Cxcl10 deficiency attenuates renal interstitial fibrosis through regulating epithelial-to-mesenchymal transition.

IFN-γ-inducible protein 10 (IP-10, CXCL10) has been widely demonstrated to be involved in multiple kidney pathological processes. However, the role of CXCL10 in renal fibrosis remains unclear. In this study, Cxcl10-deficient (Cxcl10-/-) mice were used to generate the unilateral ureteral obstruction (UUO) model. The level of renal fibrosis and inflammatory cell infiltration was examined in vivo and the effects of CXCL10 on EMT process of HK-2 cells was investigated in vitro. We observed that the injury degree of renal tissue and the collagen deposition levels were lighter and the expression of α-SMA, collagen I and fibronectin was significantly reduced in Cxcl10-/- mice, while the expression of E-cadherin was increased. However, interstitial F4/80-positive macrophages and CD4-positive T lymphocytes were unaffected by knockout of Cxcl10. Furthermore, IFN-γ or CXCL10 stimulation could obviously promote the expression of α-SMA, collagen I, fibronectin and reduce the expression of E-cadherin in HK-2 cells, which could be inhibited by transfection of Cxcl10-siRNA. Our findings suggested Cxcl10 knockout could reduce renal dysfunction and inhibit renal fibrosis through regulating EMT process of renal tubular epithelial cells in murine UUO model. These results may provide a novel insight into the mechanism and a potential therapy target of renal fibrosis.

Reversing CXCL10 Deficiency Ameliorates Kidney Disease in Diabetic Mice.

The excessive accumulation of extracellular matrix material in the kidney is a histopathologic hallmark of diabetic kidney disease that correlates closely with declining function. Although considerable research has focused on the role of profibrotic factors, comparatively little attention has been paid to the possibility that a diminution in endogenous antifibrotic factors may also contribute. Among the latter, the ELR- CXC chemokines, CXCL9, CXCL10, and CXCL11, have been shown to provide a stop signal to prevent excessive fibrosis. Although the plasma concentrations of CXCL9 and CXCL11 were similar, those of CXCL10 were markedly lower in diabetic db/db mice compared with control db/m mice. In cell culture, CXCL10 inhibited kidney fibroblast collagen production in response to high glucose and the prosclerotic growth factor, transforming growth factor-ß. In vivo, recombinant murine CXCL10 reduced mesangial and peritubular matrix expansion, albuminuria, and glomerular hypertrophy in db/db mice. In bone marrow, a major source of circulating chemokines, the concentration of CXCL10 was lower in cells derived from diabetic mice than from their nondiabetic counterparts. Silencing of CXCR3, the cognate receptor for CXCL10, abrogated the antifibrotic effects of bone marrow-derived secretions. In conclusion, experimental diabetes is a state of CXCL10 deficiency and that restoration of CXCL10 abundance prevented fibrosis and the development of diabetic kidney disease in mice.

CXCL10/CXCR3-Dependent Mobilization of Herpes Simplex Virus-Specific CD8+ TEM and CD8+ TRM Cells within Infected Tissues Allows Efficient Protection against Recurrent Herpesvirus Infection and Disease.

Herpes simplex virus 1 (HSV-1) establishes latency within the sensory neurons of the trigeminal ganglia (TG). HSV-specific memory CD8+ T cells play a critical role in preventing HSV-1 reactivation from TG and subsequent virus shedding in tears that trigger recurrent corneal herpetic disease. The CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) chemokine pathway promotes T cell immunity to many viral pathogens, but its importance in CD8+ T cell immunity to recurrent herpes has been poorly elucidated. In this study, we determined how the CXCL10/CXCR3 pathway affects TG- and cornea-resident CD8+ T cell responses to recurrent ocular herpesvirus infection and disease using a well-established murine model in which HSV-1 reactivation was induced from latently infected TG by UV-B light. Following UV-B-induced HSV-1 reactivation, a significant increase in both the number and function of HSV-specific CXCR3+ CD8+ T cells was detected in TG and corneas of protected C57BL/6 (B6) mice, but not in TG and corneas of nonprotected CXCL10-/- or CXCR3-/- deficient mice. This increase was associated with a significant reduction in both virus shedding and recurrent corneal herpetic disease. Furthermore, delivery of exogenous CXCL10 chemokine in TG of CXCL10-/- mice, using the neurotropic adeno-associated virus type 8 (AAV8) vector, boosted the number and function of effector memory CD8+ T cells (TEM) and tissue-resident memory CD8+ T cells (TRM), but not of central memory CD8+ T cells (TCM), locally within TG, and improved protection against recurrent herpesvirus infection and disease in CXCL10-/- deficient mice. These findings demonstrate that the CXCL10/CXCR3 chemokine pathway is critical in shaping CD8+ T cell immunity, locally within latently infected tissues, which protects against recurrent herpesvirus infection and disease.IMPORTANCE We determined how the CXCL10/CXCR3 pathway affects CD8+ T cell responses to recurrent ocular herpesvirus infection and disease. Using a well-established murine model, in which HSV-1 reactivation in latently infected trigeminal ganglia was induced by UV-B light, we demonstrated that lack of either CXCL10 chemokine or its CXCR3 receptor compromised the mobilization of functional CD8+ TEM and CD8+ TRM cells within latently infected trigeminal ganglia following virus reactivation. This lack of T cell mobilization was associated with an increase in recurrent ocular herpesvirus infection and disease. Inversely, augmenting the amount of CXCL10 in trigeminal ganglia of latently infected CXCL10-deficient mice significantly restored the number of local antiviral CD8+ TEM and CD8+ TRM cells associated with protection against recurrent ocular herpes. Based on these findings, a novel "prime/pull" therapeutic ocular herpes vaccine strategy is proposed and discussed.

