Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice.

Nat Commun. 2018 Dec 18;9(1):5363.

2018 Dec 18

Hancock LA, Hennessy CE, Solomon GM, Dobrinskikh E, Estrella A, Hara N, Hill DB, Kissner WJ, Markovetz MR, Grove Villalon DE, Voss ME, Tearney GJ, Carroll KS, Shi Y, Schwarz MI, Thelin WR, Rowe SM, Yang IV, Evans CM, Schwartz DA.
PMID: 30560893 | DOI: 10.1038/s41467-018-07768-9

The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF.

ROBO2 is a stroma suppressor gene in the pancreas and acts via TGF-β signalling.

Nat Commun. 2018 Nov 30;9(1):5083.

2018 Nov 30

Pinho AV, Van Bulck M, Chantrill L, Arshi M, Sklyarova T, Herrmann D, Vennin C, Gallego-Ortega D, Mawson A, Giry-Laterriere M, Magenau A, Leuckx G, Baeyens L, Gill AJ, Phillips P, Timpson P, Biankin AV, Wu J, Rooman I.
PMID: 30504844 | DOI: 10.1038/s41467-018-07497-z

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1Cre;Robo2F/F) show increased activation of Robo1+ myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1Cre;Robo2F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2low;ROBO1high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents.

Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells

Nature cell biology

2022 Jan 01

Kathiriya, JJ;Wang, C;Zhou, M;Brumwell, A;Cassandras, M;Le Saux, CJ;Cohen, M;Alysandratos, KD;Wang, B;Wolters, P;Matthay, M;Kotton, DN;Chapman, HA;Peng, T;
PMID: 34969962 | DOI: 10.1038/s41556-021-00809-4

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-β1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.

Murine Coronavirus Disease 2019 Lethality Is Characterized by Lymphoid Depletion Associated with Suppressed Antigen-Presenting Cell Functionality

The American journal of pathology

2023 Apr 05

Lee, YJ;Seok, SH;Lee, NY;Choi, HJ;Lee, YW;Chang, HJ;Hwang, JY;On, DI;Noh, HA;Lee, SB;Kwon, HK;Yun, JW;Shin, JS;Seo, JY;Nam, KT;Lee, H;Lee, HY;Park, JW;Seong, JK;
PMID: 37024046 | DOI: 10.1016/j.ajpath.2023.03.008

The disease severity of coronavirus disease 2019 (COVID-19) varies considerably from asymptomatic to serious, with fatal complications associated with dysregulation of innate and adaptive immunity. Lymphoid depletion in lymphoid tissues and lymphocytopenia have both been associated with poor disease outcomes in patients with COVID-19, but the mechanisms involved remain elusive. In this study, human angiotensin-converting enzyme 2 (hACE2) transgenic mouse models susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were used to investigate the characteristics and determinants of lethality associated with the lymphoid depletion observed in SARS-CoV-2 infection. The lethality of Wuhan SARS-CoV-2 infection in K18-hACE2 mice was characterized by severe lymphoid depletion and apoptosis in lymphoid tissues related to fatal neuroinvasion. The lymphoid depletion was associated with a decreased number of antigen-presenting cells (APCs) and their suppressed functionality below basal levels. Lymphoid depletion with reduced APC function was a specific feature observed in SARS-CoV-2 infection but not in influenza A infection and had the greatest prognostic value for disease severity in murine COVID-19. Comparison of transgenic mouse models resistant and susceptible to SARS-CoV-2 infection revealed that suppressed APC function could be determined by the hACE2 expression pattern and interferon-related signaling. Thus, we demonstrated that lymphoid depletion associated with suppressed APC function characterizes the lethality of COVID-19 mouse models. Our data also suggest a potential therapeutic approach to prevent the severe progression of COVID-19 by enhancing APC functionality.

