Lou Ellis

Endoplasmic reticulum stress-induced transcription factor, CHOP, is crucial for dendritic cell IL-23 expression

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand—cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

IRE1α mediates PKR activation in response to Chlamydia trachomatis infection

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-β production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.