Aberrant long-chain fatty acid metabolism associated with evolving systemic sclerosis-associated pulmonary arterial hypertension.

We sought to investigate differential metabolism in patients with systemic sclerosis (SSc) who develop pulmonary arterial hypertension (PAH) versus those who do not, as a method of identifying potential disease biomarkers. In a nested case-control design, serum metabolites were assayed in SSc subjects who developed right heart catheterization-confirmed PAH (n = 22) while under surveillance in a longitudinal cohort from Johns Hopkins, then compared with metabolites assayed in matched SSc patients who did not develop PAH (n = 22). Serum samples were collected at "proximate" (within 12 months) and "distant" (within 1-5 yr) time points relative to PAH diagnosis. Metabolites were identified using liquid chromatography-mass spectroscopy (LC-MS). An LC-MS dataset from SSc subjects with either mildly elevated pulmonary pressures or overt PAH from the University of Michigan was compared. Differentially abundant metabolites were tested as predictors of PAH in two additional validation SSc cohorts. Long-chain fatty acid metabolism (LCFA) consistently differed in SSc-PAH versus SSc without PH. LCFA metabolites discriminated SSc-PAH patients with mildly elevated pressures in the Michigan cohort and predicted SSc-PAH up to 2 yr before clinical diagnosis in the Hopkins cohort. Acylcholines containing LCFA residues and linoleic acid metabolites were most important for discriminating SSc-PAH. Combinations of acylcholines and linoleic acid metabolites provided good discrimination of SSc-PAH across cohorts. Aberrant lipid metabolism is observed throughout the evolution of PAH in SSc. Lipidomic signatures of abnormal LCFA metabolism distinguish SSc-PAH patients from those without PH, including before clinical diagnosis and in mild disease.NEW & NOTEWORTHY Abnormal lipid metabolism is evident across time in the development of SSc-PAH, and dysregulated long-chain fatty acid metabolism predicts overt PAH.

A novel interaction between aquaporin 1 and caspase-3 in pulmonary arterial smooth muscle cells.

Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into nonmuscular arteries. These pathological changes are predominantly due to the abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin 1 (AQP1). However, the mechanisms underlying the increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its C-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both human embryonic kidney (HEK) 293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition, respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics.NEW & NOTEWORTHY Pulmonary arterial smooth muscle cells (PASMCs) are integral to pulmonary vascular remodeling, a characteristic of pulmonary arterial hypertension (PAH). PASMCs isolated from robust animal models of disease demonstrate enhanced proliferation and migration, pathological functions associated with increased abundance of the membrane protein aquaporin 1 (AQP1). We present evidence of a novel interaction between the proteolytic enzyme caspase-3 and AQP1, which may control AQP1 abundance. These data suggest a potential new target for novel PAH therapies.

Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Pulmonary toxicity of immune checkpoint immunotherapy.

Cancer remains a leading cause of mortality on a global scale. Lung cancer, specifically non-small cell lung cancer (NSCLC), is a prominent contributor to this burden. The management of NSCLC has advanced substantially in recent years, with immunotherapeutic agents, such as immune checkpoint inhibitors (ICIs), leading to improved patient outcomes. Although generally well tolerated, the administration of ICIs can result in unique side effects known as immune-related adverse events (irAEs). The occurrence of irAEs involving the lungs, specifically checkpoint inhibitor pneumonitis (CIP), can have a profound effect on both future therapy options and overall survival. Despite CIP being one of the more common serious irAEs, limited treatment options are currently available, in part due to a lack of understanding of the underlying mechanisms involved in its development. In this Review, we aim to provide an overview of the epidemiology and clinical characteristics of CIP, followed by an examination of the emerging literature on the pathobiology of this condition.

Spatial and temporal resolution of metabolic dysregulation in the Sugen hypoxia model of pulmonary hypertension.

