While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

Alveolar macrophages produce neutrophil chemoattractants; this cellular cross-talk contributes to neutrophilic airway inflammation in chronic obstructive pulmonary disease (COPD). We have investigated the chemotaxis cross-talk mechanisms between these cells using COPD alveolar macrophages. Using conditioned media from stimulated COPD alveolar macrophages, we investigated the relative contributions of growth-related oncogene (CXCL1), interleukin-8 (CXCL8), and regulated on activation normal T cell expressed and secreted (CCL5) to neutrophil chemotaxis and evaluated the effect of blocking the chemokine receptors CXCR1 and CXCR2 on chemotaxis caused by macrophage-conditioned media. Furthermore, we evaluated whether corticosteroid treatment of stimulated alveolar macrophages inhibited the chemotaxis ability of conditioned media. Alveolar macrophages isolated from COPD (n = 8) and smoker (S) (n = 8) lungs were treated with ultra-pure lipopolysaccharide in the presence and absence of dexamethasone (1 μM). Supernatants were used for neutrophil chemotaxis assays. SB656933 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide) (CXCR2 antagonist) and Sch527123 [1-(2-chloro-3-fluorophenyl)-3-(4-chloro-2-hydroxy-3-piperazin-1-ylsulfonylphenyl)urea, 3-(2-chloro-3-fluoro-phenyl)-1-(4-chloro-2-hydroxy-3-piperazin-1-ylsulfonyl-phenyl)urea] (dual CXCR1 and CXCR2 antagonist) and blocking antibodies for CXCL8, CXCL1, and CCL5 were assessed. Conditioned media caused neutrophil chemotaxis in COPD and smokers (60.5 and 79.9% of total cells, respectively). Dexamethasone did not significantly reduce neutrophil chemotaxis in COPD or S. SB656933 and Sch527123 inhibited chemotaxis in a concentration-dependent manner, with the dual antagonist Sch527123 causing greater inhibition of chemotaxis. CXCL8 antibody inhibited neutrophil chemotaxis to basal levels, although there was no significant effect of blocking either CXCL1 or CCL5 (P>> 0.05). CXCL8 plays a major role in neutrophil chemotaxis caused by alveolar macrophage-derived conditioned media, and this is most effectively inhibited by dual antagonism of CXCR1 and CXCR2. Corticosteroids do not inhibit chemotaxis caused by macrophage-derived chemokines.

Pancreatic cancer (PC) is one of the deadliest cancers and remains a major challenge due to its invasive and metastatic nature. Increased levels of CCR5 and CCL5 have established indicators for disease status in various cancers, including PC. However, their role in invasion and metastasis of PC is not known. Here we conducted immunohistochemistry of PC tissues and found elevated epithelial staining for CCR5 and CCL5 in metastatic PC tissues compared to non-neoplastic. In vitro experiments, such as flow cytometry, immunofluorescence and western blotting with human PC cell lines (AsPc-1, BxPc-3 and MIA PaCa-2), showed higher expression levels of CCR5. The CCL5 activation of PC cells expressing CCR5 increased their invasive potential, while treatment with CCR5 inhibitor maraviroc inhibited the CCL5 activation. CCL5 induced proliferation of PC cells was mediated through F-actin polymerization, while there was marked reduction when the cells were treated with maraviroc. The direct interaction of CCR5 with CCL5 was verified using a calcium mobilization assay. Taken together, our results demonstrate that CCR5 and CCL5 are potential markers for metastatic PC cancer, and their interaction leads to the increased PC cell invasion. Thus, blocking CCR5/CCL5 axis might prove beneficial to prevent metastasis and provide a more therapeutic strategy to control PC progression.

