OBJECTIVE

Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets-Ly6C(hi) and Ly6C(lo)-to the blood and their respective role in post-ischaemic neovascularization are not clearly understood. Here, we hypothesized that distinct chemokine/chemokine receptor pathways, namely CCL2/CCR2, CX3CL1/CX3CR1, and CCL5/CCR5, differentially control monocyte subset systemic levels, and might thus impact post-ischaemic vessel growth.

RESULTS

In a model of murine hindlimb ischaemia, both Ly6C(hi) and Ly6C(lo) monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6C(hi) and Ly6C(lo) monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6C(lo) monocyte levels, whereas CCL5/CCR5 had no role. Only CCL2/CCR2 signalling influenced neovascularization, which was increased in mice overexpressing CCL2, whereas it markedly decreased in CCL2-/- mice. Moreover, adoptive transfer of Ly6C(hi)-but not Ly6C(lo)-monocytes enhanced vessel growth and blood flow recovery.

CONCLUSIONS

Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.

Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8⁺ T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4⁺ T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4, CCL5, etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

Keywords: Bioinformatics; Immunology.

BACKGROUND

Hepatitis C virus (HCV) encodes several proteins that interfere with the host cell antiviral response. Previously, the serine protease NS3/4A was shown to inhibit IFN-beta gene expression by blocking dsRNA-activated retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3)-mediated signaling pathways.

RESULTS

In the present work, we systematically studied the effect of all HCV proteins on IFN gene expression. NS2 and NS3/4A inhibited IFN gene activation. NS3/4A inhibited the Sendai virus-induced expression of multiple IFN (IFN-alpha, IFN-beta and IFN-lambda1/IL-29) and chemokine (CCL5, CXCL8 and CXCL10) gene promoters. NS2 and NS3/4A, but not its proteolytically inactive form NS3/4A-S139A, were found to inhibit promoter activity induced by RIG-I or its adaptor protein Cardif (or IPS-1/MAVS/VISA). Both endogenous and transfected Cardif were proteolytically cleaved by NS3/4A but not by NS2 indicating different mechanisms of inhibition of host cell cytokine production by these HCV encoded proteases. Cardif also strongly colocalized with NS3/4A at the mitochondrial membrane, implicating the mitochondrial membrane as the site for proteolytic cleavage. In many experimental systems, IFN priming dramatically enhances RNA virus-induced IFN gene expression; pretreatment of HEK293 cells with IFN-alpha strongly enhanced RIG-I expression, but failed to protect Cardif from NS3/4A-mediated cleavage and failed to restore Sendai virus-induced IFN-beta gene expression.

CONCLUSIONS

HCV NS2 and NS3/4A proteins were identified as potent inhibitors of cytokine gene expression suggesting an important role for HCV proteases in counteracting host cell antiviral response.

During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0.001) and CXCL8 (up to 10-fold, P < 0.001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0.001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).

Chemokines are soluble factors shown to play important roles in regulating immune cell recruitment during inflammatory responses and defense against foreign pathogens. De-regulated expression and activity of several chemokine signaling pathways have been implicated in cancer progression, including: CCL2, CCL5, CXCL1 and CXCL12. While studies in the past have focused the role of these chemokine signaling pathways in regulating immune responses, emerging studies show that these molecules regulate diverse cellular processes including angiogenesis, and regulation of epithelial cell growth and survival. New evidence indicates that chemokines are critical for cancer progression and indicate complex and diverse functions in the tumor microenvironment. This review will focus on the contributions of chemokine signaling in regulating cancer microvironment and discuss the utility of targeting or delivering chemokines in cancer therapeutics.

Platelets are inflammatory anuclear cells with a well-established role in the development and manifestation of atherosclerosis. Activated platelets secrete a plethora of chemokines including CXCL4 or platelet factor 4 (PF4), CCL5, CXCL12 or stromal cell derived factor-1α (SDF-1α), CXCL16 and others, which initiate or promote local inflammatory processes at sites of vascular injury. These processes are mainly mediated by the recruitment of circulating haematopoietic stem cells, neutrophils, monocytes or lymphocytes on vascular wall. Under acute ischemic conditions platelet-derived chemokines may promote the mobilization of bone marrow-derived progenitor cells and their homing at lesion sites. This review focuses on the role of platelet-derived chemokines in inflammation and atherosclerosis. Further, we discuss the clinical value of plasma levels of chemokines in the prognosis of atherosclerotic heart disease.

