bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mincle and TNF signaling crosstalk enhances type 1 and innate immune responses to Orientia tsutsugamushi
Authors: Fisher James Robert , Liang Yuejin , Card Galen , Gonzales Casey A , Soong Lynn 
Published: January 2023

Abstract
O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus, a life-threatening neglected disease endemic to the South-Pacific. The lungs harbor the greatest burden of infection and display strong type 1 proinflammatory responses. Here, we investigated whether the C-type lectin receptor Mincle contributes to immune recognition and inflammation. Following lethal infection in C57BL/6 mice, we performed pulmonary differential gene expression analysis via NanoString. We found 312 significantly induced genes (adj. p &lt; 0.05) at the terminal phase of disease, of which Mincle (Clec4e) was the 4th most upregulated gene. Mincle signaling partners (Fcgrs), Cxcr3, Ccr5, and their ligands, as well as Il27, were also highly upregulated. To define a role of Mincle in immune recognition, we exposed WT or Mincle−/− bone marrow-derived macrophages (MΦ) to live or inactivated bacteria and analyzed inflammatory markers via qRT-PCR. We observed infectious dose-dependent increases in Mincle transcription along with Cxcl1, Ccl2, Tnf, and Nos2 early in infection. Mincle−/− cells had abrogated responses of Cxcl1 and Ccl2. Blocking TNF signaling prior to infection markedly reduced Mincle, Ccl2, Cxcl1, Cxcl10, and Il27 transcripts. MΦ treated with rTNF-α prior to infection had greater levels of Mincle, Il27, Cxcl10, and Ccl2 expression than treatment with TNF or Orientia alone, suggesting a synergistic effect. Our study provides the first evidence for Mincle sensing O. tsutsugamushi and a role of Mincle-, TNFα-, and IL-27-related crosstalk during infection. Since there are no effective vaccines for scrub typhus, our results help understand innate immune recognition of this understudied bacterium and its disease.
Supported by grants from NIH (R01 AI132674 to LS, T32 AI007526-20 to LS, and R21 AI156536-01 to LS). JF was a recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.
Palmitic acid reprograms inflammatory responses to microbial ligands in macrophages, and mediates innate immune memory in vivo.
Authors: Seufert Amy Lorraine , Hickman James W , Traxler Ste K , Peterson Rachael M , Lashley Sydney J , Napier Ruth J , Shulzhenko Natalia , Napier Brooke A 
Published: January 2023

Abstract
Dietary saturated fatty acids (SFAs) modulate circulating lipids in mice and humans, which can alter the inflammatory capacity of innate immune cells. The most common SFA in human serum, palmitic acid (PA), is enriched in Western diets, and while it is known to induce an inflammatory response in macrophages, the impact of PA on responses to pathogenic stimuli is unknown. We developed an in vitro model to determine the impact of PA on the macrophage response to lipopolysaccharide (LPS). We find that bone marrow-derived macrophages (BMDMs) exposed to PA for 12–24 hours exhibit significantly enhanced TNF, IL-6 and IL-1β secretion following LPS exposure. BMDMs treated simultaneously with PA and the unsaturated fatty acid oleic acid (OA) show a reversal of this PA-induced hyper-inflammatory response to LPS, suggesting that OA negatively regulates the memory-like feature of PA. While monocytes and splenocytes from mice fed a PA-enriched diet exhibit unaltered basal inflammation, ex vivo LPS treatment leads to significantly enhanced pro-inflammatory cytokine expression. Mice exposed to PA for 9 days, followed by 7 days without PA, exhibit increased LPS-induced endotoxemia severity and mortality, and enhanced mRNA expression of tnf, il-6, and il-1β in blood. Rag−/− mice exposed to PA for 12-hours showed significantly reduced kidney fungal burden after Candida albicans infection. We hypothesize that PA induces a long-lasting innate immune memory program in macrophages, and reprograms their response to subsequent inflammatory challenge via metabolic and epigenetic alterations. Our findings are the first to suggest dietary PA regulates innate immune memory and is detrimental in an acute septic response, yet beneficial for pathogen clearance.
Supported by National Institute of General Medical Sciences (NIGMS) 525 grant 5R35GM133804-02 to B.A.N.
NADase activity found in bacterial TIR proteins may aid in innate immune evasion
Authors: Palm Cheyenne , Koudjra Rachida , Obiorah Trinity , Snyder Greg A , Snyder Michelle , Harberts Erin 
Published: January 2023

