Figure 4. Meningeal phagocytes do not fully acidify their late endosomes and lysosomes.

(A) Experimental strategy. Wild-type and mutant CX3CR1-Lyve1-tdT mice were implanted with chronic cranial windows and injected with dextrans subcutaneously (s.c.) 2–3 weeks later. The meninges were imaged by intravital microscopy 24h later.

(B) Intravital imaging of meningeal tdT+ macrophages in a CX3CR1-Lyve1-tdT mouse injected with Alexa 405-dextrans (70 kDa) and allowed to recover for 24h prior to imaging. Dextrans show a perinuclear distribution consistent with late endosomal and lysosomal localization.

(C) Intravital imaging of meningeal phagocytes in a C57BL/6J mouse prior to (C1) or 24h after (C2) s.c. injection with ApHID-Alexa 546-dextrans (70 kDa). Fluorescence values in meningeal phagocytes for ApHID (green channel) and Alexa 546 (red channel) measured before (Pre-s,c.) or after (Post-s.c.) dextran injection are plotted normalized to the average Pre-s.c. value (C3). To control for variations in laser power between imaging sessions, fluorescence values were additionally normalized to Nile red bead fluorescence recorded at the end of each session. Large symbols indicate integrated intensity values per mouse (n=4) and small tan circles correspond to individual segmented phagocytes. Bars indicate average fluorescence intensity ± SEM.

(D) Intravital pH imaging of acidic late endosomal and lysosomal (LE/Ly) compartments in meningeal phagocytes acquired through a cranial window in a C57BL/6J mouse. The animal was injected s.c. with ApHID-Alexa 546 dextrans (70 kDa) and allowed to recover for 24h. Vasculature was labeled by intraperitoneal (i.p.) injection of fluorescein-Alexa 546 dextrans (500 kDa) 10 min prior to imaging. The same region was imaged at various depths (D1-D3).

(E) Intravital pH imaging of meningeal phagocytes as described in (D), showing cells at the surface of the dura (E1) and others immediately adjacent to blood vessels in deeper meningeal layers (E2-E4, perivascular localization, arrowheads). Vasculature was labeled with fluorescein-Alexa 546 dextrans (500 kDa) 10 min prior to imaging. Color-coded ratiometric pH images were generated for each region (E1R-E4R). Color-coded bars indicate a pH range of 4.0 (red) to 6.0 (blue).

(F) J774A.1 murine macrophages labeled with ApHID-Alexa 546 dextrans (70 kDa), fixed and incubated in pH 5.0 buffer at 37 °C, imaged immediately following intravital imaging. A color-coded ratiometric pH image was generated as described in (E).

(G) ApHID/Alexa 546 fluorescence ratios corresponding to late endosomal and lysosomal (LE/Ly) compartments for each segmented meningeal phagocyte (D-E) were interpolated to pH using a ratio-to-pH calibration (as shown in Fig. 3I). Violin plots show integrated LE/Ly pH including all segmented phagocytes (4093 cells) from 8 mice (4 males and 4 females). Cells in close proximity to blood vessels (“Periv.”) or further from vasculature (“NP”, generally at the dural surface) were plotted separately. Superimposed dotted lines indicate median and quartiles.

(H) Mean LE/Ly pH (n=8, 4 males and 4 females) plotted according to vascular localization (“perivascular” vs “non-perivascular”) and sex. Mean LE/Ly pH between male and female mice and cells sitting in close proximity or away from vasculature were compared using a linear mixed effects model followed by Tukey’s multiple comparison test (p≥0.05 n.s.; all comparisons). Bars indicate mean LE/Ly pH ±SEM whereas individual symbols correspond to values for individual mice.

(I) Meningeal phagocytes plotted against their LE/Ly pH range (pH<5.0; pH≥5.0–<5.5; pH≥5.5–<6.0; pH≥6.0). The mean fraction of cells within each range was calculated per mouse (n=8, 4 males and 4 females) and plotted by proximity to blood vessels (perivascular vs. non-perivascular) as shown in (E). Differences in cell fraction mean ranks were assessed using the non-parametric paired Friedman test with multiple comparisons by Dunn's test (p≥0.05 n.s.; p<0.05 *; p<0.01 **). Bars indicate mean fraction of cells ±SEM whereas symbols correspond to values for individual mice.

Side panels in D, E and F show individual fluorescence channels. Scale bars: Panels (D1-D3): 50 μm. All other panels: 10 μm. Abbreviations: A405: Alexa 405; A546: Alexa 546; dex: dextran; s.c.: subcutaneously; i.p.: intraperitoneally; MΦ: meningeal phagocytes.