Immune mechanisms in fibrotic interstitial lung disease.

Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: (1) longitudinal studies emphasizing early-stage clinical disease, (2) immune mechanisms of acute exacerbations, and (3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

Leukemia inhibitory factor (LIF) receptor amplifies pathogenic activation of fibroblasts in lung fibrosis.

Fibrosis drives end-organ damage in many diseases. However, clinical trials targeting individual upstream activators of fibroblasts, such as TGFß, have largely failed. Here, we target the leukemia inhibitory factor receptor (LIFR) as a "master amplifier" of multiple upstream activators of lung fibroblasts. In idiopathic pulmonary fibrosis (IPF), the most common fibrotic lung disease, we found that lung myofibroblasts had high LIF expression. Further, TGFß1, one of the key drivers of fibrosis, upregulated LIF expression in IPF fibroblasts. In vitro anti-LIFR antibody blocking on human IPF lung fibroblasts reduced induction of profibrotic genes downstream of TGFß1, IL-4 and IL-13. Further, siRNA silencing of LIFR in IPF precision cut lung slices reduced expression of fibrotic proteins. Together, we find that LIFR drives an autocrine positive feedback loop that amplifies and sustains pathogenic activation of IPF fibroblasts downstream of multiple external stimuli, implicating LIFR as a therapeutic target in fibrosis. Significance Statement: Fibroblasts have a central role in the pathogenesis of fibrotic diseases. However, due to in part to multiple profibrotic stimuli, targeting a single activator of fibroblasts, like TGFß, has not yielded successful clinical treatments. We hypothesized that a more effective therapeutic strategy is identifying a downstream "master amplifier" of a range of upstream profibrotic stimuli. This study identifies the leukemia inhibitory factor receptor (LIFR) on fibrotic lung fibroblasts amplifies multiple profibrotic stimuli, such as IL-13 and TGFß. Blocking LIFR reduced fibrosis in ex vivo lung tissue from patients with idiopathic pulmonary fibrosis (IPF). LIFR, acting as a master amplifier downstream of fibroblast activation, offers an alternative therapeutic strategy for fibrotic diseases.

Protocol for immunophenotyping out-of-hospital cardiac arrest patients.

Immunophenotyping of out-of-hospital cardiac arrest (OHCA) patients is of increasing interest but has challenges. Here, we describe steps for the design of the clinical cohort, planning patient enrollment and sample collection, and ethical review of the study protocol. We detail procedures for blood sample collection and cryopreservation of peripheral blood mononuclear cells (PBMCs). We detail steps to modulate immune checkpoints in OHCA PBMC ex vivo. This protocol also has relevance for immunophenotyping other types of critical illness. For complete details on the use and execution of this protocol, please refer to Tamura et al. (2023).1.

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.

Single-cell transcriptomics reveal a hyperacute cytokine and immune checkpoint axis after cardiac arrest in patients with poor neurological outcome.

BACKGROUND: Most patients hospitalized after cardiac arrest (CA) die because of neurological injury. The systemic inflammatory response after CA is associated with neurological injury and mortality but remains poorly defined. METHODS: We determine the innate immune network induced by clinical CA at single-cell resolution. FINDINGS: Immune cell states diverge as early as 6 h post-CA between patients with good or poor neurological outcomes 30 days after CA. Nectin-2+ monocyte and Tim-3+ natural killer (NK) cell subpopulations are associated with poor outcomes, and interactome analysis highlights their crosstalk via cytokines and immune checkpoints. Ex vivo studies of peripheral blood cells from CA patients demonstrate that immune checkpoints are a compensatory mechanism against inflammation after CA. Interferon γ (IFNγ)/interleukin-10 (IL-10) induced Nectin-2 on monocytes; in a negative feedback loop, Nectin-2 suppresses IFNγ production by NK cells. CONCLUSIONS: The initial hours after CA may represent a window for therapeutic intervention in the resolution of inflammation via immune checkpoints. FUNDING: This work was supported by funding from the American Heart Association, Brigham and Women's Hospital Department of Medicine, the Evergreen Innovation Fund, and the National Institutes of Health.

