RANTES/CCL5, MCP-1/CCL2, TARC/CCL17 e MDC/CCL22); it is required for IL-4 production in mast cells. SCF, acting in concert with IgE, also upregulates the expression and function of CC chemokine receptors in mast cells. Structural and resident airway cells express increased levels of SCF in the bronchus of asthmatic patients. In a murine model of asthma, allergen exposure increased production of SCF by epithelial cells and alveolar macrophages, which was transient and paralleled by histamine release. SCF induced long-lived airway hyperreactivity, which was prevented by local neutralization of SCF, as well as by inhibitors of the production or activity of cysteinyl-leukotrienes. Together, these observations suggest that SCF has an important role in asthma.

To test the hypothesis that miR-155 regulates monocyte migratory potential via modulation of chemokine and chemokine receptor expression in RA, and thereby is associated with disease activity.

The miR-155 copy-numbers in monocytes from peripheral blood (PB) of healthy (n = 22), RA (n = 24) and RA SF (n = 11) were assessed by real time-PCR using synthetic miR-155 as a quantitative standard. To evaluate the functional impact of miR-155, human monocytes were transfected with control or miR-155 mimic, and the effect on transcript levels, and production of chemokines was evaluated by Taqman low-density arrays and multiplex assays. A comparative study evaluated constitutive chemokine receptor expression in miR-155-/- and wild-type murine (CD115 + Ly6C + Ly6G-) monocytes.

Compared with healthy monocytes, the miR-155 copy-number was higher in RA, peripheral blood (PB) and SF monocytes (PB P < 0.01, and SF P < 0.0001). The miR-155 copy-number in RA PB monocytes was higher in ACPA-positive compared with ACPA-negative patients (P = 0.033) and correlated (95% CI) with DAS28 (ESR), R = 0.728 (0.460, 0.874), and with tender, R = 0.631 (0.306, 0.824) and swollen, R = 0.503 (0.125, 0.753) joint counts. Enforced-expression of miR-155 in RA monocytes stimulated the production of CCL3, CCL4, CCL5 and CCL8; upregulated CCR7 expression; and downregulated CCR2. Conversely, miR155-/- monocytes showed downregulated CCR7 and upregulated CCR2 expression.

Given the observed correlations with disease activity, these data provide strong evidence that miR-155 can contribute to RA pathogenesis by regulating chemokine production and pro-inflammatory chemokine receptor expression, thereby promoting inflammatory cell recruitment and retention in the RA synovium.

Resistance to antiangiogenic therapies is a critical problem that has limited the utility of antiangiogenic agents in clinical settings. However, the molecular mechanisms underlying this resistance have yet to be fully elucidated. In this study, we established a novel xenograft model of acquired resistance to bevacizumab. To identify molecular changes initiated by the tumor cells, we performed human-specific microarray analysis on bevacizumab-sensitive and -resistant tumors. Efficiency analysis identified 150 genes upregulated and 31 genes downregulated in the resistant tumors. Among angiogenesis-related genes, we found upregulation of fibroblast growth factor-2 (FGF2) and fibroblast growth factor receptor-3 (FGFR3) in the resistant tumors. Inhibition of the FGFR in the resistant tumors led to the restoration of sensitivity to bevacizumab. Furthermore, increased FGF2 production in the resistant cells was found to be mediated by overexpression of upstream genes phospholipase C (PLCg2), frizzled receptor-4 (FZD4), chemokine [C-X3-C motif] (CX3CL1), and chemokine [C-C motif] ligand 5 (CCL5) via extracellular signal-regulated kinase (ERK). In summary, our work has identified an upregulation of a proangiogenic signature in bevacizumab-refractory HNSCC tumors that converges on ERK signaling to upregulate FGF2, which then mediates evasion of anti-VEGF therapy. These findings provide a new strategy on how to enhance the therapeutic efficacy of antiangiogenic therapy.

CONCLUSIONS

Novel xenograft model leads to the discovery of FGF as a promising therapeutic target in overcoming the resistance of antiangiogenic therapy in HNSCC.

Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)-induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra-cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3-induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro-inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti-inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC-treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein-1 (MCP)-1, MCP-3, CCL5, the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, the pro-inflammatory cytokines interleukin-6, interleukin-12, tumor necrosis factor-α, the pro-fibrotic transforming growth factorβ1, and circulating MCP-1 and MCP-3 levels decreased in CVB3 MSC mice, while LV stromal cell-derived factor-1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3-induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249-1261.