bluuebunny/crossref_metadata_2025_split huggingface dataset:
Murine Eosinophils Release IL-1β via a Non-Lytic Mechanism Dependent on Gasdermin D (GSDMD) Expression but Independent of GSDMD Proteolytic Processing
Authors: Nguyen Thu R , Diaz-Aponte Jose R , Dubyak George R 
Published: August 2023

Abstract
Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been well-characterized in macrophages. This involves GSDMD cleavage by caspase-1 to generate N-GSDMD products that assemble as macropores in the plasma membrane (PM) to mediate direct IL-1β efflux and eventual pyroptosis. Granulocytes, including neutrophils and eosinophils, are also important sources of IL-1β in multiple innate immune responses. In neutrophils, we have previously reported that GSDMD expression and proteolytic processing to N-GSDMD is required for IL-1β release but is independent from accumulation of PM N-GSDMD pores and pyroptosis due to alternative trafficking of N-GSDMD to azurophilic granules and autophagosomes; this atypical GSDMD-dependent IL-1β release is mediated in part by secretory autophagy. We have now tested whether this alternative GSDMD-dependent mechanism for non-lytic IL-1β release is also operative in eosinophils using primary murine bone marrow-derived eosinophils (BMDE) from wildtype (C57Bl/6) or Gsdmd−/−mice and stimulated with activators of NLRP3 inflammasome signaling. NLRP3 inflammasome-activated BMDE rapidly process and release IL-1β via a mechanism dependent on GSDMD expression but independent of GSDMD proteolytic processing, N-GSDMD pore accumulation in the PM, and pyroptosis. This requirement for GSDMD in IL-1β release from BMDE is operative during the initial phases (~60 min) of NLRP3 inflammasome activation but is then complemented by a yet-be-defined GSDMD-independent pathway with prolonged stimulation. These findings indicate a role for GSDMD as a chaperone rather than pore-forming protein and distinct from mechanisms defined in neutrophils.
Supported by grants from (NIH P01-AI141350, R01-EY014362).
Aryl polyenes expressed by Uropathogenic Escherichia coli contribute to pathogenesis in a mouse model with an aberrant macrophage polarization
Authors: Markley R L , Johnston Isabel , Osborn Lucas J , DeLucia Beckey , Alqudah Sara , Bobba Viharika , Nia Layan Hamidi , Ouyang Kelsey , Dester Emma , Ginter-Frankovitch Maggie , Claesen Jan 
Published: August 2023

Abstract
Uropathogenic Escherichia coli (UPEC) is a causative agent of urinary tract infections (UTIs), and one of the most common causes of pyelonephritis. Interestingly, UPEC is often found as a non-pathobiont in the gut flora. The transition from gut flora to pathogen for UPEC is dependent upon a flexible tropism, and a number of virulence factors that aid in UPEC’s colonization and invasion of the urinary tract. We recently demonstrated a role for the E. coli aryl polyenes (APEs) as a potential UPEC fitness factor. The current hypothesis is that APEs contribute to UPEC virulence in the host system. APEs are encoded by a biosynthetic gene cluster (BGC) family identified among many gram-negative bacteria. The products of this BGC family are lipids comprised of an aryl head group and conjugated double bond system, which are structurally reminiscent of established virulence factors found in gram-positives. In addition to the structural mimicry, UPEC APE expression conferred resistance to reactive oxygen species and enhanced biofilm formation. In a UTI mouse model, infection with UPEC that constitutively expressed APEs (UPECAPE+) increased pathogenicity. Transcriptomic analyses of in vitro co-incubation experiments indicates that UPECAPE+ exposure influences the macrophage gene transcription profile differently as compared to UPECWT. Taken together, our findings suggest that APE expression aids in bacterial virulence and immune modulation during infection. Therefore, targeting the biosynthetic gene product APE may be a viable therapeutic approach to reduce fitness of pathogenic populations while leaving commensal gut microflora unharmed and minimize tissue damage.
NIAID RO1AI153173 Cleveland Clinic Foundation
Defining a role for PARP1 in driving RSV induced pulmonary pathology
Authors: Mire Mohamed , Elesela Srikanth , Rasky Andrew , Morris Susan , Lukacs Nicholas 
Published: August 2023