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

CNS abnormalities can be detected during chronic human immunodeficiency virus (HIV) infection, before the development of opportunistic infections or other sequelae of immunodeficiency. However, although end-stage dementia caused by HIV has been linked to the presence of infected and activated macrophages and microglia in the brain, the nature of the changes resulting in the motor and cognitive disorders in the chronic stage is unknown. Using simian immunodeficiency virus-infected rhesus monkeys, we sought the molecular basis for CNS dysfunction. In the chronic stable stage, nearly 2 years after infection, all animals had verified CNS functional abnormalities. Both virus and infiltrating lymphocytes (CD8+ T-cells) were found in the brain. Molecular analysis revealed that the expression of several immune response genes was increased, including CCL5, which has pleiotropic effects on neurons as well as immune cells. CCL5 was significantly upregulated throughout the course of infection, and in the chronic phase was present in the infiltrating lymphocytes. We have identified an altered state of the CNS at an important stage of the viral-host interaction, likely arising to protect against the virus but in the long term leading to damaging processes.

Essential hypertension is idiopathic although it is accepted as a complex polygenic trait with underlying genetic components, which remain unknown. Our supposition is that primary hypertension involves activation of the sympathetic nervous system. One pivotal region controlling arterial pressure set point is nucleus tractus solitarii (NTS). We recently identified that pro-inflammatory molecules, such as junctional adhesion molecule-1, were over expressed in endothelial cells of the microvasculature supplying the NTS in an animal model of human hypertension (the spontaneously hypertensive rat: SHR) compared to normotensive Wistar Kyoto (WKY) rats. We have also shown endogenous leukocyte accumulation inside capillaries within the NTS of SHR but not WKY rats. Despite the inflammatory state in the NTS of SHR, transcripts of some inflammatory molecules such as chemokine (C-C motif) ligand 5 (Ccl5), and its receptors, chemokine (C-C motif) receptor 1 and 3 were down-regulated in the NTS of SHR compared to WKY rats. This may be compensatory to avoid further strong inflammatory activity. More importantly, we found that down-regulation of Ccl5 in the NTS of SHR may be pro-hypertensive since microinjection of Ccl5 into the NTS of SHR decreased arterial pressure but was less effective in WKY rats. Leukocyte accumulation of the NTS microvasculature may also induce an increase in vascular resistance and hypoperfusion within the NTS; the latter may trigger release of pro-inflammatory molecules which via paracrine signaling may affect central neural cardiovascular activity conducive to neurogenic hypertension. All told, we suggest that vascular inflammation within the brainstem contributes to neurogenic hypertension by multiple pathways.

BACKGROUND

The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)-associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.

OBJECTIVE

This study tested whether D6 regulates pulmonary chemokine levels, inflammation, or airway responsiveness during allergen-induced airway disease.

METHODS

D6-deficient and genetically matched C57BL/6 mice were sensitized and challenged with ovalbumin. ELISA and flow cytometry were used to measure levels of cytokines and leukocytes, respectively. Mechanical ventilation was used to measure airway reactivity.

RESULTS

The ability of D6 to diminish chemokine levels in the lung was chemokine concentration dependent. CCL17 and CCL22 were abundant in the airway, and their levels were attenuated by D6 when they were within a defined concentration range. By contrast, airway concentrations of CCL3, CCL5, and CCL11 were low and unaffected by D6. Allergen-challenged D6-deficient mice had more dendritic cells, T cells, and eosinophils in the lung parenchyma and more eosinophils in the airway than similarly challenged C57BL/6 mice. By contrast, D6-deficient mice had reduced airway responses to methacholine compared with C57BL/6 mice. Thus, D6 has opposing effects on inflammation and airway reactivity.

CONCLUSIONS

The ability of D6 to scavenge chemokines in the lung is dependent on chemokine concentration. The absence of D6 increases inflammation, but reduces airway reactivity. These findings suggest that inhibiting D6 function might be a novel means to attenuate airway responses in individuals with allergic asthma.