Although PAH is partially attributed to disordered metabolism, previous human studies have mostly examined circulating metabolites at a single time point, potentially overlooking crucial disease biology. Current knowledge gaps include an understanding of temporal changes that occur within and across relevant tissues, and whether observed metabolic changes might contribute to disease pathobiology. We utilized targeted tissue metabolomics in the Sugen hypoxia (SuHx) rodent model to investigate tissue-specific metabolic relationships with pulmonary hypertensive features over time using regression modeling and time-series analysis. Our hypotheses were that some metabolic changes would precede phenotypic changes, and that examining metabolic interactions across heart, lung, and liver tissues would yield insight into interconnected metabolic mechanisms. To support the relevance of our findings, we sought to establish links between SuHx tissue metabolomics and human PAH -omics data using bioinformatic predictions. Metabolic differences between and within tissue types were evident by Day 7 postinduction, demonstrating distinct tissue-specific metabolism in experimental pulmonary hypertension. Various metabolites demonstrated significant tissue-specific associations with hemodynamics and RV remodeling. Individual metabolite profiles were dynamic, and some metabolic shifts temporally preceded the emergence of overt pulmonary hypertension and RV remodeling. Metabolic interactions were observed such that abundance of several liver metabolites modulated lung and RV metabolite-phenotype relationships. Taken all together, regression analyses, pathway analyses and time-series analyses implicated aspartate and glutamate signaling and transport, glycine homeostasis, lung nucleotide abundance, and oxidative stress as relevant to early PAH pathobiology. These findings offer valuable insights into potential targets for early intervention in PAH.

Metabolic profiling of in vivo right ventricular function and exercise performance in pulmonary arterial hypertension.

Right ventricular (RV) adaptation is the principal determinant of outcomes in pulmonary arterial hypertension (PAH), however, RV function is challenging to assess. RV responses to hemodynamic stressors are particularly difficult to interrogate without invasive testing. This study sought to identify metabolomic markers of in vivo right ventricular function and exercise performance in PAH. Consecutive subjects with PAH (n = 23) underwent rest and exercise right heart catheterization with multibeat pressure volume loop analysis. Pulmonary arterial blood was collected at rest and during exercise. Mass spectrometry-based targeted metabolomics were performed, and metabolic associations with hemodynamics and comprehensive measures of RV function were determined using sparse partial least squares regression. Metabolite profiles were compared with N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) measurements for accuracy in modeling ventriculo-arterial parameters. Thirteen metabolites changed in abundance with exercise, including metabolites reflecting increased arginine bioavailability, precursors of catecholamine and nucleotide synthesis, and branched-chain amino acids. Higher resting arginine bioavailability predicted more favorable exercise hemodynamics and pressure-flow relationships. Subjects with more severe PAH augmented arginine bioavailability with exercise to a greater extent than subjects with less severe PAH. We identified relationships between kynurenine pathway metabolism and impaired ventriculo-arterial coupling, worse RV diastolic function, lower RV contractility, diminished RV contractility with exercise, and RV dilation with exercise. Metabolite profiles outperformed NT-proBNP in modeling RV contractility, diastolic function, and exercise performance. Specific metabolite profiles correspond to RV functional measurements only obtainable via invasive pressure-volume loop analysis and predict RV responses to exercise. Metabolic profiling may inform discovery of RV functional biomarkers.NEW & NOTEWORTHY In this cohort of patients with pulmonary arterial hypertension (PAH), we investigate metabolomic associations with comprehensive right ventricular (RV) functional measurements derived from multibeat RV pressure-volume loop analysis. Our results show that tryptophan metabolism, particularly the kynurenine pathway, is linked to intrinsic RV function and PAH pathobiology. Findings also highlight the importance of arginine bioavailability in the cardiopulmonary system's response to exercise stress. Metabolite profiles selected via unbiased analysis outperformed N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) in predicting load-independent measures of RV function at rest and cardiopulmonary system performance under stress. Overall, this work suggests the potential for select metabolites to function as disease-specific biomarkers, offers insights into PAH pathobiology, and informs discovery of potentially targetable RV-centric pathways.

Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.

In pulmonary artery hypertension (PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids-BCAA) are lower in the pre-capillary (i.e., RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx-MVECs; (3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.

Tumor MK2 transcript levels are associated with improved response to chemotherapy and patient survival in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

Society for Immunotherapy of Cancer (SITC) consensus definitions for immune checkpoint inhibitor-associated immune-related adverse events (irAEs) terminology.

