The M2a macrophage phenotype accompanies pulmonary granuloma resolution in Mmp12 knock-out mice instilled with multiwall carbon nanotubes

Sarcoidosis is a chronic disease with unknown etiology and pathophysiology, characterized by granuloma formation. Matrix Metalloproteinase-12 (MMP12) is an elastase implicated in active granulomatous sarcoidosis. Previously, we reported that oropharyngeal instillation of multiwall carbon nanotubes (MWCNT) into C57Bl/6 mice induced sarcoid-like granulomas and upregulation of MMP12. When Mmp12 knock-out (KO) mice were instilled with MWCNT, granuloma formation occurred 10 days post-instillation but subsequently resolved at 60 days. Thus, we concluded that MMP12 was essential to granuloma persistence. The aim of the current study was to identify potential mechanisms of granuloma resolution in Mmp12KO mice. Strikingly, an M2 macrophage phenotype was present in Mmp12KO but not in C57Bl/6 mice. Between 10 and 60 days, macrophage populations in MWCNT-instilled Mmp12KO mice demonstrated an M2c to M2a phenotypic shift, with elevations in levels of IL-13, an M2 subtype-regulating factor. Furthermore, the M2 inducer, Apolipoprotein E (ApoE), and Matrix Metalloproteinase-14 (MMP14), a promoter of collagen degradation, were upregulated in 60-day MWCNT-instilled Mmp12KO mice. In conclusion, alveolar macrophages express two M2 phenotypes in Mmp12KO mice: M2c at 10 days when granulomas form, and M2a at 60 days when granulomas are resolving. Findings suggest that granuloma resolution in 60-day Mmp12KO mice requires an M2a macrophage phenotype.ECU Open Access Publishing Support Fun

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Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells

Using graph theory to analyze biological networks

Understanding complex systems often requires a bottom-up analysis towards a systems biology approach. The need to investigate a system, not only as individual components but as a whole, emerges. This can be done by examining the elementary constituents individually and then how these are connected. The myriad components of a system and their interactions are best characterized as networks and they are mainly represented as graphs where thousands of nodes are connected with thousands of vertices. In this article we demonstrate approaches, models and methods from the graph theory universe and we discuss ways in which they can be used to reveal hidden properties and features of a network. This network profiling combined with knowledge extraction will help us to better understand the biological significance of the system

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Resolution of Chronic Granulomatous Inflammation and Fibrosis in a Murine Model of Sarcoidosis

Sarcoidosis is a chronic granulomatous disease of unknown cause. Granulomas resolve within 12-26 months in 50% of cases without therapy while 30% of patients have chronic disease that progresses to pulmonary fibrosis. The transcription factor peroxisome proliferator activated receptor gamma (PPARγ), a negative regulator of pro-inflammatory macrophage activation, is deficient in the alveolar macrophages of sarcoidosis patients. We established a chronic murine model of sarcoidosis in which multiwall carbon nanotubes (MWCNTs) are administered via oropharyngeal instillation. The expression of PPARγ is also deficient in the murine alveolar macrophages. Granulomatous lesions persist for at least 90-days post installation. Previous studies from our lab have implicated matrix metalloproteinase-12 (MMP12) and osteopontin (OPN) as key factors that work together to promote granuloma formation and persistence. We used a macrophage-specific PPARγ knockout mouse to study the effects of PPARγ deficiency on sarcoidosis pathophysiology. As expected, PPARγ mice had more robust granuloma formation at 60-days post instillation compared to C57BL/6J controls. We hypothesized that PPARγ mice would have large, persisting granulomatous structures 90-days post instillation and that levels of MMP12 and OPN would be elevated. PPARγ and C57BL/6J mice were instilled with MWCNT. Controls received PBS alone. Lung tissue from both strains were sectioned, stained, and subjected to granuloma scoring and qualitative evaluation. Tissue from PPARγ mice was also used for immunohistochemistry (IHC). Surprisingly, at 90-days post instillation, PPARγ mice had attenuated fibrosis and decreased granuloma size compared to 60-days and C57BL/6J mice. Evaluation of mediators in the canonical TGFβ signaling pathway did not explain the changes in fibrosis. Changes in MMP12 and OPN protein levels, however, were apparent. Immunohistochemistry of lung tissue showed decreased expression of both MMP12 and OPN at 90-days post instillation. These results suggest a relationship between MMP12 and OPN in the resolution of granulomatous structures. The decrease in MMP12 and OPN in tissue sections at 90-days is especially important as pulmonary granulomatous structures form and resolve within lung tissue

Resolution of Chronic Granulomatous Inflammation and Fibrosis in a Murine Model of Sarcoidosis

Sarcoidosis is a chronic granulomatous disease of unknown cause. Granulomas resolve within 12-26 months in 50% of cases without therapy while 30% of patients have chronic disease that progresses to pulmonary fibrosis. The transcription factor peroxisome proliferator activated receptor gamma (PPAR[gamma]), a negative regulator of pro-inflammatory macrophage activation, is deficient in the alveolar macrophages of sarcoidosis patients. We established a chronic murine model of sarcoidosis in which multiwall carbon nanotubes (MWCNTs) are administered via oropharyngeal instillation. The expression of PPAR[gamma] is also deficient in the murine alveolar macrophages. Granulomatous lesions persist for at least 90-days post installation. Previous studies from our lab have implicated matrix metalloproteinase-12 (MMP12) and osteopontin (OPN) as key factors that work together to promote granuloma formation and persistence. We used a macrophage-specific PPAR[gamm] knockout mouse to study the effects of PPAR[gamma] deficiency on sarcoidosis pathophysiology. As expected, PPAR[gamma] mice had more robust granuloma formation at 60-days post instillation compared to C57BL/6J controls. We hypothesized that PPAR[gamma] mice would have large, persisting granulomatous structures 90-days post instillation and that levels of MMP12 and OPN would be elevated. PPAR[gamma] and C57BL/6J mice were instilled with MWCNT. Controls received PBS alone. Lung tissue from both strains were sectioned, stained, and subjected to granuloma scoring and qualitative evaluation. Tissue from PPAR[gamma] mice was also used for immunohistochemistry (IHC). Surprisingly, at 90-days post instillation, PPAR[gamma] mice had attenuated fibrosis and decreased granuloma size compared to 60-days and C57BL/6J mice. Evaluation of mediators in the canonical TGF[beta] signaling pathway did not explain the changes in fibrosis. Changes in MMP12 and OPN protein levels, however, were apparent. Immunohistochemistry of lung tissue showed decreased expression of both MMP12 and OPN at 90-days post instillation. These results suggest a relationship between MMP12 and OPN in the resolution of granulomatous structures. The decrease in MMP12 and OPN in tissue sections at 90-days is especially important as pulmonary granulomatous structures form and resolve within lung tissue