CSF1R antagonism increases susceptibility of mice to fatal WNV infection
To determine whether CSF1R antagonism impacts survival from WNV infection, mice were fed PLX5622-embedded chow for 2 weeks, then infected with WNV-NY [42] at either 102 or 104 PFU via footpad (f.p.) inoculation. Consistent with published data [8, 49], 30% of control-treated mice survived infection with 104 PFU and 90% of control-treated mice survived infection with 102 PFU; however, mice treated with PLX5622 universally succumbed to infection with either inoculum titer (Fig. 2a). This was associated with significantly increased weight loss (Fig. 2b) and more severe clinical score (Fig. 2c, d) in PLX5622-treated compared with control-treated mice infected with 102 PFU WNV-NY. To determine whether increased mortality caused by PLX5622 treatment is associated with increased viral replication in peripheral or CNS tissues, tissue viral loads were assessed by plaque assay, and serum viral loads were determined by quantitative RT-PCR (qRT-PCR) at 2, 4, 6, and 8 days post-infection (dpi). In the CNS, virus was first detected in the olfactory bulb (Fig. 2e), then sequentially in more caudal regions including the cortex (Fig. 2f), cerebellum (Fig. 2g), brainstem (Fig. 2h), and spinal cord (Fig. 2i). Compared with control-treated animals, PLX5622-treated mice exhibited higher viral titers in the olfactory bulb and cortex at 6 and 8 dpi and in the cerebellum, brainstem, and spinal cord at 8 dpi; however, viral titers were also significantly higher in peripheral tissues of PLX5622-treated mice including the spleen at 4 dpi (Fig. 2j), kidney at 4 and 6 dpi (Fig. 2k), and serum at 2, 4, 6, and 8 dpi (Fig. 2l). PLX5622 treatment did not cause increased BBB permeability in infected mice (Additional file 5). Once WNV-NY enters the CNS via intracranial (i.c.) inoculation, PLX5622 treatment did not affect neuronal permissivity to infection (Additional file 6). Together, these data indicate a loss of immune-mediated virologic control in both peripheral and CNS tissues in CSF1R antagonist-treated mice, consistent with prior data suggesting that CSF1R signaling plays important roles in the function of both peripheral and CNS myeloid cells [50–52].
Fig. 2 PLX5622-treated mice exhibit increased mortality, increased encephalitis score, and impaired virologic control compared with control mice following peripheral infection with WNV-NY. a–d Mice were fed chow containing PLX5622 or control chow for 2 weeks, then infected with 102 or 104 PFU via footpad infection, then monitored for (a) mortality, (b) weight loss, and (c, d) encephalitis score for up to 25 dpi. a Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control-treated mice infected with either viral inoculum dose, as calculated by log-rank (Mantel-Cox) test. b Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls infected with 102 PFU as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. c Clinical scores of PLX5622 or control-treated mice infected with 102 PFU or d 104 PFU as indicated during acute illness. 0 = subclinical, 1 = hunched/ruffled fur, 2 = altered gate/slow movement, 3 = no movement, but responsive to stimuli, 4 = moribund, 5 = dead. e–k Tissue viral loads as measured by plaque assay at 2, 4, 6, and 8 dpi following infection with 102 PFU in control (black) or PLX5622-treated (red) mice. l Serum viral loads as measured by qRT-PCR. e–l Data are presented as scatter plots with each mouse represented by a dot with the SEM indicated by a line. Dotted lines in e–l indicate assay limit of detection. All data presented are the compilation of two independent experiments with 10 mice per group. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P < 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001
To determine whether microglia depletion impacts innate immune mechanisms of virologic control specifically within the CNS, mice were fed control or PLX5622-embedded chow for 2 weeks, then i.c. infected with an attenuated strain of WNV (WNV-NS5-E218A) at 104 PFU. The NS5-E218A point mutation abolishes 2′-O-methyltransferase activity, which functions to methylate the 5′ cap of viral RNA and facilitate escape of IFN-induced proteins with tetratricopeptide repeats (IFIT)-mediated suppression; thus, this attenuation limits viral replication by increasing sensitivity of the virus to type I IFN-mediated innate immune mechanisms [45, 49]. Despite this attenuation, mice treated with PLX5622 were significantly more susceptible to lethal WNV-NS5-E218A infection than those receiving control chow (Fig. 3a). Weight loss was also significantly increased in PLX5622-treated mice (Fig. 3b). Consistent with this, viral titers measured by plaque assay at 2, 4, 6, and 8 dpi in the olfactory bulb (Fig. 3d), cortex (Fig. 3e), cerebellum (Fig. 3f), brainstem (Fig. 3g), and spinal cord (Fig. 3h) were significantly higher in PLX5622-treated compared in control-treated mice infected with WNV-NS5-E218A. As virus was inoculated into the cortex, it was detected there at 2 dpi, followed by spread to the olfactory bulb by 4 dpi, and cerebellum, brainstem, and spinal cord by 6 dpi. PLX5622 treatment also led to decreased proinflammatory cytokine expression in the cortex at 8 dpi (Fig. 3c) and increased neuronal apoptosis in the hippocampus and cerebellum at 6 dpi (Fig. 3l–p) after i.c. infection with WNV-NS5-E218A.