Abstract
The mechanisms underlying how Respiratory Syncytial Virus (RSV) subverts dendritic cell (DC) immune function to enhance pathology are not well defined, but we have recently shown RSV alters DC metabolism. Analysis of Reverse Phase Protein Array (RPPA) data, which identifies phosphorylated cytoplasmic proteins (&gt;400 in the analysis), revealed one of the most differentially activated signaling molecules, PARP1, had increased activation (phosphorylation) in RSV infected DC. PARP1 is a DNA damage enzyme that, in an NAD+ depleting manner, polymerizes deposition of poly-ADP-ribosylation (PAR) units in cellular proteins, possibly altering their physiological properties. Our data shows genomic ablation of PARP1 negates the capacity of RSV to manipulate DC metabolism at the early stage of infection. Enzymatic inhibition or genomic ablation of PARP1 resulted in increased key innate immune cytokine expression including type I interferon and IL-12, which together promote a more appropriate anti-viral environment. PARP1−/− mice and PARP1 inhibitor treated mice were protected against RSV induced immunopathology including airway inflammation and mucus hypersecretion as indicated by PAS staining of lung tissue. Assessment of isolated airway epithelial cells demonstrated genomic depletion of PARP1 did not alter RSV induced innate cytokines, suggesting predominately an immune cell effect upon RSV infection. Together, these data highlight PARP1 may be a logical target for therapeutic intervention during RSV infections to diminish the immunopathology and reduce disease severity.
Supported by grants from NIH (R35 HL150682, R01 AI138348)
Fibrinogen is increased in the spleen over the course of murine endotoxemia
Authors: Nunez Kathryn Elizabeth , Hunat Junela Cecile , Falguni Mehnaz M , Harberts Erin M 
Published: August 2023

Abstract
The fibrinogen protein is composed of three subunits encoded for by the genes Fga, Fgb, and Fgg. This trimer is a main component of blood clots. Irregular blood clotting contributes to low blood pressure during septic shock, which eventually leads to death from total system organ failure. A previously published data set finds Fga, Fgb, and Fgg to be dysregulated during sepsis in the serum of both wildtype (WT) and toll-like receptor 4 (TLR4) deficient mice. Deficiency in the innate immune receptor TLR4 models immune ataxia that occurs in patients who recover from septic shock, surviving patients have an increased risk of mortality due to secondary infection. We hypothesize that fibrinogen contributes to symptoms of septic shock independent of the TLR4 pathway. In this study we confirm that fibrinogen is transcriptionally upregulated in the liver of both WT and TLR4−/− mice over the course of LPS induced endotoxemia. Western blots were completed using organ lysates from liver, spleen, kidney, and brain of WT and TLR4−/− mice from 0, 6, 12, and 18 hours after induction of endotoxemia with intraperitoneal injection of 30 mg/kg lipopolysaccharide (LPS). Western blots show that WT and TLR4−/− control mice express similar amounts of fibrinogen in organs, and that there is more fibrinogen in the liver than the spleen, which is consistent with current literature. Western blots conducted with endotoxemic samples show that over the course of disease in WT mice, fibrinogen levels are consistent in the liver but increase in the spleen. Continued experiments in TLR4−/− mice will determine the level of TLR4-independent fibrinogen activation during sepsis and provide evidence for use of serum clotting modulators in patients who have recovered from septic shock.
This study is supported by a Towson University FDRC grant awarded to EMH, a Fisher College of Science and Mathematics undergraduate research award to KEN, and the 2022 Linda Sweeting Summer research fellowship awarded to KEN by the Towson University Department of Chemistry.
Repurposing FDA-approved drugs to inhibit IL-33 mediated mast cell function
Authors: Long Dionna L , Burchett Jason R , Ryan John J 
Published: August 2023

Abstract
IL-33 is an IL-1-family cytokine that is released from fibroblasts, epithelial, endothelial, and some immune cells in response to tissue damage or inflammatory signals. IL-33 activates mast cells, innate leukocytes that play a key role in asthma. IL-33 induces production of inflammatory cytokines that worsen allergic diseases like asthma. It is essential to better understand IL-33 function and how to suppress it. Computer modeling studies using the SYBYL and GOLD algorithms to screen 3035 FDA-approved drugs for interactions with the IL-33 receptor, ST2, at the IL-33 binding site revealed several potential ST2 antagonists. In this study, we show that mouse bone marrow derived mast cells (BMMCs) treated with the antiviral drug lopinavir had diminished IL-33 responsiveness. Specifically, lopinavir (15mM) significantly reduced IL-33-mediated IL-6 and IL-13 secretion. Lopinavir effects noted when the drug was given simultaneously with IL-33 and were maximal when given 1 hour prior to IL-33 and suppression. These rapid effects suggest a direct drug-receptor interaction. We treated BMMCs with drug for up to 24 hours to assess for alternative mechanisms such as ST2 down-regulation, which was not noted. Other candidate drugs included clindamycin, which had no effect on IL-33-mediated cytokine production, and ritonavir and cobcistat, which increased cytokine secretion. Our data demonstrate that lopinavir can inhibit IL-33 mediated mast cell function, likely as an off-target effect of the drug. These data support further investigation of lopinavir and suggest the utility of repurposing FDA-approved drugs in allergy and asthma treatment.
Linking CD36-dependent lipid processing to the alveolar macrophage transcriptional program via GATA2
Authors: Jackson-Strong Morgan , Shoger Karsen E , Michalides Brandon Alan , Kruszelnicki Sonia M , Kohan Alison B , Gottschalk Rachel A 
Published: August 2023

