Eliseo F. Castillo

Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5fl/fl LysM-Cre+ mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5fl/fl LysM-Cre+ mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.

Neutral Lipid Stores and Lipase PNPLA5 Contribute to Autophagosome Biogenesis

BACKGROUND Autophagy is a fundamental cell biological process whereby eukaryotic cells form membranes in the cytoplasm to sequester diverse intracellular targets. Although significant progress has been made in understanding the origins of autophagosomal organelles, the source of lipids that support autophagic membrane formation remain an important open question. RESULTS Here we show that lipid droplets as cellular stores of neutral lipids including triglycerides contribute to autophagic initiation. Lipid droplets, as previously shown, were consumed upon induction of autophagy by starvation. However, inhibition of autophagic maturation by blocking acidification or using dominant negative Atg4(C74A) that prohibits autophagosomal closure did not prevent disappearance of lipid droplets. Thus, lipid droplets continued to be utilized upon induction of autophagy, but not as autophagic substrates in a process referred to as lipophagy. We considered an alternative model whereby lipid droplets were consumed not as a part of lipophagy, but as a potential contributing source to the biogenesis of lipid precursors for nascent autophagosomes. We carried out a screen for a potential link between triglyceride mobilization and autophagy and identified a neutral lipase, PNPLA5, as being required for efficient autophagy. PNPLA5, which localized to lipid droplets, was needed for optimal initiation of autophagy. PNPLA5 was required for autophagy of diverse substrates, including degradation of autophagic adaptors, bulk proteolysis, mitochondrial quantity control, and microbial clearance. CONCLUSIONS Lipid droplets contribute to autophagic capacity by enhancing it in a process dependent on PNPLA5. Thus, neutral lipid stores are mobilized during autophagy to support autophagic membrane formation.

Nfil3-independent lineage maintenance and antiviral response of natural killer cells

Inflammatory cytokines drive NK cell expansion in the absence of the transcription factor Nfil3, and Nfil3 is dispensable for the maintenance and function of mature NK cells.

Dendritic cells drive memory CD8 T-cell homeostasis via IL-15 transpresentation.

Interleukin-15 (IL-15) is crucial for the development of naive and memory CD8 T cells and is delivered through a mechanism called transpresentation. Previous studies showed that memory CD8 T cells require IL-15 transpresentation by an as yet unknown cell of hematopoietic origin. We hypothesized that dendritic cells (DCs) transpresent IL-15 to CD8 T cells, and we examined this by developing a transgenic model that limits IL-15 transpresentation to DCs. In this study, IL-15 transpresentation by DCs had little effect on restoring naive CD8 T cells but contributed to the development of memory-phenotype CD8 T cells. The generation of virus-specific, memory CD8 T cells was partially supported by IL-15Ralpha(+) DCs through the preferential enhancement of a subset of KLRG-1(+)CD27(-) CD8 T cells. In contrast, these DCs were largely sufficient in driving normal homeostatic proliferation of established memory CD8 T cells, suggesting that memory CD8 T cells grow more dependent on IL-15 transpresentation by DCs. Overall, our study clearly supports a role for DCs in memory CD8 T-cell homeostasis but also provides evidence that other hematopoietic cells are involved in this function. The identification of DCs fulfilling this role will enable future studies to better focus on mechanisms regulating T-cell homeostasis.

Autophagy as an immune effector against tuberculosis

The now well-accepted innate immunity paradigm that autophagy acts as a cell-autonomous defense against intracellular bacteria has its key origins in studies with Mycobacterium tuberculosis, an important human pathogen and a model microorganism infecting macrophages. A number of different factors have been identified that play into the anti-mycobacterial functions of autophagy, and recent in vivo studies in the mouse model of tuberculosis have uncovered additional anti-inflammatory and tissue-sparing functions of autophagy. Complementing these observations, genome wide association studies indicate a considerable overlap between autophagy, human susceptibility to mycobacterial infections and predisposition loci for inflammatory bowel disease. Finally, recent studies show that autophagy is an important regulator and effector of IL-1 responses, and that autophagy intersects with type I interferon pathology-modulating responses.

Dendritic cells support the in vivo development and maintenance of NK cells via IL-15 trans-presentation.

