The cytokine osteopontin modulates the severity of rotavirus diarrhea

Abstract

Osteopontin (OPN) is a sialated phosphoprotein found in tissues and secreted into body fluids. It is an integrin ligand with pleiotropic functions as an extracellular matrix protein in mineralized tissues and a cytokine that is active in cell signaling (A. B. Tuck, C. Hota, S. M. Wilson, and A. F. Chambers, Oncogene 22:1198-1205, 2003). To determine whether OPN may be important in mucosal defense against viral pathogens, we evaluated the OPN response to rotavirus infection and the extent of diarrhea manifested by infected opn null mutant (opn-/-) mice. Reverse transcription-PCR, Northern and Western blots, and immunohistochemical studies of the HT-29 intestinal epithelial cell line and murine intestine were used to evaluate OPN mRNA and product. Intestinal closed loops and diarrheal observations determined disease severity and duration. OPN mRNA levels increased after infection of HT-29 cells, peaking in 4 to 6 h. Infected cultures contained 925 microg of OPN/ml, while for controls the levels were below detection (50 microg/ml). Infection increased OPN mRNA levels in intestinal tissue between 2 and 24 h postinoculation and increased OPN protein in intestinal fluid. The cellular localization of OPN was supranuclear and apical, and responding cells were diffusely distributed on the villus surface. Three days after infection, closed intestinal loops from opn-/- mice contained more fluid than loops from controls, although secretion levels at the onset of illness were similar. Null mutant mice experienced more intense and prolonged diarrhea than controls. Rotavirus infection of intestinal epithelial cells and murine intestine caused marked increases in OPN mRNA levels and secreted OPN protein. OPN-deficient mice suffered prolonged disease.

FIG. 1.

(A) Northern blot and (B) RT-PCR mRNA amplification of OPN and β-actin mRNA in HT-29 cells at 0 to 24 h after rotavirus infection. HT-29 cells were infected with RRV (MOI of 10). RNA was extracted, and analyses for OPN and β-actin mRNA were performed. OPN mRNA levels increased after rotavirus infection of HT-29 cells, with a peak quantity reached at 4 to 6 h p.i. The level of β-actin was constant during this period.

FIG. 2.

The effect of RRV dose on OPN expression. HT-29 cell monolayers were infected with RRV (MOIs of 1 and 10), and PCR amplification of OPN mRNA (harvested at 6 h p.i.) and Western blotting of secreted OPN were performed. For the Western blot, supernatant was harvested 16 h p.i. These times were chosen based on data in Fig. 1 showing full expression of specific mRNA or protein at these times. (A) RT-PCR analysis showed that a 10-fold increase in RRV inoculum from an MOI of 1 to an MOI of 10 resulted in an increased amount of OPN mRNA. (B) Western blotting of OPN in culture supernatants showed similar increased OPN protein with increased viral inoculum. β-Actin was the internal standard for the PCR assay, while the noninfected control cells were incubated with medium only.

FIG. 3.

RT-PCR amplification of OPN and β-actin mRNA from the RRV-infected intestines of 9-day-old mouse pups. Pups were inoculated by gavage with 50 to 100 μl of RRV (10⁸ focus-forming units/ml), and the entire small intestine was removed at the indicated time points. RNA was extracted, and RT-PCR analysis for OPN and β-actin mRNA was performed. The amount of OPN mRNA increased in response to rotavirus infection between 2 and 16 h p.i., with the maximal response at 4 to 6 h.

FIG. 4.

Adult mice increase intestinal secretion of OPN in response to murine EDIM rotavirus but not simian RRV. A Western blot analysis of OPN was performed on the intestinal fluids of EDIM- and RRV-infected 5-week-old BALB/c mice as well as sham-infected controls. OPN protein was absent from the intestinal fluids of RRV-infected adult mice at any time point, while OPN protein was induced in the intestinal fluids of adult mice orally inoculated with an equivalent dose of mouse rotavirus (EDIM) at 48 to 96 h p.i. S, sham inoculated; R, rotavirus inoculated (either RRV or EDIM, as indicated).

FIG. 5.

Localization of OPN protein in the intestine. Nine-day-old BALB/c mouse pups were orally inoculated with either RRV or TNC. Samples were harvested at 24 h p.i., and immunohistochemistry using polyclonal rabbit anti-rat OPN was performed. OPN was found localized to small intestinal epithelial cells with supranuclear and luminal accentuation in RRV- but not TNC-inoculated pups. Magnification, ×20. (A) Sham-inoculated mouse small intestine. (B) RRV-infected opn^+/+ mouse intestine. (C) RRV-infected opn^+/+ mouse stained for OPN. (D) opn^−/− mouse infected with RRV and stained for OPN. Note the extensive vacuolization (compared to panel A) but the absence of OPN staining in the RRV-infected null mutants.