Joseph Kim Rathkey

All Publications

• Statin Use Is Associated With Protection Against Acute Cholangitis in Patients With Primary Sclerosing Cholangitis: A Multicenter Retrospective Cohort Study. Clinical and translational gastroenterology Kulkarni, C., Cholankeril, G., Fardeen, T., Rathkey, J., Khan, S., Murag, S., Lerrigo, R., Kamal, A., Mannalithara, A., Jalal, P., Ahmed, A., Vierling, J., Goel, A., Sinha, S. R. 2025; 16 (4): e00816

  Abstract

  Patients with primary sclerosing cholangitis (PSC) are at increased risk of acute cholangitis. The epidemiological risks of cholangitis are poorly studied despite the high morbidity associated with this infection. The aim of this study was to understand the impact of statins on acute cholangitis in PSC.This multicenter, retrospective cohort study assessed data from 294 patients with PSC at Stanford Medical Center, Baylor Medical Center, and Valley Medical Center. Clinical factors associated with the development of cholangitis were identified using multivariable Cox regression.The patients were predominantly male (68.7%) with a median age at enrollment of 48 years (interquartile range [IQR]: 31.0-60.8). Fifty patients (17.0%) were prescribed statins. The median follow-up time was 6 years (IQR: 2.0-12.0), in which 29.6% (n = 87) developed cholangitis. In multivariable analysis, statins were associated with an 81% reduction in cholangitis (HR 0.19, 95% confidence interval 0.03-0.64). Statins were associated with a lower adjusted incidence of cholangitis at 36 months compared with patients not on statin therapy (incidence of 2.8% vs 12.2%, P < 0.001). Statins were also associated with increased time-to-stricture ( P = 0.004), an outcome known to be associated with PSC complications.Statin therapy is associated with reduced risk of cholangitis in PSC, possibly by delaying the time to develop dominant or high-grade strictures. In patients with PSC, use of statin therapy may be a beneficial modality to prevent the development of cholangitis and warrants further investigation.

  View details for DOI 10.14309/ctg.0000000000000816

  View details for PubMedID 40272937

• N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis. Nature communications Karmakar, M., Minns, M., Greenberg, E. N., Diaz-Aponte, J., Pestonjamasp, K., Johnson, J. L., Rathkey, J. K., Abbott, D. W., Wang, K., Shao, F., Catz, S. D., Dubyak, G. R., Pearlman, E. 2020; 11 (1): 2212

  Abstract

  Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.

  View details for DOI 10.1038/s41467-020-16043-9

  View details for PubMedID 32371889

  View details for PubMedCentralID PMC7200749

• Human polymorphisms in GSDMD alter the inflammatory response. The Journal of biological chemistry Rathkey, J. K., Xiao, T. S., Abbott, D. W. 2020; 295 (10): 3228-3238

  Abstract

  Exomic studies have demonstrated that innate immune genes exhibit an even higher degree of variation than the majority of other gene families. However, the phenotypic implications of this genetic variation are not well understood, with effects ranging from hypomorphic to silent to hyperfunctioning. In this work, we study the functional consequences of this variation by investigating polymorphisms in gasdermin D, the key pyroptotic effector protein. We find that, although SNPs affecting potential posttranslational modifications did not affect gasdermin D function or pyroptosis, polymorphisms disrupting sites predicted to be structurally important dramatically alter gasdermin D function. The manner in which these polymorphisms alter function varies from conserving normal pyroptotic function to inhibiting caspase cleavage to disrupting oligomerization and pore formation. Further, downstream of inflammasome activation, polymorphisms that cause loss of gasdermin D function convert inflammatory pyroptotic cell death into immunologically silent apoptotic cell death. These findings suggest that human genetic variation can alter mechanisms of cell death in inflammation.

  View details for DOI 10.1074/jbc.RA119.010604

  View details for PubMedID 31988247

  View details for PubMedCentralID PMC7062166

• Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis SCIENCE IMMUNOLOGY Rathkey, J. K., Zhao, J., Liu, Z., Chen, Y., Yang, J., Kondolf, H. C., Benson, B. L., Chirieleison, S. M., Huang, A. Y., Dubyak, G. R., Xiao, T. S., Li, X., Abbott, D. W. 2018; 3 (26)

  Abstract

  Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1β, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

  View details for DOI 10.1126/sciimmunol.aat2738

  View details for Web of Science ID 000443217500005

  View details for PubMedID 30143556

  View details for PubMedCentralID PMC6462819

• Unique BIR domain sets determine inhibitor of apoptosis protein-driven cell death and NOD2 complex signal specificity SCIENCE SIGNALING Chirieleison, S. M., Rathkey, J. K., Abbott, D. W. 2018; 11 (539)

  Abstract

  The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.

  View details for DOI 10.1126/scisignal.aao3964

  View details for Web of Science ID 000438973900001

  View details for PubMedID 30018081

  View details for PubMedCentralID PMC6066185

• Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D-derived peptide inhibitor. Proceedings of the National Academy of Sciences of the United States of America Yang, J., Liu, Z., Wang, C., Yang, R., Rathkey, J. K., Pinkard, O. W., Shi, W., Chen, Y., Dubyak, G. R., Abbott, D. W., Xiao, T. S. 2018; 115 (26): 6792-6797

  Abstract

  The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

  View details for DOI 10.1073/pnas.1800562115

  View details for PubMedID 29891674

  View details for PubMedCentralID PMC6042100

• Structures of the Gasdermin D C-Terminal Domains Reveal Mechanisms of Autoinhibition. Structure (London, England : 1993) Liu, Z., Wang, C., Rathkey, J. K., Yang, J., Dubyak, G. R., Abbott, D. W., Xiao, T. S. 2018; 26 (5): 778-784.e3

  Abstract

  Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation.

  View details for DOI 10.1016/j.str.2018.03.002

  View details for PubMedID 29576317

  View details for PubMedCentralID PMC5932255

• Live-cell visualization of gasdermin D-driven pyroptotic cell death JOURNAL OF BIOLOGICAL CHEMISTRY Rathkey, J. K., Benson, B. L., Chirieleison, S. M., Yang, J., Xiao, T. S., Dubyak, G. R., Huang, A. Y., Abbott, D. W. 2017; 292 (35): 14649-14658

  Abstract

  Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.

  View details for DOI 10.1074/jbc.M117.797217

  View details for Web of Science ID 000408747500029

  View details for PubMedID 28726636

  View details for PubMedCentralID PMC5582855

• Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease JOURNAL OF BIOLOGICAL CHEMISTRY Chirieleison, S. M., Marsh, R. A., Kumar, P., Rathkey, J. K., Dubyak, G. R., Abbott, D. W. 2017; 292 (23): 9666-9679

  Abstract

  The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.

  View details for DOI 10.1074/jbc.M117.781500

  View details for Web of Science ID 000403113000017

  View details for PubMedID 28404814

  View details for PubMedCentralID PMC5465490

• People choose to run at their optimal speed. American journal of physical anthropology Rathkey, J. K., Wall-Scheffler, C. M. 2017; 163 (1): 85-93

  Abstract

  The purpose of this article is to test whether people choose to behave in a manner that reduces the amount of energy they use to travel a given distance. While this has been shown consistently for walking, it has never been tested with human running.We collected energetic data and lower limb anthropometrics on nine men running at six different running speeds. We collected on all six speeds on 3 different days and took the average of the energetic values for each speed. On each day we also asked the participants to choose the speed at which they could comfortably run for an hour, and we took the average of these preferred speeds. We then fit a 2nd order polynomial to the energetic data and compared the speed at which the minimum cost of transport (SpMinCoT) occurred with their preferred running speed.All participants showed a curvilinear relationship between speed and their cost of transport (CoT). Additionally, the preferred speed was not significantly different than the speed at the minCoT (p = 0.215), and the best fit line between the minCoT and the CoT at the preferred speed was y = x (R2  = 0.994).Humans are able to preferentially identify the speed which minimizes energy expenditure during running, as well as in walking. Over long distances, energy conservation during running would be particularly crucial so further investigations should focus on the mechanisms by which people are able to detect their 'optimal' running speeds.

  View details for DOI 10.1002/ajpa.23187

  View details for PubMedID 28195301

• Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides. Journal of immunology (Baltimore, Md. : 1950) Russo, H. M., Rathkey, J., Boyd-Tressler, A., Katsnelson, M. A., Abbott, D. W., Dubyak, G. R. 2016; 197 (4): 1353-67

  Abstract

  Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro(2+) influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro(2+) uptake in response to NG or TcdB was observed in Casp1(-/-) or Asc(-/-) BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro(2+) influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro(2+) influx and pyroptotic lysis. Extracellular La(3+) and Gd(3+) rapidly and reversibly blocked the induced Pro(2+) influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1-driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca(2+) Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1β release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1β release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1β export.

  View details for DOI 10.4049/jimmunol.1600699

  View details for PubMedID 27385778

  View details for PubMedCentralID PMC4976007