bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of novel E3 ubiquitin ligase in the regulation of TLR3 signaling pathway
Authors: Lin You-Sheng , Chang Yung-Chi , Lai Ting-Yu , Lee Chih-Yuan , Chuang Tsung-Hsien , Hsu Li-Chung 
Published: January 2023

Abstract
Toll-like receptors (TLRs) are critical players in the host’s defense against infection by recognizing pathogen-associated molecular patterns (PAMPs) derived from microbes, and subsequently induce inflammatory responses, which eventually eliminate pathogens and repair damage tissues. However, excessive inflammation is detrimental to the host, and has been associated with the pathogenesis of various inflammatory and autoimmune diseases. Ubiquitination is a crucial strategy to alter protein function, expression, or cellular localization at the post-translational level, and has been shown to participate in the regulation of innate immune responses. We previously found that a novel E3 ubiquitin ligase was induced upon endosomal TLRs (TLR3/7/9) activation. In this project, we demonstrate that this E3 ubiquitin ligase negatively regulates TLR3-driven immune responses, which is dependent on its ubiquitin ligase activity. This novel E3 ubiquitin ligase controls TLR3 trafficking from the endosomes to the lysosomes for degradation. In addition, this E3 ubiquitin ligase interacts with TLR3 and promotes the ubiquitination of TLR3. We also found that lysosomal activity and intracellular pH value were altered in macrophages lacking this E3 ubiquitin ligase after poly(I:C) stimulation. Furthermore, we showed that mice with this E3 ubiquitin ligase deficiency were resistant to encephalomyocarditis virus (EMCV) infection due to enhanced type I interferon production. Our study together reveals this novel E3 ubiquitin ligase as a negative regulator of TLR3-induced inflammatory responses and proposes a novel mechanism for the termination of TLR3 signaling and immune response.
DNA damage responses modulate macrophage transcriptional networks during microbial infection
Authors: Morales Abigail 
Published: January 2023

Abstract
In response to inflammatory stimuli, macrophages integrate myriad signaling pathways that influence the overall response to the pathogenic threat. Specifically, signals emanating from pattern recognition receptors (PRRs) activate several transcriptional networks that promote the expression of inflammatory mediators. Among the transcription factors activated downstream of PRR engagement is NF-κB, which promotes the production of pro-inflammatory cytokines and chemokines. Previous work has established that NF-κB is also activated downstream of DNA damage. In contrast to NF-κB activation following PRR engagement, this DNA damage-elicited signaling cascade originates in the nucleus and depends, in part, on Serine 85 phosphorylation of IKK regulatory subunit NEMO. During infection, macrophages produce genotoxic intermediates that are highly microbicidal. Recent work demonstrated that in macrophages, these agents damage macrophage DNA. We hypothesize that this DNA damage may modulate NF-κB activation downstream of PRR engagement. Indeed, we find that in activated macrophages, NEMO is phosphorylated at Serine 85, suggesting DNA damage-elicited NF-κB activation. Though previous reports have established that ATM is the kinase responsible for this signaling event, our data suggests that in macrophages, optimal NEMO S85 phosphorylation depends on the related DNA damage response kinase DNA-PKcs. Indeed, in the absence of functional DNA-PKcs, inflammatory cytokine production is markedly reduced in macrophages after exposure to bacterial stimuli. These findings underscore a previously underappreciated role for DNA-PKcs in modulating macrophage transcriptional networks during innate immune responses to infection.
Identifying critical immune effectors of the dermal wound healing response by innate cells using microporous-annealed-particle (MAP) hydrogel
Authors: Lempke Samantha of , Roosa Colleen , Griffin Donald , Ewald Sarah 
Published: January 2023

