Toll-like receptor 2-mediated innate immune responses against Junín virus in mice lead to antiviral adaptive immune responses during systemic infection and do not affect viral replication in the brain

Abstract

Successful adaptive immunity to virus infection often depends on the initial innate response. Previously, we demonstrated that Junín virus, the etiological agent responsible for Argentine hemorrhagic fever (AHF), activates an early innate immune response via an interaction between the viral glycoprotein and Toll-like receptor 2 (TLR2). Here we show that TLR2/6 but not TLR1/2 heterodimers sense Junín virus glycoprotein and induce a cytokine response, which in turn upregulates the expression of the RNA helicases RIG-I and MDA5. NF-κB and Erk1/2 were important in the cytokine response, since both proteins were phosphorylated as a result of the interaction of virus with TLR2, and treatment with an Erk1/2-specific inhibitor blocked cytokine production. We show that the Junín virus glycoprotein activates cytokine production in a human macrophage cell line as well. Moreover, we show that TLR2-mediated immune response plays a role in viral clearance because wild-type mice cleared Candid 1 (JUNV C1), the vaccine strain of Junín virus, more rapidly than did TLR2 knockout mice. This clearance correlated with the generation of Junín virus-specific CD8(+) T cells. However, infected wild-type and TLR2 knockout mice developed TLR2-independent blocking antibody responses with similar kinetics. We also show that microglia and astrocytes but not neurons are susceptible to infection with JUNV C1. Although JUNV C1 infection of the brain also triggered a TLR2-dependent cytokine response, virus levels were equivalent in wild-type and TLR2 knockout mice. Importance: Junín virus is transmitted by rodents native to Argentina and is associated with both systemic disease and, in some patients, neurological symptoms. Humans become infected when they inhale aerosolized Junín virus. AHF has a 15 to 30% mortality rate, and patients who clear the infection develop a strong antibody response to Junín virus. Here we investigated what factors determine the immune response to Junín virus. We show that a strong initial innate immune response to JUNV C1 determines how quickly mice can clear systemic infection and that this depended on the cellular immune response. In contrast, induction of an innate immune response in the brain had no effect on virus infection levels. These findings may explain how the initial immune response to Junín virus infection could determine different outcomes in humans.

FIG 1

JUNV C1-mediated activation of the NF-κB and Erk1/2 pathways is dependent on TLR2 signaling. (A) Primary macrophages with deletions in TLR genes were infected with JUNV C1 or treated with LPS, LPS plus polymyxin B (Pmx), or PAM, and at 2 hpi, protein lysates were prepared and analyzed by Western blotting, using antibodies against total and phosphorylated (p-Ser486) NF-κB (p65). (B) Primary macrophages were treated with UO126 for 1 h, followed by infection with JUNV C1 or LPS. At 2 hpi, cell extracts were analyzed for phosphorylated Erk1/2 by Western blotting. (C and D) At 2 hpi, RNA was isolated from cells treated as indicated, and IFN-β (C) and TNF-α (D) RNA levels were quantified by RT-qPCR. (E) Supernatants from the same cells were used to quantify TNF-α levels by ELISA. The bar graphs represent the averages and standard deviations from three technical replicates. These experiments were performed twice, with similar results.

FIG 2

Upregulation of RIG-I and MDA5 expression by JUNV C1 is TLR2 dependent. RIG-I and MDA5 levels were analyzed by Western blotting of extracts from primary macrophages infected with JUNV C1 or treated with LPS, LPS plus polymyxin, or PAM. (A) Primary macrophages derived from wild-type mice were infected with JUNV C1, and at 2 hpi, protein lysates were analyzed by Western blotting, using antibodies against RIG-I and MDA5. (B) JUNV C1-infected macrophages derived from wild-type, TLR2^−/−, and TLR4^−/− mice were harvested at 24 hpi, and the proteins were analyzed by Western blotting with antibodies against RIG-I and MDA5. A cellular protein that is detected nonspecifically by the MDA5 antibody was used as a loading control. Shown below the lanes are the relative levels of protein in each sample relative to levels in mock-treated cells. Quantification of RIG-I and MDA5 levels was done by using ImageJ software. These experiments were done twice, with similar results.

