bluuebunny/crossref_metadata_2025 huggingface dataset:
MIA-reprogramed ILC2s promote enhanced lung allergic inflammation in offspring postnatally and into adulthood
Authors: Lopez Diego A , Griffin Aleah , Beaudin Anna E
Published: August 2023

Innate lymphoid cells (ILCs) are a relatively recently identified class of specialized innate-like lymphocytes that are the topic of intense investigation for their role in immune function during homeostasis and disease states. Accumulating evidence suggests that dysregulation of ILC2s and ILC2-derived Type II cytokines plays a critical role in the development of allergic airway hyper-responsiveness and asthma. To test how prenatal inflammation impacts ILC2 establishment, I profiled changes to a committed progenitor to all ILC lineages in the fetal liver (FL). MIA induction during mid-gestation expanded FL Common helper Innate Lymphoid Progenitors, resulting in robustly expanded and hyper-proliferative neonatal ILC2s exhibiting a greater inflammatory phenotype, producing more IL-5 and IL-13 cytokine on a per-cell basis. Hyper-activated ILC2s drove remodeling of the lung immune landscape, with multiple immune cell compartments, including eosinophils and alveolar macrophages, exhibiting expansion during the first two weeks of life in MIA-treated offspring. Surprisingly, levels of IL-33, a potent ILC2 activator, significantly decreased after MIA, and ILC2s were hyper-responsive to IL-33 in ex vivo culture. Single-cell RNA sequencing of neonatal lung ILC2s revealed extensive heterogeneity between MIA and saline litters, including clusters unique to MIA-exposed litters, and transcriptional similar to ILC2 signatures in human asthma patients. Altogether, my data suggests prenatal inflammation imparts lasting changes to lung immunity by perturbing the establishment of committed ILC progenitors in the FL, affecting the generation of neonatal lung ILC2s poised to hyperactively respond to secondary immune activation.

NIH/NICHD training grant T32HD007491 The Pew Biomedical Scholars Award
B1 B cells contribute to inflammation and metabolic dysfunction in aged adipose tissue during endotoxemia
Authors: Carey Anna F , Kandikonda Pranathi , Dahlquist Korbyn , Cholensky Stephanie , Camell Christina
Published: August 2023

The elderly are at the greatest risk for severe endotoxemia and bacterial infections. The increase in low grade chronic inflammation and the decline of the immune response during aging contributes to this disease severity. Visceral adipose tissue (vWAT) exhibits immune recruitment and activation during pathogen challenge and metabolic diseases. Immune cells regulate lipolysis in vWAT which is required for the regulation of inflammation, but the intersection of bacterial inflammation and metabolism during aging is not clear. We investigated the contribution of B cells to metabolic dysfunction and inflammation during endotoxemia in vWAT. Old vWAT exhibited elevated inflammation and altered activation of resident B cells during LPS-induced endotoxemia. LPS also induced vWAT lipolysis in young mice but not in old mice. Diet induced obesity resulted in elevated LPS-induced inflammatory cytokines and B cells that failed to become activated by LPS. Deficiency of total B cells improved the homeostatic balance of immune cells within vWAT microenvironment, resulting in increased lipolysis and reduced Tnfα during endotoxemia in old mice. In contrast, B2 B cell depletion was insufficient to increase LPS-induced lipolysis or reduce inflammatory cytokines in vWAT from old mice, suggesting a driving role for B1 cells. B1 B cells from young and old vWAT were differentially activated by LPS indicating age-associated dysfunction. Our results demonstrate that endotoxemia results in immune and metabolic activation within the vWAT, both of which are dysfunctional during aging. These data reveal B cell subsets may differentially impair lipolysis and exacerbate the inflammatory response in vWAT following pathogenic challenge.

Supported by grants from NIH (R00 AG058800, R01 AG079913)
Investigating the roles of fetal-specific genes in hematopoiesis
Authors: Welsh Alia , Wilkinson Adam , Roy Anindita , Muljo Stefan Adi
Published: August 2023

Hematopoiesis, the process which generates blood cells, changes significantly during mammalian development. Fetal hematopoiesis primarily takes place in the fetal liver (FL), where hematopoietic stem cells (FL HSCs) are characterized by high self-renewal capacity and a distinctive program of multilineage differentiation. After birth, the main location of hematopoiesis is the bone marrow (BM), where stem cells (BM HSCs) are less proliferative and have a more restricted differentiation capacity. How exactly fetal hematopoiesis transitions to adult hematopoiesis and which cell-intrinsic properties or environmental factors aid this process have yet to be defined. This project aims to elucidate the cell-intrinsic mechanisms behind this transition. Studies show that FL and BM HSCs have distinct gene expression profiles, which may account for some of their phenotypic differences. Genes, such as LIN28B, which encodes an RNA binding protein, are highly expressed in the fetus, yet are absent in postnatal life. The re-expression of such fetal specific genes can confer fetal-like self-renewal and proliferation. Previous work in the Muljo lab demonstrated that LIN28B re-expression partially reprograms BM HSCs to undergo fetal-like lymphopoiesis, which is characterized by increased development of innate-like lymphocytes. I aim to fully reprogram adult BM HSCs to phenotypically resemble FL HSCs in their ability to generate cell types that develop early in life. FL HSCs, though clinically promising, are not readily available for therapeutic use. Reprogramming adult BM HSCs may be a suitable alternative that could improve current HSC transplantation procedures.