Abstract
The wound healing response is critical to maintain tissue function after injury. Inflammation is needed to sterilize the wound site, but this results in scar formation and impaired tissue function. Topical anti-microbial use, fetal wound healing, and limited studies in germ-free animals suggest that the degree of microbial contamination at a wound site positively correlates with scar formation and recovery time. However, tools to identify the molecular signals and cell types that alter tissue repair verses have been limited. Here we test the hypothesis that Danger-Associated-Molecular Patterns (DAMPs) released during sterile injury guide a fundamentally distinct wound healing response than Pathogen-Associated-Molecular-Patterns (PAMPs) using a novel microporous annealed particle hydrogel (MAP-gel). MAP-gel is an immunologically inert, injectable tissue matrix that is easily linked to a broad range of small signaling molecules. We show that subcutaneous injection of MAP-gel is a robust platform to identify innate immune signaling molecules and infiltrating cell types that control wound healing. Conjugating the model PAMP, LPS, to MAP-gel is sufficient to induce a fibrotic encapsulation response and alter infiltrating immune cell populations. To identify the candidate modulators, proteins enriched in MAP-gels isolated from non-inflammatory or LPS-driven inflammatory responses were identified by LC-MS. These candidates are being conjugated directly to MAP-gel to identify immune effectors that promote wound healing with the absence of fibrotic encapsulation. The modified MAP-hydrogel will be used to gain insight into the molecular mechanisms behind scaring and the unique role DAMPs play in tissue resolution.
Macrophages activate a MLK3/MEK signaling axis to remove pore forming toxins by microvesicle shedding
Authors: Ray Sucharit , Keyel Peter A 
Published: January 2023

Abstract
The innate immune system is critical to promoting host survival during bacterial necrotizing soft tissue infections (NSTIs). NSTI causing pathogens like Streptococcus pyogenes and Clostridium perfringens use pore-forming toxins to kill cells, including macrophages. Toxins cause rapid accumulation of Ca2+ and ceramide at the site of damage, which triggers repair responses. Ca2+ sensitive repair proteins like Annexins may clog the injured site and are spontaneously shed with toxin pores on microvesicles. However, the molecular mechanism orchestrating this repair is unknown. Pore formation activates sphingomyelinases, which generate ceramide. Ceramide signaling may promote repair, potentially via the Mixed Lineage Kinase 3 (MLK3) Mitogen Activated Protein Kinase (MAPK) pathway. Long term toxin exposure activates stress responses via the MAPK pathway but the short-term signaling during repair is unknown. We hypothesized that MLK3 activates a pro-survival kinase cascade (MEK/ERK) to activate membrane repair by microvesicle shedding in macrophages. We tested cell viability and repair responses to toxins after blocking proteins in the MAPK pathways using inhibitors or siRNA in mouse bone marrow derived macrophages and cell lines. We found MLK3 or MEK inhibition significantly reduced toxin resistance. Protective MLK3/MEK activity was downstream of Ca2+ and ceramide signaling. Inhibiting this axis reduced ANXA1/6 translocation to the site of membrane damage and decreased microvesicle shedding. Overall, these findings suggest that a MLK3/MEK axis protects macrophages from pore-forming toxins. Modulation of this axis could promote resistance to NSTIs.
Therapeutic targeting of intracellular Toll-like and interleukin-1/18 receptor (TIR) resistance domain containing proteins for protection against infection, inflammation and disease
Authors: Snyder Greg A , Lai Wendy , Brown Lindsey , Blanco Jorge , Beadenkpf Robert , wang Yajing , Vogel Stefanie , Shirey Kari Ann 
Published: January 2023

Abstract
TIR domain containing proteins are important immune associated proteins shared among Toll-like and interleukin-1/18 receptor family members. In a recent discovery select bacterial, plant and human TIR proteins exhibit enzymatic activity in binding and processing nicotinamide adenine dinucleotide (NAD+). Based on their abilities to facilitate signaling across biology, we hypothesize that TIR proteins represent unique therapeutic targets for modulating infection, inflammation and disease. Our previous structural studies of bacterial-host TIR proteins B. melitensis (TcpB) and uropathogenic E. coli CFT073 (TcpC) with human host TIRAP and MyD88 characterized peptides that negatively regulate signaling and infection. From these studies we identified interactions that are at or near reported biological TIR protein interfaces. In particular, we identified a functionally important motif found conserved on the C helix of most bacterial, human host and NAD+ consuming TIR proteins. As proof of concept for select targeting of TIR proteins and this region in particular we have used the TLR4 antagonist, TAK-242, which selectively binds within this motif. Treatment with TAK-242 or TLR4-C747S blocks LPS signaling. Additionally, TAK-242 protected mice from lethal influenza challenge similar to an extracellular TLR4 antagonist, Eritoran. Bioinformatic analysis of the region targeted by TAK242 show that it is located within the WxxxE structural motif identified to be important for protecting against microtubule destabilization and includes a catalytically essential glutamic acid (E) residue conserved among nearly all NAD+ consuming TIR proteins. These studies provide a framework for future studies targeting TIR protein function.
Physiologic Media Alters Macrophage Phenotype and Activation State
Authors: Bader Jackie E , Rathmell Jeffrey C 
Published: January 2023

