bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct effects of ethanol at relevant concentrations on conformation and ligand binding of Toll-like Receptor 3 (TLR3)
Authors: Pruett Stephen B , Lewis Edwin , Gwaltney Steven , Nanduri Bindu 
Published: January 2023

Abstract
The purpose of this study was to seek direct physical evidence that ethanol at concentrations relevant to binge drinking alters the conformation of TLR3 and decreases ligand binding. We and others have reported that TLR3-mediated responses are significantly suppressed by concentrations of ethanol that occur in binge drinkers (J. Immunol., 2004, 173: 2715–2724). This suppression can be detected at all levels of signaling that have been tested (from MAP kinases to NF-kB), but it remains possible that ethanol acts directly on TLR3 and that its other effects are secondary to altered TLR3 conformation. Purified ectodomains of TLR3 were used in this study, because they comprise most of the TLR3 molecule, and dimerization of TLR3 ectodomains is necessary for signal transduction and activation of TLR3-mediated innate immunity. Using circular dichroism (CD) to measure changes in protein conformation, we demonstrated that ethanol concentrations from 20–100 mM substantially altered the conformation of human and mouse TLR3 ectodomains complexed with a defined dsRNA ligand. Binding of poly I:C, a dsRNA analog, was significantly inhibited by ethanol. Modeling with Autodock 4 software indicated that mouse and human TLR3 contain a putative ethanol binding site with a dissociation constant consistent with the concentration dependence of the conformational changes. These results support the hypothesis that ethanol inhibits TLR3-mediated functions by directly altering the conformation of TLR3. This work was supported by NIH R01AA09505 and SBP was supported by NIH grant P20GM103646.
Targeting the TLR7 signalosome assembly by TLR7-derived decoy peptides
Authors: Toshchakov Vladimir Y , Javmen Artur 
Published: January 2023

Abstract
Toll-like receptors (TLRs) sense microbial molecules and initiate antimicrobial immune response. The members of the TLR family use cytoplasmic Toll/Interleukin-1R homology (TIR) domain to initiate intracellular signaling. Activated TLRs dimerize their TIRs and recruit adapter proteins to the dimer, through multiple interactions of receptor and adapter TIR domains. Although TLRs play an essential role in innate immunity, the aberrant TLR signaling may cause pathogenic inflammation. This study has screened a library of cell-permeable decoy peptides (CPDPs) derived from the TLR7 TIR for interference with TLR7 signaling and identified several CPDPs that inhibit TLR7 signaling. Peptides 7R1, 7R6, 7R9 and 7R11 inhibited the TLR7-induced signaling in murine and human macrophages. The most potent inhibitory peptide of the four, 7R11, significantly reduced the systemic cytokine levels elicited by administration of a TLR7 agonist, R-848, to mice. TLR7 TIR surface regions that correspond to inhibitory peptides generally corresponded to four TIR sites that mediate signalosome assembly for other TLRs. The cell-based Förster resonance energy transfer/fluorescence lifetime imaging confirmed that 7R9 and 7R11 interact with adapter TIRs. These findings clarify the molecular mechanisms that trigger the adapter recruitment to activated TLR7 and suggest that 7R9 and 7R11 have a significant translational potential as candidate or lead therapeutics for treatment of TLR7-mediated inflammation.
TLR3 co-localizes with LAMP1-labeled endosomes from the first minute following poly(I:C) stimulation of murine astrocytes
Authors: Mielcarska Matylda Barbara , Gregorczyk-Zboroch Karolina Paulina , Bossowska-Nowicka Magdalena , Szulc-Dąbrowska Lidia , Toka Felix Ngosa 
Published: January 2023

Abstract
Toll-like receptor 3 (TLR3) senses dsRNA in the acidic environment of endosomes and plays a pivotal role during infection of the brain with viruses, including herpes simplex virus type 1 (HSV-1). Studies on various cell lines corroborate TLR3 presence in LAMP1-labeled endosomes following the receptor stimulation. However, co-localization times, as well as the subjection of co-localization on the TLR3 stimulation, remain divergent depending on the cell type. In this work, we examined TLR3 residence in the LAMP1-marked endosomes in nine different time intervals following poly(I:C) stimulation of C8-D1A murine astrocytes. Interestingly, TLR3 did not localize in the lysosomal compartment in resting cells, however, the presence of the receptor was observed in LAMP1-labeled endosomes from the first minute after the addition of poly(I:C). Furthermore, 24 h following stimulation, we observed numerous lysosomes co-localizing with TLR3, which were present in all astrocytes. Our results indicate LAMP1-labeled endosomes as important organelles which may serve as the TLR3 ligand recognition sites, however, further studies on the migration of dsRNA in astrocytes are essential.
Novel Anti-bacterial and Anti-inflammatory Roles for the Macrophage Galactose-type Lectin in Pulmonary Immunity to Mycobacterium tuberculosis
Authors: Naqvi Kubra Fatima , Huante Matthew B , Endsley Mark , Gelman Benjamin , Endsley Janice J 
Published: January 2023