Absence of chemokine (C-x-C motif) ligand 10 diminishes perfusion recovery after local arterial occlusion in mice.

OBJECTIVE: In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model. APPROACH AND RESULTS: Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10(-/-) knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of α-smooth muscle actin-positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, α-smooth muscle actin-positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow-derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration. CONCLUSIONS: CXCL10 plays a causal role in arteriogenesis. Bone marrow-derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. METHODS: Cxcl10 gene-deleted (Cxcl10(-/-)) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8 weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. RESULTS: WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10(-/-) mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1ß, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPß). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. CONCLUSIONS: We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

Th1-mediated experimental autoimmune encephalomyelitis is CXCR3 independent.

Drugs that block leukocyte trafficking ameliorate multiple sclerosis (MS). Occurrences of opportunistic infection, however, highlight the need for novel drugs that modulate more restricted subsets of T cells. In this context, chemokines and their receptors are attractive therapeutic targets. CXCR3, a Th1-associated chemokine receptor, is preferentially expressed on T cells that accumulate in MS lesions and central nervous system (CNS) infiltrates of mice with experimental autoimmune encephalomyelitis (EAE). Surprisingly, mice genetically deficient in either CXCR3 or CXCL10 succumb to EAE following active immunization with myelin antigens. EAE is mediated by a heterogeneous population of T cells in myelin-immunized mice. Hence, disease might develop in the absence of CXCR3 secondary to the compensatory action of encephalitogenic CCR6(+) Th17 cells. However, in the current study, we show for the first time that blockade or genetic deficiency of either CXCR3 or of its primary ligand has no impact on clinical EAE induced by the adoptive transfer of highly polarized Th1 effector cells. Our data illustrate the fact that, although highly targeted immunotherapies might have more favorable side effect profiles, they are also more likely to be rendered ineffective by inherent redundancies in chemokine and cytokine networks that arise at sites of neuroinflammation.

Deletion of astroglial CXCL10 delays clinical onset but does not affect progressive axon loss in a murine autoimmune multiple sclerosis model.

Multiple sclerosis (MS) is characterized by central nervous system (CNS) inflammation, demyelination, and axonal degeneration. CXCL10 (IP-10), a chemokine for CXCR3+ T cells, is known to regulate T cell differentiation and migration in the periphery, but effects of CXCL10 produced endogenously in the CNS on immune cell trafficking are unknown. We created floxed cxcl10 mice and crossed them with mice carrying an astrocyte-specific Cre transgene (mGFAPcre) to ablate astroglial CXCL10 synthesis. These mice, and littermate controls, were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG peptide) to induce experimental autoimmune encephalomyelitis (EAE). In comparison to the control mice, spinal cord CXCL10 mRNA and protein were sharply diminished in the mGFAPcre/CXCL10fl/fl EAE mice, confirming that astroglia are chiefly responsible for EAE-induced CNS CXCL10 synthesis. Astroglial CXCL10 deletion did not significantly alter the overall composition of CD4+ lymphocytes and CD11b+ cells in the acutely inflamed CNS, but did diminish accumulation of CD4+ lymphocytes in the spinal cord perivascular spaces. Furthermore, IBA1+ microglia/macrophage accumulation within the lesions was not affected by CXCL10 deletion. Clinical deficits were milder and acute demyelination was substantially reduced in the astroglial CXCL10-deleted EAE mice, but long-term axon loss was equally severe in the two groups. We concluded that astroglial CXCL10 enhances spinal cord perivascular CD4+ lymphocyte accumulation and acute spinal cord demyelination in MOG peptide EAE, but does not play an important role in progressive axon loss in this MS model.

