Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis.

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

Mechanosensing regulates tissue repair program in macrophages.

Tissue-resident macrophages play important roles in tissue homeostasis and repair. However, how macrophages monitor and maintain tissue integrity is not well understood. The extracellular matrix (ECM) is a key structural and organizational component of all tissues. Here, we find that macrophages sense the mechanical properties of the ECM to regulate a specific tissue repair program. We show that macrophage mechanosensing is mediated by cytoskeletal remodeling and can be performed in three-dimensional environments through a noncanonical, integrin-independent mechanism analogous to amoeboid migration. We find that these cytoskeletal dynamics also integrate biochemical signaling by colony-stimulating factor 1 and ultimately regulate chromatin accessibility to control the mechanosensitive gene expression program. This study identifies an "amoeboid" mode of ECM mechanosensing through which macrophages may regulate tissue repair and fibrosis.

Microenvironmental sensing by fibroblasts controls macrophage population size.

Animal tissues comprise diverse cell types. However, the mechanisms controlling the number of each cell type within tissue compartments remain poorly understood. Here, we report that different cell types utilize distinct strategies to control population numbers. Proliferation of fibroblasts, stromal cells important for tissue integrity, is limited by space availability. In contrast, proliferation of macrophages, innate immune cells involved in defense, repair, and homeostasis, is constrained by growth factor availability. Examination of density-dependent gene expression in fibroblasts revealed that Hippo and TGF-ß target genes are both regulated by cell density. We found YAP1, the transcriptional coactivator of the Hippo signaling pathway, directly regulates expression of Csf1, the lineage-specific growth factor for macrophages, through an enhancer of Csf1 that is specifically active in fibroblasts. Activation of YAP1 in fibroblasts elevates Csf1 expression and is sufficient to increase the number of macrophages at steady state. Our data also suggest that expression programs in fibroblasts that change with density may result from sensing of mechanical force through actin-dependent mechanisms. Altogether, we demonstrate that two different modes of population control are connected and coordinated to regulate cell numbers of distinct cell types. Sensing of the tissue environment may serve as a general strategy to control tissue composition.

Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

Tissue Homeostasis and Inflammation.

There is a growing interest in understanding tissue organization, homeostasis, and inflammation. However, despite an abundance of data, the organizing principles of tissue biology remain poorly defined. Here, we present a perspective on tissue organization based on the relationships between cell types and the functions that they perform. We provide a formal definition of tissue homeostasis as a collection of circuits that regulate specific variables within the tissue environment, and we describe how the functional organization of tissues allows for the maintenance of both tissue and systemic homeostasis. This leads to a natural definition of inflammation as a response to deviations from homeostasis that cannot be reversed by homeostatic mechanisms alone. We describe how inflammatory signals act on the same cellular functions involved in normal tissue organization and homeostasis in order to coordinate emergency responses to perturbations and ultimately return the system to a homeostatic state. Finally, we consider the hierarchy of homeostatic and inflammatory circuits and the implications for the development of inflammatory diseases.

A neutrophil activation signature predicts critical illness and mortality in COVID-19.

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation.

Intermediate-dose anticoagulation, aspirin, and in-hospital mortality in COVID-19: a propensity score-matched analysis.

Background: Thrombotic complications occur at high rates in hospitalized patients with COVID-19, yet the impact of intensive antithrombotic therapy on mortality is uncertain. Research Question: How does in-hospital mortality compare with intermediate- versus prophylactic-dose anticoagulation, and separately with in-hospital aspirin versus no antiplatelet therapy, in treatment of COVID-19? Study Design and Methods: Using data from 2785 hospitalized adult COVID-19 patients, we established two separate, nested cohorts of patients (1) who received intermediate- or prophylactic-dose anticoagulation ("anticoagulation cohort", N = 1624), or (2) who were not on home antiplatelet therapy and received either in-hospital aspirin or no antiplatelet therapy ("aspirin cohort", N = 1956). Propensity score matching utilizing various markers of illness severity and other patient-specific covariates yielded treatment groups with well-balanced covariates in each cohort. The primary outcome was cumulative incidence of in-hospital death. Results: Among propensity score-matched patients in the anticoagulation cohort (N = 382), in a multivariable regression model, intermediate- compared to prophylactic-dose anticoagulation was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.518 [0.308-0.872]). Among propensity-score matched patients in the aspirin cohort (N = 638), in a multivariable regression model, in-hospital aspirin compared to no antiplatelet therapy was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.522 [0.336-0.812]). Interpretation: In this propensity score-matched, observational study of COVID-19, intermediate-dose anticoagulation and aspirin were each associated with a lower cumulative incidence of in-hospital death.

