Characterizing the macrophage response to immunomodulatory biomaterials through gene set analyses

Sarah E. Blatt

doi:10.17918/00000280

All implanted biomaterials influence the immune system to affect tissue repair. The primary regulators of the innate immune response are macrophages, which are essential for both the initiation and resolution phases of the tissue repair process. They accomplish these diverse functions by undergoing changes in phenotype in response to microenvironmental cues. Therefore, immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although gene expression has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. Therefore, the aim of this study was to investigate the utility of gene set analyses to facilitate characterization of the behavior of macrophages in contact with model immunomodulatory biomaterials in comparison to M1 and M2a "reference" macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the pro-inflammatory cytokine IFNg. Gene expression of a panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString at days 3, 5, and 8 of culture in vitro. An additional dataset of phenotype controls, consisting of M0, M1, and M2a macrophages derived from monocytes of three human donors was used to initially validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation of macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. To address the challenges posed with single gene analysis, ROAST was utilized both with and without incorporation of gene weights based on the genes' ability to differentiate between M1, M2a, and M0 controls. In addition to ROAST, GSVA was also utilized. Overall, ROAST with weights incorporated based on fold changes of M1 and M2a relative to M0 was slightly better than the alternative methods at distinguishing differences between phenotype control comparisons. This method revealed that macrophages cultured on collagen scaffolds inhibited an M1-like phenotype at a relatively late time point, while those cultured on IFNg-adsorbed scaffolds expressed an M1-like phenotype at all time points. In summary, this work demonstrates a powerful methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this methodology could prove beneficial for characterizing complex hybrid phenotypes.

Characterizing the macrophage response to immunomodulatory biomaterials through gene set analyses

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Characterizing the macrophage response to immunomodulatory biomaterials through gene set analyses

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