Mycobacterium avium Infection in a C3HeB/FeJ Mouse Model

Abstract

Infections caused by Mycobacterium avium complex (MAC) species are increasing worldwide, resulting in a serious public health problem. Patients with MAC lung disease face an arduous journey of a prolonged multidrug regimen that is often poorly tolerated and associated with relatively poor outcome. Identification of new animal models that demonstrate a similar pulmonary pathology as humans infected with MAC has the potential to significantly advance our understanding of nontuberculosis mycobacteria (NTM) pathogenesis as well as provide a tractable model for screening candidate compounds for therapy. One new mouse model is the C3HeB/FeJ which is similar to MAC patients in that these mice can form foci of necrosis in granulomas. In this study, we evaluated the ability of C3HeB/FeJ mice exposure to an aerosol infection of a rough strain of MAC 2285 to produce a progressive infection resulting in small necrotic foci during granuloma formation. C3HeB/FeJ mice were infected with MAC and demonstrated a progressive lung infection resulting in an increase in bacterial burden peaking around day 40, developed micronecrosis in granulomas and was associated with increased influx of CD4⁺ Th1, Th17, and Treg lymphocytes into the lungs. However, during chronic infection around day 50, the bacterial burden plateaued and was associated with the reduced influx of CD4⁺ Th1, Th17 cells, and increased numbers of Treg lymphocytes and necrotic foci during granuloma formation. These results suggest the C3HeB/FeJ MAC infection mouse model will be an important model to evaluate immune pathogenesis and compound efficacy.

Keywords: C3HeB/FeJ mouse model; Mycobacterium avium; immunity; nontuberculosis mycobacteria; pathology.

FIGURE 1

Mycobacterium avium infection in C3HeB/FeJ mice. Bacterial counts in the lungs (A), spleens (B), and livers (C) of C3HeB/FeJ mice infected with 2,000 MAC 2285 rough strain per mouse are shown. CFU were determined at 1, 20, 30, 40, 50, and 60 days after infection by plating serial dilutions of organ homogenates on nutrient 7H11 and TSA agar and quantifying CFU after 3 weeks incubation at 37°C. The C3HeB/FeJ mice after 40 days of infection showed increased bacterial burden ∼4.5 to 5.0 log₁₀ in the lungs followed by bacterial burden plateauing during chronic phase of infection ∼50–60 days (A). The C3HeB/FeJ mice showed a delay in bacterial dissemination in both the spleens (B) and liver (C) peaking after ∼50–60 days to ∼4.0 log₁₀ CFU in the spleen and ∼3.0 log₁₀ CFU in the liver. Results represent the average of two experiments (n = 5 mice per time point) and are expressed as log₁₀ CFU (±SEM).

FIGURE 2

Pulmonary pathology in Mycobacterium avium infection C3HeB/FeJ mice. Lung pathology of MAC-infected C3HeB/FeJ mice. Shown are representative photomicrographs of haematoxylin-eosin-stained (left and middle columns) and of acid-fast stain (right column) of the lungs of MAC -infected C3HeB/FeJ mice. As early as 20–30 days after infection, small granulomas are evident in C3HeB/FeJ mice. As disease progressed (day 40), significant increase in granuloma size and bacterial burden (denoted by acid-fast staining) are found. During chronic phase of infection between 50 and 60 days the number of granulomas increased with increased clusters of acid-fast-staining bacilli, accumulating in areas of necrosis (N) (arrows). Magnifications, 1X (left), 20X (middle) and 100X (right).

FIGURE 3

Kinetic influx of CD4 and CD8 T cell effector and memory cells in M. avium-infected C3HeB/FeJ mice. Increased percentages of activated effector and memory T cells were present after a moderate-dose infection of MAC 2285 rough in C3HeB/FeJ mice analyzed by flow cytometry compared to naïve controls. T cells were gated with a primary gate on viable FSC^low vs. SSC^low lymphocytes compared to the isotype controls (A–C) and then on CD3⁺ T cells, and analyzed for changes in the total mean cell number of CD3⁺CD4⁺ and CD3⁺CD8⁺ (D,G) cells over the course of infection. Shown are the numbers of activated effector T cells (CD4⁺CD44^hi and CD8⁺CD44^hi) (E,F) and memory T cells (CD4⁺CD44^hiCD127⁺ and CD8⁺CD44^hiCD127⁺) (F,I) migrating to the lungs of C3HeB/FeJ mice peaking after 40–50 days of infection. During the later stages on day 60 of infection, C3HeB/FeJ mice expressed diminished numbers of activated effector and memory T cells. Results represent the mean number of cells of five mice for each condition from two independent experiments (±SEM).

FIGURE 4

Kinetic influx of CD4 and CD8 T cells expressing IFN-γ, IL-17, and Foxp3 in M. avium-infected C3HeB/FeJ mice. Lung cells obtained from MAC-infected C3HeB/FeJ and naive control mice were analyzed by flow cytometry. (A–C) show CD4⁺ effector cells expressing IFN-γ, IL-17 and CD25^hiFoxp3. MAC-infected C3HeB/FeJ mice showed increased numbers of CD4⁺IFN-γ⁺ and CD4⁺IL-17⁺ producing cells peaking between 40 and 50 days after infection with concomitant increased numbers of CD4⁺CD25^hiFoxp3⁺ cell compared to the naïve mice. (D–F) show CD8⁺ effector cells expressing IFN-γ, IL-17, and CD25^hiFoxp3. Interestingly, CD8⁺IFN-γ⁺ effector cells demonstrated decreased migration to the lungs with a concomitant increased number of CD8⁺IL-17⁺ and CD8⁺CD25^hiFoxp3⁺ cells compared to the naïve mice. The data are expressed as the mean number of pulmonary cells in each organ ± SEM (n = 5 mice for each condition from two independent experiments (±SEM).

FIGURE 5

Kinetic influx of CD11b⁺ macrophages and CD11c⁺ dendritic cells in M. avium-infected C3HeB/FeJ mice. After the indicated times of MAC infection of C3HeB/FeJ mice and naïve mice, the lung cells obtained were analyzed by flow cytometry. Kinetic influx of CD11b⁺ macrophages and CD11c⁺ dendritic cells expressing MHC-II⁺ (A,D), IL-27⁺ (B,E) and PD-L1⁺ (C,F) of MAC infected C3HeB/FeJ mice at the indicated times. The data are expressed as the mean number of pulmonary cells in each organ ± SEM (n = 5 mice per group). Results represent the mean number of cells of five mice for each condition from two independent experiments (±SEM).