Abstract
Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with a mutation known to inhibit NADase activity. We are now using TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing RT-PCR to analyze the effects of these mutant TIRs on innate cell activation. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.
Supported by grant from NIH (R25 GM119970 04)
TGF-β and CIS inhibition synergistically enhance natural killer cell-based immunity
Authors: Souza-Fonseca-Guimaraes Fernando 
Published: January 2023

Abstract
Immune “checkpoint” inhibitor antibodies have revolutionized cancer therapy and function by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8 T cells. Recent studies have also shown similar programs in other effector lymphocyte populations such as natural killer (NK) cells. NK cells are key to cancer immunosurveillance, particularly in settings of metastasis. They are dependent on the cytokine IL-15 for their survival, development, and function, with ablation of the IL-15 signaling inhibitor CIS (Cish), shown to enhance NK cell anti-tumor immunity by increasing NK cell fitness in the tumor microenvironment (TME). The TME has also been shown to impair NK cell fitness via the production of immunosuppressive transforming growth factor (TGF)-β, even in the presence of high IL-15 signaling. Here, taking advantage of sophisticated proteomics and transcriptomics methods with computational biology analysis we identified an unexpected interaction between CIS and the TGF-β signaling pathway in NK cells. Independently, for the first time a Cish- and Tgfbr2-double-deficient NK cells were generated by our group, which displayed both hyper-responsiveness to IL-15 and hypo-responsiveness to TGF-β, with dramatically enhanced immunity against multiple mouse models of cancer. Remarkable, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development and this resistance is dependent on NK cell hyper-activity. This study contributes to the understanding on how targeting multiple suppressive pathways unveils a new immunotherapy approach that might have broad therapeutic interventions, particularly by enhancing NK cell effector functions during disease.
This work is supported by project grants from the National Health and Medical Research Council (NHMRC) of Australia (#1140406), the US Department of Defense – Breast Cancer Research Program – Breakthrough Award Level 1 (#BC200025), the ANZSA SRG Grant, and was supported by a grant (#1158085) awarded through the Priority-driven Collaborative Cancer Research Scheme and co-funded by Cancer Australia and Cure Cancer.
BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow
Authors: Sun Sarah J , Dumaine Anne , de Bree L Charlotte J , Netea Mihai G , Barreiro Luis B 
Published: January 2023

Abstract
The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.
Comparison of DNA sensing by non-immune cells and tissues
Authors: Heller Loree , Znida Katarina , Bosnjak Masa , Conniff Amanda Sales , Jesenko Tanja , Markelc Bostjan , Tur Jared , Semenova Nina , Cemazar Maja 
Published: January 2023

