bluuebunny/crossref_metadata_2025_split huggingface dataset:
GSDMD deficiency drives immunopathology in cutaneous leishmaniasis
Authors: Go Christina K , Grayczyk James , Brodsky Igor E , Scott Phillip A 
Published: January 2023

Abstract
Cutaneous leishmaniasis is a neglected tropical disease that results in severe, chronic lesions that are frequently resistant to parasite-directed therapy. Studies in patients and murine models indicate that NLRP-3 dependent IL-1β release is associated with more severe disease and treatment failure. One mechanism leading to IL-1β release as well as pyroptosis is the formation of pores by gasdermin D (GSDMD). To determine if GSDMD might contribute to IL-1β release and subsequent increased disease, we infected GSDMD knockout mice and wild-type C57BL/6 (WT) mice with Leishmania major and followed the course of infection. Contrary to our expectations, GSDMD KO mice infected mice developed more severe lesions compared with WT mice, but without a change in parasite burden. Because inflammasome activation without pyroptosis has been reported to lead to prolonged IL-1β release from hyperactivated dendritic cells, we tested whether blocking IL-1β signaling would prevent severe pathology in GSDMD KO mice. However, GSDMD KO mice treated with anti-IL-1β developed pathology similar to the untreated GSDMD KO mice, indicating that IL-1β was not mediating the increased pathology. Therefore, we next compared the immune responses in WT and GSDMD KO mice. The only significant difference found was an increase in the production of IFN-g, and our working hypothesis is that excess IFN-g may contribute to the disease severity. Why the absence of GSDMD leads to increased disease remains unclear, but previously it was found that the loss of GSDMD can have consequences beyond IL-1β release and pyroptosis. Current studies are focused on defining the mechanism(s) leading to increased disease in GSDMD KO mice.
Supported by grants from NIH (5R01AI125265-05, 1R01AI150606-01, T32AR7465).
Molecular basis for NEK7-mediated NLRP3 inflammasome activation
Authors: He Yuan , Jeltema Devon , Wang Jihong , Cai Juan , Kelley Nathan , Yang Zhe 
Published: January 2023

Abstract
The NLRP3 inflammasome is a critical component of the innate immune system that is activated by microbial infections and cellular stress signals. The molecular mechanism of NLRP3 inflammasome activation remains not fully understood. As an NLRP3-interacting partner, NEK7 has emerged as a critical mediator for NLRP3 inflammasome activation. In contrast to NEK7, NEK6, the closely related member of the NEK family, does not support NLRP3 inflammasome activation. Here, we show that the NEK7 catalytic domain, which shares a high sequence identity with the counterpart of NEK6, mediates its interaction with NLRP3 and inflammasome activation. Within their catalytic domains, a single amino acid residue at a corresponding position (R121NEK7, Q132NEK6) differentiates their function in NLRP3 inflammasome activation. Surprisingly, the substitution of the glutamine residue to arginine residue at position 132 confers NEK6 the ability of NLRP3 binding and inflammasome activation. In addition, NEK7 is reported to interact with the LRR domain of NLRP3. We show that the LRR domain self-associates independently of NEK7 and promotes inflammasome assembly and activation in macrophages at the physiological condition.
Supported by NIH R01AI148544
A TLR4-independent critical role for CD14 in intracellular LPS sensing
Authors: Vasudevan Swathy Ollikara , Russo Ashley , Kumari Puja , Vanaja Sivapriya Kailasan , Rathinam Vijay 
Published: January 2023

Abstract
Intracellular sensing of LPS is crucial for antimicrobial defense and sepsis. Noncanonical inflammasome sensing of cytosolic LPS via caspase-4/11 triggers gasdermin-D mediated pyroptosis and the release of cytokines and alarmins. During infection, LPS associated with bacteria or bacterial outer membrane vesicles gains access to the cytosol. However, free LPS can access the cytosol in vivo. The mechanism by which free LPS gains access to the cytosol and activate the caspase-11-noncanonical inflammasome in vivo mostly remains elusive. CD14, a GPI-anchored glycoprotein, binds and transfers LPS to the TLR4-MD2 complex. While the role of CD14 in TLR4-mediated host responses to extracellular LPS is clear, its role in the noncanonical inflammasome sensing of cytosolic LPS is poorly defined. Here, we found that cytosolic LPS-induced GSDMD cleavage, IL-1β, IL-18 and DAMP release, pyroptosis and lethality were remarkably impaired in CD14-deficient mice, but not in TLR4-deficient mice. By using Cd14−/− and Casp11−/− mice strains on a Tlr4−/− background, we have decoupled CD14’s role in caspase-11 activation from its known function in TLR4 signaling. Mechanistically, CD14 mediates caspase-11 activation by facilitating the internalization of LPS in a TLR4-independent manner. Overall, our findings highlight a new TLR4-independent role of CD14 in noncanonical inflammasome activation by intracellular LPS in vivo.
Secreted IgM modulates the pool of IL-10 producing B Cells
Authors: McGettigan Shannon E , Aira Lazaro E , Kumar Gaurav , Baumgarth Nicole , Debes Gudrun F 
Published: January 2023

