bluuebunny/crossref_metadata_2025_split huggingface dataset:
Macrophages and neutrophils in lymph node granulomas from Mycobacterium tuberculosis-infected macaques have immunoregulatory phenotypes
Authors: Mattila Joshua T , Simonson Andrew W , Krupinsky Kathryn C , Kirschner Denise E , Flynn JoAnne L 
Published: August 2023

Abstract
Tuberculosis (TB) is caused by infection with the Mycobacterium tuberculosis (Mtb). Lung granulomas are the defining pathologic feature of TB but Mtb can infect other organs, including lymph nodes, and causes granulomas to form in these sites. Lymph node granulomas have received less attention than lung granulomas but granulomas in these organs are sites of bacterial persistence and have implications for TB progression. Macrophage subsets and neutrophils in lymph node granulomas have not been extensively studied but the biology of these cells may yield clues that underlie the pathobiology of lymph node infection. Our objective was to describe expression of functional markers and cytokine expression by macrophage subsets and neutrophils in lymph node granulomas and to compare these myeloid cells in lymph node and lung granulomas. We used thoracic lymph node and lung granulomas from cynomolgus macaques, a nonhuman primate that experiences human-like TB pathology and course of disease, to do this work. We found that lymph node epithelioid macrophages were distinct from lymph node dendritic cells but resembled epithelioid macrophages in lung granulomas suggesting these cells share a common progenitor or exposure to common stimuli leads to a phenotypic convergence in different tissues. Macrophages expressed pro- and anti-inflammatory cytokines including type 1 interferon, a cytokine family associated with exacerbated TB. Neutrophils were present in low numbers in non-granulomatous lymph node regions but were selectively recruited to granulomas. Our data suggest that macrophages and neutrophils in lymph node granulomas have immunoregulatory properties that may contribute to local and systemic outcomes in Mtb infection.
This work is supported by NIH grants R21 AI167710, R01AI164970, and the Wellcome Trust DELTA tissue program.
Boosting NAD+ in myeloid cells upregulates Prostaglandin E2 (PGE2) and CC-chemokine receptor 7 (CCR7)-mediated migration via Sirt1-dependent transcriptional regulation
Authors: Wu Jing 
Published: August 2023

Abstract
NAD+-boosting via nicotinamide riboside (NR) confer anti-inflammatory effects. However, underlying mechanisms and therapeutic potential remain incompletely defined. Given NAD+ is a co-enzyme in oxidoreductive reactions, metabolomics analysis identified two major metabolites that were increased by NR: inosine and Prostaglandin E2 (PGE2). Both inosine and PGE2 supplementation blunted IFNβ release, however NR-mediated monocytic downregulation of IFNβ was dependent on inosine but independent of PGE2. Flow cytometric analysis of cell surface receptors in human M1 macrophage showed NR increased the expression of CC-chemokine receptor 7 (CCR7). Consequently, chemokine ligand 19 (CCL19, ligand for CCR7) - induced macrophage migration was enhanced in response to NR administration. Furthermore, NR-mediated upregulation of macrophage migration through CCL19-CCR7 axis was dependent on PGE2 synthesis. We also demonstrated that NR upregulates PGE2 synthesis through Sirt1-dependent transcriptional regulation of COX2. NR-mediated metabolic regulation appears to modulate disparate myeloid cell functions via distinct signaling intermediates and pathways.
NHLBI Division of Intramural Research
Chlamydia trachomatis infection renders M2 generation to mediated immunosuppression
Authors: Agrawal-Rajput Reena 
Published: August 2023

