Sarilumab for Relapse of Polymyalgia Rheumatica during Glucocorticoid Taper.

BACKGROUND: More than half of patients with polymyalgia rheumatica have a relapse during tapering of glucocorticoid therapy. Previous studies have suggested that interleukin-6 blockade may be clinically useful in the treatment of polymyalgia rheumatica. Sarilumab, a human monoclonal antibody, binds interleukin-6 receptor α and efficiently blocks the interleukin-6 pathway. METHODS: In this phase 3 trial, we randomly assigned patients in a 1:1 ratio to receive 52 weeks of a twice-monthly subcutaneous injection of either sarilumab (at a dose of 200 mg) plus a 14-week prednisone taper or placebo plus a 52-week prednisone taper. The primary outcome at 52 weeks was sustained remission, which was defined as the resolution of signs and symptoms of polymyalgia rheumatica by week 12 and sustained normalization of the C-reactive protein level, absence of disease flare, and adherence to the prednisone taper from weeks 12 through 52. RESULTS: A total of 118 patients underwent randomization (60 to receive sarilumab and 58 to receive placebo). At week 52, sustained remission occurred in 28% (17 of 60 patients) in the sarilumab group and in 10% (6 of 58 patients) in the placebo group (difference, 18 percentage points; 95% confidence interval, 4 to 32; P = 0.02). The median cumulative glucocorticoid dose at 52 weeks was significantly lower in the sarilumab group than in the placebo group (777 mg vs. 2044 mg; P<0.001). The most common adverse events with sarilumab as compared with placebo were neutropenia (15% vs. 0%), arthralgia (15% vs. 5%), and diarrhea (12% vs. 2%). More treatment-related discontinuations were observed in the sarilumab group than in the placebo group (12% vs. 7%). CONCLUSIONS: Sarilumab showed significant efficacy in achieving sustained remission and reducing the cumulative glucocorticoid dose in patients with a relapse of polymyalgia rheumatica during glucocorticoid tapering. (Funded by Sanofi and Regeneron Pharmaceuticals; SAPHYR ClinicalTrials.gov number, NCT03600818.).

Clinical Efficacy among Patients with Chronic Rhinosinusitis with Nasal Polyps and Clinical Features of Obstructive Lung Disease: Post Hoc Analysis of the Phase III SINUS-24 and SINUS-52 Studies.

Purpose: To provide a descriptive summary of clinical efficacy and health-related quality of life (HRQoL) measures in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and clinical features of obstructive lung disease in the Phase III dupilumab studies SINUS-24 and SINUS-52 (NCT02912468, NCT02898454). Patients and Methods: Patients met a "broad" definition of having clinical features of obstructive lung disease with any of 3 criteria: (i) pre-bronchodilator forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) <0.70 and smoking history; (ii) patient-reported medical history of chronic obstructive pulmonary disease (COPD); or (iii) asthma with >10 pack-years' smoking history. A "narrow" definition including criteria (i) or (ii) was also analyzed. CRSwNP and HRQoL measures were evaluated in all patients and lung function (FEV1; FEV1/FVC ratio) was captured and analyzed only in those patients who had a self-reported history of asthma. Results: Across both studies, 131 patients met the "broad" definition, of whom 90 also had asthma, and 115 patients met the "narrow" definition, of whom 74 had asthma. CRSwNP outcomes and HRQoL were improved with dupilumab vs placebo in both the broad and narrow subgroups. Among the 90 patients who met the broad definition and had asthma, dupilumab improved pre-bronchodilator FEV1 and FEV1/FVC ratio at Week 16 (least squares mean differences vs placebo: 0.38 L [95% confidence interval: 0.17, 0.59; p = 0.0004] and 4.8% [1.7%, 7.9%; p = 0.0024], respectively) sustained through Week 24. Similar results were seen in the "narrow" subgroup with asthma. Conclusion: In a population of patients with CRSwNP and clinical features of obstructive lung disease, dupilumab improved CRSwNP and HRQoL outcomes, and, among those with a history of asthma, also improved lung function. These results support further analyses of dupilumab in patients with evidence of type 2 inflammation and obstructive lung disease such as COPD.

