Jean K. Lim

CCR5 deficiency increases risk of symptomatic West Nile virus infection

West Nile virus (WNV) is a reemerging pathogen that causes fatal encephalitis in several species, including mouse and human. Recently, we showed that the chemokine receptor CCR5 is critical for survival of mice infected with WNV, acting at the level of leukocyte trafficking to the brain. To test whether this receptor is also protective in man, we determined the frequency of CCR5Δ32, a defective CCR5 allele found predominantly in Caucasians, in two independent cohorts of patients, one from Arizona and the other from Colorado, who had laboratory-confirmed, symptomatic WNV infection. The distribution of CCR5Δ32 in a control population of healthy United States Caucasian random blood donors was in Hardy-Weinberg equilibrium and CCR5Δ32 homozygotes represented 1.0% of the total group (n = 1,318). In contrast, CCR5Δ32 homozygotes represented 4.2% of Caucasians in the Arizona cohort (odds ratios [OR] = 4.4 [95% confidence interval [CI], 1.6–11.8], P = 0.0013) and 8.3% of Caucasians in the Colorado cohort (OR = 9.1 [95% CI, 3.4–24.8], P < 0.0001). CCR5Δ32 homozygosity was significantly associated with fatal outcome in the Arizona cohort (OR = 13.2 [95% CI, 1.9–89.9], P = 0.03). We conclude that CCR5 mediates resistance to symptomatic WNV infection. Because CCR5 is also the major HIV coreceptor, these findings have important implications for the safety of CCR5-blocking agents under development for HIV/AIDS.

Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection

The molecular immunopathogenesis of West Nile virus (WNV) infection is poorly understood. Here, we characterize a mouse model for WNV using a subcutaneous route of infection and delineate leukocyte subsets and immunoregulatory factors present in the brains of infected mice. Central nervous system (CNS) expression of the chemokine receptor CCR5 and its ligand CCL5 was prominently up-regulated by WNV, and this was associated with CNS infiltration of CD4+ and CD8+ T cells, NK1.1+ cells and macrophages expressing the receptor. The significance of CCR5 in pathogenesis was established by mortality studies in which infection of CCR5−/− mice was rapidly and uniformly fatal. In the brain, WNV-infected CCR5−/− mice had increased viral burden but markedly reduced NK1.1+ cells, macrophages, and CD4+ and CD8+ T cells compared with WNV-infected CCR5+/+ mice. Adoptive transfer of splenocytes from WNV-infected CCR5+/+ mice into infected CCR5−/− mice increased leukocyte accumulation in the CNS compared with transfer of splenocytes from infected CCR5−/− mice into infected CCR5−/− mice, and increased survival to 60%, the same as in infected CCR5+/+ control mice. We conclude that CCR5 is a critical antiviral and survival determinant in WNV infection of mice that acts by regulating trafficking of leukocytes to the infected brain.

Genetic Deficiency of Chemokine Receptor CCR5 Is a Strong Risk Factor for Symptomatic West Nile Virus Infection: A Meta-Analysis of 4 Cohorts in the US Epidemic

West Nile virus (WNV) causes disease in approximately 20% of infected humans. We previously reported that homozygosity for CCR5Delta32, a nonfunctional variant of chemokine receptor CCR5, is markedly increased among symptomatic WNV-seropositive patients from Arizona and Colorado. To confirm this, we analyzed cohorts from California and Illinois. An increase in CCR5-deficient subjects was found in both (for California, odds ratio [OR], 4.2 [95% confidence interval {CI}, 1.5-11.9] [P= .004]; for Illinois, OR, 3.1 [95% CI, 0.9-11.2] [P= .06]). A meta-analysis of all 4 cohorts showed an OR of 4.2 (95% CI, 2.1-8.3 [P= .0001]). Thus, CCR5 deficiency is a strong and consistent risk factor for symptomatic WNV infection in the United States.

Genetic Variation in OAS1 Is a Risk Factor for Initial Infection with West Nile Virus in Man

West Nile virus (WNV) is a re-emerging pathogen that can cause fatal encephalitis. In mice, susceptibility to WNV has been reported to result from a single point mutation in oas1b, which encodes 2′–5′ oligoadenylate synthetase 1b, a member of the type I interferon-regulated OAS gene family involved in viral RNA degradation. In man, the human ortholog of oas1b appears to be OAS1. The ‘A’ allele at SNP rs10774671 of OAS1 has previously been shown to alter splicing of OAS1 and to be associated with reduced OAS activity in PBMCs. Here we show that the frequency of this hypofunctional allele is increased in both symptomatic and asymptomatic WNV seroconverters (Caucasians from five US centers; total n = 501; OR = 1.6 [95% CI 1.2–2.0], P = 0.0002 in a recessive genetic model). We then directly tested the effect of this SNP on viral replication in a novel ex vivo model of WNV infection in primary human lymphoid tissue. Virus accumulation varied markedly among donors, and was highest for individuals homozygous for the ‘A’ allele (P<0.0001). Together, these data identify OAS1 SNP rs10774671 as a host genetic risk factor for initial infection with WNV in humans.

