BACKGROUND

COPD is associated with increased numbers of CD4(+) and CD8(+) lymphocytes and macrophages in the small airways and lung parenchyma. The chemokines regulating T-cell recruitment into the lung are unknown but may involve CXCR3 and CCR5 chemoattractants. The aims of this study were to determine the concentrations of CXCR3 chemokines CXCL9, CXCL10, CXCL11, and the CCR5 chemokine CCL5 in induced sputum from patients with COPD, smokers, and nonsmokers, and to examine the relationship between chemokine expression, inflammatory cells, and airway obstruction.

METHODS

Differential cell counts were performed and concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were measured in induced sputum from nonsmokers (n = 18), smokers (n = 20), and COPD patients (n = 35) using an enzyme-linked immunosorbent assay.

RESULTS

Concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were significantly increased in the sputum of patients with COPD when compared with nonsmokers but not smokers without obstruction: CXCL9 (median, 14.3 pg/mL; interquartile range [IQR], 6.5 to 99.3; vs median, 1.4 pg/mL; IQR, 0 to 10.4 [p < 0.001]; vs 8.5 pg/mL; IQR, 0 to 16.0, respectively); CXCL10 (16.9 pg/mL; IQR, 6.2 to 148.8; vs 3.7 pg/mL; IQR, 0 to 18.8 [p < 0.05]; vs 11.3 pg/mL; IQR, 3.7 to 46.7); CXCL11 (58.1 pg/mL; IQR, 34.5 to 85.3; vs 33.5 pg/mL; IQR, 23.2 to 49.7 [p < 0.05]; vs 49.8 pg/mL; IQR, 32.6 to 105.6); and CCL5 (59.9 pg/mL; IQR, 57.1 to 67.8; vs 33.5 pg/mL; IQR, 31.6 to 36.9 [p < 0.001]). CCL5 in sputum from smokers was also significantly increased compared with that from nonsmokers (median, 63.0 pg/mL; IQR, 60.8 to70.2; p < 0.001). There was a negative correlation between FEV(1) percentage of predicted, FEV(1)/FVC ratio, and percentage of macrophages, and all the chemokines analyzed. Neutrophil numbers correlated positively with the concentrations of chemokines.

CONCLUSIONS

CXCR3 chemokines and CCL5 are increased in sputum from COPD patients compared with nonsmokers, and may be important in COPD pathogenesis.

In recent years, West Nile virus (WNV) has emerged as a major cause of encephalitis in the United States. However, the neuropathogenesis of this flavivirus is poorly understood. In the present study, the authors used primary human brain cell cultures to investigate two neuropathogenic features: viral replication and induction of cytokines. Although neurons and astrocytes were found to support productive WNV infection, viral growth was poorly permissive in microglial cells. Compared to neuronal cultures that sustained viral growth for at least 2 weeks, replication peaked in astrocytes by 72 h post infection. In response to viral infection, astrocytes produced chemokines (CXCL10 and CCL5), but none of the cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, interferon alpha or gamma) tested could be detected. Although microglial cells failed to support viral replication, WNV induced production of the proinflammatory cytokines IL-6 and TNF-alpha. Microglial cells also released robust amounts of the chemokines CXCL10 and CCL2, as well as lower levels of CCL5, in response to WNV infection. WNV-induced chemokine and cytokine production by microglia was coupled with activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Inhibition of p38 MAPK decreased chemokine production in response to WNV. Taken together, these findings suggest that microglial cell responses may influence the neuropathogenesis of WNV infection.

