Kangyun Wu - Academia.edu (original) (raw)

Papers by Kangyun Wu

American Journal of Physiology-Lung Cellular and Molecular Physiology

Our study used a mouse model of post-viral lung disease to study the impact of chronic lung disea... more Our study used a mouse model of post-viral lung disease to study the impact of chronic lung disease on skeletal muscle. The model reveals a decrease in myofiber size that is selective for specific types of myofibers and an alternative mechanism for muscle atrophy that might be independent of the usual markers of protein synthesis and degradation. The findings provide a basis for new therapeutic strategies to correct skeletal muscle dysfunction in chronic respiratory disease.

Respiratory viruses, including SARS-CoV-2, can trigger chronic lung disease that persists and eve... more Respiratory viruses, including SARS-CoV-2, can trigger chronic lung disease that persists and even progresses after expected clearance of infectious virus. To gain an understanding of this process, we examined a series of consecutive fatal cases of Covid-19 that came to autopsy at 27-51 d after hospital admission. In each patient, we identify a stereotyped bronchiolar-alveolar pattern of lung remodeling with basal epithelial cell hyperplasia and mucinous differentiation. Remodeling regions also feature macrophage infiltration and apoptosis and a marked depletion of alveolar type 1 and 2 epithelial cells. This entire pattern closely resembles findings from an experimental model of post-viral lung disease that requires basal-epithelial stem cell growth, immune activation, and differentiation. The present results thereby provide evidence of possible basal epithelial cell reprogramming in long-term Covid-19 as well and thereby a pathway for explaining and correcting lung dysfunction in ...

Asthma is a common chronic disease of childhood, but for unknown reasons disease activity sometim... more Asthma is a common chronic disease of childhood, but for unknown reasons disease activity sometimes subsides as children mature. To understand why, we exposed mice across a range of ages to viral and allergic triggers of asthma exacerbations and airway pathology. We found that pathology induced by Sendai virus (SeV) or influenza A virus (IAV) occurred selectively in juvenile mice in a microbiome-independent manner, while the same phenotypes induced by allergens were insensitive to age. Age-specific responses to SeV included a juvenile bias towards type-2 airway inflammation that emerged early in infection and was lost with maturation. With aging, we observed progressive transcriptional changes to alveolar macrophages (AMs) including the acquisition of high-level MHC-II expression. Importantly, depleting AMs canceled the protective effects of maturity on post-viral airway pathology. Thus, aging of the lung-immune microenvironment influences chronic outcomes of respiratory viral infec...

Background Cell and animal models show a key role for Triggering Receptor Expressed on Myeloid Ce... more Background Cell and animal models show a key role for Triggering Receptor Expressed on Myeloid Cells (TREM)‐2 in chronic airway disease after viral infection, but comparable evidence in humans still needs to be established. Methods Lung tissue samples were obtained from lung transplant recipients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD (n = 16), nontransplantable donor lung tissues (n = 7), and resected lung tissues from patients at risk or with GOLD stage I through IV (n = 55) and were assessed for TREM‐2 and TREM‐1 messenger RNA (mRNA), protein expression, and other markers of a type 2 immune response. Results TREM2 (but not TREM1) mRNA levels were increased in GOLD stage IV COPD lung tissues compared with non‐COPD lung tissues. TREM2 mRNA was coexpressed with its signaling molecule DAP12 and the macrophage marker CD68 and M2‐macrophage markers CD206 and CHIT1. TREM‐2 protein was also increased in COPD lung tissues and was localized to CD14...

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Molecular Imaging and Biology, 2021