This work is supported by the Intramural Research Program of NIAID, NIH.

Intramural Research Program NIAID NIH
Role of walnut-derived urolithins in reducing colonic inflammation in a patient cohort elucidated by an integrated omics analysis
Authors: Moussa Marmar R , Fan Nuoxi , Nakanishi Masako , Hatano Yuichiro , Provatas Anthony , Vaziri Haleh , Birk John , Rosenberg Daniel W
Published: August 2023

Diet can directly affect colon health and cancer risk. For example, the polyphenolic ellagitannins (punicigalin) that are present in walnuts are converted by the microbiome to a panel of bioactive urolithin metabolites. In particular, urolithin A has been studied for its anti-inflammatory and anti-cancer properties. In this pilot clinical trial, we evaluate the effects of walnut consumption (2-oz per day) on urinary urolithins and blood serum inflammatory markers, as well as on the transcriptome of normal colon and polyp tissue in 38 healthy obese and non-obese subjects. Our in-depth computational analysis of metabolomics and proteomics data suggests that subjects with higher urolithin A formation show significantly lower levels of several key serological markers of inflammation, including C-Peptide, sICAM-1, sIL6-R, Ghrelin, TRAIL, sVEGFR2, MCP2, and Haptoglobin. Furthermore, RNA-Seq transcriptomic analysis of colon tissue taken from proximal and distal colon shows reduced inflammatory programs and activation of the KICSTOR complex in subjects with higher urolithin A formation. These studies indicate that higher urolithin A formation is linked to an anti-inflammatory response, warranting further studies to better understand the role of urolithins in the inflammatory process.

Supported by grants from American Institute for Cancer Research, California Walnut Commission, and National Cancer Institute (R01 CA252045).
Maternal high fat diet exposure results in maternal microbiome dependent increase in inflammatory cytokines and decrease in neurons in offspring brain
Authors: Tran Tuan Q , Niu Xinying , Wu Jun , Lu Dongmei , Leon Rachel , Minassian Berge , Mirpuri Julie
Published: August 2023

Introduction

We hypothesized that maternal high fat diet (mHFD) exposure will result in a microbiota dependent increase in inflammation and inhibition of axonal development in offspring brain.

Methods

Conventional and germ-free murine dams were exposed to 60% high fat diet (HFD) or regular diet (RD) prior to mating. Offspring brains were collected at 2-weeks of life and analyzed for inflammatory (TNF-α, IL-6, IL-17A, IL1β and TGF-β) and regulatory markers of neuronal development (Nurr1) by quantitative RT-PCR. RNA seq was performed on whole brain in conventional mice. We quantified differences in regional neurons by histology (Nissl and H&E). Behavioral studies on mHFD and RD offspring are ongoing.

Results

mHFD offspring had a significant increase in the inflammatory cytokines IL-17A and IL-6 when compared to controls. Nurr1 was increased 50-fold in mHFD offspring. In germ-free mHFD offspring, there was no difference. Quantification of neurons by Nissl staining demonstrated a decrease in neuron cell count in the cortex (20% decrease), and in hippocampal regions (22% in CA2 and 32% in CA3). Preliminary RNAseq analysis showed upregulation of specific genes involved in immunoregulation, neuron development and migration, and apoptosis in mHFD offspring.

Conclusion

mHFD exposure resulted in increased inflammatory cytokines and reduced neurogenesis in the offspring brain. This change was ameliorated in germ free mice, demonstrating a microbiome dependent diet effect. RNAseq findings demonstrate changes in specific immunoregulatory pathways which warrant further exploration. We speculate that maternal diet interacts with the maternal microbiome to increase immune activation in the fetal brain which affects neurogenesis.

Supported by grants from NIH (R01 1K12HD068369) and Children’s Medical Center Research Grant
Itaconic acid underpins hepatocyte lipid metabolism in non-alcoholic fatty liver disease
Authors: Weiss Jonathan M , Palmieri Erika M , Gonzalez-Cotto Marieli M , Bettencourt Ian A , Megill Emily L , Snyder Nathaniel W , McVicar Daniel W
Published: August 2023

Itaconate, the product of the decarboxylation of cis-aconitate, regulates numerous biological processes. We and others have revealed itaconate as a regulator of fatty acid beta-oxidation, generation of mitochondrial reactive oxygen species and the metabolic interplay between resident macrophages and tumors. In the present study, we show that itaconic acid is upregulated in human non-alcoholic steatohepatitis and a mouse model of non-alcoholic fatty liver disease. Mice deficient in the gene responsible for itaconate production (Immunoresponsive gene /Irg-1) have exacerbated lipid accumulation in the liver, glucose and insulin intolerance and mesenteric fat deposition. Treatment of mice with the itaconate derivative, 4-OI, reverses dyslipidemia associated with high fat diet feeding. Mechanistically, itaconate treatment of primary hepatocytes reduces lipid accumulation and increases their oxidative phosphorylation in a manner dependent upon fatty acid oxidation. We propose a model whereby macrophage-derived itaconate acts in trans upon hepatocytes to modulate the liver’s ability to metabolize fatty acids.

