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In situ neutrophil efferocytosis shapes T cell immunity to influenza infection

Abstract

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell–mediated immune protection.

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Fig. 1: In situ efferocytosis of neutrophils in the influenza-infected trachea.
Fig. 2: Simultaneous visualization of neutrophils and monocytes/macrophages in the influenza-infected trachea of Ly6GCre/ROSAtdTomato/Csf1r-EGFP mice.
Fig. 3: Visualizing dynamic neutrophil–phagocyte interactions during the resolution of infection.
Fig. 4: Neutrophils promote the differentiation of inflammatory monocytes into APCs for potent CD8+ T cell activation.
Fig. 5: Transcriptional analysis of the effects of neutrophil efferocytosis on the monocyte differentiation into APCs.
Fig. 6: EGF derived from apoptotic neutrophils promotes the differentiation of inflammatory monocytes into APCs.
Fig. 7: EGF released from apoptotic neutrophils promotes T cell effector functions.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank T. Hohl and A. Rivera for CCR2-DTR mouse and F. Yan for EGFR-cKO (Egfrflox/LysMCre) mouse. We especially thank M. Gunzer for CatchupIVM-Red mouse, Y. Gao for the technical assistance with IV-MPM and J. Ashton for help with transcriptomic analyses and depositing RNA-sequencing data. This project was financially supported through grants from the National Institute of Health (nos. AI143182, AI147362 and AI149775 to M.K., no. AI102851 to M.K., D.J.F. and D.J.T., no. AI138415 to H.P. and no. AI070826 to D.J.F.)

Author information

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Authors

Contributions

K.L. conducted all in vivo imaging and the mouse influenza infection experiments and analyzed data. T.H.K., A.T., A.M.A., H.P., E.C.R., D.J.F. and D.J.T. performed experiments and analyzed data. M.K. and K.L. conceived, designed and directed this study. M.K. and K.L. wrote the manuscript with suggestions from all the authors. All authors reviewed and discussed the manuscript.

Corresponding authors

Correspondence to Kihong Lim or Minsoo Kim.

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The authors declare no competing interests.

Additional information

Peer review information Jamie D. K. Wilson and Ioana Visan were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Leukocyte recruitment in the influenza-infected trachea.

a, Schematic representation of the host immune responses in the airway during influenza infection. The various waves correspond to schemes (neutrophils, monocytes, CD8+ T cells, and macrophages) that summarize the data presented in Fig. 1a,b. b, Flow cytometry analysis of the neutrophil numbers in the dLN (mediastinal lymph node) of naïve or influenza-infected mice. c, Apoptotic neutrophils from WT or LysMCre mice (upper chamber) and bone marrow (BM)-derived macrophages from ROSAtdTomato mice (bottom chamber) were co-cultured for 7 days in the cell impermeable transwell system (0.4 μm pore size). Bar, 50 μm. d, Flow cytometry analysis of the BM and dLN of the mice in the experiments described in Fig. 1j. Note the lack of tdTomato+ cells in the BM and dLN. e, Live BM cells isolated from LysMCre or WT mice (20 – 50 x 106) were injected into the peritoneum of recipient ROSAtdTomato mice. Flow cytometry analysis of the peritoneal lavage revealed tdTomato+/F4/80+ macrophages in the mice that received LysMCre cells. Representative plots of the flow cytometry data from five independent experiments are shown. f, Blood cells (day 4 post HKx31 infection) from Ly6GCre/ROSAtdTomato/Csf1r-EGFP mice were analyzed by flow cytometry.

Extended Data Fig. 2 Monocytes promote neutrophil efferocytosis.

a, In vitro fluorescence images of apoptotic neutrophil-engulfed monocytes. Monocytes (green; GFP+Ly6C+Ly6G) were sorted from influenza-infected Csf1r-EGFP mouse lungs and co-incubated with apoptotic neutrophils (red) isolated from Ly6GCre/ROSAtdTomato mice. Three hours later, Ly6GCsfr1GFP cells were sorted again and imaged by fluorescence microscopy. Arrows indicate neutrophil-engulfed monocytes. Scale bar, 25 μm. b, Numbers of monocytes (left) and total neutrophils (middle) and percentages of Annexin V+ neutrophils (right) in the influenza-infected trachea of WT or CCR2 KO mice (n ≥ 3 per group).