Abstract
Pattern recognition receptors (PRRs) are nearly ubiquitous in both immune and non-immune mammalian cells. PRRs detect pathogen invasion as well as internal damage, inducing the secretion of pro-inflammatory molecules and cell death. Members of the PRR subset that detect intracellular DNA are termed DNA sensors. We examined DNA sensing in non-immune cells and their equivalent tissues using electroporation. This physical delivery method mitigates the need for viral or chemical agents that could confound the source of PRR activation. We used RT-qPCR and/or RNA-Seq to quantify mRNAs encoding DNA sensors and their signaling pathways and western blots, ELISAs and bead arrays to quantify related proteins. Several tumor cell types and non-tumor cells, including keratinocytes, fibroblasts, and myoblasts, expressed different DNA sensors at baseline and responded to backbone plasmid DNA delivery with individual DNA sensor signatures. Some similarities were observed among all these cell types, including upregulation of the DNA sensors ZBP1, DDX60, and Ifi204, the mouse analog of human Ifi16. Each cell type secreted a range of pro-inflammatory molecules including Type I interferon. DNA electroporation to B16-F10 melanomas, skin, and skeletal muscle induced unique cytokine and chemokine signatures, paralleling cellular signatures. ZBP1, DDX60, and Ifi204 were upregulated in skin and muscle; however, no significant regulation was observed in tumors. In muscle particularly, this may be due to the homogeneity of the tissue; tumors are inherently heterogeneous, producing an assortment of individual cellular responses.
Supported by NIH grant R01CA196796.
Proteomics analysis to identify intracellular inflammatory pathways modulated by Fh15 in macrophages stimulated with LPS-E. coli
Authors: Armina-Rodriguez Albersy , Valdés-Fernández Bianca , Roche-Lima Abiel , Carrasquillo-Carrión Kelvin , Espino Ana M 
Published: January 2023

Abstract
Previous studies of our laboratory have demonstrated that Fh15, the recombinant variant of F. hepatica FABP exhibit powerful anti-inflammatory properties. The administration of a single IV dose (for NHP) or IP dose (for mouse) of Fh15 prior to or after lethal doses of E. coli-LPS, respectively is enough to significantly suppress the pro-inflammatory cytokine storm and prevent the lethal pathologic consequences of septic shock. This suggest that Fh15 has a broad spectrum of action and may modulate pro-inflammatory mechanisms. The present study aimed to identify intracellular inflammatory pathways that could be modulated by Fh15. To achieve this goal, we have applied a quantitative proteomic approach using a Tandem Mass Tag peptide labelling, which has been used to quantified and identified biological macromolecules such as proteins. Samples were analyzed using Liquid Chromatography with tandem mass spectrometry. RAW 264.7 cells were stimulated with LPS and/or Fh15, cells treated with Fh15 or LPS alone were used as control. For the bioinformatics analysis, a fold change of 1.5 and pValue ≤ 0.05, were considered for the enrichment analysis, using the Ingenuity Pathway Analysis. Using these approaches, we identified 257 proteins associated with LPS treatment: 173 were up regulated and 84 were down regulated. Furthermore, we identified 139 proteins associated with Fh15 treatment: 38 were up-regulated and 101 were down-regulated. The IPA analysis identified proteins associated with NFkB, iNOS and Th1/Th2 pathways. We also identified 8 proteins differentially abundant that are associated with inflammatory and/or infectious diseases. Currently we are evaluating these proteins with ELISA, Western Blot and/or Flow cytometry techniques.
Supported by 1SC1AI155439-01 NIAID
Adenosine deaminase-1 (ADA-1) induces maturation and skewing of cytokine and chemokine production in dendritic cells to improve adaptive immune responses.
Authors: Cusimano Gina , Chakhtoura Marita , Taramangalam Bhavani , Connors Jennifer , Kutzler Michele A , Haddad Elias 
Published: January 2023

