Sterile inflammation induced by Carbopol elicits robust adaptive immune responses in the absence of pathogen-associated molecular patterns

Abstract

HighlightsCarbopol induces Th1/IgG2a responses without PRR activation.Carbopol polymer morphology is changed by APC phagocytosis leading to ROS induction.This study highlights a potentially novel mechanism for in vivo cellular activation.

Carbomer-based Nano-Emulsion Adjuvant Enhances Dendritic Cell Cross-presentation via Lipid Body Formation Independent of Glycolysis

“…However, ADJ did not promote stabilized-HIF1α accumulation under normoxia in DCs. It has been also documented that phagolysosomal destabilization after adjuvant phagocytosis, such as Alum and Carbopol, is an important step in inflammasome activation (Gartlan et al, 2016, Hornung et al, 2008. While we have not directly characterized the intracellular location of ADJ in DCs, ADJ increased lysosomal pH, which in turn may result in lysosomal stabilization.…”

Imaging Flow Cytometry to Assess Antigen‐Presenting‐Cell Function

“…APCs [e.g., murine immature (day 6) bone marrow-derived DCs (BMDCs) or bone marrow-derived macrophages (BMDMs) prepared as in Current Protocols articles (Inaba et al, 2009;Harding, 2001)] Complete RPMI-10 medium (see recipe), 37°C Fluorescently labeled phagocytic target [e.g., zymosan A-Alexa Fluor 488 (Molecular Probes, #Z23373), CFSE-labeled apoptotic cells (Baxter et al, 2014), or Carbopol-Alexa Fluor 488 (Gartlan et al, 2016)] 5 mM EDTA in phosphate-buffered saline (PBS; containing Ca 2+ and Mg 2+ ), 37°C FACS buffer (see recipe), 4°C Surface-staining monoclonal antibody (mAb; e.g., MHC class II-Alexa Fluor 647, BioLegend, #107618, RRID: AB_493525 or CD11c-Alexa Fluor 647, BioLegend, #117312, RRID: AB_389328) 0.5 µg/ml Hoechst 33342…”

“…Optional: Prepare additional duplicate wells containing sterile 13-mm glass coverslips to enable parallel in situ confocal microscopy or scanning electron microscopy studies to be performed (Gartlan et al, 2016). 2. Incubate plate for 1 hr in a 37°C, 5% CO 2 tissue culture incubator to allow the cells to adhere/stabilize.…”

“…Imaging flow cytometry is a method that combines aspects of traditional flow cytometry and fluorescence microscopy, resulting in a platform for high‐throughput microscopy. Imaging flow cytometry has provided a novel approach to assess a number of immunological processes that have traditionally been very difficult to study, including cellular interactions such as synapse formation (Ahmed, Friend, George, Barteneva, & Lieberman, ; Markey et al., ; Markey, Gartlan, Kuns, MacDonald, & Hill, ), phagocytosis (Gartlan et al., ; Smirnov, Solga, Lannigan, & Criss, ), infection (Baxter et al., ; Haridas, Ranjbar, Vorobjev, Goldfeld, & Barteneva, ; Khoury et al., ), cell signaling and nuclear translocation (George et al., ; O. Maguire, Collins, O'Loughlin, Miecznikowski, & Minderman, ; Orla Maguire, Tornatore, O'Loughlin, Venuto, & Minderman, ), antigen presentation (Koyama et al., ), the cell cycle and mitotic division (Filby et al., ), apoptosis and DNA repair/damage (George et al., ; Parris et al., ), cell death (George et al., ), autophagy (Leveque‐El Mouttie et al., ; Pugsley, ), and morphological changes (Grimwade, Fuller, & Erber, ). The key advantage of this method is that it allows capture of not only immunofluorescence data (as is usual for standard flow cytometry) but also the spatial localization of the antibody targets of interest (as is usual for microscopy), rendering it particularly useful for study of immune function.…”

“…Phagocytosis assay acquisition and analysis. Bone marrow-derived dendritic cells (BMDCs) were cultured for 16 hr in the presence of fluorescently labeled (Alexa Fluor 488) Carbopol and then stained prior to acquisition via imaging flow cytometry and analysis using IDEAS analysis software (adapted fromGartlan et al, 2016). (A) BMDCs were selected by plotting Aspect Ratio_M01 versus Area_M01 and excluding debris (identified as having a low aspect ratio) (left panel).…”