Purified C3b triggers phagocytosis and killing of E. coli by human neutrophils via complement receptor 1
To study whether C3b can directly trigger phagocytosis of bacteria in the absence of other serum opsonins, we developed a surface chemistry method to site-specifically couple C3b onto the outer membrane of Escherichia coli (E. coli) (Fig. 1A). In short, lipopolysaccharides (LPS) were labeled via metabolic incorporation of KDO- N3 by culturing fluorescent bacteria in a medium containing azide-modified keto-deoxy-octulosonate (KDO), a major component of LPS . Subsequently, C3b was coupled to metabolically incorporated KDO-N3 via click chemistry [30, 31] (S2A). To this end, the thioester domain of C3b was site-specifically labeled with a maleimide linker  containing a dibenzocyclooctyne (DBCO) group. DBCO is the bioorthogonal partner of azide that allows covalent coupling in the absence of copper, which is compatible with living organisms [30, 31]. The fact that the DBCO group was bound to the thioester domain allowed us to label C3b molecules onto bacteria in their natural orientation, thus preserving the accessibility of binding sites essential for recognition by immune-cell receptors.
To study phagocytosis, we incubated C3b-labeled E. coli (denoted C3b-E. coli) with human neutrophils, which are the first cells recruited to sites of infection to engulf and kill invading pathogens. We observed that coupling C3b to E. coli increased the association of bacteria to neutrophils (determined by either measuring the mean fluorescence mCherry intensity (Fig. 1B, C) or the % of mCherry-positive neutrophils (S2B). Interaction of cells with bacteria prepared with the highest concentration of C3b-DBCO is comparable to the benchmark set by the physiologically opsonized bacteria (2% human serum devoid of complement protein C6 to prevent lysis) (Fig. 1C). Using confocal microscopy, we observed that C3b-coated bacteria were indeed internalized (Fig. 1D).
Next, we investigated the receptors involved in the phagocytosis of C3b-E. coli by neutrophils by pre-incubating neutrophils with either CR1 or CR3 blocking antibodies (α-CR1 and α-CR3). While CR3-blocking antibodies did not affect phagocytosis of C3b-E. coli, we observed that CR1-blocking antibodies potently reduced phagocytosis (Fig. 1E).Inhibiting CR1 reduced both the absolute number of bacteria associated with neutrophils (Fig. 1E)as well as the percentage ofGFP-positive neutrophils (S2C). Also, in confocal microscopy, we observed that α-CR1, but not α-CR3, prevented phagocytosis of C3b-E. coli.
Finally, we examined whether CR1-mediated uptake of C3b-E. coli led to intracellular killing.
During phagocytosis, bacteria are enclosed into the phagosome, which fuses with lysosomes to create a degradative environment for the enclosed target. A hallmark of this phagolysosomal stage is the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an enzymatic complex that greatly contributes to pathogen killing by producing toxic reactive oxygen species (ROS) . First, we assessed whether uptake of C3b-E. coli induced this oxidative ‘burst’ using the well-established chemiluminescence assay using the ROS-sensitive indicator luminol . C3b-E. coli was incubated with neutrophils and ROS production was measured continuously for 90 minutes (Fig. 1F). These measurements revealed that C3b-E. coli stimulated the production of ROS in a dose-dependent manner, suggesting a key role of C3b in the phagolysosomal processing of these bacteria. Remarkably, the height of the ROS plateau is comparable to the level of ROS production observed during phagocytosis of serum-opsonized bacteria. Finally, we determined whether the neutrophils kill C3b-E. coli by enumerating surviving bacteria. Consistent with the fact that C3b induced ROS formation, we observed that C3b mediated the killing of E. coli by neutrophils (Fig. 1G).
In summary, these results demonstrate that C3b can directly drive phagocytosis and killing of E. coli in the absence of other serum components.
Time-dependent cell morphology and basic mechanical features of C3b- and IgG-mediated phagocytosis are indistinguishable
Having established that purified C3b can drive phagocytosis in neutrophils, we further studied the uptake mechanism. In the seventies, Kaplan et al. introduced the idea that IgG-opsonized targets induced protruding pseudopods (“zipper model of phagocytosis”) whereas complement-opsonized targets, in which iC3b was considered determinant, were sinking without any pseudopod formation (“sinking model of phagocytosis”) . Although the results of the Kaplan report have since been disputed [35, 36], more recent single-live-cell studies have provided evidence that phagocytosis via antibodies can be different from serum-opsonized particles [5, 6]. However, these studies were performed with particles that not only have C3b on their surface, but also C4b, iC3b, and, in some cases, IgGs .