Immune-related adverse events (irAEs) associated with immune checkpoint inhibitor (ICI) therapy may vary substantially in their clinical presentation, including natural history, outcomes to treatment, and patterns. The application of clinical guidelines for irAE management can be challenging for practitioners due to a lack of common or consistently applied terminology. Furthermore, given the growing body of clinical experience and published data on irAEs, there is a greater appreciation for the heterogeneous natural histories, responses to treatment, and patterns of these toxicities, which is not currently reflected in irAE guidelines. Furthermore, there are no prospective trial data to inform the management of the distinct presentations of irAEs. Recognizing a need for uniform terminology for the natural history, response to treatment, and patterns of irAEs, the Society for Immunotherapy of Cancer (SITC) convened a consensus panel composed of leading international experts from academic medicine, industry, and regulatory agencies. Using a modified Delphi consensus process, the expert panel developed clinical definitions for irAE terminology used in the literature, encompassing terms related to irAE natural history (ie, re-emergent, chronic active, chronic inactive, delayed/late onset), response to treatment (ie, steroid unresponsive, steroid dependent), and patterns (ie, multisystem irAEs). SITC developed these definitions to support the adoption of a standardized vocabulary for irAEs, which will have implications for the uniform application of irAE clinical practice guidelines and to enable future irAE clinical trials.

MK2 nonenzymatically promotes nuclear translocation of caspase-3 and resultant apoptosis.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

Post hoc survival analyses using RNAseq data: handle with care.

With the advent of next-generation sequencing technologies, there has been a dramatic increase in the availability of paired clinical and transcriptomic data in a variety of disease states. For basic science researchers, this has provided a valuable opportunity for querying the impact of the transcript levels of a gene on disease survival in humans. However, there are a multitude of methodological and technical considerations to evaluate before embarking on these analyses. Herein, we provide a brief description of statistical considerations involved in these analyses, geared toward basic scientists who may not necessarily routinely use such statistical models as part of their studies.

Dexamethasone-Induced FKBP51 Expression in CD4+ T-Lymphocytes Is Uniquely Associated With Worse Asthma Control in Obese Children With Asthma.

Introduction: There is evidence that obesity, a risk factor for asthma severity and morbidity, has a unique asthma phenotype which is less atopic and less responsive to inhaled corticosteroids (ICS). Peripheral blood mononuclear cells (PBMC) are important to the immunologic pathways of obese asthma and steroid resistance. However, the cellular source associated with steroid resistance has remained elusive. We compared the lymphocyte landscape among obese children with asthma to matched normal weight children with asthma and assessed relationship to asthma control. Methods: High-dimensional flow cytometry of PBMC at baseline and after dexamethasone stimulation was performed to characterize lymphocyte subpopulations, T-lymphocyte polarization, proliferation (Ki-67+), and expression of the steroid-responsive protein FK506-binding protein 51 (FKBP51). T-lymphocyte populations were compared between obese and normal-weight participants, and an unbiased, unsupervised clustering analysis was performed. Differentially expressed clusters were compared with asthma control, adjusted for ICS and exhaled nitric oxide. Results: In the obese population, there was an increased cluster of CD4+ T-lymphocytes expressing Ki-67 and FKBP51 at baseline and CD4+ T-lymphocytes expressing FKBP51 after dexamethasone stimulation. CD4+ Ki-67 and FKBP51 expression at baseline showed no association with asthma control. Dexamethasone-induced CD4+ FKBP51 expression was associated with worse asthma control in obese participants with asthma. FKBP51 expression in CD8+ T cells and CD19+ B cells did not differ among groups, nor did polarization profiles for Th1, Th2, Th9, or Th17 percentage. Discussion: Dexamethasone-induced CD4+ FKBP51 expression is uniquely associated with worse asthma control in obese children with asthma and may underlie the corticosteroid resistance observed in this population.

Pretreatment Lung Function and Checkpoint Inhibitor Pneumonitis in NSCLC.

