bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδT cell subsets with distinct TCR repertoires
Authors: Mistri Somen K , Hampel Kenneth J , Sidiropoulos Nikoletta , Andretta Emma , Dienz Oliver , Boyson Jonathan 
Published: February 2023

Abstract
Innate-like T cells are unusual T cells that are enriched in mucosal tissues, and which constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. While it is known that SLAM/SAP signaling is required for the development of innate-like iNKT and MAIT αβ T cells, far less is known about the role of this pathway in the development and function of γδ T cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. We found that SAP-dependent γδNKT TCRs utilized TRGV1 paired with TRAV15N-1 or TRAV15-1/DV6-1, and we identified two distinct developmental γδNKT stages in the neonatal thymus that were distinguished by SLAMF6 and SLAMF7 expression. Moreover, we found that a significant fraction of SLAMf1+innate-like γδT17 cells utilized TRGV4 and TRGV6, γ-chains paired with TRDV2 or TRDV5 δ-chains of limited diversity. Examination of SAP-deficient neonatal thymus revealed decreased numbers of mature SLAMF1+ γδT17 cells, which was associated with lower numbers of an invariant germline-encoded TRGV4/TRDV5 clonotype. Accordingly, we observed significant alterations in the SLAMF1+ γδT17 TCR repertoire in adult lung. We found that lung γδTIFN were CD44+CD45RB+CD27+ cells that co-expressed SLAMF6and SLAMF7, and that these cells predominantly utilized a diverse TRDV7 paired with TRGV4. Interestingly, we observed a specific decrease of these TRDV7+ γδTIFN cells in SAP-deficient mice, which we confirmed using qPCR. Altogether, these data indicate a crucial link between SLAM/SAP signaling and the development of functionally distinct innate-like γδ TCR clonotypes.
Mitochondrial calcium uniporter positively regulates phagocytosis-dependent activation of NLRP3 inflammasome
Authors: Dong Hong , Zhao Bao , Wen Haitao 
Published: February 2023

Abstract
Mitochondria have been shown to play an essential role in the innate immune function and inflammatory response. Mitochondrial Ca2+ signaling, which is controlled by mitochondrial Ca2+ uniporter (MCU), is a fundamental mechanism regulating mitochondrial metabolism. Accumulative evidence suggests MCU-dependent mitochondrial Ca2+ signaling may bridge the metabolic reprogramming and regulation of immune cell function. However, the mechanism by which MCU regulates inflammation and its related disease remains elusive. Here we report a critical role of MCU in promoting phagocytosis-dependent activation of the NLRP3 inflammasome by dampening phagosome membrane repair. Myeloid deletion of MCU (McuΔmye) resulted in decreased caspase-1 cleavage and IL-1β maturation in response to the challenge with silica and alum, but not ATP and nigericin, in lipopolysaccharide (LPS)-primed macrophages. Stimulation with poly(dA:dT) or flagellin induced a similar IL-1β release between McuΔmye and wild-type (WT) macrophages. Mechanistically, we detected an enhanced phagosomal recruitment of ESCRT (endosomal sorting complex required for transport)-III complex in McuΔmye macrophages after silica or alum stimulation compared to similarly treated WT cells. ESCRT-III complex is known to be essential for repair of phagosomal membrane after damage, suggesting that deletion of MCU might lead to improved phagosomal membrane repair to limit NLRP3 inflammasome activation. Furthermore, McuΔmye mice showed attenuated inflammatory response and tissue damage in a chemically induced colitis model. In sum, our results identify a novel function and mechanism of MCU in regulating phagocytosis-dependent NLRP3 inflammatory response.
Recognition of gasdermins by proteases
Authors: Xiao Tsan Sam , Liu Zhonghua , Wang Chuanping 
Published: February 2023

Abstract
The recognition and cleavage of gasdermin family members by proteases trigger the activation of the pore-forming activities of gasdermins. A prominent example is the targeting of gasdermin D (GSDMD) by inflammatory caspases-1/4/5/11 as an essential step in initiating pyroptosis following inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD and inflammatory cytokines, but it is unclear if these are the sole determinants for caspase engagement. Here we describe structural studies of a complex between caspase-1 (CASP1) and the full-length GSDMD, which reveals that the cleavage site-containing linker in GSDMD adopts a long loop structure that engages the CASP1 active site. In addition, an exosite is observed between the caspase-1 L2 and L2′ loops and a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. The exosites endows a novel function for the GSDMD C-terminal domain as a caspase-recruitment module, in addition to its role in autoinhibition. The dual site recognition may allow stringent substrate selectivity while facilitating cleavage and pyroptosis upon inflammasome activation. The residues forming the hydrophobic pocket are conserved between human and murine GSDMD, but not in GSDME, suggesting that the exosite interface may underlie the specific recognition of GSDMD but not GSDME by inflammatory caspases. Such mode of dual site recognition may be applicable to other physiological substrates of caspases.
Defining intracellular signaling pathways that dictate a universal antiviral response to conserved viral architectures in TLR2/6-dependent manner.
Authors: Hatton Alexis Alexandria , Luu Abby , Shepardson Kelly , Goodall Cheri , Wang Yang , Elkins Morgan , Rynda-Apple Agnieszka , Douglas Trevor 
Published: February 2023