Nintedanib induces gene expression changes in the lung of induced-rheumatoid arthritis-associated interstitial lung disease mice

PloS one

2022 Jun 17

Mikami, S;Miura, Y;Kondo, S;Sakai, K;Nishimura, H;Kyoyama, H;Moriyama, G;Koyama, N;Noguchi, H;Ohkubo, H;Kanazawa, S;Uematsu, K;
PMID: 35714115 | DOI: 10.1371/journal.pone.0270056

Nintedanib is a multi-tyrosine kinase inhibitor widely used to treat progressive fibrosing interstitial lung diseases because it slows the reduction in forced vital capacity. However, the prognosis for patients treated with nintedanib remains poor. To improve nintedanib treatment, we examined the effects of nintedanib on gene expression in the lungs of induced-rheumatoid arthritis-associated interstitial lung disease model mice, which develop rheumatoid arthritis and subsequent pulmonary fibrosis. Using next-generation sequencing, we identified 27 upregulated and 130 downregulated genes in the lungs of these mice after treatment with nintedanib. The differentially expressed genes included mucin 5B and heat shock protein 70 family genes, which are related to interstitial lung diseases, as well as genes associated with extracellular components, particularly the myocardial architecture, suggesting unanticipated effects of nintedanib. Of the genes upregulated in the nintedanib-treated lung, expression of regulatory factor X2, which is suspected to be involved in cilia movement, and bone morphogenetic protein receptor type 2, which is involved in the pathology of pulmonary hypertension, was detected by immunohistochemistry and RNA in situ hybridization in peripheral airway epithelium and alveolar cells. Thus, the present findings indicate a set of genes whose expression alteration potentially underlies the effects of nintedanib on pulmonary fibrosis. It is expected that these findings will contribute to the development of improved nintedanib strategies for the treatment of progressive fibrosing interstitial lung diseases.

Hypercapnia alters stromal-derived Wnt production limiting β-catenin signaling and proliferation in alveolar type 2 cells

JCI insight

2023 Jan 10

Dada, LA;Welch, LC;Magnani, ND;Ren, Z;Han, H;Brazee, PL;Celli, D;Flozak, AS;Weng, A;Herrerias, MM;Kryvenko, V;Vadász, I;Runyan, CE;Abdala-Valencia, H;Shigemura, M;Casalino-Matsuda, SM;Misharin, AV;Budinger, GRS;Gottardi, CJ;Sznajder, JI;
PMID: 36626234 | DOI: 10.1172/jci.insight.159331

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, using a mouse model of hypercapnia exposure, cell lineage-tracing, spatial transcriptomics and 3D-cultures, we show that hypercapnia limits β-catenin signaling in AT2 cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+-fibroblasts from those maintaining AT2 progenitor activity towards those that antagonize β-catenin signaling thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality. .

In vivo visualization and molecular targeting of the cardiac conduction system

The Journal of clinical investigation

2022 Aug 11

Goodyer, WR;Beyersdorf, BM;Duan, L;van den Berg, NS;Mantri, S;Galdos, FX;Puluca, N;Buikema, JW;Lee, S;Salmi, D;Robinson, ER;Rogalla, S;Cogan, DP;Khosla, C;Rosenthal, EL;Wu, SM;
PMID: 35951416 | DOI: 10.1172/JCI156955

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.

CB1 R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky-Pudlak syndrome

Clinical and translational medicine

2021 Jul 01

Cinar, R;Park, JK;Zawatsky, CN;Coffey, NJ;Bodine, SP;Abdalla, J;Yokoyama, T;Jourdan, T;Jay, L;Zuo, MXG;O'Brien, KJ;Huang, J;Mackie, K;Alimardanov, A;Iyer, MR;Gahl, WA;Kunos, G;Gochuico, BR;Malicdan, MCV;
PMID: 34323400 | DOI: 10.1002/ctm2.471

Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder which, in its most common and severe form, HPS-1, leads to fatal adult-onset pulmonary fibrosis (PF) with no effective treatment. We evaluated the role of the endocannabinoid/CB1 R system and inducible nitric oxide synthase (iNOS) for dual-target therapeutic strategy using human bronchoalveolar lavage fluid (BALF), lung samples from patients with HPS and controls, HPS-PF patient-derived lung fibroblasts, and bleomycin-induced PF in pale ear mice (HPS1ep/ep ). We found overexpression of CB1 R and iNOS in fibrotic lungs of HPSPF patients and bleomycin-infused pale ear mice. The endocannabinoid anandamide was elevated in BALF and negatively correlated with pulmonary function parameters in HPSPF patients and pale ear mice with bleomycin-induced PF. Simultaneous targeting of CB1 R and iNOS by MRI-1867 yielded greater antifibrotic efficacy than inhibiting either target alone by attenuating critical pathologic pathways. Moreover, MRI-1867 treatment abrogated bleomycin-induced increases in lung levels of the profibrotic interleukin-11 via iNOS inhibition and reversed mitochondrial dysfunction via CB1 R inhibition. Dual inhibition of CB1 R and iNOS is an effective antifibrotic strategy for HPSPF.

