Bresciani, N;Demagny, H;Lemos, V;Pontanari, F;Li, X;Sun, Y;Li, H;Perino, A;Auwerx, J;Schoonjans, K;
PMID: 35714811 | DOI: 10.1016/j.jhep.2022.05.040
Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47hep-/- and Slc25a47-Fgf21hep-/- mice.Slc25a47hep-/- mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced in Slc25a47hep-/- mice, we generated a double Slc25a47-Fgf21hep-/- mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47hep-/- mice could not be rescued with the genetic removal of Fgf21.Collectively, our data place SLC25A47 at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of SLC25A47 in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.SLC25A47 is a liver-specific mitochondrial carrier. Slc25a47hep-/- mice are unable to maintain mitochondrial homeostasis in hepatocytes and show impaired mitochondrial respiration resulting in chronic energy deficiency, mitochondrial stress, and fibrosis in hepatocytes. Hepatic mitochondrial stress is characterized by the secretion of the mitokine FGF21 which drives a strong and systemic hypermetabolic state impacting whole-body physiology.
Todd, JL;Weber, JM;Kelly, FL;Neely, ML;Mulder, H;Frankel, CW;Nagler, A;McCrae, C;Newbold, P;Kreindler, J;Palmer, SM;
PMID: 37003354 | DOI: 10.1016/j.chest.2023.03.033
Chronic lung allograft dysfunction (CLAD) is the leading cause of death among lung transplant recipients. Eosinophils, effector cells of type 2 immunity, are implicated in the pathobiology of many lung diseases and prior studies suggest their presence associates with acute rejection or CLAD after lung transplantation.Does histological allograft injury or respiratory microbiology correlate with the presence of eosinophils in bronchoalveolar lavage fluid (BALF)? Does early posttransplant BALF eosinophilia associate with future CLAD development, including after adjustment for other known risk factors?We analyzed BALF cell count, microbiology, and biopsy data from a multicenter cohort of 531 lung recipients with 2592 bronchoscopies over the first posttransplant year. Generalized estimating equation models were utilized to examine the correlation of allograft histology or BALF microbiology with the presence of BALF eosinophils. Multivariable Cox regression was used to determine the association between ≥1% BALF eosinophils in the first posttransplant year and definite CLAD. Expression of eosinophil-relevant genes was quantified in CLAD and transplant control tissues.The odds of BALF eosinophils being present was significantly higher at the time of acute rejection and non-rejection lung injury histologies and during pulmonary fungal detection. Early posttransplant ≥1% BALF eosinophils significantly and independently increased the risk for definite CLAD development (adjusted hazard ratio 2.04, p=0.009). Tissue expression of eotaxins, IL13 related genes, and the epithelial-derived cytokines IL33 and thymic stromal lymphoprotein were significantly increased in CLAD.BALF eosinophilia was an independent predictor of future CLAD risk across a multicenter lung recipient cohort. Additionally, type 2 inflammatory signals were induced in established CLAD. These data underscore the need for mechanistic and clinical studies to clarify the role of type 2 pathway-specific interventions in CLAD prevention or treatment.
Lee, SH;Kim, N;Kim, M;Woo, SH;Han, I;Park, J;Kim, K;Park, KS;Kim, K;Shim, D;Park, SE;Zhang, JY;Go, DM;Kim, DY;Yoon, WK;Lee, SP;Chung, J;Kim, KW;Park, JH;Lee, SH;Lee, S;Ann, SJ;Lee, SH;Ahn, HS;Jeong, SC;Kim, TK;Oh, GT;Park, WY;Lee, HO;Choi, JH;
PMID: 36115863 | DOI: 10.1038/s41467-022-33202-2
Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.
Biochemical and biophysical research communications
Lin, C;Xiao, Z;Zhang, X;Wu, G;
PMID: 35430449 | DOI: 10.1016/j.bbrc.2022.04.034
Circular RNAs (circRNAs) are a class of noncoding RNAs generated by a specific type of RNA alternative splicing called backsplicing through various mechanisms. Recently, thousands of circRNAs have been identified by high-throughput RNA sequencing technologies and bioinformatics analysis. However, the functions of the majority have not been fully elucidated yet. Different tools, such as in situ hybridization, can help visualize the spatial temporal distribution of circRNA molecules, thus assisting the understanding of their biological and physiological functions. Here, we present a simple and straightforward method based on padlock probe hybridization and rolling circle amplification (RCA) for in situ detection of circRNAs. We compared our method with the commercially available BaseScope assay for the detection of Cdr1as in the mouse brain tissue. The result showed that the two methods have achieved comparable detection efficiency, thus demonstrating our padlock probe assay as an alternative yet simple circRNA in situ detection method for the research community.
