bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil Inhibitory Receptors Expression Is Distinct During Gram-Positive and -Negative Pneumonia and Affects Neutrophil Function
Authors: Pihl Riley M F , Martins Kevyn , Traber Katrina E
Published: August 2023

Rational

Despite effective antimicrobial therapy, morbidity and mortality from pneumonia remains high. Honing our understanding of immune dysfunction in pneumonia is now crucial for developing targeted therapeutics. Our objective is to understand how different lethal bacterial pneumonias affect neutrophil phenotype and function over time and identify regulators of neutrophil function.

Methods

Mice were intratracheally instilled with E. coli or S. pneumoniae (SP3) for 6, 24 or 48h and neutrophils were isolated from either bronchioloalveolar lavage fluid (BALF) or peripheral blood. Neutrophil surface marker expression (SME) was analyzed via 25-color panel run on a Cytek Aurora spectral flow cytometer (SFC). Data was analyzed in FlowJo and gated for live, single cell, CD45+Ly6G+ neutrophils, opt-SNE was run for data visualization, and Phenograph was run for unbiased clustering. For bacterial clearance assays, bone marrow neutrophils were isolated via Percoll, then incubated with blocking antibodies against inhibitor receptors (IRs) (PD-L1, SIRPα, VISTA, or CD200R), soluble ligands, or media only. Neutrophils were then cocultured with either SP3 or E. coli. Media from bacteria-neutrophil cultures was then plated on agar and the developing colonies were counted.

Results and Conclusions

Using SFC, we identified pathogen- and timepoint-specific differences in SME of the IRs PD-L1, SIRPα, VISTA and CD200R. Modulating IR signaling by blocking or stimulating them impacts neutrophils’ ability to clear bacteria. In sum, our data suggests that neutrophils modulates their response to adapt to specific pathogens. IRs may play a role in regulating these dynamic neutrophil responses, making them a potential therapeutic target.

NIH grants K08 HL130582 (KET) and R01 HL158732 (KET).
Double DAMPs – NLRP3 priming of murine bone marrow derived mast cells with IL-33 leads to robust IL-1β secretion in a Gasdermin-D independent manner
Authors: Foster Cheyanne , Lee Elizabeth , Dubyak George R
Published: August 2023

Activation of the NLRP3 inflammasome involves hierarchical activation and assembly of upstream protein complexes which can lead to a Gasdermin-D (GSDMD) dependent form of lytic cell death coined pyroptosis. Upon cleavage by Caspase-1, GSDMD oligomerizes in the plasma membrane to form pores which act as a conduit for IL-1β release – a potent proinflammatory cytokine that is implicated in a variety of physiological and pathological responses. Prior studies have utilized macrophages as a model system for interrogating the NLRP3 inflammasome, and recent studies with mast cell (MC) extrapolate these macrophage mechanisms to MCs. However, the mechanisms underlying IL-1β secretion in MCs have yet to be interrogated. We are characterizing the detailed mechanisms underlying NLRP3 activation in MCs, with a focus on GSDM-family members in the pathways of IL-1β release. Our data using primary murine MCs indicate that IL-1β is released independently of GSDMD. This correlates with low GSDMD expression in contrast to high expression of GSDME. Thus, our current studies focus on defining roles for GSDME or other mechanisms of IL-1β release from MCs. Notably, our studies demonstrate a critical role for IL-33 priming vs. LPS in the signal 1 priming of MCs for NLRP3 signaling. Taken together we demonstrate that the MC NLRP3 inflammasome signaling and IL-1β release utilize mechanisms distinct from those in macrophages.

Supported by grants from NIH (P01-AI141350)
Time Course scRNAseq Analysis Reveals Mechanisms of Impaired Lung Recovery Post Influenza Infection during Aging: Zooming into Macrophages
Authors: Wu Yue , Tang Jinyi , Gao Xiaochen , Li Chaofan , Zhu Bibo , de Almeida Santos Gislane , Narasimhan Harish , Li Ying , Zhang Ruixuan , Fain Cori E , Ayasoufi Katayoun , Johnson Aaron J , Dong Haidong , Sun Jie
Published: August 2023

Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6Chi monocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.
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