We tested whether the translocator protein (TSPO)-targeted positron emission tomography (PET) tra... more We tested whether the translocator protein (TSPO)-targeted positron emission tomography (PET) tracer, N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine ([11C]PBR28), could distinguish macrophage dominant from neutrophilic inflammation better than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in mouse models of lung inflammation and assessed TSPO association with macrophages in lung tissue from the mouse models and in patients with chronic obstructive pulmonary disease (COPD). MicroPET imaging quantified [11C]PBR28 and [18F]FDG lung uptake in wild-type (Wt) C57BL/6J or heterozygous transgenic monocyte-deficient Wt/opT mice at 49 days after Sendai virus (SeV) infection, during macrophage-dominant inflammation, and in Wt mice at 3 days after SeV infection or 24 h after endotoxin instillation during neutrophilic inflammation. Immunohistochemical staining for TSPO in macrophages and neutrophils was performed using Mac3 and Ly6G for cell identification in mouse lung sections and CD68 and neutrophil elastase (NE) in human lung sections taken from explanted lungs from patients with COPD undergoing lung transplantation and donor lungs rejected for transplantation. Differences in tracer uptake among SeV-infected, endotoxin-treated, and uninfected/untreated control mice and in TSPO staining between neutrophils and macrophage populations in human lung sections were tested using analysis of variance. In Wt mice, [11C]PBR28 uptake (% injected dose/ml lung tissue) increased significantly with macrophage-dominant inflammation at 49 days (D49) after SeV infection compared to controls (p = <0.001) but not at 3 days (D49) after SeV infection (p = 0.167). [11C]PBR28 uptake was unchanged at 24 h after endotoxin instillation (p = 0.958). [18F]FDG uptake increased to a similar degree in D3 and D49 SeV-infected and endotoxin-treated Wt mice compared to controls with no significant difference in the degree of increase among the tested conditions. [11C]PBR28 but not [18F]FDG lung uptake at D49 post-SeV infection was attenuated in Wt/opT mice compared to Wt mice. TSPO localized predominantly to macrophages in mouse lung tissue by immunostaining, and TSPO staining intensity was significantly higher in CD68+ cells compared to neutrophils in the human lung sections. PET imaging with [11C]PBR28 can specifically detect macrophages versus neutrophils during lung inflammation and may be a useful biomarker of macrophage accumulation in lung disease.

The Journal of Immunology, 2018

Clinical and experimental observations suggest that chronic lung disease is linked to respiratory... more Clinical and experimental observations suggest that chronic lung disease is linked to respiratory viral infection. However, the longterm aspect of this relationship is not yet defined using a virus that replicates at properly high levels in humans and a corresponding animal model. In this study, we show that influenza A virus infection achieves 1 3 10 6-fold increases in viral load in the lung and dose-dependent severity of acute illness in mice. Moreover, these events are followed by persistence of negative-and positivestrand viral RNA remnants for 15 wk and chronic lung disease for at least 26 wk postinfection. The disease is manifested by focal areas of bronchiolization and mucus production that contain increased levels of viral RNA remnants along with mucin Muc5ac and Il13 mRNA compared with uninvolved areas of the lung. Excess mucus production and associated airway hyperreactivity (but not fibrosis or emphysema) are partially attenuated with loss of IL-13 production or signaling (using mice with IL-13 or STAT6 deficiency). These deficiencies cause reciprocal increases in l17a mRNA and neutrophils in the lung; however, none of these disease endpoints are changed with IL-13/IL-17a compared with IL-13 deficiency or STAT6/IL-17a compared with STAT6 deficiency. The results establish the capacity of a potent human respiratory virus to produce chronic lung disease focally at sites of active viral RNA remnants, likely reflecting locations of viral replication that reprogram the region. Viral dose dependency of disease also implicates high-level viral replication and severity of acute infection as determinants of chronic lung diseases such as asthma and COPD with IL-13-dependent and IL-13/IL-17-independent mechanisms.

Journal of Clinical Investigation, 2013

Some expressions and notations related to Equations 1 and 2 were presented incorrectly. The corre... more Some expressions and notations related to Equations 1 and 2 were presented incorrectly. The correct text and equations are below. The coefficients (β) in Cox's regression model are estimated by maximizing the partial likelihood function subject to a constraint on the L1-norm of the coefficients. The lasso estimator (β) maximizes the objective function given below: (Equation 1) Here l(β) is the log partial likelihood in the Cox model; for the exact form of this function, see ref. 41. The tuning parameter, λ in Equation 1, was chosen by 10-fold cross-validation. For the implementation, we used the R package "glmnet" (39). PROVAR was defined for each of the 222 TCGA samples as the sum of the estimated coefficients multiplied by protein expression levels, as shown below. Here i represents patients (i = 1,...,222), j represents proteins with nonzero coefficients (j = 1, ..., m), β j is the lasso coefficient of the jth protein marker, and X ij is the expression level of the jth protein for the ith patient.