Abstract
Traditional cell culture media such as RPMI is consistently used in in-vitro studies in order to inform in-vivo translation. However traditional media poorly simulates physiological conditions which can lead to inaccurate interpretations of data and failed in-vivo applications. The recent development of human plasma like media (HPLM) mirrors physiologic conditions with more realistic levels of glucose, amino acids and other nutrients. We investigated bone marrow derived macrophage phenotype and activation in either RPMI or HPLM to better understand the effects of physiologic conditions on macrophages behavior. Classically activated M1 (100ng/mL LPS + 10ng/mL IFNγ) or alternatively activated M2 (20ng/mL IL-4) were investigated for metabolic activity and behavior. Macrophages cultured in HPLM exhibit a unique phenotype compared to RPMI with increased iNOS expression in response to LPS treatment and reduced CD206 after treatment with IL-4. These data suggest physiologic media can alter the behavior and function of macrophages in-vitro. Following more investigation, these unique activation states in physiologic conditions may uncover differential metabolic requirements and behavior not previously seen in traditional culture media.
Lactoperoxidase-generated hypothiocyanite efficiently kills Streptococcus pneumoniae
Authors: Rada Balazs , Gingerich Aaron , Doja Fayhaa , Thomason Rachel , Toth Eszter , Bradshaw Jessica L , McDaniel Larry S 
Published: January 2023

Abstract
Streptococcus pneumoniae (pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase, thiocyanate anion and hydrogen peroxide (H2O2). Lactoperoxidase oxidizes thiocyanate using H2O2 into the final product hypothiocyanite. While hypothiocyanite has known antimicrobial effects against a wide range of microorganisms, its effect on pneumococcus has never been studied. To test this, pneumococci were exposed to hypothiocyanite generated enzymatically in a cell-free in vitro system. Bacterial killing was determined by colony forming units on agar plates while bacteriostatic effects were measured by changes in optical density of liquid cultures. We demonstrate hypothiocyanite exerted robust killing of several pneumococcal strains. Nonencapsulated pneumococcal mutants were killed to the same extent as their parental strains, indicating no role of the capsule in protection against hypothiocyanite. Catalase, an H2O2 scavenger, inhibited pneumococcal killing by hypothiocyanite under all circumstances. Interestingly, a pneumococcal mutant deficient in pyruvate oxidase, the main bacterial H2O2 source, had enhanced susceptibility to hypothiocyanite indicating the role of this gene in partial protection of the bacterium against this oxidizing agent. Overall, numerous pneumococcal strains were found to be susceptible to lactoperoxidase-generated hypothiocyanite in vitro, which presents an opportunity to explore hypothiocyanite as a potential novel anti-pneumococcal therapy.
Cholesterol-dependent cytolysins promote immune evasion by clustering host membrane lipids
Authors: Thapa Roshan , Keyel Peter A 
Published: January 2023

Abstract
Pathogens causing necrotizing soft tissue infections, such as Streptococcus pyogenes and Clostridium perfringens, use cholesterol dependent cytolysins (CDCs) to evade immune responses by blocking pro-inflammatory TNFα production in macrophages. This evasion may be an unintended consequence of membrane repair responses to the CDC. CDCs trigger lipid-dependent sequestration and shedding of CDC pores on microvesicles. However, the molecular mechanisms by which CDCs exploit membrane repair to disrupt immune signaling is unknown. One target may be cholesterol-rich microdomains (CRMs) because they are focal points for toxin binding and immune signaling. We hypothesized that when CDCs oligomerize, they cluster membrane lipids in CRMs, along with immune receptors, into microscale assemblies that are spontaneously shed as microvesicles. We tested this hypothesis by artificially clustering sphingolipids using the sphingolipid-binding protein Ostreolysin A (OlyA), fused with tandem FKBP dimerization domains. We challenged murine bone marrow derived macrophages with and without FKBP homodimerizer and OlyA prior to LPS challenge and measured intracellular TNFα by flow cytometry. We found that oligomerization reduced OlyA binding, consistent with shedding. OlyA oligomerization further suppressed TNFα production, suggesting that microscale lipid assemblies interfere with cellular signaling. Overall, these results suggest that one cost to repair by shedding toxins on microvesicles is the loss of cellular signaling needed to promote inflammation. This may represent one strategy CDC-producing pathogens use to promote immune evasion and cause disease.
BRAF-V600E causes intrinsic hyper-responsiveness of dendritic cells to TLR4 stimulus
Authors: Minichino Danielle E , Behrens Edward M 
Published: January 2023