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

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

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

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

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

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

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

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

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

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

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

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.
What UV is what you get: skin injury by ultraviolet (UV) light results in renal cell loss and damage in the presence of a CD177high neutrophil population
Authors: Cortez Angelique , Mendyka Lindsay , Kolling Fred , Skopelja-Gardner Sladjana
Published: August 2023

Immune-mediated communication between organs has been implicated in the pathogenesis of many inflammatory and autoimmune diseases. We previously showed that skin injury by UV light triggers neutrophil migration to the kidney, stimulating expression of renal injury genes. This skin-kidney axis is highly relevant to lupus patients, for whom skin exposure to UV sunlight rays can lead to kidney disease flares. Here, we applied 10X single-cell RNA-seq on saline-perfused digested kidneys and Ly6G-enriched neutrophils of B6 mice, prior to and 2 days after a single dose of UV, to define renal cell subsets damaged by skin exposure to UV and how neutrophils mediate the injury. Dimensional reduction analysis revealed 14 kidney cell types and showed that exposure to UV led to the loss of podocytes and distal tubular cells (DTCs) and an expansion in endothelial and stromal cells. Differential gene expression analysis showed that podocytes and DTCs in UV-exposed mice upregulated genes associated with renal injury, including type I interferon genes, and downregulated genes important for cellular integrity and function. Neutrophil scRNA-seq revealed a unique activated CD177high neutrophil population in the kidneys of UV exposed mice, defined as the endpoint of the psuedotime trajectory by diffusion mapping. Ligand-receptor pair analyses showed skin injury by UV altered neutrophil interactions with renal structural cells, with expanded binding to stromal cells. While the CD177high neutrophil cluster was dominant in the kidney, it was also found in the lung, but not in the skin and blood. These studies introduce a novel model by which CD177high neutrophils may mediate podocyte, DTC, and stromal kidney injury secondary to sterile skin inflammation.

Supported by grants from NIH (1R21AR079661 – 01, 5P20GM130454-02), The Department of Defense (W81XWH2110889), and Dartmouth Health Start Up Funds.
Effector response to necroptotic cell death: an ensemble of immune and stromal cells
Authors: Hughes Lindsey D , Altun Osman , Nevin James T , Wang Mamie , Kluger Yuval , Pelorosso Facundo , Leighton Jonathan A , Rothlin Carla V , Ghosh Sourav
Published: August 2023

Cell death manifests through a panoply of molecular pathways. We postulated that the tissue effector response to cellular corpses is similarly diverse. Apoptotic cell death in the lung, in the context of helminth infection and an IL-4-rich environment, is sensed by macrophages to direct resolution of inflammation and a tissue repair gene expression program. Apoptotic cell death, however, is commonplace in the gut and integral to homeostatic tissue renewal in this organ. Another specific molecularly-executed cell death modality – necroptosis – has been reported in the gut in settings of non-resolving inflammation and tissue damage, such as in inflammatory bowel disease. Here we report the discovery of an effector response to necroptotic corpses (NC). Using a chemo-genetic approach to specifically trigger necroptosis in the intestinal epithelium in vivo, we compiled an atlas of transcriptomic changes at a single-cell level and compared this to inflammatory and resolving injury models. Fibroblasts and macrophages, upon encounter with NC, displayed an excessive inflammatory response. Fibroblasts are the major stromal cells that play an essential role in tissue healing by undergoing fibroblast to myofibroblast transition under the influence of TGF-β. This step is a critical event in wound healing as the contractile forces generated by myofibroblasts bring the two apposed edges of the wounded tissue together for wound closure. Exposure to NC prevented this myofibroblast transition. Taken together, these findings point to an invariant code that informs effector tissue responses based on the type, context, and sensor of cell death.
Plasma Derived Extracellular Vesicles Modulate Monocyte IL-18, IFN-α and IL-10 responses in Adults with HIV-related Cognitive Impairment
Authors: Yeung Stephen T , Marques de Menezes Erika G , Friday Courtney , Liu Jocelyn S , Bowler Scott A , Abdel-Mohsen Mohamed , Shikuma Cecilia , Norris Philip J , Ndhlovu Lishomwa C
Published: August 2023

Background

HIV-associated neurocognitive dysfunction persists despite suppressive antiretroviral therapy (ART). Extracellular vesicles (EVs) are emerging modulators of immunological responses. We have previously shown that EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment (CI). Whether these EV changes observed in people with HIV (PWH) influence innate cellular function among those with CI remains unclear.

