bluuebunny/crossref_metadata_2025 huggingface dataset:
Overlapping Mendelian disorders of immunity and metabolism shape CD4+ T cell immunometabolism
Authors: Patterson Andrew R , Chi Channing , Steiner KayLee K , Rathmell Jeffrey C
Published: August 2023

During the past decade it has become increasingly apparent that cellular metabolism plays a critical role in the function and regulation of the immune system. Immune cell utilization of specific metabolic pathways influences immune function and can be pharmacologically targeted to alter immune outcomes. The importance of immune cell metabolism and function is further demonstrated by the development of primary immunodeficiencies in patients deficient in certain metabolic pathways. To identify novel mechanisms governing the interplay between CD4+ T cell metabolism and function, we performed a series of CRISPR screens evaluating CD4+ T cell expansion, substrate uptake, and mitochondrial function. Specifically, we leveraged the Inborn Errors of Metabolism (IEM) and Immunity (IEI) to identify new immunometabolic regulators. Through these screens, I identified the transcription factor Bcl11b as a critical regulator in CD4+ effector T cell mitochondrial metabolism and function. Additional studies suggest Bcl11b plays a key role regulating mitochondria and metabolic stress in activated T cells. Conversely, screens of IEM genes for T cell functional defects showed that the N-linked glycosylation and the entry enzyme for de novohexosamine synthesis, Gfpt1, pathway were essential for CD4+ T cell expansion and survival. Notably, Gfpt1-deficient TH1 cells more affected than TH17 cells, which had greater capacity for salvage UDP-GlcNAc synthesis. This IEM and IEI-based CRISPR screening approach offers a unique opportunity to identify novel genes and regulatory pathways that are critical and sufficient for productive immune responses and to evaluate their effect on immunometabolism.

R01 DK105550
Th2 immune responses induced by helminth infection reprogram multilineage progenitors to produce successive waves of neutrophils, basophils, and eosinophils
Authors: Ferchen Kyle , Song Baobao , Potter Crystal , Zhang Xuan , Chou Chih-Hsing , Khodoun Marat , Finkelman Fred D , Salomonis Nathan , Grimes H Leighton
Published: August 2023

Helminths are parasitic worm pathogens known to induce innate acquired type 2 immune responses that lead to the accumulation of granulocytes in peripheral tissues. N. brasiliensis is a natural rat nematode parasite used in mouse models to elicit this immune response. Infective larvae are injected into the skin, migrate to the lung within 24–48 hours, are coughed up at 48–72 hours, and mature in the intestines in 5–6 days. Though much is known about the cytokine signaling and recruitment of granulocytes, little is known about the source of these cells. Their short half-life suggests a role for multi-lineage progenitors in the bone marrow. To test this, we exploited CITE-seq, flow cytometry, and machine learning on mouse bone marrow to build a reference atlas of transcriptional and immunophenotypic states of hematopoietic stem and progenitor cells (HSPCs). We generated an InfinityFlow object from millions of HSPCs and integrated these values with CITE-seq ADT values to allow transfer of transcriptionally defined clusters to flow cytometry-defined immunophenotypes. This enabled the generation of unsupervised gating srategies for isolation and functional validation of transcriptionally defined developmental stages. We then applied scRNA-seq and flow cytometry along a time course of N. brasiliensis infection in mice to characterize bone marrow HSPC responses to Th2-mediated immune effectors. We identified a multilineage progenitor that expands and transcriptionally reprograms at 72 hrs after infection and leads to the production of successive waves of neutrophils, basophils, and eosinophils on days 3, 7, and 10, respectively. Thus, worm infection stimulates Th2 responses that call to the marrow to specify granulocyte production.
Easy 250 EasySep&[trade] Magnet: A Novel Magnetic Platform For Large-Volume CD34+ Hematopoietic Stem and Progenitor Cell Isolation from Leukapheresis Products
Authors: Morin Peter , Toombs Eric , MacDonald Neil , Eaves Allen C , Louis Sharon A , Kokaji Andy I , de Jong Susan
Published: August 2023

We have developed a novel magnetic platform for large-volume CD34+ hematopoietic stem and progenitor cell (HPSC) isolation from both mobilized and non-mobilized leukapheresis products. As the frequency of CD34+ cells is low, and cell quality and viability can be variable between samples, it takes great effort to isolate these cells with consistently high purities and yields. Working with large volume leukapheresis products can be challenging as the sample often needs to be split and processed in parallel, which can be both time-consuming and labor-intensive.