Abstract
Alveolar macrophages (AMs) catabolize lipid-rich pulmonary surfactant to support gas exchange and have steady-state anti-inflammatory functions to limit lung tissue damage in response to minor challenge. While it is well appreciated that the lung microenvironment shapes tissue-specific AM function and that this linked with lipid metabolism, little is known about the persistent signaling events that support these functional and metabolic programs. We recently demonstrated that the absence of CISH, a suppressor of cytokine signaling (SOCS) family member, led to a lipid-laden, foamy AM phenotype and the accumulation of pulmonary surfactant. These changes were associated with increased AM expression of the transcription factor GATA2 and RNA-seq analysis of Cish−/− AMs revealed dysregulated expression of lipid-processing genes and increased expression of genes enriched for GATA2-binding motifs. While GATA TFs have been implicated in anti-inflammatory macrophage function and in dysregulation in of lipid-laden athlerosclerotic plaque macrophages, their function in AMs is largely unknown. Our preliminary data suggest that lipid uptake through the scavenger receptor CD36 enhances GATA2 activity and limits AM pro-inflammatory function. We are currently investigating mechanisms by which pulmonary surfactant lipids regulate GATA2 and AM programming, using mice with myeloid specific deletion of CD36; our data suggest that AMs from these mice are impaired in their ability to uptake DPPC, the primary lipid species in surfactant. Defining signaling processes that link CD36 with GATA2 and AM transcriptional and metabolic programs will provide insight into lung homeostasis and support our ability to manipulate AMs therapeutically.
R01 HL162658
Innate immune responses to enteric Escherichia coli dictate neonatal sepsis outcomes
Authors: Muske Josey , Greenfield Kara G , Knoop Kathryn A 
Published: August 2023

Abstract
Late-onset neonatal sepsis (LOS), a leading cause of neonatal death, can result from pathogenic bacteria disseminating to the bloodstream from the intestinal microbiota. E. coli is the second most commonly isolated pathogen causing LOS. During sepsis, there are significant immune discrepancies between infants and adults, including elevated IL-6 levels in neonates, demonstrating the need for age-specific research. We have developed a murine model of LOS that uses clinical isolates of gut-resident E. coli, which results in rapid pro-inflammatory cytokine production within 6 hours and neonatal mortality within 2–3 days post-infection. Not all enteric E. coli results in inflammation and mortality, and we have classified isolates as “commensal” or “pathogenic”. TLR4−/− mice infected with pathogenic E. coli have a delayed mortality to sepsis, suggesting innate bacterial recognition is required for acute inflammation. To further determine the source of this delay, we have begun investigating signaling pathways during bacterial infection. We found the immune signaling pathway NFκB/AP1 is consistently activated in murine macrophages by commensal and pathogenic bacterial strains. However, only pathogenic bacteria induce elevated IL-6 production by murine macrophages. Interestingly, this response required live bacteria, suggesting lipopolysaccharide alone from E. coli is not sufficient to recapitulate LOS inflammation. Our future directions will explore alternative signaling pathways that are activated uniquely during pathogenic infection and induce a robust inflammatory response. Through this work, we will determine the consequence of specific pro-inflammatory cytokines in neonatal mice during pathogenic infection.
Research supported by R03 DK122187.
The nature of the stimulant differentially impacts the innate memory response of monocytes to Mycobacterium tuberculosis
Authors: Subbian Selvakumar , Kumar Ranjeet , Kolloli Afsal , Singh Pooja 
Published: August 2023

