bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted proteomics-driven computational modeling of the mouse macrophage toll-like receptor signaling pathway
Authors: Manes Nathan Paul , Armstrong Anthony A , Fraser Iain D C , Germain Ronald , Meier-Schellersheim Martin , Nita-Lazar Aleksandra 
Published: August 2023

Abstract
The Toll-like receptor (TLR) signaling pathway is crucial for the initiation of effective immune responses. Computational modeling of biological pathways can test mechanistic hypotheses about how regulation is achieved and why it sometimes fails, causing disease (e.g., sepsis). In this investigation, mass spectrometry (LC-MS) and computational modeling are being integrated to develop a model of the TLR4 pathway.
TLR4 pathway proteins within mouse macrophages were quantitated using targeted LC-MS and stable-isotope labeled internal (phospho)peptide standards (136 unmodified peptides and 29 phosphopeptides for 54 (phospho)proteins). AlphaFold-Multimer was used to predict protein complex structures, Rosetta was used to idealize and relax the structures, and Simulation of Diffusional Association (SDA) and TransComp were used to estimate protein-protein association rates. Simmune was used to perform rule-based pathway modeling, simulation, and training.
LPS stimulation altered proteins known to be affected by TLR4 pathway activation (e.g., phosphorylated ERK1 increased from 30,000 to 250,000 copies per cell). Hundreds of protein-protein association rates were predicted. The (phospho)protein absolute abundance values and protein-protein association rates are being used as parameters for TLR4 pathway models. The parameter space is being explored to identify parameter sets that accurately reproduce the experimental data.
Experimental and computational techniques are being integrated to generate a strongly data driven model of the TLR pathway. This work was supported by the Intramural Research Program of NIAID, NIH.
This work was supported by the Intramural Research Program of NIAID, NIH.
Human microglial responses to DNA damage are mediated by the cytosolic sensor cGAS
Authors: Suptela Alex J , Marriott Ian 
Published: August 2023

Abstract
Genomic instability is a key driving force for cancer development and age-related disease. While DNA damage responses are known to regulate genome integrity and cell fates, there is mounting evidence that genomic instability can trigger inflammatory and/or antitumor responses via pattern recognition receptors (PRRs) located in the cytosol. One such PRR, cyclic GMP-AMP synthase (cGAS), canonically detects foreign viral or bacterial DNA resulting from infection, initiating a signaling cascade resulting in the production and secretion of type I interferons and inflammatory cytokines. However, we hypothesize that cGAS contributes to the initiation of innate immune responses following DNA damage in the central nervous system (CNS) outside of its canonical role of viral/microbial cytosolic DNA recognition. In the present study, we induced DNA damage using ionizing radiation or oxidative stress in a human microglial cell line, and assessed the effect of cGAS knock-down on the immune responses of these cells to these insults. DNA damage following exposure to radiation or oxidative stress was verified by fluorescent microscopy for the formation of micronuclei and expression of γH2AX. Wild-type microglia showed robust production and secretion of IL-6 and IL-8 following DNA damage as assessed specific-capture ELISA. Importantly, these inflammatory responses at 24 hours following DNA damage were significantly attenuated in human microglia-like cells following cGAS knock-down. Taken together, these data support the contention that cGAS plays a significant role in the initiation of potentially detrimental proinflammatory immune responses by human CNS cells following DNA damage outside of its canonical role in antiviral immunity.
Supported by a grant from NINDS (NS111260)
Skewing of UV-induced cell death pathways using TLR4 ligands
Authors: Williford Zachary J , Chaudhry Sumra , Kane-Jackson Michel , Smith Richard , Ernst Robert , Harberts Erin M 
Published: August 2023

