Tissue-specific deletion of the coxsackievirus and adenovirus receptor protects mice from virus-induced pancreatitis and myocarditis

Abstract

In cultured cells, infection by group B coxsackievirus (CVB) is mediated by the coxsackievirus and adenovirus receptor (CAR), but the importance of this molecule in CVB-induced disease has not been determined. We generated mice with tissue-specific ablation of CAR within each of two major CVB target organs, the pancreas and heart. In the pancreas, deletion of CAR resulted in a significant reduction in both virus titers and virus-induced tissue damage. Similarly, cardiomyocyte-specific CAR deletion resulted in a marked reduction in virus titer, infection-associated cytokine production, and histopathology within the heart. Consistent with the in vivo phenotype, CAR-deficient cardiomyocytes resisted infection in vitro. These results demonstrate a critical function for CAR in the pathogenesis of CVB infection in vivo and in virus tropism for the heart and pancreas.

Figures

Figure 1

Reduced virus titers and pancreatitis in infected Panc-KO mice. A) Mice were infected with 1×105 PFU/mouse of CVB3-H3 and blood glucose levels were measured daily. B) After 6 days, virus titers in pancreas and heart were determined by plaque assay. Each symbol represents the virus titer (PFU per gram of tissue) from an individual mouse. Means for each experimental group are indicated by horizontal black lines (n= 8–9 for each genotype). C) Virus titers in mice infected with 1×103 PFU/mouse of CVB3-H3. Titers combined from three separate experiments (n= 12–14 for each experimental group) are shown: the colored box encloses the 25th to 75th percentiles, the mean is depicted as a line, and the range for the entire group is shown as bars. Shaded regions represent the lower limits of viral detection. D) Pancreas sections stained with hematoxylin and eosin. Infected tissues were examined 6 days after infection with 1×105 PFU/mouse. Scale bars, 80 microns. E) Severity scores for pancreatitis and myocarditis (day 6) in control (n=8) and Panc-KO mice (n=9), determined as described in Experimental Procedures. Asterisks indicate a statistically significant difference between the Panc-KO and control groups by Student’s t test (*, p<0.05; ** p<0.005)

Figure 2

Reduced virus titers and myocarditis in Hrt-KO mice. A) Mice were infected with 1×105 PFU/mouse of CVB3-H3, and sacrificed 7 days post-infection. Each symbol represents the titer (PFU per gram of tissue) from an individual mouse. Means for each experimental group are indicated by horizontal black lines (n= 8 for each genotype). B) Serial titers in mice infected with 1×105 PFU (n=3–5 mice per experimental group). C) Hematoxylin and Eosin (H & E, top) and Masson’s Trichrome stain (bottom) of heart tissue sections. Uninfected samples are shown in the left-hand panels; sections from infected mice (day 7) are shown on the right. Scale bars, 80 microns. D) Severity scores for myocarditis and pancreatitis in control (n=8) and Hrt-KO mice (n=8), assessed as described in Experimental Procedures. E) Differential induction of cytokine and chemokine expression in heart tissue 7 days post infection (1×105 PFU), measured by quantitative RNA PCR. Expression is measured relative to uninfected control mice of the same genotype. Error bars are SEM, and asterisks indicate a statistical difference between the Hrt-KO and controls (*, p<0.05; ** p<0.005; #, p= 0.058).

Figure 3

CAR-deficient cardiomyocytes resist infection in vitro. A) Primary cardiomyocytes were isolated and expression of CAR (green) and the cardiomyocyte-specific marker sarcomeric myosin (red), was detected by indirect immunofluorescence. Cell nuclei were stained with DAPI (blue). Top panels show a representative field of cardiac cells isolated from control hearts; most CAR-positive cells express the cardiomyocyte marker. Bottom panels show an unusual field of Hrt-KO cardiac cells with some CAR-positive cells; note that these CAR-positive cells do not express the cardiomyocyte marker. Scale bars, 20 microns. B) CAR-negative cardiomyocytes are protected from coxsackievirus infection. Primary cardiomyocyte cultures were infected with CVB3-H3 (10 PFU/cell) for 48 hours. Expression of viral protein (green) and the cardiomyocyte-specific marker sarcomeric myosin (red) was detected by indirect immunofluorescence. Top panels show a representative field of cardiomyocytes isolated from control hearts; most of the virus-infected cells are cardiomyocytes. Lower panels show a field of Hrt-KO cardiomyocytes that contains virus-infected cells (most of the fields have no infected cells); note that the virus-infected Hrt-KO cells do not express the cardiomyocyte marker. Scale bars, 20 microns. C) Total numbers of virus-infected cardiomyocytes and non-cardiomyocytes. D) Virus titers produced by infected cardiac cells (Mean ± SEM for triplicate wells; **, p<0.005).