The present results show that microglia/macrophage depletion either by PLX5622 in mice or by immunotoxin in transgenic rats failed to abrogate the peripheral and central inflammatory response to LPS. Therefore, it was not surprising that this treatment was unable to prevent the signs of sickness that developed in response to LPS. These unexpected findings indicate that the sickness inducing effects of systemic inflammation can occur independently from microglial activation.
As already reported in previous studies on CSF-1R antagonism(10, 11, 14), administration of the CSF-1R antagonist PLX5622 for 4 weeks resulted in the near complete elimination of microglia in the brain and a significant depletion of macrophages in the spleen and liver. An alternative to the use of CSF-1R antagonism to deplete microglia is the diphtheria toxin receptor-mediated cell knockout technique. This technique is widely used to remove specific cell types in rodents engineered to express the diphtheria toxin receptor on the surface of a specific cell type(17). Several variants of this technique have already been used to efficiently deplete microglia in mice(12, 18, 19) and in rats(15, 16) by coupling the diphtheria toxin receptor to the promoter of the gene coding for the microglia/monocyte specific marker CX3CR1. Diphtheria toxin itself is generally well tolerated when administered to wild type mice(20). In the absence of diphtheria toxin Cx3cr1-Dtr transgenic rats do not show any abnormalities(15, 16). Similar to mouse models utilizing conditional diphtheria toxin receptor expression approach (12, 33, 34), administration of diphtheria toxin in Cx3cr1-Dtr rats depleted microglia by 48 h in various brain regions including the hypothalamus, with repopulation occurring by 7 days(15, 16). Although microglia depletion was associated with anorexia and weight loss, this was not due to sickness as there was no change in locomotor activity in an open-field and in two tests of anxiety, the elevated plus maze and the light-dark box(15). There was also no indication of nausea as measured by ingestion of kaolin. In addition, microglia depletion by diphtheria toxin was not associated with any evidence of impairment in learning and memory as measured by short term memory in a novel object and place recognition tasks(16). Further studies indicate that the anorexia induced by administration of diphtheria toxin to Cx3cr1-Dtr rats is actually due to disruption of the gustatory circuitry a the level of the paraventricular nucleus of the hypothalamus(15), indicating the complex role microglia play in brain functions additional to their traditional role in regulating neuroinflammation(21).
We anticipated that the elimination of microglia by PLX5622 in mice and by diphtheria toxin in Cx3cr1-Dtr rats would attenuate neuroinflammation induced by LPS and its behavioral consequences. In accordance with this prediction, there are already several publications showing that depletion of microglia by PLX5622 protects from neuroinflammation(22-25) and prevents behavioral alterations in response to cranial irradiation(25), repeated social defeat(26), partial sciatic nerve ligation(27) and experimental autoimmune encephalomyelitis(24). In addition, antibody-mediated neutralization of peripheral macrophage CSF-1R was reported to block the development of sickness behavior measured by reduced locomotor activity and body weight loss in response to CD40 activation, a model of autoimmune disease(28).
It is currently unclear why the elimination of microglia/macrophages by CSF-1R antagonism or by diphtheria toxin in the Cx3cr1-Dtr rat model failed to abrogate the inflammatory and behavioral response to LPS. At the periphery this could be due to the fact that both interventions specifically depleted tissue macrophages but did not affect pro-inflammatory monocytes recruited from the bone marrow, dendritic cells, or neutrophils which can all contribute to the peripheral inflammatory response(29). However, this cannot explain why the brain response to LPS was not only not fully abrogated in both models of microglia depletion but actually enhanced in Cx3cr1-Dtr rats. In the first study to show that CSF1 receptor antagonism eliminates microglia in a reversible way, mice were treated with a low dose of LPS (0.25 mg/kg) after only 7 days of the CSF-1R antagonist PLX3397, and brains were collected 6 h after LPS without intracardiac perfusion to eliminate residual blood (11). While this study showed that PLX3397 attenuated IL-1b and reversed TNF mRNA expression in response to LPS, it had only limited effects on other inflammatory markers, with no effect on IL-6 mRNA expression in response to LPS. In addition, a number of studies show that microglial depletion is not always neuroprotective. In mice infected with prions, administration of PLX5622 accelerated disease progression(30). In the same manner, PLX5622 increased viral load and enhanced mortality in a number of murine models of viral infection(31-33). A similar protective role of microglia was also apparent in the progression of neurodegeneration in APP-PS1 transgenic mice(34), the extent of excitotoxic injury in a model of brain injury induced by cerebral ischemia(35) and the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP)(36).