Abstract
Toll-like receptor 4 (TLR4) is a canonical innate immune receptor for the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). TLR4 deficiency has been shown to contribute to UV-induced carcinogenesis in epidermal cells. UV-induced cellular damage results in apoptosis which is characterized by a non-inflammatory cell death through the cleavage and activation of caspase-3. Utilizing bacterial enzyme combinational chemistry (BECC) we generated hypoacylated lipid A moieties from Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium which have moderately attenuated immune-stimulatory capacity. Continuously exposing TLR4 to these innate immune ligands leads to tolerization and decreased expression of the receptor in mammalian cells. We hypothesize that skewing cell death away from apoptosis using lipid A mimetics to lower TLR4 expression prior to UV exposure will reduce carcinogenic potential. To analyze the roll of TLR4 in UV-induced carcinogenesis, wildtype and TLR4-deficient bone marrow-derived macrophage (BMDM) cell lines were tolerized with hexa-acylated or penta-acylated lipid A from multiple Gram-negative species and exposed to either 10 or 25 mJ/cm2 doses of UVB radiation. UV-irradiated BMDM cells show evidence of apoptosis including cleavage of DNA creating a ladder, whereas UV-irradiated BMDM cells tolerized with lipid A mimetics show a lack of apoptosis. The data suggests that tolerization of TLR4 using canonical ligands may be a useful tool in promoting DNA repair and maintaining immune responses following UV-induced damage.
The research was supported by a Fisher College of Science and Mathematics Undergraduate Research Grant.
ALKBH5-mediated m6A RNA demethylation promotes antibacterial innate defense by intrinsically driving neutrophil generation and recruitment
Authors: Liu Yang , Cao Xuetao 
Published: August 2023

Abstract
Neutrophil generation and effective recruitment into the site of infection are essential for innate immune defense against bacterial infection. These processes that directed by extracellular signals such as growth factors or chemokines have been extensively studied, yet the intrinsic and new mechanism for determining neutrophil generation and recruitment needs further investigation. N6-methyladenosine (m6A) RNA modification and its demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, whereas their role in neutrophil generation and recruitment remains unclear. Here, using cecal ligation and puncture-induced sepsis to model systemic bacterial infection, we found that ALKBH5 is required for antibacterial innate defense by intrinsically inducing neutrophil generation and recruitment. Indeed, Alkbh5-deficient mice exhibited increased mortality, higher bacterial burden and proinflammatory cytokine production than wild-type littermates undergoing sepsis. Meanwhile, ALKBH5 depletion significantly impaired the generation of immature neutrophils in bone marrow and also reduced neutrophil recruitment into the infected site of sepsis mice. Mechanistically, ALKBH5 imprinted generation- and recruitment-promoting transcriptome signatures in both mouse and human neutrophils during bacterial infection, specially modulated the protein expression of SOCS3 and CXCR2 through altering m6A methylation-mediated decay of their mRNAs. Together, our findings reveal a previously unknown role of ALKBH5 in intrinsically driving neutrophil generation and recruitment, indicating neutrophil m6A RNA modification as potential target for controlling bacterial infections.
Supported by grants from the National Natural Science Foundation of China (81788101, 82071793), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-017), the Beijing Natural Science Foundation (7212069), and the Beijing Nova Program (20220484065).
Unraveling the cell death induced by SARS-CoV-2 in lung epithelial cells
Authors: Liang Kaixin , Barnett Katherine Camille , Xie Yuying , Oguin Thomas , Sempowski Gregory D , Lei Yu Leo , Ting Jenny P 
Published: August 2023

Abstract
The COVID-19 pandemic has continued to affect people’s lives worldwide, and SARS-CoV-2, the pathogen that causes the pandemic, has infected over 100 million people and claimed over 10 million lives globally. There is strong evidence that lung damage contributes to the inflammatory responses against SARS-CoV-2, and that diverse forms of cell death occur in myeloid cells upon infection. However, less is known about the cell death pathways activated in the primary cell target of SARS-CoV-2, lung epithelial cells. Previously, we showed that conventional pyroptosis was activated in bystander myeloid cells. Using a human epithelial cell line with the ACE2 receptor and primary human airway epithelial culture, we analyzed the impact of SARS-CoV-2 infection on cell death. The latter is grown at an air-liquid interface (ALI) and is used to model airway pseudostratified epithelium. SARS-CoV-2 infection of these cells triggers lytic cell death. Pathway analysis shows that biomarkers of necroptosis, apoptosis, and pyroptosis are activated and increase with time, but some forms of cell death are limited predominantly to cells that are co-stained with SARS-CoV-2 viral antigens while others are not. RT-PCR and single-cell RNAseq analysis revealed that cell death genes increase with high viral titer and severity of disease in patients.
R56 AI158314, R01 AI158314, AI029564, AI141333, CA232109
Ablation of Siglec-E enhances adipose tissue inflammation through CXCR3 activation and induces monocytic myeloid-derived suppressor cells
Authors: Rakib Ahmed , Kiran Sonia , Mandal Mousumi , Li Lexiao , Jiang Jianxiong , Singh Udai P 
Published: August 2023

