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Immunity. Author manuscript; available in PMC 2016 July 19. Published in final edited form as: Immunity. 2016 January 19; 44(1): 88–102. doi:10.1016/j.immuni.2015.12.002.
Apoptosis-inducing factor (AIF)-dependent mitochondrial function is required for T cell but not B cell function Sandra Milasta1, Christopher P. Dillon1, Oliver E. Sturm2, Katherine C. Verbist3, Taylor L. Brewer1, Giovanni Quarato1, Scott A. Brown1, Sharon Frase4, Laura J. Janke5, S. Scott Perry6, Paul G. Thomas1, and Douglas R. Green1 1Department
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of Immunology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
2Department
of Biology, Rhodes College, 2000 North Parkway, Memphis, TN 38112, USA
3Department
of Oncology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 4Cell
and Tissue Imaging Center, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
5Department
of Pathology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 6Department
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of Flow Cytometry, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Summary
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The role of Apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that loss of AIF in fibroblasts led to mitochondrial electron transport chain (ETC) defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deletion in T cells impacted the numbers of peripheral T cells and their ability to undergo homeostatic proliferation, while not affecting their thymic development. However, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Expression of Ndi1 rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF undergo normal death. These studies suggest that the primary role of AIF relates to complex I function, differentially affecting T and B cells.
Correspondence to: [email protected]. Author Contributions S.M. conceived the project, designed and performed most experiments, interpreted results, and co-wrote the manuscript. K.C.V, O.E.S, S.B., G.Q., L.J.J., S.F., S.S.P., C.P.D. and T.L.B. performed experiments and analysis. P.G.T provided intellectual guidance. D.R.G. conceived the project, supervised experimental designs, interpreted results, and co-wrote the manuscript.
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Introduction AIF is a mitochondrial inter-membrane space protein initially identified in an assay of mitochondrial components that cause morphological changes in isolated nuclei (Susin et al., 1999). A role for AIF in cell death was proposed based on observations that AIF is released from mitochondria during mitochondrial outer membrane permeabilization (MOMP) (Daugas et al., 2000b), a step in the mitochondrial pathway of apoptosis (reviewed in Tait and Green, 2010), and subsequently localizes to the nucleus (Daugas et al., 2000b) where it induces double stranded DNA breaks and large scale DNA condensation (Susin et al., 1999). A large number of reports have concluded that AIF mediates cell death that proceeds in the absence of caspase activity (reviewed in Candé et al., 2002; Daugas et al., 2000a).
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Germline ablation of Aif is embryonically lethal, and initial studies in vitro suggested that this lethality was due to a cavitation defect in the embryoid body (Joza et al., 2001). Subsequent studies (Brown et al., 2006) showed that the development of Aif-deficient embryos (including cavitation) is normal through embryonic day 9, followed by lethality. AIF is required for efficient assembly of complex I of the electron transport chain (ETC) and thus plays a role in maintaining normal oxidative phosphorylation (OXPHOS) (Hangen et al., 2015; Vahsen et al., 2004). Conditional deletion of AIF in muscle or liver (Pospisilik et al., 2007) confirmed this role for AIF (Pospisilik et al., 2007). AIF regulates the mitochondrial import of CHCHD4, a central component of a redox-sensitive mitochondrial inter-membrane space import machinery (Hangen et al., 2015), thus explaining its role in ETC assembly and function.
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The harlequin (Hq) mutation in mice is caused by a provirus insertion into the Aif locus, resulting in 80% loss of AIF expression (Bénit et al., 2008; Klein et al., 2002). Characterization of these animals revealed a T lymphocyte deficiency due to a failure of T cell development in the thymus, with high levels of reactive oxygen species (ROS) observed in surviving lymphocytes (Banerjee et al., 2012). Several studies in these animals (reviewed in Joza et al., 2009) suggested that cells from Hq animals are resistant to apoptosis and other forms of cell death.
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Here, we found that acute deletion of Aif in mouse embryonic fibroblasts (MEF) ablated proliferation. This effect was prevented by ectopic expression of Saccharomyces cerevisiae Ndi1, which has been shown to partially restore respiration and ETC function in mammalian cells lacking complex I activity (Santidrian et al., 2013; Seo, 1999; Seo et al., 2004). (Santidrian et al., 2013; Seo, 1999; Seo et al., 2000). To investigate the role of AIF in tissue homeostasis, we generated animals in which AIF can be ubiquitously deleted. We observed wasting and lethality upon acute deletion of AIF, accompanied by a loss of hematopoietic stem cells (HSC) and lymphocytes. However, B cells lacking AIF developed and functioned normally, despite partial deficiency in complex I. In contrast, deletion of AIF in T cells did not affect development, but profoundly impacted numbers and homeostatic proliferation of peripheral T cells in vivo. Expression of Ndi1 partially rescued the defects observed after loss of AIF. Since Aif-deficient MEF, thymocytes, and B cells displayed normal cell death, we conclude that the primary role of AIF in lymphocytes relates to complex I function and, at least in the cells studied, does not have a major role in cell death.
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Results Defects associated with loss of AIF in fibroblasts can be reversed by rescuing complex I function
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We generated SV40-immortalized MEF in which endogenous Aif is removed by 4hydroxytamoxifen (4-OHT)-mediated induction of Cre (Aifflox/y R26CreER) (Badea et al., 2003; Srinivas et al., 2001). The MEF also expressed a loxp-stop-loxp (LSL)-yellow fluorescent protein (YFP) transgene to assess recombination (Fig. S1A). Within one week after 4-OHT treatment, we observed efficient loss of AIF protein (Fig. 1A). By three weeks only YFP− cells that had not recombined the Aif locus expanded in culture (Fig. 1A, S1B). Loss of AIF expression negatively affected the expression of complexes I and IV of the ETC (Fig. 1A). An increase in mtDNA to nDNA ratio was observed following 4-OHT treatment (Fig. S1C), suggesting a compensatory effect. Consistent with this, we observed that cells lacking AIF reduced their oxygen consumption rate (OCR), and increased their extracellular acidification rate (ECAR), a consequence of lactic acid production, suggesting a switch from OXPHOS to glycolysis (Fig. 1B, S1D). Moreover, loss of AIF decreased OCR in permeabilized cells, driven by substrates for complexes I, II, and IV (Fig. 1C), consistent with diminished complex IV expression (Fig. 1A). In contrast, Aifflox/y R26CreER MEF expressing ectopic AIF displayed similar levels of complex I–IV proteins with or without deletion of endogenous AIF (Fig. 1D). Consequently, the levels of OCR and ECAR in the 4OHT-treated cells were similar to vehicle treated controls (Fig. 1E). Therefore, the changes in ETC proteins and metabolism upon ablation of AIF were due to loss of AIF protein expression.
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We then ectopically expressed Ndi1, the rotenone-insensitive yeast paralog of complex I, in Aifflox/y R26CreER MEF. The functionality of Ndi1 was confirmed by the ability of Aifflox/y R26CreER MEF expressing Ndi1 (but not vector control MEF) to clonally expand in the presence of the complex I inhibitor rotenone (Fig. S1E). In contrast to MEF without ectopic Ndi1 (Fig. 1A), the expression of Ndi1 prevented the reappearance of cells that had failed to delete Aif after 4-OHT treatment (Fig. 1D, S1F). Unlike AIF, ectopic expression of Ndi1 did not restore the expression of complex I, III and IV in Aif-deficient MEF or their substratedriven activity (Fig. 1D, S1G). Nevertheless, Ndi1 expression prevented the metabolic effects of AIF deletion. While Ndi1 elevated OCR in AIF-sufficient cells as expected (Seo, 1999; Seo et al., 2004), the change in OCR and ECAR seen upon deletion of AIF was not observed in cells expressing Ndi1 (Fig. 1E). Further, upon acute deletion of Aif by 4-OHT treatment, vector-control MEF showed a dramatic reduction in clonogenic expansion, while ectopic expression of either AIF or Ndi1 sustained such expansion (Fig. 1F).
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Unlike glucose, galactose enters glycolysis via the Leloir pathway, resulting in reduced generation of ATP via glycolysis (Qiu et al., 2013; Weinberg et al., 2010) We found that Aifdeficient MEF reconstituted with either AIF or Ndi1 expanded similarly in galactose (Fig. S1H). Therefore, the changes in cell metabolism and proliferation that occured upon deletion of AIF was prevented by expression of Ndi1, suggesting that these effects were predominantly due to loss of complex I activity. Therefore, despite its role in the import of
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several proteins involved in mitochondrial function (Hangen et al., 2015), AIF appeared to function primarily to sustain complex I activity in MEF. Cell death is largely unaffected in MEF deficient for AIF AIF was originally identified as an intermembrane space (IMS) protein involved in cell death upon MOMP (Susin et al., 1999; Wissing et al., 2004). We therefore treated Aifdeficient MEF expressing either ectopic AIF or Ndi1 with several cell death inducers with or without the pan-caspase inhibitor, qVD-oph. As shown in Fig. 1G, Aif-deficient MEF reconstituted with Ndi1 displayed normal susceptibility to apoptosis. Under conditions of caspase inhibition, cell death was diminished in all cases. Caspase-independent cell death induced by serum starvation or diazald was unaffected by AIF status, although the low level observed with STS treatment appeared to be partially dependent on AIF. Thus, AIF is not a primary mediator of cell death in MEF.
