Biology subjects

Knight, J. S.

Publications and source records attributed to Knight, J. S..

6 recordsLinked to original sources

bioRxiv2022

The IRE1α-XBP1 signaling axis promotes glycolytic reprogramming in response to inflammatory stimuli

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the ER stress sensor IRE1 and its downstream transcription factor XBP1 enhance the upregulation of glycolysis in classically activated macrophages (CAM). The IRE1-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by LPS stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1 activation; while TLR4 supports glycolysis under both conditions, TLR4 is required for activation of IRE1 in response to LPS treatment but not B. abortus infection. Though IRE1 and XBP1 are necessary for maximal induction of glycolysis in CAM, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cells metabolic response and that IRE1 activation may be a way to fine-tune metabolic reprogramming. IMPORTANCEThe immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1 by independent mechanisms. While the IRE1-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1 and XBP1 in innate immunity.

immunology↗

bioRxiv2022

Neutrophils prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal Salmonella enterica serovars

Nontyphoidal strains of Salmonella enterica are a major cause of foodborne illnesses and infection with these bacteria result in inflammatory gastroenteritis. Neutrophils are a dominant immune cell type found at the site of infection in Salmonella-infected individuals, but how they regulate infection outcome is not well understood. Here we used a co-culture model of primary human neutrophils and human intestinal organoids to probe the role of neutrophils during infection with two of the most prevalent Salmonella serovars: Salmonella enterica serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for neutrophils in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that are induced by neutrophils in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together these findings provide insight into the role of human neutrophils in modulating different host responses to pathogens that cause similar disease in humans. ImportanceNontyphoidal serovars of Salmonella enterica are known to induce robust neutrophil recruitment in the gut during early stages of infection, but the specific role of neutrophils in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of neutrophils during infection has been challenging. Here we used a co-culture model of human intestinal organoids with human primary neutrophils to study the role of neutrophils during infection of human intestinal epithelium. Using a transcriptomics approach, we define neutrophil-dependent reprogramming of the host response to Salmonella, establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by neutrophils differed between two similar nontyphoidal Salmonella serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.

immunology↗

bioRxiv2022

Cardiolipin coordinates inflammatory metabolic reprogramming through regulation of Complex II assembly and stability

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-B subunit loss through sequestration and mitophagy. Mitochondrial fission was required for lipopolysaccharide-stimulated succinate dehydrogenase-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.

immunology↗

bioRxiv2022

Human neutrophils direct epithelial cell extrusion to enhance intestinal epithelial host defense 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 then seeded with primary human polymorphonuclear leukocytes (PMN-HIOs), specifically neutrophils and analyzed for bacterial growth and host cell survival. Surprisingly, PMNs did not affect luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed intestinal epithelial cells that could be blocked by Caspase-1 or Caspase-3 inhibition. Cleaved Caspase-3 was present in epithelial cells, but expression of the inflammasome adaptor, ASC, was only detected in PMNs. Caspase inhibition also increased bacterial burden in the epithelium of the PMN-HIO, suggesting PMNs enhance activation of cell death pathways in human intestinal epithelial cells as a protective response to infection. These data support a critical function for neutrophils beyond their antimicrobial role whereby they amplify cell death and extrusion of epithelial cells from the Salmonella-infected intestinal monolayer. Significance statementNeutrophils are early responders to Salmonella intestinal infection, but how they influence infection progression and outcome is unknown. Here we use a co-culture model of human intestinal organoids and human primary neutrophils to study the contribution of human neutrophils to Salmonella infection of the intestinal epithelium. We found that neutrophils markedly enhanced epithelial defenses, including enhancing cell extrusion to reduce intraepithelial burden of Salmonella and association with the epithelium, rather than directly killing Salmonella in the HIO lumen. These findings reveal a novel role for neutrophils in the gut beyond killing invading pathogens and illuminate how neutrophils can reprogram cells in the gut environment to enhance antimicrobial defenses.

microbiology↗

bioRxiv2020

Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts.

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 {degrees}C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 {+/-} 23 nmoles.min-1.mg-1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 {+/-} 0.01 M (mean {+/-} SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naive, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.

molecular biology↗

bioRxiv2019

The IRE1α stress signaling axis is a key regulator of neutrophil antimicrobial effector function

Activation of the endoplasmic reticulum stress sensor, IRE1, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases where neutrophils are a key immune component. However, the specific role of IRE1 in regulating neutrophil effector function has not been studied. Here we show that infection-induced IRE1 activation licenses neutrophil antimicrobial capacity, including IL-1{beta} production, NET formation, and MRSA killing. Inhibition of IRE1 diminished production of mitochondrial reactive oxygen species (mROS) and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in Caspase-2 were highly susceptible to MRSA infection and failed to form NETs in a subcutaneous abscess. IRE1 activation enhanced calcium influx and citrullination of histone H3 (Cit-H3) independently of mROS production, suggesting that IRE1 coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1-Caspase-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1 in neutrophil antibacterial function.\n\nOne Sentence SummaryIRE1 controls neutrophil antimicrobial defenses

immunology↗