Abstract
Obesity is a worldwide epidemic associated with dysregulated metabolism and chronic low-grade inflammation in adipose tissue (AT). AT infiltrates with several immune cells, which secrete proinflammatory cytokines that mediate the severity of AT inflammation during obesity. During our ongoing studies, we observed the downregulation of various genes, particularly sialic acid-binding Ig-like lectin E (Siglec-E) by RNA-seq analysis in AT immune cells isolated from high-fat diet (HFD) fed mice as compared to normal diet (ND) mice. Siglec-E is mainly expressed in the cells of myeloid lineages, including macrophages, dendritic cells (DCs), and neutrophils. We confirmed the RNA-seq data of downregulation of Siglec-E in AT derived from HFD-fed mice by RT-PCR analysis. To determine the role of Siglec-E in mediating AT inflammation, we fed ND and HFD to wild-type (WT) and Siglec-E knockout mice (Siglec-E−/−), respectively. HFD consumption increased the body weight and blood glucose levels in Siglec-E−/− mice as compared to WT mice. Further, we observed increased infiltration of macrophages and decreased frequency of DCs in HFD-fed Siglec-E−/− mice AT compared to WT HFD-fed mice. We also noticed an increase in the frequency of CD8+CXCR3+ cells and monocytic myeloid-derived suppressor cells (M-MDSCs) in AT of HFD-fed Siglec-E−/− mice as compared to WT HFD-fed mice. We also noticed the increased expression of AKT, and TNF receptor-associated factor 3 (TRAF3) in HFD-fed Siglec-E−/− mice. These results suggest that Siglec-E deletion plays a role in AT inflammation and obesity through the activation of CXCR3 and M-MDSCs. This study supports the notion that modulating Siglec-E pathways protect from AT inflammation and metabolic disorders.
This research is supported by NIH grant R01 AI140405
A single-dose, tolerizing vaccine induces antigen-specific regulatory T cells and durable remission while preserving healthy immunity in a mouse model of Multiple sclerosis
Authors: Kapnick Senta , Gosselin Emily A , Tsai Shannon J , Oakes Robert S , Carey Sean T , Ackun-Farmmer Marian A , Froimchuk Eugene , Yanes Alexis A , Eppler Haleigh B , Noshin Maeesha , Tostanoski Lisa H , Black Sheneil K , Zeng Xiangbin , Bromberg Jonathan S , Jewell Christopher M 
Published: August 2023

Abstract
Multiple sclerosis (MS) is an inflammatory disorder that occurs when autoreactive lymphocytes mistakenly attack myelin, resulting in motor deficits and cognitive deterioration. Current drugs are non-curative and require life-long treatment that make compliance challenging, and existing therapeutics can leave patients vulnerable to opportunistic infections. An experimental approach to overcome these hurdles is to drive long-lasting, antigen-specific regulatory T cells (Tregs) that dampen responses to autoantigens. Here, we co-deliver antigen and rapamycin in micron-sized, diffusion-limited, polymer microparticles (MPs) directly to lymph nodes (LNs) where immune responses are orchestrated. Using adoptive transfer of transgenic T cells, we show that MPs generate antigen-specific Tregs in response to two different MP-derived peptides – evidence that MPs do not interfere with induction of peripheral tolerance in LNs. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, single-dose treatment demonstrates potent, long-lasting efficacy (up to three months) following a single treatment. Tolerizing effects are observed during chronic disease – the most challenging for treatment regimens. Efficacy is accompanied by reduced demyelination in spinal cords of treated mice, the cause of paralysis in EAE and MS. Thus, antigen-specific reprogramming of Tregs in LNs reshapes inflammation in distal tissues without need for systemic or repeated dosing. Importantly, MP-treated EAE mice mount healthy immune responses to vaccine challenge. This work advances a strategy to locally reprogram antigen-specific, tolerogenic responses that preserve healthy immunity – a hurdle faced by current MS therapeutics.
Supported by grants from NIH (R01 AI169686).
The investigation of selective sphingosine-1 phosphate receptor 1&4 modulator to treat alopecia areata mouse model
Authors: Seok Joon , Kim Su-Young , Cho Sung-Dong , Choi Yunseo , Park Yanghae , Lee Bong Yong , Kim Beom Joon , Park Su-Hyung 
Published: August 2023

