key: cord-0270716-xyl0jz0g
authors: Rubey, Kathryn M.; Mukhitov, Alexander R.; Nong, Jia; Wu, Jichuan; Krymskaya, Vera P.; Myerson, Jacob W.; Worthen, G. Scott; Brenner, Jacob S.
title: Nanoparticle-induced augmentation of neutrophils’ phagocytosis of bacteria
date: 2022-05-19
journal: bioRxiv
DOI: 10.1101/2022.05.14.491866
sha: 6336325f7172cdf8bf26e29cc0c2dec4c12f53a9
doc_id: 270716
cord_uid: xyl0jz0g

Despite the power of antibiotics, bacterial infections remain a major killer, due to antibiotic resistance and hosts with dysregulated immune systems. We and others have been developing drug-loaded nanoparticles that home to the sites of infection and inflammation via engineered tropism for neutrophils, the first-responder leukocytes in bacterial infections. Here, we examined how a member of a broad class of neutrophil-tropic nanoparticles affects neutrophil behavior, specifically questioning whether the nanoparticles attenuate an important function, bacterial phagocytosis. We found these nanoparticles actually augment phagocytosis of non-opsonized bacteria, increasing it by ~50%. We showed this augmentation of phagocytosis is likely co-opting an evolved response, as opsonized bacteria also augment phagocytosis of non-opsonized bacteria. Enhancing phagocytosis of non-opsonized bacteria may prove particularly beneficial in two clinical situations: in hypocomplementemic patients (meaning low levels of the main bacterial opsonins, complement proteins, seen in conditions such as neonatal sepsis and liver failure) or for bacteria that are largely resistant to complement opsonization (e.g., Neisseria). Additionally, we observe that; a) prior treatment with bacteria augments neutrophil uptake of neutrophil-tropic nanoparticles; b) neutrophil-tropic nanoparticles colocalize with bacteria inside of neutrophils. The observation that neutrophil-tropic nanoparticles enhance neutrophil phagocytosis and localize with bacteria inside neutrophils suggests that these nanoparticles will serve as useful carriers for drugs to ameliorate bacterial diseases.

have been developing drug-loaded nanoparticles that home to the sites of infection and 23 inflammation via engineered tropism for neutrophils, the first-responder leukocytes in 24 bacterial infections. Here, we examined how a member of a broad class of neutrophil-25 tropic nanoparticles affects neutrophil behavior, specifically questioning whether the 26 nanoparticles attenuate an important function, bacterial phagocytosis. We found these 27 nanoparticles actually augment phagocytosis of non-opsonized bacteria, increasing it by 28 ~50%. We showed this augmentation of phagocytosis is likely co-opting an evolved 29 response, as opsonized bacteria also augment phagocytosis of non-opsonized bacteria. To develop NAPs for targeted delivery to neutrophils in infectious diseases, such 93 as pneumonia, we aim here to ensure that NAPs coordinate with the key beneficial 94 functions of neutrophils, and do not negatively impact their function. Previous studies 95 have shown that nanoparticle phagocytosis may decrease neutrophil adhesion and 96 migration (Fromen et al., 2017) . Probably the most essential function of neutrophils 97 during infections like pneumonia is phagocytosis of bacteria, since phagocytosis is 98 necessary for killing of certain bacteria (Lee, Harrison and Grinstein, 2003) . Here, we 99 tested neutrophil phagocytosis of NAPs before and after neutrophil phagocytosis of the 100 common bacterial pathogen, E. coli. We found that NAPs do not negatively impact 101 neutrophil phagocytosis of bacteria and NAPs localize to neutrophils that have also 102 taken up bacteria. Quite surprisingly, NAPs, given after bacteria, enhance efficiency of 103 neutrophils' phagocytosis of bacteria that have not been opsonized by serum proteins.

This effect, termed here second particle augmentation factor (2PAF) was illustrated in 105 both: flow cytometry and microscopy. Our results suggest applicability of NAPs to two We recently developed a diverse class of nanoparticles with neutrophil-tropism:

NAPs (Myerson et al., 2022) . In the present study, we focus on a prototypical member of 119 this class, lysozyme-dextran nanogels (hereafter referred to as "nanogels" or NGs).