Abstract
Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is characterized by inflammatory pathology that is regulated by poorly understood mechanisms of innate immunity. Pattern recognition receptors (PRR) expressed on the surface of macrophages regulate the balance of inflammatory and antimicrobial functions that determine disease outcome. Our work identifies a novel role for the C-type lectin receptor Macrophage Galactose-type Lectin (MGL) PRR in a murine model of experimental tuberculosis. Bone marrow derived macrophages (BMDM) upregulate MGL-1, but not MGL-2, following in vitro exposure to both heat killed and live Mtb. Macrophages from MGL-1-deficient mice display greater lipid accumulation, and increased production of pro-inflammatory cytokines (IL-1b, IL-6 and IFN-g). Mtb growth is also increased in MGL−/− BMDM, independent of the inflammatory outcomes and production of reactive nitrogen species. TB in mice lacking MGL-1 results in increased pro-inflammatory cytokines, more diffuse granulomatous inflammation, and increased accumulation of foamy macrophages. Assessment of disease kinetics suggests that MGL has an important role for controlling mycobacterial burden during the establishment stages of TB disease, and has less influence once the chronic disease stage is established. Macrophages in murine and human TB granulomas contain abundant MGL associated with inflammatory foci. These results suggest that MGL is a potential new therapeutic target against TB.
Can Effector Molecules of Innate Immunity Target Microbial Motility?
Authors: Akahoshi Doug T , Natwick Dean E , Collins Sean R , Bevins Charles L 
Published: January 2023

Abstract
Antimicrobial peptides are key effector molecules throughout nature responsible in part for the defense against microbial pathogens and influencing the composition of colonizing microbes. Defensins are the major antimicrobial peptides produced by Paneth cells in the mammalian small intestine and are essential for maintaining homeostasis between host and microbe. While mature defensin peptides typically have microbicidal activity, human alpha defensin 6 (HD6) lacks such activity yet provides protection from invasion by bacterial pathogens. Using a transgenic mouse model expressing HD6 in Paneth cells, we reported the importance of HD6 in preventing the translocation of pathogenic Salmonella Typhimurium (STM) across the small intestinal epithelium (doi:10.1126/science.1218831); however, the mechanism underlying the inhibition of invasion is not fully understood. We hypothesize HD6 binds to extracellular bacterial protein structures, such as flagella, and inhibits swimming motility through a process of direct binding and self-assembly. Using an agar based motility assay, we show STM radially swim at a slower rate when in the presence of HD6. In ongoing studies using live-microscopy to visualize GFP-expressing STM in the presence of HD6, we observe a decrease in STM motility in a concentration dependent manner with some STM appearing to be completely immobilized, but without changes in bacterial viability. Unexpectedly, it appears the immobilization observed is influenced more by the expression of fimbriae than by the expression of flagella. With these results in mind, we plan to expand our investigation to elucidate key molecular determinants and include other microbes to test the broader relevance of this activity of HD6.
A LIVE ATTENUATED Brucella VACCINE POTENTIATES MACROPHAGE ALARMINS
Authors: Ella Bhagyaraj , Akgul Ali , Hoffman Carol , Goodwin Zakia I , Yang Xinghong , Pascual David W 
Published: January 2023