The Easy 250 EasySep™ Magnet simplifies cell isolation procedures when processing leukapheresis samples. It is based on column-free immunomagnetic EasySep™ cell isolation technology and can process sample volumes up to 225 mL. We have developed a negative selection protocol for the enrichment of CD34+ HPSCs (mean purity ± SD of 74.6 ± 10.5%; n = 8) from non-mobilized leukapheresis products. For mobilized leukapheresis products, an EasySep™ positive selection protocol has been optimized for the isolation of highly purified CD34+ HPSCs (mean purity ± SD of 92.0 ± 1.7%; n = 7).

Notably, these protocols can be completed in as little as 25 minutes, making the Easy 250 EasySep™ Magnet the fastest and simplest method for enriching highly purified HPSCs from large-volume leukapheresis products—critical for addressing the growing clinical demands for CD34+ cell-based therapies.
The role of TGF-β in the differentiation and lung specification of murine type 1 conventional dendritic cells
Authors: Romo-Perez Yuliana Maritza , Soysa Radika , Liudahl Shannon , Headley Mark
Published: August 2023

Lung-resident type 1 conventional dendritic cells (cDC1s) are critical for initiating immune responses against foreign respiratory viruses and endogenous assaults like cancer cells. Importantly, the cDCs of each tissue display unique phenotypes and functions dictated by the environment they populate. However, our understanding of the factors that regulate cDC1 development and function in the lung is minimal. The cytokines, FMS-like tyrosine kinase 3 ligand (FLT3L), and granulocyte-macrophage stimulating factor (GM-CSF) are essential for the development of tissue-resident cDC1, but in in vitro systems, these factors are insufficient to generate cDC1s with a lung-specific phenotype. RNAseq analysis of lung cDC1 identified a clear enrichment in genes associated with transforming growth factor beta (TGF-β) signaling, a factor also known to be enriched in the airspaces of the lung. TGF-β has been previously reported to maintain DC quiescence in the lung, however, our data suggested it may also play a role in differentiation and lung specification of these cells. Based on this, I hypothesized that adding TGF-β to in vitro bone marrow (BM) cultures alongside established cytokines FLT3L and GM-CSF would generate cDC1s phenotypically and functionally similar to in vivo lung cDC1s. The addition of TGF-β, but not GM-CSF, to optimized FLT3L-BMDC cultures resulted in development of cDC1 which highly resemble lung cDC1. The development of a high throughput in vitro system that differentiates functional lung cDC1s will not only inform the in vivo factors required for development of these cells but also provides a useful platform for interrogation of lung cDC1 function and a stepping-off point to begin models for human lung cDC1 production and study

Supported by grants from the University of Washington PREP program NIH (R25) and the Fred Hutchinson Cancer Center Diversity, Equity, and Inclusion Student Fellowship
Harnessing arginine metabolism overcomes hyperthermia-induced metabolic dysfunction of CAR T-cells
Authors: Kondo Taisuke , DuSold Justyn , Achar Sooraj , Adebola Serifat , Gonzalez-Menendez Pedro , Perrault Julie , Jess Jennifer , Yates Bonnie , Song Hannah , Fox Stephen , Xu Xia , Chan King , Abdelmaksoud Abdalla , Highfill Steven , Andresson Thorkell , Shah Nirali , Dardalhon Valerie , Altan-Bonnet Grégoire , Taylor Naomi
Published: August 2023