Fig. 3 CSF1R antagonism increases mortality and CNS viral burdens during i.c. infection with WNV-NS5-E218A. Mice were fed chow containing PLX5622 or control chow for 2 weeks, infected i.c. with WNV-NS5-E218A at 104 PFU, then monitored for a mortality and b weight loss for up to 25 dpi. a Survival curves show a significant increase in mortality in mice treated with PLX5622 compared with control as calculated by log-rank (Mantel-Cox) test. b Weight was measured daily during acute illness as a measure of illness. After death, the last measured weight was carried through to the end of the experiment. Significantly greater weight loss was measured in PLX5622-treated mice compared with controls as calculated by two-way ANOVA with matched values comparing group means without multiple comparisons. For a and b, data represent the compilation of two independent experiments with 10 mice per group. c At 8 dpi, cortical brain tissue was extracted and relative transcript levels in tissue homogenates were measured by SYBR qRT-PCR for indicated cytokines. Data for individual mice were normalized to Gapdh. Data are presented as mean ± SEM of three to five mice from one experiment. Statistical significance between treatment groups for each cytokine was calculated by t test. d–j Tissue viral loads were measured by plaque assay at 2, 4, 6, and 8 dpi in control (black) or PLX5622-treated (red) mice. k Serum viral loads as measured by qRT-PCR. d–k Data are presented as scatter plots with each mouse represented by a dot with the mean indicated by a line. For d–k, data represent the compilation of two-independent experiments with 10 mice per group. Dotted lines in d–k indicate assay limit of detection. l–o Representative confocal microscopic images of Tunel (red), NeuN (green), and DAPI (blue) of hippocampus (l, m) and cerebellum (n, o) of control- (l, n) or PLX5622-treated (m, o) mice infected i.c. with WNV-NS5-E218A at 6 dpi. Arrow heads point to DAPI-positive nuclei positive for both Tunel and NeuN. Asterisks denote DAPI- and Tunel-positive but NeuN-negative nuclei. p Quantification of confocal microscopic images for number of DAPI-positive nuclei positive for Tunel and NeuN in the hippocampus (Hpc), cerebellum (Cb), and brainstem (Bs). The number of Tunel+ NeuN+ nuclei per high-power field (HPF) was quantified from three to six images captured at × 40 per brain region across two separate brain sections for each of five independent mice collected in one experiment. Each symbol represents the average number for an individual mouse, with bar indicating mean ± SEM. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. For all data: ns, not significant at P < 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001
As expected, WNV-NS5-E218A was barely detected in the kidney (Fig. 3j), and the virus was steadily cleared from the serum (Fig. 3k) in both control- and PLX5622-treated mice. However, significantly higher levels of virus were detected in the spleen of PLX5622- versus control-treated, WNV-NS5-E218A-infected mice at 4 dpi (Fig. 3i), which was consistent with data obtained during f.p. infection with WNV-NY. Overall, CSF1R antagonism leads to enhanced mortality and loss of virologic control in both the periphery and CNS regardless of the WNV strain utilized, suggesting a critical role for this molecule in antiviral immunity.