Immunology and Inflammation

Cytosolic S100A8/A9 promotes Ca²⁺ supply at LFA-1 adhesion clusters during neutrophil recruitment

Matteo Napoli
Roland Immler
Ina Rohwedder
Valerio Lupperger
Johannes Pfabe
Mariano Gonzalez Pisfil
Anna Yevtushenko
Thomas Vogl
Johannes Roth
Melanie Salvermoser
Steffen Dietzel
Marjan Slak Rupnik
Carsten Marr
Barbara Walzog
Markus Sperandio
Monika Pruenster author has email address

Walter Brendel Center of Experimental Medicine, Biomedical Center, Institute of Cardiovascular Physiology and Pathophysiology, Ludwig-Maximilians-University, Planegg-Martinsried, Germany
Institute of AI for Health, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany
Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
Institute of Immunology, University of Muenster, Muenster, Germany

https://doi.org/10.7554/eLife.96810.1

Open access
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Figures and data

Cytosolic S100A8/A9 regulates leukocyte recruitment in vivo regardless of extracellular S100A8/A9
(a) ELISA measurements of S100A8/A9 levels in supernatants of WT bone marrow neutrophils stimulated for 10min with PBS, E-selectin or lysed with Triton X-100(mean+SEM, n=6 mice per group, RM one-way ANOVA, Holm-Sidak’s multiple comparison). (b) Schematic model of the mouse cremaster muscle preparation for intravital microscopy and representative picture of a vessel showing rolling and adherent cells. WT and Mrp14^-/- mice were stimulated i.s. with TNF-α 2h prior to cremaster muscle post-capillary venules imaging by intravital microscopy. Quantification of (c) number or rolling (rolling flux fraction) and (d) number of adherent neutrophils per vessel surface of WT and Mrp14^-/- mice [mean+SEM, n=5 mice per group, 25 (WT) and 30 (Mrp14^-/-) vessels, unpaired Student’s t-test]. (e) Correlation between physiological vessel shear rates and number of adherent neutrophils in WT and Mrp14^-/- mice [n=25 (WT) and 30 (Mrp14^-/-) vessels of 5 mice per group, Pearson correlation]. (f) Schematic model of sterile inflammation induced by exteriorizing WT and Mrp14^-/- cremaster muscles. (g) Analysis of number of adherent leukocytes by intravital microscopy before and after S100A8/A9^mut intra-arterial injection [mean+SEM, n=3 mice per group, 3 (WT) and 3 (Mrp14^-/-) vessels, 2way ANOVA, Sidak’s multiple comparison]. (h) Representative Giemsa staining micrographs of TNF-α stimulated WT and Mrp14^-/- cremaster muscles (representative micrographs, scale bar =30µm, arrows: transmigrated neutrophils) and (i) quantification of number of perivascular neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 55 (Mrp14^-/-) vessels, unpaired Student’s t-test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Loss of cytosolic S100A8/A9 impairs neutrophil adhesion under flow conditions without affecting β₂ integrin activation
(a) Schematic representation of blood harvesting from WT and Mrp14^-/-mice via a carotid arery catheter and perfusion into self-made flow cambers coated with E-selectin, ICAM-1, and CXCL1. Analysis of (b) number of rolling and (c) number of adherent leukocytes FOV^- ¹[mean+SEM, n=4 mice per group, 10 (WT) and 12 (Mrp14^-/-) flow chambers, paired Student’s t-test]. (d) Schematic representation of the soluble ICAM-1 binding assay using bone marrow neutrophils stimulated with PBS control or CXCL1 (10nM) assessed by (e) flow cytometry (MFI=median fluorescence intensity, mean+SEM, n=5 mice per group, 2way ANOVA, Sidak’s multiple comparison). (f) Spectroscopy fluorescence intensity analysis of percentage of adherent WT and Mrp14^-/- neutrophils, seeded for 5min on ICAM-1 coated plates and stimulated with PBS or CXCL1 (10nM) for 10min (mean+SEM, n=4 mice per group, 2way ANOVA, Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 is crucial for neutrophil spreading, crawling and post-arrest modifications under flow
(a) Representative bright-field pictures of WT and Mrp14^-/- neutrophils spreading over E-selectin, ICAM-1, and CXCL1 coated glass capillaries (scale bar=10μm). Analysis of cell shape parameters (b) area, perimeter, (c) circularity [4π * (Area/Perimeter)] and solidity [Area/Convex area] over time [mean+SEM, n=103 (WT) and 96 (Mrp14^-/-) neutrophils of 4 mice per group, unpaired Student’s t-test]. (d) Rose plot diagrams representative of migratory crawling trajectories of WT and Mrp14^-/- neutrophils in flow chambers coated with E-selectin, ICAM-1, and CXCL1 under flow (2dyne cm^-2). Analysis of (e) crawling distance, (f) directionality of migration and (g) crawling velocity of WT and Mrp14^-/- neutrophils [mean+SEM, n=5 mice per group, 113 (WT) and 109 (Mrp14^-/-) cells, paired Student’s t-test]. Western blot analysis of ICAM-1 induced (h) Pyk2 and (i) Paxillin phosphorylation of WT and Mrp14^-/- neutrophils upon CXCL1 stimulation (10nM) (mean+SEM, representative western blot of n≥4 mice per group, 2way ANOVA, Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 is crucial for neutrophil spreading, crawling and post-arrest modifications under flow