AZD5634, an inhaled ENaC inhibitor, in healthy subjects and patients with cystic fibrosis.

BACKGROUND: Epithelial sodium channel (ENaC) inhibitors may offer clinical benefit in cystic fibrosis (CF); however, data are limited. We report the outcomes of a Phase I (NCT02679729) and a Phase Ib (NCT02950805) study of AZD5634, a novel inhaled ENaC inhibitor. METHODS: A Phase I, first-in-human, single-blind, placebo-controlled, single ascending dose, sequential dose group study assessed the safety, tolerability, and pharmacokinetics of AZD5634 in healthy subjects (n=53) in part A following inhaled doses up to 1700 µg, and, in part B, following administration of single inhaled (1700 µg) and intravenous (65 µg) doses. A Phase Ib, randomized, double-blind, placebo-controlled, single-dose, 2-way cross-over study assessed the effects of a single dose (600 µg) of inhaled AZD5634 on mucociliary clearance (MCC), pharmacokinetics and safety and tolerability in patients with CF (n=11). Nasal potential difference (NPD) was assessed as an in situ target engagement exploratory biomarker. RESULTS: Absolute bioavailability of AZD5634 after inhalation was approximately 3%, indicating minimal distribution into the systemic circulation. Urinary excretion was a minor elimination pathway. Administration of inhaled AZD5634 did not improve MCC in CF patients, but AZD5634 inhibited ENaC in the nasal epithelium, as measured by NPD. AZD5634 was safe and well tolerated in both studies. CONCLUSIONS: AZD5634 showed favorable pharmacokinetics and safety in healthy subjects and patients with CF. However, despite achieving target engagement, proof of mechanism was not achieved after a single dose in patients with CF. Further evaluation into multiple dose studies is warranted to explore its therapeutic potential.

Olfactory Outcomes With Dupilumab in Chronic Rhinosinusitis With Nasal Polyps.

BACKGROUND: Loss of smell (LoS) is one of the most troublesome and difficult-to-treat symptoms of severe chronic rhinosinusitis with nasal polyps (CRSwNP). OBJECTIVE: To assess the impact of dupilumab on sense of smell in severe CRSwNP. METHODS: In the randomized SINUS-24 and SINUS-52 studies, adults with severe CRSwNP received dupilumab 300 mg subcutaneously or matching placebo every 2 weeks for 24 or 52 weeks, respectively. Smell was assessed using daily patient-reported LoS score (0-3) and University of Pennsylvania Smell Identification Test (UPSIT; 0-40). Data from the 2 studies were pooled through week 24. Relationships between patient phenotypes and smell outcomes were also assessed. RESULTS: We randomized 724 patients (286 placebo, 438 dupilumab); mean CRSwNP duration was 11 years; 63% had prior sinonasal surgery. Mean baseline LoS was 2.74. Dupilumab produced rapid improvement in LoS, evident by day 3, which improved progressively throughout the study periods (least squares mean difference vs placebo -0.07 [95% CI -0.12 to -0.02]; nominal P < .05 at day 3, and -1.04 [-1.17 to -0.91]; P < .0001 at week 24). Dupilumab improved mean UPSIT by 10.54 (least squares mean difference vs placebo 10.57 [9.40-11.74]; P < .0001) at week 24 from baseline (score 13.90). Improvements were unaffected by CRSwNP duration, prior sinonasal surgery, or comorbid asthma and/or nonsteroidal anti-inflammatory drug-exacerbated respiratory disease. Baseline olfaction scores correlated with all measured local and systemic type 2 inflammatory markers except serum total immunoglobulin E. CONCLUSIONS: Dupilumab produced rapid and sustained improvement in sense of smell, alleviating a cardinal symptom of severe CRSwNP.