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Global ablation of AIF in adult mice causes wasting
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Complete loss of AIF leads to embryonic lethality at day E12.5 (Brown et al., 2006; Joza et al., 2005). To study the role of AIF in the adult animal we treated Aifflox/y R26CreER mice with tamoxifen. Efficient deletion of the Aif allele in various tissues upon treatment with tamoxifen was confirmed by PCR (Fig. S2A). Whereas WT animals (Aifflox/y and Aifwt/y R26CreER) appeared normal after tamoxifen treatment, the littermate mutant animals (Aifflox/y R26CreER) showed a loss of body weight and subsequent mortality (Fig. 2A, B). To determine whether removal of AIF resulted in induction of apoptosis, we generated a set of mice lacking AIF and the pro-apoptotic Bcl2 family members Bax and Bak (Aifflox/y Bak−/− Baxflox/flox R26CreER). However deletion of Bak and Bax did not protect Aifdeficient mice (Fig. S2C). The observed pathology therefore proceeded independently of the intrinsic pathway of apoptosis. Histological and morphological analyses revealed loss of crypts with crypt regeneration in the intestine and thymic hypocellularity as well as weight loss in all of the tissues examined (Fig. 2C, S2B). While a wasting effect after deletion of AIF in skeletal muscle has been reported (Joza et al., 2005), we did not observe such effects (Fig. 2C), probably due to the more rapid lethality of whole body deletion in our experiments.
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We isolated lineage negative stem cells from Aifwt/y R26CreER or Aifflox/y R26CreER mice and injected these cells as a mixture with congenic WT stem cells into lethally irradiated Rag1-deficient mice. The mice were treated with tamoxifen upon reconstitution. Deletion of AIF caused diminished frequencies of T and B cells and cells with granular features as detected by flow cytometry (Fig. 2D). We also observed reduced numbers of HSC (Fig. 2E). To test whether lethality upon acute ablation of AIF was caused by the collapse of the hematopoietic system, we transferred WT BM-derived stem cells into lethally irradiated WT or Aifflox/y R26CreER animals and allowed for complete reconstitution before tamoxifen treatment. The reconstituted Aif-deficient animals lost weight similar to tamoxifen-treated Aifflox/y R26CreER animals (Fig. S2D). Thus, the loss of the hematopoietic system was not solely responsible for wasting and lethality seen in Aif-deficient animals.
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Targeted ablation of AIF has no effect on B cell development and function
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In the AIF hypomorph harlequin mouse (Hq) B cells are unaffected (Banerjee et al., 2012). To study the role of AIF in B cell development and function, we generated conditional Aif mice (Aifflox/y) with Cre-recombinase under control of the B cell specific CD19 promoter (Cd19-Cre), and a LSL-YFP transgene to label cells with active Cre (Aifflox/y Cd19-Cre LSL-YFP). Animals were born healthy and viable. PCR and immunoblot analysis confirmed deletion of the Aif allele only in the B cell lineage (Fig. 3C, S3B). We did not detect any differences in B cell development between mutant animals (Aifflox/y Cd19-Cre LSL-YFP hereafter referred to as B-AIF KO) and littermate controls (Aifwt/y Cd19-Cre LSL-YFP hereafter referred to as B-AIF WT), as frequencies and numbers of B cells in the BM and spleen did not differ between the B-AIF WT and KO mice (Fig. 3A, 3B, S3A). While we cannot exclude the possibility that the AIF protein was expressed during the early stages of development directly following Cre activation, subsequent B cell expansion and development were unaffected by AIF deletion.
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We then examined the effects of AIF loss on ETC protein expression and function in mature B cells. In agreement with other reports (Vahsen et al., 2004), AIF deficiency did not affect expression of several ETC subunits at the mRNA level, but resulted in loss of complex I, III and IV proteins (Fig. 3C, 3D, S3C). We did not observe any change in the activity of complex I, but detected increases in OCR in permeabilized cells driven by complex II and IV substrates (Fig. 3E), consistent with a compensatory increase in complex IV activity. Consequently, we found that both OCR and ECAR were comparable in the basal state and after addition of FCCP (to uncouple ATP synthesis from the ETC to determine the spare respiratory capacity) between intact WT and Aif-deficient B cells (Fig. 3F). In addition, glycolysis, fatty acid β-oxidation (FAO), glutaminolysis, and ATP content were all similar between the WT and mutant mice (Fig. S3D–G). Complex I impairment often leads to increased ROS generation (Robinson, 1998; S Pitkanen, 1996). However, measurement of ROS levels revealed no significant differences in response to loss of AIF (Fig. 3G), consistent with other reports in AIF KO liver or muscle (Chinta et al., 2009; Pospisilik et al., 2007). In vitro proliferation after lipopolysaccharide (LPS) stimulation (Fig. S3H), in vivo ovalbumin-specific antibody production (Fig. S3I), and expansion of antigen-specific antibody forming cells (AFC) after influenza infection (Fig. 3H) were not affected by AIF deletion. Therefore, B cells did not require the expression of AIF or optimal expression of mitochondrial complex I, III and IV proteins for their development and functionality. B cell death is unaffected by the absence of AIF
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As AIF does not appear to be important for survival of B cells, we examined the involvement of AIF in regulating caspase-dependent and -independent cell death in these cells. Naïve Aifflox/y Cd19-Cre LSL-YFP and WT B cells displayed equivalent susceptibility to cell death induced by STS, menadione, and diazald, as well as in response to serum withdrawal following activation with LPS (Fig. 3I), although a minor effect of AIF ablation was observed at the highest concentration of STS. The presence of qVD-oph delayed cell death in each case, but the ensuing death was unaffected by AIF deletion (Fig. 3I). These effects were also observed for spontaneous cell death of B cells in culture, represented by
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untreated controls. Thus, loss of AIF did not confer protection against caspase-dependent or –independent cell death in B cells. AIF plays a pivotal role in peripheral T cell function and survival
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We observed that acute deletion of AIF in the hematopoietic system affected T cells more rapidly than B cells (Fig. 2D). To conditionally delete Aif in T cells, we generated Aifflox/y animals expressing Cre on the proximal Lck promoter (Lck-Cre), with LSL-YFP and confirmed efficient Aif-deletion in YFP+ T cells (Fig. 4B, 4C, S4A). Loss of AIF led to a reduction in the expression of complex I, III and IV proteins. We also detected a significant increase in the expression of complex II components (Fig. 4B, C), suggesting a compensatory effect in T cells. While Aifflox/y Lck-Cre LSL-YFP (T-AIF KO) mice had similar numbers of total splenocytes as Aifwt/y Lck-Cre LSL-YFP (T-AIF WT) littermate controls, fewer CD4+, YFP+ and substantially fewer CD8+, YFP+ T cells were present (Fig. 4A, S4B). This attrition was also observed among NK1.1+ cells, but not among γδ T cells (Fig. S4C). We then examined mitochondrial function in Aif-deficient T cells. No differences were detected in ROS between the T cells isolated from T-AIF WT and T-AIF KO animals (Fig. S4D). Although basal state OCR and ECAR were similar in resting Aifdeficient T cells compared to littermate WT T cells (Fig. 4D), loss of AIF significantly affected the OCR in response to complex II and IV substrates (Fig. 4E), and reduced ATP content in naïve cells (Fig. 4F). In accordance with other reports (Wang et al., 2011), we found that T cells increased OCR and ECAR after 24 hrs of activation (Fig. 4D). AIF KO T cells, however, increased their basal OCR and uncoupled OCR to a lesser degree after activation but exhibited similar levels of ECAR compared to WT T cells (Fig. 4D). The effect on OCR was also observed in permeabilized cells provided with different substrates (Fig. 4E). These observations support the conclusion that AIF deficiency in T cells negatively affects OXPHOS.
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We then examined the effects of AIF deficiency on T cell activation. Following treatment with anti-CD3 and anti-CD28, WT and Aif-deficient T cells similarly expressed the activation markers CD25 and CD69 (Fig. S4E). Results assessing activation-induced cell proliferation in vitro were inconsistent (data not shown). It is possible, therefore, that the small numbers of Aif-deficient T cells that persist in the periphery include variable numbers of cells that have sufficiently compensated for the loss of AIF (e.g., via increased mitochondrial biogenesis) such that their function can be sustained. If so, defects might be revealed by the extensive homeostatic expansion of purified WT or Aif-deficient T cells in Rag1−/− animals in vivo. We co-injected AIF WT or AIF KO T cells with congenic T cells, and found that AIF KO T cells did not expand as well as AIF WT T cells (Fig. 4G). Notably, CD8+ T cells were more sensitive to loss of AIF than were CD4+ T cells. Similarly, we examined homeostatic expansion in vivo under non-competitive conditions. AIF KO CD4+ T cells displayed no defect in homeostatic expansion under these conditions, suggesting that these cells were able to satisfy their energetic demands, whereas AIF KO CD8+ T cells were unable to proliferate under these conditions in vivo (Fig. 4H). We thus conclude that AIF is essential for T cell function in vivo.
