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 6

Soluble CR1 (rhCD35) reduces both recognition and internalization of polymeric PGN.

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Soluble CR1 (rhCD35) reduces both recognition and internalization of pol...
Human PBMCs were incubated with 10 μg/mL NHS-opsonized, biotinylated PGN in the presence or absence of CR1 inhibitors, and the surface and internalized PGN fractions were sequentially stained using PE- and BV421-labeled streptavidins. (A) Representative biaxial plots exemplifying the effects of soluble CR1 (rhCD35) on PGN uptake by primary human monocytes (see Supplemental Figure 8A for additional plots). (B) Changes in the relative distribution of monocyte subsets containing surface and/or internalized PGN after CR1 inhibition using either the J3D3 monoclonal or increasing concentrations of the extracellular domain of CR1 (rhCD35). Data are visualized as stacked bars depicting mean ± SD (n = 12) relative frequencies of monocyte subsets per experimental group (legend: S = surface, PE-labeled PGN; I = internalized, BV421-labeled PGN). (C) Representative histogram overlays depicting the shift in fluorescence intensities of surface (left panel) and internalized (right panel) PGN in response to rhCD35 treatment. (D) Normalized changes in the surface and internalized PGN intensities associated with CR1 inhibition, illustrated as fractional difference compared with an irrelevant challenge (mIgG1k). (E) Competitive inhibition of CR1-PGN interaction by rhCD35 reduces the conformational activation of CR3, indicative of a crosstalk between the 2 CRs. Assay principle (left) and representative histogram overlay of CR3 activation depicted as the ratio between 2 CD18 reporter mAbs, the activation-dependent m24 and the activation-insensitive MEM48. (F) Changes in CR3 activation associated with CR1 inhibition in the experimental cohort (n = 12). Individual (circles) and the predicted distribution of biological responses (violin plots) are illustrated. (D and F) Differences between experimental groups were assessed by RM 2-way (D) or 1-way (F) ANOVA with Holm-Šídák correction for multiple comparisons and depicted graphically (P < 0.001; and ¶¶P < 0.0001). FMO, fluorescence minus one.