bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel dual-reporter lineage tracing model reveals phenotypic and functional heterogeneity of tissue-resident macrophages
Authors: Eddins Devon James , Kosters Astrid , Ghosn Eliver E.B.
Published: January 2023

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis, where most develop during embryogenesis independently of long-term hematopoietic stem cells (LT-HSCs) and are maintained throughout life with minimal contribution from circulating monocytes. Recent studies have shown that, in certain conditions, infiltrating monocyte-derived (MD)MΦ can supplement TRMΦ, but remain distinguishable. However, the phenotypic and functional heterogeneity of TRMΦ versus their supplemented MDMΦ counterparts in each organ system has not been established. To address this gap, we have generated a novel dual-reporter lineage tracing model to delineate TRMΦ vs MDMΦ in vivo. We used a non-genotoxic method of HSC depletion in the Runx1Cre/eGFP mouse, which allows us to transplant LT-HSCs expressing mRFP1. These dual-reporter mice exhibit normal supplementation of MDMΦ (red) to the existing TRMΦ (green) pool. We have previously identified two subsets of peritoneal MΦ that exhibit dual ontogeny. However, an in-depth examination of the phenotypic and functional differences between these two MΦ lineages is lacking. Using our new model, we performed single-cell mRNA sequencing and high-dimensional flow cytometric analyses and reveal further heterogeneity in peritoneal MΦ across lineages, supporting the notion that infiltrating MDMΦ exhibit phenotypic divergence from TRMΦ with potential functional impact. Here, we report the generation and utility of an innovative model incorporating lineage-specific fluorescent reporters in radiation-naïve mice, an important tool for understanding phenotypic and functional diversity of MΦ with different developmental origins in vivo.
Identification of a CD117+CD71+ early neutrophil progenitor population in human bone marrow
Authors: Dinh Huy , Eggert Tobias , Meyer Melissa , Zhu Yanfang , Olingy Claire , Llewellyn Ryan , Wu Runpei , Hedrick Catherine C.
Published: January 2023

Neutrophils play critical roles in health and disease. Due to their very short half-life in blood and tissue, neutrophils are constantly replenished by bone-marrow progenitors. Thus, a comprehensive understanding of bone marrow neutrophil development is of paramount importance to identify how neutrophil production is altered in disease. Recently, two novel human neutrophil progenitor populations were identified; ‘human neutrophil progenitor’ or ‘hNeP’ (Lin-CD66b+CD117+) and ‘neutrophil precursor’ or ‘preNeu’ (Lin-CD66b+CD15+CD49d+). How these subsets fit into the neutrophil lineage is unclear. By using mass and flow cytometry, we show that hNeP are a heterogenous population containing a homogeneous progenitor subset termed ‘early neutrophil progenitor’ or ‘eNeP’ (Lin-CD66b+CD117+CD71+). Surface marker and RNA expression, together with the ability to form colonies in vitro suggest that eNePs, which constitute only ~0.14% of bone marrow neutrophils, are upstream of preNeu (~5% of bone marrow neutrophils). Furthermore, we have identified novel neutrophil surface markers associated with distinct developmental stages, including CD71. Intriguingly, CD71+ characterizes proliferating neutrophils, which are expanded in the blood of melanoma and lung cancer patients and detectable in human lung tumors. Collectively, our findings i) identify CD117+CD71+ eNeP as an early neutrophil progenitor population, ii) introduce a unified model of human neutrophil bone marrow development, iii) identify novel surface markers for distinct neutrophil developmental stages and iv) provide evidence for neutrophil progenitor expansion in cancer.
Mast cells as Immune modulators of behavior and brain cellular composition
Authors: Duque-Wilckens Natalia , Zweng Sonja , Teis Robert , Robison AJ , Moeser Adam J
Published: January 2023

Mast cells (MC) are involved in peripheral disease such as allergy/anaphylaxis, but they are also present in brain where they can modulate behavior. MC deficient KitW–sh/W–sh (sash) mice have been reported to show increased anxiety-like behaviors compared to wild type (WT) mice, but these studies did not consider potential interactions with sex and/or stress, which is relevant considering that MCs are sexually dimorphic and stress-sensitive. Here, wild type (WT) and sash male and female mice were exposed to control handling or mild environmental stress and then phenotyped for behaviors relevant to human mood disorders. Stress did not alter anxiety or motivated behaviors in WT. Compared to WT, sash showed increased anxiety, which was independent of sex or stress. A measure of depressive-like behavior in rodents is preference for sucrose water over regular water. In females, sash showed reduced sucrose preference compared to WT, which was exacerbated by stress. In males, control sash and WT showed similar sucrose preference, but stress induced sucrose anhedonia only in sash. To explore the underlying mechanisms, we studied the Nucleus Accumbens, a brain area involved in anxiety and motivation. We found that sash have a reduced number of astrocytes and a two-fold higher number of cells expressing FosB, a transcription factor induced by stress and involved in synaptic plasticity and behavior. Together, these data implicate MCs as key immune modulators of brain cellular composition and activity. Our ongoing investigations aim at deeply understanding the role that MCs play on mood and cognition, which could provide significant insights into the mechanisms underlying human psychiatric disease and inform new therapeutic targets.
Contribution of CD137-CD137L pathway in sciatic nerve crush-induced behavioral hypersensitivity and functional reduction
Authors: Wakley Alexa A , Cao Ling
Published: January 2023