The chemokine CC motif receptor 5 (CCR5) and its ligands have been reported to be associated with cancer progression and metastasis. Although recent researches have demonstrated a fundamental role of hypoxia in cancer, the effect of hypoxia on the expression and function of CCR5 and its ligands in cancer cells is unknown. Here, we investigated the status of CCR5 and its ligands in cancer cells under hypoxic conditions. Quantitative polymerase chain reaction, western blotting and immunofluorescence staining showed that hypoxia induced a strong increase of CCR5 expression. Dual luciferase assay and mRNA stability analysis indicated that hypoxia-induced CCR5 mRNA expression relied on both transcriptional and posttranscriptional mechanisms. We detected the expression of CCR5 ligands and found that chemokine CC motif ligand 5 (CCL5) was induced under hypoxia. Recombinant human CCL5 stimulated cell migration rather than cell proliferation under hypoxia, and neutralization of CCL5 inhibited hypoxia-induced migration of cancer cells. Similarly, overexpression of CCR5 increased cell migration, and knockdown of CCR5 attenuated hypoxia-mediated cell migration. We further showed that hypoxia-inducible factor-1α (HIF-1α) was involved in CCR5 and CCL5 regulation under hypoxia. HIF-1α mRNA levels were highly correlated with CCR5 mRNA and CCL5 mRNA levels in clinical samples. CCR5 and CCL5 were highly expressed in breast cancer lymph nodes metastases. Taken together, our data suggest that CCR5-CCL5 interaction promotes cancer cell migration under hypoxia.

Previously, we demonstrated that membrane expression of glypican-3 (GPC3) stimulates the recruitment of macrophages into human hepatocellular carcinoma (HCC) tissues. However, functional polarization of the macrophages and the chemoattractant factors related to the recruitment has yet to be determined. In this study, to clarify the polarization (M1 or M2) of the macrophages and provide a clue as to the factors involved in the recruitment, we used xenograft models of SK-HEP-1 and SK03 cell lines with undetectable and high-level membrane expression of GPC3, respectively and analyzed the expression profiles of the relevant genes in both xenografts mainly using microarray techniques. Clustering analyses with mouse genome arrays revealed that the SK-HEP-1 and SK03 xenografts showed different expression profiles for M2 macrophage-related genes but not for M1 macrophage-related genes. Many of the M2 macrophage-related genes were upregulated in the SK03 xenografts compared to the SK-HEP-1 xenografts. Additionally, most of the macrophages infiltrating into the SK03 xenografts were positive for M2 macrophage-specific markers. Regarding the chemoattractant factors, the microarray and quantitative real-time PCR analyses revealed that, of the genes reportedly related to macrophage recruitment, CCL5, CCL3 and CSF1 were significantly upregulated in the SK03 xenograft. These findings suggest that the macrophages recruited into GPC3-overexpressing (with membrane expression) HCC are M2-polarized ones and, more specifically, M2 tumor-associated macrophages which are known to promote tumor progression and metastasis, and CCL5, CCL3 and CSF1 are possible candidate genes for the recruitment of macrophages.

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-gamma). Here, we assessed the role of CD4(+) T cells and IFN-gamma on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-gamma, CCL2 (MCP-1), CCL3 (MIP-1alpha), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-gamma had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-gamma signaling on macrophage lineage cells was assessed using transgenic mice, called "macrophages insensitive to interferon gamma" (MIIG) mice, that express a dominant-negative IFN-gamma receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4(+) T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4(+) T-cell production of IFN-gamma promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.

Retinal ischemia-reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.

Recently, we have shown that the CCL5/CCR5 axis is active in patients affected by an aggressive basal subtype of breast cancer. Using preclinical models, we have demonstrated that CCR5 promotes breast cancer invasiveness and metastatic potential, while CCR5 inhibition abrogates them. Thus, CCR5 antagonists may constitute an alternative therapeutic approach for patients affected by metastatic basal breast cancer.

The molecular mechanisms governing skin fibrosis in murine sclerodermatous graft-versus-host disease (Scl GVHD) are not known. We used Affymetrix DNA microarrays representing >14,000 mouse genes to characterize global gene expression in skin during development of Scl GVHD in lethally irradiated BALB/c (H-2d) mice transplanted with B10.D2 (H-2d) bone marrow and spleen cells. These mice develop skin thickening, whereas control mice transplanted with syngeneic BALB/c (H-2d) bone marrow and spleen cells do not develop disease. We found consistent differences between mice with Scl GVHD and controls in cytokine messenger RNAs (mRNAs) for both Th1-like (IFN-gamma) and Th2-like (IL-6, Il-10, and IL-13) inflammatory patterns. mRNAs for chemokines CCL2, CCL5, CCL17, IFN-gamma inducible chemokines (CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC), and for growth factors such as platelet-derived growth factor-c, connective tissue growth factor, fibroblast growth factor 1, epidermal growth factor, nerve growth factor-beta, vascular endothelial growth factor (VEGF)-alpha, and VEGF-beta were elevated, similar to human scleroderma. mRNAs for cell adhesion molecules, such as L-selectin (selectin lymphocyte), P-selectin (selectin platelet), E-selectin (selectin endothelium), and vascular cell adhesion molecule 1, were also upregulated. In separate experiments, we confirmed the increased synthesis of IFN-gamma and IL-2, unchanged IL-10, and absence of tumor necrosis factor-alpha, and IL-4 proteins by flow cytometry of isolated skin T cells. These constellations of immunologic changes provide a "fingerprint" for fibrosing autoimmune disease. They are useful to understand the pathogenesis of Scl GVHD, to identify markers for early diagnosis of disease, and to devise more effective strategies for intervention in early scleroderma and Scl GVHD.

