Dual microglia effects on blood brain barrier permeability induced by systemic inflammation methods
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Time course of microglia migration and changes in BBB leak
To probe the temporal relationship between microglia accumulation at blood vessels and the increases in BBB permeability, we gave daily LPS injections (1 mg/kg, intraperitoneal; i.p.) to better control the onset and development of the systemic inflammatory response,. Again, we simultaneously visualized microglia dynamics and BBB permeability, now using the Cx3cr1-GFP mice, in which microglia express GFP. Mice were injected with the differently sized fluorescent dextrans and in vivo two-photon imaging was performed daily, for 1 week prior to LPS administration to gather baseline data, and then for another week during the daily LPS (or saline) injections (Fig. ). During the baseline control week, microglia were relatively stable. Their processes occasionally made brief contact with blood vessels as part of their continued parenchymal surveillance as previously reported,....
Depletion of microglia has dual effects on BBB permeability
To clarify the consequences of microglia-vessel accumulation on BBB permeability, we partially ablated microglia using the Iba1-tetracycline transactivator (Iba1-tTA)::tetracycline operator-diphtheria toxin A (tetO-DTA) mouse (Iba1-tTA::tetO-DTA) mouse. Upon withdrawal of doxycycline from the feed (Dox-Off) of these mice, lethal diphtheria toxin A was expressed in microglia. In our conditions, Dox withdrawal (Dox-Off) predominantly reduced vessel-associated microglia (Fig. ), as measured on LPS injection Day 7, after 14 days of Dox-Off diet. The density of microglia in the parenchyma was not significantly different at this time (Fig. ). A slight increase in GFAP immunofluorescence was also seen, suggesting some possible astrocyte activation or proliferation associated with the loss of microglia and/or systemic inflammation and BBB leak (Fig., Supplementary Fig....
Characterizing vessel-associated microglia
In the SLE model mice (MRL/lpr), both parenchymal and vessel-associated microglia had significantly shorter processes and reduced branches as compared with those of WT mice, although the soma area was similar (Fig. ). These morphological changes suggest a different microglia phenotype is present in the SLE model mice. For both WT and the SLE model mice, microglia associated with vessels showed a distinct, more reactive, phenotype than those in the parenchyma (Fig. ). This prompted us to examine changes in microglia morphology during the 7-day consecutive LPS injection schedule. As microglia migrated to vessels, they adopted a different morphological phenotype (Fig. ). Quantification of these parameters revealed a gradual decrease in process length and an increase in the soma area (Fig. ). The mean process length got significantly shorter from Day 3 onwards, whi...
Characterizing vessel-associated microglia
Finally, we examined the lineage phenotype of vessel-associated microglia, to test if this population includes cells derived from systemic or perivascular macrophages, as has been suggested,. We first immunostained the cells with TMEM119, a selective microglia marker that does not stain macrophages. All the IBA1 positive cells in a field of view were also TMEM119 positive (e.g., Fig. ). We next used the Sall1-GFP mouse, where GFP is only expressed in brain resident microglia that derive from the yolk sac. Virtually all microglia expressed both SALL1 and IBA1, and the proportion of double-labeled microglia did not change after 7 days of LPS (Fig. ). Similarly, the very small numbers of IBA1+'ve and SALL1-'ve microglia did not change. Taken together, this suggests that macrophages do not contribute significantly to the vessel-associated microglia populat...
Inflammation induces expression of Claudin-5 in microglia
To probe possible interactions between microglia and the BBB during inflammation, we compared the genetic profiles of astrocytes and microglia in WT and MRL/lpr mice (Supplementary Fig. ). Numerous (>200) genes were upregulated in MRL/lpr mice, including those involved in immune responses, cell adhesion, and phagocytosis. We identified two genes and products for further study: CLDN5 (Claudin-5) and CD68. CLDN5 contributes to the tight junctions between adjacent endothelial cells in the BBB,, while CD68 is a lysosome marker expressed in phagocytic macrophages and activated microglia. We immunostained CLDN5 and CD68 in WT and MRL/lpr mice and during LPS injections in WT mice (Figs. and ). CLDN5 fluorescence showed colocalization with AQP4 expression, as expected given its association with the neurovascular unit, but also showed co-localization with microglia in MRL/lpr mi...
Microglia express phagocytic proteins in late inflammation
As indicated above, the gene analysis had also suggested expression of the phagocytic marker CD68 in MRL/lpr mice microglia (Supplementary Fig. ), so we next focused on the characteristics and functional consequences of this. Immunohistochemistry demonstrated increased CD68+'ve and IBA1+'ve cells, both in parenchymal and vessel-associated microglia of MRL/lpr mice, although to a greater extent in vessel-associated microglia (Fig. ). This CD68 expression pattern resembled the pattern of changes in morphological phenotype in MRL/lpr microglia, but contrasted with the pattern of increased CLDN5 colocalization, which was restricted to vessel-associated microglia. Indeed, the expression intensity of the two markers was negatively correlated (Fig. ) while the expression intensity of CD68 was positively correlated to that of IBA1 (Fig. ), suggesting CD68 e...