Absence of CXCL10 aggravates herpes stromal keratitis with reduced primary neutrophil influx in mice.

Herpes simplex virus 1 (HSV-1) replication initiates inflammation and angiogenesis responses in the cornea to result in herpetic stromal keratitis (HSK), which is a leading cause of infection-induced vision impairment. Chemokines are secreted to modulate HSK by recruiting leukocytes, which affect virus growth, and by influencing angiogenesis. The present study used a murine infection model to investigate the significance of the chemokine CXC chemokine ligand 10 (CXCL10; gamma interferon-inducible protein 10 [IP-10]) in HSK. Here, we show that HSV-1 infection of the cornea induced CXCL10 protein expression in epithelial cells. The corneas of mice with a targeted disruption of the gene encoding CXCL10 displayed decreases in levels of neutrophil-attracting cytokine (interleukin-6), primary neutrophil influx, and viral clearance 2 or 3 days postinfection. Subsequently, absence of CXCL10 aggravated HSK with elevated levels of interleukin-6, chemokines for CD4(+) T cells and/or neutrophils (macrophage inflammatory protein-1α and macrophage inflammatory protein-2), angiogenic factor (vascular endothelial growth factor A), and secondary neutrophil influx, as well as infiltration of CD4(+) T cells to exacerbate opacity and angiogenesis in the cornea at 14 and up to 28 days postinfection. Our results collectively show that endogenous CXCL10 contributes to recruit the primary neutrophil influx and to affect the expression of cytokines, chemokines, and angiogenic factors as well as to reduce the viral titer and HSK severity.

Astrocyte-derived CXCL10 drives accumulation of antibody-secreting cells in the central nervous system during viral encephalomyelitis.

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10(-/-) but not CXCL9(-/-) mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10(-/-) mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9(-/-) mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9(-/-) or CXCL10(-/-) mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.

Resistance to dengue virus infection in mice is potentiated by CXCL10 and is independent of CXCL10-mediated leukocyte recruitment.

CXCL10 is an IFN-inducible chemokine ligand that binds CXCR3, a receptor that is expressed on lymphocytes; CXCL10 shares the CXCR3 receptor with another two ligands, CXCL9 and CXCL11. Previously, we found that CXCL10(-/-) mice were more susceptible than wild-type (WT) mice to dengue virus (DENV) infection. In this study, we explored the mechanisms underlying this enhanced susceptibility. We found that viral loads were higher in the brains of CXCL10(-/-) mice than in WT mice. Presuming a defect in effector lymphocyte migration, we investigated whether recruitment of effector T cells and Ab-secreting cells to the infected tissues were impaired in CXCL10(-/-) mice. Unexpectedly, compared with WT, CXCL10(-/-) mice had comparable numbers of total infiltrating T cells, higher numbers of CXCR3(+) T cells, and higher numbers of Ab-secreting cells in the brain. Additionally, we found that CXCL10 was induced in neurons following DENV infection and that CXCL10 competed with DENV for binding to cell surface heparan sulfate, a coreceptor for DENV entry, thus inhibiting binding of DENV to neuronal cells. These results demonstrate that the enhanced susceptibility of CXCL10(-/-) mice to DENV infection is not due to a defect in recruitment of effector lymphocytes but rather to an antiviral activity that promotes viral clearance.

CXCR2 signaling protects oligodendrocyte progenitor cells from IFN-γ/CXCL10-mediated apoptosis.