NGs have the benefit for antibiotic delivery of prolonged nanoparticle shelf-life (years at 121 4C) and a very high drug-to-carrier mass ratio (Myerson et al., 2018 (Myerson et al., , 2019 .

In these experiments with nanogels, we again confirmed our previous findings 123 that particle uptake is enhanced by opsonization by complement proteins present in 124 serum (Myerson et al., 2022) . When complement protein C3 is depleted from serum via 125 cobra venom factor (CVF) (Haihua et al., 2018) , we see a significant decrease in the percent of neutrophils that take up nanogels (Supplemental Figure 1) . The green serum 127 nanogels (second green bar) showed ~80% positivity, as compared to the green CVF 128 nanogels (third and fifth green bars) which had ~30% positivity. It has been well 129 established that complement binding to E. coli is necessary for neutrophil phagocytosis 130 of the bacteria (Horwitz and Silverstein, 1980; Brekke et al., 2007) . We utilized serum-131 opsonization of both NAPs and E. coli bioparticles for these experiments.

One of the first tests of whether these nanocarriers can be used to augment 133 bacterial killing is whether neutrophils will take up the nanocarriers after having been 134 exposed to bacteria. In this experiment, diagrammed in Figure 1A , neutrophils were 135 incubated with heat-killed E. coli bioparticles which have surface conjugated pHrodo 136 green, a pH-sensitive dye that fluoresces green only when in the low pH environment of 137 the phagosome. After this 60-minute 37°C incubation, the neutrophils were pelleted and 138 washed to remove free bacteria. The neutrophils were then incubated with nanogels for 139 15 minutes, and subjected to flow cytometry. Before exposure to neutrophils, half the 140 samples of E. coli were serum-opsonized (hereafter referred to as "Serum EC"), while 141 the other half were not exposed to serum (simply labeled as "EC" in Fig 1) . Similarly, 142 nanogels were divided into serum-opsonized ("Serum NG") and not (simply "NG").

Flow cytometry was gated to analyze neutrophils exclusively (Supplemental 144 Figure 2 ). Representative flow cytometry dot-plots are depicted in Figure 1B and 145 quantified in n=6 biological replicates in Figure 1D . We compared the summary statistic Figure 1D . These conditions measure the fraction of neutrophils that are positive for 150 phagocytosis of E. coli that had not been exposed to serum ("EC"). When these 151 neutrophils were incubated with nanogels that had not been exposed to serum (first red 152 bar), 55% of the neutrophils were positive for E. coli phagocytosis. This percentage of 153 neutrophils positive for E. coli went up to 85% if the nanogels had been pre-opsonized 154 by serum. This means that serum-opsonized nanogels are able to augment E. coli 155 phagocytosis. This augmentation occurred even though the nanogels were delivered 156 after free E. coli had been washed away from the neutrophils. 157 We term this enhancement the "second particle augmentation factor" (2PAF) and 158 define it for this particular experiment (Fig 1) as the following ratio: (% neutrophils 159 positive for EC phagocytosis when the second delivered particle is Serum NG) / (% 160 neutrophils positive for EC phagocytosis when the second delivered particle is [non-161 serum-exposed] NG). 2PAF can be more generally defined as: (% neutrophils positive 162 for particle #1 phagocytosis, given that particle #2 is serum-opsonized) / (% neutrophils 163 positive for particle #1 phagocytosis, given that particle #2 was not exposed to serum); 164 where particle #1 refers to the particle (or microbe) neutrophils are exposed to in the 165 first incubation, and particle #2 refers to the second incubation. Thus, a 2PAF > 1 166 indicates that serum-opsonized particle #2 are able to augment phagocytosis of particle 167 #1 (as compared to the control condition, which uses particle #2 that was not exposed 168 to serum). Calculating the 2PAF for non-serum exposed E. coli (EC), we thus get 169 2PAF = 85% / 55% = 1.55, meaning that serum-opsonized nanogels increase neutrophil 170 phagocytosis of these E. coli by 50% (Fig 1D, inset, blue bar). A 2PAF > 1 is only found 171 when the E. coli have not been serum opsonized: 2PAF = 1 when using serum-opsonized E. coli, a 0% increase (Fig 1D, inset) . Thus, serum-opsonized nanogels are 173 able to augment uptake of non-opsonized bacteria, but not opsonized bacteria, the latter 174 of which are already phagocytosed so extensively (~100%) that we cannot detect 175 improvement within the dynamic range of this assay. The mechanism by which serum infections in hypocomplementemic hosts (e.g., neonatal sepsis).

