Fig 1.
Sensitivity of mononuclear phagocytes in L.donovani infected mice to RTKi treatment.
CD11c+MHCII+ MPs in d28 L. donovani infected mice were distinguished on the basis of F4/80 and CD11b expression, forward/side scatter profile and morphology into: (A) large CD11c+MHCII+F4/80hiCD11blo (F4/80hiCD11blo) cells with macrophage-like morphology, of which ∼80% harbored intracellular parasites; (B) slightly smaller CD11c+MHCII+F4/80loCD11bhi (F4/80loCD11bhi) cells with classic macrophage morphology, of which <5% harbored parasites; (C) much smaller CD11c+MHCII+F4/80loCD11blo (F4/80loCD11blo) cells with dendritic cell-like morphology and no observable parasites. Representative dot plots show pre-sorted populations with ellipsoid sort gates based on F4/80 and CD11b expression. Scale bar in micrographs = 10microns. The frequency and absolute numbers of each population is given in the right hand panels in naïve mice, infected mice and infected mice treated orally with sunitinib (Sm) for 7 days. P values = * <0.05, ** <0.008, ***<0.001, ns = not significant.
Fig 2.
Phenotypic analysis of F4/80hiCD11blocells.
Splenocytes isolated from L.donovani infected mice at 28 days post infection were stained with a panel of myeloid cell markers. CD11c+MHCII+F4/80hiCD11blo MPs were positive for CD80, CD68 and a small proportion (15%) expressed CD115 (A: isotype control, filled grey histogram). Strong SIGNR1 (white) and FITC-dextran (green) labeling co-localise in the marginal zone of naïve mice (B), whereas in infected mice FITC-dextran+ (green) cells had low expression of SIGNR1 (white). FITC-dextran+ (green) cells were negative for GR1 (white) in both naïve and infected mice (C). Scale bars = 100 microns.
Table 1.
Altered gene expression in F4/80hiCD11blo cells compared to control macrophages.
Table 2.
Comparison of growth factor mRNA abundance in F4/80hiCD11blo cells compared to other MP populations isolated from L. donovani-infected spleens.
Fig 3.
F4/80hiCD11blo MPs are located in close proximity to white pulp vasculature and possess angiogenic properties.
F4/80hiCD11blo cells (FITC-dextran, green; yellow arrows) identified in fresh frozen sections as located either in or bordering the white pulp (A,B). Red pulp F4/80+ macrophages are also shown (white). F4/80hiCD11blo cells (FITC-dextran, green) were found in close association with endothelial cells (C, E; Meca-32, magenta) but not follicular dendritic cells (D; FDCM1, red). High magnification image of area depicted by yellow circle in e (F). All sections were counterstained with DAPI (blue). Scale bars = 100 microns. F4/80hiCD11blo cells, but not other splenic MPs tested, drive SVEC4–10 endothelial cell tube formation on a gelled basement membrane extract (G). Representative images are shown. An optimised cocktail of growth factors (EGM) was used as a positive control. Quantitative analysis of SVEC4–10 mean loop area (H) and difference in tube length (I), in the presence of each MP population or control growth factors. Mean loop area in the absence of growth factors or MPs is shown as a dotted line in h. *p = 0.05, **p = 0.02. Images were analysed using WimTube software and data are expressed as mean ± SEM from at least three independent experiments.
Fig 4.
Infection-induced expression of Bdnf and Ntrk2 in spleen.
Bdnf (black line) was expressed at significantly higher levels by F4/80hiCD11blo cells compared to the other MP populations tested, as revealed by delta median fluorescence intensity levels (ΔMFI) using intracellular flow cytometry (A). Isotype control staining is highlighted by the solid grey histogram. Ntrk2 (red) expression in naïve mouse spleen is found in a subset of splenic red pulp macrophages (F4/80+, white) in naïve mouse spleen (B). Scale bar = 200microns. In L. donovani infected mice splenic white pulp vessels expressed Ntrk2 (C: red). F4/80+ (white) CD11c+ (green) MPs are found in close association with Nrtk2+ vessels (C: merge and inset). Endothelial cells (Meca-32, green) but not smooth muscle actin-positive cells (SMA, red) in white pulp express Nrtk2 (white, D and inset). All sections were stained with DAPI (blue). Scale bars = 100 microns and 50 microns in high magnification merge image.
Fig 5.
White pulp neovascularization during chronic infection is dependent on Nrtk2.
Drug treatment schedule: 21 days post infection with L. donovani, mice were treated with ANA-12, sunitinib maleate (Sm) or vehicle control (VC), daily for 7 days (A). Spleen sections from control and drug-treated mice were stained for endothelial cells (Meca-32, red) and F4/80 (green) to identify boundary of the red and white pulp (white lines). Representative whole slide scanned images are shown (B). The proportion of red pulp area (C) occupied by endothelial cells (Meca-32) and expression of Meca32 relative to total white pulp area (D) was determined by computer-assisted morphometry. Data are expressed as mean ± SEM of at least two independent experiments where n = 5 for each treatment. ** p<0.02 *p<0.05. Spleens were examined after drug treatment for parasite burdens (E) and size/body weight ratio (F).
Fig 6.
Schematic diagram of the effects of treatment with RTK inhibitors during L.donovani infection.
Cartoon to depict processes inhibited by the broad spectrum RTKi (Sm) and the selective Nrtk2 inhibitor (ANA-12) during infection-associated neovascularization of white pulp.