Infiltration of activated lymphocytes into the central nervous system (CNS) is potentially harmful by damaging resident cells through release of cytokines. Among these is IFN-γ that is secreted by activated natural killer (NK) cells and T lymphocytes and can exert a cytotoxic effect on resident glial populations including oligodendrocytes. Here we show that treatment of mouse oligodendrocyte progenitor cell (OPC)-enriched cultures with IFN-γ resulted in a dose-dependent increase in apoptosis. IFN-γ-induced apoptosis is mediated, in part, through induction of the CXC chemokine ligand 10 (CXCL10; IP-10) from cultured OPCs. Treatment of OPCs with CXCL10 resulted in cell death in a concentration-dependent manner and IFN-γ-treatment of CXCL10-/- OPCs resulted in >50% reduction in cell death. Further, treatment of CXCR3-/- OPC cultures with either IFN-γ or CXCL10 resulted in reduced cell death supporting an important role for CXCL10 signaling in IFN-γ-mediated OPC apoptosis. Data is also provided demonstrating that signaling through CXCR2 protects either IFN-γ or CXCL10-treated OPC cultures from apoptosis and this effect is abolished in CXCR2-/- OPCs. CXCR2-mediated protection from apoptosis is associated with impaired cleavage of caspase 3 and elevated expression of the anti-apoptotic protein Bcl-2. These findings demonstrate a previously unappreciated role for CXCL10 in contributing to neuropathology by promoting oligodendrocyte apoptosis and emphasize the potential relevance in targeting CXCL10 in treating human demyelinating diseases including multiple sclerosis (MS).

CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus.

The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10(-/-) ) and CXCR3-deficient (CXCR3(-/-) ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10(-/-) and CXCR3(-/-) OHSCs in response to a high (50 µM) NMDA-concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3(-/-) and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions.

CXCL10 inhibits viral replication through recruitment of natural killer cells in coxsackievirus B3-induced myocarditis.

Coxsackievirus (CV)B3 is the primary cause of viral myocarditis. We previously observed CXC chemokine ligand 10 (CXCL10) upregulation in the myocardium early in infection. However, the impact of CXCL10 in CVB3-induced myocarditis is unknown. Using isolated primary mouse cardiomyocytes we demonstrated for the first time that cardiomyocytes can express CXCL10 on interferon-gamma stimulation. To explore the role of CXCL10 in CVB3-induced myocarditis, both CXCL10 transgenic and knockout mice were used. Following CVB3 challenges, the viral titer in the hearts inversely correlated with the levels of CXCL10 at early phase of infection before visible immune infiltration. Furthermore, as compared with the control mice, the decreased virus titers in the CXCL10 transgenic mouse hearts led to less cardiac damage and better cardiac function and vice verse in the knockout mice. This antiviral ability of CXCL10 might be through recruitment of natural killer (NK) cells to the heart and increased interferon-gamma expression early in infection. At day 7 postinfection, with massive influx of mononuclear cells the expression of CXCL10 enhanced the infiltration of CXCR3(+) cells, CD4(+), and CD8(+) T cells, as well as the expression of associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines failed to alter the viral clearance and mice survival. These results suggest the protective role of CXCL10 during the early course of CVB3 infection, which is attributed to the recruitment of NK cells. Nonetheless, CXCL10-directed chemoattractant effect is not sufficient for host to clear the virus in the heart.

Plasmodium berghei ANKA infection increases Foxp3, IL-10 and IL-2 in CXCL-10 deficient C57BL/6 mice.

BACKGROUND: Cerebral malaria (CM) is a major cause of malaria mortality. Sequestration of infected red blood cells and leukocytes in brain vessels coupled with the production of pro-inflammatory factors contribute to CM. CXCL-10 a chemokine that is chemotactic to T cells has been linked to fatal CM. Mice deficient for CXCL-10 gene are resistant to murine CM, while antibody ablation of CXCL-10 enhanced the production of regulatory T cells (CD4+Cd25+Foxp3+) and IL-10 which regulate the immune system. Interleukin-2 (IL-2), a pro-inflammatory cytokine implicated in malaria pathogenesis has also been shown to be a key regulator of Foxp3. However the role of Foxp3 in resistant murine CM is not well understood. METHODS: The hypothesis that resistance of CXCL-10-/- mice to murine CM may be due to enhanced expression of Foxp3 in concert with IL-10 and IL-2 was tested. CXCL-10-/- and WT C57BL/6 mice were infected with Plasmodium berghei ANKA and evaluated for CM symptoms. Brain, peripheral blood mononuclear cells (PBMCs) and plasma were harvested from infected and uninfected mice at days 2, 4 and 8. Regulatory T cells (CD4+CD25+) and non-T regs (CD4+CD25-) were isolated from PBMCs and cultured with P. berghei antigens in vitro with dendritic cells as antigen presenting cells. Regulatory T cell transcription and specific factor Foxp3, was evaluated in mouse brain and PBMCs by realtime-PCR and Western blots while IL-10, and IL-2 were evaluated in plasma and cultured supernatants by ELISA. RESULTS: Wild type mice exhibited severe murine CM symptoms compared with CXCL-10-/- mice. Foxp3 mRNA and protein in brain and PBMC's of CXCL-10-/- mice was significantly up-regulated (p < 0.05) by day 4 post-infection (p.i) compared with WT. Plasma levels of IL-10 and IL-2 in infected CXCL-10-/- were higher than in WT mice (p < 0.05) at days 2 and 4 p.i. Ex-vivo CD4+CD25+ T cells from CXCL-10-/- re-stimulated with P. berghei antigens produced more IL-10 than WT CD4+CD25+ T cells. CONCLUSION: The results indicate that in the absence of CXCL-10, the resulting up-regulation of Foxp3, IL-10 and IL-2 may be involved in attenuating fatal murine CM.