A hypothesis to explain this enhancement is outlined in Figure 1C . The Having made the finding that nanogels can augment phagocytosis of bacteria, 195 we tested whether this was a phenomenon unique to NGs. We performed experiments 196 with NGs replaced as a "second particle" by a second E. coli particle, checking whether 197 the second bacterial particles could enhance uptake of bacteria that were delivered 198 during a first incubation. The experimental protocol was the same as Figure 1A , except 199 that particle #1 was pHrodo green E. coli, and particle #2 was pHrodo red E. coli ( Having established that both serum-opsonized NGs and serum-opsonized 212 bacteria can augment phagocytosis of non-opsonized bacteria, we questioned whether 213 NG uptake could be similarly enhanced. We used the same protocol as above, except 214 with particle #1 as NGs (green), and particle #2 as a separate sample of NGs (red) (Fig   215   3A ). Comparing the first and third green bars in Fig 3C, we see that serum-opsonized 216 NGs are minimally able to augment phagocytosis of previously delivered NGs. Thus, 217 2PAF = 1.23. These results may be attributed to difference in fluorophores of the E. coli 218 bioparticles and NGs, as NGs fluoresce equally well on the surface of neutrophils (pH 7) 219 and in phagosomes (pH 4-5). More likely though, the low levels of first incubation NG 220 fluorescence observed in the first and third green bars suggest that neutrophils do not 221 retain nanogels on their surface after washing, which would thus prevent augmentation 222 by nanogels delivered during the second incubation. Figure 3C also shows that non-223 opsonized particle #2 NG uptake (the first red bar) is higher than non-opsonized particle 224 #1 NG uptake (the first green bar). This implies that exposure of neutrophils to a first 225 particle (even one that is mostly washed away) increases the phagocytic efficiency of 226 the neutrophils for nanoparticles that are delivered later. This finding is consistent with 227 previous studies showing neutrophils are known to change their activity state after 228 phagocytosis (Bazzoni et al., 1991) . This finding suggests that the order and timing of 229 particle delivery matter significantly. 230 Finally, we administered E. coli as particle #2 after NGs as particle #1 ( Figure   231 4A). As in Figure 3 , when NGs are delivered first, their uptake is minimally augmented 232 by a second particle with 2PAF = 1.17, even when the second particle is highly (particle #1) and red (particle #2) E. coli inside of neutrophils, consistent with a large 256 fraction of particle #1 being phagocytosed at the same time and into the same 257 compartment as particle #2. Figure 5B shows data where particle #1 is NGs (green) and 258 particle #2 is E. coli (red). In this data, we observe minimal colocalization between the 259 two particles, consistent with flow cytometry data in Figure 4 showing no 2PAF effect 260 when NGs are given before E. coli. Figure 5C shows data where particle #1 is E. coli 261 (green) and particle #2 is NGs (red). The condition for which flow cytometry yielded 262 2PAF = 1.55 (EC -Serum NG; [Fig 5Cii] ), yielded imaging data with a high degree of 263 intracellular overlap between E. coli (particle #1) and NGs (particle #2). These imaging 264 data qualitatively support a key conclusion from our quantitative flow cytometry data: 265 opsonized NGs or E. coli not only augment uptake of previously delivered, non-266 opsonized bacteria, but also show localization into similar intracellular compartments.

