WNT signaling contributes to the extrahepatic bile duct proliferative response to obstruction in mice

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Abstract

Biliary obstruction and cholangiocyte hyperproliferation are important features of cholangiopathies affecting the large extrahepatic bile duct (EHBD). The mechanisms underlying obstruction-induced cholangiocyte proliferation in the EHBD remain poorly understood. Developmental pathways, including WNT signaling, are implicated in regulating injury responses in many tissues, including the liver. To investigate the contribution of WNT signaling to obstruction-induced cholangiocyte proliferation in the EHBD, we used complementary in vivo and in vitro models with pharmacologic interventions and transcriptomic analyses. To model obstruction, we used bile duct ligation (BDL) in mice. Human and mouse biliary organoids and mouse biliary explants were used to investigate the effects of WNT activation and inhibition in vitro. We observed an upregulation of WNT ligand expression associated with increased biliary proliferation following obstruction. Cholangiocytes were identified as both WNT ligand-expressing and WNT responsive cells. Inhibition of WNT signaling decreased cholangiocyte proliferation in vivo and in vitro, while activation increased proliferation. WNT effects on cholangiocyte proliferation were β-catenin-dependent, and we showed a direct effect of WNT7B on cholangiocyte growth. Our studies suggested that cholangiocyte-derived WNT ligands can activate WNT signaling to induce proliferation after obstructive injury. These findings implicated the WNT pathway in injury-induced cholangiocyte proliferation within the EHBD.

Authors

Ashley N. Calder, Mirabelle Q. Peter, John W. Tobias, Nureen H. Mohamad Zaki, Theresa M. Keeley, Timothy L. Frankel, Linda C. Samuelson, Nataliya Razumilava

Variation in HIV-1 Tat activity is a key determinant in the establishment of latent infection

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Abstract

Despite effective treatment, Human immunodeficiency virus (HIV) persists in optimally treated people as a transcriptionally silent provirus. Latently infected cells evade the immune system and the harmful effects of the virus, thereby creating a long-lasting reservoir of HIV. To gain a deeper insight into the molecular mechanisms of HIV latency establishment, we constructed a series of HIV-1 fluorescent reporter viruses that distinguish active versus latent infection. We unexpectedly observed that the proportion of active-to-latent infection depended on a limiting viral factor, which created a bottle neck that could be overcome by superinfection of the cell, T cell activation or overexpression of HIV-1 trans activator of transcription (Tat). In addition, we found that tat and rev expression levels vary amongst HIV molecular clones and that tat levels were an important variable in latency establishment. Lower rev levels limited viral protein expression whereas lower Tat levels or mutation of the Tat binding element promoted latent infection that was resistant to reactivation even in fully activated primary T cells. Nevertheless, we found that combinations of latency reversal agents targeting both cellular activation and histone acetylation pathways overcame deficiencies in the Tat-TAR axis of transcription regulation. These results provide additional insight into the mechanisms of latency establishment and inform Tat-centered approaches to cure HIV.

Authors

Francisco Gomez-Rivera, Valeri H. Terry, Cuie Chen, Mark M. Painter, Maria C. Virgilio, Marianne E. Yaple-Maresh, Kathleen L. Collins

A human-like model of aniridia-associated keratopathy for mechanistic and therapeutic studies

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Abstract

Aniridia is a rare congenital condition of abnormal eye development arising principally from heterozygous mutation of the PAX6 gene. Among the multiple complications arising in the eye, aniridia-associated keratopathy (AAK) is a severe vision-impairing condition of the cornea associated with a progressive limbal stem cell deficiency that lacks suitable treatment options. Current mouse models of aniridia do not accurately represent the onset and progression dynamics of human AAK, hindering therapy development. Here, we performed deep phenotyping of a haploinsufficient Pax6+/– small-eye (Sey) mouse model on the129Sey/SvImJ background, that exhibits key features of mild presentation at birth and progressive AAK with aging, mimicking human disease. The model exhibits a slowly progressing AAK phenotype and provides new insights into the disease including disturbed basal epithelial cell organization, function and marker expression, persistent postnatal lymphangiogenesis, disrupted corneal innervation patterns, and persisting yet altered limbal stem cell marker expression with age. The model recapitulates many of the known features of human disease, enabling investigation of underlying disease mechanisms and importantly, to access a well-defined temporal window for evaluating future therapeutics.

