Research
Abstract
The loss of integrity of the blood retina barrier (BRB) is a key pathological hallmark of vision-threatening complications in diabetic retinopathy (DR). Although DR is considered a microvascular disease, mounting evidence from mouse models and patients show that inflammation is closely connected with microvasculopathy. Inflammatory responses during retinal pathophysiology are often orchestrated by microglia, resident innate immune cells of the retina. However, the precise role of microglia activity during DR pathogenesis remains elusive. Here, we used an anti PDGFRβ antibody and inducible endothelial cell-specific PDGFB-KO during postnatal development of retinal vasculature to reproduce key features of DR pathology in mice. In addition, we applied a minocycline therapy to modulate retinal inflammation. Postnatal depletion of pericytes or loss of PDGFB in retinal vessels altered BRB integrity, triggered secretion of angiogenic and inflammatory factors with concomitant microglia reactivity, which was sustained in mature retinas. Microglia reactivity was accompanied by upregulation of disease-associated genes. Notably, minocycline attenuated the cycle of inflammatory responses in young and mature retinas, thereby preserving retinal vascular and structural integrity in mice. Together, our findings suggest that immunomodulation of microglia-driven inflammatory responses preserves retinal vasculature and maintains BRB integrity in two different mouse models of human DR.
Authors
Urbanus Muthai Kinuthia, Christoph Möhle, Ralf H. Adams, Thomas Langmann
Abstract
Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously demonstrated that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here, using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a critical component of AT2 cell dysfunction and demonstrate that targeting this druggable signaling hub can rescue the aberrant AT2 cell metabolic phenotype and mitigate lung fibrosis in vivo.
Authors
Luis R. Rodríguez, Konstantinos-Dionysios Alysandratos, Jeremy Katzen, Aditi Murthy, Willy Roque Barboza, Yaniv Tomer, Sarah Bui, Rebeca Acín-Pérez, Anton Petcherski, Kasey Minakin, Paige Carson, Swati Iyer, Katrina Chavez, Charlotte H. Cooper, Apoorva Babu, Aaron I. Weiner, Andrew E. Vaughan, Zoltan Arany, Orian S. Shirihai, Darrell N. Kotton, Michael F. Beers
Abstract
Pancreatic ductal adenocarcinoma (PDAC) has a poor survival rate due to late detection. PDAC arises from precursor microscopic lesions, termed pancreatic intraepithelial neoplasia (PanIN), that develop at least a decade before overt disease––this provides an opportunity to intercept PanIN–to–PDAC progression. However, immune interception strategies require full understanding of PanIN and PDAC cellular architecture. Surgical specimens containing PanIN and PDAC lesions from a unique cohort of five treatment-naïve patients with PDAC were surveyed using spatial-omics (proteomic and transcriptomic). Findings were corroborated by spatial proteomics of PanIN and PDAC from tamoxifen-inducible KPC (tiKPC) mice. We uncovered the organization of lymphoid cells into tertiary lymphoid structures (TLSs) adjacent to PanIN lesions. These TLSs lacked CD21+CD23+ B cells compared to more mature TLSs near the PDAC border. PanINs harbored mostly CD4+ T cells with fewer Tregs and exhausted T cells than PDAC. Peri-tumoral space was enriched with naïve CD4+ and central memory T cells. These observations highlight the opportunity to modulate the immune microenvironment in PanINs before immune exclusion and immunosuppression emerge during progression into PDAC.
Authors
Melissa R. Lyman, Jacob T. Mitchell, Sidharth Raghavan, Luciane T. Kagohara, Amanda L. Huff, Saurav D. Haldar, Sarah M. Shin, Samantha Guinn, Benjamin Barrett, Gabriella Longway, Alexei Hernandez, Erin M. Coyne, Xuan Yuan, Lalitya Andaloori, Jiaying Lai, Yun Zhou Liu, Rachel Karchin, Anuj Gupta, Ashley L. Kiemen, André Forjaz, Denis Wirtz, Pei-Hsun Wu, Atul Deshpande, Jae W. Lee, Todd D. Armstrong, Nilofer S. Azad, Jacquelyn W. Zimmerman, Laura D. Wood, Robert A. Anders, Elizabeth D. Thompson, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho, Neeha Zaidi
Abstract
The pathobiology of pulmonary hypertension (PH) is complex and multiple cell types contribute to disease pathogenesis. We sought to characterize the molecular crosstalk between endothelial and mesenchymal cells that promote PH in the tumor necrosis factor alpha transgenic (TNF-Tg) model of PH. Pulmonary endothelial and mesenchymal cells were isolated from WT and TNF-Tg mice underwent single-cell RNA sequencing. Data were analyzed using clustering, differential gene expression and pathway analysis, ligand-receptor interaction, transcription factor binding, and RNA velocity assessments. Significantly altered ligand-receptor interactions were confirmed with immunofluorescent staining. TNF-Tg mice had increases in smooth muscle cells and Col14+ fibroblasts, and reductions in general capillary (gCAP) endothelial cells, Col13+ fibroblasts, pericytes, and myofibroblasts. Pathway analysis demonstrated NF-kB, JAK/STAT, and interferon mediated inflammation, endothelial apoptosis, loss of vasodilatory pathways, increased TGF-beta signaling, and smooth muscle cell proliferation. Ligand-receptor analysis demonstrated a loss of BMPR2 signaling in TNF lungs and establishment of a maladaptive BMP signaling cascade which functional studies revealed stems from endothelial NFkB activation and subsequent endothelial SMAD2/3 signaling. This system highlights a complex set of changes in cellular composition, cell communication, and cell fate driven by TNF signaling which lead to aberrant BMP signaling which is critical for development of PH.