Intermediate-dose anticoagulation, aspirin, and in-hospital mortality in COVID-19: A propensity score-matched analysis.

Thrombotic complications occur at high rates in hospitalized patients with COVID-19, yet the impact of intensive antithrombotic therapy on mortality is uncertain. We examined in-hospital mortality with intermediate- compared to prophylactic-dose anticoagulation, and separately with in-hospital aspirin compared to no antiplatelet therapy, in a large, retrospective study of 2785 hospitalized adult COVID-19 patients. In this analysis, we established two separate, nested cohorts of patients (a) who received intermediate- or prophylactic-dose anticoagulation ("anticoagulation cohort", N = 1624), or (b) who were not on home antiplatelet therapy and received either in-hospital aspirin or no antiplatelet therapy ("aspirin cohort", N = 1956). To minimize bias and adjust for confounding factors, we incorporated propensity score matching and multivariable regression utilizing various markers of illness severity and other patient-specific covariates, yielding treatment groups with well-balanced covariates in each cohort. The primary outcome was cumulative incidence of in-hospital death. Among propensity score-matched patients in the anticoagulation cohort (N = 382), in a multivariable regression model, intermediate- compared to prophylactic-dose anticoagulation was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.518 [0.308-0.872]). Among propensity-score matched patients in the aspirin cohort (N = 638), in a multivariable regression model, in-hospital aspirin compared to no antiplatelet therapy was associated with a significantly lower cumulative incidence of in-hospital death (hazard ratio 0.522 [0.336-0.812]). In this propensity score-matched, observational study of COVID-19, intermediate-dose anticoagulation and aspirin were each associated with a lower cumulative incidence of in-hospital death.

Circulating markers of angiogenesis and endotheliopathy in COVID-19.

Increase in thrombotic and microvascular complications is emerging to be a key feature of patients with critical illness associated with COVID-19 infection. While endotheliopathy is thought to be a key factor of COVID-19-associated coagulopathy, markers indicative of this process that are prognostic of disease severity have not been well-established in this patient population. Using plasma profiling of patients with COVID-19, we identified circulating markers that segregated with disease severity: markers of angiogenesis (VEGF-A, PDGF-AA and PDGF-AB/BB) were elevated in hospitalized patients with non-critical COVID-19 infection, while markers of endothelial injury (angiopoietin-2, FLT-3L, PAI-1) were elevated in patients with critical COVID-19 infection. In survival analysis, elevated markers of endothelial injury (angiopoietin-2, follistatin, PAI-1) were strongly predictive of in-hospital mortality. Our findings demonstrate that non-critical and critical phases of COVID-19 disease may be driven by distinct mechanisms involving key aspects of endothelial cell function, and identify drivers of COVID-19 pathogenesis and potential targets for future therapies.

A neutrophil activation signature predicts critical illness and mortality in COVID-19.

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of over 3,300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, HGF, IL-8, and G-CSF, as the strongest predictors of critical illness. Neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, we define an essential role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular neutrophil markers that distinguish patients at risk of future clinical decompensation.

Circulating Markers of Angiogenesis and Endotheliopathy in COVID-19.

Despite over 9.3 million infected and 479,000 deaths, the pathophysiological factors that determine the wide spectrum of clinical outcomes in COVID-19 remain inadequately defined. Importantly, patients with underlying cardiovascular disease have been found to have worse clinical outcomes,1 and autopsy findings of endotheliopathy as well as angiogenesis in COVID-19 have accumulated.2,3 Nonetheless, circulating vascular markers associated with disease severity and mortality have not been reliably established. To address this limitation and better understand COVID-19 pathogenesis, we report plasma profiling of factors related to the vascular system from a series of patients admitted to Yale-New Haven Hospital with confirmed diagnosis of COVID-19 via PCR, which demonstrate significant increase in markers of angiogenesis and endotheliopathy in patients hospitalized with COVID-19.

Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, cross-sectional study.

BACKGROUND: An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19. METHODS: In this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan-Meier analysis was used to further explore the association between biochemical markers and survival. FINDINGS: 68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0·0001) and soluble P-selectin (15·9 ng/mL [4·8] vs 11·2 ng/mL [3·1]; p=0·0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan-Meier analysis (hazard ratio 5·9, 95% CI 1·9-18·4; p=0·0087). INTERPRETATION: Our findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19. FUNDING: This work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health.

Principles of Cell Circuits for Tissue Repair and Fibrosis.