Method

Purified EV samples from 58 HIV-infected individuals on ART with either cognitive impairment (CI, n=48) or normal cognition (NC, n=10) and 8 uninfected controls were isolated using differential centrifugation and co-cultured with PBMCs from a single uninfected donor for 18 hrs. Eight intracellular cytokines and 13 surface chemokines and myeloid differentiation markers were assessed by spectral flow cytometry. Non-parametric statistical T tests were used.

Result

Monocytes (mono) showed differential effects on multiple cytokine responses and cell surface receptors after exposure to EVs from HIV-infected individuals with or without CI. Levels of mono IL-18 were significantly lower after incubation with EVs derived from HIV-infected individuals compared to uninfected controls. Based on cognitive status, EVs from HIV-CI group significantly lower % of IL-18 mono responses and lower IFN-α and higher IL-10 responses on a per cell basis when compared to the NC group.

Conclusion

These results suggest EVs can modulate mono function irrespective of HIV status. PWH with CI exhibited altered EV driven mono cytokine responses. Our data support interventions that target EVs to reverse or restore mono perturbations and limit neuropathology in PWH on ART.

Supported by grants from NIH (R21NS106970 and R01NS117458)
Investigating a Role for Eosinophils in the Immune Response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
Authors: Snow-Smith Maryonne , Baker Paul J , Bohrer Andrea C , Castro Ehydel , Torres-Juarez Flor , Anderson Charles F , Makiya Michelle M , Sereti Irini , Klion Amy D , Mayer-Barber Katrin D
Published: August 2023

Eosinophils rapidly decrease in the peripheral blood of patients during COVID-19, and eosinopenia has been correlated with disease severity. These clinical observations suggest that eosinophils may play a functional role in the response to SARS-CoV-2 infection. Murine studies have shown that eosinophils can be recruited to the lungs in response to infection with respiratory viruses, including RSV and influenza and parainfluenza viruses, yet the role of eosinophils in the lungs in response to SARS-CoV-2 infection is unknown. Here, we used a two-pronged approach employing mouse models of SARS-CoV-2 infection to study the role of eosinophils in the antiviral immune response and viral control and clinical samples to assess eosinophilia and eosinophil activation in patients during and after COVID-19. Our initial findings indicate that eosinophils are recruited to the lungs after SARS-CoV-2 infection in mice and that eosinophil-deficient mice may have increased lung viral titers early after infection. In keeping with this hypothesis, serum levels of eosinophil granule proteins (a marker of tissue eosinophilia) were increased during active SARS-CoV-2 infection in patients participating in a long-term follow-up study of COVID-19, at a time when eosinophil counts were depressed. These preliminary data suggest that eosinophils may have a protective role in early anti-viral host defense during SARS-CoV-2 infection in mice and that further studies are required to better understand the relative roles of eosinophils in protection and pathology to SARS-CoV-2 infection.

Funding for this study was provided in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
IL-10 promotes tissue-resident memory T cells (TRM) in the respiratory tract during SARS-CoV-2 infection in rhesus macaques
Authors: Nelson Christine , Foreman Taylor , Namasivayam Sivaranjani , Kauffman Keith , Gomez Felipe , Sakai Shunsuke , Brooks Kelsie , Brenchley Jason , Via Laura , Barber Daniel L
Published: August 2023

T cells are thought to be important for control of SARS-CoV-2 infection, but little is known about the host factors that promote T cell responses in the respiratory tract. We used non-human primates to investigate the regulation of respiratory immunity during mild SARS-CoV-2 infection. We first examined the kinetics of viral replication, inflammation, and host immune responses. Foci of inflammation detected with 18FDG-PET/CT imaging peaked within 2–3 days of infection. Single cell RNA sequencing revealed an early influx of IFN-activated myeloid cells into the airways, which correlated with viral RNA loads. Antigen-specific T cell responses were first detected after inflammation and viral replication had resolved and were preferentially localized to the lower airways. We next examined the role of pro and anti-inflammatory cytokines, IFNγ and IL-10, on immunity to SARS-CoV-2 infection by blocking these cytokines with monoclonal antibodies early during infection. IFNγ blockade decreased lung inflammation but had no major impact on adaptive immune responses. In contrast, IL-10 blockade increased lung inflammation and enhanced accumulation of virus-specific T cells in the lower airways. IL-10 blockade also inhibited the differentiation of virus-specific T cells into airway CD69+CD103+ tissue resident memory cells (TRM). While virus-specific T cells were undetectable in the nasal mucosa, IL-10 blockade similarly reduced the frequency of bystander TRM cells in the nasal mucosa. Thus, in the setting of mild SARS-CoV-2 infection IL-10 has a key role in suppressing local lung inflammation and the accumulation of SARS-CoV-2-specific T cells, while also promoting TRM at respiratory mucosal surfaces.