FIG 3

Mouse macrophages sense Junín virus GPC through TLR2/6 heterodimers. (A to E) Mouse macrophage cell lines with deletions in different TLR genes were infected with JUNV C1 or treated with various TLR agonists (A to C) or infected with VSV pseudotypes bearing JUNV C1 GPC, Parodi GPC, or no GPC (Bald) (D and E). At 2 hpi, RNA was isolated, and IFN-β (A and D) and TNF-α (B and E) levels were quantified by RT-qPCR. At 6 hpi, medium from infected cells was collected and TNF-α levels were quantified by ELISA (C). The bar graphs represent the averages and standard deviations from three technical replicates. These experiments were performed three times independently, with similar results. (F) THP-1 human monocytes treated with PMA to induce macrophage differentiation were infected with JUNV C1. At 2 hpi, TNF-α RNA levels were quantified by RT-qPCR. Shown are data from duplicate experiments performed in triplicate under each condition. (G) PMA-differentiated macrophages were infected with VSV pseudotypes bearing JUNV C1 GPC, Parodi GPC, or no GPC, and TNF-α RNA levels were measured at 2 hpi. Each bar represents the average and standard deviation of data from an experiment performed in triplicate.

FIG 4

TLR2^−/− mice are more highly infected with JUNV C1 than are wild-type mice. (A) Time course of infection. Spleens were harvested on the indicated days postinfection, and viral RNA was quantified by RT-qPCR. Five mice of the corresponding phenotypes were used for each time point. Kaplan-Meier analysis showed that there was statistical significance (P < 0.05) between WT and TLR2^−/− mouse RNA level curves. The dashed line indicates the limit of detection for this RT-qPCR assay. (B and C) Additional mice were infected with JUNV C1, and at 7 days postinfection, viral RNA was quantified by RT-qPCR (B), and viral titers were determined (C). Each point represents an individual mouse. (D) WT (NR-9456) and TLR2^−/− (NR-9457) mouse macrophage cells were infected with JUNV C1 at an MOI of 0.1 and harvested at 24, 48, and 72 hpi, and viral RNA was measured by RT-qPCR. As a control, infected cells were treated with ribavirin, and the viral RNA level was quantified at 48 hpi. Two-tailed Student's t test was used to determine significance. *, P < 0.05; ns, no statistical significance.

FIG 5

Increased levels of Junín virus-specific CD8⁺ T cells but not humoral responses require TLR2 signaling. Mice were infected with JUNV C1 or LCMV. At 1 week postinfection, mouse PBMCs were collected and stained for NP-specific CD8⁺ T cells. (A and B) Percentage (A) and total number (B) of Junín virus-specific CD8⁺ T cells (per ml of blood) from mice of different TLR backgrounds. Each point represents a single mouse. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, no statistically significant difference based on one-way analysis of variance. (C and D) Mice of the indicated backgrounds (WT [C] and TLR2^−/− [D]) were infected with JUNV C1. At 2 weeks postinfection, serum was collected, and the indicated serial dilutions were incubated with JUNV C1 for 1 h prior to infection of Vero cells. Undiluted sera from mock- and LCMV-infected mice were used as negative controls. Each point represents an individual mouse.

FIG 6

JUNV C1 targets microglia and astroglia cells in the brain. Mice that received intracranial inoculations of PBS or JUNV C1 were sacrificed at 7 dpi. Brain sections from JUNV C1-infected (A, C, and E) and mock-infected (B, D, and F) mice were stained with antibodies specific for Junín virus NP and the brain cell markers Iba-1 (microglia) (A and B), GFAP (astroglia) (C and D), and NeuN (neurons) (E and F). Brain cell makers were indirectly stained with a secondary antibody labeled with Alexa 564 (red), and viral NP was indirectly stained with an Alexa 488-labeled secondary antibody (green). Cell nuclei were stained with DAPI and are shown in the merged image. The selected region of the merged image is magnified 3× (bottom right quadrant of each panel).

FIG 7

JUNV C1 induces TLR2-dependent cytokine production in the brain which does not control infection. (A and B) WT and MyD88/Mal double-knockout microglia cell lines were infected with JUNV C1 or treated with various TLR agonists. Two hours after infection of cells, total RNA was isolated and used to quantify IFN-β (A) and TNF-α (B) levels by RT-qPCR. RNA values were normalized to that of GAPDH RNA. The bar graphs represent the averages and standard deviations from three technical replicates. These experiments were performed independently three times, with similar results. (C) Virus titers from the brains of wild-type mice that received intracranial inoculations of JUNV C1 and at 3, 5, 7, 10, and 14 dpi. (D and E) RNA isolated at 5 and 7 dpi from the brains of JUNV C1-infected WT and TLR2^−/− mice was used to quantify IFN-β (D) and TNF-α (E) levels. (F) Virus titers from the brains of WT and TLR2^−/− mice at 5 dpi. Each point represents an individual mouse. **, P < 0.01; ***, P < 0.001; ns, no statistically significant difference based on 2-tailed Student's t test.