Treating COVID-19: Evolving approaches to evidence in a pandemic.

The rapid pace of the COVID-19 pandemic precluded traditional approaches to evaluating clinical research and guidelines. We highlight notable successes and pitfalls of clinicians' new approaches to managing evidence amidst an unprecedented crisis. In "Era 1" (early 2020), clinicians relied on anecdote and social media, which democratized conversations on guidelines, but also led clinicians astray. "Era 2" (approximately late 2020 to early 2021) saw preprints that accelerated new interventions but suffered from a surfeit of poor-quality data. In the current era, clinicians consolidate the evidentiary gains of Era 2 with living, online clinical guidelines, but the public suffers from misinformation. The COVID-19 pandemic is a laboratory on how clinicians adapt to an absence of clinical guidance amidst an informational and healthcare crisis. Challenges remain as we integrate new approaches to innovations made in the traditional guideline process to confront both the long tail of COVID-19 and future pandemics.

Protocol to assess performance of crisis standards of care guidelines for clinical triage.

During the COVID-19 pandemic, US states developed Crisis Standards of Care (CSC) algorithms to triage allocation of scarce resources to maximize population-wide benefit. While CSC algorithms were developed by ethical debate, this protocol guides their quantitative assessment. For CSC algorithms, this protocol addresses (1) adapting algorithms for empirical study, (2) quantifying predictive accuracy, and (3) simulating clinical decision-making. This protocol provides a framework for healthcare systems and governments to test the performance of CSC algorithms to ensure they meet their stated ethical goals. For complete details on the use and execution of this protocol, please refer to Jezmir et al. (2021).

Combating information chaos: a case for collaborative clinical guidelines in a pandemic.

The speed and scale of new information during the COVID-19 pandemic required a new approach toward developing best practices and evidence-based clinical guidance. To address this need, we produced COVIDProtocols.org, a collaborative, evidence-based, digital platform for the development and dissemination of COVID-19 clinical guidelines that has been used by over 500,000 people from 196 countries. We use a Collaborative Writing Application (CWA) to facilitate an expedited expert review process and a web platform that deploys content directly from the CWA to minimize any delays. Over 200 contributors have volunteered to create open creative-commons content that spans over 30 specialties and medical disciplines. Multiple local and national governments, hospitals, and clinics have used the site as a key resource for their own clinical guideline development. COVIDprotocols.org represents a model for efficiently launching open-access clinical guidelines during crisis situations to share expertise and combat misinformation.

Performance of crisis standards of care guidelines in a cohort of critically ill COVID-19 patients in the United States.

Many US states published crisis standards of care (CSC) guidelines for allocating scarce critical care resources during the COVID-19 pandemic. However, the performance of these guidelines in maximizing their population benefit has not been well tested. In 2,272 adults with COVID-19 requiring mechanical ventilation drawn from the Study of the Treatment and Outcomes in Critically Ill Patients with COVID-19 (STOP-COVID) multicenter cohort, we test the following three approaches to CSC algorithms: Sequential Organ Failure Assessment (SOFA) scores grouped into ranges, SOFA score ranges plus comorbidities, and a hypothetical approach using raw SOFA scores not grouped into ranges. We find that area under receiver operating characteristic (AUROC) curves for all three algorithms demonstrate only modest discrimination for 28-day mortality. Adding comorbidity scoring modestly improves algorithm performance over SOFA scores alone. The algorithm incorporating comorbidities has modestly worse predictive performance for Black compared to white patients. CSC algorithms should be empirically examined to refine approaches to the allocation of scarce resources during pandemics and to avoid potential exacerbation of racial inequities.

Protocol for assessing and predicting acute respiratory decline in hospitalized patients.