Abstract
Dendritic cells (DCs) are critical mediators of antigen-specific immunity through their ability to present antigen and drive T cell differentiation into different effector subsets such as follicular helper T cells (TFH). TFH cells provide both physical and cytokine-mediated stimuli to B cells resulting in somatic hypermutation and class-switching of B cell receptors. Molecules that target TFH cells, such as adenosine deaminase-1 (ADA-1), would improve humoral immunity. We have previously demonstrated that co-delivery of plasmid-encoded adenosine deaminase with an HIV-1 envelope and SARS-CoV-2 DNA vaccines in vivo, enhanced both humoral and cellular responses. However, the mechanism by which ADA-1 is acting as an adjuvant remains to be elucidated. To this end, we treated monocyte-derived DCs from healthy human donors in vitro with recombinant ADA-1 protein and evaluated the expression of maturation markers, cytokines and chemokines. ADA-1-treated DCs had a significantly increased expression of CD40 and CD86 as well as HLA-DR compared to their unstimulated, immature counterparts. The level of co-stimulatory marker and HLA-DR expression on ADA-1-treated DCs was similar to that on LPS/IFN-γ-treated DCs, indicating ADA-1-mediated DC maturation. ADA-1-treated DCs also exhibited a significant increase in IL-6, IL-1β and CXCL13 expression. IL-6 is a key pro-TFH cytokine and IL-1β and CXCL13 may play a role in TFH cell differentiation, function, and proliferation. Ongoing studies are aimed to drive ADA-1 overexpression in DCs and evaluate effects on antigen presentation. Overall, elucidating the mechanism of ADA-1’s adjuvanticity will allow for its progression as a clinical adjuvant.
This work was supported by funding to Dr. Elias Haddad from NIH 5RO1AI106482-01A and 1U19 AI128910-01.
dNTP catabolism is a macrophage-intrinsic gatekeeper preventing NLRP3 inflammasome hyperactivation
Authors: Zhong Zhenyu , Huo Shanshan , Liu Danhui , Wang Xiaochen , Liang Shuang 
Published: January 2023

Abstract
Aberrant activation of NLRP3 inflammasome drives the development of many diseases, including atherosclerosis, gout, osteoarthritis, Alzheimer’s disease, macular degeneration, type 2 diabetes and cancer. Our recent work demonstrated that new mitochondrial DNA (mtDNA) synthesis, initiated at the inflammasome priming step, is a prerequisite for producing oxidized mtDNA (ox-mtDNA)—an activating ligand of NLRP3. However, little is known regarding how innate immune cells, such as macrophages, manage to prevent NLRP3 inflammasome hyperactivation under physiological conditions, thereby avoiding the development of immunopathology. Here we show that SAMHD1, a cytosolic dNTP hydrolase, functions as a macrophage-intrinsic gatekeeper that restricts NLRP3 inflammasome hyperactivation. Mechanistically, inflammasome priming activates SAMHD1 to inhibit cytosolic dNTP buildup, thereby preventing their transport into mitochondria. This in turn protects mitochondria from uncontrolled new mtDNA synthesis and ox-mtDNA production, ultimately attenuating the extent of NLRP3 inflammasome activation. Consistent with these findings, SAMHD1-deficient mice exhibited NLRP3 inflammasome hyperactivation and developed exacerbated immunopathology in vivo relative to their wild-type littermates in response to various inflammatory insults. Together, these results establish cytosolic dNTP catabolism as a physiologically important protective mechanism to restrict NLRP3 hyperactivation, thereby linking dysregulation of nucleotide metabolism with the onset of NLRP3-dependent immunopathology.
Supported by NIH (K22AI135074, R35GM142654), CPRIT (#RR180014)
UTX is an epigenetic regulator of natural killer cell development and anti-viral effector function.
Authors: Cheng Mandy I , Riggan Luke , Ma Feiyang , Chin Scott , Tafti Rana , Pellegrini Matteo , O’Sullivan Timothy E , Su Maureen A 
Published: January 2023

Abstract
Natural killer (NK) cells are circulating type 1 innate lymphocytes that protect against viral infection and cancer. While it is now clear that NK cells display distinct epigenetic states during development and activation, the factors that control the epigenetic programming of NK cells during these processes are not well understood. Here, we show that the H3K27me3 histone demethylase UTX epigenetically regulates NK cells in a cell-intrinsic and demethylase-independent manner by regulating the chromatin accessibility of gene loci involved in homeostasis and effector function. As a consequence, mice with NK cell-specific UTX deficiency displayed an increase in peripheral immature NK cell cells that express higher levels of Bcl-2. Furthermore, UTX-deficient NK cells produce lower amounts of interferon (IFN)-γ and Granzyme B following MCMV infection, resulting in increased mortality. These findings reveal UTX as an essential regulator of NK cell homeostatic and effector epigenetic programs.
M.I.C. is supported by Ruth L. Kirschstein National Research Service Awards (GM007185 and AI007323), and Whitcome Fellowship from the Molecular Biology Institute at UCLA.
Several active components within MSCs secretome inhibit the IL-2-mediated effector functions of NK cells
Authors: Ko Eunhee , Lee Yoojin , Yoon Taejun , Kim Jongsun , Park Yong-Beom 
Published: January 2023

