Monocyte derived macrophages from CF pigs exhibit increased inflammatory responses at birth

Background

We sought to address whether CF macrophages have a primary functional defect as a consequence of CFTR loss and thus contribute to the onset of infection and inflammation observed in CF lung disease.

A novel function of collagen/PCL nanofiber interaction with MSCs in osteoarthritis is potentiation its immunomodulatory effect through increased ICAM expression

BackgroundThe previous studies investigated the scaffold as mechanical carrier to stem cells or growth factors as transplantation therapy. This study aimed to investigate the effect of nanofiber scaffold on immunomodulatory properties of MSCs in osteoarthritis (OA) and the mechanism of this effect.MethodsCollagen coated PCL nanofiber was prepared and examined by SEM. Mononuclear cells (MNCs) from the peripheral blood (P.B) and synovial fluid (SF) cultured with BMMSCs after stimulation by PHA using two types of co-culture (MSCs/Nano^- and MSCs/nano⁺). Using flowcytometry, CD4, CD8, CD28, activation markers CD38 and CD23, regulatory CD4CD25^high foxp3, CD14, IL17 by ELISA and cell proliferation using CCK-8 were assessed. ICAM mRNA was assessed by qRT-PCR and flowcytometry.ResultsMSCs/nano⁺ showed more significant inhibitory effect of MSCs regarding CD4, CD8, CD28, CD28 MFI, CD38, CD38 MFI, CD23, CD23 MFI, CD14, IL17 (p < 0.001) and higher level of Treg cells (p < 0.001) than nano⁻ with difference regarding cell proliferation (p = 0.275). Also, the immunomodulatory effect of MSCs culture on nano⁺ on lymphocyte separated from S·F of osteoarthritis patients more powerful than nano⁻. The secretory function of MSCs culture on nano⁺ is more beneficial than that of nano^-. Nano⁺ showed higher level of ICAM mRNA and CD54 than nano⁻.ConclusionThe present study proved a novel function of nanofiber by increasing the immunomodulatory effect of the MSCs in osteoarthritis through increased the ICAM expression and this promising for increase the success of MSCs transplantation in OA.     

CFTR plays an important role in the regulation of vascular resistance and high-fructose/salt-diet induced hypertension in mice

BackgroundThe pathophysiological roles of cystic fibrosis transmembrane-conductance regulator (CFTR) Cl^? channels in the regulation of blood pressure (BP) remain controversial. Here we studied the function of CFTR Cl^? channels in regulation of BP and in the high-fructose-salt-diet (HFSD) induced hypertension in mice.MethodsThe systolic, diastolic and mean BP (SBP, DBP and MBP, respectively) were continuously monitored from unrestricted conscious wild-type (cftr^+/+) FVB and CFTR-knockout (cftr^?/?) mice (8-week old, male). HFSD (64.7% fructose, 2% NaCl water) or control normal starch diet (CNSD, 58.9% corn starch, 0 NaCl water) was given for 8 weeks and vascular Doppler were performed. Real-time PCR and Western blot were used to examine mRNA and protein expression, respectively.ResultsThe aortic stiffness, daytime and nighttime SBP, DBP, and MBP of the cftr^?/? mice were significantly higher than those in the age- and gender-matched cftr^+/+ mice, which is consistent with the findings of increased vascular resistance in cystic fibrosis patients. The aortic stiffness, daytime and nighttime SBP, DBP, and MBP of cftr^+/+ mice fed with HFSD were all significantly higher than those fed with CNSD. Importantly, HFSD caused a significant decrease in mRNA and protein expression of WINK1, WINK4 and CFTR in aorta and mesenteric arteries, but not in the kidney, corroborating that HSFD-induced downregulation of WINKs and loss of CFTR function specifically in the arteries may mediate the increased BP.ConclusionsCFTR regulates peripheral arterial resistance and BP in vivo. HFSD-induced CFTR downregulation specifically in the arteries may be a novel mechanism for hypertension.     

Ivacaftor potentiation of multiple CFTR channels with gating mutations

BackgroundThe investigational CFTR potentiator ivacaftor (VX-770) increased CFTR channel activity and improved lung function in subjects with CF who have the G551D CFTR gating mutation. The aim of this in vitro study was to determine whether ivacaftor potentiates mutant CFTR with gating defects caused by other CFTR gating mutations.MethodsThe effects of ivacaftor on CFTR channel open probability and chloride transport were tested in electrophysiological studies using Fischer rat thyroid (FRT) cells expressing different CFTR gating mutations.ResultsIvacaftor potentiated multiple mutant CFTR forms with defects in CFTR channel gating. These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations.ConclusionThese in vitro data suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support investigation of the potential clinical benefit of ivacaftor in CF patients who have CFTR gating mutations beyond G551D.     