To quantify the data from these microscopy experiments, images of each particle 268 were thresholded via Renyi entropic filtering to identify the portions of each image that 269 were positive for either particle #1 or particle #2. Guided by DiO membrane staining, individual cells allowed us to determine the fraction of pixels in each cell that contained 273 either particle #1 or particle #2. In panels 5D, 5F, and 5H, each point in the presented 274 scatter plots represents the results of this analysis for one neutrophil. The y-axis value 275 for each point represents the percentage of pixels in the neutrophil containing particle 276 #2 and the x-axis value represents the percentage of pixels in the neutrophil containing 277 particle #1. A line was fitted to the data for each condition. Lines with slope = 1 indicate 278 that each neutrophil took up equal quantities of particle #1 and particle #2. Lines with 279 slope > 1 indicate a tendency to take up more of particle #2 than particle #1. Lines with 280 slope < 1 indicate a tendency to take up more of particle #1 than particle #2. We 281 therefore used percentage of positive pixels as a metric for levels of particle #1 and 282 particle #2 uptake in each neutrophil. To derive 2PAF values from the imaging data, we 283 subtracted from our uptake values for each cell the average uptake values for the 2PAF 284 baseline conditions (conditions where particle #2 is not opsonized). We therefore 285 determined our 2PAF value in imaging experiments as the percent increase in either 286 particle #1 or particle #2 uptake for each imaged cell vs. the expected level of uptake 287 when particle #2 is not opsonized. These findings are depicted in panels 5E, 5G, and 288 5I. Figure 2C , data in Figure 5E show a clear 2PAF effect exerted by 297 particle #2 E. coli on particle #1 E. coli. When particle #1 E. coli is not opsonized, 298 uptake of particle #1 increases by ~20% if particle #2 E. coli is opsonized, compared to 299 data where particle #2 E. coli is not opsonized. 300 Panels 5F and 5G depict analysis of imaging data where particle #1 is NGs and 301 particle #2 is E. coli. R-squared values were less than 0.1 for all lines in panel 5F 302 except that for Serum+NGs -Serum+E. coli, indicating poor linear correlation between 303 particle #1 and particle #2 uptake for the conditions in this data. This indicates that, 304 when a given neutrophil takes up NGs as particle #1, improved uptake of E. coli as 305 particle #2 cannot be predicted for that same cell. Similarly, analysis of imaging-based 306 2PAF also indicates no 2PAF effect exerted by particle #2 E. coli on particle #1 NGs.

All imaging-based 2PAF calculations showed no change in particle #1 NG uptake 308 induced by opsonized particle #2 E. coli vs. 2PAF baseline conditions with non-309 opsonized particle #2 E. coli. These data suggest that co-localization is compromised 310 under these conditions, consistent with our understanding that multiple pathways can 311 lead to phagocytosis, not all necessarily leading to accumulation in the identical 312 intracellular compartment(Sahay, Alakhova and Kabanov, 2010).

Finally, panels 5H and 5I depict analysis of imaging data where particle #1 is E. 314 coli and particle #2 is NGs. As with data in panel 5D, panel 5H shows strong positive 315 correlation between particle #1 E. coli uptake and particle #2 NG uptake. For all serum 316 treatment conditions, particle #2 NGs were more likely to be taken up in neutrophils that 317 had already taken up particle #1 E. coli. This finding contrasts with the data in panel 5F, 318 where, when NGs are given as particle #1, there was no positive correlation between 319 NG uptake and E. coli uptake in any given cell. These findings match with analysis of conditions where E. coli is particle #1 and NGs are particle #2. Again, these image 323 analysis findings agree with our flow cytometry data: When non-opsonized E. coli is 324 particle #1, we observe a 33% increase in E. coli uptake per neutrophil when particle #2

NGs are serum-treated vs. when particle #2 NGs are not serum-treated. In imaging 326 data, NGs as particle #2 exert a clear 2PAF effect enhancing uptake of E. coli as 327 particle #1.