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Interferon (IFN)-gamma plays a critical role in murine uterine spiral artery remodeling for successful pregnancy. The effect of IFN-gamma on human uterine microvasculature, however, remains poorly understood. The aim of this study was to identify the genes regulated by IFN-gamma in human uterine microvascular endothelial cells. The effect of IFN-gamma on the gene expression profile in human uterine microvascular endothelial cells was evaluated by cDNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction for the selected genes of interest. In vivo expression of the protein encoded by some of these genes in human uterine microvascular endothelial cells was evaluated by Western blotting and immunohistochemistry. Treatment with 10 ng/ml IFN-gamma for 4 h induced a significant>> or =2-fold change in 29 genes in pooled human uterine microvascular endothelial cells; a total of 20 genes were up-regulated, whereas nine genes were down-regulated. The genes significantly up-regulated included chemokines (CXCL9, CXCL10, CCL8, IL15RA, and CCL5), enzymes (GBP5, TAP1, CYP27B1, SOD2, MX1, CASP1, and PTGES), and transcription factors (TFAP2C, IRF1, NFE2L3). The genes significantly down-regulated following IFN-gamma treatment included cytokines/cytokine receptors (CSF2, IL1R2, and SPP1), and insulin-like growth factor binding proteins (WISP2 and IGFBP3). The results of the cDNA microarray analysis were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction for the selected 17 genes of interest. The immunoreactivity for the proteins encoded by IL15RA, IFI30, and MX1 was detected in human uterine microvascular endothelial cells in vivo, whereas the immunoreactivity for CCNA1 and NQO1 was not detectable. These results suggest that IFN-gamma regulates the gene expression involved in natural killer cell recruitment, embryo and trophoblast migration, endometrial decidualization, angiogenesis, angiostasis, and anti-viral infection in human uterine microvascular endothelial cells.

Current therapies for preterm labour (PTL) focus on arresting myometrial contractions but are largely ineffective, thus alternative therapeutic targets need to be identified. Leukocytes infiltrate the uterus around the time of labour, and are in particularly abundant in decidua (maternal-fetal interface). Moreover, decidual inflammation precedes labour in rat pregnancies and thus may contribute to initiation of labour. We hypothesized that chemokines mediate decidual leukocyte trafficking during preterm labour (PTL) and term labour (TL), thus representing potential targets for preventing PTL. Women were recruited into 4 groups: TL, term not in labour (TNL), idiopathic PTL and PTL with infection (PTLI). Choriodecidual RNA was subjected to a pathway-specific PCR array for chemokines. Differential expression of 12 candidate chemokines was validated by real time RT-PCR and Bioplex assay, with immunohistochemistry to confirm cellular origin. 25 chemokines were upregulated in choriodecidua from TL compared to TNL. A similar pattern was detected in PTL, however a distinct profile was observed in PTLI consistent with differences in leukocyte infiltration. Upregulation of CCL2, CCL4, CCL5, CXCL8 and CXCL10 mRNA and protein was confirmed in TL, with CCL8 upregulated in PTL. Significant correlations were detected between these chemokines and decidual leukocyte abundance previously assessed by immunohistochemical and image analysis. Chemokines were primarily expressed by decidual stromal cells. In addition, CXCL8 and CCL5 were significantly elevated in maternal plasma during labour, suggesting chemokines contribute to peripheral inflammatory events during labour. Differences in chemokine expression patterns between TL and idiopathic PTL may be attributable to suppression of chemokine expression by betamethasone administered to women in PTL; this was supported by in vitro evidence of chemokine downregulation by clinically relevant concentrations of the steroid. The current study provides compelling evidence that chemokines regulate decidual leukocyte recruitment during labour. The 6 chemokines identified represent potential novel therapeutic targets to block PTL.