This research was supported, in part, by the Intramural Research Program of the NIH, National Cancer Institute, CCR, CIL. RNA sequencing and initial data analysis were conducted at the Frederick National Laboratory for Cancer Research at the CCR Sequencing Facility, NCI Frederick. Human specimens were provided by the Clinical Biospecimen Repository and Processing Core of the Pittsburgh Liver Research Center (supported by National Institutes of Health grant 1P30DK120531). Acyl-CoA analysis was supported by R01GM132261 to NWS.
D-2-hydroxyglutarate decreases allergic sensitization in asthma
Authors: Del Carmen Camarena Maria , Tharakan Anuj , Conrad Daniel , Martin Rebecca
Published: August 2023

Allergic asthma is a chronic inflammatory disease affecting the lungs which is mediated by allergen-specific IgE and type 2 cytokines. IgE and type 2 cytokine production require Tfh13 cell and Th2 cell polarization, respectively, which must first be primed by dendritic cells (DC). Recent studies demonstrate that cellular metabolic perturbations critically regulate DC activation and subsequent T cell priming. To characterize DC metabolic mechanisms which may regulate allergic sensitization, we analyzed GWAS data to identify metabolism-related genes that are associated with asthma risk. We identified 384 single nucleotide polymorphisms (SNPs) in the D2HGDH locus that are significantly associated with childhood-onset asthma. Further, expression quantitative trait loci analysis of the 10 most significant SNPs revealed that these SNPs promote reduced expression of D2HGDH mRNA. D2HGDH is an enzyme which catalyzes the conversion of D-2-hydroxyglutarate (D-2HG) to α-ketoglutarate (aKG). D-2HG is a potent inhibitor of aKG-dependent epigenetic enzymes. Bone marrow-derived dendritic cells (BMDCs) stimulated with the fungal allergen Alternaria exhibited a significant increase in the ratio of aKG/D-2HG compared to mock or LPS treated BMDCs. Further, in vivo studies revealed that intranasal administration of cell-permeable D-2HG prior to intranasal exposure to Alternaria significantly reduced Th2 and Tfh13 polarization, impaired IgE+ germinal center B cell differentiation, and induced T follicular regulatory cell polarization in mice. Our results suggest a mechanism whereby D-2HG suppresses epigenetic modifications in DCs, which are necessary for allergic sensitization and polarization of Th2 and Tfh13 cells.

VCU CTSA UL1TR002649 as well as funding from NIAID R56AI139658
Sterol isolated from Sargassum horneri attenuated particulate matter-exacerbated allergic asthma through the Th17 response
Authors: Jin Kim Hyo , Yang Jiwon , Udayanganie Olupathage Indrachapa , Ko Mi-Ok , Jeon You-Jin , Kim Hyun Jung , Jee Youngheun
Published: August 2023

Particulate matter (PM) has become a serious health issue causing pulmonary diseases such as asthma. Due to the side effects and non-specificity of conventional drugs, it is a need to develop alternative treatment using natural product. Sargassum horneri (S. horneri) is an edible species of large brown algae inhabiting the coasts of northeastern Asia extensively. S. horneri has sterols and has been used in oriental medicine to treat allergic condition. Therefore, we investigated if sterol isolated from S. horneri ethanol extract (SHE-sterol) could mitigate the PM-exacerbated allergic asthma. To establish a mouse model of asthma, BALB/c mice were sensitized with ovalbumin (OVA, 10 mg) and challenged with PM (5 mg/m3) for 7 days consecutively. SHE-sterol (50, 200 mg/kg), Prednisone (5 mg/kg). In SHE-sterol, fucosterol was specially identified as 0.701 μg/mL. SHE-sterol attenuated the PM-aggravated granulocytes infiltration. Moreover, SHE-sterol further attenuated PM-exacerbated eosinophil infiltration in the lung, trachea, and BALF. In addition, SHE-sterol markedly mitigated the activation of mast cells and the IgE level in serum. Concomitantly, SHE-sterol further restrained the Th17 cell response in PM-exposed allergic mice through attenuating expression of relevant effector cytokine IL-17A. This resulted in mitigated neutrophil infiltration in the lung. Also, SHE-sterol significantly suppressed PM-exacerbated hypersecretion of mucus in asthmatic mice. Taken together, these results suggest that sterol isolated from SHE has therapeutic potential for treating PM-exacerbated allergic asthma.

This research was funded by the Ministry of Education (2019R1A6A1A10072987 and 2022R1I1A1A01072505).
NOX2-Deficient Pulmonary Neutrophils and Macrophages Have Lower PD-L1 Expression and May Aggravate Allergic Lung Inflammation through Affecting ILC Populations
Authors: Ling Lun-Ling , Shieh Chi-Chang
Published: August 2023

