Autophagy and Inflammatory Cell Death, Partners of Innate Immunity

As sentinels of the immune system, macrophages routinely capture and interrogate foreigners, then transmit details of their discovery to defensive specialists. To detect a broad array of pathogen-associated molecular patterns, macrophages are equipped with pattern recognition receptors. Extra-cellular contaminants are recognized by Toll-like Receptors, whereas surveillance in the cytoplasm is the work of NOD-LRR proteins, recognized by their nucleotide-binding oligomerization domains and leucine-rich repeats (reviewed in ^{1, 2}). Most often, ingested microbes are rapidly delivered to lysosomes for degradation, then their antigens are displayed. If the situation is dire, macrophages may commit pyroptosis, a pro-inflammatory programmed cell death that not only denies safe haven to intracellular microbes but also recruits other leukocytes to the site.³ More recently it has become apparent that cells also combat infection by activating autophagy,⁴ an alternate route to the digestive lysosomes that is also coupled to antigen presentation.^5,6

Insight to macrophage function has been obtained from studies of L. pneumophila, a versatile gram-negative bacterium that can replicate within a wide variety of cells, including protozoa, amoebae, and human macrophages. In contrast, mice are resistant to infection. An exception is the A/J mouse, as mutations in its NOD-LRR naip5 locus make both the animal and its isolated macrophages susceptible to L. pneumophila infection.^7–9 When ingested by resistant Naip5⁺ C57Bl/6 macrophages, single L. pneumophila can establish specialized replication vacuoles, but within hours their multiplication grinds to a halt.^10,11 As compared to permissive naip5 mutant A/J cells, resistant Naip5⁺ macrophages exhibit rapid maturation of autophagosomes, whether induced by amino acid depletion, rapamycin, or L. pneumophila.¹² Furthermore, Naip5⁺ macrophages are more sensitive to pathogen-induced death.¹¹ When exposed to numerous L. pneumophila, Naip5⁺ macrophages succumb to a death that is distinct from classical apoptosis, as judged by the morphology of their nuclei.¹¹ Moreover, the intoxicated cells rapidly discharge the inflammatory cytokine IL-1β (Molofsky, Byrne and Swanson, unpublished data). Thus, rather than commit a slow and silent apoptotic death, macrophages infected by multiple L. pneumophila likely initiate pyroptosis, a rapid caspase 1-dependent response in which active IL-1β and IL-18 are released through the cells permeable plasma membranes.³ We propose that Naip5⁺ murine macrophages can induce either autophagy or pyroptosis to restrict L. pneumophila infection.

By what mechanism might autophagy and pyroptosis be activated by the Naip5 NOD-LRR cytosolic surveillance system? The biochemical function of murine Naip5 has not been examined, nor is it known whether the A/J naip5 mutations confer a partial loss or a change of Naip5 function. Nevertheless, predictions can be drawn from studies of Naip5 homologues. By analogy to Toll-like Receptors, the Naip5 C-terminal LRR domain is predicted to bind a molecule that is common to microbes, its central NOD region to mediate oligomerization, and its N-terminal BIR (baculovirus inhibitor of apoptosis repeat) region to bind and inhibit a caspase enzyme.⁷ Thus, the Naip5 protein is equipped to regulate a caspase-dependent response to contamination of the host cytosol.

One target of NOD proteins is the assembly of inflammasomes, protein complexes in which caspase 1 is activated¹³. Indeed, a number of independent observations are consistent with the hypothesis that Naip5 regulates caspase 1. First, in murine macrophages exposed to L. pneumophila, Naip5 function correlates with susceptibility to cell death and robust autophagosome maturation.^11,12 The intracellular pathogens Salmonella typhimurium and Shigella flexneri also trigger autophagy and caspase 1-dependent inflammatory cell death.^14–17 Second, in response to L. pneumophila infection, Naip5 protein accumulates in macrophages.¹⁸ Third, in the human kidney 293 cell line, Naip function protects cells from caspase 1-mediated death and independently exerts a cytoprotective effect.¹⁹ To do so, the human Naip protein activates JNK1, a mitogen-activated protein kinase.¹⁹ Fourth, when macrophages derived from human peripheral blood monocytes are infected with L. pneumophila, JNK kinase signaling is unusually prolonged and correlates with robust L. pneumophila replication,²⁰ suggesting that the pathogen somehow perturbs JNK regulation and reduces its efficacy. Fifth, studies of monocytes manipulated by RNAi technology indicate that JNK kinase contributes to a signal transduction pathway that induces both autophagy and cell death.²¹ In some manner, Naip proteins appear to impinge on JNK and caspase 1 activity to govern autophagy and cell death.

As depicted in the Figure, we postulate that when murine macrophages ingest L. pneumophila, the Naip5 surveillance system detects microbial products that escape into the cytoplasm through pores made by the pathogen’s Type IV secretion system.²² Consequently, inflammasomes assemble and activate caspase 1 by a mechanism that requires the JNK signal transduction pathway. When the contamination is scant, macrophages elevate autophagy, a membrane trafficking pathway that captures the pathogen within membranes derived from the early secretory pathway for delivery to degradative lysosomes.^12,23–26 If autophagy fails to contain the infection, caspase 1 activity exceeds the threshold required to induce pyroptosis, a programmed cell death pathway that coordinates the maturation and release of pro-inflammatory cytokines from the permeable intoxicated macrophage.

Assuming the model proves correct, the macrophage response to intracellular pathogens provides yet another example of the regulatory link between autophagy and programmed cell death (reviewed in ²⁷). By exploiting L. pneumophila as a genetic probe of macrophage function, details of the mechanism by which autophagy and pyroptosis contribute to innate and acquired immunity can be discerned.