Abstract
Wild-type (wt) brucellae subvert host immune detection leading to their replication in the endoplasmic reticulum (ER) resulting in their persistence. Protection against Brucella infection requires a strong Th1-type immunity, characterized by the production of IFN-γ. Mucosal vaccination with a live attenuated B. abortus double mutant expressing lacZ (znBAZ) provides protection against wt B. abortus challenge via the stimulation of activated T cells. Such finding implicates rendering macrophage activation. To test the hypothesis that znBAZ diminishes subversion to enable host innate immune detection, intracellular survival of wt B. abortus, B. abortus strain 19 (S19) vaccine, and znBAZ in bone marrow-derived macrophages (BMDMs) was measured. The percentage of brucellae in LAMP1+ phagolysosomes was significantly greater in znBAZ-infected BMDMs than S19 or wt B. abortus, and znBAZ induced the alarmins, s100a8, Saa1, and Saa3. These alarmins are responsible for stimulating strong inflammatory responses by interacting with pattern recognition receptors (PRRs). Consequently, IL-1β, IL-6, IL-12, and TNFα were significantly elevated following in vitro znBAZ infection of macrophages. CD80, CD86, CD40, and MHCII expression by znBAZ-infected BMDMs was significantly increased. Analysis of lung macrophages two days after pulmonary infection reveals that znBAZ significantly increases the expression of CD86 and MHCII on interstitial and alveolar macrophages, and increased intracellular IL-1β and IL-6 by interstitial macrophages. These findings show that znBAZ enhances macrophage activation, which in turn leads to the induction of potent T cell immunity. Work is supported by NIH AI123244 & AI125516.
LncRNAs modulate immune response in myeloid cells challenged with periodontal pathogens
Authors: Naqvi Afsar , Ahmad Imran 
Published: January 2023

Abstract

Objective
Long noncoding RNAs (lncRNA) are an emerging class of regulatory RNAs. LncRNAs have been implicated in differentiation and regulation of various immune cells. However, the role of lncRNA in myeloid cell regulation, in the context of periodontal inflammation remains poorly studied. Here we examined lncRNA-mediated regulation of myeloid cells upon challenge with periopathic bacteria.

Method
CD14+ monocytes were purified from PBMCs and were differentiated into macrophage (Mϕ) or dendritic cells (DC). Monocytes were differentiated to M1, M2 Mϕ, or DC in presence of GM-CSF, M-CSF or GM-CSF+IL-4 respectively for 7 days. Mϕ and DC were challenged A. actinomycetemcomitans (Aa), P. gingivalis (Pg) or treated with LPS from Aa and Pg. Total RNA isolated from periopathogen treated cells was used for qPCR. LncRNA knockdown was achieved by siRNA transfection using Lipofectamine 2000 and cell surface markers, cytokine analysis, antigen uptake/processing and phagocytosis assays were performed.

Result
DC challenged with Pg LPS exhibit differential expression of various lncRNAs (19 were up, 2 were down). Expression of selected lncRNAs in periopathogen challenged Mϕ/DC show that lncRNA are sensitive to periopathogen challenge. Cells transfected with lncRNA targeting siRNA showed skewing of M1 or M2 Mϕ polarization markers and altered secretion of pro- or anti-inflammatory cytokines upon periopathogen challenge. Finally, lncRNA knockdown resulted in functional impairment of Mϕ and DC function as reflected by decreased antigen presentation and phagocytosis.

Conclusion
We have functionally characterized lncRNAs in myeloid cells challenged with periopathogens and demonstrated their regulatory role in shaping immune response.
Hypusination regulates the antibacterial response of macrophages
Authors: Gobert Alain P , Latour Yvonne L , Finley Jordan K , Asim Mohammad , barry Daniel P , Allaman Margaret M , Lindsey-Rose Kristie K , Piazuelo Maria B , Sierra Johanna C , Wilson Keith T 
Published: January 2023

Abstract

Introduction
Hypusine is an amino acid found only in the eukaryotic translation initiation factor 5A (EIF5A). Hypusine is synthesized by the enzyme deoxyhypusine synthase (DHPS) that transfers the N-moiety of spermidine to a lysine residue in EIF5A. This post-translational modification (hypusination) is essential to binding specific mRNAs. The EIF5AHyp/mRNA complex is translocated to the cytoplasm, reaches ribosomes, and allows for translation. We analyzed the role of hypusination in the innate response of macrophages.

Results
The gastric pathogen Helicobacter pylori stimulated DHPS expression and hypusination in murine bone marrow-derived macrophages (BMmacs). We generated myeloid-specific Dhps knockout mice (DhpsΔmye) by crossing Dhpsfl/fl mice to Lyz2-cre mice. By iTRAQ proteomics, 29 of the proteins induced by H. pylori in BMmacs from Dhpsfl/fl mice were significantly less expressed in infected DhpsΔmye mice. These included antibacterial effectors (IRG1, NOS2), cytokines (TNF-α, IL-1β), and mediators of autophagy (SQSTM, IRGM1). These findings were confirmed by Western blot. DhpsΔmye mice infected with H. pylori exhibited increased colonization (p&lt;0.01) and gastritis (p&lt;0.05) compared to Dhpsfl/fl mice. DhpsΔmye mice were also more colonized by the intestinal pathogen Citrobacter rodentium than Dhpsfl/fl mice (p&lt;0.05). EIF5AHyp and NOS2 were more expressed in gastric and colonic CD68+ macrophages from H. pylori- and C. rodentium-infected Dhpsfl/fl mice versus uninfected animals, respectively. The staining for EIF5AHyp and NOS2 was markedly reduced in infected DhpsΔmye mice.

