Milestones — Miao Lab

05

Innate Granuloma Model

Granulomas are characterized by the organization of macrophages in tissues to fight infection or wall off a foreign body. Granulomas often form around infections that cannot be cleared, and their function is thought to prevent dissemination. Pathogens can persist within these granulomas for decades. These structures are defined by the presence of macrophages, but typically they are organized by T cells of the adaptive immune response.

While studying the innate defenses against the environmental bacterium Chromobacterium violaceum, we serendipitously discovered that mice form a picture-perfect granuloma around infected lesions in the liver. This granuloma forms within 5 days and then eradicates the bacteria over the next 5–14 days — all without help from T cells or other adaptive immune cells. The C. violaceum-induced granuloma requires at least two separate defense pathways, gasdermin D and NOS2, to maintain the integrity of the granuloma architecture¹². Furthermore, CCR2 is essential for the recruitment of monocytes that become granuloma macrophages¹³. Without any of these genes, mice fail to form functional granulomas and succumb to C. violaceum infection. In wild type mice, these innate granulomas efficiently eradicate C. violaceum and the liver returns to homeostasis. This new C. violaceum-induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby eradicate infection by an environmental pathogen.

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04

Caspase-7 as a Death Facilitator

Among the apoptotic executioners, caspase-3 is sufficient for apoptosis. Caspase-7 was considered an inefficient backup with no unique function. Pyroptotic caspase-1 could activate caspase-7, but for unknown reasons. In intestinal epithelial cells (IECs), caspase-1 drives extrusion of the IEC to eject it from the intestinal monolayer. During this process, we discovered that caspase-7 activates membrane repair, which was needed to ameliorate pathology during intestinal infection by Salmonella. We discovered that caspase-7 accomplishes this by hyperactivating acid sphingomyelinase (ASM) to produce ceramide, which drives repair of gasdermin D pores. Therefore, caspase-7 is not a conventional executioner, but instead, a death facilitator that delays gasdermin D-driven lysis so that more elegant processes (e.g. extrusion) can be completed prior to cell death⁹.

Intracellular bacteria can be counteracted by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which attack infected cells. NK/CTLs use perforin pores to deliver granzyme B, which activates apoptotic caspases. Listeria was long known to be cleared by CTL-perforin attack, and we discovered that Chromobacterium violaceum can be cleared by NK-perforin attack¹⁰. We showed that NK/CTL perforin-mediated attack during Chromobacterium and Listeria infection uniquely required caspase-7, which intensified the enzymatic activity of ASM. ASM inhibition or cleavage site mutation abrogated Chromobacterium and Listeria clearance after NK/CTL attack⁹. Thus, our data suggests that after NK/CTL attack caspase-7 will preserve cell integrity, providing time to complete apoptotic processes that are essential to clear the infection. This discovery leads us to propose that caspase-7 buys a cell time to complete “bucket list” tasks before it dies¹¹. Cells undergoing regulated cell death must put their affairs in order before they die.

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03

Cytosolic LPS Sensing by Caspase-11

Caspase-11 had been poorly understood until Dr. Vishva Dixit discovered in 2011 that caspase-11 could cause pyroptosis, and that Casp1^−/− mice were actually Casp1^−/−Casp11^−/− mice due to a passenger mutation. We discovered that caspase-11 discriminates cytosolic bacteria from vacuolar/extracellular bacteria. Caspase-11 detected the cytosol-invasive bacteria Burkholderia pseudomallei and B. thailandensis. Meanwhile, caspase-11 would not detect vacuolar pathogens such as Salmonella and Legionella, but would detect mutants of these that aberrantly enter the cytosol⁶. Whereas wild type mice resist challenges of up to 20,000,000 B. thailandensis, Casp11^−/− mice were acutely susceptible to infection by as few as 100 CFUs of this environmental pathogen⁷.

We sought the mechanism by which caspase-11 detected cytosolic bacteria. LPS was long thought to be detected by a single innate immune sensor, TLR4, which was the first pattern recognition receptor discovered. We showed that in addition to TLR4 detecting extracellular and vacuolar LPS, caspase-11 detected LPS in the cytosolic compartment. This explained how caspase-11 identified cytosol-invasive bacteria. Since both TLR4 and caspase-11 are detrimental during LPS challenge in mice, we hypothesized that the two pathways work in concert, with TLR4 priming the caspase-11 pathway into a ready state. Indeed, priming with another TLR agonist caused accelerated mortality that was independent of TLR4, but required caspase-11. This was the first demonstration that TLR4 is dispensable during endotoxic shock in mice⁸. Our work demonstrated that cytosolic LPS is detected by caspase-11, and this causes pyroptosis to eliminate cells infected with cytosol-invasive bacteria.

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02

Pyroptosis and Pore-Induced Intracellular Traps

Caspase-1-dependent programmed lytic cell death (pyroptosis) was first observed in vitro in 1992; however, for the next 18 years there was no evidence for its in vivo physiologic importance. By engineering Salmonella to express flagellin, we discovered the first evidence that pyroptosis is a critical innate immune effector mechanism in vivo. We showed that pyroptosis cleared the bacteria independently from the caspase-1-driven cytokines IL-1β and IL-18⁴.

Importantly, the bacteria survive pyroptosis, but remain trapped within the cellular debris of pyroptotic macrophages. This trapping appears to be an inevitable consequence of osmotic lysis. We call the pyroptotic corpse a pore-induced intracellular trap (PIT), which is conceptually parallel to the neutrophil extracellular trap (NET) in its ability to detain bacteria. The PIT coordinates innate immune responses via complement and scavenger receptors to drive recruitment of and efferocytosis by neutrophils. Ultimately, this secondary phagocyte kills the bacteria. Bona fide intracellular bacterial pathogens, such as Salmonella, must prevent or delay pyroptosis in order to avoid being trapped in the PIT and subsequently killed by neutrophils⁵.

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01

NLRC4 Inflammasome Detects Bacterial Flagellin

NLRC4 (previously called Ipaf) forms an inflammasome that activates caspase-1, the protease that cleaves and promotes the secretion of IL-1β and IL-18, two critical cytokines involved in inflammatory and infectious disease. Before 2006, NLRC4 was known to respond to Salmonella Typhimurium in vitro, but how the bacteria were detected was unknown. We showed that macrophages use NLRC4 to detect the activity of T3SS indirectly by monitoring for the inadvertent translocation of flagellin into the macrophage cytosol¹. We later discovered that the T3S rod protein was also detected by NLRC4². Another group later also found that the T3SS needle protein is detected by human NLRC4, and we subsequently showed that this also occurs in mice³.

Remarkably, we showed that S. typhimurium evades NLRC4 detection during systemic infection in vivo by repressing flagellin and expressing a variant T3SS rod protein that is not detected^2,4. Flagellin, T3SS rod, and T3SS needle are excellent targets for innate immune detection because they are more conserved than T3SS effectors, and slow to evolve.

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