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T cells develop normally in the absence of AIF
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Ubiquitous diminution of AIF expression in the Hq mouse results in thymic arrest at the βselection stage, resulting in significantly reduced numbers of mature CD4+ and CD8+ T cells (Banerjee et al., 2012). We therefore asked if the diminished number of peripheral Aifdeficient T cells was due to defective development. Gene deletion of Aif in the thymus of Aifflox/y Lck-Cre LSL-YFP animals was confirmed by PCR analysis and immunoblot (Fig. 5A, 5B, S5A). The proportions and numbers of double negative (DN), double positive (DP) and single positive (SP) cells in the thymi of 8–12 week old mice were comparable between T-AIF KO animals and WT littermates (Fig. 5C, S5B). Although the Lck-Cre was activated between the DN2 and DN3 stage (as indicated by YFP expression) (Fig. S5C), we cannot exclude the possibility that sufficient AIF protein was expressed throughout the DN stages to account for the lack of impact on T cell development. However, we were unable to detect AIF protein in thymocytes (Fig. 5A, B). As observed in the spleen (Fig. S4C), NK1.1+ cell numbers but not γ/δ T cell numbers were affected in the thymus by the loss of AIF (Fig. S5E). AIF deletion caused a reduction in ETC subunits of complex I and III at the protein (Fig. 5A, B) but not at the mRNA level (Fig. S5D). However, these perturbations in the ETC did not manifest as defective OCR or ECAR (Fig. 5D), altered OCR in permeabilized cells supplied with different ETC substrates (Fig. 5E), increased ROS levels (Fig. S5F), or reduced ATP content (Fig. 5F). Therefore, AIF appeared to be dispensable for T cell development. AIF does not influence cell death in immature T cells
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We treated thymocytes isolated from T-AIF KO mice or control littermates with STS, menadione, diazald, or serum withdrawal to induce cell death. The AIF KO thymocytes died at comparable rates to the control cells. We obtained similar results when we pre-incubated the cells with qVD-oph (Fig. 5G). Thus, AIF does not appear to mediate caspase-dependent or –independent cell death in thymocytes under any of the conditions tested. B cells depend on glycolysis, while T cells depend on oxidative phosphorylation to fulfill their energetic demands
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It has been suggested that the mitochondrial content in T lymphocytes is developmentally regulated by autophagy, and that this is necessary for T cell survival and function, and mature T cells thereby contain fewer mitochondria than thymocytes (Jia and He, 2011; Pua et al., 2009). We thus hypothesized that peripheral T cells might be more sensitive to perturbations of mitochondrial composition due to their reduced mitochondrial content compared to B cells and thymocytes. However, B cells and T cells contained comparable amounts of mitochondria whereas thymocytes contained less (Fig. S6A–C). Because the ability of a cell to maintain its mitochondrial membrane potential (ΔΨm) can be critical for its survival (Duchen, 2004; Nicholls, 2002), we asked if the susceptibility of T cells versus B cells and thymocytes to loss of AIF is associated with levels of ΔΨm. We measured ΔΨm in WT B cells, T cells and thymocytes and found that thymocytes sustained the highest ΔΨm and T cells the lowest (Fig. S6D). Interestingly, CD8+ T cells maintain an even lower ΔΨm than CD4+ T cells, consistent with an inverse correlation between ΔΨm and increased sensitivity to loss of AIF (Fig. S6D). Indeed, when we investigated the ΔΨm in different cell
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populations in the absence of AIF, we discovered that the ΔΨm in Aif-deficient B cells, CD4+ T cells and thymocytes was indistinguishable from the controls, whereas we detected a small but significant decrease in ΔΨm in CD8+ T cells (Fig. 6A). Treatment with anti-CD3 and anti-CD28 also resulted in reduced ΔΨm in Aifflox/y Lck-Cre CD4+ T cells versus control CD4+ T cells (Fig. S6E), an effect that was diminished when the cells were activated in galactose (Fig. S6F). Despite the different levels of ΔΨm in CD4+ versus CD8+ T cells, we did not detect any differences in OCR or ECAR (Figure S6H).
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Increased mitochondrial biogenesis might explain why B cells and thymocytes, but not T cells, were able to maintain their ΔΨm in the absence of AIF. However, loss of AIF led to an increase in mitochondrial content in both B cells and T cells but not in thymocytes (Fig. 6B, C) indicating that AIF deficiency may promote mitochondrial biogenesis in the first two cell types, but not in the latter. Thus, while compensation by mitochondrial biogenesis may explain sustained function in Aif-deficient B cells and persisting T cells, it does not account for the survival and development of Aif-deficient thymocytes. We next wondered whether different metabolic requirements of T and B cells might explain their dependency on AIF. We therefore activated WT B or T cells in the presence of either glucose or galactose. T cells proliferated at comparable rates in either glucose or galactose, as reported previously (Qiu et al., 2013), whereas B cells relied almost exclusively on glucose metabolism to support expansion (Fig. S6I).
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Oligomycin is an inhibitor of the mitochondrial F0F1-ATPase, and thus elevates ΔΨm in cells with active OXPHOS. We observed that oligomycin treatment raised the ΔΨm in resting T cells to the levels observed in B cells or thymocytes, but had no effect on the ΔΨm detected in the latter two (Fig. S6G), supporting the idea that resting T cells primarily engage mitochondrial metabolism to generate energy. We therefore isolated T cells from TAIF WT or T-AIF KO animals and stimulated them with anti-CD3 and anti-CD28 in the presence of either glucose or galactose. After 72 hrs of stimulation in galactose, AIF KO T cells proliferated at a slower rate compared to the WT cells (Fig. 6D). In contrast, we observed no differences in proliferation when the cells were cultured in glucose (Fig. 6D). Thus, under conditions in which OXPHOS is required to sustain proliferation, Aif-deficient T cells were compromised. Examination of the mitochondrial ultrastructure revealed that while Aif-deficient and WT thymocytes and B cells showed normal mitochondrial morphology, resting Aif-deficient T cells often harbored enlarged mitochondria with disrupted cristae organization (Fig. 6E, F) resembling enlarged mitochondria observed in activated T cells (Fig. 6E, F)(Kamiński et al., 2012).
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Therefore, loss of AIF compromises T cells more so than B cells or thymocytes due to the requirement in the former for optimal ETC function, which is impacted by loss of AIF. This effect is more pronounced in CD8+ T cells than in CD4+ T cells. While T cells and B cells may compensate for this change in ETC capacity by increased mitochondrial biogenesis, this sustains normal B cell function but cannot do so for the majority of T cells. Those T cells that sufficiently compensate to survive can display normal T cell expansion under some conditions (glucose availability in vitro), but are nevertheless deficient in optimal homeostatic proliferation in vivo.
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Expression of Ndi1 rescues homeostatic proliferation in AIF KO T cells
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We transduced Aifflox/y or Aifwt/y Lck-cre, LSL-YFP BM with a retrovirus expressing mCherry plus wild type AIF or Ndi1, and reconstituted lethally irradiated Rag1−/− mice. We found that, ectopic AIF was selected in the Aif-deficient T cells, based on mCherry expression (Fig. 7A, B). In CD4+ AIF-null T cells, Ndi1 was also positively selected, albeit less than that observed for ectopic AIF, while this was not observed in CD8+ T cells (Fig. 7B), perhaps reflecting the more profound effects of AIF deletion in this subset (Fig. 4B). To investigate the functional consequences in Aif-deficient T cells, we again utilized homeostatic proliferation (Fig. 7C, D). Ectopic expression of AIF or Ndi1 restored homeostatic proliferation in both CD4+ and CD8+ AIF-null T cells. Therefore, the requirement for AIF in homeostatic proliferation can be replaced by the function of Ndi1, and this requirement directly corresponds to the reliance on complex I.
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Discussion We have found that acute deletion of AIF in MEF results in their demise and ectopic expression of the yeast complex I paralog Ndi1 restores survival in MEF lacking AIF, strongly supporting the idea that AIF is required for optimal assembly and function of ETC complex I. Similarly, T cells lacking AIF are deficient in homeostatic proliferation in vivo, and again expression of Ndi1 restores proliferation. Thus, Ndi1 seems to be able to fully replace the activities lost upon ablation of AIF suggesting that the primary role of AIF relates to complex I function.
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The Hq mouse, which displays reduced AIF expression (Klein et al., 2002), is viable, while we observed profound effects of acute deletion of AIF in adult animals (Fig. 2). Whereas it was described that B cell development is unaffected by the Hq mutation (Banerjee et al., 2012), our findings suggested that the complete ablation of AIF in B cells did not impact their development nor function. While Aif-ablated B cells displayed reduced levels of ETC components (Fig. 3), we observed no defects in OXPHOS, perhaps due to compensatory mitochondrial biogenesis. Thus, B cell function is sustained whether levels of AIF are reduced (Banerjee et al., 2012), or completely ablated (our results).
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The importance of AIF in T cell development was previously examined using the Hq mouse (Banerjee et al., 2012). In that study, the authors concluded that the severe decrease in peripheral T cells is due to defective thymocyte development, caused by elevated ROS. In contrast, we observed normal thymocyte development in our Lck-Cre mouse model, where Aif is ablated around the DN3 stage. Nevertheless, peripheral T cell numbers were strongly impacted in the absence of increased ROS. These contradictory results can be explained by our finding that acute AIF deficiency causes defects in HSC function (Fig. 2). As a result, T cell development in the Hq mouse due to partial loss of AIF is affected at a much earlier stage than by conditional ablation with Lck-Cre. The partial expression of AIF in the Hq mouse may explain the elevated ROS observed in the Hq animal. Our results suggest, however, that the defects observed in peripheral T cells are not due to elevated ROS production, but rather defects in ETC function.
