Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection in vivo and human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1ß heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

Th17/Treg imbalance in murine cystic fibrosis is linked to indoleamine 2,3-dioxygenase deficiency but corrected by kynurenines.

RATIONALE: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development. OBJECTIVES: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF. METHODS: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation. MEASUREMENTS AND MAIN RESULTS: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF. CONCLUSIONS: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF.

Hypoxia promotes danger-mediated inflammation via receptor for advanced glycation end products in cystic fibrosis.

RATIONALE: Hypoxia regulates the inflammatory-antiinflammatory balance by the receptor for advanced glycation end products (RAGE), a versatile sensor of damage-associated molecular patterns. The multiligand nature of RAGE places this receptor in the midst of chronic inflammatory diseases. OBJECTIVES: To characterize the impact of the hypoxia-RAGE pathway on pathogenic airway inflammation preventing effective pathogen clearance in cystic fibrosis (CF) and elucidate the potential role of this danger signal in pathogenesis and therapy of lung inflammation. METHODS: We used in vivo and in vitro models to study the impact of hypoxia on RAGE expression and activity in human and murine CF, the nature of the RAGE ligand, and the impact of RAGE on lung inflammation and antimicrobial resistance in fungal and bacterial pneumonia. MEASUREMENTS AND MAIN RESULTS: Sustained expression of RAGE and its ligand S100B was observed in murine lung and human epithelial cells and exerted a proximal role in promoting inflammation in murine and human CF, as revealed by functional studies and analysis of the genetic variability of AGER in patients with CF. Both hypoxia and infections contributed to the sustained activation of the S100B-RAGE pathway, being RAGE up-regulated by hypoxia and S100B by infection by Toll-like receptors. Inhibiting the RAGE pathway in vivo with soluble (s) RAGE reduced pathogen load and inflammation in experimental CF, whereas sRAGE production was defective in patients with CF. CONCLUSIONS: A causal link between hyperactivation of RAGE and inflammation in CF has been observed, such that targeting pathogenic inflammation alleviated inflammation in CF and measurement of sRAGE levels could be a useful biomarker for RAGE-dependent inflammation in patients with CF.

Outcome of atypical hemolytic uremic syndrome: role of triggers and complement abnormalities in the response to C5 inhibition.

BACKGROUND: Atypical-hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy often due to uncontrolled complement activation, characterized by high risk of end-stage kidney disease (ESKD). Eculizumab has improved the outcome, however, its efficacy varies among patients and its discontinuation is debated. METHODS: To identify characteristics associated with treatment response, we analyzed 244 aHUS patients referred to our center. Patients were classified according to the presence/absence of complement abnormalities and/or triggers at onset in 4 categories: (1) primary (complement abnormality without trigger), (2) secondary (trigger without complement abnormality), (3) combined (trigger and complement abnormality), (4) idiopathic (no trigger, no complement abnormality). Response to treatment was evaluated by comparing the response to eculizumab with that of conventional therapy. Short- and long-term outcomes were evaluated with the relapse rate after discontinuation of C5-inhibition. RESULTS: Patients had a better outcome with eculizumab compared to conventional treatment, with a response rate of 81.9% vs 56.9%, p < 0.001 and a long-term cumulative incidence of ESKD of 5.8% vs 22.5% (hazard ratio 0.25, 95% confidence interval: 0.10-0.80). The excellent global response was driven by the primary and combined groups (89.8% vs 54.0% and 89.3% vs 54.2%, respectively). The relapse rate following discontinuation of the C5-inhibitor was as high as 66.7% in the primary group, 18.7% in the combined, and 0% in the secondary and idiopathic groups. CONCLUSIONS: Our data show a better outcome in aHUS patients treated with C5-inhibition, particularly in the primary and combined forms, which have a high risk of relapse after discontinuation that is not observed in the secondary and idiopathic forms.

Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Characterization of CFTR mutations in people with cystic fibrosis and severe liver disease who are not eligible for CFTR modulators.