Hong, DS;Van Tine, BA;Biswas, S;McAlpine, C;Johnson, ML;Olszanski, AJ;Clarke, JM;Araujo, D;Blumenschein, GR;Kebriaei, P;Lin, Q;Tipping, AJ;Sanderson, JP;Wang, R;Trivedi, T;Annareddy, T;Bai, J;Rafail, S;Sun, A;Fernandes, L;Navenot, JM;Bushman, FD;Everett, JK;Karadeniz, D;Broad, R;Isabelle, M;Naidoo, R;Bath, N;Betts, G;Wolchinsky, Z;Batrakou, DG;Van Winkle, E;Elefant, E;Ghobadi, A;Cashen, A;Grand'Maison, A;McCarthy, P;Fracasso, PM;Norry, E;Williams, D;Druta, M;Liebner, DA;Odunsi, K;Butler, MO;
PMID: 36624315 | DOI: 10.1038/s41591-022-02128-z
Affinity-optimized T cell receptors can enhance the potency of adoptive T cell therapy. Afamitresgene autoleucel (afami-cel) is a human leukocyte antigen-restricted autologous T cell therapy targeting melanoma-associated antigen A4 (MAGE-A4), a cancer/testis antigen expressed at varying levels in multiple solid tumors. We conducted a multicenter, dose-escalation, phase 1 trial in patients with relapsed/refractory metastatic solid tumors expressing MAGE-A4, including synovial sarcoma (SS), ovarian cancer and head and neck cancer ( NCT03132922 ). The primary endpoint was safety, and the secondary efficacy endpoints included overall response rate (ORR) and duration of response. All patients (N = 38, nine tumor types) experienced Grade ≥3 hematologic toxicities; 55% of patients (90% Grade ≤2) experienced cytokine release syndrome. ORR (all partial response) was 24% (9/38), 7/16 (44%) for SS and 2/22 (9%) for all other cancers. Median duration of response was 25.6 weeks (95% confidence interval (CI): 12.286, not reached) and 28.1 weeks (95% CI: 12.286, not reached) overall and for SS, respectively. Exploratory analyses showed that afami-cel infiltrates tumors, has an interferon-γ-driven mechanism of action and triggers adaptive immune responses. In addition, afami-cel has an acceptable benefit-risk profile, with early and durable responses, especially in patients with metastatic SS. Although the small trial size limits conclusions that can be drawn, the results warrant further testing in larger studies.
Greguske, EA;Maroto, AF;Borrajo, M;Palou, A;Gut, M;Esteve-Codina, A;Barrallo-Gimeno, A;Llorens, J;
PMID: 37100209 | DOI: 10.1016/j.nbd.2023.106134
The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.
Cho, I;Chang, JB;
PMID: 35233025 | DOI: 10.1038/s41598-022-06903-3
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.
Applied In Vitro Toxicology
Neau L, Lorin C, Frentzel S, Hoeng J, Iskandar A, Leroy P, Trivedi K.
PMID: - | DOI: 10.1089/aivt.2018.0021
Abstract
Introduction: Developed by Advanced Cell Diagnostics, RNAscope™in situ hybridization technology enables detection of a target RNA in a cell-specific manner on formalin-fixed paraffin-embedded tissue sections and represents a good alternative to immunohistochemistry. The goal of this work is to illustrate an optimized protocol of the RNAscope technology to detect target genes in various human organotypic culture models (nasal, small airway, and gingival). These culture models retain the three-dimensional structure of native epithelium, mimic in vivo morphology and human physiology, and can be used as alternative sources to animal testing.
Materials and Methods: After fixation and processing of five replicates of the three different organotypic cell cultures, the tissue morphology was checked by hematoxylin and eosin staining. The RNAscope protocols were optimized based on three crucial parameters: heat pretreatment, enzymatic digestion, and signal amplification. Digital images of the RNAscope stained slides were generated using the Hamamatsu NanoZoomer 2.0 slide scanner, and images were quantified using a custom-made plugin on Definiens Tissue Studio software (Definiens AG, Munich, Germany).
Results: The tissue morphology demonstrates optimum fixation and processing for samples, while the optimized protocol for RNAscope shows preserved RNA with staining on the positive control probe with score ≥2 and no staining on the negative control probe with score <1.
Discussion and Conclusion: RNAscope combined with organotypic cell cultures is a promising tool to better understand cell-specific RNA expression while implementing 3R (replace, reduce, and refine animal testing) principles
Fe Lanfranco M, Loane DJ, Mocchetti I, Burns MP, Villapol S.
PMID: 29238736 | DOI: 10.21769/BioProtoc.2608
Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels.
Celik M�, Labuz D, Keye J, Glauben R, Machelska H
PMID: 32102987 | DOI: 10.1172/jci.insight.133093
IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, ?-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (?, ?, ?) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4-treated donors. Together, IL-4-induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain
Rodrigo Albors, A;Singer, GA;Llorens-Bobadilla, E;Frisén, J;May, AP;Ponting, CP;Storey, KG;
PMID: 36706756 | DOI: 10.1016/j.devcel.2023.01.003
The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age. Ependymal cells are, however, heterogeneous, and the biological diversity this represents and how it changes with age remain unknown. Here, we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice, identifying not only all known ependymal cell subtypes but also immature as well as mature cell states. By comparing transcriptomes of spinal cord and brain ependymal cells, which lack stem cell abilities, we identify immature cells as potential spinal cord stem cells. Following spinal cord injury, these cells re-enter the cell cycle, which is accompanied by a short-lived reversal of ependymal cell maturation. We further analyze ependymal cells in the human spinal cord and identify widespread cell maturation and altered cell identities. This in-depth characterization of spinal cord ependymal cells provides insight into their biology and informs strategies for spinal cord repair.
