ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing mRNA

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Abstract

Tuberculosis, a chronic infectious disease caused by a single pathogen, holds the highest mortality rate worldwide. RNA-binding proteins (RBPs) are involved in autophagy — a key defense mechanism against Mycobacterium tuberculosis (Mtb) infection — by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during Mtb infection remain relatively unexplored. ZNFX1, a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in Mtb-infected macrophages. Here, we aimed to explore the immune regulatory functions of ZNFX1 during Mtb infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased Mtb burden, progressive lung tissue injury, and chronic inflammation in Mtb-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing Prkaa2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to Mtb growth suppression. These findings reveal a function of ZNFX1 in establishing anti-Mtb immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster anti-tuberculosis immunotherapy.

Authors

Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma

Horizontal transmission of gut microbiota attenuates mortality in lung fibrosis

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Abstract

Pulmonary fibrosis is a chronic and often fatal disease. The pathogenesis is characterized by aberrant repair of lung parenchyma resulting in loss of physiological homeostasis, respiratory failure and death. The immune response in pulmonary fibrosis is dysregulated. The gut microbiome is a key regulator of immunity. The role of the gut microbiome in regulating the pulmonary immunity in lung fibrosis is poorly understood. Here, we determine the impact of gut microbiota on pulmonary fibrosis in C57BL/6 mice derived from different vendors (C57BL/6J and C57BL/6NCrl). We use germ free models, fecal microbiota transplantation and cohousing to transmit gut microbiota. Metagenomic studies of feces establish keystone species between sub-strains. Pulmonary fibrosis is microbiota dependent in C57BL/6 mice. Gut microbiota are distinct by β diversity (PERMANOVA P<0.001) and α diversity (P<0.0001). Mortality and lung fibrosis are attenuated in C57BL/6NCrl mice. Elevated CD4+ IL-10+ T cells and lower IL-6 occur in C57BL/6NCrl mice. Horizontal transmission of microbiota by cohousing attenuates mortality in C57BL/6J mice and promotes a transcriptionally altered pulmonary immunity. Temporal changes in lung and gut microbiota demonstrates that gut microbiota contribute largely to immunological phenotype. Key regulatory gut microbiota contribute to lung fibrosis generating rationale for human studies.

Authors

Stephen J. Gurczynski, Jay H. Lipinski, Joshua Y. Strauss, Shafiul Alam, Gary B. Huffnagle, Piyush Ranjan, Lucy H. Kennedy, Bethany B. Moore, David N. O'Dwyer

A mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition

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Abstract

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-seq comparing osteoblasts differentiated from Ezh2R684C/+ and Ezh2+/+ bone marrow mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A/6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state, and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Authors

Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner

Neuregulin 4 mediates the metabolic benefits of mild cold exposure by promoting beige fat thermogenesis

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Abstract

Interorgan crosstalk via secreted hormones and metabolites is a fundamental aspect of mammalian metabolic physiology. Beyond the highly specialized endocrine cells, peripheral tissues are emerging as an important source of metabolic hormones that influence energy and nutrient metabolism and contribute to disease pathogenesis. Neuregulin 4 (Nrg4) is a fat-derived hormone that protects mice from nonalcoholic steatohepatitis (NASH) and NASH-associated liver cancer by shaping hepatic lipid metabolism and the liver immune microenvironment. Despite its enriched expression in brown fat, whether NRG4 plays a role in thermogenic response and mediates the metabolic benefits of cold exposure remain unexplored. Here we show that Nrg4 expression in inguinal white adipose tissue (iWAT) is highly responsive to chronic cold exposure. Nrg4 deficiency impairs beige fat induction and renders mice more susceptible to diet-induced metabolic disorders under mild cold conditions. Using mice with adipocyte and hepatocyte-specific Nrg4 deletion, we reveal that adipose tissue-derived NRG4, but not hepatic NRG4, is essential for beige fat induction following cold acclimation. Furthermore, treatment with recombinant NRG4-Fc fusion protein promotes beige fat induction in iWAT and improves metabolic health in diet-induced obese mice. These findings highlight a critical role of NRG4 in mediating beige fat induction and preserving metabolic health under mild cold conditions.