The latent persistence of herpes simplex virus type 1 (HSV-1) in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltrate. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to characterize the immune cell infiltrates by this feature. We combined in situ hybridization, laser cutting microscopy, and single cell RT-PCR to demonstrate the expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T-cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8+ T-cells had features of the memory effector phenotype. The voltage-gated potassium channel Kv1.3, a marker of chronic activated memory effector cells, and the chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells. The corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all CD8 T-cells. In addition, T-cells expressed granzymes and perforin. In contrast to animal models of HSV-1 latency, hardly any FoxP3-positive regulatory T-cells were detected in human TG. Thus, HSV-1 IE genes are expressed in human TG and the infiltrating T-cells bear several characteristics that suggest viral antigenic stimulation.

CD4+ T cells are required for immunity against many viral infections, including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1, whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors, although it is not known whether these factors are produced during primary antigen-specific responses. Here, we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of naïve CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22, CCL22), and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3), macrophage inflammatory protein-1 beta (CCL4), and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus.

Viruses are associated with the majority of exacerbations of asthma and chronic obstructive pulmonary disease. Virus induction of neutrophil and lymphocyte chemokines in bronchial epithelium is important in exacerbation pathogenesis. Combined corticosteroid/beta2 agonists synergistically suppress airway smooth muscle chemokine production. Because bronchial epithelium expresses glucocorticoid and beta2 receptors, we investigated whether combination therapy can synergistically suppress rhinovirus-induced bronchial epithelial cell neutrophil (CXCL5, CXCL8) and lymphocyte (CCL5, CXCL10) chemokine production. We investigated modulation of rhinovirus- and IL-1beta-induced bronchial epithelial cell chemokine production by salmeterol and fluticasone propionate, used at therapeutic concentrations, alone and in combination. After 1 h pretreatment, combined treatment significantly inhibited rhinovirus 16, 1B, and IL-1beta-induced CCL5 and CXCL8 protein and mRNA production in BEAS-2B cells compared with fluticasone alone. When used 4 h after treatment, the combination significantly reduced virus-induced CCL5 but not CXCL8. Salmeterol alone had no effect; therefore, this inhibition was synergistic. Kinetic analysis demonstrated that combination therapy reduced by 5-fold the concentration of corticosteroid required to inhibit CXCL8 mRNA expression. In primary cells, salmeterol alone reduced rhinovirus-induced CCL5 and CXCL10 and increased CXCL5 production in a dose-dependent manner but had no effect on CXCL8. Fluticasone alone reduced CCL5, CXCL8, and CXCL10 but had no effect on CXCL5. Combination therapy augmented inhibition of CXCL8, CCL5, and CXCL10 but had no effect on CXCL5. Corticosteroids and beta2 agonists suppress rhinovirus-induced chemokines in bronchial epithelial cells through synergistic and additive mechanisms. This effect was greater for lymphocyte- than for neutrophil-related chemokines.

BACKGROUND

The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM).

METHODS

LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry.

RESULTS

The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues.

CONCLUSIONS

The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

OBJECTIVE

To define the chemokine profile in the CSF of patients with multiple sclerosis (MS) and compare it with three control groups; patients with benign headache (headache), non-inflammatory neurological diseases (NIND), and other inflammatory neurological diseases (IND). In addition, the correlations of CSF chemokine concentrations with chemokine receptor expression on CSF CD4(+) T cells and with clinical disease activity were assessed.

METHODS

Forty three patients with MS, 24 with IND, 44 with NIND, and 12 with benign headache undergoing diagnostic or therapeutic lumbar puncture were included. Supernatant fluid from CSF was analysed for four beta (CCL2, CCL3, CCL4, CCL5) and two alpha (CXCL9, CXCL10)chemokines by enzyme linked immunosorbent assay (ELISA). Chemokine receptors CCR3, CCR5, and CXCR3 on CD4(+) T cells from eight patients with MS were analysed using directly conjugated fluorescent labelled monoclonal antibodies and flow cytometry.

RESULTS

CXCL10, formerly interferon-gamma inducible protein-10 (IP-10), was significantly increased and CCL2, formerly monocyte chemoattractant protein-1 (MCP-1), was significantly reduced in the CSF of patients with MS and IND compared with those with benign headache and NIND. Concentrations of CXCL10 were significantly greater in patients with relapsing-remitting compared with secondary progressive MS and correlated significantly with CXCR3 expression on CSF CD4(+) T cells from patients with MS. Concentrations of CXCL10 decreased and CCL2 concentrations increased as time from the last relapse increased in patients with MS.