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets

BackgroundThe hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process.MethodsPeripheral blood neutrophils were isolated from newborn CFTR^+/+ and CFTR^−/− piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated.ResultsPolyunsaturated fatty acid metabolites analysis did not show any difference between CFTR^+/+ and CFTR^−/− neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR^−/− neutrophils to kill pseudomonas aeruginosa in vitro was observed.ConclusionsICM-MS demonstrated that CFTR^−/− neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.     

Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function

BackgroundIvacaftor (KALYDECO™, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function.MethodsThe effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface.ResultsIvacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance.ConclusionsThese in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.     

Second and third trimester amniotic fluid mesenchymal stem cells can repopulate a de-cellularized lung scaffold and express lung markers

Background/PurposeThis study examined the potential of amniotic fluid mesenchymal stem cells (AF-MSCs) to generate lung precursor cells in vitro and on a xenologous three-dimensional de-cellularized lung scaffold.MethodsAF-MSCs were isolated from human amniotic fluid obtained from 17–37 weeks gestation. Lung differentiation was induced on Matrigel or on de-cellularized rat lungs intra-tracheally injected with AF-MSCs by culturing with a modification of small airway growth medium (mSAGM) lacking retinoic acid (RA) and triodothyronine (T3) with addition of fibroblast growth factor-10 (FGF10). Cells and scaffolds were characterized by immunofluorescence and RT-PCR for markers of viability, proliferation, and lung distal airway differentiation (TTF-1⁺ and SPC⁺) in the absence of markers of brain (TuJ1⁻) and thyroid (Pax8⁻).ResultsAfter culture in mSAGM on either Matrigel or lung scaffolds, there were TTF-1⁺/TuJ1⁻/Pax8⁻ cells, indicating a lung precursor phenotype. In addition, SPC⁺ cells also evolved suggesting a more mature lung phenotype.ConclusionsWe demonstrate that mid- to late-trimester AF-MSCs can be induced to develop into lung precursor cells when cultured on the appropriate extracellular matrix (ECM), making them a viable source for use in cell therapy or development of an ex vivo tissue engineered lung.     

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth

BackgroundThe lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR ^−/− pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract.MethodsWe compared bone parameters of CFTR ^−/− male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (μCT), and their chemical compositions were examined using Raman microspectroscopy.ResultsThe integrity of the CFTR ^−/− bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR ^−/- pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR ^−/− trabecular bone, but not in CFTR ^−/− cortical bone.ConclusionsThe loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.     

Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation

BackgroundW1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations.MethodsWe compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors.ResultsCas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR.ConclusionAltogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.     

Macrophages from gut-corrected CF mice express human CFTR and lack a pro-inflammatory phenotype

Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftr^tm1Unc Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftr^tm1Unc Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model.     

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis

BackgroundIn cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation.MethodsGenetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated.ResultsOrganoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation.ConclusionsIn vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.     

Amplifiers co-translationally enhance CFTR biosynthesis via PCBP1-mediated regulation of CFTR mRNA

BackgroundCystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity.MethodsBiochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics.ResultsAmplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness.ConclusionsSmall molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.     

Expression of CXCR6 on CD8+ T cells was up-regulated in allograft rejection

CXCL16/SR-PSOX is a novel transmembrane-type chemokine, which was also identified as a novel scavenger receptor for oxidized low density lipoprotein. Its receptor CXCR6 expresses on activated CD8⁺ T cells, type 1-polarized CD4⁺, and constitutively expresses on NKT cells. Moreover, it has been shown that CXCL16 accumulated activated CD8⁺ T cells to sites of inflammation. To date, the effect of CXCL16 (SR-PSOX)/CXCR6 on CD8⁺ T cells and its role in allograft rejection/acceptance are not well understood. In the current study, we show that rejected allografts showed higher expressions of CXCR6 and CXCL16. More importantly, expression of CXCR6 on CD8⁺ T cells was also up-regulated by rejection. However, the blockade of CXCL16(SR-PSOX)/CXCR6 interaction could not inhibit cytotoxic activity of CD8⁺ T cells, and therefore, could not prolong the cardiac graft survival time.