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-κB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-κB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-κB pathway shows promise in OSCCs.

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

BACKGROUND

To date cancer immunotherapy has only achieved limited clinical efficacy, thus more efficient immunotherapeutic approaches need to be explored. The CC chemokine CCL5 plays a role in chemoattraction and activation of immune cells implying its potential clinical application as an adjuvant for boosting anti-tumor immunity, although an effect on carcinogenesis and tumor cell invasiveness is also reported to be associated with CCL5.

METHODS

Recent progress in exploiting CCL5 as an adjuvant for cancer prevention and treatment, and updated understanding on how CCL5 is involved in tumor invasiveness and carcinogenesis.

RESULTS

CCL5 represents a natural adjuvant for enhancing anti-tumor immune responses. However, animal experiments and clinical reports suggest that CCL5 plays a role in carcinogenesis and invasiveness of tumor cells. Therefore, a CCL5-based cancer therapeutic approach needs to avoid the CCL5-associated potential detrimental effects.

CONCLUSIONS

CCL5 has a pre-eminent role in chemotaxis and activation of a wide spectrum of immune cells. CCL5 functions as an adjuvant to boost anti-tumor immunity by diverse protocols such as co-immunization of recombinant CCL5 protein with tumor-associated antigen, vaccination with CCL-5-expressing tumor cells, or viral vector delivery of CCL5 cDNA into growing tumor. CCL5 may also promote tumor cell survival, proliferation and invasion by different mechanisms.

Vgamma9/Vdelta2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vgamma9/Vdelta2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vgamma9/Vdelta2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-alpha, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vgamma9/Vdelta2 T cells grown with IL-21 differentiated into putative central memory CD45RO(+) T cells that did not produce TNF-alpha, IFN-gamma, or IL-4, and maintained expression of CD62L, CD81, and CCR7.

Microglia are pivotal in the pathogenesis of AIDS dementia, as they serve as the major target of HIV infection in the CNS. In addition, activation of microglia correlates best with clinical dementia. Although the beta-chemokine RANTES/CCL5 is important in modulating HIV infection as well as cellular activation, no information is available regarding how its expression is regulated in microglia by HIV-1. Here we report that RANTES/CCL5 expression is induced in microglia by HIV-1, but that this requires infection by HIV-1. This conclusion was supported by (1) the delayed kinetics coinciding with viral replication; (2) the lack of effect of X4 viruses; (3) inhibition by the reverse transcriptase inhibitor AZT, and (4) the lack of effect of cytokine antagonists or antibodies. Interestingly, RANTES/CCL5 production was dependent on the viral accessory protein Vpr, in addition to Nef, demonstrating a novel role for Vpr in chemokine induction in primary macrophage-type cells. Furthermore, the specific p38 MAP kinase inhibitor SB203580 augmented chemokine expression in microglia, indicating a negative role played by p38. These data suggest unique features of RANTES/CCL5 regulation by HIV-1 in human microglial cells.

Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c+ DC subsets were identified: CD11b+ and CD11blow DCs. CD11b+CD14+ DCs were the most abundant throughout the tract. A majority of CD11c+CD14+ cells corresponded to CD1c+ myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103+ DCs, located exclusively in the endometrium, whereas DC-SIGN+ DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14+ DC-SIGN+ as well as CD14+ DC-SIGN- cells captured virus, with ∼30% of these cells representing CD1c+ myeloid DCs. CD103+ DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.

BACKGROUND

Allogeneic mesenchymal stem cells (MSCs) were immunoprivileged early after cardiac implantation and improved heart function in preclinical and clinical studies. However, long-term preclinical studies demonstrated that allogeneic MSCs lost their immunoprivilege and were rejected in the injured myocardium, resulting in recurrent ventricular dysfunction. This study identifies some of the mechanisms responsible for the immune switch in MSCs and suggests a new treatment to maintain immunoprivilege and preserve heart function.

