bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a subset of lung neutrophils that promote Acute Lung Injury resolution in mice
Authors: Tavares Luciana Padua Padua , Bruggemann Thayse , Cagnina R Elaine , Rezende Rafael M , Duvall Melody G , Nshimiyimana Robert , Serhan Charles N , Levy Bruce D 
Published: August 2023

Abstract
The acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure with limited therapeutic options. Unresolving inflammation in the lungs and alveolar-capillary barrier dysfunction are main features of ARDS. The sharp increase in the incidence of ARDS during COVID19 emphasized the need for a better understanding of immunological events in ARDS that inform novel therapeutics. Here, we used a model of self-limited acid-induced ALI by selectively instilling HCL to the left lung of adult mice. The time-course of barrier dysfunction was determined via quantification of lung consolidation by micro-computed tomography, while inflammation was assessed by histology and flow cytometry. Neutrophils, rapidly recruited into the lungs, decreased over time during resolution while macrophage numbers, reduced during initial stages, increased. Immunophenotyping of the leukocyte recruitment to the lungs during the resolution of injury uncovered a neutrophils’ subset that was expressing SiglecF. These cells were distinct from eosinophils and phenotypically, and functionally different from the classical SiglecF-negative neutrophils. The roles for SiglecF+ neutrophils were further assessed by in vitro functional studies, adoptive transfer experiments, and lipidomic analysis. Depletion of neutrophils impaired alveolar epithelial cell type II (AT2) repair, while the adoptive transfer of SiglecF+ neutrophils accelerated epithelial cell restitution. Among the mechanisms associated with the SiglecF+ neutrophils in lung repair was the production of specialized pro-resolving mediators and CSF1. In sum, we identified protective roles for a subset of tissue neutrophils during the resolution of lung injury in mice.
The Effect of Mast Cells Stimulated by Toll-Like Receptors on Keratinocytes
Authors: Kwon Ji Yun , Cho Kyung-Ah , Woo So-Youn 
Published: August 2023

Abstract
The mast cells, as located in the skin’s dermal layer, are important innate immune cells that mediate inflammation as a defense response against outer pathogens. Mast cells are activated by innate immune receptors such as Toll-like receptors receptrs (TLRs) and interact with a variety of cells including keratinocytes. This study confirmed the effect of TLRs-stimulated mast cells on the migration of keratinocytes residing in the epidermis. The HaCaT cells, a human keratinocyte cell line, was treated with the concentrated conditioned medium (CM) of the HMC-1, a human mast cell line. After that, the migratory capacity via scratch and transwell assays. In addition, we tried to confirm whether TLRs activations affect these effects of mast cells on keratinocyte migration. Thus the same experiments were carried out using the CM of HMC-1 obtained under various stimulation by TLRs agonist. As a result, HMG-1 CM promoted the migratory ability of HaCaT cells in scratch assay and transwell assay. Those effects were further increased when HMC-1 was exposed to various TLRs stimulation.
This work was supported by a grant of the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF-2021R1A2C1012551).
Kupffer cells heterogeneity and replacement by monocyte-derived KCs contributes to visceral leishmaniasis resistance
Authors: Pessenda Gabriela , Paun Andrea , Ferreira Tiago Rodrigues , Amaral Eduardo Pinheiro , Kabat Juraj , Kamenyeva Olena , Ganesan Sundar , Lee Sang Hun , Sacks David Lawrence 
Published: August 2023

