bluuebunny/crossref_metadata_2025 huggingface dataset:
Deep myeloid cell profiling provides new insights into modulators of CAR T cell expansion in patients with pediatric solid tumors
Authors: Kaczanowska Sabina , Murty Tara , Alimadadi Ahmad , Contreras Cristina F , Hedrick Catherine C , Ramakrishna Sneha , Kaplan Rosandra
Published: August 2023

Chimeric antigen receptor (CAR) T cells have shown remarkable results in hematological malignancies but limited efficacy in the setting of solid tumors. As myeloid-mediated immune suppression is known to play an important role in dampening antitumor activity, we hypothesized that myeloid cells impact CAR T cell expansion in patients with solid tumors. A phase I trial (NCT02107963) was performed to determine the feasibility and safety of administering 3rd generation GD2-CAR (GD2-CAR.OX40.28.z.ICD9) T cells in children and young adults with neuroblastoma and osteosarcoma. 76.9% of patients had stable disease by day 28, eventually all patients had disease progression. We stratified patients based on good versus poor CAR T cell expansion and evaluated peripheral immune profiles pre- and post-treatment by qPCR, ELISA, CyTOF, ATAC-seq, and RNA-seq. While A higher proportion of monocytes in pre-treatment apheresis was associated with poor CAR T cell expansion and CXCR3 expression on monocytes was the most robust marker of good CAR T cell expansion in this cohort. Longitudinal analysis demonstrated that CXCR3+ monocytes were low following treatment in both good and poor CAR T cell expanders, demonstrating a transition in myeloid populations in response to GD2-CAR T cell treatment. Together, these data suggest that GD2-CAR T cell administration is associated with changes in the myeloid cell compartment. This study provides evidence of novel myeloid-based pre-treatment biomarkers of CAR T cell expansion and rationale for the combination of CAR T cells with myeloid-modulating therapies as a strategy to improve outcomes for patients with solid tumors.

Clinical trial supported in part by: Intramural Research Program, National Cancer Institute, NIH Clinical Center, National Institutes of Health. Scientific and financial support for the CIMAC-CIDC Network are provided through the National Cancer Institute (NCI) Cooperative Agreements: U24CA224331 (to the Dana-Farber Cancer Institute CIMAC), U24CA224309 (to the Stanford University CIMAC), and U24CA224316 (to the CIDC at Dana-Farber Cancer Institute). Scientific and financial support for the Partnership for Accelerating Cancer Therapies (PACT) public-private partnership (PPP).
The landscape of glioma-infiltrated immune cells after immune checkpoint blockade treatment
Authors: Kang In , Park Janghyun
Published: August 2023

Despite the immune checkpoint blockade (ICB) having succeeded in multiple cancer therapies, high-grade gliomas such as glioblastoma multiforme (GBM) have a very low response rate to ICB, and GBM patients still do not benefit from ICB. To overcome this limitation, understanding the unique immunologic characteristics of brain tumors is needed. Here, we used single-cell RNA sequencing to analyze the macroscopic immune landscape of the tumor microenvironment (TME) under the treatment of ICB in a murine GBM model. CD8+ T cells showed both invigoration and exhaustion states, representing that CD8+ T cells show a mixed phenotype in the brain tumor. Furthermore, we found an increase in the number of anti-inflammatory macrophages in the TME after the ICB treatment, which is dependent on the increased CCL5 expression. Therefore, we hypothesized that the recruitment of anti-inflammatory macrophages mediated by CCL5 is associated with the exhaustion state of CD8 T cells and subsequent unresponsiveness of GBM against ICB. Finally, we compared our observations in the murine GBM model with publicly available data obtained from recurrent GBM patients. Our study provides critical information for the immune landscape of glioma after the ICB treatment and potentiates the development of novel immunotherapies to overcome the proposed limitations of ICB treatment.
Insights into the regulation of brain metastasis by natural killer cells using single-cell RNA sequencing and imaging mass cytometry
Authors: McMullen Timothy P , Longworth Aaron , Ma Dennis , Adam Isam , Evans Katrina T , Lawson Devon A
Published: August 2023

Metastatic brain tumors are incurable, rapidly fatal, and five times more common than primary glial or meningeal tumors of the central nervous system (CNS). Natural Killer (NK) cells are cytotoxic lymphocytes with strong in vivo anti-tumor activity, a native ability to cross the blood-brain barrier (BBB), and a demonstrated role in the regulation of metastasis in peripheral organs. NK cells perform critical roles in anti-tumor immunosurveillance via the expression of germline-encoded activation and inhibition receptors on the cell membrane. These receptors recognize ligands on both normal and transformed cells, and ultimately determine whether an NK cell will be stimulated to kill a neoplastic cell or inhibited from killing a healthy cell. Notably, NK cells are under investigation in clinical trials as both drug targets and adoptive cell therapies. We are working to characterize NK cell-mediated regulation of brain metastasis, which appears to be dysfunctional relative to the robust NK cell anti-metastatic activity observed in peripheral tissues. We seek to elucidate a molecular mechanism for this NK cell dysfunction using in vivo models of brain metastasis in conjunction with flow cytometry, single-cell RNA sequencing (scRNA-seq), and imaging mass cytometry (IMC). An improved understanding of the functional status and transcriptional programs of NK cells in brain metastasis will inform the development of neuroprotective anti-metastatic therapies aimed at modulating and potentiating the anti-tumor action of both native and adoptively-transferred NK cells.