The Journal of Immunology, 2022

Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes sub... more Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes subsides as children mature. In this study, we present clinical and animal model evidence suggesting that the age dependency of childhood asthma stems from an evolving host response to respiratory viral infection. Using clinical data, we show that societal suppression of respiratory virus transmission during coronavirus disease 2019 lockdown disrupted the traditional age gradient in pediatric asthma exacerbations, connecting the phenomenon of asthma remission to virus exposure. In mice, we show that asthmatic lung pathology triggered by Sendai virus (SeV) or influenza A virus is highly age-sensitive: robust in juvenile mice (4-6 wk old) but attenuated in mature mice (>3 mo old). Interestingly, allergen induction of the same asthmatic traits was less dependent on chronological age than viruses. Age-specific responses to SeV included a juvenile bias toward type 2 airway inflammation that emerged early in infection, whereas mature mice exhibited a more restricted bronchiolar distribution of infection that produced a distinct type 2 low inflammatory cytokine profile. In the basal state, aging produced changes to lung leukocyte burden, including the number and transcriptional landscape of alveolar macrophages (AMs). Importantly, depleting AMs in mature mice restored post-SeV pathology to juvenile levels. Thus, aging influences chronic outcomes of respiratory viral infection through regulation of the AM compartment and type 2 inflammatory responses to viruses. Our data provide insight into how asthma remission might develop in children.

Annals of the American Thoracic Society

New studies of chronic obstructive pulmonary disease (COPD) are revealing the key role of airway ... more New studies of chronic obstructive pulmonary disease (COPD) are revealing the key role of airway epithelial cells and innate immune cells in the initiation, exacerbation, and progression of airway disease. An emerging scheme focuses on expansion of airway progenitor epithelial cells that feed forward for a type 2 immune response and consequent IL-13-driven mucus production that is linked to the morbidity and mortality of COPD. Analysis of human airway progenitor epithelial cells and airway tissue shows that IL-13 signaling to MUC5AC mucin gene expression relies on specific activation of mitogen-activated protein kinase 13, providing a druggable target for attenuating mucus production in the setting of viral infection and other inhaled stimuli of airway inflammation. Moreover, structure-based drug design is delivering highly potent, selective, and nontoxic small-molecule kinase inhibitors of mitogenactivated protein kinase 13 that offer a therapeutic strategy to downregulate excess mucus production to a physiological level and thereby achieve a precision medicine solution to the major health care problem of COPD and related airway diseases.

Journal of Clinical Investigation

Conflict of interest: MJH is founder of and equity holder in NuPeak Therapeutics Inc. and is a me... more Conflict of interest: MJH is founder of and equity holder in NuPeak Therapeutics Inc. and is a member of the Data Safety Monitoring Board for AstraZeneca. KW, YZ, SPK, BJG, and MJH are inventors on patents for MAPK inhibitors and uses thereof ("Anti-mucus drugs and uses therefor," patent no. WO 2014/015056 A2; "Mitogen-activated protein kinase inhibitors, methods of making and uses thereof," patent no. WO 2019/232275; "MAPK inhibitors and uses thereof in treatment of disease due to respiratory viral infections," patent pending).

The Journal of Immunology

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell... more Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1 f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1 f/f mice and conventional Stat1 2/2 mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1 f/f and Stat1-/mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1 f/f and Stat1 2/2 mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.

Chest, Jan 7, 2017

Cell and animal models show a key role for Triggering Receptor Expressed on Myeloid Cells (TREM)-... more Cell and animal models show a key role for Triggering Receptor Expressed on Myeloid Cells (TREM)-2 in chronic airway disease after viral infection, but comparable evidence in humans still needs to be established. Lung tissue samples were obtained from lung transplant recipients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD (n = 16), nontransplantable donor lung tissues (n = 10), and resected lung tissues from patients at risk or with GOLD stage I through IV (n = 72) and were assessed for TREM-2 and TREM-1 messenger RNA (mRNA), protein expression, and other markers of a type 2 immune response. TREM2 (but not TREM1) mRNA levels were increased in GOLD stage IV COPD lung tissues compared with non-COPD lung tissues. TREM2 mRNA was coexpressed with its signaling molecule DAP12 and the macrophage marker CD68 and M2-macrophage markers CD206 and CHIT1. TREM-2 protein was also increased in COPD lung tissues and was localized to CD14(+) macrophages by flow cy...

