bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the Immunogenic Mechanisms of Merck’s Lipid Nanoparticle (LNP) Adjuvants
Authors: Sullivan Nicole L , Secore Susan , Bass Antonia , Wolf Mary , Dangizer Andrew , Thambi Nithya , Bhattacharya Shashwati , Rodriguez Silveria , O’Donnell Gregory L , Bett Andrew L
Published: August 2023

Lipid nanoparticles (LNPs) have been shown to be a safe and potent platform for vaccine delivery. When added to subunit proteins, they have the potential to induce strong immune responses capable of generating long-lasting immunity. Merck has developed a variety of LNPs utilizing different cationic lipid components including LNP A, which has a long half-life in vivo, and LNP B, which has a shorter half-life in vivo and is more biodegradable. Protein subunit vaccines containing LNP A have been shown to generate robust antigen-specific humoral and cellular responses with some dose dependent reactogenicity while vaccines formulated with LNP B elicit lower immunogenicity and increased tolerability. To gain insight into the innate signaling pathways utilized by these LNPs, we performed an immunogenicity study in MyD88 knockout mice utilizing RSV F subunit protein vaccine plus LNP A or B. Wild type mice immunized with LNP A vaccines produced significantly higher levels of pro-inflammatory and neutrophil recruitment serum cytokines than the MyD88 knockout mice. However, with LNP B, MyD88 deficiency had no effect on cytokine expression. Humoral and cellular immune readouts are ongoing. Next, we stimulated whole human blood with the LNPs to compare at cytokine production. Stimulation with LNP A produced cytokine significant upregulation of monocyte and neutrophil-specific cytokines at 24 hours while stimulation with LNP B had no effect on cytokine production. Finally, we are evaluating differential gene expression to gain insight into the cellular responses of leukocytes to stimulation with the LNPs. Together, these experiments will help mechanistically determine how these fundamentally different LNPs can activate immune responses.
Modulating IL-1β and its receptors shapes spike-specific CD4+ T cell responses to mRNA vaccination
Authors: Park Hong-Jai , Shin Min Sun , Shin Junghee J , Kim Hyoungsu , Kang Byunghyun , Par-Young Jennefer , Unlu Serhan , Afinogenova Yuliya , Catanzaro Jason , Young Juan , Kim Minhyung , Lee Sang Jin , You Sungyong , Racke Michael K , Bucala Richard , Kang Insoo
Published: August 2023

Augmenting and sustaining a durable immunity provided by COVID-19 mRNA vaccines is important. Thus, we characterized S protein-specific CD4+ T cells in healthy individuals who received COVID-19 mRNA vaccines utilizing single cell RNA-seq and mass cytometry. In humans, S protein-specific CD4+ T cells highly expressed IL-1 receptor (R) 1 and its decoy receptor IL-1R2. IL-1b promoted IFN-g expression by S protein-stimulated CD4+ T cells, which was furthered by adding anti-IL-1R2-blocking antibodies, supporting the functional implications of IL-1R1 and IL-1R2. Following the 2nd dose of vaccination, IL-1R1 expression increased while IL-1R2 expression decreased in S protein-specific CD4+ T cells. The expression levels of such cytokine receptors correlated with S-specific antibody production. Our findings provide novel insight into the role of IL-1β and its receptor system in developing COVID-19 mRNA vaccine-induced CD4+ T cell immunity.

Supported by grants from NIH (1R01AG056728 and R01AG055362) and Quest Diagnostics.
Serological assessment of the durability of vaccine-mediated protection against SARS-CoV-2 infection
Authors: Bates John T , Lirette Seth T , Farmer Andrew P , Bierdeman Michael A , Seyfarth Kristina B , Montgomery Denise D , Burnett Grace C , Ederer Dallas R , Marshall Gailen D
Published: August 2023