Abstract
Visceral leishmaniasis (VL) is a potentially fatal disease transmitted by sand fly bites and caused by Leishmania (L. donovani/L. infantum) protozoa. Kupffer cells (KCs) are the liver embryonic resident macrophages (emKCs), characterized by Clec4f and Tim4 expression and their sessile behavior within the liver sinusoids. KCs maintain their numbers via self-proliferation during homeostasis but can be replaced by monocyte-derived cells (moKCs) during inflammation. In the murine VL model, KCs are important for both initial parasite growth and granuloma formation, the latter being associated with the eventual protective response. The objective of our study is to investigate KC proliferation, migration, death, and their replacement by moKCs in VL. We found that KC proliferation was enhanced at 19 d.p.i., while at 42 d.p.i. the granulomas cores contained mixed KC clonal lineages and were located outside the liver sinusoids. At the same time point, Clec4f and Tim4 expression was reduced, and we found evidence of KC apoptosis and ferroptosis. Parabiotic mice demonstrated KC populations that bore either congenic marker, and CCR2−/− infected mice showed enhanced frequencies of emKCs, reduced frequencies of moKCs, and higher parasite loads compared to WT mice. Reduction of ferroptosis in BACH1−/− mice resulted in lower frequencies of moKCs. Collectively, our data indicates that KCs migrate to the liver parenchyma to form granulomas, facilitating their activation by other immune cells, and that KC death results in their partial replacement by moKCs, contributing to increased parasite killing. Thus, KC heterogeneity is an important hallmark of hepatic resistance in VL.
Funded by the Division of Intramural Research, NIAID.
Mycobacterium tuberculosis exploits FIP200 regulated cell-death pathways to promote replication
Authors: Dubey Neha , Woodson Reilly , Kinsella Rachel L , Nehls Michael , Naik Sumanta Kumar , Kreamalmeyer Darren , Stallings Christina L 
Published: August 2023

Abstract
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a leading health concern. The rise in antibacterial resistance has steered focus to developing host directed therapies, which requires an in-depth understanding of Mtb interactions with the host immune system. Xenophagy, the targeting of Mtb to autophagosomes within infected cells, is not effective at controlling Mtb replication in vivo, however, we have discovered that autophagy proteins function outside of xenophagy to impact Mtb pathogenesis. FIP200 is an autophagy pathway protein required for autophagosome formation. In this study, we identified a unique function of FIP200 in promoting Mtb infection in mice that is independent of its role in autophagy. We show that the loss of Fip200, but not the loss of other autophagy genes, in LysM+ innate immune cells results in lower Mtb burden in the lungs of mice by 7 dpi. Fip200fl/fl-LysM-Cre mice lungs harbor higher numbers of neutrophils, macrophages, dendritic cells (DCs), and activated CD4+ T cells relative to Fip200fl/fl mice. Depletion of either neutrophils or T cells did not revert the bacterial burdens, showing that these cells do not contribute to the enhanced control of Mtb. We show that FIP200 is specifically required in CD11c+ macrophages and/or DCs during infection. FIP200 has been reported to regulate several signaling pathways to suppress apoptosis and promote cell survival. We observed increased macrophage apoptosis in the airways of Fip200fl/fl-LysM-Cre mice compared to Fip200fl/fl controls in response to Mtb infection. Based on our data, we propose a model where loss of FIP200, but not other autophagy proteins, in CD11c+ macrophages leads to increased apoptosis and better control of Mtb replication.
NIH NIAID R01 #AI132697
NIH NIAID U19 #AI142784 ND supported with Alexander & Gertrude Berg Fellow
Disparate natural killer T cell subsets exhibit opposing regulatory effects on the cytokine storm
Authors: Torres Cameron M , Spencer Charles M 
Published: August 2023

Abstract
Inflammation is both a critical component of the immune system for effective clearance of microbial challenges and a mechanism by which some pathogens cause disease, e.g., tularemia, dengue fever, and COVID-19. The bacterium Francisella tularensis (Ft), the causative agent of tularemia, elicits a cytokine storm in which the immune system over-responds to the bacterium, releasing massive amounts of proinflammatory cytokines. Untreated, tularemia can reach a mortality rate as high as 30% in healthy adults with inhalation of as few as 10 colony forming units inducing respiratory tularemia. While Ft is spread from zoonotic sources in nature, the ease of aerosol spread and high mortality led to its weaponization and continued concern for bioterrorism applications. Natural Killer T (NKT) cells are important immune cells with the capability of modulating the inflammatory response. NKT cells are activated early following Ft infection suggesting a role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, type I and type II based on their antigen recognition, or not, of α-galactosylceramide. Literature suggests that these subsets have opposing regulatory phenotypes, however, a clear understanding of their roles in regulating inflammation remains unknown and an emerging frontier in NKT research. Our lab has observed opposing roles in suppressing the cytokine storm by NKT subtypes. Ongoing studies will elucidate the mechanism utilized to influence the cytokine storm. This project will yield a better understanding of NKT cell-mediated inflammation during Ft infection and, more broadly, characterize their involvement and potential for therapeutic targeting for a number of NIH priority pathogens.
Supported by grants from National Institute of General Medical Sciences (NIGMS) Research Initiative for Scientific Enhancement (RISE) grant R25 GM 06921-18
Acute alcohol treatment alters macrophage heterogeneity by interfering with the polarization process
Authors: Liu Liyuan , Li Lei , Stokes John V , Tan Wei , Pruett Stephen B 
Published: August 2023

