Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.     

Temporal regulation of intracellular organelle homeostasis in T lymphocytes by autophagy

The highly conserved self-degradation pathway known as autophagy plays important roles in regulating T lymphocyte homeostasis. Recently, we found that T lymphocytes lacking the autophagy-related gene Atg5 or Atg7 have defective survival and contain expanded mitochondria and endoplasmic reticulum (ER); however, whether these defects are caused by impaired autophagy or by defects in their autophagy-independent signaling capacity of Atg5 or Atg7 in T lymphocytes remains unknown. Furthermore, the function of the microtubule-associated protein L chain 3 (LC3) conjugation system in T lymphocytes remains unclear. To address these questions, we generated conditional knockout mice with specific deletion of Atg3, a ubiquitin enzyme E2-like molecule involved in the LC3 conjugation system, in T lymphocytes. Atg3-deficient T lymphocytes displayed a phenotype similar to those of Atg7- and Atg5-deficient T cells. The survival of Atg3-deficient naive CD4(+) and CD8(+) T cells was defective. Furthermore, the mitochondria and ER were expanded in Atg3-deficient T cells. Interestingly, mitochondrial and ER content did not change instantly upon inducible deletion of Atg3 in mature T lymphocytes in vitro. Instead, it began to expand 10 d after inducible deletion of Atg3 in mature T lymphocytes, and mitochondrial content continued to increase on day 18. Cell death began to increase 24 d after inducible deletion of Atg3. These data show that the LC3 conjugation system is essential for autophagy in T lymphocytes. Our data suggest that autophagy promotes T lymphocyte survival by regulating organelle homeostasis and that the decreased survival of autophagy-deficient T cells is due to the temporal accumulation of these autophagy-related defects.     

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G₂/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G₂/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G₂/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G₁/S phase, and increased COL1 expression in the G₂/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G₂/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G₂/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G₂/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G₂/M arrest.     

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTβR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.     

Self-reactive T cells selected on thymic cortical epithelium are polyclonal and are pathogenic in vivo

Positive selection of CD4+ T cells requires that the TCR of a developing thymocyte interact with self MHC class II molecules on thymic cortical epithelium. In contrast, clonal deletion is mediated by dendritic cells and medullary epithelium. We previously generated K14 mice expressing MHC class II only on thymic cortical epithelium. K14 CD4+ T cells were positively, but not negatively, selected and had significant in vitro autoreactivity. Here, we examine the function of these autoreactive CD4+ T cells in more detail. Analysis of a series of K14-derived T hybrids demonstrated that the autoreactive population of CD4+ T cells is phenotypically and functionally diverse. Purified K14 CD4+ T cells transferred into lethally irradiated wild-type B6 mice cause acute graft vs host disease with bone marrow failure. Further, these autoreactive CD4+ T cells cause hypergammaglobulinemia and the production of autoantibodies when transferred into unirradiated wild-type hosts. Thus, positive selection by normal thymic cortical epithelial cells, unopposed by negative selection, produces polyclonal CD4+ T cells that are pathologic.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter

Using a previously described human keratin 14 (K14) promoter, we created mice expressing a peptide Ag (OVAp) in epithelial cells of the skin, tongue, esophagus, and thymus. Double transgenic mice that also express a TCR specific for this Ag (OT-I) showed evidence for Ag-driven receptor editing in the thymus. Surprisingly, such mice exhibited a severe autoimmune disease. In this work we describe the features of this disease and demonstrate that it is dependent on CD8 T cells. Consistent with the Ag expression pattern dictated by the human K14 promoter, an inflammatory infiltrate was observed in skin and esophagus and around bile ducts of the liver. We also observed a high level of TNF-alpha in the serum. Given that Ag expression in the thymus induced development of T cells with dual TCR reactivity, and that dual-reactive cells have been suggested to have autoimmune potential, we tested whether they were a causal factor in the disease observed here. We found that OT-I/K14-OVAp animals on a recombinase-activating gene-deficient background still suffered from disease. In addition, OT-I animals expressing OVA broadly in all tissues under a different promoter did not experience disease, despite having a similar number of dual-specific T cells. Thus, in this model it would appear that dual-reactive T cells do not underlie autoimmune pathology. Finally, we extended these observations to a second transgenic system involving 2C TCR-transgenic animals expressing the SIY peptide Ag with the hK14 promoter. We discuss the potential relationship between autoimmunity and self-Ags that are expressed in stratified epithelium.     

Galectin-1 is expressed by thymic epithelial cells in myasthenia gravis

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the -galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.     

Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1

The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8⁺ T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.     

Nonspecific down-regulation of CD8+ T-cell responses in mice expressing human papillomavirus type 16 E7 oncoprotein from the keratin-14 promoter

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-14 (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8+ cells is down-regulated compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter. Down-regulation did not involve deletion of CD8+ T cells of high affinity or high avidity, and T-cell receptor (TCR) Vbeta-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge. We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T. Doan et al., J. Virol. 73:6166-6170, 1999). The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.     

B cell autophagy mediates TLR7-dependent autoimmunity and inflammation

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5^f/f). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

Evidence for an early role for BMP4 signaling in thymus and parathyroid morphogenesis

The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. these results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.     

A novel role for autophagy in T cell education

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.     

Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.