Abstract
Chlamydia trachomatis (C.tr) cause asymptomatic genital infections in women and is also a leading cause of preventable blindness. Limited mouse model of chronic C.tr infection are available to study the host immune response. We have developed BALB/c mice models of acute and chronic infections for C. trachomatis to explore the significance of macrophage-directed response in mediating immune activation/suppression. We observed that chronic and repeated infections accompanied abbrogated IFN-g and promoted enhanced TGF-β and IL-10 secretion. The signs of T cell exhaustion and anergy were also indicated during chronic and secondary infection. This skewed T cell response was established to be the regulated by alternatively-activated macrophages with low CD40 expression leading to sustained C. trachomatis genital infection.
Macrophages under the influence of C. trachomatis infection or its secretome demonstrate an M2-macrophage subset phenotype which responded poorly to IFN-g owing to decreased IFNg-RI and IFNg-RII expression which correlated with decreased expression/release of MHC-II, CD40, iNOS and NO even following IFN-g treatment. Alternatively-activated M2 macrophages with low CD40 expression harbored high bacterial load and rendered the generation of immunosuppressive Th2 and Treg cells. Thus, C. trachomatis modulate the innate immune cells attenuating the anti-chlamydial functions of T cells in a manner that involves alleviated CD40 expression on macrophages.
Dysregulated myeloid cell activity is associated with fatal disease in SARS-CoV-2 infected aged mice
Authors: Chanda Debarati , Ghimire Roshan , Channappanavar Rudragouda , Mack Matthias 
Published: August 2023

Abstract
Covid-19 caused by recently emerged SARS-CoV-2 is a significant threat to global public health. Although SARS-CoV-2 efficiently infects individuals of all age groups, the aged population exhibit poor clinical outcome as demonstrated by high rates of morbidity and mortality. However, the underlying mechanism driving severe pneumonia and lethal disease in the elderly is not well defined. SARS-CoV-2-induced excessive inflammation and impaired antiviral response (collectively referred to as ‘dysregulated immunity’) is believed to cause acute lung injury (ALI) and fatal pneumonia. In this study, using a novel mouse-adapted SARS-CoV-2, we evaluated disease severity in young and aged mice, and examined the mechanistic basis for age-related severe disease caused by SARS-COV-2. Similar to Covid-19, our results showed increased morbidity and mortality in the aged compared to young mice. Further examination of SARS-CoV-2 infected lungs from young and aged mice demonstrated high lung viral titers, dysregulated inflammatory cytokine expression, and downregulation of interferon (IFN) expression in aged mice compared to young cohorts. Our results also showed elevated inflammatory monocyte macrophage (IMM) and neutrophil accumulation in the aged lungs compared to young mice. To conclusively establish if robust myeloid cell activity causes severe disease, we depleted monocytes and neutrophils and found that combined depletion delayed mortality and alleviated disease severity in mice infected with a lethal dose of SARS-CoV-2. Collectively, our results establish that impaired innate antiviral response along with a robust accumulation of inflammatory myeloid cells cause severe disease in the aged hosts.
This study was supported in part by institutional research funds from Oklahoma State University—Stillwater and NIA 5P20GM103648-10 2R.C.
The extracellular tail of IFITM3 promotes SARS-CoV-2 entry into cells and is diversifying in primates
Authors: Compton Alex A , Li Tiansheng , Shi Guoli , Lai Kin Kui , Mak Nelly , Jolley Abigail , Speaks Samuel , Yount Jacob S , Yewdell Jonathan W 
Published: August 2023

