Ezrin links CFTR to TLR4 signaling to orchestrate anti-bacterial immune response in macrophages

Pietro, Caterina Di; Zhang, Ping-Xia; O’Rourke, Timothy K.; Murray, Thomas S.; Wang, Lin; Britto, Clemente J.; Koff, Jonathan L.; Krause, Diane S.; Egan, Marie E.; Bruscia, Emanuela M.

doi:10.1038/s41598-017-11012-7

Cited by 35 publications

(33 citation statements)

References 62 publications

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“…A third limitation is that this study does not demonstrate the mechanism by which ivacaftor decreases monocyte IFN-γ sensitivity. Ivacaftor may be acting by a CFTR-dependent mechanism, as has been described for other cystic fibrosis-related myeloid cell defects [11,12]. Ivacaftor could also act directly on monocytes by a CFTR-independent mechanism; recent studies observed that CFTR modulators may have immune-dampening effects on macrophages that lack modulator-susceptible CFTR mutations [12,13].…”

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confidence: 88%

Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis

et al. 2020

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Management of cystic fibrosis has been revolutionised by the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. These compounds treat the underlying molecular basis of the disease by increasing activity of defective CFTR channels, which improves many clinical parameters and enhances patient quality of life [1]. Next-generation modulators, also known as triple combination therapy, promise to be highly efficacious in up to 90% of patients [2] and will likely dramatically change the landscape of cystic fibrosis disease. Studies examining individuals before and after initiation of CFTR modulators have revealed novel functions of CFTR and shown that CFTR modulators do not reverse all disease manifestations [3-5]. Thus, knowledge of the post-modulator cystic fibrosis disease state is crucial for understanding what continued therapies will be needed for people with cystic fibrosis and what new challenges may arise. In prior work, we sought to identify immune pathways that were differentially modulated in the presence and absence of CFTR activity [6]. We isolated monocytes from subjects with G551D-CFTR mutations before and after initiation of the modulator ivacaftor. Using an unbiased proteomics approach, we identified post-ivacaftor changes in the monocyte plasma membrane proteome consistent with decreased interferon (IFN)-γ-related responses. Although both monocytes and macrophages from individuals with cystic fibrosis have been shown to manifest abnormal immune responses, myeloid IFN-γ responses had not previously been reported as altered in people with cystic fibrosis [7]. In the current study, we took advantage of a second cohort of subjects with cystic fibrosis who were initiating ivacaftor to test the hypothesis that ivacaftor dampens monocyte sensitivity to IFN-γ.

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confidence: 88%

Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis

et al. 2020

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“…The members of the ezrin/radixin/moesin (ERM) family proteins are involved in multiple physiological and pathological phenomenon by acting as cross-linkers between actin cytoskeleton and plasma membrane in polarized cells [4][5][6][7] . As the founding member of the ERM, ezrin is essential for cell migration 8 , inflammatory response regulation 9 , villus morphogenesis 10 and gastric acid secretion [11][12][13][14] . It has also been proved to be an important marker for cancer metastasis 15,16 .…”

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confidence: 99%

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Song

et al. 2020

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Chemicals and antibody. Celastrol was ordered from MedChemExpress under the item number HY-13067. Antibodies against ezrin, ezrin pT567 and α-tubulin (DM1A) were purchased from Cell Signaling Technology. Antibodies against ROCK2 and ROCK2 (phospho S1366) were purchased from Abcam. Anti-GFP antibodies were purchased from Santa Cruz Biotechnology. Anti-FLAG-tag (M2) antibody was from Sigma. plasmids. The development of bacterial expression vectors containing human ezrin fused to histidine was described previously 61. EGFP-tagged ezrin plasmids was produced as described before 62. PCR amplified ROCK2 cDNA was cloned into the 3× FLAG-Myc-CMV-24 vector (Sigma) by BamHI and RcoRI digestion. The mutanted of ezrin and ROCK2 were constructed by Fast Mutagenesis Kit (Vazyme Biotech). All plasmids were verified by sequencing (Tsingke Biological Tech). cell culture and transfection. MHCC97H was a gift from Professor Yong Chen (Fourth Military Medical University) 20. HepG2 and HEK293T cells were from American Type Culture Collection (ATCC). Huh7 cells were from National Infrastructure of Cell Line Resource. The cells were incubated according to ATCC instruction. Cells were transfected using conventional calcium phosphate method or the Lipofectamine 3,000 (Invitrogen) according to manufacturer's instructions. Determination of cell viability (MTS assay). Celastrol cytotoxicity was assessed with the use of a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega), which is a form of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Cells (5,000 cells/well) were plated in 96-well plates, treated with various concentrations of celastrol for 24 h. After celastrol treatment, cell viability was determined by adding a small amount of the One Solution Reagent directly to culture wells, incubating for 3 h and then recording the absorbance at 490 nm with a 96-well plate reader. Wound healing assay. MHCC97H, HepG2 or Huh7 cells with logarithmic growth phases, was cultivated in 12 orifices with 5 × 10 5 cells density. Each group has three replicates. After a starvation of 6 h in serum-free medium, 10 μl Tip was taken to vertical scratch "#" glyph at the bottom of the culture plate. The floating cells were washed off with PBS and the celastrol was dissolved in medium supplemented with 20% FBS. Samples were photographed at 0 h, 4 h and 8 h. Image J software was used to measure and compare the effects of celastrol on cell migration. Single cell tracking assay. The cell migration by single cell tracking assay was performed as previously described 32. Briefly, MHCC97H cells were laid in a Silian dish. After stabilized, the cells were starved with Opti-MEM for 6 h, and then cultured in DMEM containing 20% FBS. The movement track of 10 single-cells in each field of view were recorded by microscope for 6 h, with 10 min interval at once.