The T cell costimulatory molecule CD137L is involved in the development of neuropathic pain following peripheral nerve injury. Previously, our work has shown that absence or neutralization of CD137L reduces hypersensitivity, sensory and functional impairment following a sciatic nerve crush (SNC). CD137L activation utilizes both a CD137-dependent and a CD137-independent pathway. To further determine the contribution of these pathways, we examined pain-like and functional recovery-related behavioral responses following intrathecal injection (IT) of a CD137 neutralizing antibody (αCD137; daily days 3–10) after SNC in male and female B6 mice. Mice were randomly assigned to 1 of 4 groups: PBS, 0.5μg isotype control, 0.1 or 0.5μg of αCD137, and then were evaluated for sensitivity (von Frey, Hargreaves, & cold plantar test), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functions before surgery and on post-surgery days 1–77. On post-SNC day 1, all groups showed changes in sensitivity and decreased sensory and motor functions. αCD137 treatment did not alter mechanical, heat or hypersensitivity compared to PBS- or isotype-treated controls. αCD137-treated groups showed faster sensory (pin prick score) and motor functional recovery (toe spread reflex, toe spacing), as well as slightly greater improvement in grip strength test compared to controls. Together, our results indicate partial involvement of the CD137-dependent pathway in SNC-induced sensory and motor function changes following peripheral nerve injury. (Supported by NIH/NINDS R01NS098426 (Cao))
Immune response reflecting the involvement of Complement components in Gulf War illness
Authors: Brahmajothi Mulugu V
Published: January 2023

Gulf War Illness (GWI) encompasses multisymptom disorders characterized by chronic fatigue exhibiting neuromuscular involvement, affecting a considerable number of veterans who served the Persian Gulf War between 1990–91. Every war brings home soldiers with some acute/chronic disorders, but this multisymptom illness categorized as GWI is being investigated for 28 years because the treatments have been only palliative in spite of the considerable effort to treat the veterans to wellness. There are still unmet needs in understanding the pathobiology of GWI. Several studies conducted so far attribute the cause to chemical and biological exposures. From in-vivo studies, it can be deduced that the combined repetitive exposures to various neurotoxic chemicals insistently altered cellular receptors. Further, prophylactic treatments such as pyridostigmine bromide, anti-nerve gas pills, glucocorticoids, corticosterone, and vaccines not only primed the immune system but also paradoxically triggered the CNS-glymphatic system to mount a neuroinflammatory response in some individuals. This study focused on relative estimates of complement components and the convertases in the plasma, considering the immune system is the primary defense consisting of high polymorphism combined with a wide range of soluble factors. Complement components from C1 to C9 show high variability in 25 veterans with GWI compared to 20 healthy controls and other referenced neuromuscular disorders. Although C1 is the first responder of the classical pathway, consistently and significantly C1q, C5b, C7, factor D, and properdin were upregulated with the highest variability in veterans with GWI (p<0.001), indicating distinct variance in the immune complex.
Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and -independent cellular responses in Alzheimer’s disease
Authors: Zhou Yingyue , Song Wilbur M. , Andhey Prabhakar , Swain Amanda , Levy Tyler , Miller Kelly R , Poliani Pietro L. , Cominelli Manuela , Grover Shikha , Gilfillan Susan , Cella Marina , Ulland Tyler K , Zaitsev Konstantin , Miyashita Akinori , Ikeuchi Takeshi , Sainouchi Makoto , Kakita Akiyoshi , Bennett David A , Schneider Julie A , Nichols Michael R , Beausoleil Sean A , Ulrich Jason , Holtzman David M , Artyomov Maxim , Colonna Marco
Published: January 2023

Alzheimer’s disease (AD) is the most common form of dementia that is initiated by extracellular plaque deposition and intraneuronal hyperphosphorylated tau aggregates. Reactive astrocytosis and microgliosis are secondary cellular responses to pathology that have gained increasing attention. Variants of the microglia receptor TREM2 increase AD risk and activation of “disease-associated microglia” (DAM) is dependent on TREM2 in mouse models of AD. To investigate global transcriptomic changes during AD pathology, we surveyed gene expression changes associated with AD pathology and TREM2 in Ab-driven 5XFAD mouse model and human AD by single nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a novel Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less palpable in TREM2 R47H and R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.
Aberrant regulation of IL-1 during pregnancy promotes the development of autism-related neurodevelopmental disorders in mice
Authors: Lukens John R , Lammert Catherine Rose
Published: January 2023