Extended Data Fig. 3 Migration of early apoptotic neutrophil.

a, In vitro migration of Annexin V vs. Annexin V+ neutrophils on the ICAM-1 coated surface. The mean velocity, displacement, and meandering index of the neutrophils are shown. n = 3 per group. Statistical differences were assessed by a nonparametric Mann-Whitney test. b, Chemokine microarray performed with total lysates of monocytes harvested on day 5 post infection from the influenza-infected mouse lung. Chemokine expression levels are expressed as arbitrary units measured by densitometry. c, Neutrophil migration via ICAM-1 with the indicated chemokines. A single assay analyzed at least 10 cells (n = 3 assays per group). *p <0.05 compared with the control. Data were analyzed with the Kruskal-Wallis test followed by Dunns posttest.

Extended Data Fig. 4 Monocytes promote T cell activation.

a, Flow cytometry analysis of neutrophils and monocytes from influenza-infected mouse trachea with or without neutrophil depletion. Cells were stained with Ly6C antibody and CD11b antibody (mean ± SEM, n = 4 mice per group). b, Numbers of monocytes (CD11b+Ly6C+Ly6G) in the influenza-infected mouse trachea with or without neutrophil depletion. Data are presented as the mean ± SEM, n = 4 mice per group. Statistical differences were assessed by nonparametric Mann-Whitney test. c, RFP+ monocytes sorted from the trachea and lungs of HKx31-OVA virus-infected CCR2RFP/+ mice with or without neutrophil depletion were incubated with CD8+ T cells isolated from influenza-infected OT-I mouse dLNs. The monocyte-T cell conjugates were measured by microscopy. Conjugation (%); # monocytes bound to CD8+ T cells/# total monocytes. n ≥ 3 per group. Statistical differences were assessed by nonparametric Mann-Whitney test. d, Numbers of OT-I CD8+ T cells from the dLN and trachea of HKx31-OVA virus-infected WT or CCR2 KO mice. Data are presented as the mean ± SEM, n = 3 mice per group. Statistical differences were assessed by nonparametric Mann-Whitney test. e, Numbers of monocytes (CD11b+Ly6C+Ly6G) in the influenza-infected trachea of CCR2-DTR and WT mice on day 6 post infection. Diphtheria toxin was injected into both mouse strains on days -1, +1, +3, and +5 post infection. f, Flow cytometry analysis of the production of IFN-γ, TNF, and Granzyme B (Gzm B) in CD8+ T cells on day 7 post infection from WT vs. CCR2-DTR influenza-infected mice treated with diphtheria toxin. Statistical differences were assessed by nonparametric Mann-Whitney test.

Extended Data Fig. 5 Neutrophil EGF regulates DC-differentiation of monocytes.

a, Immunofluorescence image of a permeabilized neutrophil stained with Abs against Ly6G (green) and EGF (red). Scale bar, 5 μm. b, Flow cytometry analysis of EGF in permeabilized mouse neutrophils (day 4 post-infection). c, Secretion of EGF from neutrophils undergoing spontaneous apoptosis. d, Flow cytometry analysis of phagocytic activity (tdTomato) of monocytes (Csf1r-GFP) isolated from the influenza infected lung after co-incubation with tdTomato+ apoptotic neutrophils for 12 h in the presence of PBS or erlotinib (0.1 μg/ml). e, Monocytes (Csf1r-GFP) were isolated from the influenza infected lung. After co-incubation with tdTomato+ apoptotic neutrophils for 12 h in the presence of PBS or erlotinib (0.1 μg/ml), neutrophil-engulfed (tdTomato+) and non-engulfed (tdTomato) monocytes were sorted, pulsed with OVA peptide, and incubated with CD8+ T cells isolated from influenza-infected OT-I mouse dLNs. Monocyte-T cell conjugation was measured by microscopy. Data are presented as the mean ± SEM, n ≥ 3 mice per group. Statistical differences were assessed by nonparametric Mann-Whitney test.

Source data

Supplementary information

Supplementary Information

Primers used in RT–qPCR.

Reporting Summary

Supplementary Video 1

Neutrophil migration in the influenza-infected mouse trachea. 3D reconstruction of an IV-MPM image showing migration of neutrophils in the mouse trachea (Ly6GCre/ROSAtdTomato) (day 5 after influenza infection) without overt neutrophil reverse migration. Red, neutrophil; white, blood vessel (Ab CD31). Scale bar, 50 μm. See Fig. 1c,d.

Supplementary Video 2

Live image of a color switch in ROSAtdTomato macrophages cocultured with apoptotic LysMCre neutrophils. In cocultures of apoptotic LysMCre neutrophils and ROSAtdTomato macrophages, converted tdTomato+ (red) cells appeared within 24 h and lived for up to 7 d. Scale bar, 20 μm. See Fig. 1f–h.