Redox balance plays an important role in the pathogenesis of allergic asthma. We previously reported that NADPH oxidase 2 (NOX2)-derived ROS may modulate tissue inflammation through affecting neutrophil programmed cell death 1 ligand 1 (PD-L1) expression. To reveal the role of NOX2 in regulating PD-L1 expressions on pulmonary neutrophils and macrophages and in ILC differentiation, we established an allergic asthma model using NOX2-deficient (Cybb−/−) mice stimulated with the Dermatophagoides pteronyssinus (Der p) extract along with LPS. We first found that NOX2 deficiency led to increased group 2 innate lymphoid cell (ILC2) in Der p extract induced lung inflammation. The cell numbers of neutrophil, eosinophil and alveolar macrophage (AM) also increased in the stimulated Cybb−/− mice when compared with WT mice. The AM in Cybb−/− mice expressed higher levels of CD11b and the M2 marker CD163 and produced more TNF-α. Moreover, the Cybb−/− neutrophils and AMs had low PD-L1 expressions while ILC2s had significant levels of programmed cell death 1 (PD-1) expression in the inflammatory lungs. Altogether, these results indicated that NOX2 deficiency may lead to downregulation of PD-L1 expression on neutrophils and AMs while eliciting the M2 phenotype and inducing higher amounts of pro-inflammatory cytokine production. Our results suggested that the lowered PDL1 on NOX2-deficient neutrophils and AMs may aggravate allergic Inflammation through affecting ILC populations in the lung.
c-Kit signaling modulated group 3 innate lymphoid cell functions in mouse neutrophilic asthma model
Authors: Shao Jheng-Syuan , Chi Po-Yu , Chang Ya-Jen
Published: August 2023

Group 3 innate lymphoid cells (ILC3s) are involved in air pollution- and bacterial infection-associated neutrophilic asthma. Studies showed that asthmatic patients have higher stem cell factor (SCF) expression in airway and sera. Of note, ILC3s express SCF receptor, c-Kit, suggesting a potential role of SCF/c-Kit signaling in ILC3 functions and neutrophilic asthma. In this study, we aimed to determine whether SCF/c-Kit signaling could modulate ILC3 functions and neutrophilic asthma. Wild-type (WT) and c-Kit deficient mice (Kitw-sh) were intranasally treated with IL-1β/IL-23 or particulate matter (PM) 2.5 to induce neutrophilic asthma. While IL-1β/IL-23 or PM2.5 caused increases in neutrophil infiltration to bronchoalveolar lavage fluid (BALF) and airway hyperreactivity (AHR) in WT mice, Kitw-sh mice had decreased infiltration of neutrophils and ameliorated AHR. Concomitantly, flow cytometry results showed a decreased number of IL-17+ ILC3 in the lung of Kitw-sh mice compared to WT mice, which may be due to the reduced proliferation of c-Kit-deficient ILC3. In addition, we showed SCF enhanced IL-17 secretion from ILC3s in vitro. Based on re-analysis of published mouse whole lung cells scRNAseq data, we hypothesized fibroblasts are the source of SCF. Indeed, mice with SCF knockout in fibroblast exhibited decreased neutrophil infiltration and reduced IL-17+ ILC3 number in response to IL-1β/IL-23. Finally, administration of imatinib, a FDA-approved drug targeting c-Kit signaling, ameliorated AHR and ILC3 activation in the mouse neutrophilic asthma model. Taken together, our data suggested that inhibition of ILC3 activation by targeting c-Kit signaling may provide a potential therapeutic management for neutrophilic asthma.
The role of protease activity and monocyte subsets in initiating a Th2 response following allergen exposure
Authors: Meloun Audrey Rae , León Beatriz
Published: August 2023

Allergy diseases are primarily caused by aberrant T helper 2 (Th2)-driven immune responses to allergens. During the primary sensitization phase, allergen specific Th2 cells are activated and become central players in orchestrating the subsequent development of allergic inflammation following secondary exposure to the same allergen. Still, the mechanisms by which the innate immune system detects allergens and initiates Th2 responses remain unclear. Protease activity is a feature common to many allergens, including house dust mite (HDM) and papain, a protein-digesting enzyme found in latex and papaya. Using models of Th2 airway inflammation induced by the allergens HDM and papain, we show that protease activity on allergens is required for the stimulation and clonal expansion of antigen specific T cells and their polarization towards a Th2 cell profile. Mechanistically, we found that protease activity was, in particular, required for enhanced migration of allergen-carrying dendritic cells (DCs) from the lung to draining lymph nodes and the subsequent activation of T cells throughout both phases of allergic disease. Our results suggest this occurs through CCR7 and CD86 expression. Further, we observed a decrease in monocytes and monocyte derived DCs when no protease activity was present within the allergens. Our results determine the early events involved in innate immune recognition of allergens and highlight a potential role for monocytic cells in initiating Th2 responses to allergens containing protease activity.

Supported by National Institutes of Health grant 2R01AI116584 to B. León.
Chemokine CCL19 promotes type 2 helper T cell differentiation and allergic airway inflammation
Authors: Nakano Hideki , Lyons-Cohen Miranda R , Nakano Keiko , Wilkinson Christina L , Izumi Gentaro , Grimm Sara A , Whitehead Gregory S , Cook Donald N
Published: August 2023