Chemokine CCL5 induces microglial migration to vessels
We speculated that endothelial cells may respond to systemic inflammation and release molecules to trigger microglia migration and phenotype changes. To identify a potential signaling mechanism, we treated cultured endothelial cells with LPS or interferon (IFN)α and analyzed the cytokines that were released into the culture media. We found that CCL5 was significantly increased in both conditions (Fig. ) and moved to in vivo experiments to examine signaling via this chemokine pathway. We administered the CCR5 antagonist (DAPTA, 0.4 µg/mice, 1 µl) via intraventricular injection for 3 days prior to, and concurrently with 4 days of LPS (1 mg/kg, i.p.) or IFNα injection (1 × 10 5 IU/mouse, i.v.) (Fig. ). DAPTA treatment delayed the migration of microglia to vessels, resulting in a significant decrease in the number o...
Inhibiting microglial activation maintains BBB integrity
Our results suggest that microglia have dual effects on BBB integrity-initial protection of BBB integrity but then as inflammation progresses microglia develop a phagocytic phenotype to increase BBB permeability. To further test this conclusion, we inhibited reactive microglia using Minocycline (Mino),. Mino (75 mg/kg, i.p.) or vehicle was administrated before, and during, the LPS injection schedule (Fig. ). Mino prevented the decrease in process length and the increase in soma area seen during LPS injections (Fig. ), confirming an inhibition of microglial reactivity. The mean length of microglia processes in vehicle mice was longer at the end of the 7 days LPS compared with the same time in the Mino group (Fig. ). The mean cell body area was also significantly larger in the control group as compared to the Mino group, on both 4 and 7 days. Mino itself...
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To probe the temporal relationship between microglia accumulation at blood vessels and the increases in BBB permeability, we gave daily LPS injections (1 mg/kg, intraperitoneal; i.p.) to better control the onset and development of the systemic inflammatory response,. Again, we simultaneously visualized microglia dynamics and BBB permeability, now using the Cx3cr1-GFP mice, in which microglia express GFP. Mice were injected with the differently sized fluorescent dextrans and in vivo two-photon imaging was performed daily, for 1 week prior to LPS administration to gather baseline data, and then for another week during the daily LPS (or saline) injections (Fig. ). During the baseline control week, microglia were relatively stable. Their processes occasionally made brief contact with blood vessels as part of their continued parenchymal surveillance as previously reported,. About 20% of microglia made contact with blood vessels during this baseline control period, although their soma did not migrate towards vessels and the number of microglia associated with vessels did not increase (Fig. ). Within 1 day of LPS injections, however, some microglia had migrated to...
To clarify the consequences of microglia-vessel accumulation on BBB permeability, we partially ablated microglia using the Iba1-tetracycline transactivator (Iba1-tTA)::tetracycline operator-diphtheria toxin A (tetO-DTA) mouse (Iba1-tTA::tetO-DTA) mouse. Upon withdrawal of doxycycline from the feed (Dox-Off) of these mice, lethal diphtheria toxin A was expressed in microglia. In our conditions, Dox withdrawal (Dox-Off) predominantly reduced vessel-associated microglia (Fig. ), as measured on LPS injection Day 7, after 14 days of Dox-Off diet. The density of microglia in the parenchyma was not significantly different at this time (Fig. ). A slight increase in GFAP immunofluorescence was also seen, suggesting some possible astrocyte activation or proliferation associated with the loss of microglia and/or systemic inflammation and BBB leak (Fig., Supplementary Fig. ).
In the SLE model mice (MRL/lpr), both parenchymal and vessel-associated microglia had significantly shorter processes and reduced branches as compared with those of WT mice, although the soma area was similar (Fig. ). These morphological changes suggest a different microglia phenotype is present in the SLE model mice. For both WT and the SLE model mice, microglia associated with vessels showed a distinct, more reactive, phenotype than those in the parenchyma (Fig. ). This prompted us to examine changes in microglia morphology during the 7-day consecutive LPS injection schedule. As microglia migrated to vessels, they adopted a different morphological phenotype (Fig. ). Quantification of these parameters revealed a gradual decrease in process length and an increase in the soma area (Fig. ). The mean process length got significantly shorter from Day 3 onwards, while cell soma areas significantly increased from Day 5 onwards. Hence the more reactive characteristics of microglial morphology correlates to the timing when the BBB becomes leaky, but not to the period when microglia first migrate to vessels and have a protective effect on BBB permeability. Fig. 3...