Authors
ML Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, Ye Jin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman
Abstract
Dominant missense mutations in MYBPC1, the gene encoding the essential sarcomeric slow Myosin Binding Protein-C (sMyBP-C), are associated with Myotrem, a new, early-onset congenital myopathy characterized by muscle weakness, hypotonia, skeletal deformities, and myogenic tremor. Importantly, the clinical manifestation of Myotrem in mid- and late adulthood is unknown. Using the Myotrem MYBPC1 E248K Knock-In (KI) murine model, we interrogated contractile performance of soleus, gastrocnemius, and Tibalis Anterior (TA) muscles in both male and female mice in mid- (12-months) and late (24-months) adulthood. Our findings showed that the phenotypic manifestation of E248K Myotrem differs across muscle-type, sex, and age. While KI soleus muscle consistently exhibited contractile impairment across both sexes and ages, KI gastrocnemius muscle displayed preserved force production. Interestingly, TA muscle showed a sex- and age-specific impact with preserved function through 12-months in both sexes and a sharp decline at 24-months solely in males. Quantitative analysis of TA sarcomeric organization uncovered structural deficits coinciding with contractile dysfunction, supporting the notion that sMyBP-C serves a primarily structural role in skeletal muscle. Collectively, our studies revealed that aging impacts the E248K Myotrem myopathy in a muscle- and sex-dependent fashion and show that sarcomeric disorganization accompanies contractile deterioration in affected muscles.
Authors
Jennifer Megan Mariano, Humberto C. Joca, Jacob G. Kallenbach, Natasha Ranu, Julien Ochala, Christopher Ward, Aikaterini Kontrogianni-Konstantopoulos
Abstract
Androgen receptor positive prostate cancer (PC), castration resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) invariably become resistant to treatment with targeted and cytotoxic agents. Multiple pathways have been identified as being responsible for these pleotropic mechanisms of resistance. The MUC1 gene is aberrantly expressed in CRPC/NEPC in association with poor clinical outcomes; whereas, it is not known if the oncogenic MUC1-C/M1C protein drives treatment resistance. We demonstrated that MUC1-C is necessary for resistance of (i) PC cells to enzalutamide (ENZ), and (ii) CRPC and NEPC cells to docetaxel (DTX). Our results showed that MUC1-C-mediated resistance is conferred by upregulation of aerobic glycolysis and suppression of reactive oxygen species necessary for self-renewal. Dependence of these resistant phenotypes on MUC1-C for the cancer stem cell (CSC) state identified a potential target for treatment. In this regard, we further demonstrated that targeting MUC1-C with a M1C antibody-drug conjugate (ADC) is highly effective in suppressing (i) self-renewal of drug-resistant CRPC/NEPC CSCs and (ii) growth of t-NEPC tumor xenografts derived from drug-resistant cells and a patient with refractory disease. These findings uncovered a common MUC1-C-dependent pathway in treatment-resistant CRPC/NEPC progression and identified MUC1-C as a target for their treatment with a M1C ADC.