Abstract
Alopecia areata (AA) is a T cell mediated autoimmune disease characterized phenotypically by hair loss. NKG2D+CD8 +T cells is involved in the pathogenesis of AA. Sphingosine-1 phosphate (S1P) is a biologically active metabolite of cell membrane sphingolipids, which is essential for immune cell transport, and is known to be mainly involved in immune and inflammatory reactions. Due to involvement of S1P receptor (S1PR) in the immune system, S1PR modulator is being investigated for application in multiple sclerosis and other conditions. Among the five S1PRs, S1PRs 1&4 modulator (NXC736, Nextgen Bioscience, South Korea) was used to confirm its efficacy in the treatment of AA. AA lesion size measurement, disease-free ratio, immunohistochemistry and flow cytometry were used for analysis method. After oral administration of NXC736 for 84 days to AA mice, it was confirmed that the AA lesion area and disease-free ratio were significantly decreased compared to control group, and the infiltration of CD8+ T cells in the lesion skin was also significantly decreased. In addition, NKG2D level was decreased in effector CD8+ T cells. According to this result, we speculate that S1PR 1&4 modulator can be used as a promising treatment for AA.
Supported by Nextgen Bioscience
Anti-IL-6R prevents experimental type 1 diabetes
Authors: Luo Zhengkang , Hamze Nour , Berggren Elin , Chopra Saloni , Blixt Martin , Thorvaldson Lina , Espes Daniel , Carlsson Per-Ola , Sandler Stellan , Singh Kailash 
Published: August 2023

Abstract
Type 1 diabetes (T1D) is an autoimmune disease, where the insulin producing β cells are damaged by immune attacks leading to a failure of blood glucose control. The anti-inflammatory cytokine IL-35 has been shown to prevent and reverse T1D in mouse models. IL-6 signals through a homodimer of gp130 which is one of the receptors of IL-35. We thus hypothesized that blocking IL-6 signaling would facilitate IL-35 signaling and prevent T1D. We used multiple low dose streptozotocin (MLDSTZ) to induce T1D in male CD-1 mice and treated them with anti-IL-6R to study the immune response. We found that anti-IL-6R treatment prevented MLDSTZ mice from developing hyperglycemia, and preserved the ability of β cells to produce insulin. We then used flow cytometry to investigate the T cell and B cell response on day 13 after the first injection of MLDSTZ. The proportions of Treg cells were not altered by anti-IL-6R treatment in MLDSTZ treated mice, but the treatment elevated the proportions of IL-35 producing Treg cells in the spleen and thymus. It also decreased IFN-γ production in CD4, CD8, Treg and B cells in the spleen. Furthermore, anti-IL-6R treatment only tended to increase IL-17 production in CD4, CD8 and Treg cells. Altogether, our data demonstrate that anti-IL-6R treatment prevents T1D in MLDSTZ mice by maintaining the Treg cell phenotype and by reducing IFN-γ production by T and B cells.
Donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells
Authors: Graefen Bahar , Najafova Nurana , Alsiyaghy Jassam , Tauseef Mohammad , Fazal Nadeem 
Published: August 2023