The etiologies of most neurodevelopmental disorders, including autism spectrum disorder (ASD), remain incompletely understood. Interestingly, recent epidemiological and experimental evidence suggests that hyperactive maternal immune activation (MIA) impedes normal brain maturation and can promote the development of autism-related phenotypes. Indeed, our studies and work by others demonstrate that offspring born to pregnant mice that were exposed to the innate immune activator PolyI:C develop many of the defining behavioral features of ASD, including defects in social preference, communicative impairments, and repetitive/stereotyped behaviors. Although increasing evidence implicates key roles for dysregulated gestational inflammatory responses in neurodevelopmental disorders, the specific immune pathways that provoke autism-related phenotypes remain poorly described. In our studies, we demonstrate that inhibition of IL-1 signaling during gestation provides significant protection against the development of social abnormalities, communicative deficits, and repetitive/stereotyped behaviors in the MIA model of neurodevelopmental disease. Moreover, we find that treatment of pregnant mice with bioactive IL-1beta is sufficient to induce the development of autistic-like behaviors in the subsequent offspring. Our findings identify key roles for IL-1 in neurodevelopmental disorders and suggest that IL-1 may serve as a biomarker to identify at-risk pregnancies.
Kinetics and potency of T Cell-mediated cytolysis of glioblastoma
Authors: Clements Mike , Logun Meghan T , Chvatal Stacie , Hayes Heather B , Millard Daniel C , Karumbaiah Lohitash
Published: January 2023

Glioblastoma is an aggressive brain cancer with no effective treatments and a prognosis of 12–15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. Assessing the efficacy and potency of T cell therapies in vitro and with high throughput is vital for development of this promising therapy. Cellular impedance-based assays offer sensitive, label-free, and continuous monitoring of cell proliferation and immune cell-mediated cytotoxicity, revealing both the potency and kinetics of T cell killing. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma and patient-derived glioma cells.

U87MG and patient-derived glioma cells were plated at densities ranging from 10k to 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density). After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were continuously monitored for over 120 hours.

As expected, higher densities of glioma cells proliferated faster and reached a higher impedance level at 24 hours. Addition of activated human T cells resulted in a decrease in impedance consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours.

Overall, activated human T cells were highly effective for cytolysis of U87MG and patient-derived glioma cells. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios.
Mast cells as mediators of astrocyte morphology associated with neuroinflammatory disease states
Authors: Sarno Erika L
Published: January 2023

Morphological deficits observed in astrocytes, the most abundant glial cell type in the central nervous system, are associated with various neuroinflammatory diseases. The morphological and molecular astrocyte changes associated with multiple sclerosis are well-characterized and concentrated in the white matter of the brain. While the exact mechanisms behind this autoimmune disease are not fully understood, mast cells play a key role in the progression of white matter demyelination. We hypothesize mast cells orchestrate morphological astrocyte changes that are associated with neuroinflammatory disease states. To test this hypothesis, we used GFAP as a marker to visualize astrocyte morphology in white matter tracts of wild type (WT) and mast cell deficient KitW–sh/W–sh (sash) mice. Using skeleton analysis, we quantified astrocyte characteristics such as number of endpoints, branches, and branch length. Compared with WT mice, sash mice exhibited fewer branches (77 vs. 54 branches/cell) and endpoints per cell (49.5 vs. 24.5 P<0.001). These findings suggest that mast cells may drive altered astrocyte morphology and function in white matter. In order to determine whether this effect is due to altered development, studies using sash mice at time points across the lifespan are in progress. In addition, we are using diphtheria toxin in combination with novel genetic lines to ablate mast cells acutely in adult mice. Along with new molecular techniques, this will allow us to better characterize the interaction between mast cells and astrocytes in the adult and developing brain. Unraveling this interplay will be critical to understanding the role of mast cells in neurodevelopment and neuroinflammatory diseases such as multiple sclerosis.
B cell Ezrin restricts the development of allergen-induced asthma
Authors: Gupta Neetu , Stroberg Alayna , Weiss Kelly , Miao Yanling , Sammeta Ajay , Asosingh Kewal
Published: January 2023

Asthma accounts for substantial morbidity and healthcare burden all over the world. Allergic asthma is characterized by recruitment of immune cells into the lung, mucus production and exaggerated narrowing of the conducting airways. B cells are critical upstream regulators of antibody-mediated immunity and inflammation, with several reports establishing their importance as producers of IgE, presenters of antigen and promoters of the Th2 response in asthma. However, the molecular regulators of the B cell asthma response remain poorly characterized. We have previously reported that Ezrin, a plasma membrane-actin cytoskeleton linker protein regulates B cell function. Here, we show that mice with B cell-specific deficiency of ezrin (Ez-def) display increased lung B cells and eosinophils, IgE production and inflammatory cytokines, and marked deterioration in respiratory function upon sensitization and challenge with house dust mite allergens, as compared to control Mb1cre/+ mice. On a mechanistic level, we observed that Ez-def B cells express higher levels of T cell activating proteins upon antigen stimulation and have enhanced chemotactic ability. Our data suggest that B cell intrinsic ezrin restricts allergic asthma, and may act as an important checkpoint regulator of allergic airway inflammation and hyperresponsiveness by regulating B cell recruitment, activation, antibody secretion and cytokine balance in the lungs.
A novel formulation of budesonide nasal spray (Budesolv 10) potentiates clinical performance in grass pollen allergic patients
Authors: Zieglmayer isabel , Schmutz René , Lemell Patrick , Unger-Manhart Nicole , Nakowitsch Sabine , Goessl Andreas , Savli Markus , Zieglmayer René , Prieschl-Grassauer Eva
Published: January 2023

Objective

Purpose of this DBPC study was to evaluate onset of action and non-inferiority of a novel low dose aqueous solution of Budesonide (“Budesolv 10μg”) to Rhinocort® aqua 64μg (RA) in adult grass pollen allergic patients.