Chimeric antigen receptor (CAR) T-cells have shown remarkable success in the treatment of hematological malignancies, but many patients still relapse. One common adverse on-target effect of CAR T-cells is cytokine release syndrome. Importantly though, the impact of fever, a hallmark of this syndrome, on CAR T-cell function is not known. We find that exposure of CAR T-cells to hyperthermia (40°C) significantly decreased their subsequent cytotoxicity against NALM6 leukemic cells, both ex-vivo and in-vivo. This was associated with reduced secretion of IL-2, IFNg, and IL-8 by CAR T-cells and high dimensional analyses revealed the induction of a terminally differentiated T cell phenotype. Gene profiling assays highlighted a negative enrichment of mTORC1, glycolysis, and oxidative phosphorylation gene sets in CAR T-cells subjected to hyperthermia and metabolome analyses unveiled significant reductions in urea cycle metabolites. Notably, pharmacological supplementation of arginine markedly enhanced the ex-vivo and in-vivo cytotoxicity of hyperthermia-exposed CAR T-cells. Moreover, in the absence of hyperthermia, short-term arginine supplementation enhanced the metabolic fitness of CAR T-cells, augmenting their long-term in-vivo persistence and cytotoxicity. We identify arginine metabolism as a critical pathway in CAR T-cells rendered dysfunctional by exposure to hyperthermia and pharmacological arginine support will inform future iterations of CAR T-cell interventions.
Ketogenic diet influences the microbiome-immune axis with therapeutic potential in ASD
Authors: Yamamoto Brennan Y , Allan Nina , Kunihiro Braden P , Nunokawa Chandler K L , Umeda Lesley , Phankitnirundorn Krit , Peres Rafael , Maunakea Alika K
Published: August 2023

Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting social interaction and communication, associated with increased levels of inflammation and mitochondrial dysfunction. The ketogenic diet has neuroprotective affects and decreases the severity of inflammation related conditions. A cohort of 7 children aged 7 to 19 diagnosed with ASD have improved behavioral symptoms following the ketogenic diet. This study aims to identify changes to the gut microbiome and inflammatory markers that are associated with improved prognosis. Stool and plasma samples were collected before starting the diet and up to twelve months after starting the diet. Stool samples were sequenced for microbiome data and plasma samples were analyzed for inflammation factors, ketone bodies, and miRNA presence. Butyrate kinase (Buk) DNA and RNA levels were elevated in stool samples suggesting changes to the microbiome significantly affected butyrate kinase levels in the patient’s digestive tract. Metabolomic data identified the cohort had elevated butyrate levels following the ketogenic diet further characterizing the importance of butyrate kinase producing bacteria, including Roseburia. Changes to inflammatory factors identified a decrease in IL-1B, IL-12p70, and BDNF. Decreases in BDNF, an activator of the NF-kB pathway, identifies a potential pathway for decrease in IL-1B and IL-12p70. Correlation analysis identified that Roseburia species elevated Buk DNA and decreased inflammation. While limited in sample size, these findings provide important insight into key molecular changes in the microbiome-immune axis linked to brain function that could be targeted for treatment of ASD and warrants further investigation.

Supported by grants from NIH (P20GM113134)
Oral exposure to Escherichia coli toxin protects against lung allergen-induced inflammation
Authors: Salgado Caio Loureiro , Ayupe Marina Caçador , Rodrigues Juliana Falcão , Silva Luisa Menezes , De Castro Oliveira Bernardo , Moreira Francielly , Gonçalves Leonardo Mandu , da Silva Guilherme William , Ramirez Jofer Andree Zamame , De Souza Ferreira Luis Carlos , D’império-Lima Maria Regina , da Silva Henrique Borges , da Fonseca Denise Morais
Published: August 2023

