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Macrophages Storm Blood-brain Barrier, Clear Plaques—or Do They?
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6 June 2008. Blocking the signaling of TGF-β in peripheral macrophages allowed those immune-system scavengers to enter the brain and clear amyloid deposits, mitigating some symptoms in an AD mouse model, according to an advance online report in Nature Medicine. The study, by senior author Richard Flavell of Yale University School of Medicine in New Haven, Connecticut, suggests that macrophages can enter the brain in significant numbers and directly attack amyloid plaques. The finding holds out the promise of peripheral therapy, which would circumvent the troublesome requirement of broaching the blood-brain barrier. But some commentators said more work needs to be done to prove definitively that the cells responsible for the plaque clearance did indeed come from the bloodstream and that they could be deployed safely in the brain.
Flavell and his team were expecting quite different results when they devised the protocol for this study. First author Terrence Town of Cedars-Sinai Medical Center in Los Angeles, California, said he thought the interruption of TGF-β signaling would provoke an inflammatory response. That effect had already been reported in microglia, immune cells that reside in the brain (Brionne et al., 2003). Town used the CD11c-DNR mouse, in which the TGF-β signaling pathway is blocked only in macrophages, dendritic cells, and natural-killer cells. The mouse was made in Flavell’s laboratory, which specializes in studies of T cell activation and differentiation and autoimmune disease. By crossing the TGF-β-blocked mice with APP-transgenic mice, Town hoped to explore whether and how TGF-β signaling in those peripheral cells contributes to AD.
“We thought we would get mice that were just full of inflammation, and the disease would get worse,” Town said. “We were utterly surprised when we saw the opposite.”
What Town and his colleagues report instead is no evidence of an inflammatory response and a seven- to eightfold increase in the percentage of peripheral macrophages infiltrating the brains of the double-transgenic mice, as tagged by the CD45, CD11b, and CD11c markers and other measures. They also saw a reduction in plaque and soluble amyloid-β of up to 90 percent in both cerebrovascular and parenchymal tissue. “The amyloid load was strikingly reduced, not only histologically, but also biochemically,” said Town.
“These data fit very well with the novel concept that systemic innate immune cells have the capacity to fight against toxic proteins, but they do not do it in an efficient manner,” wrote Serge Rivest of Laval University in Quebec City in an e-mail. Rivest had previously suggested that macrophages could be instrumental in attacking plaque in the brain. “Improving both the infiltration and immune properties of these cells will hopefully soon be a very effective new therapy to cure AD.” (See extended comment below.)
Yet in behavioral tests, the intervention had only a modest effect on the spatial learning and memory deficits in mice that most closely resemble AD symptoms in people. There was no appreciable difference in performance in the Morris water maze, for example, and only a modest improvement in spontaneous Y-maze alternation, a measure of spatial working memory. Town and his colleagues did see a complete mitigation of the hyperactivity associated with the Tg2576 phenotype, which is thought to be brought on by cortical and/or hippocampal injury in these Alzheimer’s mice. But that hyperactivity has no established human correlate in AD.
Joseph El Khoury, for one, is not disturbed by the lackluster behavioral results. “The AD models in mice aren’t perfect,” said El Khoury, of Massachusetts General Hospital in Boston. “We should focus on the significant amelioration of pathology, rather than the behavioral effects.”
More questions have come regarding the authors’ assertion that peripheral macrophages are responsible for the plaque clearance. “We are not convinced that CD11c is a marker that can distinguish microglia from invading peripheral cells,” noted Mathias Jucker of the University of Tuebingen in an e-mail. Tony Wyss-Coray of Stanford University School of Medicine in Palo Alto, California, whose work Town credits as his inspiration, said he likewise lost faith in that marker’s ability to identify peripheral cells after reading Karen Bulloch’s recent study about CD11c cells (Bulloch et al., 2008). Furthermore, Wyss-Coray’s work shows that TGF-β has a neuroprotective effect mediated by the microglia (Wyss-Coray et al., 2001; Tesseur et al., 2006).