CONCLUSIONS

Increased CXCL10 and decreased CCL2 concentrations in the CSF are associated with relapses in MS. Although serial values from individual patients were not available, this study suggests that CXCL10 and CCL2 may return towards baseline concentrations after a relapse. Correlation of CXCL10 with CD4(+) T cell expression of CXCR3 was consistent with its chemoattractant role for activated lymphocytes. Thus CXCL10 neutralising agents and CXCR3 receptor antagonists may be therapeutic targets in MS.

OBJECTIVE

Increasing evidence suggests that interaction between the chemoattractant CXCL12/stromal cell-derived factor-1alpha and its receptor CXCR4 plays a pivotal role in the metastasis of various tumors. Our previous studies showed that multi-component Chinese herbal medicines inhibited the effects of CXCL12/CXCR4. As a result of sequential chromatographic fractionation of one herbal medicine ingredient, Lianqiao (fruit of Forsythia suspensa), we observed that tannins were, at least in part, responsible for this activity. The aim of this study was to assess the anti-CXCL12/CXCR4 activity of a commercial tannic acid and evaluate its potential to inhibit tumor cell migration and angiogenesis in vitro.

METHODS

The inhibitory effect of tannic acid on CXCL12/CXCR4 was measured by chemotaxis assay, ligand binding assay, and fluorescence-activated cell sorter analysis. The antiangiogenic effect of tannic acid was assessed by in vitro endothelial cell tube formation.

RESULTS

Tannic acid, at nontoxic concentrations, specifically inhibited CXCL12-induced human monocyte migration (IC(50), 7.5 micro g/ml) but did not inhibit CCL2-, CCL3-, CCL5-, formylmethionylleucylphenylalanine (fMLP)-, or C5a-induced migration. The compound markedly blocked CXCL12 binding to THP-1 cells (IC(50), 0.36 micro g/ml). Tannic acid also inhibited CXCL12-induced, but not epidermal growth factor-induced, migration of MDA 231 breast tumor cells. Additionally, 0.5 micro g/ml of tannic acid selectively inhibited CXCL12-mediated, but not basic fibroblast growth factor- or endothelial cell growth supplement-mediated, bovine aorta endothelial cell capillary tube formation.

CONCLUSIONS

These studies indicate that tannic acid is a novel selective CXCL12/CXCR4 antagonist and consequently may provide a mechanistic basis for the reported antitumor and anti-inflammatory properties of tannic acid.

Breast cancer (BC) is a leading cause of mortality among women in the world. To date, a number of molecules have been established as disease status indicators and therapeutic targets. The best known among them are estrogen receptor-α (ER-α), progesterone receptor (PR) and HER-2/neu. About 15%-20% BC patients do not respond effectively to therapies targeting these classes of tumor-promoting factors. Thus, additional targets are strongly and urgently sought after in therapy for human BCs negative for ER, PR and HER-2, the so-called triple-negative BC (TNBC). Recent clinical work has revealed that CC chemokine ligand 5 (CCL5) is strongly associated with the progression of BC, particularly TNBC. How CCL5 contributes to the development of TNBC is not well understood. Experimental animal studies have begun to address the mechanistic issue. In this article, we will review the clinical and laboratory work in this area that has led to our own hypothesis that targeting CCL5 in TNBCs will have favorable therapeutic outcomes with minimal adverse impact on the general physiology.

OBJECTIVE

Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant-induced polyarthritis (AIA) were investigated.

METHODS

A model of AIA in rats was used to compare the therapeutic effects of the treatment with DF2162, anti-TNF or anti-CINC-1 antibodies on joint inflammation and local production of cytokines and chemokines.

RESULTS

DF2162 prevented chemotaxis of rat and human neutrophils induced by chemokines acting on CXCR1/2. DF2162 was orally bioavailable and metabolized to two major metabolites. Only metabolite 1 retained CXCR1/2 blocking activity. Treatment with DF2162 (15 mg kg(-1), twice daily) or metabolite 1, but not metabolite 2, starting on day 10 after arthritis induction diminished histological score, the increase in paw volume, neutrophil influx and local production of TNF, IL-1beta, CCL2 and CCL5. The effects of DF2162 were similar to those of anti-TNF, and more effective than those of anti-CINC-1, antibodies. DF2162 prevented disease progression even when started 13 days after arthritis induction.