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Our data clearly indicate that a step between thymocyte maturation and the persistence of mature T cells in the periphery is largely dependent on AIF. Those T cells that survive appear to do so by a compensatory increase in their mitochondrial mass (Fig. 6), permitting normal respiration (Fig. 4) and ΔΨm (Fig. 6). These cells displayed normal overall OCR despite reduced OCR in response to complex II and IV substrates (Fig. 4) and displayed reduced levels of ΔΨm (Fig. S6) after CD3 and CD28 stimulation in vitro. Previous studies have shown that T cells rely on complex I (Yi et al., 2006) and mitochondrial metabolism (Sena et al., 2013) upon T cell activation and that ΔΨm is required for IL-2 induction (Sena et al., 2013). Supporting these reports we found AIF KO T cells to be defective in homeostatic proliferation in vivo (Fig. 4). Additionally, proliferation of these cells was impaired in vitro when we enforced respiration by culturing in galactose instead of glucose for the carbon source (Fig. 6)
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In contrast to T cells, B cell development and function was completely unaffected by AIF deficiency (Fig. 3). Resting and activated T and B cells differ in their metabolic requirements (Caro-Maldonado et al., 2014; Qiu et al., 2013), and we have found correlations between metabolic features of lymphocytes and their sensitivity to AIF ablation. Interestingly, the observed pattern of ΔΨm (thymocytes=B cells > CD4+ T cells > CD8+ T cells) corresponds to the effects of loss of AIF. Further, while T cells readily proliferate when glucose is replaced with galactose, relying on OXPHOS for energy generation (Qiu et al., 2013), B cells and Aif-deficient T cells displayed defective proliferation under this condition (Fig. 6 and S6). We found that the low ΔΨm in T cells was not due to differences in mitochondrial content (Fig. S6), and upon treatment with oligomycin could be raised to the same levels as those observed in thymocytes and B cells (Fig. S6), while such treatment did not affect ΔΨm in the latter two. This indicates that in contrast to B cells and thymocytes, ΔΨm in resting T lymphocytes is reduced due to requisite utilization of the proton gradient to fuel the production of ATP by the F0F1 ATPase (inhibited by oligomycin). Thus, loss of ETC function upon AIF ablation in T cells has a much greater effect on these cells than observed for either thymocytes or B cells.
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AIF is widely considered to be important for cell death, especially under conditions where caspase activation upon mitochondrial permeabilization is blocked or defective (Daugas et al., 2000b; Susin et al., 1999). As we have discussed, however, loss of AIF leads to ETC defects that must be compensated for cells to survive, and this compensation may have unanticipated effects on induction of cell death. In examining cells that function normally in the absence of AIF (thymocytes, B cells, MEF expressing Ndi1), we were unable to detect any consistent defects in apoptosis or caspase-independent cell death in cells lacking AIF. We suggest, at least for the cell types we have studied, that AIF has, at best, a limited role in the cell death response to a number of pro-apoptotic signals. Instead, we have found support for the idea that AIF plays important roles in the proper assembly and function of ETC components, especially complex I (Vahsen et al., 2004) and that a cell’s dependence on AIF for function and survival is dictated by its metabolic requirements. Recent studies have examined the effects of ablation of NDUFS4, an essential component of complex I which is mutated in patients with Leigh Syndrome. Mice lacking NDUFS4 developed normally, but were blind, and displayed weight loss beginning at about 25 days
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post-birth, followed by neurological and muscular abnormalities and lethality at about seven weeks of age (Kruse et al., 2008). The effects on weight and lethality closely correspond to the time course we observed following acute AIF ablation in adult mice, although the effects on hematopoiesis and T cell function were not examined in the NDUFS4-deficient animals. Daily administration of the TORC1 inhibitor rapamycin improved neuromuscular performance in these animals and extended life span (Johnson et al., 2013). It will be interesting to determine if rapamycin treatment has a similar effect in animals acutely deleted for AIF.
Methods (see Supplemental Methods)
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Mice
Aifflox (Joza et al., 2005), R26CreER (Badea et al., 2003) R26.LSL-YFP (Srinivas et al., 2001), baxflox bak−/− (Takeuchi et al., 2005), Cd19-Cre (Rickert et al., 1997) and Lck-Cre (Hennet et al., 1995) mice have been described previously. Mice were bred to generate animals with the indicated genotypes. All animal experiments were performed using age and gender matched littermates. Genotypes were confirmed by tail snip PCR as described previously. To determine the efficiency of Aif deletion, PCR was performed on DNA extracted from the respective tissues to detect the floxed and the deleted alleles of Aif as described previously (Joza et al., 2005). The St. Jude Institutional Animal Care and Use Committee approved all procedures and animal maintenance in accordance with the Guide for the Care and Use of Animals.
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Tamoxifen treatments To induce systemic deletion of Aif, animals were gavaged for six consecutive days with tamoxifen (T5648, Sigma) dissolved in sunflower seed oil (S5007, Sigma) at a concentration of 1 mg tamoxifen per 25 g of animal body weight per day. Weight loss, morbidity and mortality were assessed every second day. Metabolic assessment For intact cells the OCR and ECAR were measured in nonbuffered DMEM under basal conditions and after addition of the mitochondrial uncoupler FCCP. Complex I, II and IVsubstrate-driven OCR was determined in permeabilized cells in mitochondrial assay buffer in response to malate+pyruvate, succinate and TMPD+ascorbate respectively on the XF-24 Extracellular Flux Analyzer (Seahorse Bioscience).
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Retroviral-mediated stem cell gene transfer Retroviral transduction of murine bone marrow cells was performed as described earlier (Holst et al., 2006a; 2006b; 2008; Bettini et al., 2013). Briefly, bone marrow cells were cultured in complete DMEM with 20% FBS, murine IL-3 (20 ng/ml), human IL-6 (50 ng/ ml), and murine stem cell factor (50 ng/ml) for 24 hrs. Cells were subsequently spintransduced with filtered retroviral supernatant plus polybrene (6 mg/ml) and cytokines as
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detailed above. 2–4 × 106 transduced bone marrow cells were injected per irradiated Rag1−/− recipient mouse. Retrogenic mice were analyzed 8–12 weeks after bone marrow transplant. Cell death assay MEF were seeded at 2×105 cells /well of a 24 well plate and allowed to adhere for 5 hrs before being treated with the following reagents and concentrations: 1 µM actinomycin D (ActD; A1410, Sigma), 1 µM staurosporine (STS; S 4400, Sigma), 500 µM diazald (D28000, Sigma) or 200 µM etoposide (E1388, Sigma) in the absence or presence of 40 µM qVD-oph (A8165, Apexbio). In case of starvation, the cells were cultured in the absence of serum. Cell viability was analyzed using an Incucyte FLR imaging system (Essen Bioscience). Cells were plated in medium containing the membrane impermeant dye, Sytox Green (S7020, Invitrogen) at 25 nM, imaged continuously and analyzed using Incucyte image analysis software (Essen Bioescience).
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Homeostatic expansion in Rag1-deficient mice T cells were purified from whole spleen and lymph nodes and sorted for YFP expression. 1×106 cells were injected retro-orbitally i.v. into Rag1-deficient mice. Mice were sacrificed 28 days later and spleens removed for analysis by flow cytometry. For competitive homeostatic expansion, T cells were purified from both CD45.1 mice (Jackson Laboratory) and CD45.2, Aifwt/y Lck-Cre LSL-YFP (T-AIF WT) and Aifflox/y Lck-Cre LSL-YFP (T-AIF KO) animals and co-injected (1:1 ratio; 5×105 cells each) i.v. into Rag1-deficient mice. Statistical analysis
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p values were determined by unpaired Student`s t test unless stated otherwise. p values < 0.05 were considered significant.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments We thank Richard Cross, Greig Lennon, and Parker Ingle for all cell sorting, and Mao Yang for technical support. This work was supported by ALSAC and by grants from the US National Institutes of Health.
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Fig. 1. Mitochondrial defects caused by loss of AIF can be rescued by expression of Ndi1
(A) Aifflox/y Rosa26.CreER LSL-YFP MEF were treated with vehicle (-) or 4-OH tamoxifen (4-OHT) (+) for the indicated times. Lysates assessed by Western blot (n=3). (B) OCR and ECAR were measured under basal conditions. Values are mean ± SD of triplicates (n=3). OCR: p=0.0047; ECAR: p=0.0017. (C) Complex I (Malate+Pyruvate), II (Succinate) and IV (TMPD+Ascorbate)-driven OCR of permeabilized MEF (n=5). Data are mean ± SD. Complex I: p=0.0066; complex II: p=0.0011; complex IV: p=0.0006. (D) AIF KO MEF stably expressing vector control (vector), wild type AIF (AIF) or Ndi1 (Ndi1) were treated
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with vehicle (-) or 4-OHT (+) for two weeks, and immunoblots of lysates performed (n=3). (E) Basal OCR and ECAR were determined for groups in D. Data are mean ± SD (n=3). OCR: vector control vs vector 4-OHT, p=0.0004; vector 4-OHT vs Ndi1 4-OHT, p=0.0004; ECAR: vector control vs vector 4-OHT, p=0.0027. (F). AIF KO MEF expressing vector control (Vector), wild type AIF (AIF) or Ndi1 (Ndi1) were treated with 4-OHT for two weeks and sorted for YFP expression. For each, 3×104 cells were cultured and clonogenic survival assessed after 11 days. Representative of five independent experiments. (G) AIF KO MEF stably expressing wild type AIF or Ndi1 after at least four weeks of 4-OHT treatment were treated with STS (1 µM), ActD (1 µM), etoposide (200 µM), diazald (500 µM) or serum withdrawal (Starvation) in the absence or presence of qVD-oph (40 µM) and cell death assessed. Results are representative of three independent experiments. Mean ± SD of triplicate cultures is shown. See also Figure S1.
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Author Manuscript Author Manuscript Author Manuscript Fig. 2. AIF is required for tissue homeostasis in the adult animal
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(A–E) Aifwt/y Rosa26.CreER or Aifflox/y (AIF WT) and Aifflox/y Rosa26.CreER (AIF KO) animals were gavaged with 1mg tamoxifen for 6 consecutive days. Animals were observed over 60 days for weight loss (A) and lethality (B) n=40 for each group, *p
Immunity. Author manuscript; available in PMC 2016 July 19. Published in final edited form as: Immunity. 2016 January 19; 44(1): 88–102. doi:10.1016/j.immuni.2015.12.002.
