Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

BACKGROUND: Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. METHODS: Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. RESULTS: Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. CONCLUSIONS: We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Patient-Reported Outcomes in Calcium Pyrophosphate Deposition Disease Compared to Gout and Osteoarthritis.

OBJECTIVE: Calcium pyrophosphate deposition (CPPD) disease prevalence is similar to that of gout and osteoarthritis (OA), yet CPPD outcomes research greatly lags behind research in these other forms of arthritis. We compared validated patient-reported outcome measures in patients with CPPD vs gout and OA. METHODS: Patients with CPPD were recruited from Brigham and Women's Hospital from 2018 to 2022. Presence of CPPD manifestations (acute calcium pyrophosphate [CPP] crystal arthritis, chronic CPP inflammatory arthritis, and/or OA with CPPD) was confirmed by medical record review. Baseline surveys included the Gout Assessment Questionnaire version 2.0, modified to ask about "pseudogout" rather than "gout"; Routine Assessment of Patient Index Data 3 (RAPID-3); and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). We compared responses in patients with CPPD against published gout and OA cohort studies. RESULTS: Among 47 patients with CPPD, the mean age was 71.9 years and 51% were female. Sixty-eight percent had at least 1 episode of acute CPP crystal arthritis, 40% had chronic CPP inflammatory arthritis, and 62% had OA with CPPD. Pain visual analog scale scores during a flare were similar in CPPD (mean 6.8 [SD 1.9]) and gout (mean 6.7 [SD 2.6]; P = 0.78). Patients with CPPD reported significantly greater unmet treatment need than patients with gout (P = 0.04). RAPID-3 scores in CPPD (mean 8.1 [SD 5.6]) were lower than in gout (mean 12.1 [SD 6.2]; P < 0.01) and similar in OA (mean 6.8 [SD 6.1]; P = 0.30). Patients with CPPD had significantly worse WOMAC stiffness scores than patients with mild OA, and significantly better WOMAC function scores than patients with severe OA. CONCLUSION: Patients with CPPD may experience pain comparable to that in gout and OA and reported substantial unmet treatment needs.

Humoral and cellular immune responses in persons with rheumatoid arthritis after a third dose of mRNA COVID-19 vaccine.

OBJECTIVE: Disease-modifying anti-rheumatic drugs (DMARDs) that treat rheumatoid arthritis (RA) may reduce immune responses to COVID-19 vaccination. We compared humoral and cell-mediated immunity before and after a 3rd dose of mRNA COVID vaccine in RA subjects. METHODS: RA patients that received 2 doses of mRNA vaccine enrolled in an observational study in 2021 before receiving a 3rd dose. Subjects self-reported holding or continuing DMARDs. Blood samples were collected pre- and 4 weeks after the 3rd dose. 50 healthy controls provided blood samples. Humoral response was measured with in-house ELISA assays for anti-Spike IgG (anti-S) and anti-receptor binding domain IgG (anti-RBD). T cell activation was measured after stimulation with SARS-CoV-2 peptide. Spearman's correlations assessed the relationship between anti-S, anti-RBD, and frequencies of activated T cells. RESULTS: Among 60 subjects, mean age was 63 years and 88% were female. 57% of subjects held at least 1 DMARD around the 3rd dose. 43% (anti-S) and 62% (anti-RBD) had a normal humoral response at week 4, defined as ELISA within 1 standard deviation of the healthy control mean. No differences in antibody levels were observed based on holding DMARDs. Median frequency of activated CD4 T cells was significantly greater post- vs. pre-3rd dose. Changes in antibody levels did not correlate with change in frequency of activated CD4 T cells. CONCLUSION: Virus-specific IgG levels significantly increased in RA subjects using DMARDs after completing the primary vaccine series, though fewer than two-thirds achieved a humoral response like healthy controls. Humoral and cellular changes were not correlated.

Predictors of low spike antibody response in patients with systemic rheumatic disease after an initial course of COVID-19 vaccination.

