Alicia A. Babcock

Chemokine Expression by Glial Cells Directs Leukocytes to Sites of Axonal Injury in the CNS

Innate responses in the CNS are critical to first line defense against infection and injury. Leukocytes migrate to inflammatory sites in response to chemokines. We studied leukocyte migration and glial chemokine expression within the denervated hippocampus in response to axonal injury caused by entorhinodentate lesions. A population of Mac1/CD11b+ CD45high macrophages (distinct from CD45low microglia) was specifically detected within the lesion-reactive hippocampus by 12 hr after injury. Significant infiltration by CD3+ T cells did not occur in the denervated hippocampus until 24 hr after axotomy. A broad spectrum of chemokines [RANTES/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, interferon γ inducible protein (IP)-10/CXCL10, macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4, and MIP-2/CXCL2] was induced at this time. RANTES/CCL5 was not significantly elevated until 24 hr after axotomy, whereas MCP-1/CCL2 was significantly induced before leukocyte infiltration occurred. Neither T cells nor macrophages infiltrated the denervated hippocampus of CCR2-deficient mice, arguing for a critical role for the CCR2 ligand MCP-1/CCL2 in leukocyte migration. Both T cells and macrophages infiltrated CCR5-deficient hippocampi, showing that CCR5 ligands (including RANTES/CCL5) are not critical to this response. In situ hybridization combined with immunohistochemistry for ionized binding calcium adapter molecule (iba)1 or glial fibrillary acidic protein (GFAP) identified iba1+ microglia and GFAP+ astrocytes as major sources of MCP-1/CCL2 within the lesion-reactive hippocampus. We conclude that leukocyte responses to CNS axonal injury are directed via innate glial production of chemokines.

Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor

Microglia and infiltrating leukocytes are considered major producers of tumor necrosis factor (TNF), which is a crucial player in cerebral ischemia and brain inflammation. We have identified a neuroprotective role for microglial-derived TNF in cerebral ischemia in mice. We show that cortical infarction and behavioral deficit are significantly exacerbated in TNF-knock-out (KO) mice compared with wild-type mice. By using in situ hybridization, immunohistochemistry, and green fluorescent protein bone marrow (BM)-chimeric mice, TNF was shown to be produced by microglia and infiltrating leukocytes. Additional analysis demonstrating that BM-chimeric TNF-KO mice grafted with wild-type BM cells developed larger infarcts than BM-chimeric wild-type mice grafted with TNF-KO BM cells provided evidence that the neuroprotective effect of TNF was attributable to microglial- not leukocyte-derived TNF. In addition, observation of increased infarction in TNF-p55 receptor (TNF-p55R)-KO mice compared with TNF-p75R and wild-type mice suggested that microglial-derived TNF exerts neuroprotective effects through TNF-p55R. We finally report that TNF deficiency is associated with reduced microglial population size and Toll-like receptor 2 expression in unmanipulated brain, which might also influence the neuronal response to injury. Our results identify microglia and microglial-derived TNF as playing a key role in determining the survival of endangered neurons in cerebral ischemia.

Toll-Like Receptor 2 Signaling in Response to Brain Injury: An Innate Bridge to Neuroinflammation

Reactive gliosis is a prominent feature of neurodegenerative and neuroinflammatory disease in the CNS, yet the stimuli that drive this response are not known. There is growing appreciation that signaling through Toll-like receptors (TLRs), which is key to generating innate responses to infection, may have pathogen-independent roles. We show that TLR2 was selectively upregulated by microglia in the denervated zones of the hippocampus in response to stereotactic transection of axons in the entorhinal cortex. In mice lacking TLR2, there were transient, selective reductions in lesion-induced expression of cytokines and chemokines. Recruitment of T cells, but not macrophages, was delayed in TLR2-deficient mice, as well as in mice lacking TNFR1 (tumor necrosis factor receptor 1). TLR2 deficiency also affected microglial proliferative expansion, whereas all of these events were unaffected in TLR4-mutant mice. Consistent with the fact that responses in knock-out mice had all returned to wild-type levels by 8 d, there was no evidence for effects on neuronal plasticity at 20 d. These results identify a role for TLR2 signaling in the early glial response to brain injury, acting as an innate bridge to neuroinflammation.

Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice.

BackgroundInterleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1β is primarily neurotoxic in ischemic stroke, TNF-α may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1β and TNF-α are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.MethodsWe used flow cytometry and immunohistochemistry to examine cellular co-expression of IL-1β and TNF-α at 6, 12 and 24 hours after permanent middle cerebral artery occlusion in mice, validating the results by the use of bone marrow chimeric mice.ResultsWe found that IL-1β and TNF-α were expressed in largely segregated populations of CD11b+CD45dim microglia and CD11b+CD45high macrophages, with cells expressing both cytokines only rarely. The number of Gr1+ granulocytes producing IL-1β or TNF-α was very low, and we observed no IL-1β- or TNF-α-expressing T cells or astrocytes.ConclusionTaken together, the results show that IL-1β and TNF-α are produced by largely segregated populations of microglia and macrophages after ischemic stroke in mice. Our findings provide evidence of a functional diversity among different subsets of microglia and macrophages that is potentially relevant to future design of anti-inflammatory therapies in stroke.

Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

Reactive microgliosis is a highly characteristic response to neural injury and disease, which may influence neurodegenerative processes and neural plasticity. We have investigated the origin and characteristics of reactive microglia in the acute phase of their activation in the dentate gyrus following transection of the entorhino‐dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion‐reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (BrdU) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early‐activated resident microglia, rather than by infiltrating bone marrow‐derived cells. The number of CD34+ microglia peaked at day 3 when 67% of the resident CD11b/Mac‐1+ microglia co‐expressed CD34, and all CD34+ cells co‐expressed Mac‐1, and decreased sharply toward day 5, unlike Mac‐1, which was maximally expressed at day 5. Approximately 80% of the CD34+ cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early‐activated microglia in the facial motor nucleus following peripheral axotomy. The results suggest lesion‐reactive microglia to consist of functionally distinct subpopulations of cells; a major population of activated resident CD34+Mac‐1+ microglia with a high capacity for self‐renewal, and a subpopulation of CD34−Mac‐1+ microglia which has a mixed extrinsic and intrinsic origin and whose proliferative capacity is unknown.

NF-κB-Driven STAT2 and CCL2 Expression in Astrocytes in Response to Brain Injury

Tissue response to injury includes expression of genes encoding cytokines and chemokines. These regulate entry of immune cells to the injured tissue. The synthesis of many cytokines and chemokines involves NF-κB and signal transducers and activators of transcription (STAT). Injury to the CNS induces glial response. Astrocytes are the major glial population in the CNS. We examined expression of STATs and the chemokine CCL2 and their relationship to astroglial NF-κB signaling in the CNS following axonal transection. Double labeling with Mac-1/CD11b and glial fibrillary acidic protein revealed that STAT2 up-regulation and phosphorylation colocalized exclusively to astrocytes, suggesting the involvement of STAT2 activating signals selectively in astroglial response to injury. STAT1 was also up-regulated and phosphorylated but not exclusively in astrocytes. Both STAT2 up-regulation and phosphorylation were NF-κB -dependent since they did not occur in the lesion-reactive hippocampus of transgenic mice with specific inhibition of NF-κB activation in astrocytes. We further showed that lack of NF-κB signaling significantly reduced injury-induced CCL2 expression as well as leukocyte infiltration. Our results suggest that NF-κB signaling in astrocytes controls expression of both STAT2 and CCL2, and thus regulates infiltration of leukocytes into lesion-reactive hippocampus after axonal injury. Taken together, these findings indicate a central role for astrocytes in directing immune-glial interaction in the CNS injury response.

Signaling through MyD88 Regulates Leukocyte Recruitment after Brain Injury

Injury to the CNS provokes an innate inflammatory reaction that engages infiltrating leukocytes with the capacity to repair and/or exacerbate tissue damage. The initial cues that orchestrate leukocyte entry remain poorly defined. We have used flow cytometry to investigate whether MyD88, an adaptor protein that transmits signals from TLRs and receptors for IL-1 and IL-18, regulates leukocyte infiltration into the stab-injured entorhinal cortex (EC) and into sites of axonal degeneration in the denervated hippocampus. We have previously established the kinetics of leukocyte entry into the denervated hippocampus. We now show that significant leukocyte entry into the EC occurs within 3–12 h of stab injury. Whereas T cells showed small, gradual increases over 8 days, macrophage infiltration was pronounced and peaked within 12–24 h. MyD88 deficiency significantly reduced macrophage and T cell recruitment to the stab-injured EC and the denervated hippocampus at 5 days post-injury. Whereas macrophage and T cell entry remained impaired into the denervated hippocampus of MyD88-deficient mice at 8 days, leukocyte infiltration into the stab-injured EC was restored to levels observed in wild-type mice. Transcripts for TNF-α, IL-1β, and CCL2, which increased >50-fold after stab injury in C57BL/6 mice at the time of peak expression, were severely reduced in injured MyD88 knockout mice. Leukocyte recruitment and gene expression were unaffected in TLR2-deficient or TLR4 mutant mice. No significant differences in gene expression were observed in mice lacking IL-1R or IL-18R. These data show that MyD88-dependent signaling mediates proinflammatory gene expression and leukocyte recruitment after CNS injury.

