As the short telomere defect has been linked to the pathogenesis of IPF in some cases, our data indicate that impaired CMV immunity may be a systemic manifestation of telomere-mediated disease in these patients. Identifying this high-risk subset of lung transplant recipients has implications for risk assessment, management and potential strategies for averting posttransplant CMV morbidities.
The endogenous bronchodilator, S-nitrosoglutathione (GSNO), increases expression, maturation, and function of both the wild-type and the ⌬F508 mutant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR). Though transcriptional mechanisms of action have been identified, GSNO seems also to have post-transcriptional effects on CFTR maturation. Here, we report that 1) GSNO is only one of a class of S-nitrosylating agents that, at low micromolar concentrations, increase ⌬F508 and wild-type CFTR expression and maturation; 2) NO itself (at these concentrations) and 8-bromocyclic GMP are minimally active on CFTR; 3) a novel agent, S-nitrosoglutathione diethyl ester, bypasses the need for GSNO bioactivation by ␥-glutamyl transpeptidase to increase CFTR maturation; 4) surprisingly, expression-but not S-nitrosylation-of cysteine string proteins (Csp) 1 and 2 is increased by GSNO; 5) the effect of GSNO to increase full maturation of wild-type CFTR is inhibited by Csp silencing (si)RNA; 6) proteins relevant to CFTR trafficking are SNO-modified, and SNO proteins traffic through the endoplasmic reticulum (ER) and Golgi after GSNO exposure; and 7) GSNO alters the interactions of ⌬F508 CFTR with Csp and Hsc70 in the ER and Golgi. These data suggest that GSNO is one of a class of S-nitrosylating agents that act independently of the classic NO radical/cyclic GMP pathway to increase CFTR expression and maturation. They also suggest that the effect of GSNO is dependent on Csp and on intracellular SNO trafficking. We speculate that these data will be of relevance to the development of NO donor-based therapies for CF.Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene encoding the CF transmembrane conductance regulatory (CFTR) protein (Riordan, 1999). CFTR has several functions but is typically regarded as an apical membrane Cl Ϫ channel in epithelial cells. Its posttranslational processing involves a complex and incompletely defined series of interactions with a variety of chaperones and cochaperones that fold and glycosylate the protein and screen it for defects. The most common mutation associated with CF, ⌬F508, results in a single amino acid deletion (Drumm et al., 1991;Riordan, 1999;Gibson et al., 2003). The majority of wild-type (wt) CFTR-and virtually all ⌬F508 CFTR-is degraded before reaching the cell surface (Drumm et al., 1991). Certain agents and conditions increase expression, maturation, and (in the presence of cyclic AMP-stimulating agonists) function of ⌬F508 CFTR. Therefore, there is an interest in identifying compounds with a favorable pharmacological profile that could have this effect in vivo, reversing the molecular defect, and preventing disease progression (Denning et al., 1992;Ward and Kopito, 1994;Zeitlin et al., 2002). High-throughput screening has been used to identify 1435at ASPET Journals on May 11, 2018 molpharm.aspetjournals.org Downloaded fromcompounds that, by augmenting ⌬F508 CFTR trafficking, increase its functional expression on the surf...
Belatacept-based ISR appear to produce reasonable results in LTRs who fail CNI-based ISR. Larger prospective trials appear warranted in lung transplantation.
BackgroundAzithromycin, an antibiotic used for multiple infectious disorders, exhibits anti-inflammatory effects, but the molecular basis for this activity is not well characterized. Azithromycin inhibits IL-1β-mediated inflammation that is dependent, in part, on inflammasome activity. Here, we investigated the effects of azithromycin on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) protein, which is the sensing component of the NALP3 inflammasome, in human monocytes.MethodsTHP-1 cells were treated with azithromycin alone, LPS alone, or both. NALP3 and IL-1β protein levels were determined by immunoblotting. NLRP3 gene (encoding NALP3) transcript levels were determined by quantitative qPCR. In order to measure NLRP3 transcript decay, actinomycin D was used to impair gene transcription. THP-1 Lucia cells which contain an NF-κB responsive luciferase element were used to assess NF-κB activity in response to azithromycin, LPS, and azithromycin/LPS by measuring luminescence. To confirm azithromycin’s effects on NLRP3 mRNA and promoter activity conclusively, HEK cells were lipofected with luciferase reporter constructs harboring either the 5’ untranslated region (UTR) of the NLRP3 gene which included the promoter, the 3’ UTR of the gene, or an empty plasmid prior to treatment with azithromycin and/or LPS, and luminescence was measured.ResultsAzithromycin decreased IL-1β levels and reduced NALP3 protein levels in LPS-stimulated THP-1 monocytes through a mechanism involving decreased mRNA stability of the NALP3 – coding NLRP3 gene transcript as well as by decreasing NF-κB activity. Azithromycin accelerated NLRP3 transcript decay confirmed by mRNA stability and 3’UTR luciferase reporter assays, and yet the antibiotic had no effect on NLRP3 promoter activity in cells containing a 5’ UTR reporter.ConclusionsThese studies provide a unique mechanism whereby azithromycin exerts immunomodulatory actions in monocytes by destabilizing mRNA levels for a key inflammasome component, NALP3, leading to decreased IL-1β-mediated inflammation.
Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.
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