Dupilumab suppresses type 2 inflammatory biomarkers across multiple atopic, allergic diseases.

BACKGROUND: Type 2 inflammation is common in numerous atopic/allergic diseases and can be identified by elevated biomarker levels. Dupilumab, a fully human monoclonal antibody, blocks the shared receptor component for interleukin-4 and interleukin-13, key and central drivers of type 2 inflammation. OBJECTIVE: Assessment of dupilumab effect on type 2 inflammatory biomarkers in atopic dermatitis (AD), asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and eosinophilic esophagitis (EoE). METHODS: Data were extracted from three randomized placebo-controlled trials of dupilumab in AD (NCT02277743, N = 671; NCT02277769, N = 708; NCT02260986, N = 740); and one each in asthma (NCT02414854, N = 1902); CRSwNP (NCT02898454, N = 448); and EoE (NCT02379052, N = 47). Biomarkers assessed were serum thymus and activation-regulated chemokine (TARC), plasma eotaxin-3, serum total immunoglobulin E (IgE), serum periostin and blood eosinophil count. RESULTS: Dupilumab versus placebo significantly suppressed most type 2 inflammatory biomarker levels across all studies/indications where data were assessed. Reductions in serum TARC, plasma eotaxin-3 and serum periostin occurred rapidly, whereas reductions in serum total IgE were more gradual. Across diseases, at the end of treatment, median percentage change from baseline in TARC levels ranged from -24.8% to -88.6% (placebo +2.6% to -53.6%); -38.2% to -51.5% (placebo +8.3% to -0.16%) in eotaxin-3; -24.8% to -76.7% (placebo +8.3% to -4.4%) in total IgE; and -13.6% to -41.1% (placebo +10.1% to -6.94%) in periostin levels. Blood eosinophil responses to dupilumab varied by disease, with minimal changes in AD in the SOLO studies (median percentage change from baseline to end of treatment: 0% [95% CI: -15.8, 0]); transient increases followed by decreases to below-baseline levels in asthma (-14.6% [-20.0, -7.7]) and CRSwNP (-29.4% [-40.0, -16.3]); and significant decreases in EoE (-50.0% [-50.0, -33.3]). CONCLUSION AND CLINICAL RELEVANCE: Dupilumab reduced levels of type 2 biomarkers across clinical studies in patients with AD, asthma, CRSwNP and EoE.

Dupilumab improves upper and lower airway disease control in chronic rhinosinusitis with nasal polyps and asthma.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) and type 2 asthma share the same inflammatory pathophysiology and are frequent comorbidities. Dupilumab, a fully human monoclonal antibody, blocks the shared receptor component for interleukin 4 and interleukin 13, which are key and central drivers of type 2 inflammation. OBJECTIVE: We report the effect of dupilumab vs placebo on outcome measures of the upper and lower airways and health-related quality of life (HRQoL) in the pooled population of patients with CRSwNP and comorbid asthma from the phase 3 SINUS-24 (NCT02912468) and SINUS-52 (NCT02898454) studies. METHODS: In these randomized, double-blind, placebo-controlled trials, patients received subcutaneous dupilumab 300 mg (n = 438) or placebo (n = 286) every 2 weeks on a background of mometasone furoate nasal spray. Changes from baseline at week 24 in the upper and lower airway outcome measures are reported. RESULTS: Of the 724 patients randomized, 428 (59.1%) had comorbid asthma. In patients with asthma at week 24, dupilumab vs placebo improved the nasal polyp score (-2.04), patient-reported nasal congestion score (-1.04), Lund-Mackay computed tomography scan score (-6.43), peak nasal inspiratory flow (46.15 L/min), and 22-item sinonasal outcome test score (-21.42; all P < .001). The forced expiratory volume in 1 second and 6-item asthma control questionnaire scores were also markedly improved with dupilumab vs placebo. The most common adverse events (nasopharyngitis, headache, injection-site erythema, worsening of nasal polyposis, and asthma) were more frequent with placebo than dupilumab. CONCLUSION: Dupilumab improved upper and lower airway outcome measures and HRQoL in patients with severe CRSwNP and comorbid asthma and was well tolerated. TRIAL REGISTRATION: ClinicalTrials.gov Identifiers: NCT02912468 (SINUS-24) and NCT02898454 (SINUS-52).