(a) Representative bright-field pictures of WT and Mrp14^-/- neutrophils spreading over E-selectin, ICAM-1, and CXCL1 coated glass capillaries (scale bar=10μm). Analysis of cell shape parameters (b) area, perimeter, (c) circularity [4π * (Area/Perimeter)] and solidity [Area/Convex area] over time [mean+SEM, n=103 (WT) and 96 (Mrp14^-/-) neutrophils of 4 mice per group, unpaired Student’s t-test]. (d) Rose plot diagrams representative of migratory crawling trajectories of WT and Mrp14^-/- neutrophils in flow chambers coated with E-selectin, ICAM-1, and CXCL1 under flow (2dyne cm^-2). Analysis of (e) crawling distance, (f) directionality of migration and (g) crawling velocity of WT and Mrp14^-/- neutrophils [mean+SEM, n=5 mice per group, 113 (WT) and 109 (Mrp14^-/-) cells, paired Student’s t-test]. Western blot analysis of ICAM-1 induced (h) Pyk2 and (i) Paxillin phosphorylation of WT and Mrp14^-/- neutrophils upon CXCL1 stimulation (10nM) (mean+SEM, representative western blot of n≥4 mice per group, 2way ANOVA, Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 drives neutrophil cytoskeletal rearrangement by regulating LFA-1 nanocluster formation and Ca²⁺ availability within the clusters
(a) Representative confocal images of LFA-1 staining in WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 crawling neutrophils on E-selectin, ICAM-1, and CXCL1 coated flow chambers (scale bar=10μm). (b) Segmentation of LFA-1 signals through automatic thresholding (scale bar=10μm). (c) Size-excluded LFA-1 nanoclusters of 0.15μm² minimum size from previously thresholded images (scale bar=10μm). (d) Single cell analysis of average number of LFA-1 nanoclusters in min 0-1, 5-6 and 9-10 of analysis of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (e) Representative confocal images of Ca²⁺ signals in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils (scale bar=10μm) and (f) Ca²⁺ signals in the previously segmented LFA-1 nanoclusters (scale bar=10μm). (g) Quantification of subcellular Ca²⁺ levels in the LFA-1 nanocluster area in min 0-1, 5-6 and 9-10 in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) cells, 2way ANOVA, Sidak’s multiple comparison]. (h) Segmented LFA-1 cluster negative areas (scale bar=10μm) and (i) representative confocal images of Ca²⁺ signals in the LFA-1 cluster negative areas (scale bar=10μm). (j) Analysis of cytosolic Ca²⁺ levels in the LFA-1 cluster negative areas in min 0-1, 5-6 and 9-10 of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (k) Representative confocal micrographs of LFA-1 nanocluster spatial aggregation in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils, within 10μm² area and minimum 10 LFA-1 nanoclusters considered (≥ 10 LFA-1 nanoclusters within 10µm², yellow circles=spatial aggregation area, scale bar=10μm). (l) Analysis of spatially aggregated LFA-1 nanoclusters of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) cells, unpaired Student’s t-test]. (m) Segmentation of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophil area through Lyz2 channel automatic thresholding and (n) representative confocal images of respective F-actin signals. (o) Analysis of F-actin intensity normalized to the cell area in min 0-1, 5-6 and 9-10 of WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 74 (WT) and 66 (Mrp14^-/-) cells, 2way ANOVA, Sidak’s multiple comparison]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 drives neutrophil cytoskeletal rearrangement by regulating LFA-1 nanocluster formation and Ca²⁺ availability within the clusters
(a) Representative confocal images of LFA-1 staining in WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 crawling neutrophils on E-selectin, ICAM-1, and CXCL1 coated flow chambers (scale bar=10μm). (b) Segmentation of LFA-1 signals through automatic thresholding (scale bar=10μm). (c) Size-excluded LFA-1 nanoclusters of 0.15μm² minimum size from previously thresholded images (scale bar=10μm). (d) Single cell analysis of average number of LFA-1 nanoclusters in min 0-1, 5-6 and 9-10 of analysis of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (e) Representative confocal images of Ca²⁺ signals in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils (scale bar=10μm) and (f) Ca²⁺ signals in the previously segmented LFA-1 nanoclusters (scale bar=10μm). (g) Quantification of subcellular Ca²⁺ levels in the LFA-1 nanocluster area in min 0-1, 5-6 and 9-10 in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) cells, 2way ANOVA, Sidak’s multiple comparison]. (h) Segmented LFA-1 cluster negative areas (scale bar=10μm) and (i) representative confocal images of Ca²⁺ signals in the LFA-1 cluster negative areas (scale bar=10μm). (j) Analysis of cytosolic Ca²⁺ levels in the LFA-1 cluster negative areas in min 0-1, 5-6 and 9-10 of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (k) Representative confocal micrographs of LFA-1 nanocluster spatial aggregation in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils, within 10μm² area and minimum 10 LFA-1 nanoclusters considered (≥ 10 LFA-1 nanoclusters within 10µm², yellow circles=spatial aggregation area, scale bar=10μm). (l) Analysis of spatially aggregated LFA-1 nanoclusters of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 56 (WT) and 54 (Mrp14^-/-) cells, unpaired Student’s t-test]. (m) Segmentation of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophil area through Lyz2 channel automatic thresholding and (n) representative confocal images of respective F-actin signals. (o) Analysis of F-actin intensity normalized to the cell area in min 0-1, 5-6 and 9-10 of WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, 74 (WT) and 66 (Mrp14^-/-) cells, 2way ANOVA, Sidak’s multiple comparison]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 is dispensable for chemokine induced ER store Ca²⁺ release and for the initial phase of SOCE
(a) Average flow cytometry kinetic graphs of Ca²⁺ store release in the absence of extracellular Ca²⁺ (Ca²⁺ free medium) in WT and Mrp14^-/- neutrophils upon CXCL1 stimulation (traces are shown as mean+SEM, n=5 mice per group). (b) Rapid ER store Ca²⁺ release (MFI _peak/ MFI _0-30s) of WT and Mrp14^-/- neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. (c) Quantification of Ca²⁺ levels under baseline conditions (MFI _0-30s) [mean+SEM, n=5 mice per group, paired Student’s t-test]. (d) Average flow cytometry kinetic graphs of Ca²⁺ influx in the presence of extracellular Ca²⁺(HBSS medium, 1.5mM Ca²⁺) of WT and Mrp14^-/- neutrophils upon CXCL1 stimulation (traces are shown as mean+SEM, n=5 mice per group, double-headed arrow represents the time points of quantification). (e) Ca²⁺ levels before CXCL1 stimulation (MFI _0-30s) [mean+SEM, n=5 mice per group, paired Student’s t-test]. (f) Quantification of ER store Ca²⁺ release and calcium released activated channel (CRAC) store-operated Ca²⁺ entry (MFI _peak/ MFI _0-30s) [mean+SEM, n=5 mice per group, paired Student’s t-test]. (g) Ca²⁺ influx after CXCL1 stimulation, from peak to peak half-life (AUC _{peak – ½ peak}) of WT and Mrp14^-/- neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 is indispensable for neutrophil recruitment in vivo.
(a) Representative confocal images of S100A9 intensity in intravascular and extravascular neutrophils after TNF-α stimulation of WT cremaster muscle tissues (scale bar=50μm) and (b) quantification [mean+SEM, n=5 mice per group, 602 (intravascular) and 326 (extravasated) neutrophils, unpaired Student’s t-test]. (c) WT neutrophils’ purity assessment and gating strategy (equivalent for Mrp14^-/- neutrophils) and flow cytometry analysis of (d) CD18, (e) CD11a, (f) CD11b (g) CXCR2, (h) CD62L, (i) PSGL1 and (j) CD44 surface levels of WT and Mrp14^-/-neutrophils [mean+SEM, n=3 mice per group, unpaired Student’s t-test]. (k) Schematic model of the adhesion rescue experiments in TNF-α stimulated WT and Mrp14^-/- cremaster muscles by intra-arterial application of mutS100A8/A9 (aa exchange N70A + E79A). (l) Quantification of number of adherent WT and Mrp14^-/- leukocytes mm^-2 in the same vessel before and after mutS100A8/A9 i.v. injection [mean+SEM, n=4 mice per group, 4 (WT) and 4 (Mrp14^-/-) vessels, 2way ANOVA, Sidak’s multiple comparison]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Cytosolic S100A8/A9 is indispensable for neutrophil recruitment in vivo.
(a) Representative confocal images of S100A9 intensity in intravascular and extravascular neutrophils after TNF-α stimulation of WT cremaster muscle tissues (scale bar=50μm) and (b) quantification [mean+SEM, n=5 mice per group, 602 (intravascular) and 326 (extravasated) neutrophils, unpaired Student’s t-test]. (c) WT neutrophils’ purity assessment and gating strategy (equivalent for Mrp14^-/- neutrophils) and flow cytometry analysis of (d) CD18, (e) CD11a, (f) CD11b (g) CXCR2, (h) CD62L, (i) PSGL1 and (j) CD44 surface levels of WT and Mrp14^-/-neutrophils [mean+SEM, n=3 mice per group, unpaired Student’s t-test]. (k) Schematic model of the adhesion rescue experiments in TNF-α stimulated WT and Mrp14^-/- cremaster muscles by intra-arterial application of mutS100A8/A9 (aa exchange N70A + E79A). (l) Quantification of number of adherent WT and Mrp14^-/- leukocytes mm^-2 in the same vessel before and after mutS100A8/A9 i.v. injection [mean+SEM, n=4 mice per group, 4 (WT) and 4 (Mrp14^-/-) vessels, 2way ANOVA, Sidak’s multiple comparison]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