Recent studies suggest that the intestinal microenvironment regulate lung immune cells. Conversely, changes in the lung can modulate the gut immune system. Yet, mucosal-associated immune responses have been studied in a compartmentalized manner, as if there is no communication between different organs. Thus, the role of the gut-lung axis in mucosal immune responses is poorly understood. Using enterotoxigenic Escherichia coli (ETEC), we established a model of intestine-localized infection without translocation to any other tissue. In response to ETEC, type 2 innate lymphoid cells (ILC2s) and eosinophils accumulated in the intestinal lamina propria, even after bacterial clearance. In addition, oral ETEC transiently increased the numbers of ILC2 and eosinophils in the lung parenchyma. To test if oral ETEC promote lung immune responses to allergen, we exposed mice that recently cleared ETEC to intranasal papain/ovalbumin allergy induction model. Unexpectedly, ETEC-exposed mice were significantly resistant to allergen-induced lung inflammation, with reduced number of ILC2, Type 2 effector CD4+ T cells (Th2) and eosinophils. Paradoxically, oral ETEC-induced protection against lung allergen exposure was dependent on IL-33: IL-33KO mice developed partial lung inflammation in this model, and no protection induced by ETEC exposure was observed. Importantly, protection from allergy was achieved with ETEC heat-labile toxin (LT) oral exposure; we also found that the toxicity domain of LT is needed for this protection. Our results indicate that gastrointestinal bacterial infection mediators (toxins) negatively regulate the onset of allergic inflammation and may provide a novel immunological basis for the “hygiene hypothesis”.

R01AI170649 FAPESP 2021/15185-2
Combination immunotherapy of ibrutinib, fludarabine, and pembrolizumab for high-risk CLL patients: clinical and immunological analysis of a pilot phase II study
Authors: Mu Rui , Ahn Inhye , Sun Clare , Chen Jonathon , Singh Komudi , Wiestner Adrian
Published: August 2023

Chronic lymphocytic leukemia (CLL) is due to clonal expansion of CD5+ B cells. Immunosuppression is a hallmark of CLL and includes impaired anti-tumor T cell responses. Exhaustion of PD-1+ CD8 T cells has been implicated in disease progression, providing a rationale to test checkpoint inhibitors in the clinic. However, single-agent anti-PD-1 antibodies lacked clinical efficacy. Here, we analyzed immunologic parameters in patients enrolled on a phase II study of ibrutinib (continuous administration), fludarabine (2 cycles) and pembrolizumab (for 18 cycles after fludarabine). 13 patients were enrolled, 10 patients were evaluable, 9 patients achieved partial response (90%) and 1 patient complete response (10%). Bulk RNA-seq of CLL and T cells revealed inhibition of B-cell receptor and NF-kB signaling in tumor cells, and increased proliferation and activation gene signatures in CD3+ T cells. The proportion of Ki-67+ CD8 T cells increased in 5 out of 13 patients (immune responders) 3 weeks after the first dose of pembrolizumab. Compared with immune non-responders, responders had a significantly higher percentage of CD45RO-CD27+ and CD45RO+CD27+ CD8 T cells. At 6 months, T cell proliferation in all patients was back to baseline and there was no change in TCR clonality at 12 months. In vitro stimulation with CD3 and CD28 antibodies upregulated activation markers on CD8 T cells in all patients, increases in Ki-67 were variable. Overall, responses in patients were weaker than in healthy controls. In summary, pembrolizumab in combination with ibrutinib transiently increased CD8 T cell proliferation in a subset of CLL patients but did not improve the clinical response over ibrutinib.
Antiviral vaccines for local immunotherapy against solid tumors
Authors: Cuburu Nicolas , Sethi Shiv , Bialkowski Lukasz , Thompson Cynthia , Pontejo Sergio M , Schiller John T
Published: August 2023

Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor immune microenvironment and anti-tumor immunity. Here, we interrogate whether preexisting anti-vaccine immunity induced by licensed subunit vaccines could be leveraged for local cancer immunotherapy in the syngeneic murine tumor models. We selected Shingrix, a VZV vaccine containing the glycoprotein E (gE) antigen and adjuvant AS01B, and Gardasil-9, a HPV vaccine containing the L1 virus-like particles and alum because of the CD4 and CD8 T cell responses they respectively induce.