Efficacy and safety of dupilumab in patients with severe chronic rhinosinusitis with nasal polyps (LIBERTY NP SINUS-24 and LIBERTY NP SINUS-52): results from two multicentre, randomised, double-blind, placebo-controlled, parallel-group phase 3 trials.

BACKGROUND: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) generally have a high symptom burden and poor health-related quality of life, often requiring recurring systemic corticosteroid use and repeated sinus surgery. Dupilumab is a fully human monoclonal antibody that inhibits signalling of interleukin (IL)-4 and IL-13, key drivers of type 2 inflammation, and has been approved for use in atopic dermatitis and asthma. In these two studies, we aimed to assess efficacy and safety of dupilumab in patients with CRSwNP despite previous treatment with systemic corticosteroids, surgery, or both. METHODS: LIBERTY NP SINUS-24 and LIBERTY NP SINUS-52 were two multinational, multicentre, randomised, double-blind, placebo-controlled, parallel-group studies assessing dupilumab added to standard of care in adults with severe CRSwNP. SINUS-24 was done in 67 centres in 13 countries, and SINUS-52 was done in 117 centres in 14 countries. Eligible patients were 18 years or older with bilateral CRSwNP and symptoms despite intranasal corticosteroid use, receiving systemic corticosteroids in the preceding 2 years, or having had sinonasal surgery. Patients in SINUS-24 were randomly assigned (1:1) to subcutaneous dupilumab 300 mg or placebo every 2 weeks for 24 weeks. Patients in SINUS-52 were randomly assigned (1:1:1) to dupilumab 300 mg every 2 weeks for 52 weeks, dupilumab every 2 weeks for 24 weeks and then every 4 weeks for the remaining 28 weeks, or placebo every 2 weeks for 52 weeks. All patients were randomly assigned centrally with a permuted block randomisation schedule. Randomisation was stratified by asthma or non-steroidal anti-inflammatory drug-exacerbated respiratory disease status at screening, previous surgery at screening, and country. Patients with or without comorbid asthma were included. Coprimary endpoints were changes from baseline to week 24 in nasal polyp score (NPS), nasal congestion or obstruction, and sinus Lund-Mackay CT scores (a coprimary endpoint in Japan), done in an intention-to-treat population. Safety was assessed in a pooled population of both dupilumab groups in SINUS-52 up to week 24 and the dupilumab group in SINUS-24 and the placebo groups in both studies until week 24. The trials are complete and registered at ClinicalTrials.gov, NCT02912468 and NCT02898454. FINDINGS: Between Dec 5, 2016, and Aug 3, 2017, 276 patients were enrolled in SINUS-24, with 143 in the dupilumab group and 133 in the placebo group receiving at least one study drug dose. Between Nov 28, 2016, and Aug 28, 2017, 448 patients were enrolled in SINUS-52, with 150 receiving at least one dose of dupilumab every 2 weeks, 145 receiving at least one dose of dupilumab every 2 weeks for 24 weeks and every 4 weeks until week 52, and 153 receiving at least one dose of placebo. Dupilumab significantly improved the coprimary endpoints in both studies. At 24 weeks, least squares mean difference in NPS of dupilumab treatment versus placebo was -2·06 (95% CI -2·43 to -1·69; p<0·0001) in SINUS-24 and -1·80 (-2·10 to -1·51; p<0·0001) in SINUS-52; difference in nasal congestion or obstruction score was -0·89 (-1·07 to -0·71; p<0·0001) in SINUS-24 and -0·87 (-1·03 to -0·71; p<0·0001) in SINUS-52; and difference in Lund-Mackay CT scores was -7·44 (-8·35 to -6·53; p<0·0001) in SINUS-24 and -5·13 (-5·80 to -4·46; p<0·0001) in SINUS-52. The most common adverse events (nasopharyngitis, worsening of nasal polyps and asthma, headache, epistaxis, and injection-site erythema) were more frequent with placebo. INTERPRETATION: In adult patients with severe CRSwNP, dupilumab reduced polyp size, sinus opacification, and severity of symptoms and was well tolerated. These results support the benefits of adding dupilumab to daily standard of care for patients with severe CRSwNP who otherwise have few therapeutic options. FUNDING: Sanofi and Regeneron Pharmaceuticals.