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“…TLR4 trafficking and its regulation at the plasma membrane of MFs is altered in CF (Zhang et al, 2013). CF MFs also show blunted induction of the PI3K/AKT signaling pathway in response to LPS or P. aeruginosa (Zhang et al, 2015;Di Pietro et al, 2017). Wegiel et al (2014a) have shown that modulation of the HO-1/CO pathway or exogenous delivery of CO increases the efficiency of MFs in killing bacteria such as Escherichia coli and Enterococcus faecalis.…”

Section: Co Stimulates Cellular Host Defense Against Infections: Implmentioning

confidence: 99%

“…Several dysfunctional mechanisms may account for the blunted HO-1 induction in CF cells ( Figure 3 ). Our group has demonstrated that HO-1 is inefficiently induced in human and murine CF MΦs in response to inflammatory or infectious triggers, which correlate with exaggerated inflammation and prolonged inflammatory signaling ( Zhang et al., 2013 ; Zhang et al., 2015 ; Di Pietro et al., 2017 ). We have also shown that the defective induction of HO-1 is due to blunted activation of the PI3K/AKT pathway downstream of toll-like receptor 4 (TLR4) activation in MΦs from CF mouse models and patients with CF.…”

Section: Ho-1 Dysregulation In Cfmentioning

confidence: 99%

“…Importantly, modulation of this pathway via overexpression of HO-1 or treatment with CO-releasing molecules (discussed in the next section) was sufficient to improve the signaling cascade, thus reducing hyper-inflammation in CF MΦs ( Zhang et al., 2013 ). In investigating how loss of CFTR leads to blunted PI3K/AKT signaling, we found that ezrin, an F-actin binding protein that forms a macromolecular complex with CFTR at the plasma membrane ( Guggino and Stanton, 2006 ), links CFTR, TLR4, PI3K/AKT signaling, and HO-1 expression in MΦs ( Di Pietro et al., 2017 ).…”

Section: Ho-1 Dysregulation In Cfmentioning

confidence: 99%

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Targeting the Heme Oxygenase 1/Carbon Monoxide Pathway to Resolve Lung Hyper-Inflammation and Restore a Regulated Immune Response in Cystic Fibrosis

et al. 2020

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In individuals with cystic fibrosis (CF), lung hyper-inflammation starts early in life and is perpetuated by mucus obstruction and persistent bacterial infections. The continuous tissue damage and scarring caused by non-resolving inflammation leads to bronchiectasis and, ultimately, respiratory failure. Macrophages (MFs) are key regulators of immune response and host defense. We and others have shown that, in CF, MFs are hyper-inflammatory and exhibit reduced bactericidal activity. Thus, MFs contribute to the inability of CF lung tissues to control the inflammatory response or restore tissue homeostasis. The non-resolving hyper-inflammation in CF lungs is attributed to an impairment of several signaling pathways associated with resolution of the inflammatory response, including the heme oxygenase-1/carbon monoxide (HO-1/CO) pathway. HO-1 is an enzyme that degrades heme groups, leading to the production of potent antioxidant, anti-inflammatory, and bactericidal mediators, such as biliverdin, bilirubin, and CO. This pathway is fundamental to re-establishing cellular homeostasis in response to various insults, such as oxidative stress and infection. Monocytes/MFs rely on abundant induction of the HO-1/CO pathway for a controlled immune response and for potent bactericidal activity. Here, we discuss studies showing that blunted HO-1 activation in CFaffected cells contributes to hyper-inflammation and defective host defense against bacteria. We dissect potential cellular mechanisms that may lead to decreased HO-1 induction in CF cells. We review literature suggesting that induction of HO-1 may be beneficial for the treatment of CF lung disease. Finally, we discuss recent studies highlighting how endogenous HO-1 can be induced by administration of controlled doses of CO to reduce lung hyper-inflammation, oxidative stress, bacterial infection, and dysfunctional ion transport, which are all hallmarks of CF lung disease.

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Ezrin links CFTR to TLR4 signaling to orchestrate anti-bacterial immune response in macrophages

Cited by 35 publications

References 62 publications

Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis

Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Targeting the Heme Oxygenase 1/Carbon Monoxide Pathway to Resolve Lung Hyper-Inflammation and Restore a Regulated Immune Response in Cystic Fibrosis

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