A focused salivary gland infection with attenuated MCMV: an animal model with prevention of pathology associated with systemic MCMV infection

Abstract

While the salivary gland has been recognized as an important effector site of the common mucosal immune system, a useful model for studying anti-viral salivary gland immune responses in vivo and for exploring the role of the salivary gland within the common mucosal system has been lacking. Murine cytomegalovirus (MCMV) is a beta-herpesvirus that displays a strong tropism for the salivary gland and produces significant morbidity in susceptible mice when introduced by intraperitoneal (i.p.) inoculation. This study tested the hypothesis that MCMV morbidity and pathology could be reduced by injecting the virus directly the submandibular salivary gland (intraglandular (i.g.)), using either in vivo derived MCMV or the less virulent, tissue-culture-derived MCMV (tcMCMV). Peak salivary gland viral titers were completely unaffected by infection route (i.p vs. i.g.) after inoculation with either MCMV or tcMCMV. However, i.g. tcMCMV inoculation reduced viremia in all systemic tissues tested compared to i.p. inoculation. Furthermore, systemic organ pathology observed in the liver and spleen after i.p. inoculation with either MCMV or tcMCMV was completely eliminated by i.g. inoculation with tcMCMV. Cellular infiltrates in the salivary glands, after i.p. or i.g. inoculation were composed of both B and T cells, indicating the potential for a local immune response to occur in the salivary gland. These results demonstrate that a focused MCMV infection of the salivary gland without systemic organ pathology is possible using i.g. delivery of tcMCMV.

Figure 1

I.g. inoculation of tcMCMV limits viral titers to the salivary glands. Viral titers were determined in (A, B) salivary gland, (C, D) spleen, (E, F) liver, and (G, H) lung from Balb/c mice inoculated i.p (closed symbols, solid lines) or i.g. (open symbols, dashed lines) with MCMV (A, C, E. G) or tcMCMV (B, D, F, H) using a standard plaque assay. PFU/mg tissue sample are shown. Results are pooled averages ± standard deviation from 5–6 mice per time point from 2 – 3 independent experiments. Detection limits were 0.1 PFU/mg for salivary gland, spleen and lung, and 0.2 PFU/mg for liver. *p<0.05, i.p. MCMV vs. i.g. MCMV

Figure 2

I.g. inoculation of tcMCMV results in viral titers in the saliva but not the serum. Viral titers were determined in (A) saliva or (B) serum in Balb/c mice inoculated i.p with MCMV (closed symbols) or i.g. with tcMCMV (open symbols). PFU/ml fluid are shown. Results are pooled averages ± standard deviation from 5–6 mice per time point from 2 – 3 independent experiments. Detection limits were 100 PFU/ml of saliva, and 50 PFU/ml of serum. *p<0.05, i.p. MCMV vs. i.g. tcMCMV

Figure 3

I.g. tcMCMV inoculation of Balb/c mice reduces spleen pathology and induces salivary gland infiltrates. Spleens (A - F) and salivary glands (G - L) were isolated from sham-inoculated mice (A, D, G, J), mice inoculated i.g. with tcMCMV (B, E, H, K), or i.p. with MCMV (C, F, I, L). H&E stained sections are shown on day 14 post-infection. 10X magnification (A-C, G-I), 40X magnification of A - C are shown in D - F and 40X magnification of G - I are shown in J - L, respectively, as indicated by the boxes. Results are representative of at least 3 independent experiments for each condition. PF=primary follicle, GC=germinal center, WP=white pulp, RP=red pulp, SD=secretory duct, V=blood vessel. Arrows indicate cellular infiltrates. Arrowheads indicate MCMV-infected cells.

Figure 4

I.p. MCMV and i.g. tcMCMV inoculation result in salivary gland infiltrates composed of B and T cells. Salivary glands on day 14 from sham inoculated mice (A - C) or mice inoculated i.g. with tcMCMV (D - I) or i.p. with MCMV (J - O) were stained with Abs against B220 (A, D, G, J, M), CD4 (B, E, H, K, N), or CD8 (C, F, I, L, O). 10X magnification (A - F, J - L), 40X magnification (G - I, M - O) from the indicated boxed regions are shown. Results are representative of at least 3 independent experiments for each condition.

Figure 5

I.p. and i.g. inoculation of either MCMV or tcMCVM results in MCMV specific IgM and IgG serum Abs. BALB/c mice were infected either i.p (A, B) or i.g. (C, D) with either MCMV (A, C) or tcMCMV (B, D) and MCMV-specific Abs in serum was isotyped for IgM and IgG. Samples with OD>0.200 above background were considered positive and the last positive reciprocal dilution (log₂) is shown. Detection limit for IgM was slightly higher than IgG and values of 2 – 3 were typically obtained in control samples. Results are from 1 representative experiment each: i.p. MCMV (N=3 mice/time point), i.p. tcMCMV (N=2 mice/time point), i.g. MCMV (N=2 mice/time point), i.g. tcMCMV (N=4 mice/time point). *p<0.05, compared to same time point after i.p. inoculation with MCMV; all other time points after i.g. inoculation with tcMCMV were not significantly different from i.p. inoculation with MCMV.