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, 8 (5), e1002670

Age-dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

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Age-dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Johanna Pott et al. PLoS Pathog.

Abstract

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3(-/-)) or the adaptor molecule Trif (Trif(Lps2/Lps2)) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Age-dependent epithelial PRR expression.
(A and D) Expression levels of certain innate immune receptors as indicated in the figure were comparatively analysed by microarray analysis in IECs isolated from 3-day-old versus 21-day-old mice (A) and from 28-day-old germ-free versus conventional mice (D). (B) IECs of 2, 4, 9, 14, 20 and 27-day-old mice were analyzed by quantitative RT-PCR for the expression level of Tlr3 (n = 3/time point). The values were normalized to the expression of Gapdh. (C) Tlr3 expression levels in liver tissue (n = 3), isolated small intestinal IECs (n = 3) and immune cells (IC, 3-day-old mice: n = 10, 21-day-old mice: n = 12) were determined by quantitative RT-PCR and normalized to the expression of Gapdh. Data represent means ± SD (*p<0.05; **p<0.01; ***p<0.001, unpaired t test).
Figure 2
Figure 2. Age-dependent epithelial Tlr3 responsiveness and susceptibility towards rotavirus infection.
(A and B) Gene expression in IECs from suckling (3–7-day-old) and adult (21–28-day-old) mice following intraperitoneal injection of 50 ng poly(I∶C)/g body weight was examined. Ifn-β (A) and Isg15 (B) expression in isolated IECs 2 h after injection was analyzed by quantitative RT-PCR. (C) Viral load in IECs of suckling (n = 6) versus adult (n = 4) mice at day 4 after oral infection with the murine rotavirus strain EDIM was determined by ELISA. Data represent means ± SD (*p<0.05; ***p<0.001, unpaired t test).
Figure 3
Figure 3. Rotavirus susceptibility of TrifLps2/Lps2 and Tlr3−/− mice.
(A) Adult wild-type (wt) (n = 6), Tlr3−/− (n = 7) and TrifLps2/Lps2 (n = 5) mice were orally infected with rotavirus EDIM and shedding in fecal droppings was monitored by ELISA at day 4 p.i. in parallel with a serial dilution of a RRV virus stock with known virus titer to facilitate determination of infectious units (IU). (B) Adult wild-type (wt) and TrifLps2/Lps2 mice were orally infected with rotavirus EDIM and monitored for rotavirus antigen shedding between day 1 and day 10 p.i. (wt n = 8; TrifLps2/Lps2 n = 7). The relative OD450 nm values normalized to the values obtained from wt animals at day 4 p.i. are shown to facilitate comparison of independent experiments. (C) Suckling wt and TrifLps2/Lps2 mice were orally infected with rotavirus EDIM and colon homogenates were analyzed for IU at day 4 p.i. as described for (A). (D) OD450 nm values normalized to the values obtained from wt animals at day 5 p.i. were measured in colon homogenates of suckling wt and TrifLps2/Lps2 mice at day 5, 10 and 14 p.i. (n = 3, time point and genotype). (*p<0.05; **p<0.01, ***p<0.001, Mann-Whitney test).
Figure 4
Figure 4. Trif-deficiency in the non-hematopoitic compartment significantly contributes to rotavirus clearance.
Bone marrow chimeric wt animals reconstituted with wt bone marrow (n = 12), wt animals reconstituted with TrifLps2/Lps2 bone-marrow (n = 16), and TrifLps2/Lps2 animals reconstituted with wt bone marrow (n = 15) were generated and orally infected with rotavirus EDIM. Viral antigen shedding in feces was determined by ELISA. Values are expressed as OD450 nm relative to the absorption of the wt group at 4, 5, 6 and 7 days p.i.. Data are pooled from two independent experiments. (*p<0.05; **p<0.01, Mann-Whitney test).
Figure 5
Figure 5. Tlr3/Trif-dependent response of adult but not neonatal IECs during rotavirus infection.
(A–C) Quantitative RT-PCR analysis of mRNA prepared from IECs isolated from uninfected versus rotavirus infected adult mice at day 4 p.i.. Wt (n = 4) versus TrifLps2/Lps2 (n = 4, left panel) and wt (n = 4) versus Tlr3−/− (n = 4, right panel) mice were analyzed for expression of Ifn-λ (A), and Rantes (B). Values were normalized to the expression of Gapdh and expressed as fold-increase over the values of the wt control group. Results are representative for at least two independent experiments. (C) Suckling wt (n = 3) and TrifLps2/Lps2 (n = 3) mice were orally infected with murine rotavirus EDIM. IECs were isolated at day 4 p.i. and analyzed for the expression of Isg15 and Rantes (left and right panel, respectively). (*p<0.05; **p<0.01; ***p<0.001, unpaired t test).
Figure 6
Figure 6. Recruitment of antiviral effector cells during rotavirus infection.
(A and B) CD3 was stained in formalin-fixed and paraffin-embedded proximal parts of the small intestine of uninfected and rotavirus infected adult wild-type and TrifLps2/Lps2 mice at day 4 and 11 p.i. (A) Representative images illustrating the number of CD3+ cells in uninfected and rotavirus infected wild-type small intestinal tissue (CD3, red; wheat germ agglutinin, green; DAPI, blue; bar 50 µm) and (B) number of CD3+ cells quantified for 4 mice/time point and genotype. (C and D) Adult wt mice were orally infected with murine rotavirus EDIM and the proximal, medial and distal part of the total small intestinal tissue was analyzed at day 4 p.i. for expression levels of (C) Cd8α and (D) granzyme a (gzma). Uninfected wt mice served as controls. (E–J) The proximal part of the total small intestinal tissue of untreated or rotavirus infected wt and Tlr3−/− mice was examined by quantitative RT-PCR. The values for Cd8α and granzyme expression in wt animals are identical to the data shown for proximal expression in (A) and (B). The tissue was analyzed for expression of the following marker genes of antiviral effector cells: Cd8α (E), runt related transcription factor 2 (Runx2) (F), killer cell lectin-like receptor subfamily D, member 1 (klrd1) (G) and expression of effector molecules grzma (H), perforin 1 (prf1) (I), and nitric oxide synthase 2 (Nos2) (J). Results are representative for at least two independent experiments. (*p<0.05; **p<0.01; ***p<0.001, unpaired t test).
Figure 7
Figure 7. TLR3 and MDA5 expression in human small intestinal biopsies.
Human endoscopic samples from the duodenum of individuals between 0 and 20 years of age were analyzed by real-time PCR for the expression level of (A) TLR3 and (B) MDA5 and normalized to HPRT (age 0–5 n = 12; age 6–20 n = 28). (*p<0.05, Mann-Whitney test).

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