Elevated mitochondrial superoxide disrupts normal T cell development, impairing adaptive immune responses to an influenza challenge

Abstract

Reactive oxygen species (ROS) are critical in a broad spectrum of cellular processes including signaling, tumor progression, and innate immunity. The essential nature of ROS signaling in the immune systems of Drosophila and zebrafish has been demonstrated; however, the role of ROS, if any, in mammalian adaptive immune system development and function remains unknown. This work provides the first clear demonstration that thymus-specific elevation of mitochondrial superoxide (O(2)(•-)) disrupts normal T cell development and impairs the function of the mammalian adaptive immune system. To assess the effect of elevated mitochondrial superoxide in the developing thymus, we used a T-cell-specific knockout of manganese superoxide dismutase (i.e., SOD2) and have thus established a murine model to examine the role of mitochondrial superoxide in T cell development. Conditional loss of SOD2 led to increased superoxide, apoptosis, and developmental defects in the T cell population, resulting in immunodeficiency and susceptibility to the influenza A virus H1N1. This phenotype was rescued with mitochondrially targeted superoxide-scavenging drugs. These findings demonstrate that loss of regulated levels of mitochondrial superoxide lead to aberrant T cell development and function, and further suggest that manipulations of mitochondrial superoxide levels may significantly alter clinical outcomes resulting from viral infection.

Figure 1

SOD2 is conditionally and effectively removed from T-cells. (A) Conventional PCR analysis of floxed or excised SOD2 products from genomic DNA extracted from 6-week old SOD2^L/L or SOD2^−/− thymocytes. (B) Quantitative real-time RT-PCR analysis of SOD2 mRNA extracted from 6-week old SOD2^L/L or SOD2^−/− thymocytes. Results shown as normalized to SOD2^L/L. (C) Comparison of SOD2 protein in 6-week old SOD2^L/L or SOD2^−/− thymocytes. Beta-actin (ACTβ) shown as loading control. (D) Activity assay performed on protein extracted from 6-week old SOD2^L/L or SOD2^−/− thymocytes. At least 3 mice per experiment were analyzed; data are shown as mean and s.d. Where applicable, * = p<0.01 by Student’s t-test versus SOD2^L/L.

Figure 2

The loss of SOD2 causes superoxide specific oxidative stress in T-cells. (A) Quantification of total (entire bar) and superoxide specific (black section of bar) dihydroethidium (DHE) staining in 6-week old thymocytes. (B) The peroxide-sensitive probe dichlorofluorescein-diacetate (DCFH) demonstrated no significant change between SOD2^L/L or SOD2^−/− animals. (C) Quantification of total cellular aconitase and succinate dehydrogenase (SDH) activity in 6-week old thymocytes. Black bars indicate SOD2^L/L and open bars indicate SOD2^−/−. (D) Western blots of the iron-sulfur containing proteins aconitase 2 (ACO2) and succinate dehydrogenase B (SDHB) demonstrated no significant change in protein levels. For western blots, beta-actin (ACTβ) is displayed as loading control. At least 3 mice per experiment were analyzed; data are shown as mean and s.d. Where applicable, * = p<0.01 by Student’s t-test versus SOD2^L/L.

Figure 3

Subcellular and mitochondrial alterations are consequences of T-cell specific loss of SOD2. (A) Representative transmission electron (TEM) images of SOD2^L/L (left) and SOD2^−/− (right) 6-week old thymocytes. Open arrowheads indicate mitochondria. (B) Quantitative morphometry of thymocyte cellular area (left), mitochondrial number (center), and mitochondrial density (right). Each “X” represents one data analysis point; clear bars represent means. (C) Representative transmission electron (TEM) images of two SOD2^−/− 6-week old thymocytes. Solid arrowheads indicate apparent autophagic vesicles commonly seen in knockout animal thymocytes. (D) Western blot analysis comparing the increase of cleavage product (i.e. LC3B-II) of the autophagic marker LC3. At least 3 mice per experiment were analyzed. Where applicable, * = p<0.01 by Student’s t-test versus SOD2^L/L.