Supplementary Video 3

In vivo color switch in ROSAtdTomato phagocytes after engulfing apoptotic LysMCre neutrophils. A representative 3D image showing the in vivo color switch in ROSAtdTomato phagocytes. Neutrophils from LysMCre or WT mice were injected into the ears of ROSAtdTomato B6-Albino recipient mice. Five days later, the presence of tdTomato+ phagocytes (red) in the ears was examined by whole mount tissue staining with CD68 antibody (green) and DAPI (blue). See Fig. 1i.

Supplementary Video 4

IV-MPM of naive Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). In the steady state, 3D reconstruction of an IV-MPM image showed a main tissue-resident macrophage population (green) in the naive trachea. Scale bar, 50 μm. See Fig. 2f.

Supplementary Video 5

IV-MPM of influenza-infected Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). First part, examination of a 3D reconstruction of an IV-MPM image of the tracheal tissue on day 4 postinfection showed the presence of many GFP+ cells (monocyte/macrophage, white arrow) with engulfed tdTomato fluorescence (neutrophil). Scale bar, 20 μm. Second part, in vitro examples of tdTomato+GFP+ monocytes after incubation of tdTomato+ neutrophils and GFP+ monocytes. Scale bar, 20 μm.

Supplementary Video 6

IV-MPM of influenza-infected Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). 3D presentation showing the presence of a GFP+ cell (monocyte/macrophage) actively processing intracellular tdTomato fluorescence after engulfing neutrophils.

Supplementary Video 7

IV-MPM of influenza-infected (day 3) Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). Examination of tracheal tissue on day 3 postinfection showed the presence of both sessile and highly motile GFP+ cells (monocyte/macrophage) and actively migrating neutrophils (orange). Scale bar, 50 μm.

Supplementary Video 8

IV-MPM of influenza-infected (day 5) Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). On day 5 postinfection, most GFP+ cells became less motile, showing a morphology similar to that of tissue-resident macrophages, while most neutrophils (orange) remained highly motile. White, blood vessel (Ab CD31). Scale bar, 50 μm.

Supplementary Video 9

IV-MPM of influenza-infected (day 6) Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). On day 6 postinfection, most GFP+ cells were less motile, showing a morphology similar to that of tissue-resident macrophages, while most neutrophils (orange) remained highly motile. Note that the highly motile neutrophils were not newly extravasating cells from the blood vessels due to tissue injury. Scale bar, 50 μm.

Supplementary Video 10

IV-MPM of influenza-infected Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). 3D reconstruction and two-dimensional projection of IV-MPM images showing migration of neutrophils in the mouse trachea. Neutrophils (orange) in the influenza-infected trachea (day 6) were highly motile during the resolution phase, actively forming multiple serial encounters with the nonmigratory surrounding monocytes/macrophages (green). See Fig. 3c,d.

Supplementary Video 11

IV-MPM of influenza-infected Ly6GCreROSAtdTomatoCsf1r-EGFP mice (trachea). 3D reconstructions of IV-MPM images showing dynamic interactions between neutrophils and phagocytes (day 5 after influenza infection). First part, a highly motile neutrophil (orange, red arrow) during the resolution phase stopped at the target phagocyte (green, yellow arrow) to be engulfed. Second part, both neutrophil (orange, red arrow) and phagocyte (green, yellow arrow) actively migrated before the engulfment. See Fig. 3f.

Supplementary Video 12

T cell–monocyte-derived APC interaction in the influenza-infected trachea. IV-MPM of OT-I GFP+ CD8+ T cells in the influenza-infected trachea of CCR2-RFP mice on day 7 postinfection. OVA-specific CD8+ T cells (green) and CCR2+ monocytes (red) are shown. Scale bar, 50 μm. See Fig. 4e–h. First part, OT-I GFP+ CD8+ T cells and CCR2-RFP monocyte interaction in the HKx31-OVA virus-infected trachea. Second part, OT-I GFP+ CD8+ T cells and CCR2-RFP monocyte interaction in the HKx31-OVA virus-infected trachea. Virus-specific CD8+ T cell interactions with CCR2+ cells were assessed after i.v. injection of MHC I blocking (upper) or isotype control antibodies (lower). Third part, OT-I GFP+ CD8+ T cells and CCR2-RFP monocyte interaction in the HKx31-OVA virus-infected trachea after neutrophil depletion.

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Source Data Extended Data Fig. 5

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Lim, K., Kim, Th., Trzeciak, A. et al. In situ neutrophil efferocytosis shapes T cell immunity to influenza infection. Nat Immunol 21, 1046–1057 (2020). https://doi.org/10.1038/s41590-020-0746-x

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