Finally, we examined the lineage phenotype of vessel-associated microglia, to test if this population includes cells derived from systemic or perivascular macrophages, as has been suggested,. We first immunostained the cells with TMEM119, a selective microglia marker that does not stain macrophages. All the IBA1 positive cells in a field of view were also TMEM119 positive (e.g., Fig. ). We next used the Sall1-GFP mouse, where GFP is only expressed in brain resident microglia that derive from the yolk sac. Virtually all microglia expressed both SALL1 and IBA1, and the proportion of double-labeled microglia did not change after 7 days of LPS (Fig. ). Similarly, the very small numbers of IBA1+'ve and SALL1-'ve microglia did not change. Taken together, this suggests that macrophages do not contribute significantly to the vessel-associated microglia population. Based on the increased spatial correlation of IBA1 and AQP4 immunohistochemistry, we confirmed that LPS injections in Sall1-GFP mice induced a significant increase in the microglia population associated with vessels (Fig. ).
To probe possible interactions between microglia and the BBB during inflammation, we compared the genetic profiles of astrocytes and microglia in WT and MRL/lpr mice (Supplementary Fig. ). Numerous (>200) genes were upregulated in MRL/lpr mice, including those involved in immune responses, cell adhesion, and phagocytosis. We identified two genes and products for further study: CLDN5 (Claudin-5) and CD68. CLDN5 contributes to the tight junctions between adjacent endothelial cells in the BBB,, while CD68 is a lysosome marker expressed in phagocytic macrophages and activated microglia. We immunostained CLDN5 and CD68 in WT and MRL/lpr mice and during LPS injections in WT mice (Figs. and ). CLDN5 fluorescence showed colocalization with AQP4 expression, as expected given its association with the neurovascular unit, but also showed co-localization with microglia in MRL/lpr mice (Fig. ). The colocalization of CLDN5 and IBA1 immunoreactivity and the proportion of CLDN5+'ve microglia were higher in vessel-associated microglia in MRL/lpr mice, as compared with parenchymal microglia (Fig., Supplementary Fig. ). We next examined the temporal patterns...
As indicated above, the gene analysis had also suggested expression of the phagocytic marker CD68 in MRL/lpr mice microglia (Supplementary Fig. ), so we next focused on the characteristics and functional consequences of this. Immunohistochemistry demonstrated increased CD68+'ve and IBA1+'ve cells, both in parenchymal and vessel-associated microglia of MRL/lpr mice, although to a greater extent in vessel-associated microglia (Fig. ). This CD68 expression pattern resembled the pattern of changes in morphological phenotype in MRL/lpr microglia, but contrasted with the pattern of increased CLDN5 colocalization, which was restricted to vessel-associated microglia. Indeed, the expression intensity of the two markers was negatively correlated (Fig. ) while the expression intensity of CD68 was positively correlated to that of IBA1 (Fig. ), suggesting CD68 expression depends on the microglia activation state. Expression of CD68 was preferentially seen in vessel-associated microglia, and after LPS administration, expression increased across both parenchymal and vessel-associated microglia, masking any differences by Day 4 and Day 7 (Fig. ). Again,...
We speculated that endothelial cells may respond to systemic inflammation and release molecules to trigger microglia migration and phenotype changes. To identify a potential signaling mechanism, we treated cultured endothelial cells with LPS or interferon (IFN)α and analyzed the cytokines that were released into the culture media. We found that CCL5 was significantly increased in both conditions (Fig. ) and moved to in vivo experiments to examine signaling via this chemokine pathway. We administered the CCR5 antagonist (DAPTA, 0.4 µg/mice, 1 µl) via intraventricular injection for 3 days prior to, and concurrently with 4 days of LPS (1 mg/kg, i.p.) or IFNα injection (1 × 10 5 IU/mouse, i.v.) (Fig. ). DAPTA treatment delayed the migration of microglia to vessels, resulting in a significant decrease in the number of vessel-associated microglia compared to LPS alone after 2 days of LPS (Fig. ). DAPTA also caused an earlier onset of LPS-induced BBB leak (observed at day 2; Fig. ), together suggesting DAPTA inhibits both microglial migration and the resulting initial BBB protection. Intravenous injec...
Our results suggest that microglia have dual effects on BBB integrity-initial protection of BBB integrity but then as inflammation progresses microglia develop a phagocytic phenotype to increase BBB permeability. To further test this conclusion, we inhibited reactive microglia using Minocycline (Mino),. Mino (75 mg/kg, i.p.) or vehicle was administrated before, and during, the LPS injection schedule (Fig. ). Mino prevented the decrease in process length and the increase in soma area seen during LPS injections (Fig. ), confirming an inhibition of microglial reactivity. The mean length of microglia processes in vehicle mice was longer at the end of the 7 days LPS compared with the same time in the Mino group (Fig. ). The mean cell body area was also significantly larger in the control group as compared to the Mino group, on both 4 and 7 days. Mino itself (without LPS) did not affect microglial process lengths or soma areas (Supplementary Fig. ). Although activation of microglial were blocked by Mino, as expected, the microglia still migrated towards blood vessels and the proportion of microglial associated with vessels did not differ significa...
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