Conclusions
Hypusination in myeloid cells is critical for host innate immunity to limit the infectivity of pathogenic bacteria.
Inflammasome signalling is dynamically modulated during the course of a chronic infection by Salmonella Typhimurium
Authors: Cai David , Sad Subash 
Published: January 2023

Abstract
Salmonella is a bacterial pathogen that typically causes acute infections, however, typhoid strains may chronically persist in an asymptomatic state after the acute infection has been resolved. Inflammatory cell death plays a key role in controlling bacterial infections and inflammasomes are a key innate signalling complex that respond to Salmonella infection. Inflammasomes may be triggered by the recognition of pathogen associated molecular patterns such as virulence factors, including the flagella and type III secretion systems of Salmonella. Inflammasome activation results in caspase-1 mediated inflammation and inflammatory cell death. As a consequence, pathogens may regulate their expression of virulence factors to avoid immune recognition and encourage chronicity.
In this study, we sought to assess whether Salmonella-induced inflammasome signalling is modulated throughout the stages of infection to promote its chronic persistence. Through the use of in vivo models of chronic infection, we show that Salmonella virulence is dynamically modulated over the course of a chronic infection to circumvent inflammasome activation. Bacteria isolated during acute phases of infection exhibited an increased ability to induce cell death and inflammation whereas bacteria isolated at later time intervals displayed a progressive decrease in their ability to induce inflammasome signalling. The inflammation and inflammatory cell death observed was primarily dependent on the activation of the NLRP3 and NLRC4 inflammasomes. Together, these results demonstrate that Salmonella dynamically modulates the expression of its virulence factors over the course of an infection to evade immune recognition and promote chronic carriage.
Less may be more for Brucella bacteriophage therapy due to immune suppressive effects
Authors: Hedges Jodi F , Robison Amanda , Snyder Deann Teresa , Plewa Jack , Jutila Mark A 
Published: January 2023

Abstract
We are studying the applicability of Brucella-specific phages to counter brucellosis. With antibiotic resistance on the rise and new realizations concerning the detrimental effects of long-term antibiotic use, phage therapy may become a useful approach against bacterial infection. However, phages are frequently less effective at countering bacterial infection in vivo than is expected considering their strong anti-bacterial effects in vitro. Furthermore, the effects of phage on mammalian cells are largely unknown. We have discovered that Brucella-specific phages are capable of activating some immune components, while suppressing others. Specifically, Brucella phages can activate bovine γδ T cells directly in vitro as measured by upregulation of IL-2R, but dampen the expression of TNF-α induced by E. coli LPS. A similar effect can be measured on human THP-1 macrophages. Also, in an in vitro model of intracellular Brucella infection, low phage MOI was capable of protecting cells from infection, but, unexpectedly, use of high phage MOI diminished protection. In vivo delivery of low phage MOI to the lung was more effective at diminishing Brucella burden than higher doses of phage. Finally, combining low dose phage with innate immune stimulation was more effective at reducing Brucella bacterial burden than either treatment alone. We hypothesize that some phage preparations induce an immune suppressive response that may counteract their anti-bacterial effects. Potentially combinatorial approaches may overcome this suppressive response. Our discovery of immune suppressive effects of Brucella bacteriophages is an important consideration for using phage as a treatment. This work is supported by NIH R21 AI144496-01.
LAG-3 expression on Foxp3+ regulatory T cells is critical for controlling homeostasis and autoimmune neuroinflammation
Authors: Lee Juyeun , Min Booki 
Published: January 2023