One possibility for the conserved production of cytokines despite microglia depletion is the well-known existence of genetically defined subsets of microglia in the brain(37-39) with differential sensitivity to genetic or pharmacological depletion. The techniques used to induce microglia depletion leave intact a very small percentage of microglia in the brain, less than 1% in response to CSF-1R antagonism(40). This resistant subset of microglia has been identified as having distinct self-renewal capacity following depletion and repopulation(40). However, its ability to produce cytokines in response to neuroinflammation has not been examined, and it is difficult to imagine that it is sufficient to induce a similar and even higher inflammatory response to LPS than the whole brain microglia population.
Another possibility is the compensation of microglia functions by other brain cell types. In the study on MPTP(36), flow cytometry analysis of chemokines and proinflammatory cytokines in astrocytes from the substantia nigra and striatum revealed that PLX5622 significantly increased the IL-6 and TNF response to MPTP. These findings can be interpreted to suggest that microglia cells down regulate the astrocytic response to inflammatory insults. There is already evidence that astrocytes from mice treated chronically with the CSF-1R antagonist PLX3397 to deplete microglia still respond to LPS in vivo by developing a reactive A1 phenotype(41). This is probably facilitated by the lack of IL-10 from microglial origin as this anti-inflammatory cytokine normally lowers the proinflammatory profile of LPS- activated astrocytes(42). Activation of an astrocyte-dependent type 1 interferon response was also proposed to account for the gray matter neurodegeneration that was observed at a late stage in a model of diphtheria toxin-induced induced microglia depletion in a Cx3cr1-CreER mouse system(43). The possibility that reactive A1 astrocytes induced by LPS take over in the absence of microglia is consistent with the observation that in our study brain IL-6, a cytokine mainly produced by astrocytes during neuroinflammation(44), was the only cytokine of which the gene expression in response to LPS was enhanced by PLX5622. The increased expression of the interferon-dependent gene Oas1a in the hypothalamus of diphtheria toxin treated transgenic rats follows the same direction of change.
Another mechanism for the lack of attenuation of neuroinflammation by microglia depletion could be an enhanced trafficking of immune cells into the brain of microglia depleted mice. However, this is unlikely to account for the present results as it has been shown that PLX3397 treatment does not compromise the integrity of the blood-brain barrier, based on blue Evans coloration exclusion(11). In addition, in situations in which there was evidence of increased infiltration of lymphocytes in the brain of microglia-depleted mice, genetic elimination of lymphocytes did not modify the increased sensitivity of microglia depleted mice to neurodegeneration(36). The possible existence of a compromised blood-brain barrier has not yet been examined in the diphtheria toxin-induced transgenic model.
There has been no previous attempt to assess the effect of microglial depletion on the ability of rodents to engage in strenuous exercise, as measured by voluntary wheel running activity or by treadmill running. Our results show that PLX5622 decreased the amount of voluntary wheel running at baseline by about 20%. It is possible to interpret this finding in the context of what is already known concerning the involvement of microglia in the beneficial effects of physical exercise. In particular, microglial activation within the neurogenic niche has been shown to mediate the beneficial effects of running wheel activity on hippocampal neurogenesis in the adult or aged mouse brain(45, 46). In addition, wheel running has been reported to induce microglia proliferation in the adult murine cortex, which could play a role in the positive effects of physical exercise on neurological health(47, 48). Our observation of a significant decrease in voluntary wheel running activity in microglia depleted mice is consistent with this hypothesis.
A limitation of the current study is the absence of investigation of possible sex differences. We were unable to assess possible sex differences in the extent of microglia depletion induced by CSF-1R antagonism in mice or by immunotoxin in transgenic rats and in the effects of microglia depletion on the inflammatory and behavioral response to LPS as all the experiments were carried out in males. However, experiments carried out with PLX5622 and PLX3397 revealed no sex differences in the extent of microglia depletion induced by either of these treatments(30, 34, 49-52). In the same manner, female and male Cx3cr1-Dtr rats were found to respond identically to diphtheria toxin administration in terms of microglia depletion and body weight loss(15). This does not eliminate the possibility of an interaction between microglial depletion and the effect of the intervention, LPS in this case, as such an interaction has been described for the effects of microglial depletion by PLX3397 in rats fed a high fat diet. Microglia depletion protected only male but not female mice from the deleterious effects of a high fat diet on executive function(53).