Cardiolipin coordinates inflammatory metabolic reprogramming through regulation of Complex II disassembly and degradation.

Macrophage metabolic plasticity enables repurposing of electron transport from energy generation to inflammation and host defense. Altered respiratory complex II function has been implicated in cancer, diabetes, and inflammation, but regulatory mechanisms are incompletely understood. Here, we show that macrophage inflammatory activation triggers Complex II disassembly and succinate dehydrogenase subunit B loss through sequestration and selective mitophagy. Mitochondrial fission supported lipopolysaccharide-stimulated succinate dehydrogenase subunit B degradation but not sequestration. We hypothesized that this Complex II regulatory mechanism might be coordinated by the mitochondrial phospholipid cardiolipin. Cardiolipin synthase knockdown prevented lipopolysaccharide-induced metabolic remodeling and Complex II disassembly, sequestration, and degradation. Cardiolipin-depleted macrophages were defective in lipopolysaccharide-induced pro-inflammatory cytokine production, a phenotype partially rescued by Complex II inhibition. Thus, cardiolipin acts as a critical organizer of inflammatory metabolic remodeling.

Human neutrophil IL1ß directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins.

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids.

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.

Salmonella enterica Serovar Typhimurium SPI-1 and SPI-2 Shape the Global Transcriptional Landscape in a Human Intestinal Organoid Model System.

The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica Typhimurium. While the interaction of S Typhimurium with the mammalian host has been well studied in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestine. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells and a supporting mesenchymal cell layer. HIOs contain luminal space that supports bacterial replication, are more amenable to experimental manipulation than animals and are more reflective of physiological host responses. Here, we use the HIO model to define host transcriptional responses to S Typhimurium infection, also determining host pathways dependent on Salmonella pathogenicity island-1 (SPI-1)- and -2 (SPI-2)-encoded type 3 secretion systems (T3SS). Consistent with prior findings, we find that S Typhimurium strongly stimulates proinflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient, and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, S Typhimurium infection led to significant downregulation of host genes associated with cell cycle and DNA repair, leading to a reduction in cellular proliferation, dependent on SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as mediators of S Typhimurium-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reinforce host cell proliferation as a process targeted by SalmonellaIMPORTANCESalmonella enterica serovar Typhimurium (S Typhimurium) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, physiological human host responses driven by major virulence determinants of Salmonella have been more challenging to evaluate. Here, we use the three-dimensional human intestinal organoid model to define early responses to infection with wild-type S Typhimurium and mutants defective in the SPI-1 or SPI-2 type-3 secretion systems. While both secretion system mutants show defects in mouse models of oral Salmonella infection, the specific contributions of each secretion system are less well understood. We show that S Typhimurium upregulates proinflammatory pathways independently of either secretion system, while the downregulation of the host cell cycle pathways relies on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

tPA Deficiency in Mice Leads to Rearrangement in the Cerebrovascular Tree and Cerebroventricular Malformations.

The serine protease tissue-type plasminogen activator (tPA) is used as a thrombolytic agent in the management of ischemic stroke, but concerns for hemorrhagic conversion greatly limits the number of patients that receive this treatment. It has been suggested that the bleeding complications associated with thrombolytic tPA may be due to unanticipated roles of tPA in the brain. Recent work has suggested tPA regulation of neurovascular barrier integrity, mediated via platelet derived growth factor (PDGF)-C/PDGF receptor-α (PDGFRα) signaling, as a possible molecular mechanism affecting the outcome of stroke. To better understand the role of tPA in neurovascular regulation we conducted a detailed analysis of the cerebrovasculature in brains from adult tPA deficient (tPA(-/-) ) mice. Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls. In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1. This is intriguing since ERG is an endothelial transcription factor implicated in regulation of vascular integrity. Based on these results, we propose that the protection of barrier properties seen utilizing these tPA (-/-) mice might be due, at least in part, to these cerebrovascular rearrangements. In addition, we found that tPA (-/-) mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development. Taken together, the data presented here will advance our understanding of the role of tPA within the CNS and in regulation of cerebrovascular permeability.

Imatinib treatment reduces brain injury in a murine model of traumatic brain injury.

Current therapies for Traumatic brain injury (TBI) focus on stabilizing individuals and on preventing further damage from the secondary consequences of TBI. A major complication of TBI is cerebral edema, which can be caused by the loss of blood brain barrier (BBB) integrity. Recent studies in several CNS pathologies have shown that activation of latent platelet derived growth factor-CC (PDGF-CC) within the brain can promote BBB permeability through PDGF receptor α (PDGFRα) signaling, and that blocking this pathway improves outcomes. In this study we examine the efficacy for the treatment of TBI of an FDA approved antagonist of the PDGFRα, Imatinib. Using a murine model we show that Imatinib treatment, begun 45 min after TBI and given twice daily for 5 days, significantly reduces BBB dysfunction. This is associated with significantly reduced lesion size 24 h, 7 days, and 21 days after TBI, reduced cerebral edema, determined from apparent diffusion co-efficient (ADC) measurements, and with the preservation of cognitive function. Finally, analysis of cerebrospinal fluid (CSF) from human TBI patients suggests a possible correlation between high PDGF-CC levels and increased injury severity. Thus, our data suggests a novel strategy for the treatment of TBI with an existing FDA approved antagonist of the PDGFRα.