Division of Intramural Research at NIH
Interferon-lambda accelerates viral clearance and regulates CD8+ T cells immunity in a murine model of SARS-CoV-2 infection
Authors: Solstad Abigail , Kenney Adam D , Mahfooz Najmus Sahar , Zani Ashley , Kawahara Jeffrey , Robinson Richard T , Forero Adriana , Yount Jacob S , Hemann Emily A
Published: August 2023

SARS-CoV-2 remains a significant global health burden. A better understanding of the requirements for protective host immunity against SARS-CoV-2 will contribute to the development of novel therapeutic strategies to prevent disease. Type III interferon (IFN-lambda) limits SARS-CoV-2 infection in vitro and in murine models of infection. In clinical trials, IFN-lambda treatment accelerates viral clearance of SARS-CoV-2 and reduces risk of hospitalization. However, roles for IFN-lambda in regulating SARS-CoV-2 pathogenesis and protective immunity are not well understood. Infection of C57BL/6 (WT) and IFN-lambda receptor deficient mice (Ifnlr1−/−) with mouse-adapted SARS-CoV-2 lead to increased weight loss and viral load in Ifnlr1−/− compared to WT through day 5 post infection (pi). Global transcriptomics revealed differential regulation of proliferation and immune cell programming pathways in Ifnlr1−/− lungs compared to WT, suggesting IFN-lambda signaling regulates protective pathways outside of canonical interferon stimulated gene responses following SARS-CoV-2 infection. Histological and flow cytometric analysis confirmed an increase in proliferation in multiple cell subsets within lungs of Ifnlr1−/− lungs compared to WT following SARS-CoV-2 infection. Additionally, SARS-CoV-2 specific CD8+T cells in the lungs of Ifnlr1−/− mice are significantly reduced compared to WT on day 8 pi. Together, these studies suggest IFN-lambda signaling is required for regulation of proliferative repair pathways and CD8+T cell immunity against SARS-CoV-2. Overall, broadening the understanding of how IFN-lambda regulates SARS-CoV-2 infection, pathogenesis, and immunity may inform its therapeutic utilization.
SARS-CoV2 Decreases Human Beta-Defensin-1 and FoxI1 in the Oral Cavity Without Affecting CFTR
Authors: Ryan Lisa K , Barney Weston J , Tierney Paige K , Kim Jumin S , Robinson Tanya O , Dhaliwal Neha S , Wichman T Grant , Glover Sarah C
Published: August 2023

The ionocyte is a newly characterized cell, first identified in the airway, that is hypothesized to play a role in innate immunity. Single cell RNA seq analysis showed that ionocytes (FoxI1 and CFTR-expressing cells) are also found in the oral cavity, and highly express human beta-defensin-1 (hBD-1). We showed that hBD-1 expression is upregulated by influenza, Herpes simplex virus and Sendai virus infection of monocytes and plasmacytoid dendritic cells but is downregulated in epithelial cells. The role of hBD1 in oral ionocytes in relation to SARS-CoV-2 infection is unknown. We tested the hypothesis that SARS-CoV2 virus modulates the expression of hBD-1, and this is associated with CFTR and FoxI1 expression in ionocytes in the oral cavity. Buccal swabs from patients with Covid19 and without Covid19 (recovered from Covid19 and non-Covid19) were analyzed for gene expression via quantitative RT-PCR of hBD-1, FoxI1, and CFTR. HBD-1 and FoxI1 mRNA levels (relative to beta-actin) were significantly decreased, but CFTR mRNA remained unchanged in the Covid19 patient cohort compared with the non-Covid19 and recovered patients. This supported the in vitro observation that enveloped viruses decrease hBD-1 in epithelial cells that make up most of the buccal swab sample. Immunohistochemistry on normal minor salivary gland sections revealed that FoxI1, CFTR and hBD-1 are all associated in proximity in the duct of the minor salivary gland. However, crosslinking studies, followed by Western blot analysis, indicated that hBD-1 and CFTR were not interacting. In conclusion, SARS-CoV2 modulates innate immunity in the oral cavity, as indicated by the decrease in hBD-1, and affects ionocytes, possibly affecting pH and osmolality in salivary glands.

