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. 2022 Aug 29:13:945485.
doi: 10.3389/fimmu.2022.945485. eCollection 2022.

Microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity

Shuailong Li  1 , Isa Wernersbach  1 , Gregory S Harms  2   3 , Michael K E Schäfer  1   4   5
Affiliations

Microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity

Shuailong Li et al. Front Immunol. .

Abstract

Microglia are phagocytosis-competent CNS cells comprising a spectrum of subtypes with beneficial and/or detrimental functions in acute and chronic neurodegenerative disorders. The heterogeneity of microglia suggests differences in phagocytic activity and phenotype plasticity between microglia subtypes. To study these issues, primary murine glial cultures were cultivated in the presence of serum, different growth factors and cytokines to obtain M0-like, M1-like, and M2-like microglia as confirmed by morphology, M1/M2 gene marker expression, and nitric oxide assay. Single-cell analysis after 3 hours of phagocytosis of E.coli particles or IgG-opsonized beads showed equal internalization by M0-like microglia, whereas M1-like microglia preferably internalized E.coli particles and M2-like microglia preferably internalized IgG beads, suggesting subtype-specific preferences for different phagocytosis substrates. Time-lapse live-cells imaging over 16 hours revealed further differences between microglia subtypes in phagocytosis preference and internalization dynamics. M0- and, more efficiently, M1-like microglia continuously internalized E.coli particles for 16 hours, whereas M2-like microglia discontinued internalization after approximately 8 hours. IgG beads were continuously internalized by M0- and M1-like microglia but strikingly less by M2-like microglia. M2-like microglia initially showed continuous internalization similar to M0-like microglia but again discontinuation of internalization after 8 hours suggesting that the time of substrate exposure differently affect microglia subtypes. After prolonged exposure to E.coli particles or IgG beads for 5 days all microglia subtypes showed increased internalization of E.coli particles compared to IgG beads, increased nitric oxide release and up-regulation of M1 gene markers, irrespectively of the phagocytosis substrate, suggesting phenotype plasticity. In summary, microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity. The results suggest that prolonged phagocytosis substrate exposure enhances M1-like profiles and M2-M1 repolarization of microglia. Similar processes may also take place in conditions of acute and chronic brain insults when microglia encounter different types of phagocytic substrates.

