Murine gammaherpesvirus-68-induced interleukin-10 increases viral burden, but limits virus-induced splenomegaly and leukocytosis

Abstract

Based on its genomic sequence and its pathogenesis, murine gammaherpesvirus-68 (gammaHV-68) has been established as a tractable model for the study of viral infections caused by the human gammaherpesviruses, Epstein-Barr virus or human herpesvirus-8. Despite significant advances, the mechanisms responsible for gammaHV-68-induced alterations in the protective host response, and the accompanying virus-induced leukocytosis, are not clear. In the present study, we questioned whether viral infection resulted in endogenous interleukin-10 (IL-10) production that might alter the host response. Infection of C57BL/6 mice resulted in increased IL-10 expression, demonstrating that gammaHV-68 could induce endogenous production of this cytokine. Infected C57BL/6 mice demonstrated the characteristic splenomegaly associated with this viral infection, however, we were surprised to discover that the splenomegaly was greater in syngeneic mice genetically deficient in IL-10 (IL-10-/-). These results strongly suggested that endogenously produced IL-10 might serve to limit leukocytosis in wild-type mice. Quantification of viral burden demonstrated a significant elevation in C57BL/6 versus IL-10-/- mice, with increases in virus being observed in both the macrophage and B-lymphocyte populations. The decreased viral load in syngeneic IL-10-/- mice correlated with an increased expression of endogenous IL-12, suggesting a mechanism of protection that was IL-12 dependent. Taken together, these studies demonstrate a surprising dichotomy for endogenous IL-10 production during gammaHV-68 infection. While the lack of IL-10 results in increased IL-12 expression and a lower viral burden, IL-10-/- mice also experience an increased leukocytosis.

Figure 1

γHV-68 induced IL-10 expression in C57BL/6 mice. Groups of C57BL/6 or IL-10^−/− mice were intranasally inoculated with 1000 PFU of γHV-68 while one group received a mock infection. Fifteen days post-infection groups of animals were killed and RNA was isolated from the spleens of individual mice (a) or from purified populations of splenic macrophages, B cells, or CD4⁺ T cells (b). RNA was subjected to RT-PCR to detect IL-10 mRNA expression. Results in (a) and (b) are presented as amplified PCR products electrophoresed on ethidium-bromide-stained agarose gels, and are representative of four or two separate experiments, respectively. For quantification of IL-10 secretion, groups of C57BL/6 and syngeneic IL-10^−/− mice were inoculated intranasally with 1000 PFU. of γHV-68. Fifteen days post-infection mice were killed and sera were collected for quantification of IL-10 by ELISA (c). ELISA results are presented as the mean values of four separate mice ±standard deviations, and are representative of three separate experiments. The asterisk indicates statistical significance (P < 0·01).

Figure 2

Viral burden in the spleens of C57BL/6 or IL-10^−/− mice. Groups of C57BL/6 or IL-10^−/− mice were infected intranasally with 1000 PFU of γHV-68. Fifteen days post-infection mice were killed and splenic DNA or splenic leucocytes were isolated from individual mice. DNA was used for PCR amplification of the gene encoding viral gp150 (a). Results in (a) are shown as a PCR-amplified 462-bp DNA fragment of γHV-68 gp150 electrophoresed on ethidium bromide-stained agarose gels, and are representative of four animals per group. This experiment was performed three separate times with similar results. In addition, isolated splenic leucocytes from mock-infected or virus-infected C57BL/6 or IL-10^−/− mice were plated on a permissive monolayer of NIH-3T3 fibroblasts to quantify levels of latent virus. Results are reported as the mean±standard deviations of seven animals per group, and results are shown as the number of infectious centres per 10⁷ lymphocytes or the number of infectious centres per spleen (b). Samples with fewer than 1 PFU were considered to be below the detection limit of this assay. Asterisks indicate statistical significance (P < 0·01). This experiment was performed twice with similar results.

Figure 3

Kinetics of viral burden in the spleens of C57BL/6 or IL-10^−/− mice. Groups of C57BL/6 or IL-10^−/− mice were infected intranasally with 1000 PFU of γHV-68. At 11, 15, 19, or 23 days post-infection mice were killed and splenic DNA or splenic leucocytes were isolated from individual mice. DNA was used for PCR amplification of the gene encoding viral gp150 (a). Results in (a) are shown as a PCR-amplified 462-bp DNA fragment of γHV-68 gp150 electrophoresed on ethidium-bromide-stained agarose gels, and are representative of two animals per group. This experiment was performed twice with similar results. In addition, isolated splenic leucocytes from mock-infected or virus-infected C57BL/6 or IL-10^−/− mice were plated on a permissive monolayer of NIH-3T3 fibroblasts to quantify levels of latent virus. Results are reported as the mean±standard deviations of four animals per group, and results are shown as the number of infectious centres per 10⁷ lymphocytes (b). At all time points, levels of latent virus in C57BL/6 mice were higher than in IL-10^−/− mice (P < 0·05). This experiment was performed twice with similar results.

Figure 4

Quantification of viral latency in isolated splenic leucocyte populations of C57BL/6 and IL-10^−/− mice. Groups of C57BL/6 or IL-10^−/− mice were intranasally infected with 1000 PFU of γHV-68 and killed 15 days post-infection. Splenic macrophages (CD11b⁺), B lymphocytes (B220⁺), or leucocytes depleted of macrophages and B lymphocytes (CD11b⁻ B220⁻) were purified by magnetic cell sorting. DNA was isolated from cell populations and subjected to PCR amplification (a). Results are shown as an amplified 462-bp DNA fragment of γHV-68 gp150 electrophoresed on ethidium-bromide-stained agarose gels. This experiment was performed twice with similar results. To quantify latent virus, isolated cell populations were plated on a permissive monolayer of NIH-3T3 fibroblasts (b). Results are shown as means±standard deviations of cells isolated from three different mice. For all cell populations, latent virus in C57BL/6 mice was higher than in IL-10^−/− mice (P < 0·05). This experiment was performed twice with similar results.

Figure 5

Increased IL-12 expression in infected IL-10^−/− versus C57BL/6 mice. Groups of C57BL/6 and IL-10^−/− mice were inoculated intranasally with 1000 PFU of γHV-68. Fifteen days post-infection mice were killed and splenic RNA was isolated from two mice per group. RT-PCR was performed to quantify IL-12 p40, IFN-γ and glyceraldehyde 3-phosphate dyhydrogenase (G3PDH) mRNA expression (a). Results are shown as PCR-amplified products electrophoresed on ethidium-bromide-stained agarose gels. This experiment was performed twice with similar results. To quantify IL-12 p40/p70 secretion, sera were collected on day 15 post-infection and used for ELISA (b). ELISA results are presented as the mean values of four separate mice ±standard deviations, and are representative of two separate experiments. The asterisk indicates statistical significance (P < 0·01).

Figure 6

Spleen weight and leukocytosis of γHV-68-infected C57BL/6 and IL-10^−/− mice. Groups of C57BL/6 and IL-10^−/− mice were given one of the following treatments: (1) intranasal inoculation of 1000 PFU of γHV-68, (2) intranasal inoculation of UV-inactivated γHV-68, or (3) intranasal administration of media only. Fifteen days post-infection mice were killed and their spleens were excised and weighed (a). Single cell suspensions were then made to quantify the number of leucocytes present per spleen (b). Results from (a) and (b) represent mean values of five separate animals ±standard deviations. These studies were repeated three times with similar results. Asterisks indicate statistical significance (P < 0·01).