Abstract
Regulatory B cells (Bregs) are critical for maintaining immune tolerance, and alterations in Breg numbers has detrimental effects in inflammatory diseases, infection, and cancer. Bregs are found among most B cell subsets and act primarily by producing IL-10. Despite their importance, the signals that control Breg differentiation and maintenance are not well defined. Here, we demonstrate that mice incapable of secreting IgM (sIgM−/−) have drastically increased IL-10+ Bregs in lymphoid organs compared to wildtype (WT) mice. These IL-10+ Bregs are polyclonal and increased among all major B cell subsets. While newborn WT mice (&lt;24 hr of age) harbor detectable serum IgM, IL-10+ B cell expansion occurs early after birth in both WT and sIgM−/− mice. However, IL-10+ Bregs contract only in WT but not in sIgM−/− mice, which is in parallel to rising levels of serum IgM in WT mice. SIgM−/− B cells that develop in the presence of circulating IgM express IL-10 similar to that of WT B cells within the same mouse indicating that the expansions in IL-10+ B cell populations are not due to B cell-intrinsic effects based on the inability to secrete IgM, but rather the presence of sIgM. Mice that lack B cell-expressed high-affinity IgM receptor (FcμR) mirror the IL-10 phenotype in their B-1 and marginal zone B cells, but not follicular B cells, suggesting that secreted IgM can act as a negative regulator of IL-10 expression in B cell subsets via binding to surface-expressed FcμR, and potentially other IgM binding receptors. Our data indicate that sIgM controls IL-10 competence in B cells via IgM binding receptors thereby revealing potential novel avenues for altering IL-10+ Breg induction in disease settings. Funding: T32AI134646, R01AR067751, R01AI127389, R01AI148652.
Supported by grants from NIH (T32AI134646, R01AR067751, R01AI127389, R01AI148652)
The CD40/CD40 ligand system in linking acute neuroinflammation with chronic progressive demyelination
Authors: Sarma Jayasri Das , Saadi Fareeha , Chakravarty Debanjana , Kamble Mithila , Kumar Saurav , Shindler Kenneth S 
Published: January 2023

Abstract
CD4+ T cells play critical roles in mediating adaptive immunity and developing protective host immunity by eliminating infectious viruses. CD40 Ligand expressed on CD4+ T cells known to interact with its cognate receptor CD40 expressed on monocyte/macrophage/microglia to induce several immune functions during inflammation and host immunity. Our study aimed to delineate a potential CD4+ T cell-microglia nexus at the cellular and molecular level using CD4−/− and CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to murine-coronavirus-MHV-A59/RSA59 infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase of neuroinflammation. CD40L deficiency significantly impaired the priming, expansion, and activation of lymphoid cells in the deep cervical lymph nodes. Additionally, both CD4−/− and CD40L−/− mice presented with severe chronic phase demyelination and axonal pathology in the brain and spinal cord which was correlated with prolonged virus persistence. Results from this study provide exciting information about a critical role of CD40L in mediating RSA59 induced activation of CD4+ T cells in the CLN and their consequent infiltration into CNS in addition to affecting the numbers and activation of monocyte/macrophage/microglia during the acute-adaptive transition phase. CD40-CD40L interaction in linking the innate-acute and chronic-adaptive immune responses may suggest a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to autoimmune suppression, which holds only for autoimmune mechanisms of inflammatory demyelination.
This work was supported by a Department of Biotechnology, India, research grant (BT/PR 20922/MED/122/37/2016). We thank the Council of Scientific and Industrial Research (CSIR) India for providing fellowships to F.S. the Ministry of Human Resource Development (MHRD), India for providing fellowship to D.C; and the University Grants Commission (UGC), India, for providing fellowship to M.K.
Soluble and membrane-bound isoforms of fractalkine differentially regulate microglia activation and vascular damage in the diabetic retina
Authors: Rodriguez Derek , Church Kaira A , Pietramale Alicia N , Cardona Sandra M , Vanegas Difernando Vanegas , Nash Kevin R , Cardona Astrid E 
Published: January 2023