Abstract
Lymphocyte activation gene-3 (LAG-3) is increasingly recognized as an important immune checkpoint molecule that controls T cell activation. However, the role of LAG-3 on Foxp3+ regulatory T cell (Treg) function remains largely unknown. To test if LAG-3 expression is critical for Treg function in vivo, we generated a novel Treg-specific LAG-3 knock out mouse model (TregΔLag3). In steady state conditions, the lack of LAG-3 in Tregs significantly elevated activated memory-like phenotypes (CD62LlowCD44hi) and higher cytokine production of peripheral T cells. Following induction of experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune inflammation in the central nervous system, TregΔLag3 mice developed severe EAE compared to that of littermate controls. Flow cytometric analyses showed that TregΔLag3 mice had increased Foxp3+ Tregs in the CNS but that these Tregs expressed lower level of CD44, CD25, ICOS, and GITR, and higher inflammatory cytokines, suggesting their functions may be defective. Unlike WT Tregs, LAG-3-deficient Tregs adoptively transferred into Treg-depleted recipients were unable to rescue the mice from lethal disease, further corroborating the findings. Importantly, Tregs expressing mutant LAG-3 deficient in the cytoplasmic domain of the LAG-3 also failed to control EAE development in vivo, suggesting the key role of LAG-3 intracellular signaling on Treg function. Taken together, our study uncovers a novel Treg-intrinsic role of LAG-3 in regulating homeostasis and inflammation.
The liver controls regulatory T cell populations by enclysis, a CD4+ T cell engulfment process
Authors: Stamataki Zania , Davies Scott , Reynolds Gary , Li Xiaoyan , Grove Joe , Barnes Nicholas , Adams David , Huang Yuehua , Qureshi Omar 
Published: January 2023

Abstract

Background and Aims
Regulatory T cells (Treg) dampen inflammation and they are increased in the liver compared to blood, contributing to a tolerising environment. The swift neutralisation of Treg cells is important to enable immune effector functions to combat infection, but the mode of this is not understood. We noted that hepatocytes preferentially engulfed live Treg cells, and we set out to characterise the mechanism and outcome of this new phenomenon we termed “enclysis”.

Method
We measured enclysis, the enclosure and lysis of Treg cells, in vitro and ex vivo using perfused liver wedges, in the presence of inhibitors of known cell-in-cell processes. Reporter dyes for viability, mitochondrial activity and acidification were used to determine enclysis outcome. Cell-in-cell structures were documented by light, confocal, electron and multiphoton microscopy. FoxP3+ Treg and T-bet+ T helper cells were enumerated in donor and in end-stage disease livers of autoimmune and viral diseases.

Results
Hepatocytes preferentially engulfed live Treg cells in a process that differed in mechanism and kinetic to previously described cell-in-cell structures (efferocytosis, suicidal emperipolesis, entosis). Enclysis reflects the enclosure and specific lysis of live Treg cells inside hepatocytes, and beta-catenin-positive enclytic vesicles were connected to the endocytic pathway. Enclysis was increased in patients with autoimmune compared to viral hepatitis (p=0.0011), offering an explanation for the breach of tolerance in the liver.

Conclusion
Enclysis is the preferential engulfment and deletion of live Treg cells by hepatocytes and can be modulated pharmacologically, offering a new therapeutic target for liver immune regulation.
Interferons in Treg development and function
Authors: Sjaastad Lucy E , Owen David L , LaRue Rebecca S , Farrar Michael A 
Published: January 2023

Abstract
Thymus-derived regulatory T-cells (Tregs) are critical for controlling immune responses and preventing autoimmunity. In the setting of viral infection, Tregs are important modulators of anti-viral immune responses and help resolve inflammation. The subset of Tregs responsible for responding to viral infections and the types of antigens they recognize are not well understood. Using single-cell RNA sequencing we identified a unique subset of thymic Treg progenitors and mature Tregs that express a strong interferon stimulated gene (ISG) signature, as well as a comparable population in the spleen. Importantly, the ISG signature is much stronger than that expressed by thymocytes receiving tonic IFN stimulation in the final stages of maturation prior to thymic egress. Using flow cytometry we established that APCs in the thymus, including mTECs, as well as SIRPα+ and CD8α+ dendritic cells, require IFNα/β or IFNλ, acting via STAT1 signaling, for the expression of the canonical ISG BST2. TCR specificity was important for selection into this subset as SM1 TCR transgenic mice, which express a TCR that recognizes a non-ISG encoded antigen, have a reduced compartment of ISG-expressing CD4SP T-cells in the thymus. We are currently generating mice that specifically lack the ISG-signature Treg subset to test their function in vivo. Based on our current findings, we propose that thymic ISG antigens serve as TCR ligands for inducing the differentiation of ISG-signature Tregs that are in close physical proximity to the ISG-producing APCs. Furthermore, we hypothesize that ISG-signature Tregs are specialized for responding to ISGs induced during antiviral immune responses and play a key role in controlling IFN-driven inflammation.