Extended culture of macrophages from different sources and maturation results in a common M2 phenotype

Abstract

Inflammatory responses to biomaterials heavily influence the environment surrounding implanted devices, often producing foreign body reactions. The macrophage is a key immunomodulatory cell type consistently associated with implanted biomaterials and routinely employed in short term in vitro cell studies of biomaterials aiming to reproduce host responses. Inconsistencies within these studies, including differently sourced cells, different durations of culture, and assessment of different activation markers, lead to many conflicting results in vitro that confound consistency and conclusions. We hypothesize that different experimentally popular monocyte-macrophage cell types have intrinsic in vitro culture-specific differences that yield conflicting results. Recent studies demonstrate changes in cultured macrophage cytokine expression over time, leading to the hypothesis that changes in macrophage phenotype also occur in response to extended culture. Here, macrophage cells of different transformed and primary-derived origins were cultured for 21 days on model polymer biomaterials. Cell type-based differences in morphology and cytokine/chemokine expression as well as changes in cell surface biomarkers associated with differentiation stage, activation state, and adhesion were compared. Results reflect consistent macrophage development towards an M2 phenotype via up-regulation of the macrophage mannose receptor for all cell types following 21-day extended culture. Significantly, implanted biomaterials experiencing the foreign body response and encapsulation in vivo often elicit a shift towards an analogous M2 macrophage phenotype. In vitro “default” of macrophage cultures, regardless of lineage, to this M2 state in the presence of biomaterials at long culture periods is not recognized but has important implications to in vitro modeling of in vivo host response.

Novel Markers to Delineate Murine M1 and M2 Macrophages

“…Similarly, less than 1% of M1 macrophages were CD38 - Egr2 + at 6 days. In contrast, the CD38 - Egr2 + phenotype reached 24% within the M0 population ( S4B Fig ), consistent with the reported shift to M2 phenotype that may occur during extended in vitro conditions [ 30 , 31 , 46 ]. These data show that while the CD38 + Egr2 - phenotype is associated with M1 macrophages, the M2 CD38 - Egr2 + phenotype is linked to M2 macrophages and that both phenotypes are stable over several days.…”

Differentiated macrophages acquire a pro-inflammatory and cell death–resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1

“…CD11b is another marker of macrophage differentiation (36), which was increased during differentiation. The time required for macrophages to undergo differentiation is not clear, as macrophages have been isolated at various time intervals (37,38). Interestingly, extending the differentiation of macrophages to 14 days resulted in enhanced control of mycobacteria (39), which suggests that macrophages continue to differentiate beyond day 7 of culture.…”

Macromechanics and polycaprolactone fiber organization drive macrophage polarization and regulate inflammatory activation of tendon in vitro and in vivo

“…Their initial interaction and, more specifically, their polarization from an initial pro-inflammatory (M1-like) phenotype towards a reparative (M2-like) phenotype, is crucial for successful tissue-biomaterial integration [32]. Macrophages have the ability to polarize into a broad spectrum of phenotypes and the high degree of plasticity allows them a shift in the polarization state from M1 to M2 [33][34][35]. Therefore, 'immuno-modulatory' design characteristics of regenerative biomaterials, which induce favorable immune response upon implantation, may be a powerful tool to achieve improved healing outcomes [36,37].…”