Abstract
Alcohol abuse disorder increases the risk of a series of diseases. However, there is limited evidence to indicate how acute alcohol exposure alters immune and inflammatory responses. Macrophages are phagocytic cells with diverse functions. The heterogeneity of macrophages leads to a challenge of clear and precise identification of the functional status and mechanism by which acute alcohol alters macrophage function. To address this issue, we first establish an optimized eleven-parameter flow cytometry panel based on RAW 264.7 cells for identifying macrophage subtypes (Naïve, M1, M2a, and M2c). Then three sets of experiments have been conducted: 1) polarization stimuli were applied first, then alcohol was added to the cultures, 2) alcohol was added to the cells first then polarization stimuli, 3) alcohol and polarization stimuli were added simultaneously. A total of 32 cytokines and chemokines involved in inflammation and immunity were measured. Based on these results, we developed a mathematical expression of the M1-M2 continuum with the principal component analysis that can quantify the degree to which all the markers are consistent with an M1 phenotype or one of the M2 phenotypes, which provides a concise indicator to evaluate macrophage status. Results were consistent with previous studies in our lab using mouse peritoneal macrophages in vitro, and the results indicate that the effects of acute alcohol exposure differ depending on macrophage polarization status, and this may explain some of the effects of alcohol on macrophages that we and others have reported in vivo in a mouse model for binge drinking. The authors were supported by funding from an NIH Center of Biomedical Research Excellence (P20 GM103646-09).
The authors were supported by funding from an NIH Center of Biomedical Research Excellence (P20 GM103646-09).
Notch2 deficiency generates functionally impaired cDCs and macrophages that correlates with intestinal dysbiosis and low-grade inflammation
Authors: Cvijetic Grozdan , Mitrovic Mladen , Rodrigues Patrick Fernandes , Jacome Emily Reinoso , Mendoza-Bonilla Betsy , Conway Isabella Olive , Shi Joanne , Ottaviani Valentina , Tussiwand Roxane 
Published: August 2023

Abstract
Myeloid cell types including conventional dendritic cells (cDCs) and macrophages are key responders to innate stimuli at epithelial barriers. They are equipped with innate receptors to sense danger signals and pathogens, becoming the first line of immune defense and thereby maintaining homeostasis. The expression of selected transcription factors was shown to be essential for the development of these myeloid subsets including Batf3, Notch2, Irf4, and Irf8 among others. We performed single-cell RNA expression analysis across eight different conditional KO lines and identified multiple defects across each line that compromised the development of unique subsets and also altered expression profiles on the remaining subsets. Within CD11c expressing cells, Notch2 deficiency in particular, seems to impair not only type 2 dendritic cells (cDC2) but also macrophage subsets across several tissues. Mice defective for Notch2 were shown to be susceptible to C. rodentium infection. We observed that the lack of Notch2 was accompanied by intestinal dysbiosis, which produced low-grade inflammation. The altered microbiome could be transferred to WT mice by co-housing, which suggests that the lack of either subset enabled the growth of an opportunistic microorganism that could prevail in competitive settings. Among the dysregulated innate pathways, we identified one that appears to correlate with this dysbiosis, and its deletion was sufficient to reproduce the phenotype in preliminary experiments. Collectively, we hypothesize that Notch2 deficiency alters the functional properties of the cDCs and intestinal macrophages ultimately leading to intestinal dysbiosis and a pre-pathologic inflammatory condition.
Supported by NIH intramural funding
TLR4 dependent generation of a novel bone marrow-derived inflammatory monocyte population required for elicitation of skin contact hypersensitivity responses
Authors: Kish Danielle , Fairchild Robert L 
Published: August 2023