Abstract
Diabetic retinopathy (DR) is the leading cause of blindness worldwide due to neuronal loss, microgliosis, and vascular damage. Inflammation caused by microglia exacerbates retinal damage, resulting in glial and neuronal cell dysfunction. Microglial-neuronal crosstalk mediated by CX3CR1/FKN signaling regulates microglial responses. CX3CR1 polymorphic variants present in 25% of the population decrease FKN binding affinity to CX3CR1, and their role in microglia function in the retina during diabetes is unknown. FKN exists as a membrane-bound (mFKN) adhesion molecule. When cleaved by proteases, the soluble (sFKN) form exerts chemoattractant properties. Previous studies show that disruption of CX3CR1/FKN signaling worsens retinal pathology and can be rescued by administration of recombinant FKN. Mechanisms by which mFKN and sFKN regulate retinal function are still unknown. We hypothesize that during diabetes, the over-expression of sFKN using recombinant adeno-associated viruses (rAAVs) will prevent vascular and neuronal damage, and improve visual function. To test this hypothesis, rAAVs expressing mFKN or sFKN were delivered to FKN-KO mouse retinas. We validated the expression of FKN levels in retinal protein extracts in mice that received rAAVs expressing mFKN or sFKN. Our findings revealed that rAAV-sFKN minimizes microglial activation, reduces fibrinogen deposition, and rescues neuronal loss, compared to rAAV-mFKN. rAAV-sFKN retinas of diabetic and non-diabetic mice improved visual function in a two-choice visual discrimination task. FKN appears to be neuroprotective, serving as an alternative therapeutic approach to minimize retinal pathology and improve visual function.
Supported by NIH/NEI RO1 EY029913
The role of ionotropic AMPA receptors in T cell tolerance
Authors: Mitchell-Flack Marisa , Zheng Ying , Goldschmidt Hana , Rajkovich Kacey , Higgins Makenzie , Liu Bian , Huganir Richard , Yu Hong , Pardoll Drew 
Published: January 2023

Abstract
The ability of T cells to mount effective immune responses to pathogens and cancer while remaining tolerant to self-tissue is critical for maintaining immune homeostasis. Previously, our group identified neuritin, a neuronal protein, as a highly differentially expressed gene in anergic and regulatory T cells. Furthermore, our results indicate neuritin deficient mice exhibit enhanced autoimmunity due, in part, to dysfunctional regulatory T cells. Neuritin was recently identified as an accessory component of the ionotropic AMPA receptor (AMPAR) complex in neurons. AMPAR complexes are typically found on the post-synaptic neuronal cell and mediate glutamate dependent cation flux. Given our previous findings involving neuritin in autoimmunity and this newly identified interaction between neuritin and AMPAR, we sought to evaluate whether there is a role for the AMPAR in T cell tolerance. Here, we show that the AMPAR is expressed in CD4+ T cells and that the specific deletion of the AMPAR in T cells (AMPAR KO) leads to significantly reduced disease pathogenesis in an experimental autoimmune encephalomyelitis (EAE) model. The spinal cord inflammatory immune infiltrates from AMPAR KO mice are markedly reduced in cell number, proliferative state, and expression of proinflammatory cytokines compared to wild type (WT) mice. In addition, we have observed an increase in the CD4+FoxP3+ regulatory cell population among spinal cord infiltrates from AMPAR KO EAE mice. Consistent with the EAE model, AMPAR KO T cells also exhibit increased induced regulatory T cell development during in vitro differentiation. Taken together our results support a previously unappreciated role for ionotropic AMPA receptors in regulating T cell tolerance.
Microglia depletion elicits neuroprotective effects to alleviate vascular damage and neuronal cell loss in the diabetic retina
Authors: Church Kaira A , Rodriguez Derek , Lopez-Gutierrez Irene , Vanegas Difernando Vanegas , Cardona Sandra M , Cardonaa Astrid E 
Published: January 2023