“That doesn’t diminish what they found at all,” said Wyss-Coray, because TGF-β, like other cytokines, can have different effects on different cell types. “And whether these cells came recently from the blood or were already in the brain, I don’t care. They have a very striking effect on the pathology.”
But the questions of the provenance and brain-homing capacity of these cells do have implications for treatment in humans, where researchers have struggled to find AD therapies that can penetrate the blood-brain barrier. Town said that three different lines of evidence implicate macrophages rather than microglia in this study. In addition to the panel of immune markers, he ran tests showing that the microglia did not have the TGF-β signaling blockade, while the peripheral macrophages did. And in brain-tissue sections, the CD45+ cells appeared smooth and round, quite distinct from the process-ridden, irregularly shaped microglia, said Town. He and his colleagues saw these round cells in the brains’ blood vessels, escaping the blood vessels, surrounding amyloid deposits, and engulfing them.
“These peripheral macrophages were entering the brain and devouring the plaque,” Town said. “We caught them in the act.”
“It's pretty convincing that the cells are coming from the blood,” El Khoury agreed. He suggested parabiosis experiments of the kind performed previously (see Ajame et al., 2007; Mildner et al., 2007) to demonstrate conclusively whether the relevant factors were peripheral. To El Khoury, the big advance here is demonstrating that the immune system can be revved up to take apart amyloid deposits without simultaneously promoting potentially damaging inflammation. In the paper’s supplementary material, fluorescence-activated cell sorting characterization indicates that the vast majority of macrophages that entered the brain are of the anti-inflammatory subtype Ly-6C–.
“The question has been, how can you increase the phagocytosis and clearance of plaque without causing all this damage?” said El Khoury. “That’s what they did in this really impressive paper.”
Pritam Das of the Mayo Graduate School of Medicine in Jacksonville, Florida, is also persuaded that the cells are peripheral macrophages. In an e-mail he called the findings “fascinating” but cautioned that the effects on neurodegeneration need to be explored (see extended comment below).
“Certainly, one of the concerns of having long-term peripheral infiltration of macrophages (regardless of their phenotype), is unwanted reactions on other cell types/tissues in the brain,” Das wrote.
For his part, Flavell agreed that any clinical application of this technique would need to be approached with caution, but he also noted that antibodies to TGF-β have already been demonstrated to be safe in Phase 1 clinical trials for fibrotic disease.—Karen Wright.
Karen Wright is a freelance writer in New Hampshire, U.S.
Reference:
Town T, Laouar Y, Pittenger C, Mori T, Szekely CA, Tan J, Duman RS, Flavell RA. Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology. Nat Med. 2008 May 30; Abstract
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Comments on News and Primary Papers |
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Primary Papers: Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology.
Comment by: Serge Rivest
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Submitted 6 June 2008
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Posted 6 June 2008
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In this study, the authors have found that TGF-β signaling inhibits the natural properties of macrophages to clear Aβ and infiltrate the CNS of APP mice. Knocking out such signaling events was found to improve both the brain infiltration of bone marrow-derived macrophages/microglia and their clearance of Aβ, which prevented the cognitive decline in mouse models of AD.
These data fit very well with the novel concept that systemic innate immune cells have the capacity to fight against toxic proteins but do not do it in an efficient manner. That's probably because of anti-inflammatory signals (e.g., TGF-β), as elegantly demonstrated by Town and colleagues.
We recently reported that Toll-like receptor 2 gene deletion is also associated with Aβ42 accumulation and cognitive impairment, while TLR2 gene expression in bone marrow-derived cells rescued such a memory deficit (Richard et al., 2008). Of great interest here is that APPtg/TLR2 knockout mice had a spontaneous increase in TGF-β gene expression in immune cells adjacent to...