CXCL9 and CXCL10 mediate the recruitment of T lymphocytes and NK cells known to be important in viral surveillance. The relevance of CXCL10 in comparison to CXCL9 in response to genital HSV-2 infection was determined using mice deficient in CXCL9 (CXCL9-/-) and deficient in CXCL10 (CXCL10-/-) along with wild-type (WT) C57BL/6 mice. An increased sensitivity to infection was found in CXCL10-/- mice in comparison to CXCL9-/- or WT mice as determined by detection of HSV-2 in the CNS at day 3 postinfection. However, by day 7 postinfection both CXCL9-/- and CXCL10-/- mice possessed significantly higher viral titers in the CNS in comparison to WT mice consistent with mortality (18-35%) of these mice within the first 7 days after infection. Even though CXCL9-/- and CXCL10-/- mice expressed elevated levels of CCL2, CCL3, CCL5, and CXCL1 in the spinal cord in comparison to WT mice, there was a reduction in NK cell and virus-specific CD8+ T cell mobilization to this tissue, suggesting CXCL9 and CXCL10 are critical for recruitment of these effector cells to the spinal cord following genital HSV-2 infection. Moreover, leukocytes from the spinal cord but not from draining lymph nodes or spleens of infected CXCL9-/- or CXCL10-/- mice displayed reduced CTL activity in comparison to effector cells from WT mice. Thus, the absence of CXCL9 or CXCL10 expression significantly alters the ability of the host to control genital HSV-2 infection through the mobilization of effector cells to sites of infection.

Leukocyte extravasation into tissues is a multi-step process culminating in the migration of cells through the basement membrane. This requires the production of matrix-degrading enzymes, in particular matrix metalloproteinases (MMP). We investigated the role of chemokines in regulating MMP production in the monocytic cell line THP-1 and in peripheral blood monocytes (PBM). The CC chemokines CCL2 (MCP-1), CCL3 (MIP-1alpha), and CCL5 (RANTES) stimulated the release of monocyte MMP-9 protein in a bell-shaped dose-dependent manner. The increase in MMP-9 protein detected at 24 h was due to de novo synthesis, confirmed by Northern blotting, with MMP-9 mRNA detectable at 6-8 h. Autocrine TNF-alpha was necessary for chemokine stimulation of MMP-9. Chemokines increased TNF-alpha mRNA levels and protein release in monocytes and THP-1 cells, and neutralizing anti-TNF-alpha antibodies inhibited CCL2-induced MMP-9 release. Furthermore, the broad spectrum MMP inhibitor BB 2516, which inhibits TNF-alpha release, abrogated CCL2- and CCL5-induced MMP-9 release in both THP-1 cells and freshly isolated monocytes. Monocyte production of MMP is of major importance in the pathology of cancer, asthma, and rheumatoid arthritis. An understanding of the mechanisms by which these MMP are produced may lead to novel therapies to modulate extravasation of leukocytes in disease.

BACKGROUND

Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma. The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection. We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression.

METHODS

Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface. Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed. Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50).

RESULTS

The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively). More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively). Flow cytometric analysis confirmed the PPM. Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR.

CONCLUSIONS

Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment. Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response.

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. The WNV-induced innate immune response, including production of antiviral cytokines, is critical for controlling virus infection. The adaptor protein ASC mediates a critical step in innate immune signaling by bridging the interaction between the pathogen recognition receptors and caspase 1 in inflammasome complexes, but its role in WNV immunopathogenesis is not defined. Here, we demonstrate that ASC is essential for interleukin-1β (IL-1β) production and development of effective host immunity against WNV. ASC-deficient mice exhibited increased susceptibility to WNV infection, and reduced survival was associated with enhanced virus replication in the peripheral tissues and central nervous system (CNS). Infection of cultured bone marrow-derived dendritic cells showed that ASC was essential for the activation of caspase 1, a key component of inflammasome assembly. ASC(-/-) mice exhibited attenuated levels of proinflammatory cytokines in the serum. Intriguingly, infected ASC(-/-) mice also displayed reduced levels of alpha interferon (IFN-α) and IgM in the serum, indicating the overall protective role of ASC in restricting WNV infection. However, brains from ASC(-/-) mice displayed unrestrained inflammation, including elevated levels of proinflammatory cytokines and chemokines, such as IFN-γ, CCL2, and CCL5, which correlated with more pronounced activation of the astrocytes, enhanced infiltration of peripheral immune cells in the CNS, and increased neuronal cell death. Collectively, our data provide new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune responses to effectively control WNV infection.

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

OBJECTIVE

Novel therapeutic targets of estrogen receptor (ER)-positive breast cancers are urgently needed because current antiestrogen therapy causes severe adverse effects, nearly 50% of patients are intrinsically resistant, and the majority of recurrences have maintained ER expression. We investigated the role of estrogen-dependent chemokine expression and subsequent cancer growth in human tissues and experimental breast cancer models.