Abstract
Diabetic retinopathy (DR), an incurable eye disease, is a common complication of diabetes mellitus and the leading cause of blindness amongst working age adults. Prolonged high glucose levels damage retinal blood vessels leading to hemorrhages, ischemia and ultimately vision loss. Microglia, the resident macrophages of the central nervous system, are rapidly activated and respond to transient hyperglycemia, and reset the homeostatic threshold of the retina. As hyperglycemia persists, capillary damage occurs resulting in serum proteins and DAMPs from the periphery leaking into the retina perpetuating microgliosis, pro-inflammatory cytokine production and vascular damage that correlates with neuronal loss. Therefore, we transiently depleted microglia to determine the mechanism by which resident retinal macrophages regulate neuronal and vascular damage at early stages of diabetes. We hypothesize that transient depletion of CX3CR1CreER:R26iDTR microglia during the course of disease when microglia receive the early-stage environmental cues of retinal inflammation due to hyperglycemia will induce neuroprotective cues. Utilizing a genetic model to express diphtheria toxin receptor under the tamoxifen inducible CX3CR1-promoter, microglia become susceptible to the effects of diphtheria toxin. Our findings revealed that transient depletion of microglia induced neuroprotective cues to upregulate TUJ1+ neurons and ameliorate vascular damage in the diabetic retina. In conclusion these studies suggest that early transient microglia depletion is neuroprotective by inducing the proliferation of a homeostatic microglia cell population that induce axonal regeneration of TUJ1+ neurons and vascular repair.
Supported by grants from NIH (R01EY029913) and NIH RISE Grant (GM061655).
Chimeric antigen receptors on Regulatory T cells as a treatment strategy in autoimmune diseases
Authors: Dasari Harika 
Published: January 2023

Abstract

Research question
Can CAR-MOG Tregs help curb the neuroinflammation in a MS mouse model?

Objectives

Methods
To test this out, we sorted Tregs from human peripheral blood or murine spleens using T cell enrichment followed by flow-cytometric sorting and amplified using Dynabeads (human CD3/CD28 activator) for Tregs proliferation and stained for Treg markers to ascertain the purity of isolated Tregs. We then constructed a second-generation CAR against MOG monoclonal antibody (8.18 C5) and transduce Tregs and Tconvs to test the transduction efficacy and suppressive function of CAR-Tregs. To test the killing function of CAR-MOG-Tconv and compared this to that of CAR MOG Tregs, we set up a cytotoxicity assay against a cell line modified to express MOG. Additionally we performed suppression assays with control Tregs and CAR transduced Tregs to determine if CAR transduction affects the immunosuppressive capacity of Tregs.

Results
CAR Tconv and CAR Tregs show specificity against K562 cell line modified to express MOG, CAR Tregs have a lower killing capacity compared to CAR Tconv. CAR Tregs do not lose their immunosuppressive function and are comparable to the untransduced Tregs. CAR Tregs upon co-culture with Target cell line (K562 MOG) express activation markers and increased expression of cytokines such as IL-10, and TGF beta.
Supported by Canada Graduate Scholarships Doctoral Award (CGS D)
TGF-β signaling drives phenotypic and functional characteristics of human mast cells within epithelium
Authors: Derakhshan Tahereh Mina , Hollers Eleanor , Buchheit Kathleen , Laidlaw Tanya M , Boyce Joshua A , Dwyer Daniel 
Published: January 2023

Abstract
Mast cells (MCs) are key effector cells in the development of type 2 inflammation, such as asthma and nasal polyposis. Intraepithelial MCs are phenotypically distinct from those found within the subepithelium and preferentially expand during mucosal inflammation, yet the mechanism driving their development and their relative contribution to inflammation is not clear. We previously found that TGF-β directed the murine intraepithelial airway MC phenotype. Here, we investigate whether TGF-β has a similar effect on human MCs. Using single cell RNA-sequencing of primary nasal polyp MCs and bulk sequencing of in vitro peripheral blood-derived MCs (PB-MCs) stimulated with TGF-β, we identified a shared signature of TGF-β within human intraepithelial MCs. This included downregulation of subepithelial MC-associated granule components (NDST2, CMA1, and CTSG), differential regulation of lipid mediator biosynthetic enzymes (PTGS1, HPGDS), and upregulation of transcription factors (SKIL, FOXP1). TGF-β suppressed incorporation of the proteases chymase and cathepsin G into PB-MC granules in vitro as part of a broader intraepithelial MC phenotype, allowing direct assessment of the differential role of MC subsets during inflammation. Through this approach, we found that TGF-β modifies MC production of inflammatory mediators in response to stimulation, enhancing MC production of the lipid mediators, PGD2 and CysLTs, in response to IgE-crosslinking; selectively decreasing IL-13, TNF-α, and IL-6; and enhancing chemokine production (IL-8 and MIP-1β). Thus, we identify a central role for TGF-β signaling in driving the human intraepithelial MC phenotype and re-shaping their response to activation.
Supported by NIH (K22 AI146281, U19AI070535)
Leveraging iNKT cells to identify signals driving B cell memory commitment
Authors: Parthasarathy Raksha , Yates Jennifer , Hagglof Thomas , Dudley Elizabeth A , Leadbetter Elizabth 
Published: January 2023

