IU Indianapolis ScholarWorks :: Browsing by Author "Linnemann, Amelia K."

Browsing by Author "Linnemann, Amelia K."

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Aberrant gene expression induced by a high fat diet is linked to H3K9 acetylation in the promoter-proximal region
(Elsevier, 2021-03) Morral, Núria; Liu, Sheng; Conteh, Abass M.; Chu, Xiaona; Wang, Yue; Dong, X. Charlie; Liu, Yunlong; Linnemann, Amelia K.; Wan, Jun; Medical and Molecular Genetics, School of Medicine
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with an estimated global prevalence of 1 in 4 individuals. Aberrant transcriptional control of gene expression is central to the pathophysiology of metabolic diseases. However, the molecular mechanisms leading to gene dysregulation are not well understood. Histone modifications play important roles in the control of transcription. Acetylation of histone 3 at lysine 9 (H3K9ac) is associated with transcriptional activity and is implicated in transcript elongation by controlling RNA polymerase II (RNAPII) pause-release. Hence, changes in this histone modification may shed information on novel pathways linking transcription control and metabolic dysfunction. Here, we carried out genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet (an animal model of NAFLD), and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases.
Allergic airway recall responses require IL-9 from resident memory CD4+ T cells
(American Association for the Advancement of Science, 2022) Ulrich, Benjamin J.; Kharwadkar, Rakshin; Chu, Michelle; Pajulas, Abigail; Muralidharan, Charanya; Koh, Byunghee; Fu, Yongyao; Gao, Hongyu; Hayes, Tristan A.; Zhou, Hong-Ming; Goplen, Nick P.; Nelson, Andrew S.; Liu, Yunlong; Linnemann, Amelia K.; Turner, Matthew J.; Licona-Limón, Paula; Flavell, Richard A.; Sun, Jie; Kaplan, Mark H.; Microbiology and Immunology, School of Medicine
Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4+ T cells that secreted IL-9 as an obligate effector cytokine. IL-9-secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall-specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4+ T cell population was required for an allergen recall response.
Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics
(Nature Publishing Group, 2020-10-09) Ast, Julia; Arvaniti, Anastasia; Fine, Nicholas H. F.; Nasteska, Daniela; Ashford, Fiona B.; Stamataki, Zania; Koszegi, Zsombor; Bacon, Andrea; Jones, Ben J.; Lucey, Maria A.; Sasaki, Shugo; Brierley, Daniel I.; Hastoy, Benoit; Tomas, Alejandra; D’Agostino, Giuseppe; Reimann, Frank; Lynn, Francis C.; Reissaus, Christopher A.; Linnemann, Amelia K.; D’Este, Elisa; Calebiro, Davide; Trapp, Stefan; Johnsson, Kai; Podewin, Tom; Broichhagen, Johannes; Hodson, David J.; Pediatrics, School of Medicine
Dysfunctional β-cell autophagy induces β-cell stress and enhances islet immunogenicity
(Frontiers Media, 2025-01-29) Austin, Matthew C.; Muralidharan, Charanya; Roy, Saptarshi; Crowder, Justin J.; Piganelli, Jon D.; Linnemann, Amelia K.; Biochemistry and Molecular Biology, School of Medicine
Background: Type 1 Diabetes (T1D) is caused by a combination of genetic and environmental factors that trigger autoimmune-mediated destruction of pancreatic β-cells. Defects in β-cell stress response pathways such as autophagy may play an important role in activating and/or exacerbating the immune response in disease development. Previously, we discovered that β-cell autophagy is impaired prior to the onset of T1D, implicating this pathway in T1D pathogenesis. Aims: To assess the role of autophagy in β-cell health and survival, and whether defects in autophagy render islets more immunogenic. Methods: We knocked out the critical autophagy enzyme, ATG7, in the β-cells of mice (ATG7Δβ-cell) then monitored blood glucose, performed glucose tolerance tests, and evaluated bulk islet mRNA and protein. We also assessed MHC-I expression and presence of CD45+ immune cells in ATG7Δβ-cell islets and evaluated how impaired autophagy affects EndoC-βH1 HLA-I expression under basal and IFNα stimulated conditions. Lastly, we co-cultured ATG7Δβ-cell islet cells with diabetogenic BDC2.5 helper T cells and evaluated T cell activation. Results: We found that all ATG7Δβ-cell mice developed diabetes between 11-15 weeks of age. Gene ontology analysis revealed a significant upregulation of pathways involved in inflammatory processes, response to ER stress, and the ER-associated degradation pathway. Interestingly, we also observed upregulation of proteins involved in MHC-I presentation, suggesting that defective β-cell autophagy may alter the immunopeptidome, or antigen repertoire, and enhance β-cell immune visibility. In support of this hypothesis, we observed increased MHC-I expression and CD45+ immune cells in ATG7Δβ-cell islets. We also demonstrate that HLA-I is upregulated in EndoC β-cells when autophagic degradation is inhibited. This effect was observed under both basal and IFNα stimulated conditions. Conversely, a stimulator of lysosome acidification/function, C381, decreased HLA-I expression. Lastly, we showed that in the presence of islet cells with defective autophagy, there is enhanced BDC2.5 T cell activation. Conclusions: Our findings demonstrate that β-cell autophagy is critical to cell survival/function. Defective β-cell autophagy induces ER stress, alters pathways of antigen production, and enhances MHC-I/HLA-I presentation to surveilling immune cells. Overall, our results suggest that defects in autophagy make β-cells more susceptible to immune attack and destruction.
Editorial: Pancreas Imaging Across the Spectrum
(Frontiers Media, 2021-01-03) Linnemann, Amelia K.; Poitout, Vincent; Rutter, Guy A.; Pediatrics, School of Medicine
Efficient transduction of pancreas tissue slices with genetically encoded calcium integrators
(bioRxiv, 2025-03-25) Lazimi, Charles S.; Stis, Austin E.; Panzer, Julia K.; Hiller, Helmut; Beery, Maria L.; Linnemann, Amelia K.; Stabler, Cherie L.; Mathews, Clayton E.; Phelps, Edward A.; Pediatrics, School of Medicine
This study combines live pancreas tissue slices with adenoviral transduction of the Calcium Modulated Photoactivatable Ratiometric Integrator 2 (CaMPARI2) biosensor for high-throughput analysis of islet calcium responses. Pancreas slices preserve islets within their native microenvironment, adding tissue context to the study of islet function and pathology. A key challenge of the pancreas slice model has been efficient transgene delivery while maintaining viability and function. Here, we demonstrate a robust adenoviral gene delivery approach using targeted and universal promoters. By transducing slices with CaMPARI2 and applying 405 nm photoconverting light, we permanently marked glucose-induced calcium activity across entire islet populations while preserving the in situ tissue context. Applied to nPOD donor tissues, including from individuals with type 1 diabetes, type 2 diabetes, and non-diabetic controls, this approach demonstrated glucose responsive CaMPARI2 labeling that correlated with insulin secretion. Integrating CaMPARI2 with pancreas slices enables multiplexed analyses, linking a functional readout with spatial context through immunostaining or gene expression to advance understanding of human islet behavior.
Expanded LUXendin Color Palette for GLP1R Detection and Visualization In Vitro and In Vivo
(American Chemical Society, 2022-04-04) Ast, Julia; Novak, Alissa N.; Podewin, Tom; Fine, Nicholas H.F.; Jones, Ben; Tomas, Alejandra; Birke, Ramona; Roßmann, Kilian; Eichhorst, Jenny; Lehmann, Martin; Linnemann, Amelia K.; Hodson, David J.; Broichhagen, Johannes; Pediatrics, School of Medicine
The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615, and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology.
Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells
(American Society of Hematology, 2023) Kaczmarek, Radoslaw; Piñeros, Annie R.; Patterson, Paige E.; Bertolini, Thais B.; Perrin, George Q.; Sherman, Alexandra; Born, Jameson; Arisa, Sreevani; Arvin, Matthew C.; Kamocka, Malgorzata M.; Martinez, Michelle M.; Dunn, Kenneth W.; Quinn, Sean M.; Morris, Johnathan J.; Wilhelm, Amelia R.; Kaisho, Tsuneyasu; Munoz-Melero, Maite; Biswas, Moanaro; Kaplan, Mark H.; Linnemann, Amelia K.; George, Lindsey A.; Camire, Rodney M.; Herzog, Roland W.; Pediatrics, School of Medicine
Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.