For conditions where we observed 2PAF effects (with E. coli as both particles #1 329 and #2 or with E. coli as particle #1 and NGs as particle #2), neutrophils were also 330 examined with three-dimensional confocal imaging ( Supplementary Figures 4 and 5) .

For our confocal imaging analysis, we computed quantities of NGs and E. coli in each 332 cell as in Figure 5 , but quantities of NGs and E. coli reflected fluorescent voxels, rather 333 than pixels. Additionally, we quantified voxels that contained both particle #1 and 334 particle #2, directly assessing three-dimensional colocalization of particles #1 and #2 in 335 the confocal images.

For experiments where both particles #1 and #2 were E. coli, ~80% of E. coli 337 particle #1 signal was spatially colocalized with E. coli particle #2 signal when E. coli 338 particle #2 was opsonized (panels B-C, green bars 1 and 3 in panel C). Only ~50% of 339 E. coli particle #1 signal was spatially colocalized with E. coli particle #2 signal when E. 

This finding fits well with a central part of our hypothesis as to the 2PAF mechanism: 342 When there is a 2PAF effect, delayed phagocytosis of particle #1 is driven by coincident 343 uptake of particle #2. In our confocal data, we find that particle #1 E. coli is mostly 344 found colocalized with opsonized particle #2 E. coli inside neutrophils. This 345 colocalization is diminished under conditions where 2PAF is diminished, when particle 346 #2 E. coli is not opsonized. Under conditions with 2PAF effects, particle #2 347 colocalization with particle #1 (Supplementary Figure 4 , panel A, red bars in panel C) 348 was less than particle #1 colocalization with particle #2 (Supplementary Figure 4, panel   349 A, green bars in panel C). This can be attributed to; a) opsonized particle #2 E. coli 350 being phagocytosed independently of particle #1 at a higher frequency than events 351 where particle #1 was phagocytosed independently of particle #2; b) opsonized particle 352 #2 E. coli being taken up in neutrophils to a greater degree than particle #1 E. coli. Supplementary Figure 4 indeed indicates higher average uptake values for 354 E. coli particle #2 vs. E. coli particle #1, especially in 2PAF-affected conditions.

Our prototypical 2PAF conditions, where E. coli were particle #1 and NGs were 356 particle #2, were also examined in confocal imaging data. Here particle #1 E. coli 357 colocalized with particle #2 NGs at ~50-60% frequency when particle #2 NGs were The data presented here are consistent with a model of neutrophil phagocytosis in 482 which a non-opsonized bacteria, that is poorly phagocytosed, is nonetheless still 483 available in a compartment (likely the surface plasma membrane) from which it can be 484 subsequently taken up in response to a more phagocytic-stimulatory particle, whether it 485 be an opsonized bacteria or an opsonized nanogel. Similarly, initial exposure to a 486 bacterial-like particle further enhances subsequent uptake of nanogels, even non-487 opsonized ones. Thus, the nanogel might be used in its opsonized form, even without 488 incorporated drugs, to enhance uptake of poorly-opsonized bacteria. Furthermore, 489 under these circumstances, the bacteria and nanoparticles are found in similar 490 intracellular compartments, suggesting that delivery to specific compartments of the 491 phagocytosing neutrophil might be possible.

Acknowledgements: 494 We are grateful for the support by the grants from the National Institutes of Health F32- which both measure the % of neutrophils that phagocytosed non-serum-opsonized green E. coli 666 (EC) which were present during the first incubation. When the second incubation was also with 667 EC, only 50% of neutrophils were positive for the green EC presented during the first 668

incubation. However, when the second incubation was with Serum EC, 85% of neutrophils were 669 positive for the green EC presented during the first incubation. Thus, when the first particle is EC, 670

the "2nd particle augmentation factor (2PAF)" was ~1.65 (inset, red). While Serum EC augment 671 uptake of (non-opsonized) EC by 65%, Serum EC do not significantly augment phagocytosis of 672

Serum EC that were present in the first incubation (2PAF = 1.0, blue bar in inset). Also notable 673

is that Serum EC delivered as the first particle augments the uptake of EC delivered second 674