Allergic asthma is largely driven by the actions of allergen-specific CD4+type 2 T helper (Th2) cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4+ T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes (LNs). The molecular mechanisms underlying the induction of T helper cells have been well studied and include the migration of allergen-bearing DCs from the lung to regional LNs in a manner dependent on the chemokine receptor, CCR7. Previous studies have demonstrated that CCL21, a CCR7 ligand, is required for the accumulation of naïve T cells and activated DCs in the LNs, but the function of another ligand, CCL19, has been unknown. In the present study, we investigated the role of CCL19 in allergic airway inflammation using CCL19-deficient mice. In a mouse model of asthma, eosinophils and lymphocytes were significantly reduced in airways of CCL19-deficient mice compared with those of wildtype (WT) animals, and production of IL-4 and IL-13 in the lung was significantly diminished, suggesting that CCL19 is required for type 2 immune responses. Co-cultures of naïve WT CD4+ T cells with CCL19-deficient DCs produced less type 2 cytokines than did T cells co-cultured with WT DCs. Further, fibroblastic reticular cells (FRCs) from CCL19-deficient mouse LNs produced less Th2 cytokine production compared with WT FRCs. Addition of recombinant CCL19 to the culture of naïve CD4+ T cells increased phosphorylation of STAT5 and gene expression of Il2, Il2ra, Il2rb, Il2rg, Sta5a, Stat5b and Stat6. Together, our findings suggest that CCL19 produced by DCs and FRCs promotes Th2 differentiation through the activation of STAT5-signaling, and that this promotes allergic airway inflammation.

This work was supported by Intramural Research Program of NIEHS, NIH (ZIA ES102025-09).
The RNA-binding protein Mex3B regulates airway remodeling through modulation of TGFBR3 mRNA stability in chronic rhinosinusitis
Authors: Nan Chen Ao
Published: August 2023

Objective

To explore the pathogenic function of Mex3 RNA binding family member B (Mex3b) in chronic rhinosinusitis(CRS).

Methods

The expression of Mex3b, TGF-β receptor III (TGFBR3), TGF-β2, p-Smad2 were studied by quantitative RT-PCR, immunohistochemistry, western blotting, and ELISA. The binding of MEX3b with TGFBR3 was test by improved RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq).

Results

Compared to control subjects, mNRA and protein expression of Mex3b in nasal epithelial cells from CRSwNP patients are upregulated. And Mex3b mRNA and protein levels in cultured human nasal epithelial cells(HNECs) obtained from control subjects were upregulated by LPS, CpG and IL-1β. Incresed Mex3b downregulates TGFBR3 expression in airway epithelial cells by binding to TGFBR3 mRNA, which results in downregulated level of TGFBR3 in CRS compared to control subjects. Loss of TGFBR3 inhibits TGF-β2 induced gene transcription in nasal epithelial cells. And Mex3b suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells. Mex3b and TGFBR3 expression levels are correlated with tissue edema in CRSwNP.

Conclusions

The upregulation of Mex3b in CRSwNP suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells, and thefore regulates tissue remodeling of CRS.
Commensal probiotic provides long-term protection in a murine model of allergic lung disease by altering the host microbiota
Authors: Swartzendruber Julie A , Del Toro Rosalinda Monroy , Incrocci Ryan , Negris Olivia , McGrath Shaina
Published: August 2023

The incidence of allergic diseases is increasing, with house dust mite allergy, afflicting up to 20% of the population in developed countries. House dust mites (HDM) are a common allergen that can be difficult to avoid and triggers allergic reactions, including wheezing and shortness of breath. Bacillus subtilis is a gut-associated commensal probiotic that can prevent murine HDM-induced allergic lung inflammation. We exposed C57Bl/6 mice to B. subtilis via oral gavage and challenged with intranasal HDM for up to 8 weeks. We found that pretreatment with B. subtilis was able to provide protection from eosinophil infiltration, measured in bronchial alveolar lavage fluid (BALF), for 3 weeks. This loss of protection correlated with an increase in the eosinophil chemoattractant CCL24 levels in the BALF and reduced abundance of the bacterial phylum Verrucomicorbiota in collected feces. Additionally, we found that B. subtilis treatment altered the bacterial composition by increasing the abundance of the bacteria phylum Bacteroidetes. These results support B. subtilis as a prophylactic for preventing allergic lung disease and highlights that protection can last up to 3 weeks. This work also expands our understanding of how B. subtilis mediates protection and that in addition to modifying the immune system, it is also altering the host microbiota.

This work is supported by start-up funds and core facility outsourcing funds from Midwestern University.
Lung microbial dysbiosis during early life promotes predisposition to allergic asthma
Authors: Bloodworth Jeffrey C , Hoji Aki , Cook-Mills Joan
Published: August 2023

Background

Allergic asthma is associated with lung microbial dysbiosis. We hypothesized that the microbiome from the lungs of allergy-predisposed mouse pups is sufficient to predispose pups to development of allergy. Allergic inflammation in pups of allergic dams is blocked by α-tocopherol supplementation of the dam during gestation and nursing, but it is unknown whether α-tocopherol alters microbial composition of the lung.

Methodology

Mouse pups were predisposed to allergy by inducing allergy in the mothers. Microbiome was separated from the bronchoalveolar lavage (BAL) of pups of allergic and non-allergic mothers and transferred intranasally at postnatal day 4. 16S rRNA sequencing was performed on the Illumina MiSeq platform.