Abstract

Background
The origin of inflammation in Langerhans Cell Histiocytosis (LCH), an inflammatory disease of dendritic cells (DCs) in children, remains elusive. Over 85% of patients harbor somatic, gain-of-function mutations in the Ras-ERK signaling pathway in DCs, which leads to hyper-pERK activity. We hypothesize that DCs with hyper-pERK signaling are intrinsically hyper-responsive to TLR stimuli, resulting in increased levels of cytokines that contribute to LCH etiology.

Methods
To determine the TLR response in hyper-pERK DCs, we generated bone marrow derived DCs (BMDCs) from wild-type and CD11c-Cre:BRAF-V600E flox mice (V600E-DCs). Cells were pre-treated with or without a V600E-inhibitor and stimulated with TLR ligands. Cytokine levels were measured by ELISA and qPCR. TLR levels were measured by qPCR.

Results
V600E-DCs secreted increased LPS-induced TNFa and IL-6 compared to wild type, which was reversible with acute inhibition of V600E. Intriguingly, this was despite V600E-DCs having decreased tnfa, il-6, and tlr4 transcripts. Osteoponton (OPN) secretion was irreversibly increased in V600E-DCs both at baseline and after stimulation with LPS, CpG, and Poly(I:C); however, its gene, spp1, was not increased.

Conclusions
DCs harboring the BRAF-V600E allele have a reversible increase in LPS induced inflammatory cytokine expression, suggesting that increased pERK directly caused a hyper-response to TLR4 stimulation. The irreversible increase in OPN secretion suggests a permanent defect in V600E-DCs independent of pERK levels at time of stimulation. These data taken together suggest that the increased pool of pERK in V600E-DCs contributes to a complex disruption of cytokine secretion that may play a role in LCH etiology.
Lack of PD-1 suppressed the growth of E. multilocularis in mice
Authors: Lin Renyong , Li Zhide , Zhang Chuanshan 
Published: January 2023

Abstract
The cestode Echinococcus multilocularis (E. multilocularis) infection, a serious health problem worldwide, causes alveolar echinococcosis (AE), a tumor-like disease predominantly located in the liver. Until now, there have been few studies to explore how PD-1 contributes to parasite’s escape from immune attack and how it might be reversed. In this study, we found that liver PD-1 expression was significantly enhanced in AE patients. Compared to E. multilocularis infected wild-type mice, we found lesion’s weights in the liver were significantly deceased (101.20±43.12mg vs 393.70±123.5 mg, P&lt;0.05) and lesion’s volumes were significantly lower (82.17±28.43 mm3 vs 429.30±148.30 mm3, P&lt;0.05) in E. multilocularis infected PD-1 knockdown mice. This study demonstrates blocking PD-1 may suppress the growth of E. multulocularis in mice, and represent a possible approach to E. multilocularis immunotherapy.
Using wild mice to interrogate immunological mechanisms of asymptomatic malaria
Authors: Cornwall Douglas , Srey Mellina , Maloba Franklin , Evavold Birk , Olatunde Adesola C , Jensen Anne , Lamb Tracey , Evavold Brian 
Published: January 2023

