Biology subjects

McKee, C. M.

Publications and source records attributed to McKee, C. M..

4 recordsLinked to original sources

bioRxiv2025

NLRP3 is a thermosensor that is negatively regulated by high temperature

Inflammation is an essential response to infection and injury, but unregulated inflammation is damaging and must be limited by negative feedback signalling. Inflammasome signalling drives local inflammation and systemic responses like fever. However, our understanding of how inflammasome signalling is negatively regulated is limited. NLRP3 is activated by a vast number of stimuli and senses perturbations of cytoplasmic homeostasis. As temperature is a fundamental environmental stressor, we hypothesised that NLRP3 inflammasome signalling would be sensitive to increased temperatures and so we investigated the effects of high temperatures on NLRP3 in macrophages. Short-term incubation at high fever range temperatures significantly inhibits NLRP3 activation, while secretion of the inflammasome-independent cytokines TNF and IL-6 are much less affected. High temperature blocks NLRP3 inflammasome formation in a transcription-independent manner, and NLRP3 is highly sensitive to temperature-mediated inhibition relative to the NLRC4, AIM2, and NLRP1 inflammasomes. Using cellular assays and molecular simulations we show that the effect of high temperature on NLRP3 is protein intrinsic. NLRP3 activation is associated with a decrease in the thermal stability of the protein and multiscale molecular dynamics simulations identified a peptide in the C-terminal of the FISNA domain (COFI) that is highly flexible and undergoes a significant conformational shift at high temperature. Cellular assays demonstrate that the COFI regulates NLRP3 stability and is required for activation. Furthermore, mice exposed to high temperature display attenuated inflammatory cytokine production upon in vivo LPS challenge. Our studies thus reveal that high temperatures associated with fever limit NLRP3 activity and identify a novel role for NLRP3 as a protein thermosensor.

immunology↗

bioRxiv2025

Induced pluripotent stem cell-derived macrophages as a model for human inflammasome signaling

Macrophage models are a mainstay of inflammasome research, however current human in vitro macrophage models have significant limitations. Here we generate induced pluripotent stem cell (iPSC)-derived macrophages (iMacs) to study inflammasome signaling and benchmark them with human monocyte-derived macrophages (HMDMs). We confirm that iMacs express high levels of macrophage markers and are highly phagocytic. Whole cell proteomics analysis shows that iMacs express many inflammasome sensors and related proteins, and in functional assays iMacs respond to multiple inflammasome stimuli. The NLRP3 inflammasome is strongly activated in iMacs and we find that nigericin alone activates NLRP3. The non-canonical inflammasome does not require a priming step in iMacs as caspase-4 is constitutively expressed. High levels of NAIP/NLRC4 inflammasome activation are also observed in response to needle toxin. Finally, unlike HMDMs, iMacs activate NLRP1. Therefore, we demonstrate that iMacs are a physiologically relevant and highly tractable model to study human inflammasome signaling and regulation. MotivationiPSC-derived macrophages (iMacs) are functionally, transcriptionally, and phenotypically similar to primary human macrophages. iMacs therefore offer new opportunities to study inflammasome activity in a human macrophage model, but to date they have not been widely used. In this study, we describe a protocol to differentiate and characterize iMacs. We then describe how to activate a range of different inflammasomes within these cells and assess the inflammasome response by measuring pyroptosis, cytokine release, ASC speck formation, and processing of inflammasome-related proteins. We also benchmark iMac responses with the current gold standard primary human monocyte derived macrophage model.

immunology↗

bioRxiv2025

Plasma membrane rather than endosomal Gq signaling drives transcriptional activity by the viral chemokine receptor US28 in glioblastoma

US28 is a human cytomegalovirus-encoded chemokine receptor homologue that has high agonist-independent activity, internalizes constitutively, and plays an oncomodulatory role in glioblastoma. As G protein signaling was originally believed to strictly occur at the plasma membrane, it has been assumed that US28s constitutive Gq/11 signaling is mediated by a minor population at the plasma membrane. However, accumulating evidence shows that some GPCRs activate G proteins from intracellular organelles, such as endosomes. Importantly, endosomal rather than plasma membrane G protein signaling has been associated with transcriptional activity. Here, we demonstrate that the endosomal US28 population robustly activates Gq/11, and thus, provides the major contribution of Gq/11 signaling. Surprisingly, US28 signaling at the plasma membrane rather than from endosomes primarily drives upregulation of gene expression involved in cell proliferation and inflammatory responses that are associated with glioblastoma and cancer. Our findings highlight the crucial role of receptor signaling location in cellular responses.

cell biology↗

bioRxiv2024

Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1{beta} and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. SUMMARYNLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of a new small molecule inhibitor of NLRP3 BAL-0028 that has a distinct mechanism of action and binding site.

immunology↗