Virus-specific neutralizing antibodies are generally accepted as a correlate of protection against infection, though many questions remain about which components of the immune response protect against SARS-CoV-2 infection. We longitudinally measured Spike receptor binding domain (RBD)-specific serum IgG and nucleocapsid protein (NP)-specific serum IgG in a human cohort immunized with the Pfizer BNT162b2 vaccine. Serum samples were collected and stored at −80 °C. Antibody titers were quantitated using an endpoint dilution ELISA and reported as inverse titers. A four-fold or greater increase in NP-specific antibody titers was used as serological marker of clinical infection at some point prior to the sample collection. Using the RBD-specific IgG titer measured in participants’ serum collected prior to seroconversion for NP, we were able to calculate a protective threshold titer for RBD-specific IgG. We calculated the rate of decay of antibodies in participants who did not exhibit a four-fold increase in NP-specific IgG, i.e. had no serological evidence of infection, and found that, on average, the RBD-specific IgG response waned below the protective threshold by four months following vaccination. These data highlight the need for a more durable immune response to vaccination which may require different adjuvants, dosages, or vaccination schedules.

This work was supported by The Robert M. Hearin Foundation, The Bower Foundation, and James L. Barksdale.
Heterozygosity for Crohn’s Disease Risk Allele of Atg16L1 Protects from Salmonella Infection
Authors: Yao Xiaomin , Rudensky Eugene , Martin Patricia , Torres Victor , Cadwell Ken
Published: August 2023

Autophagy is an evolutionarily conserved pathway for the formation and trafficking of lipid vesicles in response to stress, including inflammation and microbial infection. A missense variant of the essential autophagy gene Atg16L1 (rs2241880, T300A) is carried by a majority of the human population and is linked to Crohn’s Disease (CD). Previous studies found that T300A enhances proteolytic cleavage and degradation of Atg16L1, leading to dysregulated cytokine expression and impaired control of microbial infection in knock-in mice. However, these studies have focused on homozygous animals while the plurality of the human population is heterozygous for the allele. Here we demonstrate that heterozygosity for T300A protects mice from Salmonella infection. We find the variant allele results in altered complex formation independent of cleavage, allowing for enhanced inflammasome activation but impaired conjugation of Atg8 to single membranes (CASM). In vivo, one copy of T300A is sufficient to elevate the production of inflammatory cytokines, which is required for protection from Salmonella infection. Meanwhile, mice deficient in CASM genes are susceptible to Salmonella infection and this pathway is shown to be impaired in homozygous but not heterozygous mice. Finally, we demonstrate that heterozygosity in human macrophages also results in elevated cytokine expression, while maintaining functional non-canonical autophagy. These results clarify the altered autophagy function of the CD risk allele Atg16L1T300A and suggest it can be beneficial to heterozygous carriers, providing a potential explanation for its prevalence in the human population.

NIH R01 AI121244 F31 HL149238
SPATIOTEMPORAL REGULATION OF RP105 SUBCELLULAR LOCALIZATION SHAPES ENDOSOMAL TLR4 SIGNALING
Authors: Mathmann Carmen , Schultz Thomas , Ongtengco Cherica Felize J , Dalton Emma , Domínguez Cadena Leslie C , Zamoshnikova Alina , Stow Jennifer L , Blumenthal Antje
Published: August 2023

Innate immune sensing of microbial and endogenous ligands by Toll-like receptor (TLR) 4 drives inflammation and antimicrobial mechanisms. While critical for host defense against pathogens, unchecked TLR4 activation causes organ damage and chronic disease. Molecular mechanisms that drive versus turn off TLR4 activity are being pursued as potential targets for preventing and treating infections and inflammation-driven pathologies. Mechanisms of the temporal and spatial regulation of TLR4 signaling originating from the cell surface are well established. In contrast, the regulation of TLR4 activation originating from endosomal compartments is not well known. Here we show that the TLR-related receptor, Radioprotective 105 kDa (RP105), localizes to TLR4-containing endosomal compartments in murine macrophages. RP105 has been proposed as a negative regulator of TLR4-MyD88 signaling originating from the cell surface, but whether it modulates endosomal TLR4 signaling is unknown. Our data shows that RP105-deficiency reduces LPS- and E. coli-induced type I IFN responses, suggesting that RP105 facilitates endosomal TLR4-TRIF signaling. We employed high-resolution microscopy and AI-guided quantitative analyses to interrogate the spatiotemporal relationship between RP105 and endosomal compartments. Cellular activation led to a transient reduction of RP105 from endosomal compartments. Our results suggest TLR4 activation via TRIF signaling mediates this process, but not MyD88. Moreover, our data identify phosphatidylinositol 3-kinase p110-delta and Bruton’s tyrosine kinase as critical regulators of RP105 endosomal localization. Collectively, our data identify RP105 as a positive regulator of endosomal TLR4 signaling.

