Monocyte uptake of polymeric peptidoglycan is bimodal and governed by complement C3 and C4 opsonins
Narcis I. Popescu, Jędrzej Kluza, Megan A. Reidy, Elizabeth Duggan, John D. Lambris, Linda F. Thompson, K. Mark Coggeshall
Narcis I. Popescu, Jędrzej Kluza, Megan A. Reidy, Elizabeth Duggan, John D. Lambris, Linda F. Thompson, K. Mark Coggeshall
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Research Article Immunology Infectious disease

Monocyte uptake of polymeric peptidoglycan is bimodal and governed by complement C3 and C4 opsonins

Abstract

Peptidoglycans (PGNs) are structural polymers of the bacterial cell wall and a common microbial molecular pattern encountered by the immune system daily. Low levels of PGNs are constitutively present in the systemic circulation in humans and rise during inflammatory pathologies. Since all known PGN sensors are intracellular, PGN internalization is a prerequisite for the initiation of cellular immune responses. Here, we report the mechanisms controlling the recognition and uptake of polymeric PGNs by circulating human mononuclear phagocytes. We found that complement C3 and C4 opsonins govern PGN recognition and internalization, but no single opsonin is indispensable because of multiple uptake redundancies. We observed a bimodal internalization of polymeric PGNs with distinct requirements for complement C4. At low PGN concentrations, C3 mediated PGN recognition by surface receptors while the efficient internalization of PGN polymers critically required C4. Supraphysiologic PGN concentrations triggered a secondary uptake modality that was insensitive to C4 and mediated instead by C3 engagement of complement receptors 1 and 3. To our knowledge, this is the first description of nonoverlapping C3 and C4 opsonophagocytoses working in parallel. Controlling these uptake mechanisms has the potential to modulate PGN clearance or the dysregulated immune responses during bacterial infections.

Authors

Narcis I. Popescu, Jędrzej Kluza, Megan A. Reidy, Elizabeth Duggan, John D. Lambris, Linda F. Thompson, K. Mark Coggeshall

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Figure 5

Bimodal internalization of polymeric PGN requires C3 and C4 opsonins.

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Bimodal internalization of polymeric PGN requires C3 and C4 opsonins. Bi...
Biotinylated PGN particles were preopsonized with pooled NHS or sera depleted of C3 or C3/C4 opsonins before incubation with human PBMCs. Recognition and uptake of polymeric PGN by primary monocytes were assessed by sequential labeling of surface and internalized PGN using PE- and BV421-labeled streptavidins, respectively. (A and B) Effect of PGN concentration on uptake of NHS-opsonized polymeric PGN. Representative biaxial plots (A) and mean relative distribution of PGN+ monocyte subsets (B, n = 14) containing surface, PE-labeled, and/or internalized, BV421-labeled, polymeric PGN. At low concentrations, PGN-biotin is detected almost exclusively intracellularly, while at high concentrations, most monocytes contain surface and internalized PGN. (C and D) Confocal analysis of subcellular localization of NHS-opsonized PGN particles (10 μg/mL) after 30 minutes’ exposure to PBMCs. Surface-exposed biotinylated PGN-FITC particles were stained with Alexa Fluor 647–conjugated streptavidin, and the colocalized fluorescent signals were assessed in Imaris (yellow, arrowheads). Internalized PGN-FITC particles were protected from streptavidin staining and detected as FITC fluorescence only (green, arrows). (C) Data depict an optical section highlighting monocytes (white, CD14) containing surface and internalized PGN particles. PGN also interacted with, but was not taken up by, CD14– mononuclear cells with rounder nuclei (*). (D) Mean relative distribution of PGN+ monocyte subsets assessed by confocal microscopy. Data depict group averages ± SD of relative frequencies from 6 donors (circles). RM 1-way ANOVA with Holm-Šídák correction for multiple comparisons (*P < 0.05). (E and F) Opsonin requirements for PGN recognition and uptake by primary human monocytes. (E) Representative biaxial plots, similar to A, exemplifying the recognition and uptake of 10 μg/mL PGN-biotin by human monocytes in the presence or absence of C3 and/or C4 opsonins. (F) Mean relative distribution of PGN+ monocyte subsets after uptake of polymeric PGN preopsonized by NHS or sera depleted of C3 and/or C4. Stacked bars depict group averages ± SD (n = 12) of relative frequencies of monocyte subsets, color-coded according to B.