Cerebral microglia recruit monocytes into the brain in response to tumor necrosis factoralpha signaling during peripheral organ inflammation

Abstract

In inflammatory diseases occurring outside the CNS, communication between the periphery and the brain via humoral and/or neural routes results in central neural changes and associated behavioral alterations. We have recently identified another immune-to-CNS communication pathway in the setting of organ-centered peripheral inflammation: namely, the entrance of immune cells into the brain. In our current study, using a mouse model of inflammatory liver injury, we have confirmed the significant infiltration of activated monocytes into the brain in mice with hepatic inflammation and have defined the mechanism that mediates this trafficking of monocytes. Specifically, we show that in the presence of hepatic inflammation, mice demonstrate elevated cerebral monocyte chemoattractant protein (MCP)-1 levels, as well as increased numbers of circulating CCR2-expressing monocytes. Cerebral recruitment of monocytes was abolished in inflamed mice that lacked MCP-1/CCL2 or CCR2. Furthermore, in mice with hepatic inflammation, microglia were activated and produced MCP-1/CCL2 before cerebral monocyte infiltration. Moreover, peripheral tumor necrosis factor (TNF)alpha signaling was required to stimulate microglia to produce MCP-1/CCL2. TNFalpha signaling via TNF receptor 1 (TNFR1) is required for these observed effects since in TNFR1 deficient mice with hepatic inflammation, microglial expression of MCP-1/CCL2 and cerebral monocyte recruitment were both markedly inhibited, whereas there was no inhibition in TNFR2 deficient mice. Our results identify the existence of a novel immune-to-CNS communication pathway occurring in the setting of peripheral organ-centered inflammation which may have specific implications for the development of alterations in cerebral neurotransmission commonly encountered in numerous inflammatory diseases occurring outside the CNS.

Figure 1.

Monocytes infiltrate the brains of BDR mice. A, B, Representative flow cytometry profiles from sham (A) and BDR (B) mouse at day 10 postsurgery. Resident microglia were identified as CD45^low CD11b⁺ cells (bottom right quadrant). Cerebral infiltrating monocytes were identified as CD45^high CD11b⁺ cells (top right quadrant) and were clearly distinguishable from the CD45^low CD11b⁺ cells. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺. C, Solid bars represent the percentage of total brain mononuclear cells isolated from day 10 sham and BDR mice that are CD45^high CD11b⁺. Hatched bars represent the total number of CD45^high CD11b⁺ cells in the brains of day 10 sham and BDR mice. Error bars represent mean ± SEM of data from 9 sham and 9 BDR mice; *p < 0.05 versus sham controls.

Figure 2.

Detection of CFSE-labeled monocytes in brains of day 10 BDR mice. CFSE-labeled monocytes were observed within the parenchyma, most prominently in the periventricular and perivascular regions, in brains of day 10 recipient BDR mice. A, CFSE-labeled monocytes present in the lumen of the choroid plexus and also within the cerebral parenchymal area surrounding the choroid plexus. B, CFSE-labeled monocytes present in the cerebral parenchyma in the subfornical organ region of the brain (both the choroid plexus and the subfornical organ are potential routes used for leukocyte transmigration into brain parenchyma). C, Immunohistochemical staining for vWF (labels cerebral vascular endothelium) demonstrated that CFSE-labeled monocytes were present within the cerebral parenchyma (closed arrow) in areas clearly distinct from cerebral blood vessels, and were also observed in close proximity to cerebral blood vessels (open arrow). A, B, C, Brain sections from n = 3 day 10 BDR mice. C, A blood vessel in the periventricular region in a day 10 BDR mouse. Scale bars: A, C, 5 μm; B, 20 μm. BV, Blood vessel.

Figure 3.

Region-specific detection of CFSE-labeled monocytes in BDR mice. Large numbers of CFSE-labeled monocytes were most readily detected in the cerebral parenchyma of BDR mice in specific regions of the brain, including the basal ganglia (A), hippocampus (B) and motor cortex (C). Immunostaining for vWF was used to identify blood vessels in all three panels. Positive VWF staining is depicted by red immunofluorescence. A, B, C, Brain sections from n = 3 day 10 BDR mice. Scale bar, 20 μm. BV, Blood vessel.

Figure 4.

