Extended Data Fig. 6: Neutrophils deposit collagen and regulate collagen fibres in the skin.
From: Matrix-producing neutrophils populate and shield the skin
a, Mechanism of collagen tagging by CNA35–mCherry inside cells. This strategy was used to generate the Rosa26LSL; CNA35-mCherry reporter model, which allows Cre-dependent expression of a fluorescent form of CNA35, a collagen-binding protein of bacterial origin. Cell-specificity in this model is permitted because the fluorescent protein lacks an export signal and cannot be sorted out of the endoplasmic reticulum unless it binds trimeric collagen, thereby preventing unspecific labelling of collagen produced by other cells. b, Neutrophils sorted from naive lungs were cultured overnight for 16 h on fibronectin-coated glass coverslips. Left, representative images (top) and reconstructions (bottom) of a Col+ lung neutrophil stained for DAPI, Col1/3, and wheat germ agglutinin (WGA). White arrows show deposits of extracellular collagen. In contrast ColNEG neutrophils do not show extracellular deposits (middle panels). Right, quantification of extracellular collagen signal area (top) and the number of collagen specks, within a 100 μm perimeter of individual neutrophils (n = 5 ColNEG cells, 7 Col+ cells). Data are presented as mean values ± s.e.m. c, Representative images (top) and reconstructions (bottom) of a Col+ lung neutrophil (determined by positive CNA35–GFP signal). White arrowheads indicate the extracellular deposits positive for CNA35–mCherry and CNA35–GFP, indicating collagen deposition by the cell. These deposits are not found around CNA35–GFPNEG neutrophils (middle panels). Right, quantification of extracellular CNA35–mCherry+ area (top) and number of specks in a 100 µm x 100 µm perimeter around individual neutrophils. Data show mean ± s.e.m. from 4 GFPNEG cells and 4 GFP+ cells. P-values were determined by a two-sided unpaired Student’s t-test. d, Representative image from the skin of Ly6GCre; Rosa26LSL/CNA35-mCherry mice showing extracellular deposits of CNA35–mCherry. e, Top and side views of the skin from Rosa26LSL/CNA35-mCherry reporter mice crossed with CreNEG control mice (n = 7), and Ly6GCre (n = 11) or DptCreERT2 (n = 4) driver lines to induce CNA35–mCherry expression in neutrophils and fibroblasts. Ly6GCre targets mature neutrophils, while DptCreERT2 is expressed by 60% of fibroblasts. Arrows show distances of CNA35-collagen to the epidermis. f, Volume per cent of CNA35–mCherry in the naive skin of the neutrophil (n = 3) and fibroblast (n = 3) reporter lines. g, Distance to the epidermis of collagen produced by neutrophils or fibroblasts. P-value determined by two-way ANOVA.