Methods

Budesonide, a poorly water-soluble corticosteroid, currently marketed as suspension, was reformulated to enhance solubility and local concentration on the target mucosa. Subjects received Budesolv, the market comparator RA and placebo once daily for 8 days. Allergic symptoms were induced by 6h grass pollen challenges in the Vienna Challenge Chamber at days 1 and 8 of each treatment period. First dose was applied after 1:45h to evaluate onset of action of either treatment. Nasal, ocular, asthma symptoms and objective measures of nasal secretion and nasal obstruction were evaluated. A 95% confidence interval (CI) was calculated for difference in means between active treatments. Non-inferiority was stated if the upper limit of the CI did not exceed 115% of the reference. Onset of action was defined as first time point when the difference in nasal symptoms change from baseline between active treatment and placebo was p<0.05.

Results

75 patients concluded the study per protocol. The primary endpoint (TNSS; sum of obstruction, itch, sneeze and rhinorrhoea) was met proving non-inferiority of Budesolv compared to RA. A significant difference between Budesolv and placebo was shown for TNSS 2.45 h and for total asthma score 2 h after first dose. On day 8 Budesolv 10μg was significantly better than placebo for all parameters evaluated.

Conclusion

Non-inferiority of Budesolv compared to Rhinocort® was shown on day 8 of treatment. Early onset of action within 3h after first dose was evident for Budesolv only.
Oral administration of Sargassum horneri extract containing Mojabanchromanol attenuates PM-exacerbated allergic airway response in murine model of asthma
Authors: Youngheun Jee , Herath Kalahe Hewage Iresha Nadeeka Madushani , Kim Hyo Jin , Jeon You Jin , Kim Hyun Jung
Published: January 2023

Epidemiological studies indicate correlation with particulate matter (PM) and the pathogenesis of airway inflammatory lung diseases. We sought to identify whether Sargassum horneri ethanol extract containing mojabanchromanol (SHE) confers maximal health benefit against PM exacerbated allergic asthma. Using BALB/c mice undergoing sensitization and challenge with ovalbumin (OVA, 10 μg) we developed a PM exacerbated mouse model of asthma. After 2 weeks of sensitization, mice were challenged with PM (5 mg/m3) for 7 days consecutively together with oral administration of SHE (200, 400 mg/Kg), Prednisone (5mg/Kg) or PBS. SHE restrained Th2 polarization by attenuating transcription factors GATA3 and Signal transducer and activator of transcription (STAT) 5. This resulted in mitigated eosinophil infiltration in airway and balf and secretion of Th2 cytokines interleukin (IL)-4, IL-5, IL-13 in lung homogenates of PM exacerbated asthmatic mice. Immunoglobulin (Ig) E secretion in serum and activation of mast cells in trachea were markedly attenuated. Concomitantly, SHE further restrained the Th17 cell response in PM exposed allergic mice through attenuating expression of transcription factors RORγT, STAT3 and expression of relevant effector cytokines IL-17a. This further mitigated CD11b+ Ly6G+ neutrophil population in lung. Taken together, SHE significantly suppressed PM exacerbated histopathological changes, hyper secretion of mucus in asthmatic mice. These results suggest that SHE might portend a natural candidate against against PM exacerbated allergic asthma via concomitant inhibiting of Th2/Th17 responses. This research was supported by the Ministry of Oceans and Fisheries, Korea (No. 20170285).
TNFSF14 (LIGHT) Induces Airway Smooth Muscle Contraction
Authors: Miki Haruka , Kiosses William B , Herro Rana , Croft Michael
Published: January 2023