Results

Compared to pups born to non-allergic dams and dams administered with a-tocopherol supplementation, mouse pups born to allergic dams had elevated abundance of several genera of the γ-Proteobacteria, Firmicutes, and Verrucomicrobia (p<0.05). There was decreased abundance of genera from Bacteroides, Firmicutes, and α-Proteobacteria (p<0.05). Transfer of lung microbiome from neonates of allergic dams to neonates of non-allergic dams was sufficient to confer responsiveness to allergen in the recipient pups. Neonates of allergic dams were not protected from development of allergy by transfer of donor lung microbiome from neonates of non-allergic dams or neonates of αT-supplemented allergic dams. The donor microbiome from pups of allergic mothers colonized the pup lung, suggesting that early life detrimental bacterial species persists.

Conclusion

The lung microbiome from allergy-predisposed pups is dominant and sufficient for enhanced neonate responsiveness to allergen.

Supported by grants from NIH (R01AI127695)
Impact of the LPS from Cyanobacteria Brasilonema sp. on Lung Inflammation and Glucocorticoid Expression
Authors: Swanson-Mungerson Michelle Anne , Del Toro Rosalinda Monroy , Jiang Kallie , Incrocci Ryan , Mayer Alejandro M S , Williams Philip , Swartzendruber Julie A
Published: August 2023

Inhaled glucocorticoids (GCs) are the first-line treatment for asthma. However, patients with asthma characterized by recruitment of neutrophils show poor sensitivity to GC therapy. A potential etiology of this GC-resistant asthma is aerosolized cyanobacterial lipopolysaccharide (LPS) from cyanobacterial blooms. Previously, we demonstrated that inhalation of LPS from Geitlerinema sp. of cyanobacteria induces neutrophilic lung infiltration in mice. Our current data shows that the LPS from another cyanobacteria, Brasilonema sp., also induces lung inflammation after inhalation. Flow cytometric data demonstrate that Brasilonema sp. LPS induces neutrophil infiltration and ELISA identified increases in pro-inflammatory cytokine levels in bronchoalveolar lung fluid after Brasilonema sp. LPS exposure. Furthermore, IHC data demonstrate that mice exposed to Brasilonema sp. LPS show an increased level of lung epithelial GCRβ, a glucocorticoid receptor (GR) analog that inhibits GC activity. Conversely, expression of GCRα, the GR isoform responsible for glucocorticoid sensitivity, remains unchanged. These data help to elucidate the mechanisms of Brasilonema sp. LPS-induced lung-inflammation and potential glucocorticoid resistance, while highlighting the importance of the toxin in promoting human disease near cyanobacterial blooms.

This work is supported by the Kenneth A. Suarez Fellowship and Intramural Funds at Midwestern University.
Probiotic-derived exopolysaccharide interferes with TH2 priming and inhibits allergic inflammation
Authors: Hollinger Maile Kananiokala , Velez Tania Estefania , Zamora-Pineda Jesus , Knight Katherine L , Sperling Anne I
Published: August 2023

Allergic asthma is an increasing burden in industrialized countries, where treatments are limited to targeting symptoms and end-stage effectors rather than root causes. In recent years, the use of prophylactic, inhaled probiotic cocktails has shown promise in preventing allergic asthma. However, the mechanisms by which such probiotics prevent allergic symptoms and the exact bacterial product(s) responsible for probiotic activity remain to be elucidated. In this study, we focused on exopolysaccharide (EPS) from Bacillus subtilis, a ubiquitous soil bacterium that is commonly found as a dietary supplement. We found that in a mouse model of allergic airway disease, EPS treatment, prior to allergic sensitization and challenge, limits lung mucus hypersecretion and eosinophilia. Furthermore, EPS treatment inhibited accumulation of TH2, but not TH1 and TH17 subsets. To determine whether EPS was affecting TH2 cell priming by dendritic cells (DCs), we compared the composition and phenotype of DCs in the lung and mediastinal lymph nodes (medLN) after EPS treatment. Surprisingly, pre-treatment with EPS induced DC maturation as measured by an increase in costimulatory molecules and a decrease in the ability to uptake allergen and migrate to the medLN. DC maturation by EPS was TLR4-dependent, as TLR4 KO DCs did not respond to EPS. Together, our data suggest that EPS inhibits allergic sensitization by inducing DC maturation prior to allergen exposure, thereby leading to decreased TH2 priming and allergic inflammation. Thus, we provide a potential mechanism by which inhalation of a single probiotic-derived molecule can prevent the development of allergic inflammation.

Supported by grants from NIH (3R01 AI125644-05S1, 1F31 HL156659-01A1)
Loss of microRNA-29 in B cells leads to atopic skin phenotype in mice
Authors: Borbet Timothy C , Hines Marcus J , Koralov Sergei B
Published: August 2023

Developing B cells are highly dependent on microRNA (miRNA) regulation. We have previously demonstrated the miR-29 family of miRNAs contributes to the regulation of B cell survival through fine-tuning of PTEN expression, a negative regulator of B cell receptor (BCR) signaling. When the miR-29 alleles are selectively deleted in B cells, this leads to reduced survival which can be rescued by ablating one copy of Pten to restore normal BCR signaling. We observed that mice with B cells lacking miR-29 also develop an inflammatory skin phenotype associated with lesions, leukocyte infiltration into the skin, and persistent pruritis. Nearly 50% of miR-29-deficient animals die within 6 months of birth, and ablation of Pten to restore BCR signaling did not improve the survival rate. This suggests that additional targets of miR-29 in B lymphocytes are involved in promoting this inflammatory skin condition. Epidermal thickening, granulocyte plasma cell infiltration in the skin, and high serum IgE titers suggest that miR-29 may contribute to the regulation of IgE-driven immune responses and that in the absence of this miRNA family, animals develop atopic dermatitis. Our bioinformatic analysis revealed that miR29 is predicted to regulate the expression of the Il4Ra transcript, and in vitro differentiation assays revealed that miR-29-deficient B cells have a higher propensity for IgE class switch recombination in response to IL-4 and/or IL-13 stimulation compared to wild-type control B cells. We conclude that miR-29 is critical in the regulation of both Pten and Il4ra expression in mature B cells thereby influencing the survival and function of peripheral B lymphocytes, which can have profound repercussions for the development of autoimmune disease.