Here we combine our methods to prepare C3b-coated targets with flow cytometry and single-live-cell/single target assay [5, 38] to study C3b-mediated phagocytosis. Since polystyrene beads are more suitable for our dual-micropipette experiments [5, 38] than bacteria, we first coupled C3b to beads, now with the thioester domain labeled with biotin  (Fig. 2A, B). Indeed, we observed that beads associate with neutrophils in a C3b dose-dependent manner (Fig. 2C, S3A). Consistent with the C3b-E. coli data, we observed that the association of C3b-beads to neutrophils was blocked by antibodies blocking CR1, but not CR3 (Fig 2D, S3B).
Next, we employed single-live-cell imaging to examine the time-dependent behavior of human neutrophils during one-on-one encounters with C3b-beads. In each dual-micropipette experiment, we brought a non-adherent, initially passive neutrophil into contact with a target bead (Fig. 3A, B) and recorded time-lapse videos of the ensuing cell response (Fig. 3C, Movie S1). These single-cell experiments confirmed that human neutrophils readily recognize C3b-coated targets upon contact. The C3b beads consistently drew a strong adhesive and phagocytic response by neutrophils. The typical engulfment time, estimated from the first visible cell deformation triggered by the captured bead to the closure of the phagocytic cup, was approximately 1-2 min. In many (but not all) experiments, the overall cell shape remained roughly axisymmetric throughout the engulfment process, and the bead stayed within, or close to, the microscope’s focal plane, allowing us to visualize the time-dependent cell morphology in exceptional detail (Fig. 3C). The typical cell deformation consisted of the formation of a phagocytic cup that advanced around the bead without significantly pushing the bead itself outwards, followed by the inward motion of the bead as the main cell body gradually resumed a spherical shape (Movie S1). The same phagocytosis morphology—lacking an initial outward push of the target—previously had been observed during antibody-mediated phagocytosis [5, 38]. Indeed, when we repeated the current single-cell experiments with IgG-coated beads that had a similar size (5 µm) as the C3b beads, we were unable to detect obvious morphological differences between the phagocytic uptake of C3b- and IgG-coated beads (Movie S2). In contrast, serum-opsonized targets such as zymosan particles  and fungal pathogens  previously were found to experience a noticeable outward displacement at the onset of phagocytosis experiments, underlining the discriminative power of this single-cell assay.
In addition to the above qualitative inspection of the phagocytosis morphology, we quantified key biomechanical aspects of target internalization, such as the trajectory of the target bead and the neutrophils’ cortical tension during engulfment. Quantifying these features allows us to assess how the neutrophil remodels its cytoskeleton to achieve the deformations accompanying phagocytosis.
Typical example trajectories of C3b-beads during phagocytosis are shown in Fig. 3D, E (blue curves), depicting the recorded bead positions as a function of time. The resulting bead traces allow us to quantify the amount of initial target push-out and the speed at which each particle is pulled into the cell (Fig. 3D). The shown trajectories confirm that C3b beads did not experience a significant outward push at the onset of phagocytosis, in contrast to the typical motion of serum-opsonized zymosan particles , which traditionally have been considered to be prototypical targets for the study of complement-mediated phagocytosis (Fig. 3E). We also have included in Fig. 3E a representative trajectory of an endospore of the fungus Coccidioides posadasii , illustrating that the initial target push-out by neutrophils is common to serum-opsonized zymosan and fungi. On the other hand, considering natural cell-to-cell variability, the trajectories of C3b beads were indistinguishable from those of IgG-coated beads (red curves in Fig. 3E; Movie S2). This similarity bears out in the detailed analysis of the pull-in speeds (defined in Fig. 3D) of these two types of target bead. Our comparison of the speeds measured in N = 35 experiments with C3b beads and N = 53 experiments with IgG beads revealed no significant difference (Fig. 3F).
Finally, we also compared the maximum cortical tensions during the uptake of C3b- and IgG-coated beads. The cortical tension is a measure of the cell’s resistance to expansion of its apparent surface area. An initially round cell inevitably increases its surface area during phagocytosis. The resulting rise of the cortical tension reflects the mechanical effort expended by the cell during this process. In our experiments, the cell first tries to recruit part of the needed surface area by retracting its projection from inside the holding pipette. The operator prevents this by increasing the pipette-aspiration pressure, aiming to keep the projection length constant. We recorded the aspiration pressure throughout each experiment and converted it to an estimate of the cortical tension using a well-known mathematical relationship based on Laplace’s law . This analysis did not find a significant difference between the maximum tensions measured during the phagocytosis of C3b beads (N = 51) and IgG beads (N = 53) (Fig. 3G).