Authors
Keisuke Shigeta, Tatsuaki Daimon, Hiroshi Hongo, Sheng-Yu Ku, Hiroki Ozawa, Naoki Haratake, Atsushi Fushimi, Ayako Nakashoji, Atrayee Bhattacharya, Shinkichi Takamori, Michihisa Kono, Masahiro Rokugo, Yuto Baba, Takeo Kosaka, Mototsugu Oya, Justine Jacobi, Mark D. Long, Himisha Beltran, Donald W. Kufe
Abstract
IL-33 is a key driver of type-2 inflammation and implicated in pathology of COPD and asthma. However, the mechanism for IL-33 secretion and regulation in the context of chronic airway disease is poorly understood. We previously reported an airway disease-associated isoform IL-33Δ34 that escapes nuclear sequestration and is tonically secreted from epithelial cells. Here, we describe how this IL-33Δ34 isoform interacts with HSP70 within cells and is targeted to secretory organelles through coordinated binding to phosphatidylserine (PS), and delivered to compartments for unconventional protein secretion (CUPS). Once secreted, extracellular HSP70 (eHSP70) in complex with IL-33Δ34 stabilizes cytokine by inhibiting oxidation and degradation, which results in enhanced IL-33Δ34-receptor binding and activity. We further find evidence that IL-33 along with mediators of the proteostasis network HSP70, HSP90 and the Chaperonin Containing TCP1 (CCT) complex are dysregulated in human chronic airway disease. This phenomenon is reflected in the differential extracellular vesicle (EV) proteome in bronchial wash from COPD and asthma samples, which could mark disease activity and potentiate IL-33 function. This study confirms proteostasis intermediates, chiefly HSP70, as a chaperone for non-canonical IL-33 secretion and activity that may be amenable for therapeutic targeting in the chronic airway diseases COPD and asthma.
Authors
Omar A. Osorio, Heather E. Raphael, Colin E. Kluender, Ghandi F. Hassan, Lucy S. Cohen, Deborah F. Steinberg, Ella Katz-Kiriakos, Morgan D. Payne, Ethan M. Luo, Jamie L. Hicks, Derek E. Byers, Jennifer Alexander-Brett
Abstract
High myopia (HM) and posterior staphyloma (PS) are major causes of vision loss worldwide. Genetic and environmental factors, especially light exposure, influence myopia. This study shows that LRP2 (Low-density lipoprotein-related receptor type 2) levels are decreased in the vitreous of patients with HM and PS, and that in human donor eyes affected by PS, LRP2 expression was reduced in the neural retina and retinal pigment epithelium (RPE), with morphologic changes similar to those observed in the Foxg1-Cre-Lrp2lox/lox mouse that also develops PS. In human iPSc-derived RPE cells (iRPE), LRP2 silencing regulated genes involved in eye and neuronal development, visual perception, tissue remodeling, hormone metabolism and RPE structure. Its expression increased under light exposure, particularly red light, but was downregulated by cortisol. These findings establish a link between LRP2, myopization, and environmental factors, highlighting its crucial role in nonsyndromic HM and PS. LRP2 appears to be a promising therapeutic target for high myopia treatment.
Authors
Kimberley Delaunay, Emilie Picard, Patricia Lassiaz, Laurent Jonet, Vidjea Cannaya, José Maria Ruiz-Moreno, Kentaro Kojima, Henrik Vorum, Bent Honoré, Jorge R. Medrano, Lasse Jørgensen Cehofski, Eric Pussard, Renata Kozyraki, Alicia Torriglia, Olivier Cases, Francine Behar-Cohen
Abstract
Prion diseases are fatal, infectious and incurable neurodegenerative conditions affecting humans and animals, caused by the misfolding of the cellular prion protein (PrPC) into its pathogenic isoform, PrPSc. In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form. Recently, we demonstrated that treatment with the FDA-approved anti-HIV drug Efavirenz (EFV) significantly reduced PrPSc and extended survival of scrapie prion-infected mice. Among other effects, EFV activates the brain cholesterol metabolizing enzyme, CYP46A1, which converts cholesterol into 24S-hydroxycholesterol (24S-HC). However, drugs effective against scrapie prions often fail in human prion diseases, and a relation of the anti-prion effects of EFV to CYP46A1 activation is not established. Thus, we evaluated EFV treatment in mice overexpressing human PrPC infected with human sCJD prions. Oral, low-dose EFV treatment starting at 30- or 130-days post-infection significantly slowed disease progression and extended their survival. At early clinical stage, we observed reduced PrPSc accumulation, decreased cholesterol and lipid droplet content, and elevated CYP46A1 and 24S-HC levels in EFV-treated mice. Overexpression of CYP46A1 in prion-infected neuronal cells reduced PrPSc levels and increased 24S-HC, indicating that anti-prion effects of EFV correlate with CYP46A1 activation. These findings highlight EFV as a safe and efficacious therapeutic candidate for human prion diseases.
Authors
Tahir Ali, Jessica Cashion, Samia Hannaoui, Hanaa Ahmed-Hassan, Hermann M. Schatzl, Sabine Gilch
Abstract
The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with initiation and progression of lung fibrosis.
Authors
Jeremy A. Herrera, Mark Maslanka, Rachel Z. Blumhagen, Rachel Blomberg, Nyan Ye Lwin, Janna Brancato, Carlyne D. Cool, Jonathan P. Huber, Jonathan S. Kurche, Chelsea M. Magin, Kirk C. Hansen, Ivana V. Yang, David A. Schwartz
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