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In this study, the authors have found that TGF-β signaling inhibits the natural properties of macrophages to clear Aβ and infiltrate the CNS of APP mice. Knocking out such signaling events was found to improve both the brain infiltration of bone marrow-derived macrophages/microglia and their clearance of Aβ, which prevented the cognitive decline in mouse models of AD.
These data fit very well with the novel concept that systemic innate immune cells have the capacity to fight against toxic proteins but do not do it in an efficient manner. That's probably because of anti-inflammatory signals (e.g., TGF-β), as elegantly demonstrated by Town and colleagues.
We recently reported that Toll-like receptor 2 gene deletion is also associated with Aβ42 accumulation and cognitive impairment, while TLR2 gene expression in bone marrow-derived cells rescued such a memory deficit (Richard et al., 2008). Of great interest here is that APPtg/TLR2 knockout mice had a spontaneous increase in TGF-β gene expression in immune cells adjacent to the senile plaques. We can therefore propose that macrophages are not properly activated and do not efficiently infiltrate the CNS of APP mice. This natural innate immune mechanism against endogenously produced toxic elements may prevent chronic diseases, such as AD (see Soulet and Rivest, 2008). Improving both the infiltration and immune properties of these cells will hopefully soon be an effective new therapy to cure AD. The debate about the physiological relevance of these cells in the CNS will be over once patients are cured.
 View all comments by Serge Rivest |
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Primary Papers: Blocking TGF-beta-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology.
Comment by: Pritam Das
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Submitted 6 June 2008
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Posted 6 June 2008
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In this study, Town et al. present some fascinating findings with regard to the role of peripheral macrophages and Aβ amyloid clearance from the brains of Tg2576 mice. The authors genetically interrupted TGF-β signaling specifically in peripheral macrophages of Tg2576 mice and then evaluated Aβ pathology during aging of these mice. To the authors’ surprise, Aβ deposits were significantly attenuated in both brain parenchymal and cerebral blood vessels in these mice. Based on their data (both in vivo and in vitro), the authors suggest that the mechanism for this reduction in Aβ deposition may be due to increased infiltration of these altered peripheral macrophages into the brain and around cerebral blood vessels, resulting in increased Aβ phagocytosis. Although there are much recent data for the role of resident microglial cells in enhancing microglial-mediated phagocytosis of Aβ plaques, this is the first report to directly indicate peripheral macrophages in Aβ phagocytosis and clearance mechanisms.
Undoubtedly, these interesting results will facilitate future investigations...
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In this study, Town et al. present some fascinating findings with regard to the role of peripheral macrophages and Aβ amyloid clearance from the brains of Tg2576 mice. The authors genetically interrupted TGF-β signaling specifically in peripheral macrophages of Tg2576 mice and then evaluated Aβ pathology during aging of these mice. To the authors’ surprise, Aβ deposits were significantly attenuated in both brain parenchymal and cerebral blood vessels in these mice. Based on their data (both in vivo and in vitro), the authors suggest that the mechanism for this reduction in Aβ deposition may be due to increased infiltration of these altered peripheral macrophages into the brain and around cerebral blood vessels, resulting in increased Aβ phagocytosis. Although there are much recent data for the role of resident microglial cells in enhancing microglial-mediated phagocytosis of Aβ plaques, this is the first report to directly indicate peripheral macrophages in Aβ phagocytosis and clearance mechanisms.
Undoubtedly, these interesting results will facilitate future investigations in this area; however, several questions from this report need further clarifications. Certainly, the gold standard for such experiments will be studies in chimeric mice to evaluate the effects of such altered peripheral macrophages on Aβ pathology. However, caution is warranted with regard to consequences of long-term peripheral inhibition of TGF-β signaling on macrophages. What is the effect on peripheral T cells in this scenario? Do peripheral T cells infiltrate into the CNS, and what is the phenotype of these cells? Although the authors evaluated extensively Aβ pathology in these studies, they missed an opportunity to evaluate any effects on neurodegeneration. One of the concerns of having long-term peripheral infiltration of macrophages, regardless of their phenotype, is unwanted reactions on other cell types/tissues in the brain.