Supported by grants from the National Cancer Institute (R01CA237376-01A1), the UCI Stem Cell Research Center, and the UCI Medical Scientist Training Program
High-dimensional profiling of circulating dendritic cells and monocytes in atopic dermatitis patients by mass cytometry
Authors: Jeong Soyoung , Bang Yoon Ji , Lee Brian H , Lee Dong Gun , Lee Yu Jin , Kim Hyun Jee , Kang Hoon , Park Chung-Gyu , Lee Dong Hun , Kim Jung Eun , Kim Hyun Je
Published: August 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disorder, with a multifactorial pathophysiology, including barrier dysfunction, dysbiosis and immune dysregulation. Although AD has been characterized by a T helper type 2 cell response, the role of the myeloid populations in the pathogenesis of AD still remains unclear. Here, we used mass cytometry, a high-dimensional analysis tool, primarily focusing on these cell populations to identify differences in the immune phenotypes of circulating dendritic cells and monocytes in AD. Peripheral blood mononuclear cells (PBMCs) from 48 Korean AD patients and 48 age-, sex-matched healthy controls were profiled. We report significant differences in the frequency and phenotypic markers of dendritic cells and monocytes. The frequency of CD141+ CLEC9A+ cDC1 was significantly decreased in AD, and its frequency negatively correlated with disease severity and serum IgE levels. This suggests that cDC1 may have preferentially migrated to the lesional skin to control the inflammation, especially in severe AD. CLEC9A expression was significantly reduced, while FceRIa expression was significantly increased in cDC1. The expression of FceRIa was also elevated in cDC2, pDC, and Axl+ DC. In cDC2, HLA-DR expression was decreased, and CD163 expression was increased. These results indicate changes in the immunophenotypes of circulating dendritic cells in AD compared with healthy controls. Although further investigation is needed, our results suggest a potential role of dendritic cells in modulating AD pathophysiology.
Regulation of eosinophils by the transcription factor Ikaros
Authors: Schjerven Hilde , Vestbøstad Johanne , Howell William
Published: August 2023

Immune cell development is a tightly controlled process that enables a homeostatic level of innate and adaptive immune cells of the myeloid and lymphoid lineages, respectively. Imbalance in immune cells can lead to diseases due to immune defects or over-activation. such as immunodeficiency, allergies or autoimmunity. Ikaros is a transcription factor known to be critically important for lymphoid development, with recurrent mutations in different human diseases, but less is known about the role of Ikaros in myeloid development. Ikaros is encoded by the Ikzf1 gene, and contains four DNA-binding zinc fingers (ZnF) and two ZnFs mediating protein dimerization. The four DNA-binding ZnFs (ZnF1 through ZnF4) are encoded by three exons, with the two central ZnF2 and ZnF3 encoded by exon 5 being required for binding to the core DNA recognition site. The two flanking ZnFs 1 and 4 are encoded by exon 4 and exon 6, and can modulate DNA binding by contacting the flanking DNA. Interestingly, Ikaros is highly alternatively spliced, creating multiple isoforms with different combinations of the DNA-binding ZnFs. We study the role of Ikaros in immune cell development using Ikzf1-mutant mouse models, and analysis of mice with deletions of exon 4 or exon 6 (encoding ZnF1 and ZnF4, respectively) has revealed differential phenotypes in the two mutant mouse models. Here, we describe a new role of Ikaros in regulation of eosinophils. We have found that Ikaros is required for limiting eosinophil production in bone marrow (BM), as both mutants show increased levels of BM eosinophils. Interestingly, the levels of peripheral tissue eosinophils differ between the two mutants, suggesting a second role of Ikaros in regulating migration, tissue recruitment or retention.
Metformin treatment of CD8 T cells decreases pro-inflammatory cytokine production and shifts their metabolic state toward oxidative metabolism
Authors: Molloy Caitlyn Therese , Alwarawrah Yazan , MacIver Nancie
Published: August 2023

Obesity is a systemic metabolic disorder that affects over 40% of the U.S. adult population. Among many other serious health consequences, obesity leads to increased morbidity and mortality from select infections and impaired immunological memory responses to these infections. CD8 T cells mediate the clearance of pathogens and establish immunological memory. Many studies have established that T cell metabolism and function are linked, and that changes in T cell metabolism can alter T cell responses; however, it is currently unknown how obesity impacts CD8 T cell metabolism and their ability to respond to pathogens. Furthermore, it is also unknown if drugs used to reverse the metabolic effects of obesity can alter CD8 T cell metabolism and impact CD8 T cell response to infection. To test this, we first isolated splenic CD8 T cells from lean or diet-induced obese (DIO) mice, activated them in the presence or absence of the drug metformin, and measured T cell function and metabolism using flow cytometry and extracellular flux analysis. CD8 T cells activated in the presence of metformin showed significantly decreased production of IFN-γ and Granzyme B and decreased oxidative metabolism. To note, we saw a similar decrease in oxidative metabolism in splenic CD8 T cells isolated from DIO mice that were treated with metformin systemically for six weeks. These results demonstrate that metformin treatment either in vitro or in vivo affects CD8 T cell metabolism and function. To determine if metformin treatment improves immunological memory in the context of infectious diseases, future studies will include immunological analyses of CD8 T cells from lungs of influenza virus-infected lean and DIO mice that have been treated with out without metformin.