Research supported by a grant from NIH / NIDCR R01 DE031928 and the University of Louisville School of Dentistry Summer Research Scholar Program
STAT3 operates as a rheostat regulating sensitivity of NK cells to inflammatory cytokines
Authors: Grassmann Simon , Fan Sherry , Kim Ray , Beroshvili Giorgi , Sun Joseph Chai-Yuen
Published: August 2023

NK cells are innate lymphocytes that respond to a variety of cytokines during viral infection. Several homeostatic and inflammatory cytokines bind to receptors that signal via STAT3. STAT3 can form homodimers or heterodimers. To understand the role of STAT3 homodimers and heterodimers, we made use of knockout models in which NK cells lack STAT3 or specific cytokine receptors. We found that NK cells deficient in STAT3 expand less than wildtype NK cells during mouse cytomegalovirus (MCMV) infection. IL-21R and IL-10R are the only receptors expressed on NK cells that signal through STAT3 homodimers. However, IL-21R or IL-10R-deficient NK cells showed no or little expansion differences, suggesting additional cytokine receptors may be signaling through STAT3.

To evaluate whether STAT3 regulates the sensitivity of NK cells to cytokines that signal through heterodimerization partners STAT1 and STAT5, we performed cytokine titrations. We found that sensitivity towards IFN-a and IL-15 was reduced in STAT3-deficient NK cells. NK cells require inflammatory cytokines for antiviral expansion but proliferate poorly in hyper-inflammatory environments. Because STAT3 appears to regulate the sensitivity of NK cells to inflammatory cytokines, we hypothesized that STAT3-deficient NK cells would be shielded in settings of hyper-inflammation. Indeed, using high-dose MCMV infection, we observed that STAT3-deficient NK cells expanded better than WT NK cells. RNA-seq analysis further corroborated that in high-dose infection STAT3-deficient NK cells are nearly indistinguishable from WT NK cells during regular-dose infection. Thus, we believe STAT3 operates as a rheostat, modulating the sensitivity of NK cells to their inflammatory environment.

Supported by the Cancer Research Institute, Donald J. Gogel Postdoctoral Fellowship, CRI393
Interferon Regulatory Factor 7-mediated induction of interferon-α is required for survival from alphavirus encephalomyelitis
Authors: Nguyen Benjamin H , Troisi Elizabeth , Griffin Diane E
Published: August 2023

Alphaviruses are a group of arthropod-borne viruses that have the capacity to cause encephalomyelitis, which is inflammation of the brain and spinal cord due to the immune response to viral infection, and can result in fever, headache, and neurologic disease such as cognitive impairment, seizures, ataxia, and paralysis. Neurons are the primary target cells of Sindbis virus (SINV), the prototypic alphavirus; however, due to the indispensable nature of neurons, mechanisms of viral clearance from the central nervous system (CNS) during recovery cannot depend on immune-mediated killing of infected neurons and is therefore specialized. We have previously shown that interferon (IFN) regulatory factor (IRF) 7, a transcription factor important for amplification of type I IFN (mainly IFNα), is required for survival from infection with SINV. Mice deficient in IRF7 succumb to infection – despite local production of IFNβ – while wild type (WT) mice recover. Treatment of Irf7−/− mice with IFNα rescues the WT phenotype, suggesting that IRF7-mediated induction of IFNα and subsequent downstream signaling is required for survival from SINV infection. Additionally, Ifnb−/− mice do not phenocopy Irf7−/− mice, further suggesting that IFNα is required for survival while IFNβ is dispensable despite usage of the same receptor. Interestingly, Ifnb−/− mice have similar viral titers to Irf7−/− mice, implying that the protective effects of IFNα are independent from restriction of viral replication. These findings show subtype-specific differences in protection by type I IFN and can inform treatment options for alphavirus-induced encephalomyelitis.

These works have been funded by the U.S. National Institutes of Health (R01 NS087539 and T32 AI007417).