Figure 4

Significant T-cell developmental aberrations are observed in SOD2^−/− animals. (A) Left, total thymic cellularity counts at different ages of T-cell development for SOD2^L/L (blue circles) and SOD2^−/− (red diamonds) animals. Clear bars show SOD2^L/L averages, while solid bars show SOD2^−/− averages. Middle, total cell counts for three peripheral lymphoid organs at 6-weeks of age. Right, total thymic cellularity counts for 6-week old mice that were created using the Vav-iCre promoter. No significant change in thymic numbers is noted when knocking-out SOD2 at a different stage in T-cell development. (B) Left, representative flow scatter diagrams of SOD2^L/L (left) and SOD2^−/− (right) 6-week old thymocytes for the developmental markers CD4 and CD8. Right, quantification of CD4+ and CD8+ 6-week old thymocytes. Colored arrowheads (left) correlate to respective colored quantification (right); Solid bars and arrowheads indicate SOD2^L/L and open bars and arrowheads indicate SOD2^−/−. (C) Left, Quantification of flow cytometric analysis of γδ+ T-cells, as well as for markers indicative of T-cell activation MEL14 and CD44 on splenic CD4+ cells (Middle) and CD8+ cells (Right). In all graphs, solid bars indicate SOD2^L/L and open bars indicate SOD2^−/−. At least 3 mice per experiment were analyzed; data are shown as mean and s.d. Where applicable, * = p<0.01 by Student’s t-test versus SOD2^L/L.

Figure 5

Apoptosis explains the decrease in SOD2^−/− thymocytes, which may be rescued by superoxide scavenger supplementation. (A) Representative annexin V and propidium iodide flow scatter diagrams of 6-week old thymocytes from vehicle treated SOD2^L/L (left), vehicle treated SOD2^−/− (middle), and Mito-CTPO treated SOD2^−/− (right) mice. (B) Left, total thymic cellularity with and without pharmaceutical anti-oxidant supplementation. Middle, quantification of annexin V+ and propidium iodide+ thymocytes with and without pharmaceutical anti-oxidant supplementation. Colored quantification correlates to respective colored arrowheads in part (A). Right, regression analysis of total thymic cellularity versus apoptotic fraction. At least 3 mice per experiment were analyzed; data are shown as mean and s.d. Where applicable, * = p<0.01 by Student’s t-test versus vehicle treated SOD2^L/L. Where applicable, ¥ = p<0.01 by Student’s t-test versus vehicle treated SOD2^−/−.

Figure 6

SOD2^−/− mice are immunologically susceptible to influenza virus, but may be rescued by superoxide scavenging pharmaceuticals. (A) Left, Kaplan-Meier analysis of mice succumbing to IAV, H1N1 infection during a two-week period after administration with and without Mito-CTPO administration. Right, relative weight loss of mice over a two-week period after infection with IAV with and without Mito-CTPO supplementation. Black squares/Solid line: Vehicle treated SOD2^L/L, Black triangles/Dotted line: Vehicle treated SOD2^−/−, Red circles/Dotted line: Mito-CTPO treated SOD2^−/−. (B) Left, total lymphocyte counts harvested from lungs of both SOD2^L/L and SOD2^−/− animals 8 days post influenza virus infection. Also, flow cytometric analysis of CD8+ influenza-specific NP366 and PA224 MHC-tetramer positive cells (Middle) and analysis of NP366 and PA224-specific IFNγ+/TNFα+ (Right) CD8+ T-cells isolated from lungs 8 days post IAV, H1N1 infection. At least 6 mice per experiment were analyzed; data are shown as mean and s.d. For weights and graphs, * = p<0.01 by Student’s t-test versus SOD2^L/L. For mortality, * = p<0.01 by log-rank analysis versus SOD2^L/L.