In any event, these new findings do provide a unique peripheral approach for attenuating Aβ deposition in the brain and certainly warrant further investigations.
View all comments by Pritam Das |
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Comment by: Terrence Town
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Submitted 10 June 2008
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Posted 12 June 2008
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I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while...
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I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while opposing the proinflammatory response, both of which likely exist as a continuum of innate immune cell activation profiles (Town et al., 2005). But, if we can accomplish this, will amyloid-reducing therapies ultimately be successful AD therapeutics? As stated by Dave Morgan and others on this forum, the first test of the amyloid cascade hypothesis of AD in humans will likely be the Aβ vaccine. We anxiously await whether the hypothesis holds up and delivers an efficacious AD therapy. If it does, then the floodgates will open for a whole host of amyloid-targeted AD therapeutics—both immune and non-immune.
About the issue raised by Mathias Jucker and Tony Wyss-Coray of CD11c as a marker for blood-borne innate immune cells/macrophages versus microglia, I should mention that we initially thought that CD11c would be a microglial marker in the context of AD. However, after examining numerous brain sections from various ages of wild-type versus Tg2576 or mutant APP/PS1 doubly transgenic mice for CD11c expression, we concluded that while microglia in the parenchyma around Aβ deposits were CD11b, CD45, MHC II, F4/80 Ag, and CD68 positive, they were negative for CD11c. However, we did observe a small number of round, non-process bearing CD11c positive cells within the lumen of blood vessels in both Tg2576 and APP/PS1 mice, consistent with Stalder and colleagues’ report of invading hematopoietic cells in brains of aged Tg2576 mice. At the time that we were checking for CD11c expression in AD mice, Alon Monsonego and Harold Weiner published a review in Science where they mentioned (as data not shown) that plaque-associated microglia were CD11c positive. I called Alon and asked him about the methodological details. However, after trying various tissue handling techniques, antibodies, and confocal settings, I was unable to reproduce this despite getting microglia in day 20 MOG-EAE brain sections to light up like a Christmas tree with CD11c. I came away thinking that it is possible to acutely activate microglia with the necessary vigor to promote CD11c expression, for example, in the context of EAE. However, I believe that this form of activation does not occur in AD mice, where the profile more closely resembles a chronic, persistent, low-level inflammation.
I have recently read the paper by Bulloch and coworkers with great interest, which shows the presence of CD11c/EYFP “dendritic-like” mouse microglia in multiple stages of life. However, because the authors did not quantify their observations, it is unclear how prevalent these cells are in the brain, and/or whether these cells arose from the blood or were long-term CNS residents. Further, the authors had difficulty in co-staining these cells with CD11c antisera in tissue sections, raising a possibility that those who work with transgenics are all too aware of: expression of transgenes is often more promiscuous than expected. In our study, we demonstrated a seven- to eightfold increase in CD45+CD11b+CD11c+CD68+Ly-6C- cells (presumed “anti-inflammatory” macrophages initially immunophenotyped by Littman’s group in Geissmann et al., 2003) in our crossed mice, and immunohistochemical approaches revealed prominent vascular cuffing, where these cells appeared to be entering the brain via cerebrovessels. Regarding the questions from Joseph El Khoury and Pritam Das about the origin of these brain macrophages, we agree that the “acid test” of whether the macrophage-like cells that we see in and around cerebral vessels and β amyloid plaques arise from the periphery or from within the CNS would either be a chimeric approach or parabiosis. We moved away from the chimeric approach following recent reports in Nature Neuroscience (Ajami et al., 2007; Mildner et al., 2007) showing that the act of irradiating the mice leads to brain infiltration of monocytes/macrophages—the very dependent variable that we are interested in testing. However, we believe that 1) parabiosis of AD mice with GFP+CD11c-DNR mice or 2) chemical methods of ablating hematopoietic cells in AD mice followed by reconstitution with GFP+CD11c-DNR bone marrow containing or depleted of macrophages represent possible strategies that we are currently pursuing.