Cystic-fibrosis-related liver disease (CFLD) is a variable phenotype of CF. The severe CFLD variant with cirrhosis or portal hypertension has a poor prognosis and life expectancy. CFTR modulator therapies are now available for people with CF and eligibility for such treatment is based on their CFTR genotype. We evaluated the genetic eligibility for elexacaftor, tezacaftor, ivacaftor (ETI), and ivacaftor (IVA) monotherapy in a previously reported CF cohort of 1591 people with CF of whom 171 with severe CFLD. Based on their CFTR mutations, 13% (N=184/1420) of subjects without CFLD and 11% (N=19/171) of those with severe CFLD are not eligible for either ETI or IVA therapy. The non-eligible patients without CFLD or with severe CFLD can currently not take advantage of the potential benefits of these new treatments. Although this study cannot provide any data regarding the effect of ETI or IVA on the progression of severe CFLD, the consequences for ineligibility of patients with extreme liver phenotype may be even more significant because of their poorer disease risk profile.

Complement System as a New Target for Hematopoietic Stem Cell Transplantation-Related Thrombotic Microangiopathy.

Thrombotic microangiopathy (TMA) is a complication that may occur after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) and is conventionally called transplant-associated thrombotic microangiopathy (TA-TMA). Despite the many efforts made to understand the mechanisms of TA-TMA, its pathogenesis is largely unknown, its diagnosis is challenging and the case-fatality rate remains high. The hallmarks of TA-TMA, as for any TMA, are platelet consumption, hemolysis, and organ dysfunction, particularly the kidney, leading also to hypertension. However, coexisting complications, such as infections and/or immune-mediated injury and/or drug toxicity, together with the heterogeneity of diagnostic criteria, render the diagnosis difficult. During the last 10 years, evidence has been provided on the involvement of the complement system in the pathophysiology of TA-TMA, supported by functional, genetic, and therapeutic data. Complement dysregulation is believed to collaborate with other proinflammatory and procoagulant factors to cause endothelial injury and consequent microvascular thrombosis and tissue damage. However, data on complement activation in TA-TMA are not sufficient to support a systematic use of complement inhibition therapy in all patients. Thus, it seems reasonable to propose complement inhibition therapy only to those patients exhibiting a clear complement activation according to the available biomarkers. Several agents are now available to inhibit complement activity: two drugs have been successfully used in TA-TMA, particularly in pediatric cases (eculizumab and narsoplimab) and others are at different stages of development (ravulizumab, coversin, pegcetacoplan, crovalimab, avacopan, iptacopan, danicopan, BCX9930, and AMY-101).

Eculizumab treatment in atypical hemolytic uremic syndrome: correlation between functional complement tests and drug levels.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is characterized by platelet consumption, hemolysis, and renal injury. Eculizumab, a humanized antibody that blocks complement activity, has been successfully used in aHUS, but the best treatment schedule has not yet been clearly defined. METHODS: Herein we report our experience with eculizumab maintenance treatment, in which the interval between subsequent doses was adjusted based on classical complement pathway (CCP) activity, targeted to < 30% for the prevention of relapses. Trough circulating levels of free eculizumab were determined by an immunoenzymatic method. Genetic and serologic characteristics of the patients were also assessed. RESULTS: We report on 38 patients with aHUS with a median age of 25.0 years (range 0.5-60.0 years) treated with eculizumab. Once stable disease remission was obtained, the interval between eculizumab doses was extended based on target CCP activity. With this approach, presently, 22 patients regularly receive eculizumab infusion every 28 days and 16 receive it every 21. During a median observation period of 32.3 months (range 4.0-92.4 months) and a cumulative period of 1295 months, no patient relapsed. An inverse correlation between CCP activity and eculizumab circulating levels was present (r = - 0.690, p = 0.0001), with CCP activity being inhibited as long as free eculizumab was measurable in serum. CONCLUSIONS: In patients with aHUS on eculizumab maintenance treatment, complement activity measurement can be used as a proxy for circulating levels of the drug. Monitoring complement activity allows for safe tailoring of the frequency of eculizumab administration, thus avoiding excessive drug exposure while keeping the disease in remission.

Endometrial microbiome: sampling, assessment, and possible impact on embryo implantation.

