Fig 1.
Monocytes patrol the brain postcapillary venules under resting conditions and are substantially recruited after C. neoformans infection.
(A) A series of IVM images showing a GFP+ cell crawling inside a brain postcapillary venule of a naïve CX3CR1gfp/+ mouse. See also S1 Video. (B) Representative IVM images showing a substantial recruitment of GFP+ cells to brain postcapillary venules of CX3CR1gfp/+ mice 24 h after i.v. infection with 20x106 C. neoformans as compared to naïve mice. See also S3 Video. (C) The number of GFP+ cells recruited to brain postcapillary venules of CX3CR1gfp/+ mice (n = 5 per time point) at various time points after i.v. infection with 20x106 C. neoformans. The number of GFP+ cells in each postcapillary venule of 300 μm in length were enumerated. (D) Representative flow cytometry plots of GFP+ cells in the brain of naïve and infected CX3CR1gfp/+ mice 24 h post infection with 20x106 C. neoformans. (E) The recruitment of GFP+ cells in postcapillary venules comparing to precapillary arterioles in the brain of CX3CR1gfp/+ mice (n = 5) 24 h post infection with C. neoformans. Left panel: representative IVM images; right panel: quantification. (F) Representative flow cytometry histograms showing CD11b expression on CD45+GFP+ cells (blue solid line) and CD45+GFP- cells (red filled) isolated from the brain of naïve and infected CX3CR1gfp/+ mice 24 h post infection with 20x106 C. neoformans. The percentages of CD11b+ cells out of CD45+GFP+ cells were shown in the figure. (G) Representative flow cytometry plots showing NK1.1 expression on a small portion of GFP+ cells in naïve and infected mice 24 h after infection. Initially, total CD45+ brain leukocytes were gated. Scale bars: 10 μm. Data are expressed as mean ± SEM and representative of 2 independent experiments. ***, p<0.001.
Fig 2.
Recruited monocytes are mainly Ly6Clow populations.
(A) IVM images showing the expression of CX3CR1 and CCR2 on recruited monocytes in brain postcapillary venules of CX3CR1gfp/+CCR2rfp/+ mice 24 h after infection with 20x106 C. neoformans (Green: CX3CR1, Red: CCR2). (B) Expression of Ly6C on recruited monocytes (arrow) in brain postcapillary venules of CX3CR1gfp/+CCR2rfp/+ mice 24 h after infection with 20x106 C. neoformans. For labeling of Ly6C, mice were i.v. injected with 2 μg AF647-anti-Ly6C mAb through the tail vein 5 min before imaging. (C) Representative flow cytometry plots of CX3CR1+Ly6Chi and CX3CR1+Ly6Clow monocyte subsets in the brain of naïve and infected C57BL/6 mice. Gated on CD45+ cells. The expression of CD11b on CX3CR1- (filled) and CX3CR1+ (solid) populations was shown on the right. C57BL/6 mice were i.v. infected with 20x106 C. neoformans and 24 h later brain leukocytes were isolated and analyzed by flow cytometry. (D) Flow cytometry analysis of the numbers of different subsets of cells recruited to the brain of C57BL/6 mice (n = 4 per group) 24 h after infection. Ly6Chi monocytes were defined as CD45+Ly6G-NK1.1-CD11b+CX3CR1+Ly6Chi, Ly6Clow monocytes as CD45+Ly6G-NK1.1-CD11b+CX3CR1+Ly6Clow, NK cells as CD45+NK1.1+, neutrophils as CD45+CD11b+Ly6G+. (E) A representative IVM image showing the recruitment of RFP+ monocytes in brain postcapillary venules of CCR2rfp/rfp mice (CCR2 deficient) 24 h after i.v. infection with 20x106 C. neoformans. See also S4 Video. (F) Flow cytometry analysis of the numbers of Ly6Chi and Ly6Clow monocytes and neutrophils recruited to the brain of WT and CCR2-/- mice (n = 4 per group) 24 and 48 h post i.v. infection with 20x106 C. neoformans. Scale bars: 20 μm. Data are expressed as mean ± SEM and representative of 3 independent experiments. * p<0.05.
Fig 3.
VCAM1 and VLA4 interaction mediates the recruitment of monocytes to the brain vasculature.