AZD7624, an Inhaled p38 Inhibitor, Demonstrates Local Lung Inhibition of LPS-Induced TNFα with Minimal Systemic Exposure.

Inhaled drugs generally aim to drive a local pharmacological effect in lung, at the same time minimizing systemic exposure, in order to obtain efficacy in lung disease without unwanted systemic effects. Here, we demonstrate that inhaled delivery of a p38 inhibitor (AZD7624) can provide superior pharmacokinetic exposure and superior pharmacodynamic lung effects. In rats, inhaled AZD7624 had a five times higher dose-adjusted lung exposure compared with intravenous dosing. In healthy volunteers, lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) in sputum has been shown to be significantly reduced (85%) by means of inhaled AZD7624. Here, we demonstrate that this effect is associated with a mean unbound plasma concentration, gained from in vitro and ex vivo LPS-challenge protocols, significantly below potencies obtained for AZD7624, suggesting that lung exposure is probably much higher than systemic exposure. This assessment was made for the unbound potency (pIC50u), e.g., the potency remaining after adjustment for plasma protein binding and blood plasma ratio. Hence, the unbound potency of AZD7624 to inhibit LPS-induced TNFα in human mononuclear cells, in whole blood as well as in alveolar macrophages in vitro, was 8.4, 8.7 (full inhibition), and 9.0 (partial inhibition), respectively. The pIC50u in whole blood ex vivo was 8.8, showing good in vitro/in vivo potency correlations. Thus, a mean unbound AZD7624 plasma concentration of 0.3 nmol/l, which was associated with a decrease in LPS-induced sputum TNFα by 85%, is much lower than the pIC50u. This demonstrates that AZD7624 can achieve significant local lung pharmacodynamic effects with concomitant sub-pharmacological systemic exposure.

A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial.