Intratumoral injection of Shingrix alone or with immune checkpoint blockade (CTLA-4), in prevaccinated mice delayed tumor growth and often led to complete regression. These responses were associated with the induction of CD8+ T cell responses against tumor associated antigen, to tumor immune activation and alteration of the myeloid compartment. The injection of selected MHC-II-restricted gE minimal peptide epitopes combined with polyI:C also led to durable remission suggesting a contribution of gE-specific CD4 T cells. In contrast, Gardasil-9 i.t. injection did not delay tumor growth or cause tumor rejection which suggests inefficient class I cross-presentation of native VLP in the tumor cells. However, the injection of MHC-I-restricted L1 minimal peptide epitopes led to complete and durable remissions suggesting efficient tumor control by L1-specific CD8 T cells.

Our results suggest that anti-viral licensed vaccines can be leverage as a new class of immunotherapeutics for local cancer therapy and intrinsic immunogenicity properties of vaccines should be considered as these effects appeared to be vaccine specific.

NIH Intramural program.
Uncovering the role of the local environment in shaping antiviral immunity of the female reproductive tract
Authors: Segrist Elisha , Belkaid Yasmine
Published: August 2023

The female reproductive tract (FRT) is a unique barrier site. It tolerates foreign entities like sperm and the fetus for reproduction but must also be immunologically active enough to control pathogens, like viruses. Millions of women use antibiotics or anti-retrovirals, and these treatments alter the environment of the FRT by changing the microbiota or inhibiting endogenous retroviruses, but the impact of this on FRT immunity and function are understudied. Human cohort data has revealed that the vaginal microbiota profoundly influences risk of virus infection, but the mechanisms for this are not well understood. Endogenous retroviruses (ERV) are necessary for reproduction, as an ERV envelope protein is essential for placental formation, but how these elements shape immunity of the FRT is barely characterized. Preliminarily, I have found that both the microbiota and ERVs shape innate and adaptive immune cell populations and functions in the FRT. Additionally, I found that microbial derived ligands such as LPS are sufficient to induce ERV expression in the uterine epithelium. Therefore, the main hypothesis tested by my project is that the vaginal microbiota induces ERV expression in the epithelium of the reproductive tract which in turn activates host nucleic acid sensing pathways and interferon production in the epithelium and that this response promotes local antiviral protection.

Y.B. was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID; ZIAAI001115-09, ZIAAI001132-07, ZIAAI001133-08). E.S. was supported by the Training Office and OITE of the National Institute of Allergy and Infectious Diseases (Intramural AIDS Research Fellowship).
Molecular heterogeneity of antiviral resident memory CD8 T cell in the female reproductive tract
Authors: Hasan Mohammad H , Mithila Farha J , Lin Ying , Wallace Joselynn , Pacia Jessica , Zhang Nu , Beura Lalit K
Published: August 2023

Resident memory CD8 T cells (TRM) in the female reproductive tract (FRT) represent a primary defense mechanism against viruses. Presence of CD8 TRM in the reproductive mucosa is associated with accelerated viral control and rapid establishment of an antiviral state. However, little is known about the factors that control and maintain TRM in this important barrier organ. Here, using single-cell RNA-sequencing (scRNA-seq) and genetic methods, we characterized development of distinct FRT CD8 TRM subsets. Our scRNA-seq studies revealed that most FRT CD8 TRM belong to two transcriptionally distinct subsets that are marked by mutually exclusive surface expression of CD103 and Ly6C. CD103+ TRM were dependent on the cytokine TGF-b for development, as CD8 T cells lacking TGF-b receptors were significantly impaired at giving rise to CD103+ TRM. Local activation of TGF-b by av integrins was important for the maintenance of these FRT TRM. In comparison to the CD103+ TRM which resembled mature TRM, the Ly6C+ TRM exhibited features of effector cells. Ly6C+ TRM were regulated by the transcription factor T-bet and developed under distinct priming conditions compared to CD103+ TRM. Upon in vivo antigenic recall, the CD103+ subset elaborated greater cytotoxicity than the Ly6C+ subset. Taken together, our findings provide key insights into molecular and developmental heterogeneity of FRT CD8 TRM which has implications for improving vaccination and local immunotherapy approaches.