This protocol aids both new and experienced researchers in designing retrospective clinical and translational studies of acute respiratory decline in hospitalized patients. This protocol addresses (1) the basics of respiratory failure and electronic health record research, (2) defining patient cohorts as "mild, progressive, or severe" instead of "ICU versus non-ICU", (3) adapting physiological indices, and (4) using biomarker trends. We apply these approaches to inflammatory biomarkers in COVID-19, but this protocol can be applied to any progressive respiratory failure study. For complete details on the use and execution of this protocol, please refer to Mueller et al. (2020).

Simulated laparoscopic sigmoidectomy training: responsiveness of surgery residents.

PURPOSE: This study aimed to evaluate the responsiveness of surgery residents to simulated laparoscopic sigmoidectomy training. METHODS: Residents underwent simulated laparoscopic sigmoidectomy training for previously tattooed sigmoid cancer with use of disposable abdominal trays in a hybrid simulator to perform a seven-step standardized technique. After baseline testing and training, residents were tested with predetermined proficiency criteria. Content validity was defined as the extent to which outcome measures departed from clinical reality. Content-valid measures of trays were evaluated by two blinded raters. Simulator-generated metrics included path length and smoothness of instrument movements. Responsiveness was defined as change in performance over time and was assessed by comparing baseline testing with unmentored final testing. RESULTS: For eight weeks, eight postgraduate year 3/4 residents performed 34 resections. Overall operating time (67 vs. 37 min; P = 0.005), flexure (10 vs. 5 min; P = 0.005), inferior mesenteric vessel (8 vs. 5 min; P = 0.04), and ureter (7 vs. 1 min; P = 0.003) times improved significantly. Content-valid measures from trays remained unchanged. Path length (27,155.2 mm) and smoothness (3,575.5 cm/s3) of instrument movement remained unchanged. There were two bowel perforations and 19 anastomotic leaks. Leak rate decreased from 87% to 12.5%. Strong correlation was found between path length and smoothness of instrument movements (r = 0.9; P < 0.001). There was no correlation between simulator-generated metrics and content-valid outcome measures. Interrater reliability was 1.0 for all measures except anastomotic leak (k = 0.56). There was a linear relationship between residents' clinical advanced laparoscopic case volume and responsiveness (r = -0.7; P = 0.04). CONCLUSIONS: Simulated laparoscopic sigmoidectomy training affected responsiveness in surgery residents with significantly decreased operating time and anastomotic leak rate.

Risk factors for conversion of laparoscopic to open cholecystectomy.

BACKGROUND: Laparoscopic cholecystectomy (LC) has become the treatment of choice for symptomatic gallstones; however conversion to open cholecystectomy (OC) remains a possibility. Unfortunately, preoperative factors indicating risk of conversion are unclear. Therefore, we aimed to identify risk factors associated with conversion of LC to OC. PATIENTS AND MATERIALS: Records of 564 patients undergoing LC in 1995 and 1996 were reviewed. Patients were assigned to one of two groups: (1) acute cholecystitis defined by the presence of gallstones, fever, leukocyte count >10(4), and inflammation on ultrasound or histology; (2) chronic cholecystitis that included all other symptomatic patients. Demographics, history, and physical, laboratory, and radiology data, operative note, and the pathology report were reviewed. RESULTS: 161 of 564 patients, had acute and 403 patients had chronic cholecystitis; 16 acute cholecystitis patients (10%) were converted from LC to OC and 17 chronic cholecystitis patients (4%) had LC converted to OC. Patients having open conversion were significantly older, had greater prevalence of cardiovascular disease, and were more likely to be males. LC conversion to OC in acute cholecystitis patients was associated with a greater leukocyte count; in gangrenous cholecystitis patients, 29% had open conversion. CONCLUSIONS: Importantly, these risk factors-older men, presence of cardiovascular disease, male gender, acute cholecystitis, and severe inflammation-are determined preoperatively, permitting the surgeon to better inform patients about the conversion risk from LC to OC. While acute cholecystitis was associated with more than a twofold increased conversion rate, only 10% of these patients could not be completed laparoscopically. Therefore, acute cholecystitis alone should not preclude an attempt at laparoscopic cholecystectomy.