The Bernard Levine Fellowship
Bispecific antibody Armed activated T cells exhibit time-dependent co-expression patterns of inhibitory ligands and gene expression programs
Authors: Barnes Rob , Thakur Archana , Dolatshahi Sepideh , Lum Lawrence
Published: August 2023

Immunotherapy success has been limited in treating solid tumors. Bispecific antibody Armed activated T cell (BATs) therapy activates, expands, and arms patient T cells ex vivo with bispecific antibodies (BiAb) to produce a “drug” that combines non-MHC restricted cytotoxicity of T cells and specificity of BiAb. BATs therapy promises to improve survival in patients with metastatic breast, prostate, and pancreatic cancer without dose limiting toxicities. However, the anti-tumor potency of BATs varies between patients and decreases with time. Understanding the mechanisms behind this decrease of potency will help identify therapeutic combinations and dosing strategies to maximize patient response.

Low effector:target coculture conditions allowing target cell escape from BAT killing were identified. BAT function was measured by anti-tumor cytotoxicity, flow cytometry, and RNA sequencing. Samples were taken at set timepoints to produce a dynamic representation of BAT functional changes in target escape conditions. We observed time-dependent co-expression of inhibitory ligands in BATs, including increased co-expression of PD-1 and LAG3 during the first 24h of coculture, followed by increased co-expression of TIGIT and LAG3 at later timepoints. RNA-seq revealed upregulation of genes associated with T cell dysfunction, including NR4A and checkpoint proteins TIGIT, LAG3, and PD-1, as early as 1 hr in coculture. These findings were integrated into a mechanistic model of BAT-target cell interactions. Our model demonstrated time-dependent emergence of therapeutically targetable inhibitory receptors, suggesting optimizing dosing targets and administration times may improve synergistic immunotherapy combination outcomes.
Examining methotrexate’s safety and efficacy in combination with immune checkpoint inhibitors to control immune related inflammatory arthritis in cancer
Authors: Papadopoulos Theodore , Santinon Francois , Hudson Marie , Del Rincon Sonia Victoria
Published: August 2023

Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8+T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8+T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8+T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.
Generation of novel anti-tumor chimeric antigen receptors incorporating downstream T-cell signaling proteins
Authors: Angeles Benjamin C , Moreno Taylor , Balagopalan Lakshmi , Yi Jason , Qin Haiying , Alvinez Neriah , Yamane Hide , Lee Mariah , Pallikkuth Sandeep , Tran Andy , McIntire Katherine , Taylor Naomi , Samelson Lawrence E
Published: August 2023

Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.
Hinging on Success: Leveraging the Power of CAR T-Cell Therapy through In-Silico Modeling of Hinge Length and Epitope Location
Authors: Mirazee Justin M , Aranganathan Akashnathan , Achar Sooraj , Jia Dongya , Chen Xiang , Vani Bodhi , Chien Christopher D , Pouzolles Marie , DeDe Kniya , Youkharibache Philippe , Walters Kylie , Tiwary Pratyush , Altan-Bonnet Grégoire , Taylor Naomi
Published: August 2023

Genetic engineering of T-cells using chimeric antigen receptors (CARs) has shown promise in the treatment of relapsed/refractory B-cell leukemia. However, a significant percentage of patients will eventually relapse, often due to the emergence of tumor cells expressing low levels of the target antigen. Previous research has demonstrated that the hinge domain of the CAR plays a crucial role in regulating the cytotoxic capabilities of these cells. In this study, we assessed the potential to model optimal hinge length for a given single-chain variable fragment (scFv) in relation to the location of the targeted epitope. We designed an array of hinges originating from CD8α and incorporated these hinges into CARs targeting CD19, CD22, CD33, and CD20 antigens. We found that the location of the epitope is critical in determining the optimal hinge; a narrow range of short hinges is more effective against membrane-distal epitopes while long hinges are more effective against membrane-proximal epitopes. To build an in-silico model, we used AlphaFold2 (AF2), reducing the length of the multiple sequence alignment (MSA) clusters to introduce stochasticity and increase the conformational diversity of the structural predictions for the CAR hinges and target proteins. We discovered that CARs with a predicted intermembrane distance of 15nm between the CAR and ligand were particularly effective against antigen-low leukemic cells. Our findings suggest that intermembrane distance can predict CAR responsiveness against a specific epitope, enabling the rapid optimization of CARs by adjusting hinge length. The development of this modeling strategy will facilitate the design of CARs with optimized cytotoxic potential against a wide range of novel targets.

