Cell Elasticity Determines Macrophage Function

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function

Novel Developments in the Epidemic of Human Immunodeficiency Virus and Tuberculosis Coinfection

Tuberculosis (TB) disease remains one of the highest causes of mortality in HIV-infected individuals, and HIV–TB coinfection continues to grow at alarming rates, especially in sub-Saharan Africa. Surprisingly, a number of important areas regarding coinfection remain unclear. For example, increased risk of TB disease begins early in the course of HIV infection; however, the mechanism by which HIV increases this risk is not well understood. In addition, there is lack of consensus on the optimal way to diagnose latent TB infection and to manage active disease in those who are HIV infected. Furthermore, effective point-of-care testing for TB disease remains elusive. This review discusses key areas in the epidemiology, pathogenesis, diagnosis, and management of active and latent TB in those infected with HIV, focusing attention on issues related to high- and low-burden areas. Particular emphasis is placed on controversial areas where there are gaps in knowledge and on future directions of study

A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial

Background The phe508del CFTR mutation causes cystic fibrosis by limiting the amount of CFTR protein that reaches the epithelial cell surface. We tested combination treatment with lumacaftor, an investigational CFTR corrector that increases trafficking of phe508del CFTR to the cell surface, and ivacaftor, a CFTR potentiator that enhances chloride transport of CFTR on the cell surface. Methods In this phase 2 clinical trial, we assessed three successive cohorts, with the results of each cohort informing dose selection for the subsequent cohort. We recruited patients from 24 cystic fibrosis centres in Australia, Belgium, Germany, New Zealand, and the USA. Eligibility criteria were: confirmed diagnosis of cystic fibrosis, age at least 18 years, and a forced expiratory volume in is (FEV1) of 40% or more than predicted. Cohort 1 included phe508del CFTR homozygous patients randomly assigned to either lumacaftor 200 mg once per day for 14 days followed by addition of ivacaftor 150 mg or 250 mg every 12 h for 7 days, or 21 days of placebo. Together, cohorts 2 and 3 induded phe508del CFTR homozygous and heterozygous patients, randomly assigned to either 56 days of lumacaftor (cohort 2: 200 mg, 400 mg, or 600 mg once per day, cohort 3: 400 mg every 12 h) with ivacaftor 250 mg every 12 h added after 28 days, or 56 days of placebo. The primary outcomes for all cohorts were change in sweat chloride concentration during the combination treatment period in the intention-to-treat population and safety (laboratory measurements and adverse events). The study is registered with ClinicalTrials.gov, number NCT01225211, and EudraCT, number 2010-020413-90. Findings Cohort 1 induded 64 participants. Cohort 2 and 3 combined contained 96 phe508del CFTR homozygous patients and 28 compound heterozygotes. Treatment with lumacaftor 200 mg once daily and ivacaftor 250 mg every 12 h decreased mean sweat chloride concentration by 9.1 mmol/L (p<0.001) during the combination treatment period in cohort 1. In cohorts 2 and 3, mean sweat chloride concentration did not decrease significantly during combination treatment in any group. Frequency and nature of adverse events were much the same in the treatment and placebo groups during the combination treatment period; the most commonly reported events were respiratory. 12 of 97 participants had chest tightness or dyspnoea during treatment with lumacaftor alone. In pre-planned secondary analyses, a significant decrease in sweat chloride concentration occurred in the treatment groups between day 1 and day 56 (lumacaftor 400 mg once per day group -9.1 mmol/L, p<0.001; lumacaftor 600 mg once per day group -8.9 mmol/L, p<0.001; lumacaftor 400 mg every 12 h group -10.3 mmol/L, p=0.002). These changes were significantly greater than the change in the placebo group. In cohort 2, the lumacaftor 600 mg once per day significantly improved FEV1 from day 1 to 56 (difference compared with placebo group: +5.6 percentage points, p=0.013), primarily during the combination period. In cohort 3, FEV1 did not change significantly across the entire study period compared with placebo (difference +4.2 percentage points, p=0.132), but did during the combination period (difference +7.7 percentage points, p=0.003). Phe508del CFTR heterozygous patients did not have a significant improvement in FEV1. Interpretation We provide evidence that combination lumacaftor and ivacaftor improves FEV, for patients with cystic fibrosis who are homozygous for phe508del CFTR, with a modest effect on sweat chloride concentration. These results support the further exploration of combination lumacaftor and ivacaftor as a treatment in this setting

Bacterial PAMPs (LPS) and cytokines (IFNγ) increase macrophage phagocytosis and cell elasticity.

