Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging

<div><p>Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the “professional phagocyte” of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity <i>in vivo</i>. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.</p></div

Monoclonal antibodies capable of modulating efferocytosis are identified using high content image-based screening.

<p>Purified hybridoma supernatants were tested in a 384-well format using day 7 MΦ’s co-cultured with apoptotic Jurkat cells then analyzed on the Operetta High Content Imager. Results are expressed as PI (z-axis) and % positive cells (y-axis) for each sample (A) with controls represented as follows: cytochalasin D treated MΦ’s with apoptotic cells negative control (green squares); live cell, baseline phagocytosis (dark red squares); apoptotic cells with no antibody (blue squares); dexamethasone treated MΦ’s with apoptotic cells positive control (orange squares). Samples from each plate are grouped and represented by colored squares. The antibody panel contains molecules that exhibit positive and negative effects on MΦ efferocytosis as revealed by comparison of PI of treated cells to controls (B). Each diamond represents the PI data from an individual hybridoma and each is compared to the PI of MΦ’s incubated with apoptotic cells in absence of antibody (red line) which is geometric mean of 4 replicates 9.995 ± 7.54. Data shown are from testing antibodies on macrophages from a single donor, a second assay using cells from a different donor was also performed yielding similar results.</p

The majority of antibodies enhance apoptotic cell phagocytosis through recognition of unidentified surface molecules.

<p>Antibodies having a PI ≥ average PI + 1 SD (standard deviations) for cultures with no antibody treatment are considered as enhancing. Those having a PI≤ average PI—1 SD are considered inhibitory. All other antibodies having PI within 1 SD of average calculated for untreated MΦ’s are considered as having no effect. Frequency of each category of antibody is calculated as percent of total number of antibodies in the panel (left). Binding to known molecules could be detected in a small number of clones (right). Antibodies were evaluated by ELISA and readings that were ≥ 5-fold over background were considered positive.</p

Intact apoptotic cells are detectable within macrophages.

<p>Macrophages were co-cultured with apoptotic Jurkat cells labeled with pHrodo dye for 30 minutes. Excess Jurkat cells were washed away then macrophages were fixed and stained with a cocktail of antibodies consisting of anti-CD14-APC, anti-CD64-APC and anti-CD11b-APC. Macrophages (APC⁺) appear green in the image and apoptotic Jurkat cells (Cy3⁺) appear red. On the left is a representative image from a single field of 6 total image fields taken per well of a 96-well plate (40X objective) and has been enlarged to show macrophages that have engulfed apoptotic Jurkat cells. Image on the right showing macrophages co-cultured with live Jurkat cells includes all 6 fields of a well to demonstrate the paucity of engulfed cells. Images were captured using the Operetta High Content Imager. Confirmation of active phagocytosis during the 30 minute incubation is demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.s005" target="_blank">S1 Movie</a>.</p

Phagocytic Index (PI) provides a direct measure of macrophage function.

<p>Comparison of data presented as either PI (A) or representing the frequency of macrophages containing apoptotic cells expressed as “percent (%) positive cells” (B) within the same population in each well of a 96 well plate. The PI was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.g003" target="_blank">Fig 3</a>. Calculation of the % positive cells is based on the assumption that macrophages (APC⁺) containing apoptotic cells (Cy3⁺) appear fluorescent for both fluorophores and calculated as follows: % positive cells = (# APC⁺ Cy3⁺ cells / # APC⁺ cells) * 100. Ratios listed along the X-axis represent the (Target) T: MΦ ratios. Baseline engulfment was determined using MΦ’s co-cultured with live cells (Live); Negative controls consisted of cytochalasin D treated MΦ’s co-cultured with apoptotic cells (Neg). Test samples consisted of apoptotic cells co-cultured with MΦ’s (Apop).</p

Approximately half of the hybridomas retain MΦ binding following expansion and purification.

<p>Supernatants from hybridomas that were expanded and purified were screened on IFN-γ treated MΦ’s using MSD as the readout. All 700 hybridomas were tested, shown above is representative data from two 96-well plates. Histograms are laid out in a 96-well plate format such that the X-axis represents the column numbers and the Z-axis represents the row identities. Each sample was run as a single replicate and log transformed signal ≥ 0.5 was considered positive.</p

Dexamethasone treatment impacts the ability of macrophages to engulf apoptotic cells in a dose-dependent manner.

<p>Macrophages were treated with varying doses of dexamethasone then imaged following 30 minute incubation with apoptotic Jurkat cells as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.g002" target="_blank">Fig 2</a>. Phagocytic Index (PI) was calculated at each dose from 1 field/well collected using the Operetta High Content Imager. PI = (Total # engulfed cells/Total # counted MΦ) * (# MΦ’s containing engulfed cells/Total # counted MΦ) * 100 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.ref049" target="_blank">49</a>].</p

Characteristics of SelecT generated macrophages and apoptotic status of staurosporine-treated cells.

<p>Macrophages were prepared by culture with M-CSF as described in materials and methods. Following treatment with IFN-γ, the majority of macrophages express markers found on both M1 and M2 populations (A). Cells were gated by scatter then expression of markers was assessed on live cells. M1-M2 intermediate phenotype macrophages are CD64⁺CD16⁺ (B). Jurkat cells were treated with staurosporine for 3 hours at 37°C as described in materials and methods. Cells were stained for active caspase 3 then quantitated using flow cytometry (C) or stained with Magic Red then measured using high content imaging (D).</p

Mice immunized with human macrophages produce antibodies that recognize molecules involved in efferocytosis.

<p>Sera collected from mice immunized with human M1-M2 MΦ’s treated with IFN-γ were tested for titers to LRP-1, Bai1, Stabilin 2, MFG-E8 and SIRPα by ELISA. Data are shown for 5 immunized mice with each mouse represented by a filled symbol, and compared to sera from an unimmunized (naïve) mouse represented by the open circles.</p