Immunohistochemical detection of Ly6C-stained endothelium and CFSE-labeled monocytes in the brains of day 10 BDR mice. A–C, A Ly6C-positive CFSE-labeled monocyte present in the perivascular region in the brain of a day 10 BDR mouse. D–F, A Ly6C-positive CFSE-labeled monocyte that appears to be adherent to the cerebral blood vessel endothelium. G–I, A Ly6C-positive CFSE-labeled monocyte that appears to be adherent to the blood vessel (open arrow) and a Ly6C-positive CFSE-labeled monocyte that is present within the cerebral parenchyma (closed arrow). J–L, A Ly6C-immunostained CFSE-labeled monocyte that appears to be adherent to the blood vessel wall or in the process of transmigrating out of the blood vessel and into the brain parenchyma. All panels are representative of blood vessels in the periventricular region in brain sections from n = 3 day 10 BDR mice. CFSE-labeled monocytes are indicated in the panels on the left (A, D, G, J). Ly6C-immunostained cerebral endothelium and monocytes are indicated by red fluorescence in the middle panels. Combined images of Ly6C-immunostained CFSE-labeled monocytes (i.e., images from respective left and middle panels were superimposed) are demonstrated in yellow (panels on the right, C, F, I, L). Scale bar, 5 μm. BV, Blood vessel.

Figure 5.

MCP-1/CCL2 via its actions on CCR2 mediates monocyte recruitment into the brains of BDR mice. A, Solid bars represent the percentage of total peripheral blood monocytes (PBMs)-expressing CCR2 in day 10 sham and BDR mice. Hatched bars represent the total number of CCR2-expressing PBMs isolated from day 10 sham and BDR mice. Error bars represent mean ± SEM of data from 6 sham and 9 BDR mice; *p < 0.05 versus sham controls. B, Solid bars represent the percentage of total brain microglia that express CCR2 in day 10 sham and BDR mice. Hatched bars represent the total number of CCR2-expressing microglia isolated from the brains of day 10 sham and BDR mice. Error bars represent the mean ± SEM of data from 6 sham and 9 BDR mice; *p < 0.05 versus sham controls.

Figure 6.

Cerebral monocyte recruitment in day 10 BDR mice is inhibited in the absence of MCP-1/CCL2 or CCR2. Representative flow cytometry profiles from day 10 WT BDR (A) versus MCP-1/CCL2 KO BDR (B) mice. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ (i.e., infiltrating monocytes). C, Solid bars represent the percentage of isolated brain mononuclear cells that are CD45^high CD11b⁺ in day 10 WT BDR and MCP-1/CCL2 KO BDR mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells isolated from the brains of day 10 WT BDR and MCP-1/CCL2 KO BDR mice. Error bars represent the mean ± SEM of data from 4 WT BDR and 3 MCP-1/CCL2 KO BDR mice; *p < 0.05 versus WT BDR mice. Representative flow cytometry profiles from day 10 WT BDR (D) versus CCR2 KO BDR (E) mice. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺. F, Solid bars represent the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ in day 10 WT BDR and CCR2 KO BDR mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells isolated from the brains of day 10 WT BDR and CCR2 KO BDR mice. Bars represent the mean ± SEM of data from 4 WT BDR and 4 CCR2 KO BDR mice; *p < 0.05 versus WT BDR mice.

Figure 7.

Cerebral sources of MCP-1/CCL2 in day 10 BDR mice. Solid bars represent the total number of microglia expressing MCP-1/CCL2 in the brains of day 10 BDR and sham mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells (i.e., infiltrating monocytes) isolated from the brains of day 10 sham and BDR mice that express MCP-1/CCL2. Error bars represent mean ± SEM of data from 7 sham and 10 BDR mice; *p < 0.05 versus sham controls.

Figure 8.

Microglia are stimulated to produce MCP-1/CCL2 before monocytes are recruited into the brains of BDR mice. A, B, Representative flow cytometry profiles from day 5 sham (A) and BDR (B) mice. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ (i.e., infiltrating monocytes). C, Solid bars represent the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ in day 5 sham and BDR mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells isolated from the brains of day 5 sham and BDR mice. Error bars represent mean ± SEM of data from 5 sham and 5 BDR mice, p > 0.05 versus sham mice. D, Solid bars represent the percentage of total brain microglia that express MCP-1/CCL2 in day 5 sham and BDR mice. Hatched bars represent the total number of cerebral microglia expressing MCP-1/CCL2 in day 5 sham and BDR mice. Error bars represent mean ± SEM of data from 5 sham and 5 BDR mice; *p < 0.05 versus sham controls.

Figure 9.

Immunohistochemical staining for Iba1-positive microglia in day 5 BDR mice. In general, microglia in sham mice (A, C, E) have a resting ramified morphology with longer processes while microglia in BDR mice (B, D, F) are more rounded with retracted and thicker processes typical of activated microglial cells. Compared with sham controls, in BDR mice there was a dominance of activated microglia in the periventricular region [sham (C); BDR (D)] and in the area close to the blood vessels [sham (E); BDR (F)]. A, B, Microglia in the periventricular regions in respective day 10 sham and BDR mice and demonstrate the typical morphology (indicated by arrows) seen for microglia in sham (A) and BDR (B) mice. C, D, The periventricular region in sham (C) and BDR (D) mice. E, F, An area close to the blood vessel in sham (E) and BDR (F) mice. All pictures are representative of brain sections from 3 sham and 3 BDR mice. Scale bars: A, B, 5 μm; C–F, 20 μm. BV, Blood vessel.