Finally, Pritam Das raises the interesting questions of the long-term consequences of inhibiting TGF-β signaling on peripheral macrophages and the effects on T cells. We did not observe increased peripheral numbers of innate immune cells (including macrophages and dendritic cells), CD4+ or CD8+ T cells, or B cells in CD11c-DNR mice alone or in Tg2576xCD11c-DNR crossed mice, suggesting that an autoimmune state was not generated and that the increased abundance of macrophages in the brains of our crossed mice was β amyloid-directed. We also quantified T cells in brains of our crossed mice versus singly transgenic animals, and detected that about 4-5 percent of brain hematopoietic cells were TcRαβ positive (presumed T cells), and they were divided about equally between CD4+ and CD8+ subsets—however, these numbers were similar amongst wild-type, CD11c-DNR, APP/PS1, and APP/PS1xCD11c-DNR mice, suggesting that neither the CD11c-DNR nor the APP/PS1 transgenes were able to modify brain entry of T cells. Finally, regarding the issue of assessing neurodegeneration, we are currently pursuing this line of investigation by quantitative synaptophysin immunohistochemistry and hope to answer this question in the near future.
References:
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007 Dec;10(12):1538-43. Abstract
Bulloch K, Miller MM, Gal-Toth J, Milner TA, Gottfried-Blackmore A, Waters EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC, Steinman RM, McEwen BS. CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain. J Comp Neurol. 2008 Jun 10;508(5):687-710. Abstract
Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003 Jul;19(1):71-82. Abstract
Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nat Neurosci. 2007 Dec 1;10(12):1544-53. Abstract
Monsonego A, Weiner HL. Immunotherapeutic approaches to Alzheimer's disease. Science. 2003 Oct 31;302(5646):834-8. Abstract
Richard KL, Filali M, Préfontaine P, Rivest S. Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J Neurosci. 2008 May 28;28(22):5784-93. Abstract
Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract
Town T, Nikolic V, Tan J. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2005 Oct 31;2:24. Abstract
Wyss-Coray T, Masliah E, Mallory M, McConlogue L, Johnson-Wood K, Lin C, Mucke L. Amyloidogenic role of cytokine TGF-1 in transgenic mice and in Alzheimer's disease. Nature. 1997 Oct 9;389(6651):603-6. Abstract
View all comments by Terrence Town |
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Comment by: Milan Fiala (Disclosure)
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Submitted 13 August 2008
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Posted 14 August 2008
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I am glad that the researchers studying transgenic models are finally confirming our results published in 2002 (Fiala et al., 2002), which showed transmigration of macrophages across the brain vessel wall and clearance of plaques by these large macrophages.
The migrating macrophages broke through ZO-1 tight junction barrier and aggregated around brain vessels similarly as in HIV encephalitis. This has been followed by a recent publication in PNAS (Fiala et al., 2007). The animal studies cannot resolve the crucial question: are macrophages of patients with AD different from those of control subjects? The answers for interested readers are available in our PNAS article and more current work presented at ICAD. Not only macrophages penetrate across the blood-brain barrier but also clear oligomeric amyloid-β from neurons.
References:
Fiala M, Liu QN, Sayre J, Pop V, Brahmandam V, Graves MC, Vinters HV. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier. Eur J Clin Invest. 2002 May;32(5):360-71. Abstract
Fiala M, Liu PT, Espinosa-Jeffrey A, Rosenthal MJ, Bernard G, Ringman JM, Sayre J, Zhang L, Zaghi J, Dejbakhsh S, Chiang B, Hui J, Mahanian M, Baghaee A, Hong P, Cashman J. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12849-54. Abstract
View all comments by Milan Fiala |
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