BACKGROUND: The phe508del CFTR mutation causes cystic fibrosis by limiting the amount of CFTR protein that reaches the epithelial cell surface. We tested combination treatment with lumacaftor, an investigational CFTR corrector that increases trafficking of phe508del CFTR to the cell surface, and ivacaftor, a CFTR potentiator that enhances chloride transport of CFTR on the cell surface. METHODS: In this phase 2 clinical trial, we assessed three successive cohorts, with the results of each cohort informing dose selection for the subsequent cohort. We recruited patients from 24 cystic fibrosis centres in Australia, Belgium, Germany, New Zealand, and the USA. Eligibility criteria were: confirmed diagnosis of cystic fibrosis, age at least 18 years, and a forced expiratory volume in 1 s (FEV1) of 40% or more than predicted. Cohort 1 included phe508del CFTR homozygous patients randomly assigned to either lumacaftor 200 mg once per day for 14 days followed by addition of ivacaftor 150 mg or 250 mg every 12 h for 7 days, or 21 days of placebo. Together, cohorts 2 and 3 included phe508del CFTR homozygous and heterozygous patients, randomly assigned to either 56 days of lumacaftor (cohort 2: 200 mg, 400 mg, or 600 mg once per day, cohort 3: 400 mg every 12 h) with ivacaftor 250 mg every 12 h added after 28 days, or 56 days of placebo. The primary outcomes for all cohorts were change in sweat chloride concentration during the combination treatment period in the intention-to-treat population and safety (laboratory measurements and adverse events). The study is registered with ClinicalTrials.gov, number NCT01225211, and EudraCT, number 2010-020413-90. FINDINGS: Cohort 1 included 64 participants. Cohort 2 and 3 combined contained 96 phe508del CFTR homozygous patients and 28 compound heterozygotes. Treatment with lumacaftor 200 mg once daily and ivacaftor 250 mg every 12 h decreased mean sweat chloride concentration by 9.1 mmol/L (p<0.001) during the combination treatment period in cohort 1. In cohorts 2 and 3, mean sweat chloride concentration did not decrease significantly during combination treatment in any group. Frequency and nature of adverse events were much the same in the treatment and placebo groups during the combination treatment period; the most commonly reported events were respiratory. 12 of 97 participants had chest tightness or dyspnoea during treatment with lumacaftor alone. In pre-planned secondary analyses, a significant decrease in sweat chloride concentration occurred in the treatment groups between day 1 and day 56 (lumacaftor 400 mg once per day group -9.1 mmol/L, p<0.001; lumacaftor 600 mg once per day group -8.9 mmol/L, p<0.001; lumacaftor 400 mg every 12 h group -10.3 mmol/L, p=0.002). These changes were significantly greater than the change in the placebo group. In cohort 2, the lumacaftor 600 mg once per day significantly improved FEV1 from day 1 to 56 (difference compared with placebo group: +5.6 percentage points, p=0.013), primarily during the combination period. In cohort 3, FEV1 did not change significantly across the entire study period compared with placebo (difference +4.2 percentage points, p=0.132), but did during the combination period (difference +7.7 percentage points, p=0·003). Phe508del CFTR heterozygous patients did not have a significant improvement in FEV1. INTERPRETATION: We provide evidence that combination lumacaftor and ivacaftor improves FEV1 for patients with cystic fibrosis who are homozygous for phe508del CFTR, with a modest effect on sweat chloride concentration. These results support the further exploration of combination lumacaftor and ivacaftor as a treatment in this setting. FUNDING: Vertex Pharmaceuticals, Cystic Fibrosis Foundation Therapeutics Development Network.

Vitamin D rescues impaired Mycobacterium tuberculosis-mediated tumor necrosis factor release in macrophages of HIV-seropositive individuals through an enhanced Toll-like receptor signaling pathway in vitro.

Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV(+)) persons. Alveolar macrophages from HIV(+) persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV(+) macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV(+) U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV(+) persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV(+) persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV(+) persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV(+) persons.

Cell elasticity determines macrophage function.

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation.

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.

MyD88-dependent TLR4 signaling is selectively impaired in alveolar macrophages from asymptomatic HIV+ persons.

Alveolar macrophages (AMs) are the predominant effector cell in the lungs and contribute to a critical first line of defense against bacterial pathogens through recognition by pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4-mediated tumor necrosis factor alpha (TNFalpha) release is significantly impaired in HIV(+) macrophages, but whether HIV impairs myeloid differentiation factor 88 (MyD88)-dependent and/or MyD-independent TLR4 signaling pathways in human macrophages is not known. Comparing human U937 macrophages with HIV(+) U1 macrophages (HIV-infected U937 subclone), the current study shows that HIV infection is associated with impaired macrophage TLR4-mediated signaling, specifically targeting the MyD88-dependent TLR4-mediated signaling pathway (reduced MyD88-interleukin-1 receptor-associated kinase [IRAK] interaction, IRAK phosphorylation, nuclear factor [NF]-kappaB nuclear translocation, and TNFalpha release) while preserving the MyD88-independent TLR4-mediated signaling pathway (preserved STAT1 phosphorylation, interferon regulatory factor [IRF] nuclear translocation, and interleukin-10 [IL-10] and RANTES release). Extracellular TLR4 signaling complex was intact (similar levels of CD14 and MD2), and similar patterns of response were observed in clinically relevant AMs from healthy and asymptomatic HIV(+) persons at high clinical risk of pneumonia. Taken together, these data support the concept that chronic HIV infection is associated with specific and targeted disruption of critical macrophage TLR4 signaling, which in turn may contribute to disease pathogenesis of bacterial pneumonia.