Abstract
The Cholinergic Anti-inflammatory Pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Donepezil, a centrally acting reversible acetylcholinesterase inhibitor, has been used in the palliative treatment of Alzheimer’s disease (AD). We hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Endothelial permeability was evaluated by using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. Our data show that 25mM glucose concentration increased endothelial permeability and abrogated the wound healing process. Donepezil at the dose of 5 μM and 10 μM significantly prevented alteration of endothelial barrier function and enhances wound healing process in the presence of high glucose concentration.
Comparative genomics identified TLR-dependent fine tuning of the dendritic cell immune response by melatonin
Authors: Hira Sumit Kumar , Paladhi Ankush , Mondal Indrani , Das Purna Chandra , Manna Partha Pratim 
Published: August 2023

Abstract
Melatonin, a neuro hormone, has been shown to activate human monocytes and induce their cytotoxic properties in addition to the production of IL-1, IL-6, and TNF-α. The molecular mechanisms involved in melatonin and dendritic cell interactions are largely unknown. In this study, we performed a comprehensive RNA sequencing (RNA-seq)-based analysis of monocyte-derived human dendritic cells across different melatonin stimulations. We profiled gene expression changes among naïve DCs along with DCs generated with melatonin (MDCs) and DCs matured with melatonin (DCMs). We found that melatonin significantly promoted the morphological maturation of MDCs and increased the expression of DC-related surface markers. Furthermore, MDCs increased the general immune response by promoting the secretion of IL-12, IL-6 and IFN-γ cytokines and enhancing the proliferation of TH cells, while DCM did not. Genome-wide comparative transcriptomic profiling showed that the expression of 7417 genes was significantly upregulated, and 3325 genes were significantly downregulated in MDCs. We observed the spatiotemporal activity of TLR 2-, 4-, 6- and 8-specific genes in MDCs. Interestingly, we found that the expression of TLR 10-specific signaling genes was significantly higher in DCMs. Further in vitro validation showed that along with TLRs, the upregulation of IRAK, GRB2, CREB and TNFRSF was significantly associated with MDCs. Overall, we found that during the differentiation of dendritic cells, the presence of melatonin generates a unique population of dendritic cells with activated TLR4 signaling. DCs generated through this unique culture process may act as good candidates for immunomodulation during cancer and inflammation.
Supported by grants fromDST-SERB, GOI (CRG/2019/001296)
Paradoxical role of IL-10 during influenza A virus and MRSA coinfection
Authors: McKelvey Michael , Uddin Md Bashir , Palani Sunil , Shao Shengjun , Sun Keer 
Published: August 2023

Abstract
Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung injury characterized by an excessive and dysregulated cytokine response. Viral or bacterial pneumonia is the most common cause of ARDS, but the events that trigger hypercytokinemia are unclear. Thus, no ARDS mechanism-specific treatments exist. Secondary methicillin-resistant Staphylococcus aureus (MRSA) pneumonia has contributed significantly to morbidity and mortality in recent influenza pandemics. By using a mouse model of influenza A virus (IAV) and MRSA coinfection, we have shown that IAV infection inhibits phagocyte oxidative burst-dependent MRSA clearance but nevertheless lethal oxidative lung damage ensues following MRSA infection. Cytokines regulate the balance between immune protection and immunopathology. Accordingly, we have shown that an IFN-γ and TNF-α dominant cytokine storm promotes lethal lung inflammation following IAV/MRSA coinfection. IL-10 is a potent anti-inflammatory cytokine; it counteracts many biological effects of IFN-γ. Therefore, we hypothesize that IL-10 is required to limit lung immunopathology during IAV/MRSA coinfection. Indeed, we found that IL-10-deficient mice are highly susceptible to coinfection, correlating with their IFN-γ hyperproduction. Interestingly, mice lacking IL-10 receptor alpha in myeloid cells (Lyz2CreIl0rafl/fl) are also highly susceptible to coinfection, despite their improved bacterial clearance. This increased susceptibility is associated with hyperproduction of inflammatory cytokines. Ongoing studies will examine the impact of IL-10 on neutrophils and mononuclear phagocytes to elucidate their respective contribution to bacterial clearance and immunopathology.
R01 HL118408