IL-15 is a key component that regulates the development and homeostasis of NK cells and is delivered through a mechanism termed trans-presentation. During development, multiple events must proceed to generate a functional mature population of NK cells that are vital for tumor and viral immunity. Nevertheless, how IL-15 regulates these various events and more importantly what cells provide IL-15 to NK cells to drive these events is unclear. It is known dendritic cells (DC) can activate NK cells via IL-15 trans-presentation; however, the ability of DC to use IL-15 trans-presentation to promote the development and homeostatic maintenance of NK cell has not been established. In this current study, we show that IL-15 trans-presentation solely by CD11c+ cells assists the in vivo development and maintenance of NK cells. More specifically, DC-mediated IL-15 trans-presentation drove the differentiation of NK cells, which included the up-regulation of the activating and inhibitory Ly49 receptors. Although these cells did not harbor a mature CD11bhigh phenotype, they were capable of degranulating and producing IFN-γ upon stimulation similar to wild-type NK cells. In addition, DC facilitated the survival of mature NK cells via IL-15 trans-presentation in the periphery. Thus, an additional role for NK-DC interactions has been identified whereby DC support the developmental and homeostatic niche of NK cells.

Secretory versus Degradative Autophagy: Unconventional Secretion of Inflammatory Mediators

Autophagy (macroautophagy) is often defined as a degradative process and a tributary of the lysosomal pathway. In this context, autophagy carries out cytoplasmic quality control and nutritional functions by removing defunct or disused organelles, particulate targets and invading microbes, and by bulk digestion of the cytoplasm. However, recent studies indicate that autophagy surprisingly affects multiple secretory pathways. Autophagy participates in extracellular delivery of a number of cytosolic proteins that do not enter the conventional secretory pathway via the Golgi apparatus but are instead unconventionally secreted directly from the cytosol. In mammalian cells, a prototypical example of this manifestation of autophagy is the unconventional secretion of a major proinflammatory cytokine, IL-1β. This review examines the concept of secretory autophagy and compares and contrasts the role of autophagy in the secretion of IL-1α and IL-1β. Although IL-1α and IL-1β have closely related extracellular inflammatory functions, they differ in intracellular activation, secretory mechanisms and how they are affected by autophagy. This example indicates that the role of autophagy in secretion is more complex, at least in mammalian cells, than the simplistic view that autophagosomes provide carriers for unconventional secretion of cytosolic proteins.

Regulating the immune system via IL-15 transpresentation.

Transpresentation has emerged as an important mechanism mediating IL-15 responses in a subset of lymphocytes during the steady state. In transpresentation, cell surface IL-15, bound to IL-15Rα is delivered to opposing lymphocytes during a cell-cell interaction. The events most dependent on IL-15 include the development and homeostasis of memory CD8 T cells, Natural Killer cells, invariant Natural Killer T cells, and intraepithelial lymphocytes. As lymphocyte development and homeostasis involve multiple steps and mechanisms, IL-15 transpresentation can have diverse roles throughout. Moreover, distinct stages of lymphocyte differentiation require IL-15 transpresented by different cells, which include both hematopoietic and non-hematopoietic cell types. Herein, we will describe the points where IL-15 transpresentation impacts these processes, the specific cells thought to drive IL-15 responses, as well as their role in the course of development and homeostasis.

Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation.

Invariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα(-/-) mice, CD44(High)NK1.1(+) iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-γ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation.

Coordinate activation of inflammatory gene networks, alveolar destruction and neonatal death in AKNA deficient mice.

Gene expression can be regulated by chromatin modifiers, transcription factors and proteins that modulate DNA architecture. Among the latter, AT-hook transcription factors have emerged as multifaceted regulators that can activate or repress broad A/T-rich gene networks. Thus, alterations of AT-hook genes could affect the transcription of multiple genes causing global cell dysfunction. Here we report that targeted deletions of mouse AKNA, a hypothetical AT-hook-like transcription factor, sensitize mice to pathogen-induced inflammation and cause sudden neonatal death. Compared with wild-type littermates, AKNA KO mice appeared weak, failed to thrive and most died by postnatal day 10. Systemic inflammation, predominantly in the lungs, was accompanied by enhanced leukocyte infiltration and alveolar destruction. Cytologic, immunohistochemical and molecular analyses revealed CD11b+Gr1+ neutrophils as major tissue infiltrators, neutrophilic granule protein, cathelin-related antimicrobial peptide and S100A8/9 as neutrophil-specific chemoattracting factors, interleukin-1β and interferon-γ as proinflammatory mediators, and matrix metalloprotease 9 as a plausible proteolytic trigger of alveolar damage. AKNA KO bone marrow transplants in wild-type recipients reproduced the severe pathogen-induced reactions and confirmed the involvement of neutrophils in acute inflammation. Moreover, promoter/reporter experiments showed that AKNA could act as a gene repressor. Our results support the concept of coordinated pathway-specific gene regulation functions modulating the intensity of inflammatory responses, reveal neutrophils as prominent mediators of acute inflammation and suggest mechanisms underlying the triggering of acute and potentially fatal immune reactions.