Abstract
Previously we found that conserved viral architecture, namely the repeating protein subunit pattern (RPSP) of virus capsids is recognized by the cell-surface receptor TLR2/6. This RPSP is common to most, if not all virus capsids (vc) potentially serving as a conserved mechanism of extracellular recognition for viral pathogen-associated molecular patterns (PAMPs). TLR2/6-dependent recognition of vcRPSP induced an antiviral state that resulted in recruitment of immune cells and induction of type I IFN which we found to be required for reduced susceptibility to subsequent bacterial infections (BSI) in murine model of respiratory S. aureus infections. The improved BSI clearance after RPSP recognition was also seen in vcRPSP-treated PBMC-derived human macrophages, indicating the RPSP response is likely similar in humans. TLR2 is unique in that it recognizes an array of PAMPs, signals from the cell surface or endosome (proinflammatory or type I IFN response, respectively), and interacts with co-receptors, including CD14. Indeed, using bone marrow macrophages (BMMs) deficient in CD14, we found that CD14 was required for improved BSI clearance post-vcRPSP recognition. Interestingly, while blocking endosome maturation and acidification did not reduce RPSP-dependent improved clearance of BSI, using knockout BMMs we also found that MyD88, a canonical cell surface TLR signaling molecule, but also TRAM that is associated with endosomal signaling, were both required for improved BSI clearance post-vcRPSP recognition. This indicates that CD14, and the TLR signaling adaptors, MyD88 and TRAM play a role in the recognition and response to RPSP for a TLR2/6-vcRPSP mediated antiviral response but that endosome is not involved in the response.
TurboID biotin-tagging mass spectrometry identifies specific caspase-11-associated proteins regulating non-canonical inflammasome activation
Authors: Rabinovich-Ernst Orna , Bradfield Clinton , Yoon SungHwan , Armstrong Anthony , Katz Samuel , Nita-Lazar Aleksandra , Fraser Iain 
Published: February 2023

Abstract
While it has been demonstrated that cytosolic LPS can directly activate caspase11, the cellular processes regulating the non-canonical inflammasome response remain poorly defined. Caspase11 and caspase1 show substantial structural similarity, however, unlike the activation of caspase1 by NLR inflammasomes, there are no sensor or adaptor proteins known to be involved in transmitting cytosolic LPS signal to caspase11. Also, while caspase11 has been shown to associate with LPS, it lacks a characteristic LPS binding domain as observed in many other LPS binding proteins such as MD2 and LBP. Thus, we hypothesized that other effectors may be required to facilitate cytosolic LPS recognition. Moreover, the pathway is likely to be tightly regulated as caspase11 activation leads to highly inflammatory cell death.
To identify novel regulators of caspase11, we generated immortalized macrophages expressing a caspase11-TurboID-DHFR chimeric protein. The destabilizing domain was included to avoid cell death induced by caspase11 over-expression. We used a TurboID biotin-tagging MS assay to detect proteins in close proximity to caspase11 pre and post cytosolic LPS introduction. Importantly, the TurboID assay permits recognition of transient interactions, typically missed by traditional IP. To validate relevance of putative hits, we used siRNA knockdown in BMDM.
We’ve identified novel regulators specific for cytosolic LPS triggering. Several proteins interact with caspase11 only in the resting state, suggesting negative regulation to prevent pyroptosis. Among the identified regulators are kinases and proteins with pyrin and LRR domains, both common NLR features.
This work was supported by the Intramural Research Program of NIAID, NIH.
-Itaconate confers tolerance to late NLRP3 inflammasome activation
Authors: Bambouskova Monika , Potuckova Lucie , Paulenda Tomas , Kerndl Martina , Mogilenko Denis A. , Lizotte Kate , Swain Amanda , Hayes Sebastian , Sheldon Ryan D. , Kim Hyeryun , Kapadnis Unnati , Ellis Abigail E. , Isaguirre Christine , Burdess Samantha , Laha Anwesha , Amarasinghe Gaya K. , Chubukov Victor , Roddy Thomas P. , Diamond Michael S. , Jones Russell J. , Simons Donald M. , Artyomov Maxim N. 
Published: February 2023