Abstract
Malaria is a significant world health problem, with billions of people at risk and over 200 million clinical cases per year that result in over 200,000 deaths. However, the majority of the population in malaria endemic areas (&gt;60%) is asymptomatic (without overt symptoms), even in high transmission areas. Although identified by Plasmodium-infected red blood cells in the circulation, the term asymptomatic malaria is a misnomer with individuals experiencing mild anemia, vascular activation and increased susceptibility to co-morbidities such as non-typhoidal Salmonella infections. The immunological mechanisms governing the development and maintenance of asymptomatic malaria are poorly understood and the influence of host genetics in endemic populations is unknown. Currently, no immunologically intact animal model is available to understand how host genetics influence the asymptomatic nature of Plasmodium infection. Here, we use wild-derived Mus musculus to show that the genetic diversity seen in the wild host population leads to increased variation in anemia upon infection with Plasmodium yoelii XNL with a subpopulation of mice experiencing asymptomatic malaria with mild anemia. Plasma TNF-a was positively correlated with the level of anemia, as might be expected from studies showing a positive correlation of TNF-a with anemia in children. Also, there was a negative correlation between monocyte populations and anemia experienced by the mice, which has also been observed in asymptomatic children in eastern Uganda. These results show that the wild derived mice can model asymptomatic malaria and allow us to interrogate the different immune profiles that lead to the range of phenotypes to malarial disease.
A novel variant in ubiquinone biosynthesis highly prevalent in Papua New Guinea children increases mortality following bacterial pneumonia
Authors: Walker Emma C , Ramani Rashmi , Javati Sarah , Todd Elizabeth , Chandra Pallavi , Matlam John-Paul , Anaya Edgar , Pomat William , Morley Sharon Celeste 
Published: January 2023

Abstract
To identify immune variants predisposing to severe pneumonia, we performed whole exome sequencing in a pediatric population highly susceptible to acute lower respiratory infections, identifying a candidate novel variant in the ubiquinone (CoQ10) biosynthetic pathway. To evaluate the effect of this variant on immune function during bacterial pneumonia, we generated a mouse line using CRISPR-Cas9 that expresses the homologous aspartate to tyrosine variant in the enzyme COQ6. Intra-tracheal S. pneumoniae infection leads to increased bacteremia and mortality in mice homozygous for the variant despite similar numbers of immune cells in the lung. Mechanistic studies show that macrophages expressing the variant have decreased mitochondrial activity at the ubiquinone-dependent reduction of cytochrome c by complex III, as well as decreased maximum respiratory capacity in response to acute stimulation. Variant-expressing macrophages also exhibit impaired generation of mitochondrial reactive oxygen species (mROS) causing a direct, intrinsic defect in intracellular killing of internalized bacteria. Thus, the novel variant in CoQ10 biosynthesis leads to changes in macrophage mitochondria and an intrinsic inability to kill internalized bacteria. As alveolar macrophages are the first responders in the lung to bacterial challenge, the inability of these macrophages to mount a sufficient immune response can explain the observed increase in mortality following bacterial pneumonia. Because variants in CoQ10 biosynthesis can be supplemented with CoQ10, a readily available therapy may be able to correct this defect and improve survival in children with this variant
Association of gut microbiota with spontaneous autoimmune uveitis studied by human flora reconstitution of germ-free mice
Authors: Zhang Amy , Horai Reiko , Salvador Ryan S. , O’hUigin Colm , Badger Jonathan , Yuan Wuxing , Thovarai Vishal , Sudo Katsuko , Atarashi Koji , Honda Kenya , Caspi Rachel R. 
Published: January 2023

Abstract
Autoimmune uveitis is a T cell driven, intraocular inflammation that affects the neuroretina and can lead to blindness. In a mouse model of spontaneous autoimmune uveitis (R161H), retina-specific T cells are primed in the gut through their TCR and trigger disease (PMID: 26287682). To support the relevance to human disease, we examined development of uveitis and its association with bacterial taxa in gnotobiotic R161H mice harboring human flora. Specifically, fecal samples from three healthy human donors were inoculated into germ-free R161H mice and their wild type (WT) littermates in separate isolators. Fecal pellets from reconstituted mice and their offspring were collected periodically and subjected to 16S amplicon sequencing. Uveitis scores were determined in R161H offspring by histology at termination of the experiment. Our results show that healthy human gut commensals can support uveitis in R161H mice, but to a lesser extent than native mouse flora from the NIH facility. Diversity analyses indicate that human flora-reconstituted mice retained a distinct but simplified gut microbial community compared to the original sample, and R161H mice tended to have a more complex microbiome profile than WT littermates. Among R161H offspring, mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Differentially abundant bacterial taxa were identified between mice with different disease scores, which may associate with intensity of autoreactive T cell activation and development of autoimmunity. R161H associated with human gut flora could provide a more translatable platform than mouse flora to explore microbial candidates and products that may modulate autoreactive T cells in uveitis.