AAI Careers in Immunology Fellowship, 2022–2023
Mkp-1 deficiency strengthens immune defense against fungal infection
Authors: Liu Yusen , Mager Carli E , Shelton Evan D , Crowell Sara A , Tang Juan , Kim Victoria Y , Nelin Leif D , Zhang Jian
Published: August 2023

Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1−/− mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1−/− mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1−/− mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1−/− bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1−/− BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens.

*The studies were supported by NIH grants (AI113930 and AI124029 to Y.L., and AI121196 and AI123253 to J.Z.).
Aromatic amino acid metabolites influence interferon signaling and Influenza infectivity
Authors: Anand Gautam , Clark-Dinovo Colin , Perry Alexandra M , St. Raymond Emma , Sakleshpur Sonia , Steed Ashley L
Published: August 2023

The gut microbiota extensively metabolizes the three aromatic amino acids. Previously we found that a tyrosine metabolite can alter type I interferon (IFN) signaling. Herein we screened 17 metabolites of these amino acids for effects on IFN signaling alone and with LPS, IFN-β, and Influenza utilizing IFN reporter human lung epithelial cells (A549) and monocytes (THP1). While the tryptophan metabolites reduced IFN signaling in both cell types, tyrosine and phenylalanine metabolites had varied effects, depending on cell type. The cells were also treated with pooled metabolites from each family and a composite treatment with all three family metabolites. The composite treatment reduced IFN signaling in both cell types, suggesting a tryptophan metabolite effect dominance. Strikingly, when pretreated with composite treatment and infected with influenza, cells had reduced viral recovery and IFN signaling in both cell types. RNA sequencing validated earlier observation of reduced viral loads and IFN signaling. Gene silencing utilizing single siRNAs for upregulated genes identified (EGR2, ATP6V0D2, SPOCK1, and IL31RA) did not completely abrogate the repressed IFN signaling. However, these targets demonstrated linkage to TGF-beta signaling, and treatment with a TGF-beta inhibitor and gene silencing of all four gene targets led to a significant reversal of IFN signaling suppression. Our in vivo work demonstrated that intranasal administration of these metabolites prior to influenza infection leads to reduced animal weight loss, viral titers, and histological inflammation. Our work implicates that microbial metabolites alter IFN signaling through TGF-beta signaling and can promote beneficial outcomes during influenza infection.

The study was supported by grant from NIH (K08 AI135097-02) and Burroughs Wellcome Fund, awarded to Dr. Ashley L. Steed
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

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

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

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

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

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

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

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

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

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

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

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

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
UBL5 Modulates Expression of Crucial TLR Pathway Genes and Inflammasome Activation in Human Macrophage Cells
Authors: John Sinu P , Jean-Pierre Makheni , Katz Samuel , Fraser Iain D C
Published: August 2023

We conducted a genome-wide siRNA screen for regulators of LPS-driven TNF expression in macrophages. We used a splicing-independent reporter for the screen, and while we identified very few core spliceosome components among the strongest screen hits, we observed a clear enrichment for peripheral splicing regulators. Among these, a novel putative splicing regulator, UBL5, gave a strong screen phenotype, and a UBL5 shRNA-expressing cell line showed a selective defect in LPS-driven gene expression. While many genes were induced normally by LPS in the UBL5 deficient cells, a subset of genes, including core components of the LPS-activated TLR4 signaling pathway, were substantially diminished in UBL5-deficient cells. We conducted both Illumina and Pacbio long-read sequencing, and we observed marked defects in the LPS-induced splicing of numerous genes, however the primary impact on known components of the TLR4 pathway was observed at the level of overall gene expression rather than alternative splicing. Our data suggests that the putative splicing regulator UBL5 also has key roles in the maintenance and induction of gene expression for several core components of the TLR4 signaling pathway. Key role of UBL5 in the induction of TLR4 signaling pathway was further evidenced by the defective NLRP3 inflammasome activation and protection of OC43 coronavirus infection in UBL5-deficient cells.