Figure 10.

Peripheral TNFα signaling stimulates microglia to produce MCP-1/CCL2 in day 5 BDR mice. Solid bars represent the percentage of total cerebral microglia that express MCP-1/CCL2 in day 5 BDR mice that had received either normal rabbit serum (NRS) or anti-TNFα serum intraperitoneally on days 2 and 4 postsurgery. Hatched bars represent the total number of MCP-1/CCL2-expressing cerebral microglia in day 5 BDR mice treated with either NRS or anti-TNFα serum on days 2 and 4 postsurgery. Error bars represent mean ± SEM of data from 4 NRS-treated BDR and 3 anti-TNFα serum-treated BDR mice; *p < 0.05 versus NRS-treated BDR mice.

Figure 11.

TNFα signaling via TNFR1 stimulates microglia in BDR mice to produce MCP-1/CCL2 and to subsequently recruit monocytes into the brain. A, B, Representative flow cytometry profiles from day 10 WT BDR (A) and TNFR1 KO BDR (B) mice. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ (i.e., infiltrating monocytes). C, Solid bars represent the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ in day 10 WT BDR and TNFR1 KO BDR mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells isolated from the brains of day 10 WT BDR and TNFR1 KO BDR mice. Error bars represent mean ± SEM of data from 9 WT BDR and 6 TNFR1 KO BDR mice; *p < 0.05 versus WT BDR mice. D, Solid bars represent the percentage of total cerebral microglia that express MCP-1/CCL2 in day 10 WT BDR and TNFR1 KO BDR mice. Hatched bars represent the total number of cerebral microglia that express MCP-1/CCL2 in day 10 WT BDR and TNFR1 KO BDR mice. Error bars represent mean ± SEM of data from 9 WT BDR and 6 TNFR1 KO BDR mice; *p < 0.05 versus WT BDR mice. E, F, Representative flow cytometry profiles from day 10 WT BDR (E) and TNFR2 KO BDR (F) mice. The inserted number shows the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ (i.e., infiltrating monocytes). G, Solid bars represent the percentage of total brain mononuclear cells that are CD45^high CD11b⁺ in day 10 WT BDR and TNFR2 KO BDR mice. Hatched bars represent the total number of CD45^high CD11b⁺ cells isolated from the brains of day 10 WT BDR and TNFR2 KO BDR mice. Error bars represent mean ± SEM of data from 5 WT BDR and 5 TNFR2 KO BDR mice; p > 0.05 versus WT BDR mice.

Figure 12.

Cerebral recruitment of monocytes in BDR mice contributes significantly to the development of hepatic inflammation associated sickness behavior. Sickness behavior was assessed by quantifying (1) the duration of social exploratory behavior exhibited by an adult mouse toward a congenic juvenile mouse placed in close proximity (2), the total number of interactions of the adult test mouse with the juvenile mouse, and (3) the total time the test mouse remained immobile during the observation period. A, Total duration of social exploration in seconds during a 5 min observation period. Error bars represent mean ± SEM of data from 5 sham mice, 5 anti-P selectin and anti-α₄ integrin-treated BDR mice, and 5 isotype IgG-treated control BDR mice; **p < 0.001 IgG-treated BDR versus sham controls; **p < 0.001 antibody-treated BDR versus IgG-treated BDR mice; **p < 0.001 antibody-treated BDR versus sham mice. B, Total number of interactions of the adult test mouse with a juvenile mouse during the 5 min observation period. Error bars represent mean ± SEM of data from 5 sham mice, 5 anti-P selectin and anti-α₄ integrin-treated BDR mice, and 5 isotype IgG-treated control BDR mice; *p < 0.05 IgG-treated BDR versus sham mice; **p < 0.01 antibody-treated BDR versus IgG-treated BDR mice. C, Total time in seconds that the adult test mouse remained immobile during the 5 min observation period after the introduction of the juvenile mouse into the test mouse home cage. Error bars represent mean ± SEM of data from 5 sham mice, 5 anti-P selectin and anti-α₄ integrin-treated BDR mice, and 5 isotype IgG-treated control BDR mice; **p < 0.001 IgG-treated BDR versus sham mice; **p < 0.001 antibody-treated BDR versus IgG-treated BDR mice; *p < 0.05 antibody-treated BDR versus sham mice. Ab, Antibody.