Supported by grants from NIH (P20GM121298)
SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease
Authors: Alsiyaghy Jassam , Graefen Bahar , Aliyeva Leyla , Lnu Arina , Tauseef Mohammad , Fazal Nadeem
Published: August 2023

Sphingosine kinases (SPHKs) catalyze the formation of sphingosine 1 phosphate (S1P) from sphingosine. S1P, a lipid mediator, was shown to be one such factor promoting endothelial barrier function. S1P binds to S1P receptor-1 (S1PR1) in endothelial cells, leading to activation of heterotrimeric G proteins (Gi), and signals enhancement of endothelial barrier function by the activation of small GTPase Rac1. We hypothesized that SPHK1 via generation of S1P induces reannealing of adherens junctions and thereby prevents thrombin-induced endothelial permeability. To understand the role of SPHK1 pathway, we depleted SPHK1 protein in endothelial cells using SPHK1 specific small interference (si) RNA. Depletion of SPHK1 significantly disrupted the adherens junction, VE-cadherin and increased the gap formation of endothelial cells. Upon depletion of SPHK1 we found decrease cell proliferation and migration, but no change in the expression of Sphingosine 1 Phosphate Receptor 1 (S1PR1) expression in endothelial cells. To determine if in the absence of SPHK1, does S1P has any pharmacological role, we treated the cells with Sphingosine 1 Phosphate (S1P) at the dose of 1μM. We found that S1P re-anneal the adherens junctions and improve cell proliferation and migration in SPHK1 depleted endothelial cells. This indicates that SPHK1 via generation of S1P prevents endothelial permeability through S1PR1 receptor activation. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process.

CTRE grant to Professor Nadeem Fazal, MD, PhD
Identification of p-eIF4E as a new regulator of regulatory T-cell activity
Authors: Santinon François , Papadopoulos Theodoros , Henderson-Berg Meagan-Helen , Gonçalves Christophe , Abrahams Madelyn , Gagnon Natascha , Gupta Vrinda , Chou Hsiang , Miller Wilson H , del Rincon Sonia Victoria
Published: August 2023

Objective

Phosphorylated Eukaryotic translation initiation factor 4E (p-eIF4E) is a critical regulator of protein synthesis and is phosphorylated by MNK1/2 to promote the translation of a mRNA subset. We have shown that blocking the eIF4E phosphorylation increased the anti-tumor immune response, especially the CD8 T cell activation. However, the role of p-eIF4E in CD4 T cell subsets, and in particular regulatory T cells (Tregs) is still unknown. The aim of this study was to explore the impact that the absence of p-eIF4E could have on the Treg activity as well as in an inflammatory context.

Methods

To investigate the role of p-eIF4E on Tregs activity, we used genetically modified mice expressing a non-phosphorylatable form of eIF4E (KI mice). First, we analyzed Treg activity in WT and KI mice and the same mice subjected to dextran sulfate sodium (DSS)-induced colitis. We also analyzed the colonic immune cell infiltration using spectral flow cytometry and CODEX technology.

Results

Using our mouse models, we observed that the p-eIF4E lack led to a decrease in Treg stability as well as a decrease in their ability to control the helper T cell (Th) proliferation. In the colitis context, we observed an increase in the disease severity in KI compared to WT mice characterized by an increase in colonic immune infiltration. Moreover, our mesenteric lymph node and colon immunophenotyping revealed a significant decrease in Treg, and an increase in Th expressing IFNγ in KI compared to WT mice. Finally, we observed a decrease in KI Treg ability to migrate to the lymph nodes.

Conclusion

These results demonstrate for the first time the preponderant role of p-eIF4E in the control of Treg stability and Treg migration but also in their ability to regulate inflammation.
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

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

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

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

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

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

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

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

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 >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

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

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

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.

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