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

Reviewer #1 (Public Review):

Summary:

In this manuscript by Napoli et al, the authors study the intracellular function of Cytosolic S100A8/A9 a myeloid cell soluble protein that operates extracellularly as an alarmin, whose intracellular function is not well characterized. Here, the authors utilize state-of-the-art intravital microscopy to demonstrate that adhesion defects observed in cells lacking S100A8/A9 (Mrp14-/-) are not rescued by exogenous S100A8/A9, thus highlighting an intrinsic defect. Based on this result subsequent efforts were employed to characterize the nature of those adhesion defects.

Strengths:

The authors convincingly show that Mrp14-/- neutrophils have normal rolling but defective adhesion caused by impaired CD11b activation (deficient ICAM1 binding). Analysis of cellular spreading (defective in Mrp14-/- cells) is also sound. The manuscript then focuses on selective signaling pathways and calcium measurements. Overall, this is a straightforward study of biologically important proteins and mechanisms.

Weaknesses:

Some suggestions are included below to improve this manuscript.

Reviewer #2 (Public Review):

Summary:

Napoli et al. provide a compelling study showing the importance of cytosolic S100A8/9 in maintaining calcium levels at LFA-1 nanoclusters at the cell membrane, thus allowing the successful crawling and adherence of neutrophils under shear stress. The authors show that cytosolic S100A8/9 is responsible for retaining stable and high concentrations of calcium specifically at LFA-1 nanoclusters upon binding to ICAM-1, and imply that this process aids in facilitating actin polymerisation involved in cell shape and adherence. The authors show early on that S100A8/9 deficient neutrophils fail to extravasate successfully into the tissue, thus suggesting that targeting cytosolic S100A8/9 could be useful in settings of autoimmunity/acute inflammation where neutrophil-induced collateral damage is unwanted.

Strengths:

Using multiple complementary methods from imaging to western blotting and flow cytometry, including extracellular supplementation of S100A8/9 in vivo, the authors conclusively prove a defect in intracellular S100A8/9, rather than extracellular S100A8/9 was responsible for the loss in neutrophil adherence, and pinpointed that S100A8/9 aided in calcium stabilisation and retention at the plasma membrane.

Weaknesses:

(1) Extravasation is shown to be a major defect of Mrp14-/- neutrophils, but the Giemsa staining in Figure 1H seems to be quite unspecific to me, as neutrophils were determined by nuclear shape and granularity. It would have perhaps been more clear to use immunofluorescence staining for neutrophils instead as seen in Supplementary Figure 1A (staining for Ly6G or other markers instead of S100A9).

(2) The representative image for Mrp14-/- neutrophils used in Figure 4K to demonstrate Ripley's K function seems to be very different from that shown above in Figures 4C and 4F.

(3) Although the authors have done well to draw a path linking cytosolic S100A8/9 to actin polymerisation and subsequently the arrest and adherence of neutrophils in vitro, the authors can be more explicit with the analysis - for example, is the F-actin co-localized with the LFA-1 nanoclusters? Does S100A8/9 localise to the membrane with LFA-1 upon stimulation? Lastly, I think it would have been very useful to close the loop on the extravasation observation with some in vitro evidence to show that neutrophils fail to extravasate under shear stress.