CONCLUSIONS

DF 2162, a novel orally-active non-competitive allosteric inhibitor of CXCR1 and CXCR2, significantly ameliorates AIA in rats, an effect quantitatively and qualitatively similar to those of anti-TNF antibody treatment. These findings highlight the contribution of CXCR2 in the pathophysiology of AIA and suggest that blockade of CXCR1/2 may be a valid therapeutic target for further studies aiming at the development of new drugs for treatment of rheumatoid arthritis.

Inflammation has long been implicated as a contributor to pathogenesis in many CNS illnesses, including Lyme neuroborreliosis. Borrelia burgdorferi is the spirochete that causes Lyme disease and it is known to potently induce the production of inflammatory mediators in a variety of cells. In experiments where B. burgdorferi was co-cultured in vitro with primary microglia, we observed robust expression and release of IL-6 and IL-8, CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta) and CCL5 (RANTES), but we detected no induction of microglial apoptosis. In contrast, SH-SY5Y (SY) neuroblastoma cells co-cultured with B. burgdorferi expressed negligible amounts of inflammatory mediators and also remained resistant to apoptosis. When SY cells were co-cultured with microglia and B. burgdorferi, significant neuronal apoptosis consistently occurred. Confocal microscopy imaging of these cell cultures stained for apoptosis and with cell type-specific markers confirmed that it was predominantly the SY cells that were dying. Microarray analysis demonstrated an intense microglia-mediated inflammatory response to B. burgdorferi including up-regulation in gene transcripts for TLR-2 and NFkappabeta. Surprisingly, a pathway that exhibited profound changes in regard to inflammatory signaling was triggering receptor expressed on myeloid cells-1 (TREM1). Significant transcript alterations in essential p53 pathway genes also occurred in SY cells cultured in the presence of microglia and B. burgdorferi, which indicated a shift from cell survival to preparation for apoptosis when compared to SY cells cultured in the presence of B. burgdorferi alone. Taken together, these findings indicate that B. burgdorferi is not directly toxic to SY cells; rather, these cells become distressed and die in the inflammatory surroundings generated by microglia through a bystander effect. If, as we hypothesized, neuronal apoptosis is the key pathogenic event in Lyme neuroborreliosis, then targeting microglial responses may be a significant therapeutic approach for the treatment of this form of Lyme disease.

Over half of breast-cancer-related deaths are due to recurrence 5 or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.

BACKGROUND

Polyclonal antithymocyte globulins (ATG) induce T-cell depletion and functional impairment of nondeleted lymphocytes. Interference of ATG with the main leukocyte surface molecules involved in cellular adhesion and leukocyte-endothelium interaction was investigated in the present study.

METHODS

In three rabbit ATG, the authors measured antibodies to integrins, beta2-integrin ligands, and chemokine receptors by flow cytometry; chemotactic responses; and down-modulation of cell surface expression on lymphocytes, monocytes, and neutrophils.

RESULTS

Antibodies to CD11a/CD18 (leukocyte function-associated antigen-1 [LFA-1]) present in ATG induced a dose-dependent down-modulation of cell surface expression of this beta2 integrin on lymphocytes, monocytes, and neutrophils. In contrast, anti-LFA-1 monoclonal antibodies did not induce LFA-1 modulation unless cross-linked by a second antibody. ATG also contained functional antibodies to the beta1 integrin CD49d/CD29 (VLA-4), the alpha4beta7 integrin, CD50, CD54, and CD102 but not to CD62L. ATG were shown to bind to CXCR4 and CCR7 on lymphocytes, CXCR4, and CCR5 on monocytes; to down-modulate cell surface expression of CCR7; and to decrease monocyte chemotactic response to CCL5 (RANTES) and lymphocyte chemotactic response to CCL19 (MIP-3beta).

CONCLUSIONS

These results show that ATG may interfere with leukocyte responses to chemotactic signals but mostly inhibit the expression of integrins required for firm cellular adhesion. The latter property of inhibition is not shared by monoclonal antibodies, and it may contribute to decreasing graft cellular infiltration during acute rejection and possibly after postischemic reperfusion.