Abstract
Itaconate is a unique regulatory metabolite that is induced upon toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLRP3 inflammasome activation after long LPS priming which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with iNOS and the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation which, if uncontrolled, can result in pyroptotic cell death and tissue damage.
Citrin, a mitochondrial transporter, interacts with NLRP3 and regulates inflammasome activity
Authors: Shuvarikov Andrey A , Davis Michael , Esser-Nobis Katharina , White Sean , Gale Michael 
Published: February 2023

Abstract
The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including microbial infection, metabolic dysfunction, and cell-internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death. While multiple regulators of the NLRP3 inflammasome have been described, specific ligands of NLRP3 and its mechanism of activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of mitochondrial solute carrier proteins. Co-IP experiments verified that four of these carriers, namely CTP, ANT3, OGCP, and Citrin, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of Citrin, a calcium-binding aspartate/glutamate carrier, in inflammasome function. When ectopically expressed, Citrin induced IL-1β release in cells, while CRISPR/Cas9 knockout of Citrin from THP-1 macrophages significantly abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. This regulatory activity of Citrin was dependent on its calcium-sensing N-terminal domain. Importantly, this regulation was specific to NLRP3 and did not affect the AIM2 inflammasome or other cell death pathways. Altogether, our studies reveal a novel role for Citrin in regulation of the NLRP3 inflammasome and inflammatory cytokine release.
Liver-localized CD8 T cells do not need Levy walks to be efficient killers
Authors: Ganusov Vitaly V , Rajakaruna Harshana , O’Connor James , Cockburn Ian A 
Published: February 2023

Abstract
Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate antigen either in the lymph node or infected tissue. To optimize the search for rare targets it has been proposed that T cells might perform a random walk with long displacements called Levy walks enabling superdiffusive behavior and shorter search times which may be evolutionary selected. We examined the behavior of activated CD8 T cells in the liver where both the movement of the cells and the underlying structural constraints can be clearly defined. We show that Plasmodium-specific liver-localized CD8 T cells perform short displacement, Brownian-like walks and yet display transiently superdiffusive displacement, the cardinal feature of efficient Levy walks. Because liver-localized CD8 T cells are mainly associated with liver sinusoids, simulations of Brownian or Levy walkers in structures derived from the liver sinusoids illustrate that structure together with preferred forward movement results in superdiffusive movement. Interestingly, Brownian walkers require less time to find a rare target when T cells search for the infection in physiologically-derived liver structures. Our results strongly suggest that observed patterns of movement of CD8 T cells are likely to result from a combination of a cell-intrinsic movement program, physical constraints imposed by the environmental structures, and other environmental cues. Future work needs to focus on quantifying the relative contributions of these factors to the overall observed movement patterns of biological agents.
Immunopathology and Meningitis During Late-Onset Gut-Originating Neonatal Sepsis
Authors: Yokanovich Lila T , Greenfield Kara , Knoop Kathryn 
Published: February 2023

Abstract
Late-onset neonatal sepsis (LOS) is a bacterial bloodstream infection that occurs during the first two months of life, and remains a significant contributor to neonatal mortality. In addition to the number of clinical challenges these patients face in the short term, neonates who survive LOS can experience a number of long-term adverse outcomes, such as impaired cognitive development. A major obstacle in LOS research remains the lack of an animal model that successfully recapitulates clinical LOS. We have developed a mouse model that replicates the clinical finding that a subset of LOS infections results from enterally-derived pathogens translocating from the intestine. In this model, pups develop sepsis-like disease upon oral gavage of virulent E. coli species. This translocation results in immune cell infiltration and bacterial presence in many peripheral sites, including the meninges. Specifically, we found infiltration of monocytes, and neutrophils into the meninges, along with increased numbers and activation status of both αβ+ and γδ+ T cells. Meningitis could be replicated by LPS alone, suggesting systemic bacterial presence contribute to infiltration into the meninges. Interestingly, mice older than 10 days did not develop meningitis in response to LPS, suggesting neonatal pups are uniquely susceptible to bacterial-initiated meningitis. Furthermore, this meningitis is induced partially by adaptive immune cell components, as no neutrophil or monocyte infiltration was seen in RAG KO mice following LPS injection. This study provides novel insights into the clinical reported complication of meningitis during LOS and suggests a unique contribution of adaptive lymphocytes in the neonate during this systemic infection.
Mycobacterium tuberculosis modulates DC-T cell cross-talk and Th17 polarization by dampening Notch signaling
Authors: Enriquez Ana B , Sia Jonathan K , Dhkar Hedwin K , Goh Shu Ling , Quezada Melanie J , Rengarajan Jyothi 
Published: February 2023

