Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice

Abstract

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Fig. 1.. Lrp1-depleted MEFs are resistant to RVFV infection.

(A) Schematics showing the generation of mouse embryonic fibroblasts (MEFs) from embryonic day (E) 14.5 embryos obtained by timed mating of Lrp1^f/+ mice. dpi, days postinfection. (B) Western blot of Lrp1^f/f MEFs infected with adenovirus expressing an empty adenovirus vector (Ad) and the cre recombinase enzyme (Ad^cre). The blot was probed for lipoprotein receptor–related protein 1 (Lrp1) and β-tubulin. Lrp1-sufficient and Lrp1-deficient MEFs were infected with Rift Valley fever virus (RVFV) MP12^GFP at a multiplicity of infection (MOI) of 2 for 16 hours. (C) Fluorescence images show green fluorescent protein (GFP), DAPI (4′,6-diamidino-2-phenylindole), and merged panels. Images were taken at ×20 magnification (scale bars, 200 μm). (D and E) Representative flow cytometry histograms and corresponding analysis of flow cytometry histogram data from MEF infections. Experiment was performed five times in duplicates. Student’s t test was used to determine significance; ****P < 0.0001.

Fig. 2.. Elimination of Lrp1 in hepatocytes significantly delays time to death after RVFV infection.

(A) Lrp1^f/f and Lrp1^f/fAlb-Cre mice were infected with 20 plaque-forming units (PFUs) of RVFV ZH501 by footpad and monitored for survival (n = 61 animals, data were derived from three experiments). Clinical signs of (B) Lrp1^f/f mice and (C) Lrp1^f/fAlb-Cre mice highlight the frequency of neurological symptoms (note that mice found deceased are not included in this scoring). (D) Percentages of mice across three studies that experienced neurological signs of disease including tremors and paralysis (light blue) on the day of euthanasia in Lrp1^f/f and Lrp1^f/fAlb-Cre mice. Mice that were found deceased could not be clinically scored and were not included in analysis in (D). Kaplan-Meier survival analysis was completed to assess for significant differences between genotypes. ****P < 0.0001.

Fig. 3.. Lrp1 KO in hepatocytes leads to a significant reduction in viral burden in the liver at 3 dpi.

(A) Lrp1^f/fAlb-Cre mice (n = 16) have significantly less (A) infectious virus and (B) viral RNA (vRNA) in the liver at 3 dpi than Lrp1^f/f controls (n = 15). (C) RVFV nucleoprotein (N) (magenta) and active caspase-3 (green) staining in the liver at 3 dpi in Lrp1^f/f and Lrp1^f/fAlb-Cre livers (40×). (D) Interferon α (IFNα) and (E) IFNβ relative mRNA expression in the liver of uninfected and RVFV-infected Lrp1^f/f and Lrp1^f/fAlb-Cre mice at 3 dpi. Bars represent SEM. Statistics were determined by an unpaired t test or a one-way analysis of variance (ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant. Scale bars, 250 μm. Dotted lines indicate the limit of detection.

Fig. 4.. Lrp1 KO in hepatocytes reduces RVFV in the liver at time of euthanasia.

(A) vRNA titers in the liver on the day of euthanasia show a significant negative correlation in Lrp1^f/fAlb-Cre mice from 3 to 14 dpi. LOD, limit of assay detection. (B) Gross pathology of liver tissue obtained from the indicated genotypes at 5 dpi. DOA, deceased on arrival. (C) RVFV N antigen staining in liver tissue of Lrp1^f/f mice compared to Lrp1^f/fAlb-Cre mice at 5 and 7 dpi (40×). (D) Interleukin-1β (IL-1β), (E) IL-6, and (F) MCP-1 protein levels in the liver tissue homogenate on the day of euthanasia from 3 to 14 dpi. Scale bars, 250 μm. A linear regression was used to determine whether the slope was significantly nonzero. **P < 0.01; ***P < 0.001.

Fig. 5.. Mice with Lrp1 KO in hepatocytes succumb to RVF neurological disease.

(A) vRNA titers in the brain at euthanasia show a significant positive correlation in Lrp1^f/fAlb-Cre mice from 3 to 14 dpi. (B) RVFV staining in the brain tissue of Lrp1^f/fAlb-Cre mice compared to Lrp1^f/f mice from 5, 7, and 14 dpi (40×). (C) Brain tissue from a Lrp1^f/fAlb-cre mice at 12 dpi stained for RVFV N (magenta), β-tubulin III (green), and DAPI (blue; 10×). (D) IL-1β, (E) IL-6, and (F) MCP-1 protein in the brain tissue at euthanasia from 3 to 14 dpi. Scale bars, 250 μm. A linear regression was used to determine whether the slope was significantly nonzero. **P < 0.01.