Supported by grants from NIH (R01-DK106090)
Sargassum horneri suppresses PM-induced oxidative DNA damage in alveolar macrophage (AM) of allergic asthma: Relevance to PM-mediated M1/M2 AM polarization
Authors: Jin Kim Hyo , Yang Jiwon , Herath Kalahe Hewage Iresha Nadeeka Madushani , Jeon You-Jin , Kim Hyun Jung , Jee Youngheun
Published: August 2023

Particulate matter (PM) exposure causes oxidative damage to the lung by attaching to the alveolar macrophages (AM) and alveolar epithelial cells (AEC). Sargassum horneri can be a promising source to mitigate PM-induced oxidative damage. This study investigated the role of Sargassum horneri ethanol extract (SHE) against PM-induced oxidative DNA damages in allergic asthma mice. We discovered that PM triggered prolonged oxidative stress leading to lipid peroxidation in AEC and AM. PM-induced oxidative stress in AM caused the increase of CD80+ AM (M1 phenotype) and CD206+ AM (M2 phenotype) populations, especially, the number of M2-polarized AM, producing skewed AM polarization. Furthermore, we observed high expression of 8-OHdG, an oxidative DNA damage marker, in AM, the accumulation of which caused apoptotic cell death. However, SHE treatment, however, attenuated PM-induced lipid peroxidation and recovered balance of antioxidant enzymes in AEC and AM of allergic asthma mice. It resulted in the decrease of AM polarization mitigating the oxidative stress. More importantly, SHE salvaged AM from apoptotic cell death by attenuating oxidative DNA damage. Therefore, we demonstrated that SHE ameliorates oxidative DNA damage of M1- and M2-polarized AM in PM-induced allergic asthma mice in this study.

This research was funded by the Ministry of Education (2019R1A6A1A10072987) and the Ministry of Oceans and Fisheries, Republic of Korea (Project NO. 20172085).
Pulmonary ILC2-derived serotonin mediates RSV-evoked airway hyperreactivity in mice
Authors: Luo Chia-Hui , Chang Ya-Jen
Published: August 2023

Respiratory syncytial virus (RSV) infection is epidemiologically linked to asthma. During RSV infection, lung epithelial cell-released IL-33 is elevated and promotes group 2 innate lymphoid cells (ILC2s) activation, leading to type 2 cytokines production and asthma development. Serotonin is a neurotransmitter involved in numerous biological functions in the central nervous system as well as peripheral tissues. Previous studies have found elevated serotonin levels in plasma and bronchoalveolar lavage fluid (BALF) from asthma patients, implying that serotonin may involve in the pathogenesis of asthma. Therefore, we aimed to elucidate the role of ILC2-released serotonin in the regulation of RSV-induced airway hyperreactivity (AHR). To investigate whether ILC2 would secrete serotonin upon activation, pulmonary ILC2s were sorted from C57BL/6 mice and then treated with IL-33 in vitro, and we found an increase in the production of serotonin from IL-33-activated ILC2s. Furthermore, elevated BALF serotonin levels were also found in RSV-infected mice. Moreover, we clarified that ILC2 as a major source of serotonin during RSV infection. Next, to determine the role of serotonin in RSV-evoked AHR, we found that the treatment of Clozapine (serotonin receptor inhibitor) significantly attenuated AHR and decreased IL-13 expression. Meanwhile, we demonstrated that the administration of exogenous serotonin exacerbated RSV-caused AHR. Furthermore, serotonin enhanced IL-5 and IL-13 production from IL-33-activated ILC2 in vitro. Taken together, our results indicated that pulmonary ILC2-derived serotonin contributes to RSV-evoked AHR and exacerbates type 2 cytokines through a positive regulation of ILC2 function.
Identifying the expression and function of Blimp-1 in ILC2s in allergic asthma
Authors: Zheng Yibo , Zhang Jinyi , He Kun , Poholek Amanda C
Published: August 2023

Allergic asthma is driven by type 2 inflammation from Th2 cell and Type 2 innate lymphoid cells (ILC2s). We recently demonstrated a critical role for Blimp-1, a transcriptional repressor, in Th2 cells to promote allergic asthma. However, the expression and function of Blimp-1 in ILC2s remains undefined. Using genetically modified mouse strains that report Blimp-1 expression via YFP, we surveyed Blimp-1 expression in allergic asthma and other type 2 immune models across various tissues. We found that Blimp-1 is induced in ILC2s in the lung in response to the typical alarmins IL-33 and IL-25, as well as house dust mite (HDM). However, ILC2 activation did not upregulate Blimp-1 in the skin or gut in response to peanut allergen, suggesting a tissue-specific requirement for Blimp-1 in ILC2s. IL-33, as known to be a potent activator of ILC2s, was not required for Blimp-1 expression upon HDM stimulation. Interestingly, IL-10, a potent driver of Blimp-1 in Th2 cells, induced Blimp-1 expression in ILC2s, suggesting IL-10 but not IL-33 may be important for a Blimp-1+ ILC2 state. Intriguingly, Blimp-1 expression did not correlate with cytokine production, potentially representing a unique functional state of ILC2 cells that are high for Blimp-1. To evaluate Blimp-1 function, we deleted Blimp-1 in ILC populations using IL-7Ra Cre crossed to Blimp-1 floxed mice and generated mixed bone marrow chimeras with IL-7Ra Cre control donors. Blimp-1 knockout ILC2s in response to HDM exhibited an alteration in markers of ILC2 activation including ST2, KLRG1 and GATA3, suggesting Blimp-1 plays a role in promoting ILC2 differentiation. Collectively, our study has identified a lung-specific state of ILC2s expressing Blimp-1 that are found in a model of allergic asthma.
Autocrine pro-inflammatory IL-10 initiates lung-specific Th2 responses to inhaled allergen to induce allergic asthma
Authors: He Kun , Xu Zhongli , MacDonald William A , Ray Anuradha , Chen Wei , Lambrecht Bart N , Poholek Amanda
Published: August 2023