Abstract
Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of a novel population of Gr-1+CXCR2+ cells expressing FasL and perforin. The goal of these studies was to further characterize this novel population of myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The myeloid cells were purified from the bone marrow of hapten sensitized mice 2 hrs after hapten challenge by depletion of CD3+, B220+, NK1+, F4/80+, class II MHC+ and Ly6G+ cells. Flow cytometry analyses indicate &gt;90% of the remaining bone marrow cells were CD11c+, Ly6C+, CCR2+, expressing FasL+ and perforin. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers 2 hrs. after hapten challenge of sensitized WT and Rag−/−mice and T-cell depleted WT mice. Generation of the myeloid cells was absent in hapten sensitized and challenged TLR4-deficient mice and was inhibited by treatment with anti-TNFa mAb at the time of hapten challenge. Whereas generation of neutrophils is inhibited by anti-G-CSF antibody, this treatment further increased numbers of these myeloid cells in the bone marrow following hapten challenge. These results indicate that generation of bone marrow-derived Ly6C+CXCR2+ cells expressing FasL and perforin in response to inflammation requires TNFa and TLR4 signaling that occurs independently of T cell activation.
Effect of Interleukin-2 Inducible Kinase (ITK) on the Temporal Dynamics of TCR Signaling in CD8 T cells
Authors: Hunkins Josh , Perrenoud Loni , Schoenbach Mary , Ono Masahiro , Berg Leslie J 
Published: August 2023

Abstract
Antigen affinity has been linked to the rate of downstream TCR activation and the activation of transcription factors such as NFAT and NF-kb. Interleukin-2 inducible T cell kinase (ITK) plays a key role in the differential rate and magnitude of TCR-dependent transcription factor activation, especially when T cells are stimulated with lower affinity ligands. The Nr4a3-Tocky Timer mouse model carries a construct for a fluorescent protein controlled by the Nr4a3 promoter. Nr4a3 is highly expressed following TCR activation and thus is a good metric for TCR signaling. When first produced, the Tocky protein fluoresces “blue,” at peak wavelength 466nm and then, after ~7hrs, degrades to a “red” fluorescence at peak wavelength 583nm. Splenocytes were harvested from OT-I RAG1KO mice crossed to Nr4a3-Tocky mice and stimulated using varying affinities and doses of ova peptide over 7 days in vitro. Cells were exposed to the ITK inhibitor, PRN694, to observe the effect of ITK on the rate of expression of Tocky fluorescence along with activation surface markers CD69, CD25, CD44, and CD122. With ITK inhibition, CD8 T cells expressed the “Blue” protein slower and in fewer cells compared to OT-I cells stimulated without PRN694 at 4hrs. At 48hrs after stimulation, sustained signaling was observed in control cells, whereas a subset of ITK-inhibited cells showed evidence of halted TCR-induced Tocky transcription. This decrease in the longevity of TCR signaling was more evident when lower peptide doses or lower affinity peptides were used for stimulation. These results suggest that ITK plays a role in amplifying the initial TCR signal, and is also required for sustained signaling after TCR activation in CD8 T cells, resulting in the tuning of early gene expression.
Supported by grants from the NIH (Dissecting the pathways controlling tunable responses to TCR signaling: 5R01AI132419-06)
Isoprenylation Inhibition Suppresses IL-33-mediated Mast Cell Activation of Allergic Asthma
Authors: Pryor Destiny T , Jackson Kaitlyn , Ryan John J 
Published: August 2023