(A) A representative IVM image showing the labeling of monocytes by anti-CX3CR1 mAb in brain postcapillary venules of C57BL/6 mice 24 h after i.v. infection with 20x106 C. neoformans. Mice were i.v. injected with 2 μg AF647-anti-CX3CR1 mAb to label monocytes 5 min before imaging. (B) IVM analysis of the effect of blocking selectins on CX3CR1+ monocyte recruitment to brain postcapillary venules of C57BL/6 mice (n = 5 per group) 24 h after infection with 20x106 C. neoformans. Mice were i.v. injected with 100 μg selectin-blocking mAbs or control Ab and 2 μg AF647 conjugated anti-CX3CR1 mAb (to label monocytes) 20 min and 5 min, respectively, before imaging. (C) The quantification of VCAM1 mRNA expression in the brain of mice (n = 5 per time point) before and after infection with C. neoformans using quantitative PCR. (D) Evaluation of the effect of anti-VCAM1 and anti-VLA4 mAbs on the number of total recruited CX3CR1+ monocytes to brain postcapillary venules of mice 24 h after i.v. infection with 20x106 C. neoformans. Left panel: representative images of infected CX3CR1gfp/+CCR2rfp/+ mice showing monocyte recruitment before and after i.v. injection with 100 μg anti-VCAM1 or anti-VLA4 mAbs. Right panel: infected WT mice (n = 5 per group) were i.v. injected with 100 μg anti-VCAM1 mAb, anti-VLA4 mAb or control Ab 20 min before imaging. (E) IVM determination of the number and percentage of CX3CR1+ monocytes rolling on and adhering to brain postcapillary venules of C57BL/6 mice (n = 5 per group) 24 h after infection. Infected mice were i.v. injected with 100 μg anti-VCAM1 blocking mAb, anti-VLA4 blocking mAb or control Ab 20 min before imaging. (F) Flow cytometry determination of the numbers of Ly6Chi and Ly6Clow monocytes in the brain of naïve and infected C57BL/6 mice (n = 4 per group). The infected mice were euthanized 24 h post infection. Infected mice were i.v. injected with 100 μg anti-VCAM1 blocking mAb, anti-VLA4 blocking mAb, or control Ab 20 min before euthanasia. Scale bars: 10 μm. Data are expressed as mean ± SEM and representative of 2 independent experiments. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 4.
ICAM1 and CD11a but not CD11b are partially involved in monocyte recruitment to the brain vasculature.
(A) Representative IVM images showing the recruitment of CX3CR1+ monocytes in brain postcapillary venules of WT, ICAM1-/-, CD11a-/-, and CD11b-/- mice 24 h after i.v. infection with 20x106 C. neoformans. The infected mice were i.v. injected with 2 μg AF647-anti-CX3CR1 mAb to label monocytes 5 min before imaging. (B) IVM quantification of CX3CR1+ monocytes recruited to brain postcapillary venules of WT, ICAM1-/-, CD11a-/-, and CD11b-/- mice (n = 5 per group) 24 h after infection. (C) IVM determination of the percentage of CX3CR1+ monocytes rolling on and adhering to brain postcapillary venules of WT, ICAM1-/-, CD11a-/-, and CD11b-/- mice (n = 5 per group) 24 h after infection. (D) Flow cytometry determination of the numbers of Ly6Chi and Ly6Clow monocytes recruited to the brain of WT, ICAM1-/-, CD11a-/-, and CD11b-/- mice (n = 4 per group) 24 h and 48 h after infection. Scale bar: 10 μm. Data are expressed as mean ± SEM and representative of 2 independent experiments. *, p<0.05, ***, p<0.001.
Fig 5.
TNFR signaling is crucial for monocyte recruitment to the brain by enhancing monocyte VLA4 expression.