RESULTS

Rat MSC immunoprivilege was mediated by prostaglandin E2 (PGE2)-induced secretion of 2 critical chemokines, CCL12 and CCL5. These chemokines stimulated the chemoattraction of T cells toward MSCs, suppressed cytotoxic T-cell proliferation, and induced the production of T regulatory cells. MSCs treated with 5-azacytidine for 24 hours differentiated into myogenic cells after 2 weeks, which was associated with decreased PGE2 and chemokine production and the loss of immunoprivilege. Treatment of differentiated MSCs with PGE2 restored chemokine levels and preserved MSC immunoprivilege. In a rat myocardial infarction model, allogeneic MSCs (3 × 10(6) cells/rat) were injected into the infarct region with or without a biodegradable hydrogel that slowly released PGE2. Five weeks later, the transplanted MSCs expressed myogenic lineage markers and were rejected in the control group, but in the PGE2-treated group, the transplanted cells survived and heart function improved.

CONCLUSIONS

Allogeneic MSCs maintained immunoprivilege by PGE2-induced secretion of chemokines CCL12 and CCL5. Differentiation of MSCs decreased PGE2 levels, and immunoprivilege was lost. Maintaining PGE2 levels preserved immunoprivilege after differentiation, prevented rejection of implanted MSCs, and restored cardiac function.

Pulmonary CMV infection (CMVI) and disease (CMVD) is associated with reduced long-term survival post-lung transplantation, however, the specific biologic mechanisms remain unclear. We have demonstrated a role of CC chemokines during lung allograft dysfunction. Based on these findings, we hypothesized that pulmonary CMV upregulates the expression of multiple CC chemokines that leads to allograft dysfunction and decreased long-term survival. We performed a nested case control study in lung transplant recipients to investigate alterations in CC chemokine biology during pulmonary CMV. Levels of CC chemokines were measured in bronchoalveolar lavage fluid (BALF) from recipients with CMVI (n = 33), CMVD (n = 6), and in healthy lung transplant controls (n = 33). We found a trend toward increased levels of MIP-1alpha/CCL3 during pulmonary CMVI. Levels of MCP-1/CCL2 and RANTES/CCL5 were significantly elevated during pulmonary CMV. Interestingly, elevated levels of CCL3 in BALF were protective with regards to survival. Importantly, elevated levels of CCL2 in BALF predicted the development of BOS, while elevated levels of CCL5 in BALF predicted an increase in mortality post-lung transplant. Altered levels of specific CC chemokines during pulmonary CMV are associated with future clinical outcomes. These results suggest a possible utility of BALF CC chemokines as biomarkers for guiding risk assessment during pulmonary CMV post-lung transplantation.

OBJECTIVE

A prior study showed that mice deficient in IFN-gamma (GKO) are more susceptible to experimental autoimmune uveitis (EAU) than are wild-type (WT) mice. Histopathology of uveitic eyes revealed that the ocular infiltrate in GKO mice was dominated by neutrophils and eosinophils rather than by mononuclear cells, as in WT mice. The present study was conducted to explore the differential expression of chemokine(s) likely to account for the distinct inflammatory cell composition in uveitic eyes of WT and GKO mice.

METHODS

Mice were immunized to induce EAU. Lymph nodes draining the site of the immunization and the eyes were collected at different time points for chemokine analysis. Microarray, real-time PCR and protein analyses were performed to examine the expression of chemokines in WT and GKO mice.

RESULTS

Many chemokines were differentially upregulated in GKO versus WT mice. Expression of the Th1-associated chemokines CXCL10, CXCL9, CCL5, and CXCL11 was elevated in WT mice, whereas the Th2-associated chemokines CCL11, CCL17, and CCL1 and the Th17-associated chemokines CCL22 and CXCL2 were elevated in the GKO mice. Depletion of granulocytes abrogated EAU in both WT and GKO mice.

CONCLUSIONS

These results suggest that Th1-associated chemokines play a critical role in the attraction of mononuclear cells to the eyes in the presence of IFN-gamma, while in the absence of this cytokine, Th2- and Th17-related chemokines may be the key elements for influx of granulocytes.