The Journal of experimental medicine, Jan 4, 2015

Viral infections and type 2 immune responses are thought to be critical for the development of ch... more Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infectio...

Nucleic Acids Research, 2012

The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL... more The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL) exhibits a range of activities including template-dependent de novo primer synthesis, primer extension and template-independent terminal nucleotidyl transfer using either rNTPs or dNTPs. Remarkably, the enzyme is able to synthesize products far longer than templates in vitro. Here we show that the long products resulted from template-dependent polymerization across discontinuous templates (PADT) by SsoPriSL. PADT was initiated through either primer synthesis or terminal transfer, and occurred efficiently on templates containing contiguous dCs. Template switching took place when the 3 0-end of a growing strand synthesized on one template annealed to another template directly or following the terminal addition of nucleotides, and was subsequently extended on the new template. The key to PADT was the ability of SsoPriSL to promote strand annealing. SsoPriSL catalyzed PADT with either dNTPs or rNTPs as the substrates but preferred the latter. The enzyme remained active in PADT but became inefficient in primer synthesis in vitro when temperature was raised from 55 C to 70 C. Our results suggest that SsoPriSL is capable of bridging noncomplementary DNA ends and, therefore, may serve a role in double-strand DNA break repair in Archaea.

Nature, 2009

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits t... more Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis (Mtb). Given that proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-ones kill non-replicating Mtb and act as selective suicide-substrate inhibitors of the Mtb proteasome by cyclo-carbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose H-bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-ones to Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Molecular Microbiology, 2007

The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes... more The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes long RNA and DNA products in vitro. How primer synthesis by primase is coupled to primer extension by DNA polymerase in this organism is unclear. Here we show that the small subunit of the clamp loader replication factor C (RFC) of S. solfataricus interacted with both the catalytic and non-catalytic subunits of the primase by yeast two-hybrid and coimmunoprecipitation assays. Further, the primase-RFC interaction was also identified in the cell extract of S. solfataricus. Deletion analysis indicated that the small subunit of RFC interacted strongly with the N-terminal domain of the catalytic subunit of the primase. RFC stimulated dinucleotide formation but decreased the amount of primers synthesized by the primase. The inhibition of primer synthesis is consistent with the observation that RFC reduced the affinity of the primase for DNA templates. On the other hand, primase stimulated the ATPase activity of RFC. These findings suggest that the primase-RFC interaction modulates the activities of both enzymes and therefore may be involved in the regulation of primer synthesis and the transfer of primers to DNA polymerase in Archaea.

PloS one, 2012

Host factors that microbial pathogens exploit for their propagation are potential targets for the... more Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove usef...

Bioorganic & Medicinal Chemistry Letters, 2011

We report the identification of new, structurally diverse inhibitors of interferon-induced, doubl... more We report the identification of new, structurally diverse inhibitors of interferon-induced, double-stranded RNA-activated protein kinase (PKR) using a combined experimental and computational approach. A training set with which to build a predictive statistical model was generated by screening a set of 80 known Ser/Thr kinase inhibitors against recombinant human PKR, resulting in the identification of 28 compounds from 18 chemical classes with &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.1 μM ≤ IC(50) ≤ 20 μM. The model built with this data was used to screen a database of 5 million commercially available compounds in silico to identify candidate inhibitors. Testing of 128 structurally diverse candidates resulted in the confirmation of 20 new inhibitors from 11 chemical classes with 2 μM ≤ IC(50) ≤ 20 μM. Testing of 34 analogs in the newly identified pyrimidin-2-amine active series provided initial SAR. One newly identified inhibitor, N-[2-(1H-indol-3-yl)ethyl]-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-amine (compound 51), inhibited intracellular PKR activation in a dose-dependent manner in primary mouse macrophages without evident toxicity at effective concentrations.