In summary, our highly discriminatory single-live-cell assay has been unable to discern differences between the immunomechanical responses of human neutrophils to C3b- and IgG-coated beads. Thus, neutrophils appear to utilize the same machinery of phagocytosis mechanics during interactions with these two types of targets. We, therefore, speculate that downstream of IgG- and C3b-specific receptor recognition and signaling, the respective biochemical response paths partially merge to induce a common mechanical cell behavior.
Internalization of C3b-beads is less efficient than IgG-beads
Although the above single-live-cell assays did not reveal morphological differences between phagocytosis of C3b- and IgG-coated targets, it was still unclear whether phagocytosis of these targets occurred with the same efficiency. In micropipette experiments, it is difficult to assess the efficiency of uptake since the target is artificially held in contact with the neutrophil by the operator. Therefore, we extended the bulk internalization assay with DNA-based quenching . Briefly, target beads were labeled with an Atto647 dye that is coupled to a single-stranded oligonucleotide. The fluorescence of non-internalized beads can be quenched by counter-staining with a BlackBerry Quencher (BBQ) that is coupled to the reverse complementary oligonucleotide. The unquenched condition provides information regarding the interaction of neutrophils with C3b-beads (adherence + internalization), while in the quenched condition only the fluorescent signal of internalized beads is present (Fig. 4A, S4).
We first studied the internalization of IgG1-labeled beads. Oligo-Atto647-beads were labeled with DNP-PEG-biotin and human anti-DNP IgG1 . Next, IgG-labeled beads were incubated with human neutrophils for 10 minutes before adding the quencher. We observed that the quencher marginally affected the Atto647 fluorescence of the neutrophil population (Fig. 4B, C), indicating that most IgG-beads had been internalized, a conclusion that was supported by confocal microscopy (Fig. 4D).
Next, we optimized the labeling of Atto647 beads with C3b (S5) and evaluated their internalization. In contrast to IgG-beads, we observed that the quencher significantly reduced the Atto647 fluorescence of neutrophils incubated with C3b-beads, suggesting that only a fraction of C3b-beads was internalized (Fig. 4B). In contrast, the percentage of neutrophils interacting with beads was minimally affected by the quenching treatment, confirming that most cells interacting with C3b-beads also internalized at least one bead (Fig. 4C). This result was confirmed by confocal microscopy, in which externally adherent beads were found on the cell surface(Fig. 4E). In accordance with previous results, both the adherence and the internalization of C3b-beads to neutrophils were severely impaired upon CR1-blocking treatment, confirming the important role of CR1 in mediating the interaction between neutrophils and C3b targets (Fig. 4B, C).
In all, our quenching method shows that although C3b coupling to beads leads to a strong CR1-dependent interaction with the cell membrane, only a fraction of adherent C3b-beads is internalized. In contrast, neutrophil interaction with IgG-beads leads to internalization.
Neutrophils spread slower on C3b-coated surfaces in frustrated phagocytosis assays
Having found that C3b-coated beads are phagocytosed much less than IgG-coated beads, we sought to determine whether this difference could be due to a change in cell spreading dynamics in response to C3b vs. IgG. To specifically probe the speed and the extent of spreading, we utilized a frustrated phagocytosis assay in which neutrophils were exposed to glass coverslips coated with either IgG or C3b (Fig. 5A, S6).
After depositing human neutrophils onto these surface, we monitored the time course of spreading by imaging the contact region between individual cells and the coverslip with high resolution using reflection interference contrast microscopy (RICM). First, we quantified the total fraction of cells that adhered and/or spread over a 30-minute time window. In agreement with the bulk phagocytosis assays, we found that while most cells adhered to either surface, the fraction of cells that spread was significantly smaller on C3b-coated than on IgG-coated surfaces (Fig. 5B).
We then assessed spreading dynamics by analyzing the contact area of individual spreading cells as a function of time (Fig. 5C).Plots of the average contact area versus time indicate that neutrophils spread slower on C3b-coated than IgG-coated surfaces (Fig. 5D). Indeed, our analysis of the spreading curves revealed that the mean cell spreading speed on C3b was significantly lower than on IgG (Fig. 5E). On the other hand, the extent of spreading as indicated by the maximum contact area appeared to be similar for both conditions (Fig. 5F). This implies that the difference in target internalization observed in the bulk phagocytosis assay is not due to a difference in the extent of phagocytic spreading, but instead appears to be a consequence of a lower likelihood of cells initiating spreading on C3b-coated surfaces.