Abstract
Intense scrutiny of the process of memory B cells (MBC) over the last decade has improved our understanding of MBC development especially for vaccines against viruses capable of rapid diversification like SARS-CoV-2. Most memory responses develop in germinal centers (GC), but the constellation of specific signals which commit B cells to a memory fate is poorly understood. Identification of discrete T and B cell factors leading to MBC commitment is hindered by a lack of murine models with essential controls. We report a unique system harnessing iNKT cell help for B cells to create two conditions, both of which expand NP+ B cells in a class-switched primary response, but only one of which produces a MBC response. In the first case, NP+ B cells receive exclusive cognate iNKT cells upon immunization with the Nitrophenyl-haptenated iNKT cell agonist alphaGalactosylCeramide (NP-aGC) leading to an expansion of NP+ GC B cells, but not MBCs. In contrast, NP+ B cells helped by conventional T cells adjuvanted by iNKT cell activation by vaccinating with haptenated protein (NP-KLH) plus aGC drives a conventional GC B cell expansion and development of NP+ MBCs. Interestingly, cognate antigen which does not induce B memory also elicits fewer pre-memory B cells, compared to non-cognate antigen. These different immunization regimens allow a unique comparison of activated B cells recognizing identical epitopes and producing similar primary humoral responses but with contrasting memory outcomes. Importantly, this also controls for the GC environment which is unaccounted for in other model systems. Examination of these B cell populations will better define the transcriptional landscape of MBC and pinpoint key signals which dictate MBC fate.
Supported by AAI Careers in Immunology Fellowship 2021
Mechanism of Tim-3 regulation of CD8+ T cell function
Authors: Manandhar Priyanka , Landy Emily Elizabeth , Murter Benjamin , Banerjee Hridesh , Workman Andrea , Bosco Isabella , Kane Lawrence P 
Published: January 2023

Abstract
Tim-3 or transmembrane immunoglobulin and mucin domain-3 is a type I membrane protein expressed by various immune cell types, and has been shown to have a co-stimulatory role in T cells through the PI3K pathway. Tim-3 comprises extracellular, transmembrane and intracellular domains, the latter of which contains five tyrosine residues. We and others have shown that these tyrosines participate in TCR-mediated signaling pathways. Using LCMV infection, we also found that Tim-3 expression influences the formation of short-lived effector and memory precursor CD8+ T cells. We hypothesize that Tim-3 signaling through the tyrosine residues in its cytoplasmic domain modulates CD8+ T cell activation and differentiation. To test this hypothesis, we have used LCMV-specific TCR transgenic (P14) mice crossed to truncated version of Tim-3 and CD8+ conditional deletion of Tim-3. CD8+ T cell activation and memory were analyzed by flow cytometry after infection with LCMV Armstrong to induce an acute infection. The effects of Tim-3 on CD8+ T cells were found to be most prominent at the effector stage of infection and RNA sequencing has shown that loss of Tim-3 signaling promotes acquisition of a memory-like phenotype. Transcription factors T-bet and Blimp-1 were found to be lower in Tim-3 KO CD8+ T cells, while Tcf-1 maintained at higher levels in the absence of Tim-3 signaling. Cytokine production by effector CD8+ T cells was also found to be lower when Tim-3 was absent. Thus, we conclude that Tim-3 signaling via its cytoplasmic tyrosine residues plays a role in CD8+ T cells in an acute infection. These results may lead to a better understanding of CD8+ T cell activation and memory development in different settings.
Supported by R01 AI138504-01A1