METHODS

For in vivo sampling of human chemokines, microdialysis was used in breast cancers of women or normal human breast tissue before and after tamoxifen therapy. Estrogen exposure and targeted therapies were assessed in immune competent PyMT murine breast cancer, orthotopic human breast cancers in nude mice, cell culture of cancer cells, and freshly isolated human macrophages. Cancer cell dissemination was investigated using zebrafish.

RESULTS

ER(+) cancers in women produced high levels of extracellular CCL2 and CCL5 in vivo, which was associated with infiltration of tumor-associated macrophages. In experimental breast cancer, estradiol enhanced macrophage influx and angiogenesis through increased release of CCL2, CCL5, and vascular endothelial growth factor. These effects were inhibited by anti-CCL2 or anti-CCL5 therapy, which resulted in potent inhibition of cancer growth. In addition, estradiol induced a protumorigenic activation of the macrophages. In a zebrafish model, macrophages increased cancer cell dissemination via CCL2 and CCL5 in the presence of estradiol, which was inhibited with anti-CCL2 and anti-CCL5 treatment.

CONCLUSIONS

Our findings shed new light on the mechanisms underlying the progression of ER(+) breast cancer and indicate the potential of novel therapies targeting CCL2 and CCL5 pathways.

The infiltration of various immune cell populations, including monocytes/macrophages, natural killer (NK), NKT cells and T cells, is a central pathogenic feature following acute- and chronic liver injury. Chemotactic cytokines, chemokines, are small-protein mediators that direct the migration of immune cells. Several hepatic cell populations, including hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells, can secrete chemokines upon activation. Samples from liver-disease patients and animal models of experimental injury highlight multiple activated chemokine pathways during initiation, maintenance or resolution of liver pathology. Monocyte chemoattractant protein-1 (Chemokine [C-C motif] ligand [CCL]2) can attract monocytes via CCR2. Infiltrating monocytes probably have functions in both disease progression and resolution of damage. RANTES (CCL5) may promote infiltration of NK (via CCR1) and T cells (via CCR5). Dissecting the exact functional contribution of immune cell subsets, chemokines and chemokine-receptor pathways in liver injury will hopefully identify novel targets for the treatment of acute liver failure, liver fibrosis or cirrhosis.

The developmental and oncogenic roles of MYC proteins are well established, but the transcriptional targets mediating their functions remain elusive. Using small interfering RNA-mediated knockdown in breast and cervix carcinoma cell lines, which overexpress c-MYC, we show that c-MYC independently controls metabolism and cell proliferation, and can, depending on the cells, promote or inhibit migration. We identified new c-MYC target genes in these cell lines, and show that selective regulation of some targets correlates with the phenotypic responses of these different cell lines to c-MYC depletion. Notably, we show that a positive regulation of the WNT signalling pathway contributes to c-MYC pro-mitogenic effects in breast and cervix carcinoma cells. We also show that repression of CCL5/RANTES accounts for c-MYC anti-migratory effects in specific breast cancer cells. Our combined genomic and phenotypic analysis indicates that c-MYC functions are cellular-context-dependent and that selectively regulated genes are responsible for its differential properties.

BACKGROUND

Chemokines play an important role in atherogenesis and in ischemic injury and repair; however, prospective data on individual chemokines in unstable angina pectoris (UAP) are scarce. Therefore, we assessed chemokine patterns in a prospective cohort of patients with UAP.

RESULTS

Plasma samples of 54 patients with Braunwald class IIIB UAP were examined at baseline for 11 chemokines and 5 inflammatory mediators via multiplex analysis. Levels of CC chemokine ligand (CCL)-5 (also known as RANTES [regulated on activation, normally T-cell expressed, and secreted]; 32.7 versus 23.1 ng/mL, P=0.018) and CCL18 (also known as PARC [pulmonary and activation-regulated chemokine]; 104.4 versus 53.7 ng/mL, P=0.011) were significantly elevated in patients with refractory ischemic symptoms versus stabilized patients. Temporal monitoring by ELISA of CCL5, CCL18, and soluble CD40 ligand (sCD40) levels revealed a drop in CCL5 and sCD40L levels in all UAP patients from day 2 onward (CCL5 12.1 ng/mL, P<0.001; sCD40L 1.35 ng/mL, P<0.05), whereas elevated CCL18 levels were sustained for at least 2 days, then were decreased at 180 days after inclusion (34.5 ng/mL, P<0.001). Peripheral blood mononuclear cells showed increased protein expression of chemokine receptors CCR3 and CCR5 in CD3+ and CD14+ cells at baseline compared with 180 days after inclusion, whereas mRNA levels were downregulated, which was attributable in part to a postischemic release of human neutrophil peptide-3-positive neutrophils and in part to negative feedback. Finally, elevated CCL5 and CCL18 levels predicted future cardiovascular adverse events, whereas C-reactive protein and sCD40L levels did not.