Apoptosis-inducing factor (AIF)-dependent mitochondrial function is required for T cell but not B cell function Sandra Milasta1, Christopher P. Dillon1, Oliver E. Sturm2, Katherine C. Verbist3, Taylor L. Brewer1, Giovanni Quarato1, Scott A. Brown1, Sharon Frase4, Laura J. Janke5, S. Scott Perry6, Paul G. Thomas1, and Douglas R. Green1 1Department
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of Immunology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
2Department
of Biology, Rhodes College, 2000 North Parkway, Memphis, TN 38112, USA
3Department
of Oncology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 4Cell
and Tissue Imaging Center, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
5Department
of Pathology, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 6Department
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of Flow Cytometry, St. Jude Children`s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Summary
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The role of Apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that loss of AIF in fibroblasts led to mitochondrial electron transport chain (ETC) defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deletion in T cells impacted the numbers of peripheral T cells and their ability to undergo homeostatic proliferation, while not affecting their thymic development. However, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Expression of Ndi1 rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF undergo normal death. These studies suggest that the primary role of AIF relates to complex I function, differentially affecting T and B cells.
Correspondence to: [email protected]. Author Contributions S.M. conceived the project, designed and performed most experiments, interpreted results, and co-wrote the manuscript. K.C.V, O.E.S, S.B., G.Q., L.J.J., S.F., S.S.P., C.P.D. and T.L.B. performed experiments and analysis. P.G.T provided intellectual guidance. D.R.G. conceived the project, supervised experimental designs, interpreted results, and co-wrote the manuscript.
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Introduction AIF is a mitochondrial inter-membrane space protein initially identified in an assay of mitochondrial components that cause morphological changes in isolated nuclei (Susin et al., 1999). A role for AIF in cell death was proposed based on observations that AIF is released from mitochondria during mitochondrial outer membrane permeabilization (MOMP) (Daugas et al., 2000b), a step in the mitochondrial pathway of apoptosis (reviewed in Tait and Green, 2010), and subsequently localizes to the nucleus (Daugas et al., 2000b) where it induces double stranded DNA breaks and large scale DNA condensation (Susin et al., 1999). A large number of reports have concluded that AIF mediates cell death that proceeds in the absence of caspase activity (reviewed in Candé et al., 2002; Daugas et al., 2000a).
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Germline ablation of Aif is embryonically lethal, and initial studies in vitro suggested that this lethality was due to a cavitation defect in the embryoid body (Joza et al., 2001). Subsequent studies (Brown et al., 2006) showed that the development of Aif-deficient embryos (including cavitation) is normal through embryonic day 9, followed by lethality. AIF is required for efficient assembly of complex I of the electron transport chain (ETC) and thus plays a role in maintaining normal oxidative phosphorylation (OXPHOS) (Hangen et al., 2015; Vahsen et al., 2004). Conditional deletion of AIF in muscle or liver (Pospisilik et al., 2007) confirmed this role for AIF (Pospisilik et al., 2007). AIF regulates the mitochondrial import of CHCHD4, a central component of a redox-sensitive mitochondrial inter-membrane space import machinery (Hangen et al., 2015), thus explaining its role in ETC assembly and function.
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The harlequin (Hq) mutation in mice is caused by a provirus insertion into the Aif locus, resulting in 80% loss of AIF expression (Bénit et al., 2008; Klein et al., 2002). Characterization of these animals revealed a T lymphocyte deficiency due to a failure of T cell development in the thymus, with high levels of reactive oxygen species (ROS) observed in surviving lymphocytes (Banerjee et al., 2012). Several studies in these animals (reviewed in Joza et al., 2009) suggested that cells from Hq animals are resistant to apoptosis and other forms of cell death.
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Here, we found that acute deletion of Aif in mouse embryonic fibroblasts (MEF) ablated proliferation. This effect was prevented by ectopic expression of Saccharomyces cerevisiae Ndi1, which has been shown to partially restore respiration and ETC function in mammalian cells lacking complex I activity (Santidrian et al., 2013; Seo, 1999; Seo et al., 2004). (Santidrian et al., 2013; Seo, 1999; Seo et al., 2000). To investigate the role of AIF in tissue homeostasis, we generated animals in which AIF can be ubiquitously deleted. We observed wasting and lethality upon acute deletion of AIF, accompanied by a loss of hematopoietic stem cells (HSC) and lymphocytes. However, B cells lacking AIF developed and functioned normally, despite partial deficiency in complex I. In contrast, deletion of AIF in T cells did not affect development, but profoundly impacted numbers and homeostatic proliferation of peripheral T cells in vivo. Expression of Ndi1 partially rescued the defects observed after loss of AIF. Since Aif-deficient MEF, thymocytes, and B cells displayed normal cell death, we conclude that the primary role of AIF in lymphocytes relates to complex I function and, at least in the cells studied, does not have a major role in cell death.
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Results Defects associated with loss of AIF in fibroblasts can be reversed by rescuing complex I function
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We generated SV40-immortalized MEF in which endogenous Aif is removed by 4hydroxytamoxifen (4-OHT)-mediated induction of Cre (Aifflox/y R26CreER) (Badea et al., 2003; Srinivas et al., 2001). The MEF also expressed a loxp-stop-loxp (LSL)-yellow fluorescent protein (YFP) transgene to assess recombination (Fig. S1A). Within one week after 4-OHT treatment, we observed efficient loss of AIF protein (Fig. 1A). By three weeks only YFP− cells that had not recombined the Aif locus expanded in culture (Fig. 1A, S1B). Loss of AIF expression negatively affected the expression of complexes I and IV of the ETC (Fig. 1A). An increase in mtDNA to nDNA ratio was observed following 4-OHT treatment (Fig. S1C), suggesting a compensatory effect. Consistent with this, we observed that cells lacking AIF reduced their oxygen consumption rate (OCR), and increased their extracellular acidification rate (ECAR), a consequence of lactic acid production, suggesting a switch from OXPHOS to glycolysis (Fig. 1B, S1D). Moreover, loss of AIF decreased OCR in permeabilized cells, driven by substrates for complexes I, II, and IV (Fig. 1C), consistent with diminished complex IV expression (Fig. 1A). In contrast, Aifflox/y R26CreER MEF expressing ectopic AIF displayed similar levels of complex I–IV proteins with or without deletion of endogenous AIF (Fig. 1D). Consequently, the levels of OCR and ECAR in the 4OHT-treated cells were similar to vehicle treated controls (Fig. 1E). Therefore, the changes in ETC proteins and metabolism upon ablation of AIF were due to loss of AIF protein expression.
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We then ectopically expressed Ndi1, the rotenone-insensitive yeast paralog of complex I, in Aifflox/y R26CreER MEF. The functionality of Ndi1 was confirmed by the ability of Aifflox/y R26CreER MEF expressing Ndi1 (but not vector control MEF) to clonally expand in the presence of the complex I inhibitor rotenone (Fig. S1E). In contrast to MEF without ectopic Ndi1 (Fig. 1A), the expression of Ndi1 prevented the reappearance of cells that had failed to delete Aif after 4-OHT treatment (Fig. 1D, S1F). Unlike AIF, ectopic expression of Ndi1 did not restore the expression of complex I, III and IV in Aif-deficient MEF or their substratedriven activity (Fig. 1D, S1G). Nevertheless, Ndi1 expression prevented the metabolic effects of AIF deletion. While Ndi1 elevated OCR in AIF-sufficient cells as expected (Seo, 1999; Seo et al., 2004), the change in OCR and ECAR seen upon deletion of AIF was not observed in cells expressing Ndi1 (Fig. 1E). Further, upon acute deletion of Aif by 4-OHT treatment, vector-control MEF showed a dramatic reduction in clonogenic expansion, while ectopic expression of either AIF or Ndi1 sustained such expansion (Fig. 1F).
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Unlike glucose, galactose enters glycolysis via the Leloir pathway, resulting in reduced generation of ATP via glycolysis (Qiu et al., 2013; Weinberg et al., 2010) We found that Aifdeficient MEF reconstituted with either AIF or Ndi1 expanded similarly in galactose (Fig. S1H). Therefore, the changes in cell metabolism and proliferation that occured upon deletion of AIF was prevented by expression of Ndi1, suggesting that these effects were predominantly due to loss of complex I activity. Therefore, despite its role in the import of
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several proteins involved in mitochondrial function (Hangen et al., 2015), AIF appeared to function primarily to sustain complex I activity in MEF. Cell death is largely unaffected in MEF deficient for AIF AIF was originally identified as an intermembrane space (IMS) protein involved in cell death upon MOMP (Susin et al., 1999; Wissing et al., 2004). We therefore treated Aifdeficient MEF expressing either ectopic AIF or Ndi1 with several cell death inducers with or without the pan-caspase inhibitor, qVD-oph. As shown in Fig. 1G, Aif-deficient MEF reconstituted with Ndi1 displayed normal susceptibility to apoptosis. Under conditions of caspase inhibition, cell death was diminished in all cases. Caspase-independent cell death induced by serum starvation or diazald was unaffected by AIF status, although the low level observed with STS treatment appeared to be partially dependent on AIF. Thus, AIF is not a primary mediator of cell death in MEF.