Tissue repair is a protective response after injury, but repetitive or prolonged injury can lead to fibrosis, a pathological state of excessive scarring. To pinpoint the dynamic mechanisms underlying fibrosis, it is important to understand the principles of the cell circuits that carry out tissue repair. In this study, we establish a cell-circuit framework for the myofibroblast-macrophage circuit in wound healing, including the accumulation of scar-forming extracellular matrix. We find that fibrosis results from multistability between three outcomes, which we term "hot fibrosis" characterized by many macrophages, "cold fibrosis" lacking macrophages, and normal wound healing. This framework clarifies several unexplained phenomena including the paradoxical effect of macrophage depletion, the limited time-window in which removing inflammation leads to healing, and why scar maturation takes months. We define key parameters that control the transition from healing to fibrosis, which may serve as potential targets for therapeutic reduction of fibrosis.

Changes in inflammatory and immune drivers in response to immunomodulatory therapies in COVID-19.

As the global community strives to discover effective therapies for COVID-19, immunomodulatory strategies have emerged as a leading contender to combat the cytokine storm and improve clinical outcomes in patients with severe disease. Systemic corticosteroids and selective cytokine inhibitory agents have been utilized both as empiric therapies and in clinical trials. While multiple randomized, placebo controlled trials have now demonstrated that corticosteroids improve survival in patients with COVID-19,1, 2 IL-6 inhibition, which gained significant early interest based on observational studies, has not demonstrated reliable efficacy in randomized, placebo controlled trials.3, 4 To better understand the mechanistic basis of immunomodulatory therapies being implemented for treatment of COVID-19, we assessed longitudinal biochemical changes in response to such approaches in hospitalized patients with COVID-19. We demonstrate broad suppression of multiple immunomodulatory factors associated with adverse clinical outcomes in COVID-19 in patients who received corticosteroids, but no such response was seen in patients who either received tocilizumab or no immunomodulatory therapy. Our findings provide early insights into molecular signatures that correlate with immunomodulatory therapies in COVID-19 which may be useful in understanding clinical outcomes in future studies of larger patient cohorts.

No Time for Silence: An Urgent Need for Political Activism Among the Medical Community.

Despite being a major stakeholder in the U.S. health care system, the medical community has remained relatively mute in the debate over the future of the Patient Protection and Affordable Care Act (ACA). If the ACA were repealed, tens of millions of Americans would be in danger of losing their insurance, resulting in a significant increase in mortality. Because misinformation about the ACA is rampant, it is imperative that health care providers explain to the public what exactly the ACA is and how repeal will affect patients. Traditionally, many in the medical community have abstained from political advocacy for multiple reasons, including compromising the doctor-patient relationship, financial incentives, lack of experience with activism due to an absence of training in that area, and fear of political retaliation. Encouragingly, there are indications that the medical community is beginning to become more vocal. Medical students are one example, having formed a grassroots response to repeal. However, students need more guidance and support from experienced mentors to most effectively serve as patient advocates. This is no time for silence: On this life-or-death issue, the medical community cannot afford to remain mute.

The immune cell infiltrate populating meningiomas is composed of mature, antigen-experienced T and B cells.

BACKGROUND: Meningiomas often harbor an immune cell infiltrate that can include substantial numbers of T and B cells. However, their phenotype and characteristics remain undefined. To gain a deeper understanding of the T and B cell repertoire in this tumor, we characterized the immune infiltrate of 28 resected meningiomas representing all grades. METHODS: Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes. A molecular analysis of the immunoglobulin variable region of tumor-infiltrating B cells was used to characterize their antigen experience. Flow cytometry of fresh tissue homogenate and paired peripheral blood lymphocytes was used to identify T cell phenotypes and characterize the T cell repertoire. RESULTS: A conspicuous B and T cell infiltrate, primarily clustered in perivascular spaces, was present in the microenvironment of most tumors examined. Characterization of 294 tumor-infiltrating B cells revealed clear evidence of antigen experience, in that the cardinal features of an antigen-driven B cell response were present. Meningiomas harbored populations of antigen-experienced CD4+ and CD8+ memory/effector T cells, regulatory T cells, and T cells expressing the immune checkpoint molecules PD-1 and Tim-3, indicative of exhaustion. All of these phenotypes were considerably enriched relative to their frequency in the circulation. The T cell repertoire in the tumor microenvironment included populations that were not reflected in paired peripheral blood. CONCLUSION: The tumor microenvironment of meningiomas often includes postgerminal center B cell populations. These tumors invariably include a selected, antigen-experienced, effector T cell population enriched by those that express markers of an exhausted phenotype.