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

Characterization of patients seeking care at a Sexual Health Clinic who report engaging in exchange sex

Thesis (Master's)--University of Washington, 2021Background: People who exchange sex (PWES) for money or drugs are at increased risk of HIV and other sexually transmitted infections (STIs) and may need tailored prevention and care services. Our objective was to characterize patients in the Public Health - Seattle & King Country (PHSKC) Sexual Health Clinic who reported engaging in exchange sex and identify opportunities for improved service. Methods: We conducted a cross-sectional analysis of patient encounters for new problem visits October 2010 - March 2020 with a completed computer assisted self-interview, including sex assigned at birth, gender identity, and receipt of money or drugs for sex, ever or in the past year. Individual patient data was the unit of analysis. We analyzed demographics, STIs, HIV history, Hepatitis C (HCV) testing and treatment history, HIV Pre-exposure Prophylaxis (PrEP) use, and reason for visit, stratified by gender. Our analysis focused on people who reported a lifetime history of exchange sex because the characteristics of this group represented people who reported exchanging sex in the past year. We compared characteristics of PWES ever vs. never using chi-square tests. Results: During the study period, 30,327 patients attended the clinic. Of these, 1,611 (5%) were PWES and 700 (2%) reported exchange sex in the past 12 months. Compared to people who never exchanged sex, PWES were more likely to report homelessness (29% vs. 7%, p<0.001), injection drug use (IDU) (39% vs. 4%, p<0.001), STIs in the past 12 months (36% vs. 19%, p<0.001), prior HIV diagnosis (13% vs. 5%, p<0.001), and prior HCV diagnosis (13% vs. 2%, p<0.001). PWES were less likely to report HIV viral suppression (67% vs. 73%, p=0.04) and HCV treatment (24% vs. 37%, p<0.001) than people who never exchanged sex. Among PWES there were 981 (61%) cisgender men, 545 (34%) cisgender women, and 85 (5%) transgender/genderqueer/non-binary persons. Compared to cisgender men and transgender/genderqueer/non-binary persons, cisgender women were more likely to report homelessness (41%) and prior HCV diagnosis (17%) but less likely to have received HCV treatment (42%). Cisgender men were more likely to report multiple sexual partners (48%-50%), IDU (37%), STIs in the past 12 months (42%), and prior HIV diagnosis (20%) than cisgender women and transgender/genderqueer/nonbinary persons. Among PWES, the most common reasons for coming to the clinic were wanting an STI test (60%) or HIV test (45%) and having STI symptoms (38%). At the clinic visit included in the analysis, evaluation of the clinic visit outcome for PWES showed 320 (20%) were diagnosed with one or more STI, 15 (1%) were newly diagnosed with HIV, and 15 (1%) initiated PrEP. Conclusion: Many PWES in the Sexual Health Clinic had complex barriers to care, including homelessness and IDU, and a higher prevalence of previously diagnosed STIs, HIV, and HCV. Clinic visits are an opportunity to increase HIV viral suppression, HCV treatment, and PrEP uptake for PWES

Changes in cell elasticity from physiologic substrate rigidity and stretch modulate macrophage phagocytosis.

<p><b>A.</b> Increased cell spreading and filopodial projections of macrophages cultured on a rigid surface. Differential interference contrast (DIC) microscopy images of RAW 264.7 macrophages cultured on a less rigid (<i>E</i> = 1.2 kPa) versus rigid (<i>E</i> = 150 kPa) polyacrylamide gel coated with poly-L-lysine to facilitate attachment. <b>B.&C.</b> Increased elasticity of macrophages cultured on a rigid versus less rigid substrate. Optical Magnetic Twisting Cytometry (OMTC) measurement of mouse RAW macrophages (B.) and human alveolar macrophages (AM) (C.) cultured on less rigid (<i>E</i> = 1.2 kPa) versus rigid (<i>E</i> = 150 kPa) substrate. Data is displayed as relative elastic modulus (REM) as compared to less rigid condition. *P<0.001 (Mann Whitney U test) Results depict data from 1 of 3 representative experiments with >100 magnetic beads assayed for each experiment. Each experiment had similar results <b>D.</b> Increased phagocytosis of beads by macrophages cultured on a rigid versus less rigid surface. Merged DIC and fluorescent images of RAW macrophages cultured on a less rigid versus rigid surface, and exposed to blue-green fluorescent IgG coated latex beads for two hours at standard culture conditions. Unbound beads have been washed away, showing overall fewer beads per cell in soft versus stiff substrate. Nuclei have been stained blue with Hoechst stain. <b>E&F.</b> Quantification of phagocytosis in RAW macrophages, counting beads per cell in soft versus stiff macrophages. Results show significantly fewer beads per cell in macrophage cultured on less rigid substrate in both IgG-opsonized (*P = 0.03, n = 5; Mann Whitney U test), and unopsonized (*P = 0.03, n = 5; Mann Whitney U test) beads <b>G.</b> Phagocytosis of IgG-coated latex beads is also reduced in human alveolar macrophages (AM) comparing rigid versus less rigid surface. *P = 0.004, n = 5 (Mann Whitney U test) <b>H.</b> Substrate stretch decreases macrophage elasticity. Relative elasticity over time after a single 10% stretch of RAW macrophages shows initial 80% reduction in relative elasticity (REM) that returns back to steady state (prestretch) elasticity by about 8 seconds. P<0.0001 for stiffness over time using Spearman Rank Test. Results depict data from 1 of 3 representative experiments with >100 magnetic beads assayed for each time point. <b>I.</b> Periodic stretch reduces phagocytosis of uncoated latex beads on either less rigid (1.2 kPa) or more rigid substrate (150 kPa). Results show less phagocytosis (normalized to unstretched cells) in stretched cells on both more rigid and less rigid surface. *P<0.001 (Mann Whitney U test).</p

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

Macrophage elasticity affects response to LPS and LPS tolerance.