S100A8/A9 deficient cells are more susceptible to increasing shear stress compared to WT cells.
(a) Analysis of number of adherent WT and Mrp14^-/- neutrophils under flow as percentage related to the initial number of adherent neutrophils in E-selectin, ICAM-1, and CXCL1 coated flow chambers at indicated shear stress levels. Shear stress was increased every 30sec. [mean+SEM, n=3 mice per group, 3 (WT) and 3 (Mrp14^-/-) flow chambers, unpaired Student’s t-test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

S100A8/A9 deficient cells display higher frequencies but shorter duration of Ca²⁺ waves compared to WT cells.
(a) Analysis of basal Ca²⁺ levels normalized to the cell area of bone marrow derived WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils seeded on poly-L-lysine coated slides [mean+SEM, n=234 (WT) and 192 (Mrp14^-/-) neutrophils of 5 mice per group, paired Student’s t-test]. (b) Representative western blot images and quantification of total calmodulin levels normalized to GAPDH signal of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, representative western blot of n=4 mice per group, unpaired Student’s t-test]. (c) Schematic representation of the Ripley’s K used to determine spatial LFA-1 nanocluster correlation. (d) Representative western blot images and quantification of total actin levels (β-Actin) of WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, representative western blot of n≥4 mice per group, unpaired Student’s t-test]. (e) Histogram of Ca²⁺ event frequency distribution and (f) quantification of Ca²⁺ event mean frequency in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. (g) Histogram of Ca²⁺ event duration and (h) quantification of average Ca²⁺ event duration in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

S100A8/A9 deficient cells display higher frequencies but shorter duration of Ca²⁺ waves compared to WT cells.
(a) Analysis of basal Ca²⁺ levels normalized to the cell area of bone marrow derived WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils seeded on poly-L-lysine coated slides [mean+SEM, n=234 (WT) and 192 (Mrp14^-/-) neutrophils of 5 mice per group, paired Student’s t-test]. (b) Representative western blot images and quantification of total calmodulin levels normalized to GAPDH signal of WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, representative western blot of n=4 mice per group, unpaired Student’s t-test]. (c) Schematic representation of the Ripley’s K used to determine spatial LFA-1 nanocluster correlation. (d) Representative western blot images and quantification of total actin levels (β-Actin) of WT ^Lyz2xGCaMP5and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, representative western blot of n≥4 mice per group, unpaired Student’s t-test]. (e) Histogram of Ca²⁺ event frequency distribution and (f) quantification of Ca²⁺ event mean frequency in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. (g) Histogram of Ca²⁺ event duration and (h) quantification of average Ca²⁺ event duration in WT ^Lyz2xGCaMP5 and Mrp14^-/- ^Lyz2xGCaMP5 neutrophils [mean+SEM, n=5 mice per group, paired Student’s t-test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Microvascular parameters in vivo
Number of mice, number of vessels, vessel diameter, centerline velocity, wall shear rate and WBC of TNF-α stimulated WT and Mrp14^-/- mice, as well as of WT and Mrp14^-/- mice treated with mutS100A8/A9 without any prior stimulation (trauma model) and also of TNF-α stimulated WT and Mrp14^-/- mice treated with mutS100A8/A9 (mean+SEM; unpaired student’s t-test).

Microvascular parameters ex vivo
Number of mice, number of flow chambers, cells per FOV and WBC of ex vivo flow chamber assay (Mean+SEM, unpaired student’s t-test).

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