Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages

Abstract

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we used in vitro models with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneously in vivo to investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1- and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1->M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneous in vivo model. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels with and without drug caused markers of both M1 and M2 phenotypes to be highly expressed, but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such as Col5a1 and Col6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.

Evaluation of the inflammatory responses to sol–gel coatings with distinct biocompatibility levels

“…At initial stages of inflammatory responses, the M1 is the most prevalent but, with time, macrophages undergo a transition to the M2 phenotype. However, the extent of the diversity of the M2 phenotype is not completely understood, and several M2 subtypes have been described (M2a, M2b, M2c, and M2d) 28 . These phenotypes attenuate acute and chronic inflammation through different mechanisms and signals 29 even though this classification still fails to cover the wide range of signals and functions related to M2 macrophages 30 .…”

The inflammatory speech of fibroblasts

“…Consequently, macrophages do not exist in a binary M1/M2 state but in a spectrum of phenotypes, sometimes with hybrid phenotypes exhibiting M1 and M2 characteristics. 169,170 Again, scRNAseq, fate mapping and linage tracing tools have supplemented flow cytometry analysis to annotate specific macrophage populations in various adult human tissues, for example, mapping the normal human liver uncovered two distinct populations of intrahepatic CD68 + macrophages. 171 One population is enriched for expression of the LYZ, SCTA, and CD74 and suggested to represent the pool of inflammatory intrahepatic macrophages.…”

Biomaterials-Mediated Regulation of Macrophage Cell Fate

“…For example, surface hydrophilicity can be used to overcome non-specific protein absorption and reduce FBGC formation (Quinn et al, 1997;Jenney and Anderson, 1999;Voskerician et al, 2003;Collier et al, 2004). However, growing evidence has shown that macrophage engagement during implantation can be harnessed to improve implant success rates (Spiller et al, 2014;Yu et al, 2016a) and that a timely transition from M1 to M2 phenotype benefits tissue remodeling (Badylak et al, 2008;Brown et al, 2012;Spiller et al, 2014;Yu et al, 2016a;Witherel et al, 2020). Many approaches to promote this M1-M2 shift, like a sequential delivery of immunomodulatory cytokines IFN-γ and IL-4, have achieved some positive outcomes (Mokarram et al, 2012;Spiller et al, 2015).…”