Abstract
Tuberculosis (TB) is among the leading causes of death worldwide. While IFN-γ and CD4+ Th1 T cell responses are necessary to mount an immune response to Mycobacterium tuberculosis (Mtb), the causative agent of TB, they are not sufficient to provide protection. Studies from several groups, including our own, have highlighted an important role for IL-17 and Th17 responses in immunity to Mtb infection. Previous research from the laboratory has shown that Mtb restricts Th17 responses by dampening dendritic cell (DC) responses and has identified CD40-mediated co-stimulation as critical for generating Th17 responses in response to Mtb. We showed that exogenous CD40 engagement on Mtb-infected DCs enhances Th17 polarization and reduces Mtb burden in the lungs in a vaccination model. However, the DC mechanisms that mediate CD40-dependent Th17 polarization and protection have not been defined. Here we show that Notch signaling in DCs modulates DC-T cell crosstalk and influences T cell polarization during infection. Engaging the CD40 pathway on Mtb-infected DCs increases the mRNA and protein expression of Notch ligands DLL4 and Jagged1 over the course of infection. Blockade of Jagged1 during a DC-T cell co-culture lowers Th1 responses while blockade of both DLL4 and Jagged1 significantly limits Th17 polarization. These results reveal that during infection, Mtb restricts expression of Notch ligands, which impedes Th17 responses during TB. Insights from this study could be applied to designing more efficacious TB vaccines and adjuvants.
The infection-chronic disease link: chronic kidney disease abate immunity in Porphyromonas gingivalis infection leading to pathogen overgrowth and kidney degeneration
Authors: Adamowicz Karina , Ribeiro Andrea , Wadowska Marta , Potempa Jan , Koziel Joanna , Lech Maciej 
Published: February 2023

Abstract

Objectives:
Various studies provide evidence confirming that chronic diseases can derive from infections. Vice versa, chronic disorders impair immune responses and affect the incidence and severity of infections.

Methods:
We investigated the progress of Porphyromonas gingivalis (P. g) infection during chronic kidney disease (CKD) and the effects of P.g-overgrowth on CKD. Two infectious models: the alveolar bone-loss or chamber model were applied in CKD mice. We stimulated primary macrophages, B-lymphocytes as well as osteoclasts with P. g in the presence of uremic toxins to characterize immune responses.

Results:
We showed that CKD significantly increased survival and organ-distribution of P.g. We observed a decreased IgGs in sera of CKD mice infected with P.g, indicating an immune depression caused by uremia. Levels of pro-inflammatory factors indicated changes in inflammation and homeostasis. The bone-loss was significantly increased in the CKD group, indicating that uremia affects bone formation. In vitro results revealed that microbiome/pathogen-derived indoxyl sulfate significantly inhibited the formation of TRAP-positive multinucleated osteoclasts. These effects of indoxyl sulfate might be associated with a higher rate and severity of the gingival infections observed in the CKD group. Moreover, infected mice showed a significantly higher progression of CKD.

Conclusion:
This study represents a link between infection and chronic disease. It shows that controlling pathogens’ overgrowth might reduce the morbidity of preexisting chronic kidney disease. Moreover, the efficient removal of indoxyl sulfate by dialysis could be a therapeutic approach to restore local homeostasis and immune responses.
The origin and fate of malaria-associated atypical memory B cells
Authors: Bunnik Evelien M , Batugedara Gayani , Braddom Ashley , Sullivan Richard , Gonzales Jake , Reyes Raphael , Ssewanyana Isaac , Greenhouse Bryan , Bol Sebastiaan 
Published: February 2023

Abstract
Chronic and frequently recurring infectious diseases, such as malaria, are commonly associated with expanded populations of atypical memory B cells (atMBCs, here defined as CD19+CD20+CD21−CD27− B cells). While the phenotype and conditions leading to the generation of atMBCs in malaria-experienced individuals have extensively been studied, the origin and fate of these cells remain largely elusive. Using B cell receptor (BCR) sequencing to track clonally related cells present before and after a malaria episode in 5-year-old Ugandan children, we observed that IgM+ atMBCs were predominantly derived from naïve B cells, while IgG+ atMBCs present after a malaria episode mainly originated from FcRL5+IgG+ classical MBCs (cMBCs, CD19+CD20+CD21+CD27+ B cells). IgM+ atMBCs showed fewer connections with other B cell subsets and higher turnover than IgG+ atMBCs. Single-cell transcriptomics analysis revealed that IgG+ atMBCs could be divided into two distinct clusters, one of which was maintained up to 6 months after infection. These results underscore the heterogeneity among atMBCs and highlight differences in their longevity. Finally, we observed that atMBCs from malaria-naïve and malaria-experienced donors showed many similarities in BCR characteristics, such as levels of somatic hypermutation, suggesting that atMBCs undergo similar differentiation pathways in response to different types of pathogens. In terms of BCR characteristics, IgM+ atMBCs of all donors closely resembled NBCs, while IgG+ atMBCs were more similar to IgG+ cMBCs. Collectively, our results highlight differences between unswitched and switched atMBC populations and shed light on the origin and fate of atMBCs in malaria-experienced individuals.