Abstract
Innate immune cells, such as monocytes, can generate a non-specific ‘memory’ response (a.k.a. trained immunity) upon stimulation with an antigen/bacterium (e.g., LPS or BCG), which confers heterologous protection against another infectious agent. However, the association between the nature of stimulants and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the response of macrophages “trained” by various stimulants to subsequent infection by different clinical Mycobacterium tuberculosis (Mtb) strains remains unknown. Here, we report the trained immunity profile of the blood-derived monocytes (BDM) of BCG-vaccinated adults and monocytic THP-1 cells after stimulation with BCG, LPS, Purified Protein Derivative (PPD), heat-killed MtbHN878 or MtbCDC1551 strains. The response of monocytes trained by these agents to subsequent infection with clinical Mtb isolates HN878 and CDC1551, which differ in their virulence potential, was evaluated using standard molecular and cellular immunologic assays. The BCG- and LPS-trained macrophages produced higher proinflammatory, antimicrobial responses, and autophagy induction than unstimulated, PPD, HK-HN878, or CDC1551-stimulated cells. The HN878-infected trained THP-1 cells elicited a more robust inflammatory response than the CDC1551 infection. A significantly reduced intracellular Mtb growth was noted in the BCG and LPS-trained THP-1 cells, which was augmented by adding rapamycin, an autophagy inducer. A similar immunological response was noted in the BDM of BCG-vaccinated adults infected with these Mtb strains. Thus, the nature of the stimulant affects the macrophages’ trained immunity profile and its response to Mtb infection.
Supported by grants from NIH (R01 AI161822) and the Bill and Melinda Gates Foundation (INV-009099; formerly OPP1157210).
Investigating the role of BHLHE40 in host responses to Mycobacterium tuberculosis infection and myeloid cell development
Authors: Hendrix Skyler V , Mreyoud Yassin , Nehls Michael , Smirnov Asya , Bradstreet Tara , Webber Ashlee , Edelson Brian T , Stallings Christina L 
Published: August 2023

Abstract
We currently lack a complete understanding of the immune responses required to control Mycobacterium tuberculosis (Mtb) infection. Our lab has shown that the transcription factor BHLHE40 is essential during Mtb infection. Deletion of Bhlhe40 in CD11c+ lung macrophages and dendritic cells (DCs) is sufficient to increase mouse susceptibility to Mtb infection, though the role of BHLHE40 in these cells during infection is unknown. Murine bone marrow cultured in the presence of GM-CSF, an abundant cytokine in the lung, develop into populations of CD11c+ macrophages, DCs, and granulocytes, all of which express Bhlhe40. Using this system, we examined cytokine and transcriptional responses before and after exposure to heat-killed Mtb to understand how BHLHE40 impacts cell development and inflammatory responses. We found that in WT mice, GM-CSF-cultured cells develop into 9 distinct populations and expression of Bhlhe40 was required for GM-CSF-mediated pro-inflammatory cytokine and transcriptional programs. Additionally, loss of Bhlhe40 led to decreased DCs accompanied by upregulation of the transcription factor C/EBPβ in DC progenitor populations, which negatively impacts DC development. ChIP-seq analysis revealed BHLHE40 binds directly to C/EBPβ’s gene locus, indicating that BHLHE40 may directly repress Cebpb. In contrast, loss of Bhlhe40 did not affect macrophage development, but led to a transcriptional shift towards an anti-inflammatory state. These findings demonstrate that in the presence of GM-CSF, BHLHE40 is required for DC development and inflammatory macrophage polarization. Defects in either of these aspects could contribute to the essential role for BHLHE40 in lung macrophages and DCs during Mtb infection in vivo.
Determining the fate of pulmonary iILC2s in helminth infection
Authors: Kashyap Amita , Miller Mindy M , Reinhardt R Lee 
Published: August 2023