INTRODUCTION: Checkpoint inhibitor pneumonitis (CIP) is a serious toxicity of anti-programmed death-(ligand) 1 immunotherapy. Whether pretreatment differences in pulmonary function exist in patients who develop CIP is unknown. We analyzed the pulmonary function tests (PFTs) of patients with NSCLC treated with immune checkpoint inhibitors (ICIs) to evaluate whether pretreatment lung function was associated with CIP development. METHODS: Patients were included if they completed greater than or equal to 1 PFT within 2 years preceding ICI initiation. CIP status (CIP+: developed CIP, CIP-: did not develop CIP) was determined clinically. Generalized estimating equation-based linear regression was used to evaluate the effects of time and CIP on lung function. Primary outcomes included the following: percent-predicted forced expiratory volume in 1 second (FEV1pp), percent-predicted forced vital capacity (FVCpp), and FEV1/FVC. RESULTS: A total of 43 patients (34 CIP-, 9 CIP+) with 79 PFTs (59 CIP-, 20 CIP+) were included. CIP+ patients had a 21.7% lower pretreatment FEV1pp compared with the CIP- group (95% confidence interval: -38.6 to -4.7). No statistically significant differences in FVCpp or FEV1/FVC were observed. The prevalence of obstructive lung disease was similar in both groups at 67% and 62% for the CIP+ and CIP- cohorts, as was the prevalence of current/former smoking at 100% and 93%, respectively. CONCLUSIONS: Pretherapy differences in lung function were evident between patients who did and did not develop CIP, though the prevalence of obstructive lung disease was similar. Prospective studies are needed to validate these findings, inform potential risk factors for CIP, and investigate the effects of ICI treatment and CIP on pulmonary function in patients with NSCLC.

Comparison of polynomial fitting versus single time point analysis of ECIS data for barrier assessment.

Electrical cell-substrate impedance sensing (ECIS) is an in vitro methodology for measuring the barrier integrity of a variety of cell types, including pulmonary endothelial cells. These experiments are frequently used for in vitro assessment of lung injury. The data derived from ECIS experiments consists of repeated measures of resistance across an endothelial monolayer. As such, these data reflect the dynamic changes in electrical resistance that occur over time. Currently methodologies for assessing ECIS data rely on single point assessments of barrier function, such as the maximal drop in trans-endothelial electrical resistance (TERMax ). However, this approach ignores the myriad of changes in resistance that occur before and after the TERMax data point. Herein, we utilize polynomial curve fitting on experimentally generated ECIS data, thus allowing for comparing ECIS experiments by examining the mean polynomial coefficients between groups. We show that polynomial curves accurately fit a variety of ECIS data, and that concordance between TERMax and coefficient analysis varies by type of stimulus, suggesting that TERMax differences may not always correlate with a significant difference in the overall shape of the ECIS profile. Lastly, we identify factors that impact coefficient values obtained in our analyses, including the length of time devoted to baseline measurements before addition of stimuli. Polynomial coefficient analysis is another tool that can be used for more comprehensive interrogation of ECIS data to better understand the biological underpinnings that lead to changes in barrier dysfunction in vitro.

Acetazolamide prevents hypoxia-induced reactive oxygen species generation and calcium release in pulmonary arterial smooth muscle.

Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 µM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.

Steroid-refractory PD-(L)1 pneumonitis: incidence, clinical features, treatment, and outcomes.

BACKGROUND: Immune-checkpoint inhibitor (ICI)-pneumonitis that does not improve or resolve with corticosteroids and requires additional immunosuppression is termed steroid-refractory ICI-pneumonitis. Herein, we report the clinical features, management and outcomes for patients treated with intravenous immunoglobulin (IVIG), infliximab, or the combination of IVIG and infliximab for steroid-refractory ICI-pneumonitis. METHODS: Patients with steroid-refractory ICI-pneumonitis were identified between January 2011 and January 2020 at a tertiary academic center. ICI-pneumonitis was defined as clinical or radiographic lung inflammation without an alternative diagnosis, confirmed by a multidisciplinary team. Steroid-refractory ICI-pneumonitis was defined as lack of clinical improvement after high-dose corticosteroids for 48 hours, necessitating additional immunosuppression. Serial clinical, radiologic (CT imaging), and functional features (level-of-care, oxygen requirement) were collected preadditional and postadditional immunosuppression. RESULTS: Of 65 patients with ICI-pneumonitis, 18.5% (12/65) had steroid-refractory ICI-pneumonitis. Mean age at diagnosis of ICI-pneumonitis was 66.8 years (range: 35-85), 50% patients were male, and the majority had lung carcinoma (75%). Steroid-refractory ICI-pneumonitis occurred after a mean of 5 ICI doses from PD-(L)1 start (range: 3-12 doses). The most common radiologic pattern was diffuse alveolar damage (DAD: 50%, 6/12). After corticosteroid failure, patients were treated with: IVIG (n=7), infliximab (n=2), or combination IVIG and infliximab (n=3); 11/12 (91.7%) required ICU-level care and 8/12 (75%) died of steroid-refractory ICI-pneumonitis or infectious complications (IVIG alone=3/7, 42.9%; infliximab alone=2/2, 100%; IVIG + infliximab=3/3, 100%). All five patients treated with infliximab (5/5; 100%) died from steroid-refractory ICI-pneumonitis or infectious complications. Mechanical ventilation was required in 53% of patients treated with infliximab alone, 80% of those treated with IVIG + infliximab, and 25.5% of those treated with IVIG alone. CONCLUSIONS: Steroid-refractory ICI-pneumonitis constituted 18.5% of referrals for multidisciplinary irAE care. Steroid-refractory ICI-pnuemonitis occurred early in patients' treatment courses, and most commonly exhibited a DAD radiographic pattern. Patients treated with IVIG alone demonstrated an improvement in both level-of-care and oxygenation requirements and had fewer fatalities (43%) from steroid-refractory ICI-pneumonitis when compared to treatment with infliximab (100% mortality).

Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by exuberant vascular remodeling leading to elevated pulmonary arterial pressure, maladaptive right ventricular remodeling, and eventual death. The factors controlling pulmonary arterial smooth muscle cell (PASMC) and endothelial cell hyperplasia and migration, hallmark features of the vascular remodeling observed in PAH, remain poorly understood. We previously demonstrated that hypoxia upregulates the expression of aquaporin 1 (AQP1), a water channel, in PASMCs, and that this upregulation was required for hypoxia-induced migration and proliferation. However, whether the same is true in a model of severe PAH and in pulmonary microvascular endothelial cells (MVECs) is unknown. In this study, we used the SU5416 plus hypoxia (SuHx) rat model of severe pulmonary hypertension, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in PASMCs and MVECs and contributed to a hyperproliferative/hypermigratory phenotype. Rats received a single injection of SU5416 (20 mg/kg) and then were placed in 10% O2 for 3 weeks, followed by a return to normoxic conditions for an additional 2 weeks. We found that AQP1 protein levels were increased in both PASMCs and MVECs from SuHx rats, even in the absence of sustained hypoxic exposure, and that in MVECs, the increase in protein expression was associated with upregulation of AQP1 mRNA levels. Silencing of AQP1 had no significant effect on PASMCs from control animals but normalized enhanced migration and proliferation observed in cells from SuHx rats. Loss of AQP1 also reduced migration and proliferation in MVECs from SuHx rats. Finally, augmenting AQP1 levels in MVECs from control rats using forced expression was sufficient to increase migration and proliferation. These results demonstrate a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in pulmonary vascular cells from a rodent model that reflects many of the features of human PAH.

Estradiol resolves pneumonia via ERß in regulatory T cells.

Current treatments for pneumonia (PNA) are focused on the pathogens. Mortality from PNA-induced acute lung injury (PNA-ALI) remains high, underscoring the need for additional therapeutic targets. Clinical and experimental evidence exists for potential sex differences in PNA survival, with males having higher mortality. In a model of severe pneumococcal PNA, when compared with male mice, age-matched female mice exhibited enhanced resolution characterized by decreased alveolar and lung inflammation and increased numbers of Tregs. Recognizing the critical role of Tregs in lung injury resolution, we evaluated whether improved outcomes in female mice were due to estradiol (E2) effects on Treg biology. E2 promoted a Treg-suppressive phenotype in vitro and resolution of PNA in vivo. Systemic rescue administration of E2 promoted resolution of PNA in male mice independent of lung bacterial clearance. E2 augmented Treg expression of Foxp3, CD25, and GATA3, an effect that required ERß, and not ERα, signaling. Importantly, the in vivo therapeutic effects of E2 were lost in Treg-depleted mice (Foxp3DTR mice). Adoptive transfer of ex vivo E2-treated Tregs rescued Streptococcus pneumoniae-induce PNA-ALI, a salutary effect that required Treg ERß expression. E2/ERß was required for Tregs to control macrophage proinflammatory responses. Our findings support the therapeutic role for E2 in promoting resolution of lung inflammation after PNA via ERß Tregs.