There is growing interest on the potential clinical relevance of the endometrial microbiome. However, insufficient attention has been given to the methodology of sampling. To minimize contamination, we advocate the use of the double-lumen catheters commonly employed for the embryo transfer. Endometrial fluid samples obtained from 53 women scheduled for IVF were studied for microbiome characterization. Control samples from the vagina of these same women were concomitantly obtained. Samples were analysed by V3-V4-V6 regions of 16S rRNA gene sequencing with Next Generation Sequencing technique. Endometrial Lactobacillus-dominant cases were uncommon compared to previous evidence, being observed in only 4 (8%) women. Taxonomy markedly differed between the endometrial and vaginal microbiomes composition. The most common bacterial genera coincided in only 4 (8%) women. The comparison between women who did and did not subsequently become pregnant failed to identify any microorganism associated with the success of the procedure. However, the endometrial biodiversity resulted higher among pregnant women. Shannon's Equitability index in pregnant and non pregnant women was 0.76 [0.57-0.87] and 0.55 [0.51-0.64], respectively (p = 0.002). In conclusion, the use of embryo transfer catheters for testing the endometrial microbiome is promising. The scant concordance with vaginal samples supports the validity of this approach. Moreover, our study highlighted a possible beneficial role of a higher biodiversity on endometrial receptivity.

Risk of Atypical HUS Among Family Members of Patients Carrying Complement Regulatory Gene Abnormality.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is mainly due to complement regulatory gene abnormalities with a dominant pattern but incomplete penetrance. Thus, healthy carriers can be identified in any family of aHUS patients, but it is unpredictable if they will eventually develop aHUS. METHODS: Patients are screened for 10 complement regulatory gene abnormalities and once a genetic alteration is identified, the search is extended to at-risk family members. The present cohort study includes 257 subjects from 71 families: 99 aHUS patients (71 index cases + 28 affected family members) and 158 healthy relatives with a documented complement gene abnormality. RESULTS: Fourteen families (19.7%) experienced multiple cases. Over a cumulative observation period of 7595 person-years, only 28 family members carrying gene mutations experienced aHUS (overall penetrance of 20%), leading to a disease rate of 3.69 events for 1000 person-years. The disease rate was 7.47 per 1000 person-years among siblings, 6.29 among offspring, 2.01 among parents, 1.84 among carriers of variants of uncertain significance, and 4.43 among carriers of causative variants. CONCLUSIONS: The penetrance of aHUS seems a lot lower than previously reported. Moreover, the disease risk is higher in carriers of causative variants and is not equally distributed among generations: siblings and the offspring of patients have a much greater disease risk than parents. However, risk calculation may depend on variant classification that could change over time.

Urinary Extracellular Vesicles and Salt-Losing Tubulopathies: A Proteomic Approach.

Renal tubular cells release urinary extracellular vesicles (uEV) that are considered a promising source of molecular markers for renal dysfunction and injury. We investigated uEV proteomes of patients with hereditary salt-losing tubulopathies (SLTs), focusing on those caused by Gitelman and Bartter (BS) syndromes, to provide potential markers for differential diagnosis. Second morning urine was collected from patients with genetically proven SLTs and uEV were isolated by the ultracentrifugation-based protocol. The uEV proteome was run through a diagonal bidimensional electrophoresis (16BAC/SDS-PAGE), to improve hydrophobic protein resolution. Sixteen differential spots from the proteome of two variants (BS2 and BS3) were analysed by nLC-ESI-MS/MS after in-gel tryptic digestion. A total of 167 protein species were identified from 7 BS2 spots and 9 BS3 spot. Most of these proteins were membrane-associated proteins, in particular transmembrane proteins, and were related to typical renal functions. The differential content of some uEV was then validated by immunoblotting. Our work suggests that uEV proteomics represents a promising strategy for the identification of differential SLT proteins. This could play a role in understanding the pathophysiological disease mechanisms and may support the recognition of different syndromes.

High-sensitive microarray substrates specifically designed to improve sensitivity for the identification of fetal paternally inherited sequences in maternal plasma.

BACKGROUND: The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application. METHODS: A combined approach based on innovative microarray slides coated with a special copolymer was developed for the identification of three polymorphisms located in the causative gene for cystic fibrosis (CF). This technique was applied to the analysis of fetal DNA in maternal plasma from four couples that carried different allelic variants. RESULTS: The use of highly sensitive slides correctly identified fetal paternally inherited alleles without any enrichment strategy. CONCLUSIONS: Our results confirm that the high fluorescence signal provided by the optimized substrate may be applied to the identification of any fetal paternally inherited sequence. This helps extend the application of non-invasive prenatal diagnosis to genetic diseases caused by predominant mutations or minor rare molecular defects.

When and how ruling out cystic fibrosis in adult patients with bronchiectasis.