Abstract
The interferon-induced transmembrane (IFITM) proteins confer cells with resistance to diverse virus infections by altering membrane biomechanics and disfavoring virus-cell membrane fusion. Human IFITM3 strongly restricts SARS-CoV and MERS Spike-mediated infections in endosomes. However, work from our labs demonstrated that IFITM3 has opposing effects on SARS-CoV-2 (SCV2) infection: IFITM3 in endosomes restricts SCV2 infection, but IFITM3 at the cell surface enhances SCV2 infection. In the present study, we investigated the determinants governing the enhancement of SCV2 infection by human IFITM3. Using clinical isolates of SCV2 and pseudoviruses bearing SCV2 Spike, we found that the penultimate and ultimate amino acid residues of IFITM3, which are predicted to extend into the extracellular space, are essential for enhancement of SCV2 Spike-mediated infection. Deletion of these residues in human IFITM3 resulted in loss of SCV2 infection enhancement. On the other hand, we found that certain non-human IFITM3 proteins enhanced SCV2 infection to a greater extent than human IFITM3, and this mapped to sequence divergence in the penultimate and ultimate residues of the IFITM3 extracellular tail. Transferring the last two amino acids of macaque IFITM3 to human IFITM3 resulted in further enhancement of SCV2 infection. These results indicate that certain non-human primate IFITM3 proteins may be poised to act as potent enhancers of SCV2 infection in those species. Genomic analyses revealed that the IFITM3 extracellular tail is diversifying rapidly and recurrently in primates over evolutionary time, raising the possibility that variation at these sites is adaptive in nature and allows for escape from co-option by coronaviruses.
NIH Intramural Research Program NIAID Intramural Targeted Anti-COVID19 Program Award
Intracellular complement proteins are tumor-promoting effectors and poor prognosis biomarkers in renal cancer
Authors: Zarantonello Alessandra , Revel Margot , Rybkine Tania Robe , Bourdin Antonin , Armento Angela , Fridman Catherine Sautés , Ueffing Marius , Fridman Wolf Hervé , Roumenina Lubka 
Published: August 2023

Abstract
Clear cell renal cell carcinoma (ccRCC) tumors highly express proteins of the complement system, a major innate immune defense mechanism. Exploring the spatial transcriptome and making an atlas of staining patterns by ultra-high multiplex imaging of ccRCC tumor sections, we revealed presence of regions with co-regulated overexpression of complement components, including C3 and Factor H (FH). The strong staining for these proteins correlated with poor prognosis in a ccRCC patient cohort. These proteins interacted physically in the lysosomes of ccRCC tumor cells in situ, as evidenced by proximity ligation assay. We discovered previously unrecognized intracellular functions of complement C3 and FH in renal tumor cells. C3 knockdown resulted in formation of more compact tumorospheres than control cells and hampered energy metabolism, while FH knockdown perturbed sphere-forming capacity due to decreased viability and proliferation and enhanced the Krebs cycle. For these functions the two proteins act together intracellularly, FH being a negative regulator of C3. C3 and FH also function independently of each other, regulating gene transcription. Exploring the FH interactome by mass spectrometry, we found that FH interacted with transcription factors to regulate the cell cycle and with proteins, controlling actin cytoskeleton remodeling, explaining altered cell morphology and reduced motility of the FH-deficient cells. In conclusion, we discovered that intracellular C3 and FH are multitasking effectors, impacting multiple hallmarks of cancer - enabling replicative immortality, stimulating proliferation, resisting cell death, and deregulating cellular metabolism, bearing potential as new therapeutic targets.
The main author was supported by DFF-1025-00015B Independent Research Fund Denmark (IRFD or DFF)
Effects of TcpC’s NADase Activity on Inhibition of Innate Immune Signaling
Authors: Erickson Britney C , Sumilang Angelique , Palm Cheyenne , Snyder Greg , Snyder Michelle 
Published: August 2023

Abstract
The innate immune system is activated by pattern recognition receptors such as Toll-like receptors (TLRs), which differentiate between self and non-self by recognizing distinct molecular structures on pathogens. As this process threatens pathogen survival in the host, bacteria have developed mechanisms to combat this. Uropathogenic Escherichia coli (UPEC) express the Toll/Interleukin-1 Receptor (TIR) domain-containing protein TcpC, which inhibits the function of TLRs by binding to the signaling adapter protein myeloid differentiation factor 88 (MyD88). Bacterial TIR proteins, including TcpC, recently have been found to also exhibit NADase activity. We hypothesize that the ability of TcpC to block TLR signaling and its NADase function are interrelated and that the TcpC EA mutant, which has been shown to have diminished NADase activity will not bind efficiently to MyD88. We performed in vitro translation assays to first produce both WT and EA mutant versions of TcpC that contain both a streptavidin and histidine tag. Then we conducted pull-down experiments to determine if the TcpC EA mutant could successfully bind to streptavidin beads, as well as MyD88, potentially resulting in clustering with the TIR signaling protein and blocking the signaling pathway entirely. While we have successfully produced tagged WT and EA TcpC proteins and pulled down the proteins using streptavidin beads, we are working to optimize conditions to determine if the NADase-deficient TcpC EA mutant is able to bind MyD88, as has been shown previously for WT TcpC. The resulting information will give us a closer look into TcpC’s effect on virulence and the suppression of the innate immune response via TLR and MyD88 related signaling pathways.
This research was partially funded by the USM LSAMP program, supported by NSF LSAMP Award #1619676, the Towson OSPR Seed Funding for Grant Seekers, and the Bridges to Doctorate Scholars Program, NIH-R25GM119970 04.
Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K+-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells
Authors: Dubyak George R , Horan Katherine , Miller Brandon A 
Published: August 2023