Keywords: brain; immune response; inflammation; live imaging; microglia; phagocytosis; plasticity; polarization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Microglia subtypes differ in morphology and expression of M1/M2 protein markers. (A) Confocal images showing anti-Iba1 immunostaining of M0-, M1-, and M2-like microglia subtypes in primary glia cultures at 7 div, cultivated the presence of serum and/or growth factors and cytokines (GM-CSF/IFNγ for M1-like or M-CSF/IL-4 for M2-like) (B) Histograms showing differences in mean cell size and (C) circularity of microglia subtypes. (D) Confocal images showing triple-immunostaining using antibodies specific to the pan-marker Iba1, the M1-marker MHC2, or the M2-marker MRC1. M1- or M2-like microglia show increased expression of MHC2 or MRC1, respectively. (E, F) Histograms showing the percentage of Iba1+ microglia expressing MHC2 or MRC1 as determined by cell counts after triple immunostaining using antibodies specific to Iba1, MHC2, or MRC1. Data are expressed as mean ± SEM (n = 5, independent biological replicates are shown) and were tested for significant differences by one-way ANOVA (post-hoc correction Holm-Šídák) or Kruskal-Wallis test (post-hoc correction Dunnett), *p < 0.05, **p < 0.01, ****p < 0.0001 ns, not significant.
Figure 2
Figure 2
Microglia subtypes differ in M1/M2 gene marker expression and metabolism. (A–E) Gene expression analyses of the pan-marker Aif1, M1-markers Mhc2, Nos2 and M2 markers Arg1, and Mrc1 demonstrating differential expression by microglia subtypes. Ppia was used as a reference gene. (F) Alterations in cellular arginine metabolism were detected by colorimetric Griess assay, with M1-like microglia releasing significantly more NO than M0- or M2-like microglia. (G) Scatter plot showing positive correlation between NO and Nos2 expression (non-parametric Spearman correlation, r = 0.8842, p < 0.0001). Values are expressed are mean ± SEM from 5 individual experiments (biological replicates), one-way ANOVA (post-hoc correction Holm-Šídák), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.
Figure 3
Figure 3
M1- and M2-like microglia show substrate-specific phagocytosis preference and capacities. (A–C) Confocal images showing anti-Iba1 immunostaining of M0-, M1- or M2-like microglia subtypes along with rhodamine-E.coli particles (red, E.coli) or IgG-FITC beads (green, IgG) after 3 hours of phagocytosis substrate exposure. Histograms showing percentage of microglia with internalized E.coli particles or IgG beads. M0-like microglia showed no phagocytosis preference, whereas M1- and M2-like microglia showed opposing phagocytosis preferences for E.coli particles or IgG beads, respectively. (D) Co-localization analyses showing the relative occupancy of Iba1 immunostained cell areas by E.coli particles or IgG beads. M1- and M2-like microglia displayed opposing phagocytosis capacities for E.coli particles or IgG beads, respectively. Data are expressed as mean ± SEM from 5 independent biological replicates (A–C) or 5 cells from each of 5 independent biological replicate (D). Data are means ± SEM. ****p < 0.0001, ns, not significant, Student’s t-test.
Figure 4
Figure 4
Microglia subtypes show substrate-specific phagocytosis capacities and dynamics for E.coli particles over 16 hours (A) Single frame images and (B) image enlargements from live imaging movies of anti-CD68 live-immunolabelled microglia (blue) after addition of E.coli-rhodamine particles (red) at time-points 0h and 16h, scale bar, 100µm. Note pronounced E.coli particle accumulation in M0- and M1- but not in M2-like microglia at 16h after addition of E.coli particles. (C) Time-series plot showing the mean number of E. coli-rhodamine particles internalized (averaged from 8-10 ROIs for each time interval) by microglia subtypes over 16 hours. Simple linear regression calculation indicate different slopes of phagocytic capacities of M0- (black) (r2: 0,6902, p < 0,0001), M1- (pink) (r2: 0,5364, p < 0,0001), and M2-like (cyan) (r2: 0,3691, p < 0,0001), respectively. Note that M2-like microglia discontinued internalization at about 9 hours after addition of E.coli particles. (D) Mean number of internalized particles over 16 hours (averaged from 8-10 ROIs for each time interval). (E) Number of imaged anti-CD68 immunolabelled microglia encountering E.coli particles over 16 hours. Data are expressed as means ± SEM, one-way ANOVA (post-hoc correction Holm-Šídák test, *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.
Figure 5
Figure 5
Microglia subtypes show different phagocytosis capacities and dynamics for IgG-FITC beads over 16 hours. (A) Single frame images and (B) image enlargements from live imaging movies of anti-CD68 live-immunolabelled microglia (blue) after addition of IgG-FITC beads (green) at time-points 0h and 16 h, scale bar, 100µm. Note pronounced IgG bead accumulation in M0- and M2- but not in M1-like microglia at 16h after addition of E.coli particles. (C) Time-series plot showing the mean number of IgG beads internalized (averaged from 8-10 ROIs for each time interval) by microglia subtypes over 16 hours. Simple linear regression calculation indicate different slopes of phagocytic capacities of M0- (black) (r2: 0,1637, p < 0,0001), M1- (pink) (r2: 0,2733, p < 0,0001), and M2-like (cyan) (r2: 0,07045, p < 0,0001), respectively. Note that M2-like microglia discontinued internalization at about 9 hours after addition of IgG-FITC beads. (D) Mean number of internalized particles over 16 hours (averaged from 8-10 ROIs for each time interval). (E) Number of imaged anti-CD68 immunolabelled microglia encountering IgG-FITC beads over 16 hours. Data are expressed as means ± SEM, one-way ANOVA (post-hoc correction Holm-Šídák test, ****p < 0.0001. ns, not significant.
Figure 6
Figure 6
Microglia subtypes show M1-like features after prolonged phagocytosis substrate exposure (A–C) Confocal images showing anti-Iba1 immunostaining of M0-, M1- or M2-like microglia subtypes along with rhodamine-E.coli particles (red, E.coli) or IgG-FITC beads (green, IgG) after 5 days of phagocytosis substrate exposure. Histograms showing percentage of microglia with internalized E.coli particles or IgG beads. Microglia subtypes showed phagocytosis preference for E.coli particles rather than IgG beads. (D) Scatter plots showing similar correlation (non-parametric Spearman correlation) between NO levels and Nos2 gene expression for M0- (r = 0.6536, p=0.0099), M1- (r = 0.8214, p = 0.0003), and M2-like (r = 0.8857, p < 0.0001) microglia. Ppia was used as a reference gene. (E–G) Co-localization analyses showing the relative occupancy of Iba1 immunostained cell areas by E.coli particles or IgG beads. Microglia subtypes showed phagocytosis preference for E.coli particles rather than IgG beads. Data are expressed as mean ± SEM from 5 independent biological replicates (A–C) or 4-6 cells from each of 5 independent biological replicate (D). Data are means ± SEM. ****p < 0.0001, ns, not significant, Student’s t-test.
Figure 7
Figure 7
Microglia subtypes show M1-like gene expression after prolonged phagocytosis substrate exposure (A–R) Gene expression analyses of microglia subtypes after prolonged phagocytosis substrate exposure (E.coli-rhodamine particles, IgG-FITC beads) for the pan-marker Aif1, M1-markers Mhc2, Nos2 and M2 markers Arg1, Mrc1 as well as the glia activation marker Mmp9. Ppia was used as a reference gene. (A–E) In M0-like microglia, conditions with E.coli particles or IgG beads show up-regulation of M1 markers Mhc2 and Nos2 as well as up-regulation of M2 marker Mrc1 as compared to M0-like microglia not exposed to phagocytosis substrate (control). (F–J) In M1-like microglia, conditions with E.coli particles show downregulation of microglia pan-marker Aif1 and up-regulation of M1 markers Mhc2 and Nos2 but down-regulation of M2 markers Arg1 and Mrc1 and conditions with IgG show up-regulation of Nos2 as compared to M1-like microglia not exposed to phagocytosis substrate (control). (K–O) In M2-like microglia, conditions with E.coli particles or IgG beads show up-regulation of M1 markers Mhc2 and Nos2 as compared to M2-like microglia not exposed to phagocytosis substrate (control). Down-regulation of M2 marker Arg1 was observed in conditions with E.coli particles as well as down-regulation of Mrc1 in conditions with E.coli particles or IgG beads as compared to M2-like microglia not exposed to phagocytosis substrate (control). (P–R) The glia activation marker Mmp9 was up-regulated in M0- and M2-like microglia both in conditions with E.coli particles of IgG beads as compared to microglia subtypes not exposed to phagocytosis substrate (control). Multiple comparisons were performed dependent on data distribution by one-way Anova (post-hoc correction Holm-Šídák) or Kruskal-Wallis test (post-hoc correction Dunnett), if F achieved the necessary level of statistical significance p < 0.05. Data points are shown for biological replicates and expressed as mean ± SEM, p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001.
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References

