Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1 mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1 mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1 mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1Mlkl macrophages, but not in wild-type, Mlkl or Ripk1Ripk3 macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. Methods:We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate , 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate , 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/ 449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).Keywords: miRNA; asthma; airway epithelium Asthma is an inflammatory condition of the lung characterized by episodic airway obstruction and remodeling of airway tissues (1). The airway epithelium is increasingly recognized as an important participant in asthma pathophysiology (2, 3). Airway epithelial cells produce mucus, which is a major contributor to airway obstruction in fatal asthma (4), and even patients with stable mild and moderate asthma have increased epithelial mucin stores (5). Previous human, animal model, and cell culture studies demonstrate that the Th2 cytokine IL-13 is a critical inducer of airway epithelial abnormalities in asthma (6-8). IL-13 stimulation of epithelial cells leads to an increase in the number of mucous cells (mucous metaplasia) and a decrease in the number of ciliated cells (9). IL-13 stimulation of airway epithelial cells activates signal transduction and transcription factor 6, leading to changes in expression of mRNAs that...

Background Previously, we found that mast cell tryptases and carboxypeptidase A3 (CPA3) are differentially expressed in the airway epithelium in asthmatic subjects. We also found that asthmatic subjects can be divided into 2 subgroups (“TH2 high” and “TH2 low” asthma) based on epithelial cell gene signatures for the activity of TH2 cytokines. Objectives We sought to characterize intraepithelial mast cells (IEMCs) in asthma. Methods We performed gene expression profiling in epithelial brushings and stereology-based quantification of mast cell numbers in endobronchial biopsy specimens from healthy control and asthmatic subjects before and after treatment with inhaled corticosteroids (ICSs). We also performed gene expression and protein quantification studies in cultured airway epithelial cells and mast cells. Results By means of unsupervised clustering, mast cell gene expression in the airway epithelium related closely to the expression of IL-13 signature genes. The levels of expression of mast cell genes correlate positively with lung function improvements with ICSs. IEMC density was 2-fold higher than normal in subjects with TH2-high asthma compared with that seen in subjects with TH2-low asthma or healthy control subjects (P = .015 for both comparisons), and these cells were characterized by expression of tryptases and CPA3 but not chymase. IL-13 induced expression of stem cell factor in cultured airway epithelial cells, and mast cells exposed to conditioned media from IL-13–activated epithelial cells showed downregulation of chymase but no change in tryptase or CPA3 expression. Conclusion IEMC numbers are increased in subjects with TH2-high asthma, have an unusual protease phenotype (tryptase and CPA3 high and chymase low), and predict responsiveness to ICSs. IL-13–stimulated production of stem cell factor by epithelial cells potentially explains mast cell accumulation in TH2-high asthmatic epithelium.

Background-Chitinolytic enzymes play important roles in the pathophysiology of allergic airway responses in mouse models of asthma. Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) have chitinolytic activity, but relatively little is known about their expression in human asthma.