Abstract
Allergic asthma is an inflammatory disease of the airway associated with sensitization to allergens and symptoms such as sneezing, wheezing, and airway hyperresponsiveness. The severity of this disease varies between sex, age, and race; therefore, understanding signaling mechanisms is integral to developing targeted therapeutics. Mast cells (MC), an important effector cell in allergic disease, can be activated by interleukin (IL)-33, an alarmin released after contact with cellular stressors including common allergens. In response to IL-33, MC release inflammatory cytokines and lipid mediators. We previously found that statins suppress IgE-mediated MC activation by inhibiting protein isoprenylation. Therefore, we hypothesized that directly targeting the isoprenylation enzymes farnesyl transferase (FT) and geranylgeranyl transferase (GGT) would suppress IL-33-mediated MC function. We show that the dual transferase inhibitor FGTI-2734 decreased IL-33-mediated inflammatory cytokine secretion, effects that were mimicked by simvastatin. In contrast to the dual inhibitor, suppressing only FT or GGT had no effect on cytokine secretion. These data suggest that inhibiting farnesyl and geranylgeranyl transferases is a potential treatment for IL-33-mediated mast cell activation and allergic disease.
Supported by grants from NIH (RO1 AI138495 and RO1 AI164710)
Imbalanced receptors, gene signatures, and function of NK subsets predicts poor clinical outcomes in AML
Authors: Costa Amanda , Kuznetsova Valeriya , Marani Leticia O , Lopes Izabela A , Binelli Larissa S , Scheucher Priscila S , Schiavinato Josiane L , Madeira Maria Isabel A , Pagnano Katia B , Duarte Bruno K , Glória Ana Beatriz F , Rego Eduardo M , Traina Fabíola , Figueiredo-Pontes Lorena L , Welner Robert 
Published: August 2023

Abstract
NK cells mediate anti-leukemic immunosurveillance in AML. We hypothesized that impaired NK cells predict poor outcomes in AML. We analyzed the expression of receptors on CD56 NK cell subsets and their corresponding ligands on the CD45dim blast by flow cytometry in 100 AML and 17 healthy bone marrows. Risk stratification, complete remission, relapse, measurable residual disease, overall survival, relapse-free survival, NK cell ligand expression, NK function, and degranulation were evaluated. Additionally, we mapped the transcriptional landscape of AML-NK cells using single-cell RNA-seq. The expression of activating receptors by CD56dim NK was decreased in adverse risk, relapse, and MRD+ patients. Inhibitory receptors were increased in adverse risk, relapse, and MRD+. NK cell activating ligands were decreased in patients with higher expression of the corresponding receptor while inhibitory ligands were increased. Patients with a lower frequency of CD56dim NK showed less degranulation and killing rates. Transcriptionally, we observed an enrichment of inflammatory-associated pathways and an upregulation of inhibitory receptors in AML, especially in patients with the worst prognosis. Our data indicate that NK exhaustion, with a high frequency of inhibitory receptors, leads to impaired cytotoxicity and can predict poor outcomes in AML. Also, a selective pressure in the expression of NK cell ligands on the AML blast together with imbalanced receptors may vary in both NK and leukemic cells. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy.
“division funds” - 3117175
Single-cell atlas of the liver myeloid compartment with cure of chronic viral hepatitis
Authors: Alatrakchi Nadia , Cui Ang , Li Bo , Gonnye Anna , Wallace Michael S , Patel Hailey , Oetheimer Christopher , Tonnerre Pierre , Holmes Jacinta A , Lieb David , Ma Aileen , Damasio Marcos , Brown Joelle , Sade-Feldman Moshe , Aneja Jasneet , Gustafson Jenna , Butler Michael Dillon , Epstein Eliana T , Thabet Ashraf , Kim Arthur , Lauer Georg M , Hacohen Nir , Chung Raymond T 
Published: August 2023

Abstract

Background/Aims
Chronic viral infections present serious public health challenges. Direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with the hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in humans.

Methods
To leverage this opportunity, we used plate-based single-cell RNA-seq (scRNA-seq) to deeply profile myeloid cells from liver fine needle aspirates in HCV patients before and after DAA treatment. We characterized liver neutrophils, eosinophils, mast cells, conventional dendritic cells (cDCs), plasmacytoid DCs, classical/non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types.

Results
We discovered cell-type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ cDCs, which may support restoration from chronic exhaustion. We observed an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host’s immune system. We found an upregulation of PD-L1 expression in ISG-high neutrophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programs shared by multiple cell types, distilling core functions of the myeloid compartment.

Conclusion
This comprehensive scRNA-seq atlas of human liver myeloid cells in response to a cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights.

The single-cell sorting was done at the MGH Flow and Mass Cytometry Research Core, which was supported by NIH Shared Instrumentation program grants 1S10OD012027-01A1, 1S10OD016372-01, 1S10RR020936-01, and 1S10RR023440-01A1. Additional sorting was done at the Immunology Core Facility of the Ragon Institute, which is part of the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354).