BACKGROUND: Patients with rheumatic disease may mount a suboptimal serologic response to COVID-19 vaccination. We evaluated predictors of low antibody response in a clinic-based cohort. METHODS: We conducted a cross-sectional study using electronic health record (EHR) data at Brigham and Women's Hospital, Boston, MA. Patients with systemic rheumatic disease that had SARS-CoV-2 spike antibody (Ab) tested using the Roche Elecsys immunoassay, February-August 2021, after 2 doses of mRNA vaccine or 1 dose of adenovirus vector vaccine were identified. Demographics, systemic rheumatic disease, vaccination dates, and disease-modifying antirheumatic drugs (DMARDs) were extracted. The primary outcome was low spike Ab (≤ 200 U/mL). Logistic regression models estimated predictors of low spike Ab. RESULTS: Among 382 patients, the mean age was 57 years, 77% were female, and 37% had low spike Ab. Older age (OR 1.03, 95% CI [1.02, 1.05]), SLE (OR 4.81 [2.08, 8.43], reference: inflammatory arthritis), prednisone (OR 1.67 [1.03, 2.74]), and rituximab (OR 22.91 [9.85, 53.29]) were significantly associated with higher odds of low spike Ab. Use of csDMARD monotherapy (OR 0.12 [0.04, 0.33]) and JAK inhibitors (OR 0.41 [0.18, 0.92]) were associated with significantly lower odds for low spike Ab. After adjusting for systemic rheumatic disease and DMARDs, SLE and rituximab remained significantly associated with low spike Ab. CONCLUSIONS: Over a third of patients with systemic rheumatic disease with spike Ab tested in routine care had low spike Ab after 2 doses of mRNA or 1 dose of adenovirus vector COVID-19 vaccine. SLE and rituximab were significant risk factors for low spike Ab. KEY POINTS: • More than one-third of patients with systemic rheumatic disease that had spike Ab tested in routine care had low spike Ab after 2 doses of mRNA or 1 dose of adenovirus vector COVID-19 vaccine. • Diagnosis of SLE, use of prednisone, and use of rituximab were significantly associated with greater odds of low spike antibodies. • These data underscore the importance of additional doses of COVID-19 vaccine and prophylactic Evusheld in immunosuppressed patients with systemic rheumatic disease as recommended by the US Centers for Disease Control.

In silico Drug Screening Approach Using L1000-Based Connectivity Map and Its Application to COVID-19.

Conventional drug screening methods search for a limited number of small molecules that directly interact with the target protein. This process can be slow, cumbersome and has driven the need for developing new drug screening approaches to counter rapidly emerging diseases such as COVID-19. We propose a pipeline for drug repurposing combining in silico drug candidate identification followed by in vitro characterization of these candidates. We first identified a gene target of interest, the entry receptor for the SARS-CoV-2 virus, angiotensin converting enzyme 2 (ACE2). Next, we employed a gene expression profile database, L1000-based Connectivity Map to query gene expression patterns in lung epithelial cells, which act as the primary site of SARS-CoV-2 infection. Using gene expression profiles from 5 different lung epithelial cell lines, we computationally identified 17 small molecules that were predicted to decrease ACE2 expression. We further performed a streamlined validation in the normal human epithelial cell line BEAS-2B to demonstrate that these compounds can indeed decrease ACE2 surface expression and to profile cell health and viability upon drug treatment. This proposed pipeline combining in silico drug compound identification and in vitro expression and viability characterization in relevant cell types can aid in the repurposing of FDA-approved drugs to combat rapidly emerging diseases.

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ.

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

COS-derived GPP relationships with temperature and light help explain high-latitude atmospheric CO2 seasonal cycle amplification.

In the Arctic and Boreal region (ABR) where warming is especially pronounced, the increase of gross primary production (GPP) has been suggested as an important driver for the increase of the atmospheric CO2 seasonal cycle amplitude (SCA). However, the role of GPP relative to changes in ecosystem respiration (ER) remains unclear, largely due to our inability to quantify these gross fluxes on regional scales. Here, we use atmospheric carbonyl sulfide (COS) measurements to provide observation-based estimates of GPP over the North American ABR. Our annual GPP estimate is 3.6 (2.4 to 5.5) PgC · y-1 between 2009 and 2013, the uncertainty of which is smaller than the range of GPP estimated from terrestrial ecosystem models (1.5 to 9.8 PgC · y-1). Our COS-derived monthly GPP shows significant correlations in space and time with satellite-based GPP proxies, solar-induced chlorophyll fluorescence, and near-infrared reflectance of vegetation. Furthermore, the derived monthly GPP displays two different linear relationships with soil temperature in spring versus autumn, whereas the relationship between monthly ER and soil temperature is best described by a single quadratic relationship throughout the year. In spring to midsummer, when GPP is most strongly correlated with soil temperature, our results suggest the warming-induced increases of GPP likely exceeded the increases of ER over the past four decades. In autumn, however, increases of ER were likely greater than GPP due to light limitations on GPP, thereby enhancing autumn net carbon emissions. Both effects have likely contributed to the atmospheric CO2 SCA amplification observed in the ABR.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

AIMS: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. METHODS AND RESULTS: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. CONCLUSIONS: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Soil exchange rates of COS and CO18O differ with the diversity of microbial communities and their carbonic anhydrase enzymes.