    1. Norden DM, Fenn AM, Dugan A, Godbout JP. TGFβ produced by IL-10 redirected astrocytes attenuates microglial activation. Glia. (2014) 62(6):881–95. doi: 10.1002/glia.22647 - DOI - PMC - PubMed
    1. Butovsky O, Weiner HL. Microglial signatures and their role in health and disease. Nat Rev Neurosci (2018) 19(10):622–35. doi: 10.1038/s41583-018-0057-5 - DOI - PMC - PubMed
    1. Waisman A, Ginhoux F, Greter M, Bruttger J. Homeostasis of microglia in the adult brain: Review of novel microglia depletion systems. Trends Immunol (2015) 36(10):625–36. doi: 10.1016/j.it.2015.08.005 - DOI - PubMed
    1. Prinz M, Masuda T, Wheeler MA, Quintana FJ. Microglia and central nervous system-associated macrophages-from origin to disease modulation. Annu Rev Immunol (2021) 39:251–77. doi: 10.1146/annurev-immunol-093019-110159 - DOI - PMC - PubMed
    1. Loane DJ, Kumar A. Microglia in the TBI brain: The good, the bad, and the dysregulated. Exp Neurol (2016) 275(Pt 3Pt 3):316–27. doi: 10.1016/j.expneurol.2015.08.018 - DOI - PMC - PubMed

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