Allergic asthma remains a significant health burden for both children and adults. CD4+ Th2 cells are critical drivers of disease, yet the mechanisms that support initiation of the Th2 cell response to environmental allergens are not well understood. We demonstrated a distinct requirement for the transcriptional repressor Blimp-1 to promote Th2 cells in the lung to inhaled but not systemically or subcutaneously delivered allergens. Using temporal, spatial and single cell transcriptomic tracking of house dust mite (HDM) specific T cell responses, we demonstrate that inhalation of HDM drove early Blimp-1 expression necessary for GATA3 upregulation and subsequent Th2 differentiation in the lung that coincides with IL2Rα expression. Blimp-1 expression remains confined to HDM-specific Treg and Th2 cells that traffic to the lung but importantly is dispensable for Th2 cell maintenance. We found that inhaled allergens induce a pattern of STAT activation with transient pSTAT5 concomitant with sustained pSTAT3 within GATA3+ allergen-specific T cells, which is critical for the induction of Blimp-1 during the earliest phase of T cell priming in the lymph node. IL2Rα cooperates with IL10Rα signaling acting directly on allergen specific T cells to drive Th2 cell differentiation. Furthermore, IL-10 derived from allergen specific T cells was sufficient to induce Th2 cells suggesting an autocrine or paracrine loop of IL-10 supports Blimp-1 to regulate GATA3 upregulation at the T-B border and subsequent Th2 differentiation. These data shed light on the steps initiating Th2 responses to inhaled allergens and identify an unexpected pro-inflammatory requirement for IL-10 and Blimp-1 driving inflammatory T cell responses to environmental antigens.
Low intensity vibration increases proliferation in human primary T cells: a non-invasive strategy to reduce “vein-to-vein” time in autologous cell therapies
Authors: Ashdown Christopher P , Chan Meilin , Rubin Clinton
Published: August 2023

Chimeric antigen receptor T cell therapy (CAR-T therapy), is revolutionizing cancer treatment. Nevertheless, autologous CAR-T therapy remains an inefficient process; CAR-T manufacturing is often prohibitively slow, requiring up to 4 weeks from white blood cell acquisition to infusion (“vein-to-vein” time). Our goal is to leverage the innate mechanosensitivity of human T cells through low intensity vibration (LIV) to promote proliferation and shorten the expansion phase of autologous CAR-T therapy. LIV is characterized as a low-magnitude (0.7g, where g=9.8ms−2) and high-frequency (30Hz) signal. LIV was delivered to human primary T cells in culture for two one hour bouts per day, with each bout separated by a 2h rest period. Following 5d, each group was analyzed for proliferation rates and activation status/exhaustion.

We found that subjecting T cells to LIV enhanced proliferation by 29% relative to sham controls (p<0.004). This indicates a significant increase in proliferation over a relatively short period of time. Overstimulation of T cells can cause the development of a phenotype commonly known as “exhaustion”, which is characterized by decreased effector function. We used flow cytometry to examine the effect of LIV on T cell activation and exhaustion and found that there were no significant differences in surface receptors linked with T cell activation (CD25, CD62L, CD69) or T cell exhaustion (TIM-3, LAG-3, and PD-1) between control and LIV groups after 5d exposure to LIV.

These data suggest that LIV could be used to shorten the vein-to-vein time for CAR-T therapy. Furthermore, the lack of exhaustion is equally important because it suggests that LIV expanded cells are able to proliferate faster while maintaining their functionality.

Supported by grants from NIH REACH (U01) HL127522 and SUNY Technology Accelerator Fund (TAF), with additional support from the Snyder Family Fellowship (SBU Cancer Center).
CAR T-cell intrinsic PD1 Dominant Negative Receptor (PD1DNR) functions by both cell-intrinsic and extrinsic mechanisms
Authors: Amador Alfredo , Hou Zhaohua , Chintala Navin , Adusumilli Prasad S
Published: August 2023

Objectives

We have shown that chimeric antigen receptor (CAR) T cells with T-cell intrinsic PD1 dominant negative receptor (PD1DNR) prolong functional persistence. Herein, we further define T-cell intrinsic and extrinsic mechanistic functions of PD1DNR.

Methods

Human T cells were retrovirally transduced with mesothelin targeted, CD28 co-stimulated CARs– either M28z (M) or M28z with PD1DNR (MDNR). Cytotoxicity, proliferation, phenotype, cytokine and effector and inhibitory protein expression were assessed by chromium release, cell count, flow cytometry, olink and mass spectrometry, respectively. MDNR extrinsic activity was measured by the ELISA for soluble PD1 and assessing cytotoxic functions of M cells following co-culture with MDNR supernatant. CAR T-cell functional persistence was assessed in vitro by repeated antigen stimulation and in vivo by tumor treadmill test and mice survival.