Intramural Research Program of the National Cancer Institute (Bethesda, MD)
Functional and transcriptome analysis of peripheral blood lymphocytes in pancreatic cancer patients before and after chemotherapy
Authors: Brugiapaglia Silvia , Bulfamante Sara , Curcio Claudia , Follia Laura , Spadi Rosella , Cristiano Carmen , Bonello Lisa , Cappello Paola , Novelli Francesco
Published: August 2023

Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 11% due to its high resistance to chemotherapy (CT).

Understanding how to better induce an effector response following tumor associated antigen (TAA) stimulation is critical to set up an efficient tailored immunotherapy. Peripheral blood mononuclear cells (PBMC) from PDA patients, before and after gemcitabine-based CT, were in vitro stimulated with four TAA (ENO1, FUBP1, K2C8 and G3P) in presence or absence of immune-check point blockade (ICB). Gemcitabine increased the number of TAA recognized by patient T cells, which positively correlated with survival, a high-rate of TAA-induced proliferative responses and high IFN-γ production. By contrast, ICB suppressed TAA-induced proliferative responses before or after CT. Transcriptional analysis revealed that after CT only, responding patients displayed more differentially expressed genes than non-responding patients, suggesting that Gemcitabine modulates gene expression. The clonotypic analysis of ENO1-stimulated T cells from patients showed that TCRβ repertoire was modulated by CT as some clonotypes. These data indicate that Gemcitabine alone, as opposed to ICB, shifted the immunological tone toward a more effector phenotype. The expansion of V-J rearrangements due to TAA stimulation was enhanced by CT, with a stronger reactivity of precise TAA-specific T cell clones after CT, which support the development of precision immunotherapy in selected patient groups, based on CT combined with TAA vaccination.

Supported by grants from AIRC (ID 19931)
Super2-IL-33 armored CAR T cells reshape the tumor microenvironment to universally suppress solid tumor growth
Authors: Ferry Shannon Lynn , Brog Rachel A , Weyburne Emily , Murad Joana , Huang Yina H
Published: August 2023

Tumor associated immunosuppressive cells limit the efficacy of CAR T cells against solid tumors. To alter immunosuppression in the tumor microenvironment, CAR T cells were armored with an IL-2 superkine (Super2) and alarmin IL-33 and adoptively transferred into immunocompetent mice with established primary or metastatic B16F10 melanoma or intradermal MC38 colon carcinoma. We show that Super2-IL-33 CAR T cells universally promoted the control of solid tumors in mice. Analysis of tumor infiltrating lymphocytes (TILs) revealed that Super2 and IL-33 expanded CAR T cells and endogenous lymphocytes cells, resulting in an increased ratio of CD8 effector T cells to regulatory T cells. The expression of the CAR or effector molecules was dispensable for CAR T cell-induced tumor control, underscoring the contribution of endogenous immune cells in mediating therapeutic efficacy. We next evaluated the effects of Super2-IL-33 CAR T cells in bone, liver, and thymus (BLT) humanized mice to assess efficacy against human tumors in an immunocompetent model. BLT mice were intradermally inoculated with Caki1, a human renal cell carcinoma in which non-treated tumors are highly enriched for regulatory T cells. To determine the impact of Super2-IL-33 armored CAR T cells on human tumors and infiltrating immune cells, tumor growth and TILs were analyzed. Super2-IL-33 armored human CAR T cells recruit endogenous immune cells to the tumor that facilitate the suppression of tumor growth. Thus, the Super2-IL-33 cytokine combination may serve as a universal platform capable of overcoming immunosuppression to promote broad anti-tumor immunity.

Supported by grants from NIH (R43-CA271880 and R01-CA254042)
Engineering Antibody Fusion Proteins for Targeted Intracellular Therapeutic Delivery
Authors: Kaeo Kyle J , Ludwig Seth , Spangler Jamie
Published: August 2023

Intracellular (IC) proteins make up a significant portion of the proteome, leading to an interest in achieving immune modulation via IC targeted therapeutics. Most IC protein-targeted drugs are small molecules, which can easily pass through the cell membrane but are hindered by a lack of specificity and short half-life. Antibodies, thanks to their greater contact surface area, can have superior target specificity, and persist longer in the blood. Fusing a therapeutic to an antibody presents a promising solution, but antibodies lack a good path into the cell.

We recently discovered a multiparatopic antibody that binds multiple epitopes on the immune checkpoint protein programed death-ligand 1 (PD-L1) to induce its downregulation. This platform promotes endocytosis through clustering receptors together on the surface and enhancing internalization. We sought to exploit this mechanism for IC delivery by introducing endosomalytic peptides to induce escape of the multiparatopic antibody into the cytosol. We inserted two different endosomalytic peptides (EPs) into the downregulating antibody, one before and after the hinge region. We found that these EPs do not hinder downregulation and observed decreased lysosomal accumulation of the antibodies.

To determine the efficacy of this modified antibody as a vehicle for an IC therapeutic, we fused Cas9 to the C-terminus of the heavy chain (HC) of our anti-PD-L1 multiparatopic antibody. We further demonstrated robust PD-L1 downregulation and endosomal escape for these fusion proteins. These data generate proof-of-concept results, establishing a promising new vehicle for IC delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.