CONCLUSIONS

We are the first to report that CCL18 and CCL5 are transiently raised during episodes of UAP, and peak levels of both chemokines are indicative of refractory symptoms. Because levels of both chemokines, as well as of cognate receptor expression by circulating peripheral blood mononuclear cells, are increased during cardiac ischemia, this may point to an involvement of CCL5/CCL18 in the pathophysiology of UAP and/or post-UAP responses.

Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-alpha is a major inducer of proinflammatory mediators in keratinocytes. We have recently observed that epidermal growth factor receptor (EGFR) signaling affects TNF-alpha-driven chemokine expression in epidermal keratinocytes, and its functional impairment increases the levels of crucial chemoattractants such as CCL2/MCP-1, CCL5/RANTES, and CXCL10/IFN-gamma-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/2 activity is implicated in this mechanism. Abrogation of ERK1/2 activity with specific inhibitors increased chemokine expression in keratinocytes by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/2 inhibitor. In contrast, impairment of p38alpha beta MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/2 activity in keratinocytes takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.

CCL5 (previously called RANTES) is in the CC-chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found CCL5 increased the migration and cell surface expression of alphavbeta3 integrin in human lung cancer cells (A549 cells). CCL5 stimulation increased phosphorylation of the p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) and serine 473 of Akt. Also, we found that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed CCL5-induced migration activities and integrin expression of A549 cells. Transfection of cells with p85 or Akt mutant also reduced CCL5-mediated cancer migration. In addition, treatment of A549 cells with CCL5 induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the CCL5-mediated increases in p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Taken together, our results suggest that CCL5 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activation of alphavbeta3 integrin and contributing to the migration of human lung cancer cells.

Leukocyte access into the central nervous system (CNS) parenchyma is tightly regulated by the blood-brain barrier (BBB). Leukocyte migration through the endothelial cell wall into the perivascular space is well characterized; however, mechanisms regulating their penetration through the glia limitans into the parenchyma are less well studied, and the role of monocytes relative to neutrophils is poorly defined. Acute viral encephalitis was thus induced in CCL2-deficient (CCL2(-/-)) mice to specifically abrogate monocyte recruitment. Impaired monocyte recruitment prolonged T cell retention in the perivascular space, although no difference in overall CNS accumulation of CD4 or CD8 T cells was detected by flow cytometry. Delayed penetration to the CNS parenchyma was not associated with reduced or altered expression of either matrix metalloproteinases (MMP) or the T cell chemoattractants CXCL10 and CCL5. Nevertheless, decreased parenchymal leukocyte infiltration delayed T cell-mediated control of virus replication as well as clinical disease. These data are the first to demonstrate that the rapid monocyte recruitment into the CNS during viral encephalitis is dispensable for T cell migration across the blood vessel endothelium. However, monocytes facilitate penetration through the glia limitans. Thus, the rapid monocyte response to viral encephalitis constitutes an indirect antiviral pathway by aiding access of effector T cells to the site of viral infection.

The peritoneal macrophage (Mphi) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11b(high) cells, with the remaining 5% being 12/15-LOX(-). 12/15-LOX(+) cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX(-) cells are a dendritic cell population. Resident peritoneal Mphi numbers were significantly increased in 12/15-LOX(-/-) mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mphi from 12/15-LOX(-/-) mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX(-/-) mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX(-/-) adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX(+) cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX(-/-) mice showed elevated TGF-beta1, along with increased immigration of monocytes/Mphi, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-alpha. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MPhi population and regulates both the recruitment of monocytes/Mphi and cytokine response to bacterial products in vivo.