Differentiating the contributions of photosynthesis and respiration to the global carbon cycle is critical for improving predictive climate models. Carbonic anhydrase (CA) activity in leaves is responsible for the largest biosphere-atmosphere trace gas fluxes of carbonyl sulfide (COS) and the oxygen-18 isotopologue of carbon dioxide (CO18O) that both reflect gross photosynthetic rates. However, CA activity also occurs in soils and will be a source of uncertainty in the use of COS and CO18O as carbon cycle tracers until process-based constraints are improved. In this study, we measured COS and CO18O exchange rates and estimated the corresponding CA activity in soils from a range of biomes and land use types. Soil CA activity was not uniform for COS and CO2, and patterns of divergence were related to microbial community composition and CA gene expression patterns. In some cases, the same microbial taxa and CA classes catalyzed both COS and CO2 reactions in soil, but in other cases the specificity towards the two substrates differed markedly. CA activity for COS was related to fungal taxa and ß-D-CA expression, whereas CA activity for CO2 was related to algal and bacterial taxa and α-CA expression. This study integrates gas exchange measurements, enzyme activity models, and characterization of soil taxonomic and genetic diversity to build connections between CA activity and the soil microbiome. Importantly, our results identify kinetic parameters to represent soil CA activity during application of COS and CO18O as carbon cycle tracers.

Clinical Practice Guideline for the Management of Infantile Hemangiomas.

Infantile hemangiomas (IHs) occur in as many as 5% of infants, making them the most common benign tumor of infancy. Most IHs are small, innocuous, self-resolving, and require no treatment. However, because of their size or location, a significant minority of IHs are potentially problematic. These include IHs that may cause permanent scarring and disfigurement (eg, facial IHs), hepatic or airway IHs, and IHs with the potential for functional impairment (eg, periorbital IHs), ulceration (that may cause pain or scarring), and associated underlying abnormalities (eg, intracranial and aortic arch vascular abnormalities accompanying a large facial IH). This clinical practice guideline for the management of IHs emphasizes several key concepts. It defines those IHs that are potentially higher risk and should prompt concern, and emphasizes increased vigilance, consideration of active treatment and, when appropriate, specialty consultation. It discusses the specific growth characteristics of IHs, that is, that the most rapid and significant growth occurs between 1 and 3 months of age and that growth is completed by 5 months of age in most cases. Because many IHs leave behind permanent skin changes, there is a window of opportunity to treat higher-risk IHs and optimize outcomes. Early intervention and/or referral (ideally by 1 month of age) is recommended for infants who have potentially problematic IHs. When systemic treatment is indicated, propranolol is the drug of choice at a dose of 2 to 3 mg/kg per day. Treatment typically is continued for at least 6 months and often is maintained until 12 months of age (occasionally longer). Topical timolol may be used to treat select small, thin, superficial IHs. Surgery and/or laser treatment are most useful for the treatment of residual skin changes after involution and, less commonly, may be considered earlier to treat some IHs.

Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance.

OBJECTIVES: Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. CONCLUSION: ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

A multi disciplinary obstetric emergency training programme.

The Rotunda Hospital (Dublin) obstetric emergency training programme (RHOET) was designed, in 2008, to meet the ongoing education and training needs of the local multidisciplinary team. Prior to its implementation, senior midwives attended the advanced life support in obstetrics (ALSO) course, and many of the obstetricians attended the Management of obstetric emergencies and trauma (MOET) and/or ALSO courses. Attendance at these off site courses meant that the only opportunity for team training was the informal and ad hoc 'drills and skills' that took place in the birthing suite. This paper documents our journey since RHOET was implemented.

Rho activity critically and selectively regulates endothelial cell organization during angiogenesis.

The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants. In this animal model, dominant negative N19RhoA selectively impaired assembly of ECs into new blood vessels; and, in contrast, active V14RhoA stimulated ECs to form blood vessels with functional lumens. In vitro, dominant negative N19RhoA reduced EC actin stress fibers and prevented ECs from contracting and reorganizing into precapillary cords within collagen gels. In contrast, active V14RhoA promoted EC stress fiber formation, contractility, and organization into cords. Neither N19RhoA nor V14RhoA significantly affected EC proliferation or migration in vitro; and, similarly, neither mutant significantly affected EC density during angiogenesis in vivo. Thus, these studies identify a critical and selective role for Rho activity in regulating EC assembly into new blood vessels, and they identify both negative and positive manipulation of Rho activity, respectively, as strategies for suppressing or promoting the organizational stages of neovascularization.

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.