Reduced surface toll-like receptor-4 expression and absent interferon-γ-inducible protein-10 induction in cystic fibrosis airway cells.

ABSTRACT As part of the innate and adaptive immune system, airway epithelial cells secrete proinflammatory cytokines after activation of Toll-like receptors (TLRs) by pathogens. Nevertheless, cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. The authors have shown that in CF bronchial epithelial cells, a reduced surface expression of TLR-4 causes a diminished interleukin (IL)-8 and IL-6 response upon lipopolysaccharide (LPS) stimulation. However, there is no information regarding activation of the MyD88 (myeloid differentiation primary response gene 88)-independent TLR-4 signaling pathway by LPS, which results in the activation of adaptive immune responses by secretion of the T cell-recruiting chemokine interferon-γ-inducible protein (IP)-10. Therefore, the authors investigated the induction of IP-10 in CF bronchial epithelial cell line CFBE41o- and its CFTR-corrected isotype under well-differentiating conditions. TLR-4 surface expression was significantly reduced in CFBE41o- by a factor of 2, compared to the CFTR-corrected cells. In CFTR-corrected cells, stimulation with LPS increased IP-10 secretion. Incubating cells with siRNA directed against TLR-4 inhibited the LPS stimulated increase of IP-10 in CFTR-corrected cells. The reduced TLR-4 surface expression in CF cells causes the loss of induction of IP-10 by LPS. This could compromise adaptive immune responses in CF due to a reduced T-cell recruitment.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

CXCL10 expressing hematopoietic-derived cells are requisite in defense against HSV-1 infection in the nervous system of CXCL10 deficient mice.

The chemokine CXCL10 is crucial for the control of viral replication through the regulation of mobilization of antigen-specific T cells to sites of infection. CXCL10 is highly expressed both at sites of inflammation as well as constitutively within lymphoid organs by both bone marrow (BM)-derived and non-BM-derived cells. However, the relative immunologic importance of CXCL10 expressed by these divergent sources relative to HSV-1 infection is unknown. Using mouse chimeras reconstituted with either wild type or CXCL10 deficient mouse BM, we show BM-derived, radiation-sensitive cells from wild type mice were solely responsible for resistance to HSV-1 in the trigeminal ganglia and brain stem. The resistance was not reflected by a deficiency in the recruitment of effector cells to sites of inflammation or expression of chemokines or IFN-gamma and likely results from additional, yet-to-be-determined factors emanating from wild type, BM-derived cells.

Effective immunotherapy against murine gliomas using type 1 polarizing dendritic cells--significant roles of CXCL10.

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis.

Regional differences in inflammation during viral infections of the CNS suggest viruses differentially induce patterns of chemoattractant expression, depending on their cellular targets. Previous studies have shown that expression of the chemokine CXCL10 by West Nile virus (WNV)-infected neurons is essential for the recruitment of CD8 T cells for the purpose of viral clearance within the CNS. In the current study we used mice deficient for the CXCL10 receptor, CXCR3, to evaluate its role in leukocyte-mediated viral clearance of WNV infection within various CNS compartments. WNV-infected CXCR3-deficient mice exhibited significantly enhanced mortality compared with wild-type controls. Immunologic and virologic analyses revealed that CXCR3 was dispensable for control of viral infection in the periphery and in most CNS compartments but, surprisingly, was required for CD8 T cell-mediated antiviral responses specifically within the cerebellum. WNV-specific, CXCR3-expressing T cells preferentially migrated into the cerebellum, and WNV-infected cerebellar granule cell neurons expressed higher levels of CXCL10 compared with similarly infected cortical neurons. These results indicate that WNV differentially induces CXCL10 within neuronal populations and suggest a novel model for nonredundancy in chemokine-mediated inflammation among CNS compartments.