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Global ablation of AIF in adult mice causes wasting
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Complete loss of AIF leads to embryonic lethality at day E12.5 (Brown et al., 2006; Joza et al., 2005). To study the role of AIF in the adult animal we treated Aifflox/y R26CreER mice with tamoxifen. Efficient deletion of the Aif allele in various tissues upon treatment with tamoxifen was confirmed by PCR (Fig. S2A). Whereas WT animals (Aifflox/y and Aifwt/y R26CreER) appeared normal after tamoxifen treatment, the littermate mutant animals (Aifflox/y R26CreER) showed a loss of body weight and subsequent mortality (Fig. 2A, B). To determine whether removal of AIF resulted in induction of apoptosis, we generated a set of mice lacking AIF and the pro-apoptotic Bcl2 family members Bax and Bak (Aifflox/y Bak−/− Baxflox/flox R26CreER). However deletion of Bak and Bax did not protect Aifdeficient mice (Fig. S2C). The observed pathology therefore proceeded independently of the intrinsic pathway of apoptosis. Histological and morphological analyses revealed loss of crypts with crypt regeneration in the intestine and thymic hypocellularity as well as weight loss in all of the tissues examined (Fig. 2C, S2B). While a wasting effect after deletion of AIF in skeletal muscle has been reported (Joza et al., 2005), we did not observe such effects (Fig. 2C), probably due to the more rapid lethality of whole body deletion in our experiments.
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We isolated lineage negative stem cells from Aifwt/y R26CreER or Aifflox/y R26CreER mice and injected these cells as a mixture with congenic WT stem cells into lethally irradiated Rag1-deficient mice. The mice were treated with tamoxifen upon reconstitution. Deletion of AIF caused diminished frequencies of T and B cells and cells with granular features as detected by flow cytometry (Fig. 2D). We also observed reduced numbers of HSC (Fig. 2E). To test whether lethality upon acute ablation of AIF was caused by the collapse of the hematopoietic system, we transferred WT BM-derived stem cells into lethally irradiated WT or Aifflox/y R26CreER animals and allowed for complete reconstitution before tamoxifen treatment. The reconstituted Aif-deficient animals lost weight similar to tamoxifen-treated Aifflox/y R26CreER animals (Fig. S2D). Thus, the loss of the hematopoietic system was not solely responsible for wasting and lethality seen in Aif-deficient animals.
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Targeted ablation of AIF has no effect on B cell development and function
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In the AIF hypomorph harlequin mouse (Hq) B cells are unaffected (Banerjee et al., 2012). To study the role of AIF in B cell development and function, we generated conditional Aif mice (Aifflox/y) with Cre-recombinase under control of the B cell specific CD19 promoter (Cd19-Cre), and a LSL-YFP transgene to label cells with active Cre (Aifflox/y Cd19-Cre LSL-YFP). Animals were born healthy and viable. PCR and immunoblot analysis confirmed deletion of the Aif allele only in the B cell lineage (Fig. 3C, S3B). We did not detect any differences in B cell development between mutant animals (Aifflox/y Cd19-Cre LSL-YFP hereafter referred to as B-AIF KO) and littermate controls (Aifwt/y Cd19-Cre LSL-YFP hereafter referred to as B-AIF WT), as frequencies and numbers of B cells in the BM and spleen did not differ between the B-AIF WT and KO mice (Fig. 3A, 3B, S3A). While we cannot exclude the possibility that the AIF protein was expressed during the early stages of development directly following Cre activation, subsequent B cell expansion and development were unaffected by AIF deletion.
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We then examined the effects of AIF loss on ETC protein expression and function in mature B cells. In agreement with other reports (Vahsen et al., 2004), AIF deficiency did not affect expression of several ETC subunits at the mRNA level, but resulted in loss of complex I, III and IV proteins (Fig. 3C, 3D, S3C). We did not observe any change in the activity of complex I, but detected increases in OCR in permeabilized cells driven by complex II and IV substrates (Fig. 3E), consistent with a compensatory increase in complex IV activity. Consequently, we found that both OCR and ECAR were comparable in the basal state and after addition of FCCP (to uncouple ATP synthesis from the ETC to determine the spare respiratory capacity) between intact WT and Aif-deficient B cells (Fig. 3F). In addition, glycolysis, fatty acid β-oxidation (FAO), glutaminolysis, and ATP content were all similar between the WT and mutant mice (Fig. S3D–G). Complex I impairment often leads to increased ROS generation (Robinson, 1998; S Pitkanen, 1996). However, measurement of ROS levels revealed no significant differences in response to loss of AIF (Fig. 3G), consistent with other reports in AIF KO liver or muscle (Chinta et al., 2009; Pospisilik et al., 2007). In vitro proliferation after lipopolysaccharide (LPS) stimulation (Fig. S3H), in vivo ovalbumin-specific antibody production (Fig. S3I), and expansion of antigen-specific antibody forming cells (AFC) after influenza infection (Fig. 3H) were not affected by AIF deletion. Therefore, B cells did not require the expression of AIF or optimal expression of mitochondrial complex I, III and IV proteins for their development and functionality. B cell death is unaffected by the absence of AIF
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As AIF does not appear to be important for survival of B cells, we examined the involvement of AIF in regulating caspase-dependent and -independent cell death in these cells. Naïve Aifflox/y Cd19-Cre LSL-YFP and WT B cells displayed equivalent susceptibility to cell death induced by STS, menadione, and diazald, as well as in response to serum withdrawal following activation with LPS (Fig. 3I), although a minor effect of AIF ablation was observed at the highest concentration of STS. The presence of qVD-oph delayed cell death in each case, but the ensuing death was unaffected by AIF deletion (Fig. 3I). These effects were also observed for spontaneous cell death of B cells in culture, represented by
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untreated controls. Thus, loss of AIF did not confer protection against caspase-dependent or –independent cell death in B cells. AIF plays a pivotal role in peripheral T cell function and survival
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We observed that acute deletion of AIF in the hematopoietic system affected T cells more rapidly than B cells (Fig. 2D). To conditionally delete Aif in T cells, we generated Aifflox/y animals expressing Cre on the proximal Lck promoter (Lck-Cre), with LSL-YFP and confirmed efficient Aif-deletion in YFP+ T cells (Fig. 4B, 4C, S4A). Loss of AIF led to a reduction in the expression of complex I, III and IV proteins. We also detected a significant increase in the expression of complex II components (Fig. 4B, C), suggesting a compensatory effect in T cells. While Aifflox/y Lck-Cre LSL-YFP (T-AIF KO) mice had similar numbers of total splenocytes as Aifwt/y Lck-Cre LSL-YFP (T-AIF WT) littermate controls, fewer CD4+, YFP+ and substantially fewer CD8+, YFP+ T cells were present (Fig. 4A, S4B). This attrition was also observed among NK1.1+ cells, but not among γδ T cells (Fig. S4C). We then examined mitochondrial function in Aif-deficient T cells. No differences were detected in ROS between the T cells isolated from T-AIF WT and T-AIF KO animals (Fig. S4D). Although basal state OCR and ECAR were similar in resting Aifdeficient T cells compared to littermate WT T cells (Fig. 4D), loss of AIF significantly affected the OCR in response to complex II and IV substrates (Fig. 4E), and reduced ATP content in naïve cells (Fig. 4F). In accordance with other reports (Wang et al., 2011), we found that T cells increased OCR and ECAR after 24 hrs of activation (Fig. 4D). AIF KO T cells, however, increased their basal OCR and uncoupled OCR to a lesser degree after activation but exhibited similar levels of ECAR compared to WT T cells (Fig. 4D). The effect on OCR was also observed in permeabilized cells provided with different substrates (Fig. 4E). These observations support the conclusion that AIF deficiency in T cells negatively affects OXPHOS.
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We then examined the effects of AIF deficiency on T cell activation. Following treatment with anti-CD3 and anti-CD28, WT and Aif-deficient T cells similarly expressed the activation markers CD25 and CD69 (Fig. S4E). Results assessing activation-induced cell proliferation in vitro were inconsistent (data not shown). It is possible, therefore, that the small numbers of Aif-deficient T cells that persist in the periphery include variable numbers of cells that have sufficiently compensated for the loss of AIF (e.g., via increased mitochondrial biogenesis) such that their function can be sustained. If so, defects might be revealed by the extensive homeostatic expansion of purified WT or Aif-deficient T cells in Rag1−/− animals in vivo. We co-injected AIF WT or AIF KO T cells with congenic T cells, and found that AIF KO T cells did not expand as well as AIF WT T cells (Fig. 4G). Notably, CD8+ T cells were more sensitive to loss of AIF than were CD4+ T cells. Similarly, we examined homeostatic expansion in vivo under non-competitive conditions. AIF KO CD4+ T cells displayed no defect in homeostatic expansion under these conditions, suggesting that these cells were able to satisfy their energetic demands, whereas AIF KO CD8+ T cells were unable to proliferate under these conditions in vivo (Fig. 4H). We thus conclude that AIF is essential for T cell function in vivo.