Asthma is a chronic inflammatory disorder characterized by reversible airway obstruction and airway hyperresponsiveness (AHR). The precise mechanism responsible for AHR is not known but it may involve excess mucus production and alterations in airway smooth muscle cell (SMC) activity. SMC from asthmatic patients are thought to generate more force and contract to a greater extent to allergens or certain inflammatory stimuli, but which inflammatory stimuli in asthmatics drive contraction is still not clear. Previously, we showed that the tumor necrosis factor superfamily member LIGHT (TNFSF14) promotes airway remodeling and AHR in mouse models of chronic asthma, despite having little effect on airway eosinophilia. Exogenous administration of rLIGHT to the mouse airways also induced smooth muscle hyperplasia. We found that LTβR and HVEM, the receptors for LIGHT, are expressed on both human and mouse SMC from the lungs, with LTbR primarily expressed on human cells, suggesting a direct effect of this T cell-derived cytokine. From studies in vitro with primary human SMC, we found that rLIGHT administration induced contraction of SMC in collagen-gels in 3D structures, and migration of SMC in monolayers. rLIGHT induced both actin polymerization and phosphorylation of myosin light chain which leads to actomyosin formation to induce contraction. rLIGHT promoted both canonical and non-canonical NF-kB in airway SMC, and these signals activated the small GTPase Rac1 and P21-Activated Kinase 1 (PAK1) which are involved in actin rearrangement and myosin light chain kinase activity. These results reveal direct evidence that LIGHT can drive SMC contraction and may directly regulate airway hyperresponsiveness relevant to asthma pathogenesis.
Allergic sensitization upon transdermal exposure to gluten and oral anaphylaxis in an adjuvant-free mouse model of wheat gluten allergy
Authors: Gangur Venugopal , Jorgensen Rick , Gao Haoran , Othman Aqilah , Raghunath Rajsri , Wade Labrawn , Chandra Shivam , Ng Perry KW
Published: January 2023

Wheat allergies are growing globally, similar to many other major food allergies. Exposure to wheat in sensitized subjects can be life-threatening because of the high risk of anaphylaxis. Wheat contains both gluten and non-gluten protein allergens. Previous animal model studies have shown that gluten, when co-administered with powerful adjuvants like alum, can elicit IgE responses and anaphylaxis upon systemic injections. Currently, it is unknown whether skin exposure to gluten, without adjuvants, can cause sensitization for oral anaphylaxis. Here, we tested the hypothesis that transdermal exposure to alcohol-soluble wheat gluten (ASWG) will sensitize mice for oral gluten-induced anaphylaxis. Balb/c mice were produced and maintained on a wheat-free diet during this study. Groups of mice were exposed to ASWG prepared from durum wheat, without adjuvant, using a novel transdermal exposure (TDE) method. Mice were sensitized by nine weekly exposures. A significant and robust ASWG-specific IgE antibody response was observed by the 4th TDE. Anaphylaxis upon oral challenge with ASWG after the 9th TDE was quantified by measuring elevation of plasma MMCP-1, which is a specific immune marker of IgE-mediated anaphylaxis in mice. Mice showed significant elevation of MMCP-1, confirming IgE-mediated anaphylaxis. These data demonstrate for the first time that skin exposure to ASWG, without an adjuvant, can clinically sensitize mice for oral gluten-induced anaphylaxis.
Attenuation of mast cell degranulation and pro-inflammatory cytokine release by rosemary extract and carnosic acid
Authors: Crozier Robert W.E. , Yousef Michael , Hicks Natalie J. , Tsiani Evangelia , MacNeil Adam J.
Published: January 2023

Mast cells are sentinel immune cells that drive normal and pathological inflammatory responses, most notably allergic inflammation. Allergen-induced mast cell activation results in rapid degranulation of preformed pro-inflammatory mediators in the early phase, and sustained release of newly synthesized pro-inflammatory mediators in the late phase. Plant compounds known as polyphenols, have been examined in models of pathology due to their inhibition of pro-inflammatory mediator release. Rosemary extract (RE) and three polyphenolic constituents: carnosic acid (CA), carnosol (CO) and rosmarinic acid (RA) have been found to inhibit signaling cascades required for pro-inflammatory mediator release. The objective of this study was to establish the inhibitory effects of RE in a mast cell model and dissect the polyphenolic mixture, focusing on CA, CO, and RA, evaluating their potential as novel therapeutics. Sensitized mast cells were stimulated with allergen and treated with RE (5–50 μg/ml), CA, CO or RA (1–100 μM). The β-hexosaminidase release assay was used to measure degranulation, and cytokine release was measured by ELISA. Degranulation was inhibited dose dependently to 10% when treated with RE, 14% CA and 17% CO (p<0.001), while RA had no effect. ELISA analysis showed RE and CA inhibit release of IL-6 (p<0.05, p<0.0001), TNF (p<0.05, p<0.0001), and IL-13 (p<0.05, p<0.0001) while CO and RA had no effect. These novel findings identify RE and CA as potent regulators of mast cell functional responses. Further dissecting the mechanism behind RE and CA mediated inhibition of pro-inflammatory mediator release, will support the establishment of these natural plant products as effective anti-inflammatory therapeutics.
A novel neuroinvasive infection modality for Francisella tularensis elicits neuroinflammation resulting in cellular damage
Authors: Muniz Mireya G Ramos , Spencer Charles T
Published: January 2023