Abstract
NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K+] being the best-characterized signal. The required K+ efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K+ efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/− BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates.
Supported by grants from the NIH (P01-AI141350 and R01-EY014362)
Cell painting assays for profiling responses in Mycobacterium tuberculosis-infected macrophages after metabolic perturbation
Authors: Lane Daniel Scot , Mattila Joshua T 
Published: August 2023

Abstract
Tuberculosis (TB) is the leading infectious disease killer in the world, and a lack of accessible, easy treatments contributes to its growing burden. Repurposing approved treatments for other conditions, including metabolic inhibitors under consideration for use in cancer therapy, as host-directed therapies may improve TB treatments. Detailed preliminary studies to identify mechanisms that underly drug function need to be performed before these drugs can be used in patients but there is a lack of tools to investigate metabolic perturbations in target cells. This problem is particularly acute for treatments that have uncharacterized effects on host cells or host-pathogen interactions, or for adherent cells where cell morphology and migration patterns may be factors that are modulated by treatments. Cell painting assays are microscopy-based analyses that use quantitative imaging data to generate insight into how drugs and infections perturb host cells. Here, we sought to optimize a cell painting assay to investigate changes in macrophage morphology and antimicrobial activity after metabolic perturbation and Mycobacterium tuberculosis (Mtb) infection. Our objective was to apply the quantification and analysis pipelines to macrophages that were infected with fluorescent Mtb reporter strains to identify cellular responses after inhibition of fatty acid oxidation, oxidative phosphorylation, or glycolysis. Moreover, we compared inhibitors that target different points in the glycolysis pathway to define how these points differentially affect macrophage antimicrobial activity. Our work provides a framework for using cell painting assays as tools for dissecting host-pathogen interactions in metabolically perturbed cells.
Supported by grants from NIH grants AI167710 and #LINK#AI164970#ENDLINK#
Beta-hydroxybutyrate regulation of metabolic stress responses to Staphylococcus aureus infection in macrophages
Authors: Kim Min Joo , Faneuff Eden , Serezani C Henrique 
Published: August 2023

Abstract
Macrophages orchestrate the initiation, intensity, and duration of the inflammatory response induced during Staphylococcus aureus infection. The pleiotropic role of macrophages is dictated by the capacity to adapt to different metabolic cues. During acute inflammation, macrophages are highly glycolytic; during resolution, these cells shift to oxidative metabolism. While glycolysis drives the expression of pro-inflammatory genes, oxidative phosphorylation leads to fatty acid oxidation and synthesis of ketone bodies, such as beta-hydroxybutyrate (BHB). Therefore, identifying the events that drive the shift from glycolytic to oxidative phosphorylation could provide clues for inflammation and homeostasis. If exogenous BHB influences macrophage responses to S. aureus infection is not well understood. We hypothesize that BHB is a bridge between glycolysis and oxidative phosphorylation during S. aureus infection. BHB increases mitochondrial respiration in S. aureus-infected immortalized bone-marrow-derived macrophages (iBMDM). During infection, treatment with BHB increased PDHA1 (Pyruvate Dehydrogenase E1 Subunit Alpha 1) but decreased HK1 (Hexokinase 1) expression in iBMDMs. Furthermore, BHB decreased macrophage phagocytic capacity in S. aureus infected peritoneal macrophages, indicating a decrease in glycolytic activity. Cumulatively, these findings suggest that BHB may cause a shift toward aerobic respiration and increase the anti-inflammatory capacity of macrophages. Therefore, exogenous BHB or ketogenic diets could be detrimental during S. aureus infection by decreasing both antimicrobial effector functions and inflammatory responses.
NIH R01 HL124159-09 The SyBBURE Searle Undergraduate Research Program
Sphingosine 1 phosphate production is an important mediator of skin inflammation and host defense in hyperglycemic mice
Authors: Salina Ana , Penteado Leticia Aquino , Blackman Amondrea , Serezani C Henrique 
Published: August 2023