Fluorescently conjugated annular fibrin clot for multiplexed real-time digestion analysis
(Royal Society of Chemistry, 2021-12) Zeng, Ziqian; Nallan Chakravarthula, Tanmaye; Muralidharan, Charanya; Hall, Abigail; Linnemann, Amelia K.; Alves, Nathan J.; Emergency Medicine, School of Medicine
Impaired fibrinolysis has long been considered as a risk factor for venous thromboembolism. Fibrin clots formed at physiological concentrations are promising substrates for monitoring fibrinolytic performance as they offer clot microstructures resembling in vivo. Here we introduce a fluorescently labeled fibrin clot lysis assay which leverages a unique annular clot geometry assayed using a microplate reader. A physiologically relevant fibrin clotting formulation was explored to achieve high assay sensitivity while minimizing labeling impact as fluorescence isothiocyanate (FITC)-fibrin(ogen) conjugations significantly affect both fibrin polymerization and fibrinolysis. Clot characteristics were examined using thromboelastography (TEG), turbidity, scanning electron microscopy, and confocal microscopy. Sample fibrinolytic activities at varying plasmin, plasminogen, and tissue plasminogen activator (tPA) concentrations were assessed in the present study and results were compared to an S2251 chromogenic assay. The optimized physiologically relevant clot substrate showed minimal reporter-conjugation impact with nearly physiological clot properties. The assay demonstrated good reproducibility, wide working range, kinetic read ability, low limit of detection, and the capability to distinguish fibrin binding-related lytic performance. In combination with its ease for multiplexing, it also has applications as a convenient platform for assessing patient fibrinolytic potential and screening thrombolytic drug activities in personalized medical applications.
Identification of a Hypothalamic Neural System That Can Reduce Body Weight and Adipose Mass in Diet-Induced Obesity
(2024-11) Basu, Rashmita; Flak, Jonathan N.; Linnemann, Amelia K.; Witczak, Carol A.; Sheets, Patrick L.; Jerde, Travis J.
Dynamic hypothalamic circuits balance energy intake with expenditure to protect individuals from obesity. Lasting negative energy balance, however, triggers a compensatory decrease in energy expenditure, hindering progressive weight loss. While we understand some key players underlying energy balance, the detailed neural underpinnings remain unclear. Here I will delineate the functional efferent circuitry from the ventromedial hypothalamic nucleus (VMN) that facilitates weight loss and prevents rebound weight gain. VMN neurons have long been linked to a role in energy balance. Both vesicular communication by VMN steroidogenic factor 1 (SF1) neurons and pituitary adenylate cyclase activating peptide (PACAP) release from VMN neurons are essential for maintaining body weight and activating VMNSf1 neurons curbs diet-induced obesity without altering food intake. However, the exact pathway of this VMN signal is unclear because the VMN does not directly communicate with preganglionic sympathetic neurons, indicating signal transmission through an efferent node. Of the few brain sites they communicate with, VMNSf1 neurons sends the densest projections to the caudal preoptic area (POA) and the anterior bed nucleus of stria terminalis (BNST). Stimulating VMNPACAP axonal fibers in the caudal POA, but not anterior BNST, induced thermogenesis in brown and beige adipose tissues in both sexes of mice. To identify caudal POA populations in body weight regulation, I activated excitatory (glutamatergic) and inhibitory (GABAergic) caudal POA cells in diet-induced obese male mice and found that both glutamatergic and GABAergic caudal POA neurons can reduce diet-induced obesity through separate means. While there is intra-POA communication, my data supports efferent communication with separate downstream circuits by glutamate and GABA caudal POA cells in ameliorating diet-induced obesity. Because the POA and BNST are extremely complex regions with diverse functions, I then employed deep transfer learning to pinpoint obesity and diabetes risk-associated cell subsets in the POA and BNST. Using single nuclei RNA sequencing on >200,000 nuclei from both sexes of mice, I identified 6 specialized sets of caudal POA and BNST neuronal subtypes that increased in obese and glucose-intolerant mice on a high-fat diet. Targeting these newly identified pathways and neuron subtypes could lead to future obesity and diabetes therapeutics.

Browsing by Author "Linnemann, Amelia K."

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