Impaired M. tuberculosis-mediated apoptosis in alveolar macrophages from HIV+ persons: potential role of IL-10 and BCL-3.

The mechanism of increased MTb disease susceptibility in HIV+ persons remains poorly understood. Apoptosis of macrophages in response to MTb represents a critical host defense response, and decreased apoptosis may represent a mechanism of increased susceptibility to MTb in HIV. In the current study, MTb-mediated apoptosis of human AM was reduced in HIV+ subjects compared with healthy subjects in a TNF-alpha-dependent manner. IL-10 levels in BALF from HIV+ persons were significantly elevated compared with HIV- persons, and exogenous IL-10 reduced MTb-mediated apoptosis in healthy AM, suggesting that IL-10 could mediate decreased apoptosis observed in HIV. Further study showed that IL-10 reduced TNF release in response to MTb in AM through a reduction in TNF mRNA levels, and exogenous TNF could partially reverse IL-10-associated effects on AM apoptosis. IL-10 did not influence p-IRAK, IkappaB degradation, or NF-kappaB p65 nuclear translocation in response to MTb, but IL-10 did increase levels of AM BCL-3, an inhibitor of NF-kappaB nuclear activity. BCL-3 knockdown in human macrophages increased MTb-mediated TNF release. Importantly, BCL-3 levels in AM from HIV+ subjects were higher compared with healthy subjects. Taken together, these data suggest that elevated lung levels of IL-10 may impair MTb-mediated AM apoptosis in HIV through a BCL-3-dependent mechanism. BCL-3 may represent a potential therapeutic target to treat or prevent MTb disease in HIV+ persons.

Constitutive activation of phosphatidylinositol 3-kinase signaling pathway down-regulates TLR4-mediated tumor necrosis factor-alpha release in alveolar macrophages from asymptomatic HIV-positive persons in vitro.

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis.

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.

HIV impairs TNF-alpha release in response to Toll-like receptor 4 stimulation in human macrophages in vitro.

The molecular mechanisms for increased risk of bacterial pneumonia in HIV+ persons remain incompletely understood. Recognizing the critical role of Toll-like receptor (TLR) signaling in host defense, this study showed that human U937 macrophage stimulation by the TLR4-specific ligand, lipid A (biologically active component of bacterial LPS), promoted TNF-alpha release through extracellular regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase phosphorylation. In contrast, HIV+ U1 macrophages had significantly reduced TNF-alpha release (despite preserved TLR4 expression) and reduced ERK1/2 phosphorylation, whereas TNF-alpha release was intact via a TLR4-independent pathway. In HIV+ U1 cells, reduced ERK1/2 phosphorylation was not due to reduced upstream MEK1/2 activation, but was associated with a reciprocal induction of MAP kinase phosphatase-1 (MKP-1). HIV nef protein was sufficient to reduce TNF-alpha release and induce MKP-1 in healthy macrophages. Pharmacologic inhibition of endogenous cellular phosphatases increased ERK1/2 phosphorylation and partially restored TLR4-mediated TNF-alpha release in HIV+ macrophages. Furthermore, targeted gene silencing of MKP-1 partially restored lipid A-mediated TNF-alpha release in HIV+ U1 cells. Similar results were observed using clinically relevant human alveolar macrophages, comparing healthy to asymptomatic HIV+ persons at clinical risk for bacterial pneumonia. Thus, reduced TLR4-mediated TNF-alpha release through altered ERK1/2 regulation by HIV may impair an effective innate immune response to bacterial challenge. Inhibition of cellular phosphatases may serve as a potential therapeutic target in the management of bacterial pneumonia in HIV+ persons.

Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.

Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)-kappaB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)-1beta, IL-6, or tumor necrosis factor (TNF)-alpha (important cytokines in the host response to Pneumocystis) and did not induce IL-1beta, IL-6, or TNF-alpha mRNA transcripts. These findings were not attributed to Pneumocystis-induced cytopathic changes, as these same AM released IL-8 and matrix metalloproteinase-9 in response to Pneumocystis. NF-kappaB-mediated IL-8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL-1beta, IL-6, and TNF-alpha release and mRNA induction were preserved in response to lipopolysaccharide or serum-opsonized Pneumocystis. The absence of IL-1beta, IL-6, and TNF-alpha release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF-alpha release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll-like receptor 4-mediated TNF-alpha release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.

The influence of diagnostic bronchoscopy on clinical outcomes comparing adult autologous and allogeneic bone marrow transplant patients.

STUDY OBJECTIVES: To review our experience with diagnostic bronchoscopy in the evaluation of pulmonary infiltrates in adult hematopoietic stem cell transplantation (HSCT) recipients in the era of Pneumocystis prophylaxis and cytomegalovirus antigen testing. The study focused on diagnostic yields and the influence of bronchoscopic findings on pharmacologic therapy and mortality, comparing allogeneic (allo) HSCT patients to autologous (auto) HSCT patients. DESIGN: Case series review. SETTING: Tertiary care academic urban medical centers. PATIENTS: All adult allo-HSCT and auto-HSCT patients undergoing bronchoscopy for the evaluation of pulmonary infiltrates from January 1997 to September 2001. MEASUREMENTS AND RESULTS: The review identified 169 bronchoscopies that had been performed on HSCT patients, representing 12.5% of all HSCT patients (allo-HSCT patients, 125 bronchoscopies; auto-HSCT patients, 44 bronchoscopies). Bronchoscopy was requested more often in allo-HSCT patients (18.7%) compared to auto-HSCT patients (6.6%). Findings at bronchoscopy provided a specific diagnosis more frequently in allo-HSCT patients (50%) compared to auto-HSCT patients (34%). For both allo-HSCT and auto-HSCT patients, most diagnoses were obtained by BAL alone, whereas transbronchial biopsy (TBBx) provided additional specific information in < 10% of cases. For select patients (n = 27), surgical lung biopsy following bronchoscopy provided unique diagnoses in 47 to 50% of cases. Information from bronchoscopy influenced clinical decisions more often in allo-HSCT patients (50%) than in auto-HSCT patients (36%), and allowed for the discontinuation or addition of antimicrobial, corticosteroid, or antineoplastic agents to treatment. Complications from bronchoscopy occurred in 9% of all HSCT patients (n = 15), and were associated with higher in-hospital mortality rates in allo-HSCT patients (82%; n = 9) compared to auto-HSCT patients (50%; n = 2). The overall in-hospital mortality rates for allo-HSCT and auto-HSCT patients having bronchoscopy was similar (38% vs 27%, respectively; p = 0.25), and establishing a specific diagnosis by bronchoscopy did not improve the in-hospital mortality rate for allo-HSCT or auto-HSCT patients. CONCLUSIONS: Bronchoscopy may provide clinically useful information in the evaluation of adult allo-HSCT and auto-HSCT recipients with pulmonary infiltrates. The results of testing BAL fluid samples alone suggested an etiology in most cases, whereas the findings of TBBx provided unique diagnoses infrequently. Further studies are warranted to improve the utility of diagnostic bronchoscopy in the evaluation of HSCT patients.