Figure 4.

Impact of polarization on macrophage functional responses. (A–D) Monocyte-derived macrophages were polarized by cytokine culture to either M1, M2 polarization states, or resting (M0). The LPS stimulated cytokine responses of polarized macrophages over 48h in culture following polarization were assessed using cytokine ELISA. (A) IL-10 release (B) TNF-α release (C) IL-8 release (D) MCP-1. (E) Nitric oxide (NO) production in response to LPS stimulation was measured by Griess reaction assay. (F) The impact of macrophage polarization on bactericidal activity was assessed by killing of S. pseudointermedius, bactericidal assay was performed with 1 h for phagocytosis followed by 3 h of bacterial killing activity. Bar graph shows percent killing as ratio of 3-h bacterial growth compared to baseline, as colonies in CFU/mL. (G) The effects of polarization on bacterial phagocytosis were measured over time using labeled S. aureus and quantified by Incucyte instrument. For the phagocytosis assay, M0 macrophages are depicted in blue, M1 macrophages were depicted in red, and M2 macrophages were depicted in green. The x-axis denotes incubation time in hours. Data are depicted graphically as mean ± SD. Statistical differences were determined using one-way ANOVA, followed by Tukey's multiple comparisons adjustment (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001), ns for non-significant.