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T cells develop normally in the absence of AIF
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Ubiquitous diminution of AIF expression in the Hq mouse results in thymic arrest at the βselection stage, resulting in significantly reduced numbers of mature CD4+ and CD8+ T cells (Banerjee et al., 2012). We therefore asked if the diminished number of peripheral Aifdeficient T cells was due to defective development. Gene deletion of Aif in the thymus of Aifflox/y Lck-Cre LSL-YFP animals was confirmed by PCR analysis and immunoblot (Fig. 5A, 5B, S5A). The proportions and numbers of double negative (DN), double positive (DP) and single positive (SP) cells in the thymi of 8–12 week old mice were comparable between T-AIF KO animals and WT littermates (Fig. 5C, S5B). Although the Lck-Cre was activated between the DN2 and DN3 stage (as indicated by YFP expression) (Fig. S5C), we cannot exclude the possibility that sufficient AIF protein was expressed throughout the DN stages to account for the lack of impact on T cell development. However, we were unable to detect AIF protein in thymocytes (Fig. 5A, B). As observed in the spleen (Fig. S4C), NK1.1+ cell numbers but not γ/δ T cell numbers were affected in the thymus by the loss of AIF (Fig. S5E). AIF deletion caused a reduction in ETC subunits of complex I and III at the protein (Fig. 5A, B) but not at the mRNA level (Fig. S5D). However, these perturbations in the ETC did not manifest as defective OCR or ECAR (Fig. 5D), altered OCR in permeabilized cells supplied with different ETC substrates (Fig. 5E), increased ROS levels (Fig. S5F), or reduced ATP content (Fig. 5F). Therefore, AIF appeared to be dispensable for T cell development. AIF does not influence cell death in immature T cells
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We treated thymocytes isolated from T-AIF KO mice or control littermates with STS, menadione, diazald, or serum withdrawal to induce cell death. The AIF KO thymocytes died at comparable rates to the control cells. We obtained similar results when we pre-incubated the cells with qVD-oph (Fig. 5G). Thus, AIF does not appear to mediate caspase-dependent or –independent cell death in thymocytes under any of the conditions tested. B cells depend on glycolysis, while T cells depend on oxidative phosphorylation to fulfill their energetic demands
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It has been suggested that the mitochondrial content in T lymphocytes is developmentally regulated by autophagy, and that this is necessary for T cell survival and function, and mature T cells thereby contain fewer mitochondria than thymocytes (Jia and He, 2011; Pua et al., 2009). We thus hypothesized that peripheral T cells might be more sensitive to perturbations of mitochondrial composition due to their reduced mitochondrial content compared to B cells and thymocytes. However, B cells and T cells contained comparable amounts of mitochondria whereas thymocytes contained less (Fig. S6A–C). Because the ability of a cell to maintain its mitochondrial membrane potential (ΔΨm) can be critical for its survival (Duchen, 2004; Nicholls, 2002), we asked if the susceptibility of T cells versus B cells and thymocytes to loss of AIF is associated with levels of ΔΨm. We measured ΔΨm in WT B cells, T cells and thymocytes and found that thymocytes sustained the highest ΔΨm and T cells the lowest (Fig. S6D). Interestingly, CD8+ T cells maintain an even lower ΔΨm than CD4+ T cells, consistent with an inverse correlation between ΔΨm and increased sensitivity to loss of AIF (Fig. S6D). Indeed, when we investigated the ΔΨm in different cell
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populations in the absence of AIF, we discovered that the ΔΨm in Aif-deficient B cells, CD4+ T cells and thymocytes was indistinguishable from the controls, whereas we detected a small but significant decrease in ΔΨm in CD8+ T cells (Fig. 6A). Treatment with anti-CD3 and anti-CD28 also resulted in reduced ΔΨm in Aifflox/y Lck-Cre CD4+ T cells versus control CD4+ T cells (Fig. S6E), an effect that was diminished when the cells were activated in galactose (Fig. S6F). Despite the different levels of ΔΨm in CD4+ versus CD8+ T cells, we did not detect any differences in OCR or ECAR (Figure S6H).
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Increased mitochondrial biogenesis might explain why B cells and thymocytes, but not T cells, were able to maintain their ΔΨm in the absence of AIF. However, loss of AIF led to an increase in mitochondrial content in both B cells and T cells but not in thymocytes (Fig. 6B, C) indicating that AIF deficiency may promote mitochondrial biogenesis in the first two cell types, but not in the latter. Thus, while compensation by mitochondrial biogenesis may explain sustained function in Aif-deficient B cells and persisting T cells, it does not account for the survival and development of Aif-deficient thymocytes. We next wondered whether different metabolic requirements of T and B cells might explain their dependency on AIF. We therefore activated WT B or T cells in the presence of either glucose or galactose. T cells proliferated at comparable rates in either glucose or galactose, as reported previously (Qiu et al., 2013), whereas B cells relied almost exclusively on glucose metabolism to support expansion (Fig. S6I).
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Oligomycin is an inhibitor of the mitochondrial F0F1-ATPase, and thus elevates ΔΨm in cells with active OXPHOS. We observed that oligomycin treatment raised the ΔΨm in resting T cells to the levels observed in B cells or thymocytes, but had no effect on the ΔΨm detected in the latter two (Fig. S6G), supporting the idea that resting T cells primarily engage mitochondrial metabolism to generate energy. We therefore isolated T cells from TAIF WT or T-AIF KO animals and stimulated them with anti-CD3 and anti-CD28 in the presence of either glucose or galactose. After 72 hrs of stimulation in galactose, AIF KO T cells proliferated at a slower rate compared to the WT cells (Fig. 6D). In contrast, we observed no differences in proliferation when the cells were cultured in glucose (Fig. 6D). Thus, under conditions in which OXPHOS is required to sustain proliferation, Aif-deficient T cells were compromised. Examination of the mitochondrial ultrastructure revealed that while Aif-deficient and WT thymocytes and B cells showed normal mitochondrial morphology, resting Aif-deficient T cells often harbored enlarged mitochondria with disrupted cristae organization (Fig. 6E, F) resembling enlarged mitochondria observed in activated T cells (Fig. 6E, F)(Kamiński et al., 2012).
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Therefore, loss of AIF compromises T cells more so than B cells or thymocytes due to the requirement in the former for optimal ETC function, which is impacted by loss of AIF. This effect is more pronounced in CD8+ T cells than in CD4+ T cells. While T cells and B cells may compensate for this change in ETC capacity by increased mitochondrial biogenesis, this sustains normal B cell function but cannot do so for the majority of T cells. Those T cells that sufficiently compensate to survive can display normal T cell expansion under some conditions (glucose availability in vitro), but are nevertheless deficient in optimal homeostatic proliferation in vivo.
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Expression of Ndi1 rescues homeostatic proliferation in AIF KO T cells
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We transduced Aifflox/y or Aifwt/y Lck-cre, LSL-YFP BM with a retrovirus expressing mCherry plus wild type AIF or Ndi1, and reconstituted lethally irradiated Rag1−/− mice. We found that, ectopic AIF was selected in the Aif-deficient T cells, based on mCherry expression (Fig. 7A, B). In CD4+ AIF-null T cells, Ndi1 was also positively selected, albeit less than that observed for ectopic AIF, while this was not observed in CD8+ T cells (Fig. 7B), perhaps reflecting the more profound effects of AIF deletion in this subset (Fig. 4B). To investigate the functional consequences in Aif-deficient T cells, we again utilized homeostatic proliferation (Fig. 7C, D). Ectopic expression of AIF or Ndi1 restored homeostatic proliferation in both CD4+ and CD8+ AIF-null T cells. Therefore, the requirement for AIF in homeostatic proliferation can be replaced by the function of Ndi1, and this requirement directly corresponds to the reliance on complex I.
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Discussion We have found that acute deletion of AIF in MEF results in their demise and ectopic expression of the yeast complex I paralog Ndi1 restores survival in MEF lacking AIF, strongly supporting the idea that AIF is required for optimal assembly and function of ETC complex I. Similarly, T cells lacking AIF are deficient in homeostatic proliferation in vivo, and again expression of Ndi1 restores proliferation. Thus, Ndi1 seems to be able to fully replace the activities lost upon ablation of AIF suggesting that the primary role of AIF relates to complex I function.
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The Hq mouse, which displays reduced AIF expression (Klein et al., 2002), is viable, while we observed profound effects of acute deletion of AIF in adult animals (Fig. 2). Whereas it was described that B cell development is unaffected by the Hq mutation (Banerjee et al., 2012), our findings suggested that the complete ablation of AIF in B cells did not impact their development nor function. While Aif-ablated B cells displayed reduced levels of ETC components (Fig. 3), we observed no defects in OXPHOS, perhaps due to compensatory mitochondrial biogenesis. Thus, B cell function is sustained whether levels of AIF are reduced (Banerjee et al., 2012), or completely ablated (our results).
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The importance of AIF in T cell development was previously examined using the Hq mouse (Banerjee et al., 2012). In that study, the authors concluded that the severe decrease in peripheral T cells is due to defective thymocyte development, caused by elevated ROS. In contrast, we observed normal thymocyte development in our Lck-Cre mouse model, where Aif is ablated around the DN3 stage. Nevertheless, peripheral T cell numbers were strongly impacted in the absence of increased ROS. These contradictory results can be explained by our finding that acute AIF deficiency causes defects in HSC function (Fig. 2). As a result, T cell development in the Hq mouse due to partial loss of AIF is affected at a much earlier stage than by conditional ablation with Lck-Cre. The partial expression of AIF in the Hq mouse may explain the elevated ROS observed in the Hq animal. Our results suggest, however, that the defects observed in peripheral T cells are not due to elevated ROS production, but rather defects in ETC function.