Results

Following repeat antigen stimulation, MDNR cells demonstrate retained cytotoxicity, proliferation, accumulation, and enrichment of TNF-related cytokines (≥1× Log2), and overexpression of T-cell activation and proliferation proteins (GZMA, GNLY, IFNAR2, MFAP4). Soluble PD1 was 10-fold higher in MDNR supernatants. Co-culture of M cells with MDNR supernatant demonstrated higher cytotoxicity (p<0.01). Mice treated with a single dose of MDNR show enhanced survival compared to M (median survival 68.5 vs. 27 days, P<0.01). MDNR displayed also superior in-vivo functionality against advanced stage disease (median survival 62 vs. 12 days, P<0.01).

Conclusions

PD1DNR expression in CAR T cells functions by both cell-intrinsic and extrinsic functions that are beneficial in solid tumors adoptive cell therapy.
LKB1 controls inflammatory potential through CRTC2-dependent epigenetic remodeling
Authors: Compton Shelby Elizabeth , DeCamp Lisa M , Kitchen-Goosen Susan M , Lau Kin , Dahabieh Michael S , Guak Hannah , VanderArk Alexandra , Vos Matthew , Williams Kelsey S , Shi Xiaobing , Rothbart Scott B , Krawczyk Connie M , Jones Russell G
Published: August 2023

Deregulated inflammation is a critical feature driving tumor progression in tumors harboring mutations in the Liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

Supported by the National Institute of General Medical Sciences (NIGMS, R35GM124736), the National Institute of Allergy and Infectious Diseases (NIAID, R21AI153997), the Paul G. Allen Frontiers Group Distinguished Investigator Program, the National Institute of Allergy and Infectious Diseases (NIAID, R01AI165722), and Van Andel Institute.
The effects of cannabinoid 1 receptor blockade on oxazolone-induced contact hypersensitivity mouse model
Authors: Han Ji Hye , Kim Wook
Published: August 2023

Contact hypersensitivity (CHS) is an experimental model of human allergic contact dermatitis, one of the representative skin diseases, where NLRP3 inflammasome plays a crucial role. Although the regulation of NLRP3 inflammasome activity by cannabinoid 1 receptor (CB1R) has been shown in metabolic diseases such as obesity and type 2 diabetes, the role of CB1R in CHS is unclear. Here, we investigate the putative role of CB1R in CHS by using a selective CB1R antagonist rimonabant. Pharmacological blockade of CB1R by rimonabant ameliorated oxazolone-induced CHS by suppressing hyperproliferation of keratinocytes and expression of pro-inflammatory cytokines in a TLR2/4-dependent manner. The inhibitory effect of rimonabant in oxazolone-induced CHS was not observed in NLRP3-deficient mice. These results suggest that CB1R could control TLR2/4 signaling pathway via NLRP3 inflammasome in CHS.

Supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2022R1I1A1A01071661)
Intercellular mitochondria transfer rescues aerobic respiration in metabolically compromised macrophages in vitro and in vivo
Authors: Brestoff Jonathan R , Giwa Rocky , Jia Wentong , Field Rachael L
Published: August 2023

Mitochondria evolved through an ancient endosymbiotic process and are essential for eukaryotic cellular metabolism. An emerging body of literature indicates that some cells export mitochondria for delivery to other cells, a process termed intercellular mitochondria transfer. Recently, we demonstrated that adipocytes transfer mitochondria to macrophages in vivo and that this process maintains white and brown adipose tissue homeostasis (Brestoff et al., Cell Metab, 2021; Rosina et al., Cell Metab, 2022; Borcherding/Jia et al., Cell Metab, 2022). It is believed that macrophages degrade exogenous mitochondria as a form of licensed mitophagy that supports adipocyte mitochondria quality control, however it is unclear whether externally-derived mitochondria are able to respire in macrophages once captured. We administered purified mitochondria to wildtype (WT) or NDUFS4 KO mice, which lack an enzyme required for mitochondrial Complex I activity. Although WT peritoneal macrophages quickly degraded exogenous mitochondria, KO peritoneal macrophages captured, retained, and utilized exogenous mitochondria to rescue cell-intrinsic defects in aerobic respiration. Similarly, healthy BV2 microglial cells quickly degraded captured mitochondria in vitro, but BV2 microglial cells exposed to rotenone and Antimycin A captured and utilized exogenous WT mitochondria to rescue the toxin-induced defects in aerobic respiration. Purified mitochondria from NDUFS4 KO mice failed to rescue aerobic respiration in toxin-stressed BV2 cells. These data suggest that macrophages with defective mitochondrial metabolism can obtain and utilize exogenous, cell-free mitochondria from their environment to overcome a metabolic crisis.

Supported by a grant from the Burroughs Wellcome Fund (CAMS #1019648) to JRB.
A novel role of EphA4/Stat6/Mertk signaling in cleaning up the brain after trauma
Authors: Soliman Eman , Gershenson Ilana , Basso Erwin Gudenschwager , Leonard John , Mills Jatia , Ju Jing , Tartaglino Chiara , Elhassanny Mohamed , Chen Michael , Wang Xia , Theus Michelle
Published: August 2023