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)-derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF-α transgenic (hTNFtg) mice. A microarray analysis of WNT5A-treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS-purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF-α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A-treated osteoblasts identified cytokines and chemokines as targets. The induction of IL-1β, IL-6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF-κB, mitogen-activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS-induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A-treated BMSC was increased by 25% and 20%, respectively. This study underlines the critical role of BMSC-derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.

Background: Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks.

Methods: A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You'an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4-7 days over 1 month per patient using a bioplex multiplex immunoassay.

Results: We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases.

Conclusion: Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.

Keywords: Adaptive immunity; COVID-19.

A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs), including professional antigen-presenting cells such as dendritic cells (DCs), play a central role in T-cell biology. Here, we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell-derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes, but cell death was significantly increased after monocyte depletion. Furthermore, monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin, and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES), where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively, the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders.

BACKGROUND

CC chemokines mediate leukocyte infiltration into inflamed tissue. We have recently shown that blockade of the CC chemokine receptor CCR1 reduces interstitial inflammation and fibrosis in murine obstructive nephropathy. However, it is not known whether CCR 1 blockade is protective in progressive renal injury associated with severe proteinuria. We therefore studied the effect of the small-molecule CCR1 antagonist BX471 in a murine model of adriamycin-induced focal segmental glomerulosclerosis (FSGS) with nephrotic syndrome and progressive interstitial inflammation and fibrosis.

METHODS

Adriamycin nephropathy with persistent proteinuria was induced in male BALB/c mice by two intravenous injections of adriamycin (13 mg/kg) at day 0 and 14. BX471 treatment was started at day 14 when proteinuria and interstitial inflammation had developed. At 6 weeks, renal histology was studied by morphometry and immunohistochemistry.

RESULTS

At week 6, adriamycin-treated mice showed FSGS, associated with tubulointerstitial injury consisting of tubular dilation and atrophy, interstitial leukocyte infiltration, and fibrosis. The mRNA expression of CCR1 and CC chemokines, including the CCR1 ligands CCL3 (MIP-1alpha) and CCL5 (RANTES), was up-regulated in diseased kidneys, with a prominent interstitial expression of CCL5. Compared to vehicle-treated controls BX471 significantly reduced the amount of macrophages and T lymphocytes in interstitial lesions by 51% and 22%, respectively. Markers of renal fibrosis such as interstitial fibroblasts (48%) and interstitial volume (23%) were significantly reduced by BX471 treatment. In contrast, the extent of proteinuria and glomerular sclerosis was not affected by BX471 treatment.

CONCLUSIONS

Blockade of CCR1 substantially reduced interstitial leukocyte accumulation and the subsequent renal fibrosis in a murine model of nephrotic syndrome and FSGS. These findings support a role for CCR1 in interstitial leukocyte recruitment and suggest that CCR1 blockade might be a new therapeutic strategy in progressive nephropathies such as FSGS.

Atherosclerosis is a chronic inflammatory disease of the arterial wall and an increasing body of evidence suggests that the immune system actively participates in the initiation, progression and persistence of atherosclerosis. Different types of leukocytes such as T and B lymphocytes, natural killer cells (NK) and NKT cells, macrophages, dendritic cells and mast cells have been found within atherosclerosis-prone aortas. The mechanisms of monocyte recruitment have been partially characterized and involve P-selectin, E-selectin, VCAM-1, ICAM-1 and JAM-A. CXCL1, CCL5, CXCL4, CXCL7 and MIF are also implicated in monocyte trafficking into aortas. Recently it has been reported that Ly6C(high) and Ly6C(low) monocyte subsets differently use CCL2, CX3CL1 and CCL5 for their homing into atherosclerotic aortas. T and B lymphocytes constitutively migrate into the normal and atherosclerotic aortic wall in an L-selectin-dependent manner. Recent studies suggest an important role of CCL5, CXCL10, CXCL16, CXCR6 and MIF in T cell influx into the atherosclerotic wall. However, there is little information available on the mechanisms of recruitment of other types of the immune cells such as NK, NKT and mast cells. In this review we shall summarize what is known about leukocyte recruitment into the aortic wall during atherosclerosis with a focus on mouse model systems.