<p><b>A.</b> Decreasing macrophage elasticity using actin inhibitors increases response to LPS. Human, PMA-differentiated U937 macrophage like cells were cultured on plastic tissue culture plate. Control cells, cells treated with Cytochalasin D (10 µM), or Latrunculin A (1 µM), were cultured for 24 hours with or without LPS (1 µg/ml). Culture supernatants were assayed for TNF-α via ELISA. *P = 0.015 versus control, (n = 6) Mann-Whitney U test. **P = 0.015 versus control, (n = 6), Mann-Whitney U test <b>B.</b> LPS tolerance is reduced by actin inhibition on U937 macrophages. Differentiated human U937 macrophages were stimulated with LPS, and had culture supernatant examined for TNFα via ELISA. Levels were compared to cells that were rechallenged with LPS for another 24 hours after 24 hours of initial LPS challenge, or treated Latrunculin A (1 µM) for 30 minutes before rechallenge with LPS. Results show that latrunculin enchanced LPS response with rechallenge. (n = 6) *P = 0.015 <b>C.</b> The WASP inhibitor, wiskostatin, increases LPS-mediated TNF-α release. Differentiated human U937 macrophages were stimulated with LPS with or without pretreatment with wiskostatin. Culture supernatants were assessed for TNFα release via ELISA. Results show a dose dependent increase in TNFα release in cells stimulated with LPS with wiskostatin, versus LPS alone. *P<0.01 (n = 6) <b>D.</b> Increasing substrate rigidity decreases TNF-α release in response to LPS in RAW macrophages. RAW macrophages were cultured in a 96 well plate in wells containing poly-L-lysine coated gels of increasing elastic modulus from 0.3 kPa-76.8 kPa, or on poly-L-lysine coated glass for 24 hours. Cells were then stimulated with LPS (1 µg/ml) for 24 hours and culture supernatants were assayed for TNF-α via ELISA. P<0.001 for correlation of TNF level with substrate rigidity. Spearman Rank Test. <b>E.</b> Increasing substrate rigidity decreases TNF-α release in response to LPS in human U937 macrophage-like cells. Protocol similar to D, except PMA-differentiated U937 cells are used. P<0.01, Spearman Rank Test.</p

Macrophage gene expression is determined by duration of culture on substrate and substrate rigidity.

<p>Gene expression profile over time in RAW macrophages increasingly correlates with substrate rigidity. RAW macrophages, after removing from a plastic tissue culture dish, were plated on less rigid (E = 1.2 kPa) or more rigid (E = 150 kPa) substrate for 2 hours, 6 hours, and 18 hours, and RNA was extracted for microarray analysis at each time point. <b>A.</b> Principle component analysis (PCA) reveals a PC1 that well correlates with duration of culture on substrate and describes the largest variance (34.8%), while a PC2 distinguished 6 hours culture time from 2 hours and 18 hours culture times. <b>B</b>. The third larges source of variance was PC3 (9.9% of variance) that distinguishes by substrate rigidity increasingly over time.</p

MyD88-dependent TLR4 signaling is selectively impaired in alveolar macrophages from asymptomatic HIV+ persons

Alveolar macrophages (AMs) are the predominant effector cell in the lungs and contribute to a critical first line of defense against bacterial pathogens through recognition by pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4-mediated tumor necrosis factor α (TNFα) release is significantly impaired in HIV+ macrophages, but whether HIV impairs myeloid differentiation factor 88 (MyD88)–dependent and/or MyD-independent TLR4 signaling pathways in human macrophages is not known. Comparing human U937 macrophages with HIV+ U1 macrophages (HIV-infected U937 subclone), the current study shows that HIV infection is associated with impaired macrophage TLR4-mediated signaling, specifically targeting the MyD88-dependent TLR4-mediated signaling pathway (reduced MyD88–interleukin-1 receptor–associated kinase [IRAK] interaction, IRAK phosphorylation, nuclear factor [NF]–κB nuclear translocation, and TNFα release) while preserving the MyD88-independent TLR4-mediated signaling pathway (preserved STAT1 phosphorylation, interferon regulatory factor [IRF] nuclear translocation, and interleukin-10 [IL-10] and RANTES release). Extracellular TLR4 signaling complex was intact (similar levels of CD14 and MD2), and similar patterns of response were observed in clinically relevant AMs from healthy and asymptomatic HIV+ persons at high clinical risk of pneumonia. Taken together, these data support the concept that chronic HIV infection is associated with specific and targeted disruption of critical macrophage TLR4 signaling, which in turn may contribute to disease pathogenesis of bacterial pneumonia