BACKGROUND: Bronchiectasis is the final result of different processes and most of the guidelines advocate for a careful evaluation of those etiologies which might be treated or might change patients' management, including cystic fibrosis (CF). MAIN BODY: CFTR mutations have been reported with higher frequency in bronchiectasis population. Although ruling out CF is considered as a main step for etiological screening in bronchiectasis, CF testing lacks of a standardized approach both from a research and clinical point of view. In this review a list of most widely used tests in CF is provided. CONCLUSIONS: Exclusion of CF is imperative for patients with bronchiectasis and CFTR testing should be implemented in usual screening for investigating bronchiectasis etiology. Physicians taking care of bronchiectasis patients should be aware of CFTR testing and its limitations in the adult population. Further studies on CFTR expression in human lung and translational research might elucidate the possible role of CFTR in the pathogenesis of bronchiectasis.

Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3).

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal-recessive disorder due to mutations in the ATP-binding cassette, subfamily B, member 4 gene (ABCB4). ABCB4 is the liver-specific membrane transporter of phosphatidylcholine, a major and exclusive component of mammalian bile. The disease is characterized by early onset of cholestasis with high serum gamma-glutamyltranspeptidase activity, which progresses into cirrhosis and liver failure before adulthood. Presently, about 20 distinct ABCB4 mutations associated to PFIC3 have been described. We report the molecular characterization of 68 PFIC3 index cases enrolled in a multicenter study, which represents the largest cohort of PFIC3 patients screened for ABCB4 mutations to date. We observed 31 mutated ABCB4 alleles in 18 index cases with 29 distinct mutations, 25 of which are novel. Despite the lack of structural information on the ABCB4 protein, the elucidation of the three-dimensional structure of bacterial homolog allows the three-dimensional model of ABCB4 to be built by homology modeling and the position of the mutated amino-acids in the protein tertiary structure to be located. In a significant fraction of the cases reported in this study, the mutation should result in substantial impairment of ABCB4 floppase activity. The results of this study provide evidence of the broad allelic heterogeneity of the disease, with causative mutations spread along 14 of the 27 coding exons, but with higher prevalence on exon 17 that, as recently shown for the closely related paralogous ABCB1 gene, could contain an evolutionary marker for mammalian ABCB4 genes in the seventh transmembrane segment.

A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling.

IL-1 receptor antagonist ameliorates inflammasome-dependent inflammation in murine and human cystic fibrosis.

Dysregulated inflammasome activation contributes to respiratory infections and pathologic airway inflammation. Through basic and translational approaches involving murine models and human genetic epidemiology, we show here the importance of the different inflammasomes in regulating inflammatory responses in mice and humans with cystic fibrosis (CF), a life-threatening disorder of the lungs and digestive system. While both contributing to pathogen clearance, NLRP3 more than NLRC4 contributes to deleterious inflammatory responses in CF and correlates with defective NLRC4-dependent IL-1Ra production. Disease susceptibility in mice and microbial colonization in humans occurs in conditions of genetic deficiency of NLRC4 or IL-1Ra and can be rescued by administration of the recombinant IL-1Ra, anakinra. These results indicate that pathogenic NLRP3 activity in CF could be negatively regulated by IL-1Ra and provide a proof-of-concept evidence that inflammasomes are potential targets to limit the pathological consequences of microbial colonization in CF.

A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene.

We report the case of a patient with an apparent homozygosity for the D1152H mutation located in exon 18 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The parents had no personal history of cystic fibrosis (CF) and referred to our laboratory after the diagnosis of fetal bowel hyperechogenicity. The proband presented with meconium ileus and normal sweat chloride test. Sequencing of the CFTR exon 18 together with quantitative genomic assays, such as real-time PCR and the multiplex ligation probe amplification (MLPA) techniques, were performed and revealed that the father was heterozygous for the D1152H mutation and the mother carried a large deletion of the CFTR gene encompassing the genomic sequence including the same mutation. The child inherited D1152H from his father and the large deletion of the CFTR gene from his mother. We suggest that D1152H likely acts as a mild mutation with a dominant effect on the severe deletion of exon 18, considering that after 3 years of clinical examinations the child shows no classical signs and symptoms of CF. Not testing for large deletions in subjects with apparent homozygosity for a mutated CFTR allele could lead to the misidentification of CFTR mutation carrier status.