The expression of chemokines within the heart during experimental infection of susceptible mice with the Colombiana strain of Trypanosoma cruzi was characterized in an attempt to determine a functional role for these molecules in both host defense and disease. Analysis of chemokine transcripts revealed that CXC chemokine ligand 9 (CXCL9) and CXCL10, as well as CC chemokine ligand 2 (CCL2) and CCL5, were prominently expressed during acute disease, whereas transcripts for CXCL9, CXCL10, and CCL5 remained elevated during chronic infection. Inflammatory macrophages present within the heart were the primary cellular source of these chemokines following T. cruzi infection. Peak chemokine expression levels coincided with increased gamma interferon expression and inflammation within the heart, suggesting a role for these molecules in both host defense and disease. Indeed, simultaneous treatment of T. cruzi-infected mice with neutralizing antibodies specific for CXCL9 and CXCL10 resulted in an increased parasite burden that was sustained out to 50 days p.i. Antibody targeting either CXCL10 or CCL5 did not change either T. cruzi burden within the heart nor attenuate the severity of cardiac inflammation at any time point examined, while targeting CXCL9 in combination with CXCL10 resulted in increased parasite burden. Collectively, these studies imply that CXCL9 and CXCL10 signaling enhances immune responses following parasite infection. However, antibody targeting of CXCL9 and CXCL10, or CXCL10 alone, or CCL5 alone does not directly modulate the inflammatory response within the heart, suggesting that other proinflammatory factors are able to regulate inflammation in this tissue in response to T. cruzi infection.

Large B cell lymphomas can comprise numerous CD14+ cells in the tumor stroma, which raises the question of whether monocytes can support B cell survival and proliferation. We show that the coculture of monocytes with B cells from peripheral blood or from diffuse large B cell lymphoma enabled prolonged B cell survival. Under these conditions, diffuse large lymphoma B cells proliferated, and addition of B cell-activating factor of the TNF family (BAFF) and IL-2 enhanced cell division. Monocytes and dendritic cells (DC) had similar antiapoptotic activity on healthy B cells but displayed differences with respect to B cell proliferation. Monocytes and cord blood-derived CD14+ cells promoted B cell proliferation in the presence of an anti-CD40 stimulus, whereas DC supported B cell proliferation when activated through the BCR. DC and CD14+ cells were able to induce plasmocyte differentiation. When B cells were activated via the BCR or CD40, they released the leukocyte attractant CCL5, and this chemokine is one of the main chemokines expressed in diffuse large B cell lymphoma. The data support the notion that large B cell lymphoma recruit monocytes via CCL5 to support B cell survival and proliferation.

Viral DNA induces potent antiviral immunity by activating dendritic cells; however, the mechanism governing viral DNA-mediated triggering or aggravation of glomerulonephritis is unknown. Glomerular endothelial cells (GEnCs) do not express toll-like receptor (TLR)9, the only DNA-specific TLR. We therefore hypothesized that DNA could activate GEnCs via the recently discovered TLR-independent viral DNA recognition pathway. Indeed, double-stranded non-CpG (B-) DNA activated GEnCs to produce interleukin-6, CCL5/RANTES, CCL2/MCP-1, CXCL10/IP10, interferon (IFN)-alpha, and IFN-beta when cationic lipids facilitated intracellular DNA uptake. This cytokine production was inhibited by chlorpromazine, suggesting that clathrin-dependent endocytosis is required for B-DNA entry. However, chloroquine and MyD88 inhibition did not affect GEnC activation, suggesting TLR-independent DNA recognition. In addition, IFN-beta activated cytokine and chemokine mRNA expression, although only CXCL10/IP10 was induced at the protein level, and type I IFN did not activate GEnC in an autocrine-paracrine auto-activation loop. B-DNA complexes induced intercellular adhesion molecule-1 expression at the GEnC surface and increased intercellular adhesion molecule-1-dependent leukocyte adhesion and microvascular extravasation in vivo. Furthermore, B-DNA complexes increased albumin permeability of GEnC monolayers in culture or microvascular dextran leakage in vivo. In addition, B-DNA complexes impaired GEnC proliferation. Thus, complexed B-DNA activates GEnC to produce cytokines, chemokines, and type I IFNs, increases leukocyte adhesion and microvascular permeability, and reduces GEnC proliferation via a MyD88-independent cytosolic DNA recognition pathway. This innate antiviral response program suggests a novel pathomechanism regulating DNA virus-mediated induction or aggravation of glomerulonephritis.