Authors

Dina Javidjam, Petros Moustardas, Mojdeh Abbasi, Ava Dashti, Yedizza Rautavaara, Neil Lagali

TFAP2C is a key regulator of intrauterine trophoblast cell invasion and deep hemochorial placentation

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Abstract

Transcription factor AP-2 gamma (TFAP2C) has been identified as a key regulator of the trophoblast cell lineage and hemochorial placentation. The rat possesses deep placentation characterized by extensive intrauterine trophoblast cell invasion, which resembles human placentation. Tfap2c is expressed in multiple trophoblast cell lineages, including invasive trophoblast cells situated within the uterine-placental interface of the rat placentation site. Global genome-editing was used to explore the biology of Tfap2c in rat placenta development. Homozygous global disruption of Tfap2c resulted in prenatal lethality. Heterozygous global disruption of Tfap2c was associated with diminished invasive trophoblast cell infiltration into the uterus. The role of TFAP2C in the invasive trophoblast cell lineage was explored using Cre-lox conditional mutagenesis. Invasive trophoblast cell-specific disruption of Tfap2c resulted in inhibition of intrauterine trophoblast cell invasion and intrauterine and postnatal growth restriction. The invasive trophoblast cell lineage was not impaired following conditional monoallelic disruption of Tfap2c. In summary, TFAP2C contributes to the progression of distinct stages of placental development. TFAP2C is a driver of early events in trophoblast cell development and reappears later in gestation as an essential regulator of the invasive trophoblast cell lineage. A subset of TFAP2C actions on trophoblast cells are dependent on gene dosage.

Authors

Esteban M. Dominguez, Ayelen Moreno-Irusta, Regan L. Scott, Khursheed Iqbal, Michael J. Soares

Repurposing of lonafarnib as a treatment for SARS-CoV-2 infection

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus disease 2019 (COVID-19), has emerged as a global pandemic pathogen with high mortality. While treatments have been developed to reduce morbidity and mortality of COVID-19, more antivirals with broad-spectrum activities are still needed. Here we identified lonafarnib (LNF), a Food and Drug Administration (FDA)-approved drug inhibitor of cellular farnesyltransferase (FTase), as an effective anti-SARS-CoV-2 agent. LNF inhibited SARS-CoV-2 infection and acted synergistically with known anti-SARS antivirals. LNF was equally active against diverse SARS-CoV-2 variants. Mechanistic studies suggested that LNF targeted multiple steps of viral life cycle. Using other structurally diverse FTase inhibitors and LNF-resistant FTase mutant, we demonstrated a key role of FTase in SARS-CoV-2 life cycle. To demonstrate in vivo efficacy, we infected SARS-CoV-2 susceptible humanized mice expressing human angiotensin-converting enzyme 2 (ACE2) and treated them with LNF. LNF at clinically relevant dose suppressed viral titer in the respiratory tract and improved pulmonary pathology and clinical parameters. Our study demonstrated that LNF, an approved oral drug with excellent human safety data, is a promising antiviral against SARS-CoV-2 that warrants further clinical assessment for treatment of COVID-19 and potentially other viral infections.

Authors

Mohsin Khan, Parker Irvin, Seung Bum Park, Hannah M. Ivester, Inna Ricardo-Lax, Madeleine Leek, Ailis Grieshaber, Eun Sun Jang, Sheryl L. Coutermarsh-Ott, Qi Zhang, Nunziata Maio, Jian-Kang Jiang, Bing Li, Wenwei Huang, Amy Q. Wang, Xin Xu, Zongyi Hu, Wei Zheng, Yihong Ye, Tracey Rouault, Charles M. Rice, Irving C. Allen, T. Jake Liang

Mast cell activation by NGF drives the formation of trauma-induced heterotopic ossification

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Abstract

Soft tissue trauma can cause immune system disturbance and neuropathological invasion, resulting in heterotopic ossification (HO) due to aberrant chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanisms behind the interaction between the immune and nervous systems in promoting HO pathogenesis are unclear. In this study, we found that mast cell-specific deletion attenuated localized tissue inflammation, with marked inhibition of HO endochondral osteogenesis. Likewise, blockage of nerve growth factor (NGF) receptor, known as tropomyosin receptor kinase A (TrkA), led to similar attenuations in tissue inflammation and HO. Moreover, while NGF-TrkA signaling did not directly affect MSCs chondrogenic differentiation, it modulated mast cell activation in traumatic soft tissue. Mechanistically, lipid A in lipopolysaccharide binding to TrkA enhanced NGF-induced TrkA phosphorylation, synergistically stimulating mast cells to release neurotrophin-3 (NT3), thereby promoting MSCs chondrogenic differentiation in situ. Finally, analysis of single-cell datasets and human pathological specimens confirmed the important role of mast cell-mediated neuroinflammation in HO pathogenesis. In conclusion, NGF regulates mast cells in soft tissue trauma, and drives HO progression via paracrine NT3. Targeted early inhibition of mast cells holds substantial promise for treating traumatic HO.