(A) The level of TNF-α mRNA in the brain of mice (n = 5 per time point) before and after i.v. infection with 20x106 C. neoformans. (B) IVM analysis of monocyte recruitment to brain postcapillary venules of TNFR-/- mice (n = 5) as compared to WT mice (n = 5) 24 h after i.v. infection with 20x106 C. neoformans. The infected mice were i.v. injected with 2 μg AF647-anti-CX3CR1 mAb to label monocytes 5 min before imaging. Left panel: representative images, right panel: quantification of monocytes. (C) Flow cytometry determination of the numbers of Ly6Chi and Ly6Clow monocytes and neutrophils in the brain of TNFR-/- and WT mice (n = 5 per group) 24 h (upper panel) and 48 h (lower panel) after i.v. infection with 20x106 C. neoformans. (D) The gating strategy for brain endothelial cells. Endothelial cells were defined as CD45-CD11b-CD31+ population which demonstrated expression of CD102. (E) Flow cytometry determination of the percentage of brain endothelial cells expressing VCAM1 in WT and TNFR-/- mice (n = 4 per group) 24 h after infection of 20x106 C. neoformans as compared to naïve mice. Left panel: representative plots, right panel: quantification. (F) Flow cytometry analysis of the expression of VLA4 on Ly6Clow monocytes from the brain of WT and TNFR-/- mice (n = 5 per group) 24 h after infection with 20x106 C. neoformans as compared to naïve mice. Left panel: representative histograms, right panel: quantification. (G) Isolated monocytes from WT and TNFR-/- mice (CD45.2 background) were stained by lipophilic dye CellVue and PKH26 respectively and mixed at 1:1 ratio. The mixed monocytes (2x106) were transferred into CD45.1 recipient mice (n = 5 mice) and the mice were i.v. infected with 20x106 C. neoformans for 24 h. Flow cytometry was performed to analyze the recruitment of adoptively transferred monocytes to the brain (left, gated on CD45.2+CD45.1- donor monocytes). The quantification of recruited donor monocytes was shown in the right panel. Scale bar: 10 μm. Data are expressed as mean ± SEM and representative of 2 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
Fig 6.
Ly6Clow monocytes engulf C. neoformans and carry the organism as they crawl on and adhere to the vessel wall and migrate to the brain parenchyma.
(A) A series of IVM representative images showing that a GFP+ monocyte (green) carrying an ingested C. neoformans (red) was crawling on the luminal side of a postcapillary venule in the brain of infected CX3CR1gfp/+ mice. IVM was performed on the brain of mice 18 h after infection with 20x106 C. neoformans. See also S10 Video. (B) A representative IVM image showing that a GFP+ monocyte (green) carrying C. neoformans (red) was attached to the luminal side of a postcapillary venule in the brain of infected CX3CR1gfp/+ mice. IVM was performed 18 h post infection with 20x106 C. neoformans and the blood vessel (blue) was labeled with AF647-conjugated BSA. See also S11 Video. (C) Representative IVM images showing that GFP+ monocytes (green) carrying C. neoformans (red) were in the process of crossing the vessel wall (left panel) or were located outside the postcapillary venule (right panel). IVM was performed on the brain in CX3CR1gfp/+ mice 18 h post infection with C. neoformans and the blood vessel (yellow) was labeled with AF647-conjugated BSA. (D) Monocytes (green, arrowhead) were seen in close proximity to C. neoformans (red, arrow) in capillary vessels (blue, upper panel), or attaching to C. neoformans (middle panel), or engulfing C. neoformans within capillary vessels (lower panel). Immunohistochemistry was performed for the brain tissues of C57BL/6 mice 18 h after infection with 20x106 C. neoformans. (E) Representative IVM images showing GFP+ monocytes (green) carrying C. neoformans (red, arrow) were Ly6C (blue, AF647-anti-Ly6C mAb) negative. IVM was performed on the brain of CX3CR1gfp/+ mice 18 h after infection with C. neoformans. (F) Representative flow cytometry histograms showing the expression level of Ly6C on monocytes carrying C. neoformans. Brain leukocytes were purified from CX3CR1gfp/+ mice (n = 5) 18 h after infection with 20x106 Uvitex 2B labeled C. neoformans. Monocytes carrying C. neoformans were defined as CD45+Uvitex 2B+GFP+ cells (upper panel). The expression of Ly6C on monocytes carrying C. neoformans (CD45+Uvitex 2B+GFP+) was compared to Ly6C expression by Ly6Chi monocytes, neutrophils, and Ly6Clow monocytes (lower panel). Scale bars: 10 μm.