This work was supported by the Intramural Research Program of NIAID, NIH

Intramural Research Program of NIAID, NIH
Characterization of Calcium signals and oscillations in dendritic cells in vivo in response to pathogens
Authors: Groot N , Sathuvalli Pranav , Barbet Gaëtan
Published: August 2023

Dendritic cells (DCs) are disseminated throughout organisms and serve as sentinels, organizing immune responses to infection, tissue damage, and cancer. DCs play a crucial role bridging innate and adaptive immune systems and elicit a tailored adaptive immune response to the types of threats they encounter. Manipulation of DCs is critical for vaccination, and DC-based immunotherapies have been tested in clinical trials as anti-cancer therapeutics and for treatment of infectious diseases. Calcium, a ubiquitous secondary messenger, regulates many critical effector functions of DCs like phagocytosis, maturation, migration, and cytokine production. However, regulation and mobilization of calcium in DCs remains poorly understood. Using microscopy, our preliminary experiments unveiled for the first time an oscillatory calcium signal in DCs in response to whole bacteria; our in vitro studies show that oscillatory calcium signaling patterns are specific to the type of bacteria. To decipher calcium signals induced by bacterial stimuli in DCs intravitally, we generated CD11c-Salsa6f reporter mice that express the fluorescent protein (FP) construct tdTomato fused to GCaMP6f in CD11c expressing cells. Hence, expression of TdTomato, a red FP, highlights cell shape, and GCaMP6f, which fluoresces green upon calcium binding, visualizes intracellular calcium levels. We are using CD11c-Salsa6f mice to observe calcium signals in DCs in the periphery and in secondary lymphoid organs using two photon microscopy. This model, perfectly suited for intravital two photon microscopy, allows us to unveil bacteria-specific calcium signals in DCs in in their natural microenvironment.

Supported by a grant from the NIH (1T32GM139804-01)
Pharmacological activation of STING agonists blocks schistosome egg-mediated pro-inflammatory cytokine production
Authors: Liu Pengyu , Jump Kaile , Kalantari Parisa
Published: August 2023

Infection with the helminth parasite Schistosoma mansoni causes morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens. Here we demonstrated that low-pathology BL/6 mice lacking Stimulator of Interferon genes (STING) exhibited markedly enhanced hepatic granulomatous inflammation associated with significantly increased Th17 and diminished Th2 cytokine responses. Additionally, CBA mice tend to have a severe form of liver granulomatous inflammation due to a profound decrease of type I Interferon production. Since CBA bone marrow-derived dendritic cells have lower expression of STING, we hypothesized that STING agonists such as diABZI-3 and DMXAA would control the pro-inflammatory cytokine production and inflammation by activating STING expression and activation. Both diABZI-3 and DMXAA induced robust Interferon (IFN)β production, while IL-1β and IL-17 induced by schistosome eggs were impaired in egg-stimulated BMDCs. Our study suggest that STING agonists may serve as a novel therapeutic strategy to restrain schistosome immunopathology. This work is supported by NIAID grant R01 AI148656 to PK.

This work is supported by NIAID grant R01 AI148656 to PK.
Distinct regulation of CD4+ T-cell function in cancer and autoimmunity by canonical NF-kappaB subunits
Authors: Lautraite Raphaelle , Lalle Guilhem , Klein Ulf , Ghosh Sankar , Grinberg-Bleyer Yenkel
Published: August 2023

Whereas CD4+ effector T cells (Teff cells) are critical to limit tumor progression, uncontrolled Teff cell activation can also lead to autoimmunity; however the molecular basis of these functions are elusive. Here we investigated the selective functions of distinct NF-kappaB transcription factors in the function of Teff cells.