Phagocytosing neutrophils produce and release high amounts 500 of the neutrophil-activating peptide 1/interleukin 8'

Nanoparticle uptake by circulating leukocytes: A major barrier 503 to tumor delivery

Bad bugs, no drugs: No ESKAPE! An update from the 506

Infectious Diseases Society of America

The role of complement C3 opsonization, C5a receptor, and 509 CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18

CR3), phagocytosis, and oxidative burst in human whole blood

Antibiotic resistance threats in the United 513

S Department of Health and Human services

Complement proteins bind to nanoparticle protein corona and 516 undergo dynamic exchange in vivo

ICAM-1 targeted nanogels loaded with dexamethasone 519 alleviate pulmonary inflammation

Burden of community-acquired pneumonia in North 522 American adults

Antimicrobial mechanisms of 525 phagocytes and bacterial evasion strategies

Neutrophil-Particle Interactions in Blood Circulation Drive 528

Particle Clearance and Alter Neutrophil Responses in Acute Inflammation

Persistent bacterial infections, antibiotic tolerance, 531 and the oxidative stress response', Virulence

Cobra Venom Factor-induced complement depletion protects 533 against lung ischemia reperfusion injury through alleviating blood-air barrier damage

Influence of the Escherichia coli capsule on 536 complement fixation and on phagocytosis and killing by human phagocytes

Modulatory Role of Surface Coating of Superparamagnetic Iron 539

Oxide Nanoworms in Complement Opsonization and Leukocyte Uptake HHS Public 540

The neonatal immune system

Phagocytosis by neutrophils', Microbes and Infection

Mechanisms and treatment of organ failure in 546 sepsis

Lysozyme -Dextran Core -Shell nanogels prepared via a green 548 process', Langmuir

Off to a slow start: Under-551 development of the complement system in term newborns is more substantial following 552 premature birth

CXCL5 Regulates Chemokine Scavenging and Pulmonary Host 555 Defense to Bacterial Infection

Complement activation turnover on surfaces of 558 nanoparticles

Neutrophil granule contents in 560 the pathogenesis of lung injury

Flexible Nanoparticles Reach Sterically Obscured 563

Endothelial Targets Inaccessible to Rigid Nanoparticles', Advanced Materials, 30(32)

Cross-linker-Modulated Nanogel Flexibility Correlates with 566

Tunable Targeting to a Sterically Impeded Endothelial Marker

Supramolecular arrangement of protein in nanoparticle 569 structures predicts nanoparticle tropism for neutrophils in acute lung inflammation

Blood sample collection in 572 small laboratory animals

With friends like these: The complex role of neutrophils in the 575 progression of severe pneumonia

Spread of antibiotic-resistance gene does not spell bacterial 580 apocalypse -yet'

Nanomedicine to fight infectious disease

Endocytosis of nanomedicines

Neonatal immunology

Role of complements C3 and C5 in the phagocytosis of 589 liposomes by human neutrophils', Pharmaceutical research

Challenges of antibacterial discovery

Dexamethasone modulates immature neutrophils and interferon 595 programming in severe COVID-19'

Multidrug resistant bacteria in critically ill patients: a step further 598 antibiotic therapy

Immunoglobulin deposition on biomolecule corona determines 601 complement opsonization efficiency of preclinical and clinical nanoparticles'

Some aspects of the humoral immunity and the phagocytic 604 function in newborn infants

Mortality in the United States

The lung is a host defense niche for immediate neutrophil-609 mediated vascular protection

NETosis: how vital is it?', Blood

Transepithelial migration of 614 neutrophils: Mechanisms and implications for acute lung injury

Factors That Influence Infant Immunity

Vaccine Responses

NG"). Importantly, before 626 exposure to the neutrophils, select aliquots of nanogels and E. coli were opsonized by serum and 627 thoroughly washed before exposure to neutrophils. B) Representative dot-plots of the neutrophils 628 subjected to flow cytometry after the second incubation, examining for green fluorescence (x-axis, 629 indicating phagocytosis of the E. coli) and red (y-axis, nanogels)

are represented as dots to the right of the vertical bar in the middle of the plot, with that 634 threshold determined by measuring fluorescence of E