Abstract
Group 2 Innate Lymphoid Cells (ILC2s) are innate counterparts to Th2 cells and key early responders in helminth infection. ILC2s have two subpopulations: tissue-resident “natural” nILC2s, present in the lungs at homeostasis, and “inflammatory” iILC2s, which appear transiently in the lungs during the response to helminth infection. We aim to determine the fate of pulmonary iILC2s and hypothesize that pulmonary iILC2s acquire an nILC2-like phenotype and make biologically relevant long-term contributions to the tissue-resident ILC2 pool. Early studies demonstrated that iILC2s can convert to an nILC2 phenotype, but the prevailing view in the field is that the nILC2 population is maintained through self-renewal. However, fate-mapping models show an external contribution to the pulmonary ILC2 population which corresponds with an influx of iILC2s. We have further characterized the pulmonary ILC2 population during helminth infection and found an intermediate-phenotype population 8 days post infection. Preliminary flow cytometry and scRNA-seq data from congenic transfer experiments indicate that iILC2s may acquire an nILC2 phenotype in vivo. Preliminary multiple infection experiments indicate that mice lacking a pulmonary iILC2 response have smaller nILC2 compartments compared to wildtype counterparts after 3 infections. We are further testing our hypothesis using fate-mapping models and congenic transfer experiments in conjunction with single-cell sequencing approaches to more rigorously confirm these findings. Understanding the contribution of iILC2s to the long-term tissue-resident ILC2 population could lead to therapies in diseases with ILC2 involvement including helminth infection and steroid-resistant asthma.
Supported by NIH grant R01 AI56901-0
Leukocyte associated immunoglobulin-like receptor 1 modulates immune response to Staphylococcus aureus infection
Authors: Dorando Hannah K , Rich Helen , Warheit-Niemi Helen , Mutic Evan , Tomaszewski Kelly , Tian Ling , Wurtz Mellisa , Quinn Chaz , Veis Deborah , Wardenburg Juliane Bubeck , Moore Bethany B , Payton Jacqueline E 
Published: August 2023

Abstract
Immunity is a balance of maximal pathogen defense with minimal host damage. Inhibitory immune receptors are important for maintaining this balance. We recently identified elevated levels of leukocyte-associated immunoglobulin (Ig)-like receptor protein1 (LAIR1), an inhibitory immune receptor, in cutaneous lymphoma patients, in whom invasive Staphylococcus aureus (S. aureus) infections are frequent. We therefore tested Lair1 knock-out (KO) mice in two models of invasive S. aureus infection: pneumonia and subcutaneous skin infection. In WT mice, skin infections resolve by 14 days post-infection (dpi), while pneumonia infections cause &gt;50% lethality by 2dpi. In both models, Lair1 KO mice showed consistent patterns of myeloid hyper-activation. Lair1 KO had consistently larger skin lesions than WT throughout the 14 days, but no difference in bacterial clearance. Lair1 KO with pneumonia had higher survival (100% KO vs. 33% WT) despite comparable measures of lung injury and immune cell recruitment. In both models, KO mice produced higher levels of proinflammatory cytokines (IL-6, IL-1b) and neutrophil chemokines (CCL2, 3, 4) in lung, bronchoalveolar lavage, and skin by multiplex cytokine array. KO bone marrow-derived macrophages infected with S. aureus in vitro also secreted higher levels of proinflammatory cytokines (IL-6) and neutrophil chemokines (CCL2, 3, 4). Together, our results demonstrate that LAIR1 loss enhances myeloid activation, resulting in excessive tissue damage in skin infection, yet is protective against death in pneumonia. We posit that LAIR1 plays a crucial role in balancing myeloid immune response; LAIR1 activity can be beneficial or detrimental to the host depending on the site and type of infection.
Antibacterial functions of neutrophils against cystic fibrosis isolates of Staphylococcus aureus are compromised by short chain fatty acids
Authors: Rada Balazs , Miller Arthur , Fantone Kayla , Tucker Samantha L , Goldberg Joanna B 
Published: August 2023

Abstract
Short chain fatty acids (SCFA) are generated by anaerobic bacteria as byproducts of their anaerobic fermentation at mucosal sites. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF). In CF airways SCFAs are present at millimolar concentrations. CF is characterized by lung inflammation dominated by neutrophils and polymicrobial bacterial infections made up of facultative anaerobes, including Staphylococcus aureus. In mammalian organisms, neutrophils represent the main arm of the immune system to fight S. aureus. The reason why neutrophils are unable to clear respiratory pathogens including S. aureus, in CF remains though unclear. Based on published data and our prior results, we hypothesized that SCFAs compromise the ability of human neutrophils to kill S. aureus in vitro. SCFAs do not affect the viability of neutrophils or in vitro S. aureus growth. Our data show that SCFAs do not stimulate release of neutrophil extracellular traps in human neutrophils either. On the other hand, superoxide production, another important effector function of activated neutrophils against S. aureus, was significantly inhibited by SCFAs. SCFAs also compromised the ability of neutrophils to kill some CF isolates of S. aureus in vitro. Overall, our results provide new knowledge on the interactions between SCFAs and the innate immune system, and suggest that SCFAs produced by anaerobic bacteria in the CF lung could interfere with the ability of neutrophils to kill S. aureus, a prominent respiratory pathogen in this disease.
This work was supported by grants provided by the National Institutes of Health (R01HL136707 to B.R. and R21AI154343 to B.R. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.