Abstract
Mesenchymal stem cells (MSCs) are well-known for their immunomodulatory functions in innate and adaptive immunity. Their functional diversity was verified in various disease model through paracrine mechanisms. MSCs-derived secretome consists of a variety of bioactive components such as nucleic acids, cytokines, chemokines, and small molecules. However, the effect of MSCs on natural killer (NK) cells activity is still controversial. In this study, we tried to identify active components within MSCs secretome possessing ability to affect NK cells activity and to investigate their action mechanisms. We found that MSCs secretome had no effect on survival and proliferation of NK-92 cells but specifically diminished cell activity, as evidenced by lower secretion of cytotoxic granules and effector cytokines and also reducing killing ability. This effect was reproduced in human peripheral NK cells equally. Moreover, seven candidates for modulating inflammation within MSCs secretome were identified via LC-MS/MS proteomic analysis. Interestingly, they are totally different factors from those known as MSC’s immunomodulatory factor. After treatment of neutralizing antibodies to each candidate, we observed that only three of the groups with neutralizing antibodies recovered cytokine secretion and cytotoxic ability of NK-92 cells. These findings suggest that MSCs secretome can inhibit the effector functions of NK cells via some particular molecules, which are increasing expression of inhibitory receptor CD96 on NK cells, consequently activating inhibitory signal.
This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2021R1F1A1049630).
Preclinical in vivo assessment of replacing linezolid for spectinamide-1599 in the Nix-TB regimen
Authors: Ali Malik Zohaib , Dutt Taru S , Walz Amanda , Pearce Camron , Lee Richard E , Robertson Gregory T , Hickey Anthony J , Meibohm Bernd , Gonzalez-Juarrero Mercedes 
Published: January 2023

Abstract
Tuberculosis (TB) patients diagnosed as drug-resistant TB have limited treatment options. Nix-TB trial is testing three-oral-drugs, namely Bedaquiline (B), Pretomanid (Pa) and Linezolid (L) (BPaL regimen) against drug-resistant TB. The BPaL regimen shows excellent favorable outcomes, but toxic effects are observed due to the long-term administration of Linezolid. Spectinamide-1599 (1599) is a potent protein inhibitor of Mycobacterium tuberculosis (Mtb) without known adverse effects and cross-resistance with other TB drugs. We hypothesized that inhaled 1599 could replace L in the BPaL regimen and provide similar or higher efficacy as BPaS regimen. Therefore, we tested BPaL and BPaS regimens in Balb/c (TB resistant) and C3HeB/FeJ (TB susceptible) mice infected with low-dose Mtb, and evaluated bacterial burden using colony-forming units (CFUs). Compared to untreated mice, CFUs in lungs of BPaL and BPaS treated Balb/c mice were decreased by &gt;1 and 5 logs after 2- and 4-weeks treatment, respectively. In C3HeB/FeJ mice, 4-weeks of BPaL and BPaS treatment decreased CFUs by &gt;3 logs compared to untreated control. However, no statistically significant differences were observed between treatment groups in both the mice strains. Cellular profiling using flow cytometry showed increased monocytes (CD11bhi CD14hi CCR2hi) and neutrophils (CD11b+ Ly6G+) in the bone marrow of BPaS mice compared to BPaL. These results along with cytokine profiling of the two treatment groups showed enhanced inflammatory response in BPaS mice than BPaL. We concluded that inhaled 1599 is a potential replacement for Linezolid if combined as BPaS regimen, and further studies are warranted.
This project was funded by National Institute of Health under the funding number NIH-NIAID-Allergy and Infect Diseases AI120670.