When these neutrophils encounter Serum NG, they phagocytose not just the 640 nanogels, but also the E. coli (third neutrophil image), causing the E. coli to fluoresce in the low 641 pH of the phagosome (fourth neutrophil). D) Quantification of the flow cytometry experiments with 642 n=6 biological replicates for each sample. The bottom axis lists for the 1st and 2nd incubations 643 whether the E. coli and nanogels were first exposed to serum or not. The y-axis lists the % of 644 neutrophils that were positive for phagocytosed E. coli (recalling that pH-sensitive E.coli on the 645 surface of neutrophils will not fluoresce)

Serum NG only augment phagocytosis of non-opsonized E. coli

Representative flow cytometry dot-plots. C) Quantification of n=6 biological replicate. The most 698 notable comparison again is the first and third green bars, which indicate that serum

This is clinically relevant 702 because it means that if a neutrophil phagocytoses a nanogel and then later encounters a 703 bacterium

Microscopy confirms that serum-opsonized nanogels improve internalization of 708 non-opsonized E. coli, and that they then share significant colocalization within 709 neutrophils. A) Example images of neutrophils given two different labeled E. coli doses in 710 sequence, for different serum pretreatment conditions applied to the E. coli. B) Example images 711 of neutrophils given lysozyme-dextran nanogels (NGs) prior to E. coli, for different serum 712

The 715 horizontal coordinate indicates quantity of green E. coli signal and the vertical coordinate 716 indicates quantity of red E. coli signal. Higher slope indicates that cells generally have more

All conditions showed strong positive correlation 718 between red and green signal, indicating that cells taking up green E. coli, given first, were likely 719 to take up red E. coli, given subsequently. E) 2PAF enhancement of first particle (green) E. coli 720 by addition of serum to second particle (red) E. coli., in imaging experiments as in (A)

Data is as presented in (D-E), but for imaging experiments as in (B), wherein NGs were 725 given to neutrophils before E. coli. There was poor correlation between per-cell NG signal and 726

for all serum pretreatment conditions, indicating that neutrophil uptake of NGs 727 does not predict subsequent uptake of E. coli. G) indicates no 2PAF effect exerted by second 728 particle E. coli on NGs. H-I) Data is as presented in (D-E) and (F-G), but for imaging 729 experiments as in (C), wherein E. coli was given to neutrophils before NGs. In (H), there was 730 strong correlation between NG and E. coli uptake in any given cell, when E. coli was taken up 731 before neutrophil exposure to NGs. In (I)

 Figure 3 . Serum-opsonized nanogels do not augment neutrophil association with 678 previously delivered nanogels. A) Similar to the paradigm described in Figure 1 , neutrophils 679were exposed to green nanogels, followed by washing to remove unbound neutrophils, then 680 exposed to red nanogels, washed, and then the neutrophils were subjected to flow cytometry. B) 681Representative flow cytometry dot-plots. C) Quantification of n=6 biological replicates. The most 682 notable comparison is the first and third green bars, which indicate that serum-opsonized 683 nanogels ("Serum NGs") do not augment the fraction of neutrophils that are positive for binding 684 to non-serum-exposed nanogels ("NGs"). Notably, unlike the pH-sensitive E. coli used in Figs 1 685 & 2, nanogels fluoresce both when bound to the neutrophil surface and when in the 686 phagosome. Thus, it is possible that some NGs are bound to the neutrophil surface and then 687internalized after exposure to Serum NGs, but this assay cannot detect such internalization 688 events. Another major result of this set of conditions is that Serum NGs have uniformly high 689 uptake into neutrophils regardless of whether the neutrophils were first exposed to other nanogels 690(NGs or Serum NGs), suggesting the neutrophils do not saturate their uptake of Serum NGs in 691 this dynamic range (they do not "get full"). 692 693