Francisella tularensis (Ft.) is a gram-negative coccobacillus bacterium that causes the zoonotic disease tularemia in humans. Ft. causes the most severe form of the disease through inhalation, but is most commonly transmitted through direct contact with infected animal carcasses such as rodents and rabbits. Due to its extremely low infectious dose, high mortality rate, and potential use as a biological warfare agent, Ft. is classified by the CDC as a Tier 1 select agent. Ft. infection triggers an overactive inflammatory response, termed a “cytokine storm”, which produces excessive tissue damage of vital organs, leading to rapid death of the host before its adaptive immunity responses can be brought to bear against the infection. In the periphery, macrophages, paradoxically the target cell for Ft., provide innate immune defense against foreign material, including bacteria. Microglia, the resident macrophage-like cells of the central nervous system, rapidly respond to pathological changes, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. In the United States, several clinical cases of tularemic meningitis were diagnosed since 1931; however, there is no further research exploring the condition. We hypothesize that, after peripheral inoculation of Ft., the bacteria infiltrate the central nervous system using a Trojan horse-type mechanism and infect microglia, leading to overproduction of pro-inflammatory cytokines in the brain which then cause damage to surrounding neurons, ultimately leading to death.
Identification of antimicrobial molecules released by Th17 targeting both Gram positive and Gram negative bacteria
Authors: Agak George W , Mouton Alice , Teles Rosane M. B , Weston Thomas , Morselli Marco , Pellegrini Matteo , Modlin Robert L
Published: January 2023

TH17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of TH17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the TH17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known TH17 secreted protein with direct antimicrobial activity. We generated C. acnes-specific antimicrobial TH17 clones (AMTH17) with varying antimicrobial activity against C. acnes. A combination of RNA-seq and informatics analysis unraveled the transcriptional and antimicrobial gene profiles present in modules associated with the AMTH17 vs. non-antimicrobial TH17 cells. The expression of antimicrobial genes such as GNLY, GZMB, HIST2H2B also correlated with secreted antimicrobial proteins as measured by ELISA, and antimicrobial activity as determined in CFU assays. Scanning electron microscopy reveal that AMTH17s can release antimicrobial molecules that kill C. acnes and other Gram-positive and Gram-negative bacteria. Our study identifies a functionally distinct subpopulation of TH17 cells with an ability to secrete antimicrobial proteins in order to capture and kill bacteria.
Repeated febrile malaria exposure remodels monocytes/macrophages toward a regulatory/tolerogenic phenotype
Authors: Guha Rajan , Mathioudaki Anna , Arora Gunjan , Li Shangping , Siddiqui Shafiuddin , Doumtabe Didier , Doumbo Safiatou , Ongoiba Aissata , Traore Boubacar , Zaugg Judith , Crompton Peter D
Published: January 2023

In malaria-naïve individuals, P. falciparum (Pf) infection results in numerous Pf-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, repeatedly infected individuals in endemic areas are often asymptomatic and have low levels of iRBCs, even children who have yet to acquire reliably protective antibodies. The molecular mechanisms underlying these clinical observations are unclear. PBMCs collected from Malian children before the malaria season responded to iRBCs by producing pyrogenic, pro-inflammatory mediators such as IL-1β, IL-6 and TNF. However, following febrile malaria there was a marked shift in the response to iRBCs with the same children’s PBMCs producing lower levels of those cytokines. These data suggest that malaria-induced epigenetic reprogramming of innate immune cells may play a role in immunity to malaria. Accordingly, age-stratified analysis of monocytes collected before the malaria season showed an inverse relationship between age and pro-inflammatory cytokine production capacity. Monocytes of Malian adults expressed higher levels of CD163, CD206, Arginase-1, TGM2, molecules which are known to be associated with regulatory phenotype. These observations were recapitulated with an in vitro system of monocyte-macrophage differentiation whereby re-exposure to iRBCs was associated with diminished expression of pro-inflammatory mediators and a corresponding decrease in epigenetic markers of active gene transcription (i.e. H3K4me3) at the TSS of the same pro-inflammatory mediators. Together these data support the hypothesis that epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype contributes to clinical immunity to malaria.
Vps33B is a crucial regulator of Type I Interferon response downstream of the cGAS-STING pathway
Authors: Meibers Hannah , McDaniel Margaret , Pasare Chandrashekhar
Published: January 2023

Vacuolar protein sorting-associated protein 33B (Vps33B) plays a critical role in the trafficking of endosomal contents to lysosomes in macrophages, particularly following Toll-like receptor (TLR) activation. Upon activation by microbial ligands, TLRs are internalized into endosomes, then trafficked to lysosomes for degradation. We have previously shown that endosomal to lysosomal trafficking, and subsequent degradation of TLR4, is mediated by Vps33B. Without this crucial degradation step, TLRs remain trapped within altered endosomes, leading to an enhanced inflammatory response. Since Vps33B is critical for termination of signaling following TLR ligation, we wanted to determine if it had a conserved role in regulating other pattern recognition receptor (PRR) signaling pathways. Specifically, we investigated the role of Vps33B in regulating cytosolic DNA sensing by the cGAS-STING pathway. Absence of Vps33B in macrophages and mouse embryonic fibroblasts resulted in an exaggerated type 1 interferon and interferon-stimulated gene response after stimulation with dsDNA and other STING ligands. Our findings suggest that Vps33B has a conserved role as a regulator of innate inflammatory responses downstream of both plasma membrane as well as cytosolic PRRs. Mechanistically, we hypothesize that Vps33B plays a role in STING degradation, similarly to how it permits TLR4 degradation, through mediating endo-lysosomal trafficking. This would suggest a broad and critical role for Vps33B in PRR-induced vesicular transportation and receptor degradation. Future work will investigate the mechanisms by which Vps33B regulates STING-mediated signaling as well as its importance for anti-viral immunity.
Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation
Authors: Zanoni Ivan , Di Gioia Marco
Published: January 2023

Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide (LPS). While cells activated by LPS rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine and favor the accumulation of oxaloacetate in the cytoplasm: this metabolite potentiates IL-1beta production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.
Human monocyte chromatin accessibility variations identify inflammaging heterogeneity
Authors: Singh Amit K , Mazan-Mamczarz Krystyna , Roy Roshni , Kaileh Mary , Sarantopoulou Dimitra , Yin Jinhu , Bektas Arsun , De Supriyo , Zukley Linda , Weng Nan-Ping , Sen Ranjan
Published: January 2023

Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly population. The innate immune system is implicated as master regulator of this process via expression of cytokines and other key mediators. We used ATAC-seq to identify chromatin signatures in monocytes obtained from healthy donors of the GESTALT cohort. State of differentially open chromatin (DOC) between young (<60y) and older (>60y) donors were identified. Older donors segregated in two clusters, young-like and distinct old-like cluster. Old-like DOC’s enriched for intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. The relationships of age-associated DOC to gene expression and DNA methylation reveled enrichment of cytokine mediated inflammatory pathways. Ongoing studies of cytokine expression using high-dimensional flowcytometry will be used to determine the functional consequences of chromatin structural changes associated with age.
Sex specific Schistosomiasis induced metabolic modulation of the myeloid lineage
Authors: Fairfax Keke C , Cortes-Selva Diana , Gibbs Lisa , Rajwa Bartek , Amiel Eyal , Cox James E
Published: January 2023

There is a known inverse correlation between chronic helminth infections and metabolic diseases. Despite strong evidence that prior helminth infection exerts a protective effect against the development of metabolic disease, a major gap exists in understanding the mechanism(s) underlying this protection. We recently published that S. mansoni infection induces dramatic transcriptional alterations in hepatic macrophage metabolism, and that this correlates with protection from high fat diet (HFD) induced atherosclerosis and glucose intolerance in male ApoE−/− mice. Since it has previously been shown that during S. mansoni infection the majority of macrophages are monocyte derived, and that schistosome antigens circulate systemically, we sought in the present study to determine the broad effects of S. mansoni exposure on the myeloid lineage using the ApoE−/− HFD model. Surprisingly, we discovered that macrophages derived from the bone marrow of S. mansoni infected male mice have dramatically increased oxygen consumption and mitochondrial mass. This shift is accompanied by increased glucose shuttling into TCA cycle intermediates and decreased cholesterol esters. When we examined the role of biological sex in schistosome induced modulation. We found that S. mansoni infection does not reliably protect ApoE−/− female mice from HFD induced weight gain or glucose intolerance. The sex-dependent effect of infection extends to myeloid precursors, where bone marrow derived macrophages from infected females display the opposite metabolic phenotype as those from infected males. Overall, these data present the first evidence that S. mansoni systemically modulates the myeloid compartment in a sex-dependent manner.
Speckled Protein (SP)140, an immune-specific chromatin reader, contains a CARD and chromatin-interacting domains that dictate subnuclear localization and macrophage identity
Authors: Fraschilla Isabella Rose , Jeffrey Kate L
Published: January 2023

Chromatin regulators that “read” histone post-translational modifications are desirable therapeutic targets in inflammation and cancer. The Speckled Protein (SP) family of chromatin “readers” consists of SP100, SP110, SP140 and SP140L and share high homology with autoimmune regulator (AIRE). SPs possess a caspase activation and recruitment domain (CARD) and multiple chromatin “reader” domains (Plant Homeodomain (PHD), Bromodomain, and SAND). SP140 is preferentially expressed in immune cells and loss-of-function single nucleotide polymorphisms (SNPs) within SP140 significantly associate with immune disorders, such as Crohn’s disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). We have previously shown that SP140 is a critical orchestrator of macrophage identity and function by occupying and repressing silenced lineage-inappropriate genes. We also demonstrated that PBMCs from patients carrying SP140 SNPs lose SP140 expression and are hypo-responsive to TLR stimulation. How SP140 functions to interact with or maintain heterochromatin for cell identity and function, what SP140 protein domains drive activity, and how disease variants alter SP140 function remain to be determined. Here we show that human SP140 localizes to promyelocytic leukemia (PML) nuclear bodies, sites of transcriptional repression, and we delineate the SP140 protein domains required for this. Furthermore, we determined the impact of SP140 disease-associated SNPs on PML localization and macrophage identity and functional responses. Collectively, our work provides functional characterization of this important chromatin protein and how SP140 disease variants contribute to immunological disease.
Circulating Complement C3-Alpha Chain Levels Predict Survival of Septic Shock Patients
Authors: Cheng Tzu Hsuan , Puskarich Mike , Li Xiang , Fang Zhou , Xu Fou , Chen Yong , Jiang Xian-Cheng , Worat Samrat , Jones Alan E. , Zhang Ming
Published: January 2023