Authors

Zhimin Chen, Peng Zhang, Tongyu Liu, Xiaoxue Qiu, Siming Li, Jiandie D. Lin

Hypoxia-activated prodrug and anti-angiogenic therapy cooperatively treat pancreatic cancer but elicit immunosuppressive G-MDSC infiltration

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Abstract

We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived model of pancreatic adenocarcinoma (PDAC) to examine the tumor response and adaptive resistance mechanisms involved in response to two established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast to prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8 T cell response. Bulk-tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid derived suppressor cells (G-MDSC) through CCL9 secretion. Blockade of the CCL9-CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302 and anti-VEGFR-2 with ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization, and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.

Authors

Arthur Liu, Seth T. Gammon, Federica Pisaneschi, Akash Boda, Casey R. Ager, David Piwnica-Worms, David S. Hong, Michael A. Curran

Lactate and Immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs

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Abstract

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for four weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transients measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.

Authors

Kalina J. Rossler, Willem J. De Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge

Spatial transcriptomics identifies candidate stromal drivers of benign prostatic hyperplasia

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Abstract

Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the “reawakening” of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were Insulin Like Growth Factor 1 (IGF1) and C-X-C Motif Chemokine Ligand 13 (CXCL13), which we confirmed by RNA in situ hybridization to be co-expressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in three-dimensional culture. Our findings partially support historic speculations on the etiology of BPH, and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.

Authors

Anna S. Pollack, Christian A. Kunder, Noah Brazer, Zhewei Shen, Sushama Varma, Robert B. West, Gerald R. Cunha, Laurence S. Baskin, James D. Brooks, Jonathan R. Pollack

Disruption of CFAP418 interaction with lipids causes widespread abnormal membrane-associated cellular processes in retinal degenerations

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Abstract

Syndromic ciliopathies and retinal degenerations are large heterogeneous groups of genetic diseases. Pathogenic variants in the CFAP418 gene may cause both disorders, and its protein sequence is evolutionarily conserved. However, the disease mechanism underlying CFAP418 mutations has not been explored. Here, we apply quantitative lipidomic, proteomic, and phosphoproteomic profiling and affinity purification coupled with mass spectrometry to address the molecular function of CFAP418 in retinas. We show that CFAP418 protein binds to lipid metabolism precursor phosphatidic acid (PA) and mitochondrion-specific lipid cardiolipin but does not form a tight and static complex with proteins. Loss of Cfap418 in mice disturbs membrane lipid homeostasis and membrane-protein association, which subsequently causes mitochondrial defects and membrane remodeling abnormalities across multiple vesicular trafficking pathways in photoreceptors, especially the endosomal sorting complexes required for transport (ESCRT) pathway. Ablation of Cfap418 also increases the activity of PA-binding protein kinase Cα in the retina. Overall, our results indicate that membrane lipid imbalance is a pathological mechanism underlying syndromic ciliopathies and retinal degenerations, which is associated with other known causative genes of these diseases.

Authors

Anna M. Clark, Dongmei Yu, Grace Neiswanger, Daniel Zhu, Junhuang Zou, J. Alan Maschek, Thomas Burgoyne, Jun Yang

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression

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Abstract

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

Authors

Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Busra Ozbek, Tracy Jones, Ajay M. Vaghasia, Tatianna C. Larman, Rulin Wang, Mark C. Markowski, Samuel R. Denmeade, Kenneth J. Pienta, Ralph H. Hruban, Emmanuel S. Antonarakis, Anuj Gupta, Chi V. Dang, Srinivasan Yegnasubramanian, Angelo M. De Marzo

DLL4-Notch3-WNT5B axis mediates bi-directional pro-metastatic crosstalk between melanoma and lymphatic endothelial cells

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Abstract

Despite strong indications that melanoma interaction with lymphatic vessels actively promotes melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk we established human primary lymphatic endothelial cell (LEC) co-cultures with human melanoma cell lines. Here, we show that co-culture with melanoma cells induced transcriptomic changes in LECs and led to multiple alterations in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found contributing to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the co-culture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC co-culture escaped efficiently from the primary site to the proximal tumor draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3-WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bi-directional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Authors

Sanni Alve, Silvia Gramolelli, Joonas Jukonen, Susanna Juteau, Anne Pink, Atte A. Manninen, Satu Hänninen, Elisa Monto, Madeleine H. Lackman, Olli Carpén, Pipsa Saharinen, Sinem Karaman, Kari Vaahtomeri, Päivi M. Ojala