Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection

Abstract

Among infectious agents, measles virus (MV) remains a scourge responsible for 1 million deaths per year and is a leading cause of childhood deaths in developing countries. Although MV infection itself is not commonly lethal, MV-induced suppression of the immune system results in a greatly increased susceptibility to opportunistic bacterial infections that are largely responsible for the morbidity and mortality associated with this disease. Despite its clinical importance, the underlying mechanisms of MV-induced immunosuppression remain unresolved. To begin to understand the basis of increased susceptibility to bacterial infections during MV infection, we inoculated transgenic mice expressing the MV receptor, CD46, with MV and Listeria monocytogenes. We found that MV-infected mice were more susceptible to infection with Listeria and that this corresponded with significantly decreased numbers of macrophages and neutrophils in the spleen and substantial defects in IFN-gamma production by CD4(+) T cells. The reduction in CD11b(+) macrophages and IFN-gamma-producing T cells was due to reduced proliferative expansion and not to enhanced apoptosis or to altered distribution of these cells between spleen, blood, and the lymphatic system. These results document that MV infection can suppress both innate and adaptive immune responses and lead to increased susceptibility to bacterial infection.

Figure 1

Concurrent MV infection impairs the clearance of LM from the host. YAC-CD46 transgenic mice were either untreated (naive), infected with MV alone, infected with LM alone, or infected with MV and 5 days later coinfected with LM. (a) LM-induced splenomegaly is reduced during acute MV infection. Representative spleens were photographed 8 days after LM infection (i.e., 13 days after MV infection). Based on 6 to 21 samples per group, the average spleen weights (± SD) were as follows: naive, 0.13 ± 0.02 g; MV, 0.18 ± 0.06 g; LM, 0.40 ± 0.15 g; and MV and LM, 0.29 ± 0.09 g. Concurrent infection with MV and LM resulted in significantly reduced splenomegaly as compared with LM infection alone (P = 0.009, Student’s t test). The scale on the left side of the figure is in centimeters. (b) Five days after MV infection, YAC-CD46 transgenic mice were coinfected with LM, and bacterial titers in the spleen were compared with those of mice that received LM in the absence of MV infection. Eight days after LM challenge, there was a sixfold increase in LM CFUs/g of spleen in mice concurrently infected with MV. This difference is statistically significant (P = 0.001, Student’s t test), indicating that coinfection with MV resulted in an increased persistence of bacteria during secondary infection. The results show the average (± SEM) of six mice per group. Equivalent results were observed in two additional experiments.

Figure 2

MV infection alters the recruitment of innate effector cells into the spleen after secondary infection with LM. Mice were either untreated (naive), infected with MV alone, infected with LM alone, or infected with MV and 5 days later coinfected with LM. Cell numbers were quantitated by flow cytometry 8 days after LM infection (i.e., 13 days after MV infection). The total numbers of cells expressing (a) CD11b⁺ (macrophages), (b) CD11c⁺ (dendritic cells), (c) DX5⁺CD3^– (NK cells), and (d) Gr-1⁺CD3^– (neutrophils) were calculated per spleen, and the average (± SEM) of four to eight mice per group is shown. Statistical significance was determined using Student’s t test. Equivalent results were observed in two additional experiments.

Figure 3

Minor alterations in apoptosis occur during concurrent MV infection. CD46 transgenic mice were infected with MV, LM, or MV and LM, and the levels of apoptosis occurring in vivo were determined 8 days after secondary bacterial infection (i.e., day 13 after primary MV infection). The degree of apoptosis was measured by quantitating the number of annexin V⁺/7AAD^– cells in (a) CD11b⁺, (b) CD11c⁺, (c) Gr-1⁺, and (d) CD3⁺ T cells. The results show the average (± SEM) of three mice per group.

Figure 4

MV-induced immunosuppression results in reduced proliferative expansion of CD11b⁺ and CD11c⁺ APCs and CD3⁺ T cells. Studies were performed using transgenic mice expressing the MV receptor CD46 (7). These mice were infected with MV, LM, or MV and LM and given BrdU from days 4–6 after LM infection (days 9–11 after MV infection) (a–c), or BrdU was administered from days 6–8 after LM infection (d). Spleen cells were stained for CD11b (a), CD11c (b), or CD4 and CD8 (c and d) before permeabilization and staining for BrdU that was incorporated into the DNA of proliferating cells. The results show the average (± SEM) of three mice per group.

Figure 5

MV-induced immunosuppression results in a decreased frequency of IFN-γ–producing T cells. T cell responses from naive mice were compared with mice infected with MV, infected with LM, or infected with MV and 5 days later coinfected with LM. T cell–mediated cytokine production was analyzed 8 days after LM infection (i.e., 13 days after MV infection) by intracellular cytokine staining after 6 hours of direct ex vivo culture with anti-CD3. Less than 0.4% of CD8⁺ or CD4⁺ T cells were IFN-γ producing after 6 hours of in vitro culture without anti-CD3 stimulation (data not shown). Only a small percentage of CD8⁺ T cells or CD4⁺ T cells from naive or MV-infected mice produced IFN-γ after anti-CD3 stimulation. In contrast, LM infection resulted in a large percentage of T cells that could be identified by IFN-γ production after anti-CD3 stimulation. MV and LM coinfection resulted in a slight decrease in IFN-γ–producing CD8⁺ T cells (P = 0.09) but a significant reduction in the percentage of IFN-γ–producing CD4⁺ T cells (P = 0.02). Equivalent results were observed in two additional experiments.