BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.

Impaired Myofibroblast Proliferation is a Central Feature of Pathologic Post-Natal Alveolar Simplification.

Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFß) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFß signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFß signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.

IRE1α drives lung epithelial progenitor dysfunction to establish a niche for pulmonary fibrosis.

After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase-IRE1α-a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)-or conditional epithelial IRE1α gene knockout-both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvß6, a key activator of transforming growth factor ß (TGF-ß) in pulmonary fibrosis, corresponding to decreased TGF-ß-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19CreERT2-lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.

ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR.

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

Management and Outcomes of Critically-III Patients with COVID-19 Pneumonia at a Safety-net Hospital in San Francisco, a Region with Early Public Health Interventions: A Case Series.

Background: Following early implementation of public health measures, San Francisco has experienced a slow rise and a low peak level of coronavirus disease 2019 (COVID-19) cases and deaths. Methods and Findings: We included all patients with COVID-19 pneumonia admitted to the intensive care unit (ICU) at the safety net hospital for San Francisco through April 8, 2020. Each patient had ≥15 days of follow-up. Among 26 patients, the median age was 54 years (interquartile range, 43 to 62), 65% were men, and 77% were Latinx. Mechanical ventilation was initiated for 11 (42%) patients within 24 hours of ICU admission and 20 patients (77%) overall. The median duration of mechanical ventilation was 13.5 days (interquartile range, 5 to 20). Patients were managed with lung protective ventilation (tidal volume ≤8 ml/kg of ideal body weight and plateau pressure ≤30 cmH2O on 98% and 78% of ventilator days, respectively). Prone positioning was used for 13 of 20 (65%) ventilated patients for a median of 5 days (interquartile range, 2 to 10). Seventeen (65%) patients were discharged home, 1 (4%) was discharged to nursing home, 3 (12%) were discharged from the ICU, and 2 (8%) remain intubated in the ICU at the time of this report. Three (12%) patients have died. Conclusions: Good outcomes were achieved in critically ill patients with COVID-19 by using standard therapies for acute respiratory distress syndrome (ARDS) such as lung protective ventilation and prone positioning. Ensuring hospitals can deliver sustained high-quality and evidence-based critical care to patients with ARDS should remain a priority.

Small Molecules to Improve ER Proteostasis in Disease.

Abnormally high levels of misfolded proteins in the endoplasmic reticulum (ER) lumen result in a stress state that contributes to the progression of several pathological conditions including diabetes, cancer, neurodegeneration, and immune dysregulation. ER stress triggers a dynamic signaling pathway known as the unfolded protein response (UPR). The UPR enforces adaptive or cell death programs by integrating information about the intensity and duration of the stress stimuli. Thus, depending on the disease context, ER stress signaling can be beneficial or detrimental. We discuss current efforts to develop small molecules to target distinct components of the UPR, and their possible applications in treating human disease, focusing on neurodegenerative diseases, metabolic disorders, and cancer.

Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung.

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing.

To use microRNAs to downregulate mRNA targets, cells must first process these ~22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors.

FoxP3(+) regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes. NOD Tregs did show a defect in standard in vitro T cell suppression assays, particularly at low suppressor/effector ratios. Gene expression profiling revealed the vast majority of transcripts constituting the "Treg signature" to be normally distributed in NOD Tregs versus CD4(+) T conventional (Tconv) cells, although there were a few differences affecting one or the other population. According to results from criss-cross experiments, the functional inefficacy was not rooted in NOD Tregs, which suppressed as well as their C57BL/6 (B6) counterparts, but rather in NOD Tconv, which were less prone to suppression than were B6 Tconv cells. They also responded more effectively to anti-CD3/28 monoclonal antibody (mAb) stimulation in vitro or to a natural pancreatic antigen in vivo. This difference was independent of autoimmune inflammation, did not map to the idd3 region, and was not due to the overproduction of interleukin-21 in NOD mice. That the immune dysregulation in this T1D model is rooted in the ability of effector T cells to be regulated, rather than in Tregs themselves, has implications for proposed therapeutic interventions.

Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors.

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.

A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta 19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta F/F) approximately 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta F agreed well with those for epibatidine-induced currents, but were shifted approximately 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha gamma-binding site than to the alpha delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta F monitors an event linked to binding specifically at the alpha delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta F occurs within milliseconds, i.e., during activation. At low [ACh] (< or = 10 microM), a phase of Delta F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta F is complete before the slowest phase of recovery from desensitization (time constant approximately 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.