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) skin infections are characterized by the initial recruitment of neutrophils followed by the establishment of a localized abscess. An abscess comprises a fibrous capsule, viable and apoptotic neutrophils, and bacteria in the abscess core. Along with eliminating bacteria, the clearance of dead neutrophils (efferocytosis) is crucial to restoring tissue homeostasis. Efferocytosis is a tightly regulated process initiated when dying cells secrete “find-me” signals such as Sphingosine 1 phosphate (S1P) that trigger phagocyte migration resulting in apoptotic neutrophil clearance. S1P binds to five different G protein-coupled receptors (S1PR1–5). Our data show that obese and hyperglycemic mice are more susceptible to MRSA skin infection, characterized by increased lesion size, bacterial loads, and poor abscess formation. In hyperglycemic mice, we also observed increased dead cells on days 1 and 9 post-infection with MRSA. When we assessed in vivo efferocytosis, we observed a lower ratio of ingestion of dead cells in the skin of hyperglycemic mice compared to euglycemic mice. Imaging mass spectrometry and lipidomic analysis revealed that while S1P levels are decreased, phosphoethanolamine (a product of the S1P degradation) is increased before and on day 1 post-infection in obese mice. We found that S1pr3 is significantly increased, whereas S1pr4 and S1pr5 are decreased in the infected skin obese. Topical S1P treatment reduced lesion size and bacterial load in the skin of hyperglycemic mice. Our data suggest that S1P deficiency during skin infections culminates in less efferocytosis, accumulation of dead cells, uncontrolled infection, and delay in the resolution of the infection.
Visualizing cutaneous CD8+ T cells in multiple disease states to uncover common effector mechanisms
Authors: Droppo Joanna M , Pei Luxin , Hickman Heather D 
Published: August 2023

Abstract
Cytotoxic CD8+ T cells are important for the control of many different pathologies in the skin, including cutaneous viral infections and skin cancers. One skin cancer, melanoma, is responsible for many deaths each year in the US alone. Despite this, we still have much to learn regarding the mechanisms that prevent and eliminate cutaneous cancers. Our laboratory has had a long-standing interest in better understanding the biology of T cells in the skin responding to the poxvirus vaccinia (VACV). To better understand global mechanisms of CD8+ T cell biology and effector function, we also established a melanoma model based on other published studies to compare with VACV infection. To do this, we adoptively transferred Rag2−/−Il2rg−/− mice with OT-I CD8+ T cells expressing dsRed, allowing us to easily visualize T cells using both frozen tissue sections and intravital microscopy. We then either 1) infected with VACV expressing or lacking cognate antigen (the SIINFEKL peptide) or 2) injected ovalbumin-expressing B16 melanoma cells into the outer ear pinnae. Primary analyses of this model via confocal microscopy of pinnae sections revealed OT-I T cells accumulating around the periphery of the melanoma lesion with a few cells infiltrating the tumor. Similarly, OT-I T cells accrued at the borders of foci of viral replication in the epidermis. Ongoing studies using this model will further characterize the immune response by visualizing the location of immune cells such as monocytes and neutrophils in relation to both tumors and virally infected cells. Comparisons between these two disease states may reveal shared mechanisms of interaction between effector CD8+ T cells and innate myeloid cells that traffic into the skin during pathology.