<p><b>A.</b> LPS and IFN-γ increase phagocytosis by macrophages on a less rigid (1.2 kPa) substrate in proportion to changes in elasticity. Control, LPS-stimulated (1 µg.ml), or IFN-γ-stimulated (200 U/ml) macrophage were cultured on asubstrate rigidity corresponding to the lungs (1.2 kPa) for 24 hours, and then exposed to IgG-coated latex beads for 2 hours. Beads per cell were manually counted using confocal microscope images. *P = .008 LPS vs. control (n = 5), *P = 0.004, IFN-γ vs control (n = 5) Mann-Whitey U test. <b>B.</b> LPS and IFN-γ increase macrophage elasticity on a less rigid (1.2 kPa) substrate. Control unstimulated (US), LPS (1 µg/ml) stimulated, IFN-γ (200 U/ml) stimulated RAW macrophages were cultured on a less rigid substrate for 24 hours, and cell elasticity was measured via Optical Magnetic Twisting cytometry (OMTC). Data is shown as Relative Elastic Modulus (REM). Results depict data from 1 of 3 representative experiments with >100 magnetic beads assayed for each experiment. *P<0.0001 compared to control for both, Mann-Whitney U test. <b>C.</b> Macrophage elasticity is decreased by actin polymerization inhibitor. Optical Magnetic Twisting Cytometry (OMTC) measurement of RAW macrophages cultured on more rigid (150 kPa) surface and treated with the myosin inhibitor blebbistatin (Bleb, 50 µM) or the actin polymerization inhibitor latrunculin A (Lat A, 1 µM). Data is shown as Relative Elastic Modulus (REM). Results depict data from 1 of 3 representative experiments with >100 magnetic beads assayed for each experiment. *P<0.001, Mann Whitney U test <b>D.</b> LPS stimulation of macrophages on a less rigid (1.2 kPa) substrate increases cell spreading, filapodial projections, actin polymerization. RAW macrophages were cultured on less rigid substrate for 24 hours with or without LPS (1 µg/ml) stimulation for 24 hours. Cells were fixed, stained for polymerized actin (alexa-fluor phalloidin), and DNA (Hoechst nuclear stain), and visualized via confocal microscopy. Images represent collapsed stack of 7 confocal slices. <b>E.</b> LPS and IFN-γ increase polymerized actin in macrophages. RAW macrophages in suspension were treated with LPS (1 µg/ml), IFN-γ (200 U/ml), or latrunculin A (1 µM). After 24 hours, cells were fixed, stained for actin with Alexa-fluor phalloidin, and fluorescence was quantified via flow cytometry. Data is show as relative change in mean fluorescence (RMF) from control untreated cells (US). *P = 0.028, n = 4 compared to control, Mann-Whitney U test. **P = 0.028 compared to similar condition without Latrunculin A for each, n = 4, Mann-Whitney U test.</p

Macrophage elasticity is modulated by actin polymerization and rhoGTPase acitivity.

<p><b>A.</b> Decreased polymerized actin staining and filapodia in macrophages cultured on a less rigid surface. Collapsed stack confocal (total of 7 slices) image of RAW macrophages cultured on less rigid (1.2 kPA) or rigid substrate (150 kPa). Green color represents actin staining (alexa-fluor 488 phalloidin) with blue stained nuclei (Hoechst). <b>B.</b> Decreased actin polymerization in macrophages cultured on less rigid substrate. RAW macrophages were cultured on less rigid versus more rigid substrate for 24 hours, and then lifted from surface using trypsin/EDTA solution, and immediately fixed, stained for polymerized actin (alexi-fluor-phalloidin), and staining was quantified using flow cytometry. Data displayed as changes in relative mean fluorescence. *P = 0.029, n = 4, Mann-Whitney U test. <b>C.</b> Decreased actin-rich filapodial projections and actin fibers in macrophages treated with rhoGTPase inhibitor. RAW macrophages were cultured on more rigid (150 kPa) substrate with or without C. difficile toxin (400 pM) for 24 hours. Cells were then fixed, stained for polymerized actin with alexi-fluor phalloidin and visualized via confocal microscopy. Image represent a collapsed stack of 7 confocal slices. <b>D.</b> RhoGTPase inhibitor decreases macrophage elasticity. OMTC measurement of RAW macrophages cultured on more rigid substrate (150 kPa) with or without C. diff toxin (400 pM). *P<0.0001, Mann Whitney U test. Representative experiment for >4 independent observations. <b>E.</b> Attachment to substrate leads to activation of cdc42. RAW macrophages in suspension were allowed to adhere to plastic tissue culture dish and protein lysates were assayed for cdc42 activation via ELISA after labeled incubation times. *P = 0.028, n = 4, Mann Whitney U test.</p