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Our data clearly indicate that a step between thymocyte maturation and the persistence of mature T cells in the periphery is largely dependent on AIF. Those T cells that survive appear to do so by a compensatory increase in their mitochondrial mass (Fig. 6), permitting normal respiration (Fig. 4) and ΔΨm (Fig. 6). These cells displayed normal overall OCR despite reduced OCR in response to complex II and IV substrates (Fig. 4) and displayed reduced levels of ΔΨm (Fig. S6) after CD3 and CD28 stimulation in vitro. Previous studies have shown that T cells rely on complex I (Yi et al., 2006) and mitochondrial metabolism (Sena et al., 2013) upon T cell activation and that ΔΨm is required for IL-2 induction (Sena et al., 2013). Supporting these reports we found AIF KO T cells to be defective in homeostatic proliferation in vivo (Fig. 4). Additionally, proliferation of these cells was impaired in vitro when we enforced respiration by culturing in galactose instead of glucose for the carbon source (Fig. 6)
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In contrast to T cells, B cell development and function was completely unaffected by AIF deficiency (Fig. 3). Resting and activated T and B cells differ in their metabolic requirements (Caro-Maldonado et al., 2014; Qiu et al., 2013), and we have found correlations between metabolic features of lymphocytes and their sensitivity to AIF ablation. Interestingly, the observed pattern of ΔΨm (thymocytes=B cells > CD4+ T cells > CD8+ T cells) corresponds to the effects of loss of AIF. Further, while T cells readily proliferate when glucose is replaced with galactose, relying on OXPHOS for energy generation (Qiu et al., 2013), B cells and Aif-deficient T cells displayed defective proliferation under this condition (Fig. 6 and S6). We found that the low ΔΨm in T cells was not due to differences in mitochondrial content (Fig. S6), and upon treatment with oligomycin could be raised to the same levels as those observed in thymocytes and B cells (Fig. S6), while such treatment did not affect ΔΨm in the latter two. This indicates that in contrast to B cells and thymocytes, ΔΨm in resting T lymphocytes is reduced due to requisite utilization of the proton gradient to fuel the production of ATP by the F0F1 ATPase (inhibited by oligomycin). Thus, loss of ETC function upon AIF ablation in T cells has a much greater effect on these cells than observed for either thymocytes or B cells.
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AIF is widely considered to be important for cell death, especially under conditions where caspase activation upon mitochondrial permeabilization is blocked or defective (Daugas et al., 2000b; Susin et al., 1999). As we have discussed, however, loss of AIF leads to ETC defects that must be compensated for cells to survive, and this compensation may have unanticipated effects on induction of cell death. In examining cells that function normally in the absence of AIF (thymocytes, B cells, MEF expressing Ndi1), we were unable to detect any consistent defects in apoptosis or caspase-independent cell death in cells lacking AIF. We suggest, at least for the cell types we have studied, that AIF has, at best, a limited role in the cell death response to a number of pro-apoptotic signals. Instead, we have found support for the idea that AIF plays important roles in the proper assembly and function of ETC components, especially complex I (Vahsen et al., 2004) and that a cell’s dependence on AIF for function and survival is dictated by its metabolic requirements. Recent studies have examined the effects of ablation of NDUFS4, an essential component of complex I which is mutated in patients with Leigh Syndrome. Mice lacking NDUFS4 developed normally, but were blind, and displayed weight loss beginning at about 25 days
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post-birth, followed by neurological and muscular abnormalities and lethality at about seven weeks of age (Kruse et al., 2008). The effects on weight and lethality closely correspond to the time course we observed following acute AIF ablation in adult mice, although the effects on hematopoiesis and T cell function were not examined in the NDUFS4-deficient animals. Daily administration of the TORC1 inhibitor rapamycin improved neuromuscular performance in these animals and extended life span (Johnson et al., 2013). It will be interesting to determine if rapamycin treatment has a similar effect in animals acutely deleted for AIF.
Methods (see Supplemental Methods)
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Mice
Aifflox (Joza et al., 2005), R26CreER (Badea et al., 2003) R26.LSL-YFP (Srinivas et al., 2001), baxflox bak−/− (Takeuchi et al., 2005), Cd19-Cre (Rickert et al., 1997) and Lck-Cre (Hennet et al., 1995) mice have been described previously. Mice were bred to generate animals with the indicated genotypes. All animal experiments were performed using age and gender matched littermates. Genotypes were confirmed by tail snip PCR as described previously. To determine the efficiency of Aif deletion, PCR was performed on DNA extracted from the respective tissues to detect the floxed and the deleted alleles of Aif as described previously (Joza et al., 2005). The St. Jude Institutional Animal Care and Use Committee approved all procedures and animal maintenance in accordance with the Guide for the Care and Use of Animals.
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Tamoxifen treatments To induce systemic deletion of Aif, animals were gavaged for six consecutive days with tamoxifen (T5648, Sigma) dissolved in sunflower seed oil (S5007, Sigma) at a concentration of 1 mg tamoxifen per 25 g of animal body weight per day. Weight loss, morbidity and mortality were assessed every second day. Metabolic assessment For intact cells the OCR and ECAR were measured in nonbuffered DMEM under basal conditions and after addition of the mitochondrial uncoupler FCCP. Complex I, II and IVsubstrate-driven OCR was determined in permeabilized cells in mitochondrial assay buffer in response to malate+pyruvate, succinate and TMPD+ascorbate respectively on the XF-24 Extracellular Flux Analyzer (Seahorse Bioscience).
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Retroviral-mediated stem cell gene transfer Retroviral transduction of murine bone marrow cells was performed as described earlier (Holst et al., 2006a; 2006b; 2008; Bettini et al., 2013). Briefly, bone marrow cells were cultured in complete DMEM with 20% FBS, murine IL-3 (20 ng/ml), human IL-6 (50 ng/ ml), and murine stem cell factor (50 ng/ml) for 24 hrs. Cells were subsequently spintransduced with filtered retroviral supernatant plus polybrene (6 mg/ml) and cytokines as
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detailed above. 2–4 × 106 transduced bone marrow cells were injected per irradiated Rag1−/− recipient mouse. Retrogenic mice were analyzed 8–12 weeks after bone marrow transplant. Cell death assay MEF were seeded at 2×105 cells /well of a 24 well plate and allowed to adhere for 5 hrs before being treated with the following reagents and concentrations: 1 µM actinomycin D (ActD; A1410, Sigma), 1 µM staurosporine (STS; S 4400, Sigma), 500 µM diazald (D28000, Sigma) or 200 µM etoposide (E1388, Sigma) in the absence or presence of 40 µM qVD-oph (A8165, Apexbio). In case of starvation, the cells were cultured in the absence of serum. Cell viability was analyzed using an Incucyte FLR imaging system (Essen Bioscience). Cells were plated in medium containing the membrane impermeant dye, Sytox Green (S7020, Invitrogen) at 25 nM, imaged continuously and analyzed using Incucyte image analysis software (Essen Bioescience).
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Homeostatic expansion in Rag1-deficient mice T cells were purified from whole spleen and lymph nodes and sorted for YFP expression. 1×106 cells were injected retro-orbitally i.v. into Rag1-deficient mice. Mice were sacrificed 28 days later and spleens removed for analysis by flow cytometry. For competitive homeostatic expansion, T cells were purified from both CD45.1 mice (Jackson Laboratory) and CD45.2, Aifwt/y Lck-Cre LSL-YFP (T-AIF WT) and Aifflox/y Lck-Cre LSL-YFP (T-AIF KO) animals and co-injected (1:1 ratio; 5×105 cells each) i.v. into Rag1-deficient mice. Statistical analysis
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p values were determined by unpaired Student`s t test unless stated otherwise. p values < 0.05 were considered significant.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments We thank Richard Cross, Greig Lennon, and Parker Ingle for all cell sorting, and Mao Yang for technical support. This work was supported by ALSAC and by grants from the US National Institutes of Health.
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Fig. 1. Mitochondrial defects caused by loss of AIF can be rescued by expression of Ndi1
(A) Aifflox/y Rosa26.CreER LSL-YFP MEF were treated with vehicle (-) or 4-OH tamoxifen (4-OHT) (+) for the indicated times. Lysates assessed by Western blot (n=3). (B) OCR and ECAR were measured under basal conditions. Values are mean ± SD of triplicates (n=3). OCR: p=0.0047; ECAR: p=0.0017. (C) Complex I (Malate+Pyruvate), II (Succinate) and IV (TMPD+Ascorbate)-driven OCR of permeabilized MEF (n=5). Data are mean ± SD. Complex I: p=0.0066; complex II: p=0.0011; complex IV: p=0.0006. (D) AIF KO MEF stably expressing vector control (vector), wild type AIF (AIF) or Ndi1 (Ndi1) were treated
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with vehicle (-) or 4-OHT (+) for two weeks, and immunoblots of lysates performed (n=3). (E) Basal OCR and ECAR were determined for groups in D. Data are mean ± SD (n=3). OCR: vector control vs vector 4-OHT, p=0.0004; vector 4-OHT vs Ndi1 4-OHT, p=0.0004; ECAR: vector control vs vector 4-OHT, p=0.0027. (F). AIF KO MEF expressing vector control (Vector), wild type AIF (AIF) or Ndi1 (Ndi1) were treated with 4-OHT for two weeks and sorted for YFP expression. For each, 3×104 cells were cultured and clonogenic survival assessed after 11 days. Representative of five independent experiments. (G) AIF KO MEF stably expressing wild type AIF or Ndi1 after at least four weeks of 4-OHT treatment were treated with STS (1 µM), ActD (1 µM), etoposide (200 µM), diazald (500 µM) or serum withdrawal (Starvation) in the absence or presence of qVD-oph (40 µM) and cell death assessed. Results are representative of three independent experiments. Mean ± SD of triplicate cultures is shown. See also Figure S1.
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Author Manuscript Author Manuscript Author Manuscript Fig. 2. AIF is required for tissue homeostasis in the adult animal
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(A–E) Aifwt/y Rosa26.CreER or Aifflox/y (AIF WT) and Aifflox/y Rosa26.CreER (AIF KO) animals were gavaged with 1mg tamoxifen for 6 consecutive days. Animals were observed over 60 days for weight loss (A) and lethality (B) n=40 for each group, *p