Targeted alveolar regeneration with Frizzled-specific agonists

Cell

2023 Jun 14

Nabhan, AN;Webster, JD;Adams, JJ;Blazer, L;Everrett, C;Eidenschenk, C;Arlantico, A;Fleming, I;Brightbill, HD;Wolters, PJ;Modrusan, Z;Seshagiri, S;Angers, S;Sidhu, SS;Newton, K;Arron, JR;Dixit, VM;
PMID: 37321220 | DOI: 10.1016/j.cell.2023.05.022

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

Insulin-like growth factor receptor / mTOR signaling elevates global translation to accelerate zebrafish fin regenerative outgrowth

Developmental biology

2023 Jun 06

Lewis, VM;Le Bleu, HK;Henner, AL;Markovic, H;Robbins, AE;Stewart, S;Stankunas, K;
PMID: 37290497 | DOI: 10.1016/j.ydbio.2023.05.008

Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR)/mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.

Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

Nature communications

2023 Feb 15

Li, Q;Zhang, XX;Hu, LP;Ni, B;Li, DX;Wang, X;Jiang, SH;Li, H;Yang, MW;Jiang, YS;Xu, CJ;Zhang, XL;Zhang, YL;Huang, PQ;Yang, Q;Zhou, Y;Gu, JR;Xiao, GG;Sun, YW;Li, J;Zhang, ZG;
PMID: 36792623 | DOI: 10.1038/s41467-023-36521-0

To explore the mechanism of coadaptation and the potential drivers of pancreatic ductal adenocarcinoma (PDAC) metastasis to the liver, we study key molecules involved in this process and their translational value. Premetastatic niche (PMN) and macrometastatic niche (MMN) formation in a mouse model is observed via CT combined with 3D organ reconstruction bioluminescence imaging, and then we screen slit guidance ligand 2 (SLIT2) and its receptor roundabout guidance receptor 1 (ROBO1) as important factors. After we confirm the expression and distribution of SLIT2 and ROBO1 in samples from PDAC patients and several mouse models, we discover that SLIT2-ROBO1-mediated coadaptation facilitated the implantation and outgrowth of PDAC disseminated tumour cells (DTCs) in the liver. We also demonstrate the dependence receptor (DR) characteristics of ROBO1 in a follow-up mechanistic study. A neutralizing antibody targeting ROBO1 significantly attenuate liver metastasis of PDAC by preventing the coadaptation effect. Thus, we demonstrate that coadaptation is supported by the DR characteristics in the PMN and MMN.

Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal & ventral adult neurogenesis

eLife

2021 Jul 14

Cebrian Silla, A;Nascimento, MA;Redmond, SA;Mansky, B;Wu, D;Obernier, K;Romero Rodriguez, R;Gonzalez Granero, S;García-Verdugo, JM;Lim, D;Álvarez-Buylla, A;
PMID: 34259628 | DOI: 10.7554/eLife.67436

The ventricular-subventricular zone (V-SVZ), on the walls of the lateral ventricles, harbors the layrgest neurogenic niche in the adult mouse brain. Previous work has shown that neural steym/progenitor cells (NSPCs) in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. The molecular signatures that underlie this regional heterogeneity remain largely unknown. Here we present a single-cell RNA-sequencing dataset of the adult mouse V-SVZ revealing two populations of NSPCs that reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. These regional differences in gene expression were further validated using a single-nucleus RNA-sequencing reference dataset of regionally microdissected domains of the V-SVZ and by immunocytochemistry and RNAscope localization. We also identify two subpopulations of young neurons that have gene expression profiles consistent with a dorsal or ventral origin. Interestingly, a subset of genes are dynamically expressed, but maintained, in the ventral or dorsal lineages. The study provides novel markers and territories to understand the region-specific regulation of adult neurogenesis.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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