Biology subjects

Rauch, I.

Publications and source records attributed to Rauch, I..

10 recordsLinked to original sources

bioRxiv2026

Unbiased proteomics following inflammasome activation identifies caspase targets in primary intestinal epithelial cells

Inflammasomes are cytosolic innate immune sensors that, once activated by a pathogenic threat, lead to activation of the inflammatory Caspase-1. Inflammasome activation and its consequences have been studied extensively in myeloid cells and in overexpression systems. Recent studies have identified cell type specific effects that are not fully explained by the known cleavage targets of Caspase-1. Here, we identified targets of caspase cleavage using mass spectrometry in primary intestinal epithelial cells by specifically activating the NAIP-NLRC4 inflammasome. We have taken an unbiased approach and developed a novel method for analyzing mass spectrometry data for evidence of caspase activity. Our approach can also be applied to existing proteomic datasets to establish the presence of caspase activity under various biological conditions. These results lay the groundwork for future studies on mechanisms of caspase-induced processes such as intestinal epithelial cell extrusion.

immunology↗

bioRxiv2025

Discovery Stack Pilot: Feasibility and Outcomes of a Scientist-Designed Peer Review Model Separating Quality and Impact

Peer review serves as the cornerstone of scientific quality control. Yet, the current journal-centric system is hindered by long timelines, high publication costs, inconsistent review quality, systemic biases, and editorial gatekeeping. Notably, the system relies on misaligned measures of impact that are tethered to journal branding and conflate scientific rigor (Quality) with perceived significance (Impact). Here, we report findings from the Discovery Stack Pilot Study, which tested a scientist-designed, journal-independent peer review model. The Discovery Stack model integrates in-line reviewer comments to promote constructive feedback and separately evaluates scientific Quality and Impact using defined criteria. To examine feasibility and effectiveness, manuscripts were reviewed in parallel with traditional journal review. A total of 162 reviews were completed, and survey data from 86 participants were analyzed. The results showed that reviewers effectively evaluated Quality and Impact as separate dimensions, with Quality scores being more consistent across reviewers than Impact scores. Participants strongly supported the core elements of the Discovery Stack model and expressed enthusiasm for its broader adoption to enhance transparency, efficiency, and value in peer review. Future studies will explore integrating this model into a digital platform for reviewing and curating scientific discoveries to improve the production and dissemination of high-quality research.

scientific communication and education↗

bioRxiv2024

Interferon Regulatory Factor 6 Determines Intestinal Epithelial Cell Development and Immunity

Intestinal epithelial cell (IEC) responses to interferon (IFN) favor antiviral defense with minimal cytotoxicity, but IEC-specific factors that regulate these responses remain poorly understood. Interferon regulatory factors (IRFs) are a family of nine related transcription factors, and IRF6 is preferentially expressed by epithelial cells, but its roles in IEC immunity are unknown. In this study, CRISPR screens found that Irf6 deficiency enhanced IFN-stimulated antiviral responses in transformed mouse IECs but not macrophages. Furthermore, KO of Irf6 in IEC organoids resulted in profound changes to homeostasis and immunity gene expression. Irf6 KO organoids grew more slowly, and single-cell RNA sequencing indicated reduced expression of genes in epithelial differentiation and immunity pathways. IFN-stimulated gene expression was also significantly different in Irf6 KO organoids, with increased expression of stress and apoptosis-associated genes. Functionally, the transcriptional changes in Irf6 KO organoids were associated with increased cytotoxicity upon IFN treatment or inflammasome activation. These data indicate a previously unappreciated role for IRF6 in IEC biology, including regulation of epithelial development and moderation of innate immune responses to minimize cytotoxicity and maintain barrier function.

immunology↗

bioRxiv2023

NAIP-NLRC4 Inflammasome Activation in Tuft Cells Activates a PGD2-ILC3 Signaling Circuit that Protects Against Enteric Infection.

Intestinal epithelial cells (IEC) use innate sensing pathways to distinguish pathogens from commensals. One such pathway, the NAIP--NLRC4 inflammasome, initiates extrusion of infected IEC and mediator release upon cytosolic bacterial sensing. Tuft cells are primarily known for their function in anti-parasite immunity. We previously reported that activation of the inflammasome in tuft cells leads to release of prostaglandin D2 (PGD2). We test the hypothesis that tuft cell specific release of PGD2 after inflammasome activation initiates antibacterial responses. NAIP--NLRC4 inflammasome activation in tuft cells leads to a type 3 antimicrobial response with increased IL-22 and antimicrobial protein levels within the small intestine, which is dependent on PGD2 signaling. A subset of ILC3 express the PGD2 receptor CRTH2 and we show them as the source of the increased IL-22. Inflammasome activation in tuft cells also leads to better control of Salmonella Typhimurium. These data support that intestinal tuft cells can also induce antibacterial responses. SummaryPGD2 release after NAIP--NLRC4 inflammasome activation in tuft cells signals onto ILC3s and mediates host defense mechanisms against Salmonella Typhimurium within the small intestine. Tuft cells therefore not only promote immune reactions against parasites, but also bacteria.

immunology↗

bioRxiv2021

Salmonella Typhimurium induces NAIP/NLRC4- and NLRP3/ASC-independent, caspase-1/4-dependent inflammasome activation in human intestinal epithelial cells

Salmonella enterica serovar Typhimurium is a gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SSs) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of Salmonella by cytosolic immune sensors. Upon detecting translocated Salmonella ligands, these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to proinflammatory cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella. While these processes are studied quite well in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human intestinal epithelial cell lines and organoids. Additionally, the NLRP3 inflammasome and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a partial requirement for caspase-1, and a necessity for caspase-4 and GSDMD pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4, and also do not use NLRP3/ASC. Instead, they primarily use caspases-1 and -4 to mediate early inflammasome responses to SPI-1-expressing Salmonella.

microbiology↗

bioRxiv2021

Pyroptosis-dependent and -independent cross-priming of CD8+ T cells by intestinal epithelial cell-derived antigen

The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens are cross-presented to CD8+ T cells. However, activation of CD8+ T cells by IEC-derived antigen only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (cDC1), whereas cross-priming in the presence of pyroptosis did not. These data suggest the existence of parallel pyroptosis-dependent and pyroptosis-independent but cDC1-dependent pathways for cross-presentation of IEC-derived antigens.

immunology↗

bioRxiv2020

NAIP-NLRC4-deficient mice are susceptible to shigellosis

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Shigellosis develops upon oral ingestion of as few as 100 bacteria, but million-fold higher doses fail to cause disease in mice. The lack of a physiologically relevant mouse model of shigellosis has impeded our understanding of this important human disease, but why mice are resistant is unknown. Here we show that in human cells, but not in mice, Shigella evades detection by the NAIP-NLRC4 inflammasome, an immune sensor present in intestinal epithelial cells (IECs). We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis, including bacterial replication in IECs and neutrophilic inflammation of the colon. Confirming a role for bacterial replication in IECs in our new model, a Shigella mutant lacking IcsA, a factor required for cell-to-cell spread among IECs, is attenuated in otherwise susceptible NAIP-NLRC4-deficient mice. Although inflammasome-mediated cell death is widely held to promote Shigella infection and pathogenesis, we instead demonstrate that IEC-specific NAIP-NLRC4-induced cell death is sufficient to protect the host from shigellosis. Thus, NAIP-NLRC4-deficient mice are a physiologically relevant and experimentally tractable model for shigellosis. More broadly, our results suggest that the lack of an inflammasome response in IECs may help explain the extreme susceptibility of humans to shigellosis.

microbiology↗

bioRxiv2019

New mutant mouse models clarify the role of NAIPs, phosphorylation, NLRP3, and tumors in NLRC4 inflammasome activation

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates Caspase-1 and initiates potent downstream immune responses. Structural, biochemical, and genetic data all demonstrate that the NAIP proteins act as receptors for specific bacterial ligands, while NLRC4 is a downstream adaptor protein that multimerizes with NAIPs to form a macromolecular structure called an inflammasome. However, several aspects of NLRC4 biology remain unresolved. For example, in addition to its clear function in responding to bacteria, NLRC4 has also been proposed to initiate anti-tumor responses, though the underlying mechanism is unknown. NLRC4 has also been shown to be phosphorylated on serine 533, and this modification was suggested to be important for NLRC4 function. In the absence of S533 phosphorylation, it was further proposed that another inflammasome component, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line as well as mice encoding phosphomimetic S533D and non-phosphorylatable S533A NLRC4 proteins. Using these genetic models in vivo and in vitro, we fail to observe a role for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results simplify and clarify our understanding of the mechanism of NAIP/NLRC4 activation and its biological functions.

immunology↗

bioRxiv2019

Defective cell death of distinct microglial subsets contributes to ADHD-like behavior in mice

Microglia are resident immune cells in the central nervous system that play essential roles to maintain homeostasis and neuronal function. Microglia are heterogeneous cells but the mechanisms by which they contribute to normal brain development remain unclear. Here,we show that microglia in the developing striatum and thalamus undergo pyroptosis,a type of lytic cell death that occurs as a result of Caspase-1 (CASP1) activation downstream of inflammasomes. We observe that pyroptosis occurs in a spatiotemporally regulated and Casp1-dependent manner during fetal brain development. Mice lacking Casp1 or the inflammasome regulating molecules, NLRP3, IL-1R, and Gasdermin D exhibit behavior changes characterized by hyperactivity, inattention, and impulsivity that are similar to attention-deficit/hyperactivity disorder (ADHD). Furthermore, re-expression of Casp1 in Cx3cr1+ cells including microglia restores normal behavior and cell death. We demonstrate that injection of an NLRP3 inhibitor into pregnant wild-type mice is sufficient to induce ADHD-like behaviors in offspring. These data suggest that microglial inflammasome activation and pyroptosis are essential for normal brain development and that genetic and pharmacological disruptions in this pathway may represent new ADHD risk factors.

neuroscience↗

bioRxiv2018

Transcriptome profiling of pathogen-specific CD4 T cells identifies T-cell-intrinsic caspase-1 as an important regulator of Th17 differentiation

One sentence summaryOur study revealed that DCs shape distinct pathogen-specific CD4 T cell transcriptome and from which, we discovered an unexpected role for T-cell-intrinsic caspase-1 in promoting Th17 differentiation.\n\nABSTRACTDendritic cells (DCs) are critical for priming and differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells leading to effector heterogeneity remains elusive. Here we have used an in vitro approach to prime naive CD4 T cells by DCs stimulated with distinct pathogens. We have found that such pathogen-primed CD4 T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. In contrast to cytokine-polarized Th17 cells that display signatures of terminal differentiation, pathogen-primed Th17 cells maintain a high degree of heterogeneity and plasticity. Further analysis identified caspase-1 as one of the genes upregulated only in pathogen-primed Th17 cells but not in cytokine-polarized Th17 cells. T-cell-intrinsic caspase-1, independent of its function in inflammasome, is critical for inducing optimal pathogen-driven Th17 responses. More importantly, T cells lacking caspase-1 fail to induce colitis following transfer into RAG-deficient mice, further demonstrating the importance of caspase-1 for the development of pathogenic Th17 cells in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.

immunology↗