Septic shock is a critical clinical condition with a high mortality rate. Previous observations have suggested that circulating complement C3 fragments, released during septic shock, might contribute to the development of complications such as profound hypotension and disseminated intravascular coagulation, resulting in a more severe disease course and a poorer outcome. However, there are conflicting reports regarding the role of C3 in the course of septic shock. Because circulating C3 exists in different sizes through enzymatic processing or spontaneous hydrolysis, these previous studies, which used ELISA-based methods, would not have distinguished among different C3 species. We have used Western blotting, with its associated protein size differentiation feature, to investigate the forms of circulating C3 in two cohorts of septic shock patients: a discovery cohort of 24 patents and a validation cohort of 181 patients. In both cohorts there were significantly lower levels of the C3-alpha chain in non-survivors than in survivors. In addition, the levels of the C3-alpha chain significantly correlated with days of survival. ROC and AUC analyses showed an AUC=0.65 (95% confidence interval [CI]: 0.56–0.75). At a best cutoff value (Youden) of 970.6 mg/ml, the test demonstrated a sensitivity of 68.5% and specificity of 61.5%. At this cutoff point, Kaplan-Meier survival analysis showed that patients with lower levels of C3-alpha chain had significantly lower survival than those with higher levels (p < 0.001). Thus, circulating C3-alpha chain levels in septic shock patients may predict survival rates.
Essential Role of Indoleamine 2, 3-dioxygenase 1 (IDO1) in Metabolic Programming of Innate Immune Responses in Aged Lung during Influenza
Authors: Stout-Delgado Heather W , Harris Rebecca , Lee Stefi , Yang Jianjun , Hong Kyung Sook , Cho Soo Jung
Published: January 2023

Influenza remains a leading cause of morbidity and mortality, with highest incidence of hospitalization and death occurring in persons >65 years of age. The goal of this study was to interrogate biochemical profiles manifested in lung tissue originating from young adult and aged adult mice over the course of infection, with the aim of characterizing metabolic migration in lung during influenza. There was heightened inflammation, cellular infiltration, and alveolitis present in the aged lung that corresponded with increased viral titers, morbidity, and mortality. Focusing on the biochemical and pathway changes illustrated an important role for kynurenine and tryptophan metabolism in modulating lung specific immune responses to influenza. There was a significant decrease in kynurenine production in aged alveolar macrophages by day 7 post influenza infection that was associated with increased inflammatory and diminished regulatory responses. Despite a rapid increase in IDO1 expression in young, there was impaired expression and activity in aged macrophages and lung during the course of infection. Age-associated mitochondrial dysfunction contributed to changes in IDO1 expression and decreased kynurenine production in aged lung and alveolar macrophages and resulted in alternate immunometabolic signaling and heightened pro-inflammatory responses. Treatment with a mitochondrial targeted antioxidant improved IDO1 mediated production of kynurenine as well as ameliorated mortality in aged mice. Taken together, our data provide additional evidence as to why older persons are more susceptible to influenza and suggest a possible therapeutic to improve immunometabolic responses in this population.
Dynamic metabolic reprogramming in innate immune cells: an early response to influenza infection that is essential for effector function
Authors: Smallwood Heather S , Rezinciuc Svetlana , Bahadoran Azadeh , Bezavada Lavanya , DeVincenzo John , Cormier Stephania , McGargill Maureen Ann , Green Douglas R , Thomas Paul Glyndwr
Published: January 2023

Influenza infection (Flu) is a cause of morbidity and mortality that poses increased risk to children and lacks consistent treatment. Modulating immunometabolism has therapeutic potential. Immune metabolism in response to TLR agonists is well characterized compared to Flu. Clinical reports indicate Flu induces hypermetabolism in the lungs and draining lymph nodes. We demonstrated epithelial cells may be a contributing factor as Flu significantly increased glycolysis, glucose uptake, and lactate excretion and reports suggest these conditions diminish immune function. Dendritic cells (DC) and macrophages (MO) are thought to have rigid metabolic profiles that dictate inflammatory phenotypes after TLR stimulation. We found during Flu epithelial cells, DC, and MO have similar metabolism through shared and distinct molecular mechanisms that allow innate cells to retain metabolic plasticity. We found DC and MO significantly increase both glycolysis and respiration in response to Flu while TLR agonists induce glycolysis with respiratory defects. DC from murine lungs during Flu had the same metabolic phenotype. However, when DC metabolism was constrained motility and T cell priming were diminished. We identified a metabolic drug that significantly improved infection outcomes in vivo and reduced Flu in a clinical trial. Its antiviral mechanism is unknown, but we found it cell specifically modulates metabolic regulators and effector function. To investigate this in humans we collected upper airway cells and secretions from children with respiratory viral infections and found glycolysis and respiration were significantly increased. Thus, we find Flu induces hypermetabolism in vitro, in vivo, and in children’s respiratory tract.