Organ-Specific Innate Immune Responses in a Mouse Model of Invasive Candidiasis

In a fatal mouse model of invasive candidiasis (IC), fungal burden changes with variable dynamics in the kidney, brain, spleen, and liver and declines in all organs except for the kidney, which inexorably loses function. Since leukocytes are required to control Candida, we hypothesized that differential leukocyte infiltration determines organ-specific outcome of the infection. We defined leukocyte accumulation in the blood, kidney, brain, spleen, and liver after infection using fluorescent-activated cell sorting (FACS) and immunohistochemistry. Accumulation of Ly6cintCD11b+ neutrophils predominated in all organs except the brain, where CD45intCD11b+CD11c– microglia were the major leukocytes detected, surrounding foci of invading Candida. Significantly more neutrophils accumulated in the spleen and liver than in the kidney during the first 24 h after infection, when neutrophil presence is critical for Candida control. Conversely, at later time points only the kidney continued to accumulate neutrophils, associated with immunopathology and organ failure. The distribution of neutrophils was completely different in each organ, with large abscesses exclusively forming in the kidney. Candida filamentation, an essential virulence factor, was seen in the kidney but not in the spleen or liver. IC induced Ly6chiCD11b+ inflammatory monocyte and NK1.1+ cell expansion in the blood and all organs tested, and MHCII+F4/80+CD11c– macrophage accumulation, mainly in the spleen and liver. This study is the first detailed analysis of leukocyte subsets accumulating in different target organs during IC. The results delineate immune responses to the same pathogen that are highly idiosyncratic for each organ tested. The work provides novel insights into the balance between effective host defense and immunopathology in IC.

Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity

One antibody for all and all antibodies for one Antibodies against related flavi-viruses such as dengue (DENV) and West Nile (WNV) can cross-react with Zika virus (ZIKV) and could thereby increase disease severity. Bardina et al. tested whether DENV and WNV antibodies from humans, or even yellow fever vaccination, could enhance ZIKV infection. In a mouse model, low titers of DENV and WNV antibodies enhanced ZIKV viremia, especially in the spinal cord and testes, whereas high titers remained protective. Generally, WNV antibodies were less disease-enhancing than DENV antibodies, and, in macaques, yellow fever vaccination had very little effect. Science, this issue p. 175 Antibodies against dengue and West Nile viruses cross-react with anti–Zika virus antibodies to enhance infection and fever in mice. Zika virus (ZIKV) is spreading rapidly into regions around the world where other flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are endemic. Antibody-dependent enhancement has been implicated in more severe forms of flavivirus disease, but whether this also applies to ZIKV infection is unclear. Using convalescent plasma from DENV- and WNV-infected individuals, we found substantial enhancement of ZIKV infection in vitro that was mediated through immunoglobulin G engagement of Fcγ receptors. Administration of DENV- or WNV-convalescent plasma into ZIKV-susceptible mice resulted in increased morbidity—including fever, viremia, and viral loads in spinal cord and testes—and increased mortality. Antibody-dependent enhancement may explain the severe disease manifestations associated with recent ZIKV outbreaks and highlights the need to exert great caution when designing flavivirus vaccines.

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

CCR5 Deficiency Is a Risk Factor for Early Clinical Manifestations of West Nile Virus Infection but not for Viral Transmission

BACKGROUND West Nile virus (WNV) is a neurotropic flavivirus transmitted to humans by mosquito vectors. Homozygosity for CCR5Delta32, a complete loss-of-function mutation in CC chemokine receptor 5 (CCR5), has been previously associated with severe symptomatic WNV infection in patients who present with clinical disease; however, whether it acts at the level of initial infection or in promoting clinical progression is unknown. METHODS Here, we address this gap in knowledge by comparing CCR5Delta32 distribution among US blood donors identified through a comprehensive blood supply screening program (34,766,863 donations from 2003 through 2008) as either WNV true positive (634 WNV-positive cases) or false positive (422 WNV-negative control participants). All subjects self-reported symptoms occurring during the 2 weeks following blood donation using a standardized questionnaire. RESULTS No difference was observed in CCR5Delta32 homozygous frequency between the WNV-positive cases and WNV-negative control participants. However, CCR5Delta32 homozygosity was associated in cases but not controls with clinical symptoms consistent with WNV infection (P = .002). CONCLUSIONS CCR5 deficiency is not a risk factor for WNV infection per se, but it is a risk factor for both early and late clinical manifestations after infection. Thus, CCR5 may function normally to limit disease due to WNV infection in humans.

Chemokine Receptor Ccr2 Is Critical for Monocyte Accumulation and Survival in West Nile Virus Encephalitis

West Nile virus (WNV) is a re-emerging pathogen responsible for outbreaks of fatal meningoencephalitis in humans. Previous studies have suggested a protective role for monocytes in a mouse model of WNV infection, but the molecular mechanisms have remained unclear. In this study, we show that genetic deficiency in Ccr2, a chemokine receptor on Ly6chi inflammatory monocytes and other leukocyte subtypes, markedly increases mortality due to WNV encephalitis in C57BL/6 mice; this was associated with a large and selective reduction of Ly6chi monocyte accumulation in the brain. WNV infection in Ccr2+/+ mice induced a strong and highly selective monocytosis in peripheral blood that was absent in Ccr2−/− mice, which in contrast showed sustained monocytopenia. When a 1:1 mixture of Ccr2+/+ and Ccr2−/− donor monocytes was transferred by vein into WNV-infected Ccr2−/− recipient mice, monocyte accumulation in the CNS was not skewed toward either component of the mixture, indicating that Ccr2 is not required for trafficking of monocytes from blood to brain. We conclude that Ccr2 mediates highly selective peripheral blood monocytosis during WNV infection of mice and that this is critical for accumulation of monocytes in the brain.

Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

Invasive candidiasis is the 4th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1lo to Ccr1high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1+/+ and Ccr1−/− donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.