Traumatic brain injury (TBI) is a major cause of disabilities and deaths from acute injuries in the US and worldwide. Neuroinflammation and neuronal apoptosis are hallmarks of TBI; however, the mechanism of clearance of apoptotic debris by efferocytosis remains ill-defined. Our previous studies demonstrated that EphA4 receptor in peripheral-derived macrophage (PDM) mediates the pro-inflammatory response after injury. Here, we aimed to evaluate whether PDM-specific EphA4 restricts efferocytosis following TBI. We generated wild-type (WT) and EphA4-knockout (KO), GFP+ bone marrow (BM) chimeric mice and performed Controlled cortical impact injury. We observed that KO mice displayed improved motor function, cerebral blood flow, reduced cortical cell death as measured by TUNEL, and enhanced efferocytosis compared to WT mice. The percentage of GFP+/Iba1− expressing PDMs that engulf NeuN+ or TUNEL+ cells was significantly higher in KO mice (p<0.05, n=5). We further performed an in vitro efferocytosis assay using pHrodo-labeled apoptotic Jurkat cells co-cultured with GFP+ WT and KO BM-derived macrophages. We found a significant increase in efferocytosis efficiency in KO compared to WT cells (p<0.05, n=4). In addition, mRNA isolated from KO cells show increased expression of the efferocytosis receptor Mertk and its ligands Gas6 and Pros1 and phospho-array analysis shows increased p-Stat6, a key regulator of Mertk/Gas6. Interestingly, the use of Stat6 and Mertk inhibitors attenuated efferocytosis effects in KO macrophages in-vitro. Our findings implicate the EphA4/Stat6/Mertk axis as a novel regulator of apoptotic debris clearance that may be targeted to augment resolution of inflammation following TBI.

National Institute of Neurological Disorders and Stroke of the National Institutes of Health, NS096281, NS119540, and NS121103 (MHT)
CD8α identifies human NK cells with altered proliferation, metabolism, and IL-15 signaling
Authors: Cubitt Celia , Wong Pamela , Tran Jennifer , Foltz Jennifer Ann , Marin Nancy D , Marsala Lynne , Foster Mark , Schappe Timothy , Berrien-Elliott Melissa M , Fehniger Todd A
Published: August 2023

NK cells are cytotoxic innate lymphoid cells that can differentiate into memory-like (ML) NK cells following IL-12/15/18 stimulation. Previous work identified that CD8α expression on donor ML NK cells was negatively associated with outcome for cellular therapy of AML. However, the role of CD8α on human NK cells remains poorly studied, and murine NK cells are CD8α-precluding study in mice. We found that CD8α-NK cells had greater proliferation and survival in vitro and in vivo. Interestingly, we observed that CD8α expression was dynamic, and a subset of CD8α-NK cells upregulated CD8α (“new” CD8+) after IL-15 stimulation. Notably, the “new” CD8+ NK cells were the most proliferative in vitro and in vivo, and had significantly greater signaling following IL-15 stimulation. Strikingly, we also found that “new” CD8+ NK cells had significantly higher glucose uptake, nutrient receptor expression, and an enhanced capacity for glycolysis and oxidative phosphorylation. These results indicate that recent CD8α acquisition marks the ability of NK cells to respond to IL-15 signals to engage in the robust metabolic activity required for proliferation. We also investigated the role of CD8α in NK cell activation, given that CD8α can bind HLA and associate with Lck. In the absence of CD8α, NK responses (CD107a, IFNg, TNF) following activating receptor ligation were enhanced for several receptors, suggesting an intrinsic role for CD8α in inhibiting NK cell activation. Together, these results identify a novel association of CD8α with treatment outcome for NK cellular therapy and identify a previously unappreciated role of CD8α on human NK cell proliferation, metabolism, and response through activating receptors.

Supported by grants from NIAID F30AI16131
“The role of macrophage scavenger receptors in engineered biomaterial scaffold remodeling”
Authors: Faust Mondreakest T , Sadtler Kaitlyn , Destefano Sabrina , Fertil Daphna , Josyula Aditya
Published: August 2023

CD206, also known as mannose receptor C type 1, is used frequently as a marker to identify the M2 polarized macrophages in the investigation of immune responses to biomaterials. M2 macrophages help with tissue repair, inflammation reduction, building immune tolerance, and defense against excessive inflammation. CD206 is found on the surface of M2 macrophages and dendritic cells, and functions as a pattern recognition receptor for a variety of connective tissues in health, including pulmonary tuberculosis, liver fibrosis, and others. Previous research has shown a role of CD206 in internalization of collagen fragments in tumors. In this study, we aim to determine whether CD206 is more than just an M2 marker in response to biomaterials. We have found that adaptive immune cells as well as cross-presenting dendritic cells are critical for CD206 induction in biomaterial-treated muscle injury. In Batf3−/− mice, where CD206 expression is lost, we find hemosiderin deposition suggesting defects in phagocytosis. In future studies, we will use CD206-deficient to directly evaluate its role in scaffold remodeling at the site of injury.
TNF receptors orchestrate differential neutrophil responses critical to immunity against Staphylococcus aureus skin infection
Authors: Youn Christine , Pontaza Cristina , Wang Yu , Dikeman Dustin A , Joyce Daniel P , Alphonse Martin P , Nolan Sabrina J , Ahmadi Michael , Young Jeremy , Tocaj Aron , Garza Luis A , Miller Lloyd S , Archer Nathan K
Published: August 2023

As the leading cause of skin and soft tissue infections, Staphylococcus aureus is a major health burden and a continued threat to human health due to the emergence of antibiotic-resistance. Through this current study, we aimed to better characterize the protective immune mechanisms against S. aureus skin infection to identify novel targets for the development of host-directed immunotherapeutics as alternatives to antibiotics. Tumor necrosis factor (TNF) is a master regulatory cytokine whose inhibition is associated with the increased risk of S. aureus skin colonization and infection in humans. Despite the clinical implications, the contribution of TNF and its cognate receptors, TNFR1 and TNFR2, to immunity against S. aureus skin infection is unclear. Utilizing an in vivo mouse model of S. aureus intradermal infection, we determined that immune cell-derived TNF engages with both TNFR1 and TNFR2 to promote protection against S. aureus in the skin. Neutrophil-intrinsic TNFR1 and TNFR2 signaling directed differential antimicrobial mechanisms against S. aureus skin infections. Mechanistically, TNFR1 mediated neutrophil recruitment to the skin, whereas TNFR2 directed neutrophil activation (e.g.-NOX2 activation, ROS production, and PAD4-dependent neutrophil extracellular trap (NET) formation) and prevented systemic dissemination of skin infection. Collectively, our findings revealed non-redundant functions and cellular mechanisms mediated by TNFR1 and TNFR2 in neutrophils for immunity against S. aureus.

This study was funded in part by grants R01AR073665 (NKA), R01AR069502 (NKA), R01AI111205 (NKA), and R01AI146177 (NKA) and from the United States National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and National Institute of Allergy and Infectious Diseases (NIAID).
SARS-CoV-2 Envelope Protein Tolerizes Macrophage Response to Secondary Inflammatory Stimuli
Authors: Pierce Stephen H , Mclennan Rebecca , Khanal Santosh , Menden Heather , Sampath Vankatesh , Bradley Todd
Published: August 2023

Exposure of innate immune cells to bacteria or viruses may result in epigenetic rewiring that either enhances or weakens innate immune responses to a secondary challenge. One primary mechanism of innate immune cell function is through toll-like receptors (TLRs) that sense a variety of bacterial and viral molecules and cause immune activation. SARS-CoV-2 Envelope protein (E protein) has been shown to mediate innate immune activation, through engagement of TLR2. In this study, we determined the secretome of chemokines, cytokines and growth factors from monocytes in response to E protein stimulation and compared this response with other viral antigens and TLR agonists. Next, we stimulated monocytes with E protein followed by secondary stimulation with lipopolysaccharide (LPS) on monocyte-derived macrophages (MDM) 1 week later. Reduced expression of CCL3, CCL4, CCL5, CXCL10, TNF-A, and IL-12 upon secondary LPS or autologous E protein stimulation were statistically reflecting a more tolerant MDM state that is imprinted after E protein exposure. Finally, we demonstrated that immunizing neonatal mice with SARS-CoV-2 E protein induced proinflammatory cytokine secretion and lung tissue inflammation pathology and demonstrated a long-term impact on immune cell function and secondary response to LPS. Thus, E protein induces macrophage proinflammatory networks that lead to tissue inflammation, but also induce a more tolerant state after return to a resting state that could impact the ability to respond to secondary infection. Further understanding of the molecular pathways that drive this immune activation and tolerance could identify treatment targets to restore immunity after SARS-CoV-2 infection.
Understanding the role of CD22 on Macrophage and Dendritic Cell Function
Authors: Zastrow Alexi , Friedman David J , Crotts Sydney B , Rajcula Matthew , Hammer Brady , Elissa Mai , Shapiro Virginia Smith
Published: August 2023

At the terminal position of many glycan chains are unique sugars known as sialic acids. Sialic acids bind sialic acid immunoglobin-like lectins (Siglecs). Our lab is interested in the role of CD22 (Siglec-2), which binds to α2,6-linked sialic acid generated by ST6GalI. CD22 can associate with itself or other molecules on the same cells in a “cis” configuration or bind in “trans” with ligands on other cells. CD22 contains four ITIMs within its cytoplasmic tail and is primarily known to function as an inhibitory co-receptor of the B cell receptor (BCR). CD22-ligand interactions restrain CD22 and BCR association which dampens BCR signaling. While CD22 is usually characterized as a B cell specific protein, we found that CD22 is highly expressed in hybrid macrophages (CD11b+ CD11c+ F4/80+) and expression decreases with activation, implying that CD22 may modulate myeloid cell responses. Using novel macrophage and dendritic cell-specific CD22 cKO mouse models, we will explore how CD22 impacts myeloid cell activation. We propose that CD22 impacts myeloid cell function and modulating this interaction may improve immune responses.
Inflammation-induced development and migration of a novel monocyte-like dendritic cell population that regulates antiviral CD8 T cell immunity
Authors: Oliva Kimberly , Reagin Katie , Klonowski Kimberly D
Published: August 2023

We recently discovered a CD11c+MHC-II+CD103+CD11b+ monocyte-like dendritic cell (moDC) population that colocalizes with CD8 T cells in the draining lymph node (dLN) early after respiratory infection. Importantly, depletion of these cells enhances the formation of CD8+ tissue resident memory T cells (TRM) in the lung. Given the importance of TRMafter respiratory challenge, we aimed to understand the origin and migration of these moDCs. To this end, we intranasally (i.n.) infected C57Bl/6 mice with influenza virus A HK-×31 (×31) or with vesicular stomatitis virus (VSV), the latter which allows us to compare with identical systemic infection. moDCs were detected in the bone marrow (BM) ~2 days post infection, one day prior to their peak recovery from the dLN. BM moDCs phenotypically resembled dLN moDCs, including expression of the inflammatory monocyte-associated markers CCR2 and Ly6C. Adoptive transfer of BM Ly6C+ monocytes into congenically mismatched recipients showed a meaningful percentage of moDCs were monocyte-donor derived. Additionally, type I IFNs, which can induce monocyte differentiation and are crucial in early antiviral immunity, were required for moDC development. We showed that moDCs are decreased in the BM, and consequently the dLN of Ifnar1−/− mice compared to WT mice following respiratory infection. Lastly, moDCs were detected in the BM after intravenous (i.v.) VSV but not in the dLN. This could be attributed to the increased expression of CCL2, MIP-1β, and IL-12p40 in the dLN of i.n. VSV or ×31 infected mice compared to naïve or i.v. VSV infected mice, where inflammation is dispersed. Overall, we provide potential targets in the moDC development and migration process that could enhance antiviral CD8 T cell immunity.

Supported by grant from NIH (R21 AI131093)
Surfactant lipids inhibit PI3K-dependent signaling pathways induced by IL-4 in alveolar macrophages
Authors: Casals Cristina , García-Fojeda Belen , Tenreiro Paula , Minutti Carlos M
Published: August 2023

Alveolar macrophages (AMs) are less able to respond to IL-4 in vivo than macrophages from the peritoneal cavity, due to a still-unknown factor of the lung environment. The aim of this study is to investigate whether surfactant lipids, which are continuously endocytosed by AMs, could influence IL-4-mediated alternative activation and proliferation of AMs. To that end, AMs were preincubated with surfactant lipids and stimulated with IL-4 in the presence or absence of surfactant protein SP-A, an amplifier of IL-4 actions. We found that alveolar lipids reduced IL-4- and IL-4+SP-A-dependent arginase activity, the expression of genes associated with alternative activation, and proliferation of AMs. Mechanistically, endocytosed lipids decreased IL-4- and IL-4+SP-A-induced activation of the PI3K-Akt-mTORC1 signaling axis, but not the IL-4-dependent STAT6 axis. Lipid-dependent inhibition of the Akt/mTORC1 signaling axis is consistent with reduced IL-4+SP-A-driven glycolysis and mitochondrial respiration as well as decreased ATP citrate lyase expression and histone acetylation stimulated by IL-4. We conclude that surfactant lipids inhibit PI3K-dependent signaling pathways and suggest that decrease of surfactant lipids in chronic lung diseases might augment IL-4-dependent fibrotic responses.

This research is funded by the Spanish Ministry of Science and Innovation through Grant PID2021-123044OB-I00 to C. Casals
Identifying and targeting a subpopulation of dendritic cells with enhanced activation of adaptive responses - identifying first responders
Authors: Esser-Kahn Aaron P , Ung Trevor Wai-Ming
Published: August 2023

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of “first responders” (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and T cell responses in vivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.

7U01AI124286-03, 5 F32 AI147517
NF-κB activation and pro-inflammatory cytokine production is modulated by a single point-mutation in the RSV fusion protein
Authors: Santhin Austin Bruce , Waldstein Kody , Varga Steven M
Published: August 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide. Clinical studies have associated elevated levels of pro-inflammatory cytokines with severe RSV-induced disease. However, the viral mechanisms that control disease severity remain undefined. Infection with the RSV 2–20 strain resulted in increased IL-1β, IL-6, and TNF levels compared to infection with RSV A2. RSV 2–20 also induced significantly increased NF-κB activation 6hr and 12hr post-infection as compared to RSV A2. A recombinant A2 virus expressing the 2–20 fusion (F) protein induced pro-inflammatory cytokines and NF-κB activation to similar levels as wild-type 2–20. Specific amino acids in a region of the RSV F protein likely to interact with host cells contributed to these strain-dependent differences. A single point-mutation at amino acid position 63 of the RSV F protein significantly affected IL-1β, IL-6, and TNF cytokine levels as well as NF-κB activation. Our results demonstrate that the amino acid position 63 in the RSV F protein plays a critical role in the induction of pro-inflammatory cytokines and modulating disease severity in the animal model and may contribute to differential RSV pathogenesis.

Supported by grants from NIH NIAID (RO1) and the University of Iowa Immunology T32 training grant.
Defining parameters that influence susceptibility and protection to murine respiratory beta-coronavirus MHV-1 infection
Authors: Silva Elvia E , Moioffer Steven , Van De Wall Stephanie , Berton Roger , Meyerholz David , Harty John T , Badovinac Vladimir
Published: August 2023

Patients infected with SARS-CoV-2 can experience variable susceptibility and sepsis-like immune dysfunction. Also, patients with comorbidities such as previous septic events are more likely to be hospitalized and succumb to COVID19 complications. However, the extent to which respiratory beta-coronavirus pathology is dose dependent and produces sepsis-like dysregulation in an experimental murine model are not completely defined. We utilized an established SARS-like murine model by infecting Murine Hepatitis Virus 1 (MHV-1) susceptible C3H/HeJ mice intranasally and analyzed responses over time after primary and/or secondary challenges. Immunologically naïve C3H mice undergo pathological changes defined by dose-dependent changes in disease severity and lung infiltrate/edema. MHV-infected mice also experience sepsis-like lymphopenia and cytokine dysregulation in the blood. Importantly, a low/surviving dose (5×102 PFU) was identified that yielded no morbidity/mortality after infection of C3H hosts. However, this low dose provides antibody dependent but not CD4/CD8 circulatory T cell dependent protection against a lethal high dose MHV-1 re-challenge. In addition, while C3H hosts with history of prior infections were more resistant, C3H mice that survived surgical sepsis showed increased susceptibility to MHV-1 infection over their surgical sham controls. Together, these data define how infection dose, immunological status, and/or comorbidities influence outcome of primary and secondary beta-coronavirus infections in highly susceptible hosts.

Supported by grants from NIH (R35GM134880, T32AI007511)