Authors

Tao Jiang, Xiang Ao, Xin Xiang, Jie Zhang, Jieyi Cai, Jiaming Fu, Wensheng Zhang, Zhenyu Zheng, Jun Chu, Minjun Huang, Zhongmin Zhang, Liang Wang

Longitudinal clinical and proteomic diabetes signatures in post-gestational diabetes women

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Abstract

We characterized the longitudinal serum protein signatures of women 6 and 10 years after gestational diabetes mellitus (GDM) to identify factors associated with the development of type 2 diabetes mellitus (T2D) and prediabetes in this at-risk post-GDM population, aiming to discover potential biomarkers for early diagnosis and prevention of T2D. Our study identified 75 T2D-associated serum proteins and 23 prediabetes-associated proteins, some of which were validated in an independent T2D cohort. Machine learning (ML) performed on the longitudinal proteomics highlighted protein signatures associated with progression to post-GDM diabetes. We also proposed prognostic biomarker candidates, that were differentially regulated in healthy participants at 6 years postpartum who later progressed to T2D. Our longitudinal study revealed T2D-risk factors for post-GDM populations, who are relatively young and healthy, providing insights for clinical decisions and early lifestyle interventions.

Authors

Heaseung Sophia Chung, Lawrence Middleton, Manik Garg, Ventzislava A. Hristova, Rick B. Vega, David J. Baker, Benjamin G. Challis, Dimitrios Vitsios, Sonja Hess, Kristina Wallenius, Agneta Holmäng, Ulrika Andersson-Hall

Tamm-Horsfall protein augments neutrophil NETosis during urinary tract infection

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Abstract

Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THP-deficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared to WT mice. Furthermore, THP-deficient mice displayed impaired urinary NETosis during UTI. To investigate the impact of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways, enhances NETosis in an ROS-dependent manner, and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Authors

Vicki Mercado-Evans, Holly Branthoover, Claude Chew, Camille Serchejian, Alexander B. Saltzman, Marlyd E. Mejia, Jacob J. Zulk, Ingrid Cornax, Victor Nizet, Kathryn A. Patras

Rapamycin improves satellite cells autophagy and muscle regeneration during hypercapnia

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Abstract

Abstract: Both CO2 retention, or hypercapnia, and skeletal muscle dysfunction predict higher mortality in critically ill patients. Mechanistically, muscle injury and reduced myogenesis contribute to critical illness myopathy, and while hypercapnia causes muscle wasting, no research has been conducted on hypercapnia-driven dysfunctional myogenesis in vivo. Autophagy flux regulates myogenesis by supporting muscle stem cell -satellite cell- activation, and previous data suggests that hypercapnia inhibits autophagy. We tested whether hypercapnia worsens satellite cell autophagy flux and myogenic potential, and if autophagy induction reverses these deficits. Satellite cell transplantation and lineage tracing experiments showed that hypercapnia undermines satellite cells activation, replication, and myogenic capacity. Bulk and single cell sequencing analyses indicated that hypercapnia disrupts autophagy, senescence, and other satellite cells programs. Autophagy activation was reduced in hypercapnic cultured myoblasts, and autophagy genetic knockdown phenocopied these changes in vitro. Rapamycin stimulation led to AMPK activation and downregulation of the mTOR pathway, which are both associated with accelerated autophagy flux and cell replication. Moreover, hypercapnic mice receiving rapamycin showed improved satellite cells autophagy flux, activation, replication rate, and post transplantation myogenic capacity. In conclusion, we have shown that hypercapnia interferes with satellite cell activation, autophagy flux and myogenesis, and systemic rapamycin administration improved these outcomes.

Authors

Joseph Balnis, Emily L. Jackson, Lisa A. Drake, Diane V. Singer, Ramon Bossardi Ramos, Harold A. Singer, Ariel Jaitovich.

Myeloid Drp1 deficiency limits revascularization in ischemic muscles via inflammatory macrophage polarization and metabolic reprograming

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Abstract

Macrophage plays a crucial role in promoting perfusion recovery and revascularization after ischemia through anti-inflammatory polarization, a process essential for the treatment of peripheral arterial disease (PAD). Mitochondrial dynamics, particularly regulated by the fission protein DRP1, are closely linked to macrophage metabolism and inflammation. However, the role of DRP1 in reparative neovascularization remains unexplored. Here we show that DRP1 expression was increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Mice lacking Drp1 in myeloid cells exhibited impaired limb perfusion recovery, angiogenesis and muscle regeneration post-HLI. These effects were associated with increased pro-inflammatory M1-like macrophages, p-NFkB and TNFα, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1–/– mice. In vitro, Drp1-deficient macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction, and excessive mitochondrial ROS production, resulting in increased pro-inflammatory M1-gene and reduced anti-inflammatory M2-gene expression. Conditioned media from HSS-treated Drp1–/– macrophages exhibited increased pro-inflammatory cytokine secretion, leading to suppressed angiogenesis in endothelial cells. Thus, macrophage DRP1 deficiency under ischemia drives pro-inflammatory metabolic reprogramming and macrophage polarization, limiting revascularization in experimental PAD.

Authors

Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai