Neutrophils dominate in opsonic phagocytosis of P. falciparum blood-stage merozoites and protect against febrile malaria

Antibody-mediated opsonic phagocytosis (OP) of Plasmodium falciparum blood-stage merozoites has been associated with protection against malaria. However, the precise contribution of different peripheral blood phagocytes in the OP mechanism remains unknown. Here, we developed an in vitro OP assay using peripheral blood leukocytes that allowed us to quantify the contribution of each phagocytic cell type in the OP of merozoites. We found that CD14 + +CD16− monocytes were the dominant phagocytic cells at very low antibody levels and Fc gamma receptor (FcγR) IIA plays a key role. At higher antibody levels however, neutrophils were the main phagocytes in the OP of merozoites with FcγRIIIB acting synergistically with FcγRIIA in the process. We found that OP activity by neutrophils was strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India. Our results demonstrate that peripheral blood neutrophils are the main phagocytes of P. falciparum blood-stage merozoites.

In this study, the authors developed an in vitro opsonic phagocytosis assay using peripheral blood leukocytes and clearly demonstrate that CD14++CD16-monocytes are the dominant phagocytic cells at very low antibody levels. They found that at higher antibody levels neutrophils are the main phagocytes in the opsonic phagocytosis of merozoites. In the case of monocytes mediated phagocytosis (FcγR) IIA plays a key role while neutrophils mediated phagocytosis FcγRIIIB acting synergistically with FcγRIIA. They also observed that opsonic phagocytosis activity by neutrophils is strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India and conclude that neutrophils are the main phagocytes of P. falciparum blood-stage merozoites.
This work is technically sound and the claims are supported by the experimental data. I recommend the consideration of this manuscript for publication.

Reviewer #2 (Remarks to the Author):
This is an interesting and well-designed work describing in detail the role of neutrophils and monocytes in the clearance of P. falciparum merozoites in vitro and in vivo. The in vitro experiments are carefully performed and analyzed. The data is well explained and supports the conclusions. The second part performing experiments with plasma samples of malaria patients, is an essential component, suggesting the in vitro results represent the real disease. This last part requires more clarifications, since the authors rely too much on previous descriptions without even giving a brief summary of some of the important data here. However, the results are solid and support the conclusions of the study.
Minor points: In Fig. 3A the difference between ati-CD16 and anti-CD16+antiCD32 appears to be small in absolute numbers, although it is 61% proportionally it doesn't seem to be an important component of the phagocytic response. Error bars should be plotted for every condition relative to isotype control.
Line 156: This sentence is confusing since it doesn't explain that the phagocytic responses were measured in neutrophils from blood donors, not from Ghanaian children "…we used data and samples from a well-established longitudinal cohort survey (LCS) performed in Ghanaian children [19]. A wide range of phagocytic responses were observed for both neutrophils…" Line 176: "Next, we repeated the phagocytosis assays using highly purified neutrophils (>99%) and merozoites opsonized with the same plasma". It is not clear what plasma are they referring to, the high phagocytosis-inducing plasma? All plasma in the cohort? Both cohorts? These data may be more clearly understood if presented in a table comparing both cohorts.
Line 194: the abbreviation ADCI is not spelled out in the manuscript.

Reviewer #1 (Remarks to the Author):
In this study, the authors developed an in vitro opsonic phagocytosis assay using peripheral blood leukocytes and clearly demonstrate that CD14++CD16-monocytes are the dominant phagocytic cells at very low antibody levels. They found that at higher antibody levels neutrophils are the main phagocytes in the opsonic phagocytosis of merozoites. In the case of monocytes mediated phagocytosis (FcγR) IIA plays a key role while neutrophils mediated phagocytosis FcγRIIIB acting synergistically with FcγRIIA. They also observed that opsonic phagocytosis activity by neutrophils is strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India and conclude that neutrophils are the main phagocytes of P. falciparum blood-stage merozoites.
This work is technically sound and the claims are supported by the experimental data. I recommend the consideration of this manuscript for publication.
We thank the Reviewer for acknowledging our work and recommending the consideration of this manuscript for publication.

Reviewer #2 (Remarks to the Author):
This is an interesting and well-designed work describing in detail the role of neutrophils and monocytes in the clearance of P. falciparum merozoites in vitro and in vivo. The in vitro experiments are carefully performed and analyzed. The data is well explained and supports the conclusions. The second part performing experiments with plasma samples of malaria patients, is an essential component, suggesting the in vitro results represent the real disease. This last part requires more clarifications, since the authors rely too much on previous descriptions without even giving a brief summary of some of the important data here. However, the results are solid and support the conclusions of the study.
We thank this Reviewer for appreciating our work and highlighting some important points which have been addressed now. Please see our point by point response below. Necessary modifications have been introduced in the revised manuscript.

Comment
Response 1) In Fig. 3A the difference between ati-CD16 and anti-CD16+antiCD32 appears to be small in absolute numbers, although it is 61% proportionally it doesn't seem to be an important component of the phagocytic response. Error bars should be plotted for every condition relative to isotype control. Figure 3 has been modified to show the comparison of every condition relative to the respective isotype control. Figure 3 legend has been modified accordingly.

Figure 3
2) Line 156: This sentence is confusing since it doesn't explain that the phagocytic responses were measured in neutrophils from blood donors, not from Ghanaian children "…we used data and samples from a well-established longitudinal cohort survey (LCS) performed in Ghanaian children [19]. A wide range of phagocytic responses were observed for both neutrophils…" PBLs and purified neutrophils were obtained from Danish blood donors. This has been clarified throughout the manuscript.
Lines 156-157 "The PBLs used here in phagocytosis assays were obtained from a Danish blood donor." Lines 178-180 "Next, we repeated the phagocytosis assays using highly purified neutrophils (>99%; obtained from a Danish blood donor) and merozoites opsonized with the same Ghanaian plasma samples (n=140) [19] (Supplementary Table  1)." Lines 187-189 "In general, neutrophils in PBL preparations (obtained from a Danish blood donor) showed higher phagocytosis activity than monocytes (Figure 4c)." Lines 341-342 "Peripheral blood leukocytes (PBLs) were isolated from whole blood samples from Danish donors by centrifugation at 800 g for 25 min"

Line 465-468 "Paired aligned dot plots coupled to box and whiskers plots showing the number of EtBr-positive neutrophils and monocytes present in PBLs from Danish blood donors which were used as effector cells to test the phagocytosis mediating ability of antibodies present in Ghanaian (a) and
Indian (c) cohort plasma samples." 3) Line 176: "Next, we repeated the phagocytosis assays using highly purified neutrophils (>99%) and merozoites opsonized with the same plasma". It is not clear what plasma are they referring to, the high phagocytosis-inducing plasma? All plasma in the cohort? Both cohorts? These data may be  (Figure 4c)." 4) Line 194: the abbreviation ADCI is not spelled out in the manuscript This has been corrected. Lines 200-201 "The antibody-dependent cellular inhibition (ADCI) assay measures the parasite killing capacity of antibodies in collaboration with human monocytes." 5) Fig. 5 and the text show that there is no correlation between phagocytosis and ADCI. The title of this section states "IgG mediated merozoite-phagocytosis and ADCI, both independently correlate with protection against We agree. The title for this section has been changed to better reflect the text and Figure 5: Lines 198-199 "Relationship between antibodymediated merozite-phagocytosis by PBLs and clinical malaria." However, this is not even mentioned in the text or in Fig. 5.