In this study, we aimed to determine whether morphine alone or in combination with HIV-1 Tat or gp120 affects the expression of Toll-like receptors (TLRs) by astrocytes and to assess whether TLRs expressed by astrocytes function in the release of inflammatory mediators in vitro. TLR profiling by immunofluorescence microscopy, flow cytometry, in-cell westerns, and RT-PCR showed that subpopulations of astrocytes possessed TLR 2, TLR3, TLR4, and TLR9 antigenicity. Exposure to HIV-1 Tat, gp120, and/or morphine significantly altered the proportion of TLR-immunopositive and/or TLR expression by astroglia in a TLR-specific manner. Subsets of astroglia displayed significant increases in TLR2 with reciprocal decreases in TLR9 expression in response to Tat or gp120 ± morphine treatment. TLR9 expression was also significantly decreased by morphine alone. Exposing astrocytes to the TLR agonists LTA (TLR2), poly I:C (TLR3), LPS (TLR4) and unmethylated CpG ODN (TLR9) resulted in increased secretion of MCP-1/CCL2 and elevations in reactive oxygen species. TLR3 and TLR4 stimulation increased the secretion of TNF-α, IL-6, and RANTES/CCL5, while activation of TLR2 caused a significant increase in nitric oxide levels. The results suggest that HIV-1 proteins and/or opioid abuse disrupt the innate immune response of the central nervous system (CNS) which may lead to increased pathogenicity.

Within the brain, quinolinic acid (QUIN) is an important neurotoxin, especially in AIDS dementia complex (ADC). Its production by monocytic lineage cells is increased in the context of inflammation. However, it is not known whether QUIN promotes inflammation. Astrocytes are important in immunoregulation within the brain and so we chose to examine the effects of QUIN on the astrocyte. Using purified primary human fetal astrocyte cultures, we determined chemokine production using ELISA assays and RT-PCR and chemokine receptor expression using immunocytochemistry and RT-PCR with QUIN in comparison to TNFalpha, IL-1beta, and IFNgamma. We found that QUIN induces astrocytes to produce large quantities of MCP-1 (CCL2) and lesser amounts of RANTES (CCL5) and IL-8 (CXCL8). QUIN also increases SDF-1alpha (CXCL12), HuMIG (CXCL9), and fractalkine (CX(3)CL1) mRNA expression. Moreover, QUIN leads to upregulation of the chemokine receptor expression of CXCR4, CCR5, and CCR3 in human fetal astrocytes. Most of these effects were comparable to those induced by TNFalpha, IL-1beta, and IFNgamma. The present work represents the first evidence that QUIN induces chemokine and chemokine receptor expression in astrocytes and is at least as potent as classical mediators such as inflammatory cytokines. These results suggest that QUIN may be critical in the amplification of brain inflammation, particularly in ADC.

OBJECTIVE

Butyrate, produced by colonic fermentation of dietary fibers is often hypothesized to beneficially affect colonic health. This study aims to assess the effects of butyrate on inflammation and oxidative stress in subjects with chronically mildly elevated parameters of inflammation and oxidative stress.

METHODS

Thirty-five patients with ulcerative colitis in clinical remission daily administered 60 ml rectal enemas containing 100mM sodium butyrate (n=17) or saline (n=18) during 20 days (NCT00696098). Before and after the intervention feces, blood and colonic mucosal biopsies were obtained. Parameters of antioxidant defense and oxidative damage, myeloperoxidase, several cytokines, fecal calprotectin and CRP were determined.

RESULTS

Butyrate enemas induced minor effects on colonic inflammation and oxidative stress. Only a significant increase of the colonic IL-10/IL-12 ratio was found within butyrate-treated patients (p=0.02), and colonic concentrations of CCL5 were increased after butyrate compared to placebo treatment (p=0.03). Although in general butyrate did not affect colonic glutathione levels, the effects of butyrate enemas on total colonic glutathione appeared to be dependent on the level of inflammation.

CONCLUSIONS

Although UC patients in remission were characterized by low-grade oxidative stress and inflammation, rectal butyrate enemas showed only minor effects on inflammatory and oxidative stress parameters.