Using mice carrying conditional ablation of the canonical NF-kB subunits RelA and c-Rel, in Teff cells, we found that RelA, rather than c-Rel, shaped the transcriptome of Teff cells in the steady-state and their ability to polarize toward the TH17 lineage, by directly binding to genes encoding cytokines and other transcription factors. Similar conclusions could be reached using CRISPR/Cas9-edited primary human Teff cells. Consistently, RelA – but not c-Rel-deficient mice were fully protected against neuro-inflammation in a murine model of multiple sclerosis. Mechanistically, TH17 priming and in situ reactivation, were impaired in RelA-deficient mice. Next, we investigated the roles of NF-kB in cancer immunity. Strikingly, Teff-restricted ablation of c-Rel, but not RelA, impaired their accumulation and function in the microenvironment of melanoma, resulting in enhanced cancer burden. In addition, c-Rel-deficiency abrogated the therapeutic effect of PD-1-blockade. Accordingly, in melanoma patients, the expression of c-Rel-dependent genes was associated with prolonged survival and better response to immunotherapy.

Together, our data highlight novel, context-dependent, regulators of Teff cell function. We propose a division of labor between the different subunits of the NF-kB pathway, paving the way to subunit-targeted immunotherapies in autoimmunity and cancer.
LncRNA U90926 is induced in activated macrophages, is protective in endotoxic shock, and encodes a novel secreted protein
Authors: Sabikunnahar Bristy , Varnum Stella , Caldwell Sydney , Hogan Tyler , Cousens Phoebe , Ballif Bryan , Krementsov Dimitry
Published: August 2023

The most commonly transcribed non-coding RNA class is long non-coding RNAs (lncRNAs) in mammalian genomes, yet the majority of them remain understudied. In our studies, we have identified a mouse lncRNA of unknown function named U90926, whose expression is greatly induced upon TLR-stimulation in macrophages in a Myd88- and p38 MAPK-dependent manner. To characterize this gene, we have generated a global U90926-knockout (U9-KO) mice in C57BL/6J background using CRISPR-Cas9. Surprisingly, we found minimal effects of U90926 deficiency on gene expression and basic immune effector function in cultured LPS-stimulated macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol and associates with ribosomes. Using an epitope-tagged construct, we found that U90926 contains a functional open reading frame that encodes a novel glycosylated protein (U9-ORF), which is secreted from the cell. We have confirmed the identity of U9-ORF protein by mass spectrophotometry. We generated a U9-ORF-specific monoclonal antibody which detected the endogenous U9-ORF protein in LPS-stimulated macrophages. In further characterization, we found that U9-KO mice exhibited increased sickness responses and mortality in an in vivo model of LPS-endotoxemia. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that inflammation induced expression of U90926 decreases endotoxic shock susceptibility, potentially mediated by the novel U9-ORF protein secreted by activated macrophages.

NIH NIAID (R21AI151116)
LKB1 regulates macrophage metabolism and functional polarization in immunomodulation
Authors: Yang Jing , Xu Chengxian , Lechner Joseph , Walls Haley , Yang Kai
Published: August 2023

Macrophages play critical roles in maintaining tissue homeostasis and modulating immune responses. In response to microenvironmental cytokines, macrophages coordinate cellular signaling networks and diverse transcriptional programs to dictate their phenotypical and functional heterogeneity. For instance, LPS/IFNγ and IL-4 induce classically (M1) and alternatively (M2) activated macrophages, respectively. Remodeling cellular metabolism has been highlighted a key process underlying macrophage functional polarization. However, the precise mechanisms coordinating macrophage metabolism and polarization remain elusive. We report here that the kinase LKB1, a well-known negative regulator of mTOR signaling pathway, serves as a metabolic checkpoint that connects cellular metabolic reprogramming to functional polarization in macrophages. We found that genetic depletion of LKB1 in macrophages polarized their differentiation towards M2-like macrophages, characterized by enhanced expression of M2-associated markers, independently of mTOR and STAT6 signaling pathways. In contrast, loss of LKB1 had no substantial impact on M1-like macrophages. Moreover, LKB1-defienct macrophages orchestrated reprogramming of mitochondrial metabolism to facilitate M2 polarization and tumor immune evasion. Collectively, our studies establish a previously unappreciated role for LKB1 in connecting macrophage metabolism and functional polarization in modulating tumor immunity.

This work was partially supported by the Herman B Wells Center and Riley Children’s Foundation.