Accelerated microglial pathology is associated with Aβ plaques in mouse models of Alzheimer's disease.

Microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network. Here, we used a digital tool for the quantitative morphometric characterization of fine cortical microglial structures in mice, and the changes they undergo with aging and in Alzheimers‐like disease. We show that, compared with microglia in young mice, microglia in old mice are less ramified and possess fewer branches and fine processes along with a slightly increased proinflammatory cytokine expression. A similar microglial pathology appeared 6–12 months earlier in mouse models of Alzheimers disease (AD), along with a significant increase in brain parenchyma lacking coverage by microglial processes. We further demonstrate that microglia near amyloid plaques acquire unique activated phenotypes with impaired process complexity. We thus show that along with a chronic proinflammatory reaction in the brain, aging causes a significant reduction in the capacity of microglia to scan their environment. This type of pathology is markedly accelerated in mouse models of AD, resulting in a severe microglial process deficiency, and possibly contributing to enhanced cognitive decline.

Cytokine-producing microglia have an altered beta-amyloid load in aged APP/PS1 Tg mice.

Beta-amyloid (Aβ) plaques and chronic neuroinflammation are significant neuropathological features of Alzheimers disease. Microglial cells in aged brains have potential to produce cytokines such as TNF and IL-1 family members (IL-1α, IL-1β, and IL-1Ra) and to phagocytose Aβ in Alzheimers disease, however the inter-relationship between these processes is poorly understood. Here we show that % Aβ plaque load followed a sigmoidal trajectory with age in the neocortex of APPswe/PS1ΔE9 Tg mice, and correlated positively with soluble Aβ40 and Aβ42. Aβ measures were moderately correlated with mRNA levels of CD11b, TNF, and IL-1Ra. Cytokine production and Aβ load were assessed in neocortical CD11b(+)(CD45(+)) microglia by flow cytometry. Whereas most microglia in aged mice produced IL-1Ra, relatively low proportions of microglia produced TNF, IL-1α, and IL-1β. However, microglial production of these latter cytokines was generally increased in APP/PS1 Tg mice. Microglia that phagocytosed endogenously-produced Aβ were only observed in APP/PS1 Tg mice. Differences in phagocytic index and total Aβ load were observed in microglia with specific cytokine profiles. Both phagocytic index and total Aβ load were higher in IL-1α(+) and IL-1Ra(+) microglia, than microglia that did not produce these cytokines. In contrast, total Aβ load was lower in IL-1β(+) and TNF(+) microglia, compared to IL-1β(-) and TNF(-) microglia, and TNF(+) microglia also had a lower phagocytic index. Using GFP bone marrow chimeric mice, we confirmed that the majority of neocortical CD11b(+)(CD45(+)) microglia were resident cells (GFP(-)) in APP/PS1 Tg mice, even after selectively analysing CD11b(+)CD45(high) cells, which are typically considered to be infiltrating cells. Together, our data demonstrate that cytokine expression is selectively correlated with age and Aβ pathology, and is associated with an altered Aβ load in phagocytic microglia from APP/PS1 Tg mice. These findings have implications for understanding the regulation of microglial cytokine production and phagocytosis of Aβ in Alzheimers disease.

Enhanced microglial clearance of myelin debris in T cell-infiltrated central nervous system

Acute multiple sclerosis lesions are characterized by accumulation of T cells and macrophages, destruction of myelin and oligodendrocytes, and axonal damage. There is, however, limited information on neuroimmune interactions distal to sites of axonal damage in the Tcell-infiltrated central nervous system. We investigated T-cell infiltration, myelin clearance, microglial activation, and phagocytic activity distal to sites of axonal transection through analysis of the perforant pathway deafferented dentate gyrus in SJL mice that had received T cells specific for myelin basic protein (TMBP) or ovalbumin (TOVA). The axonal lesion of TMBP-recipient mice resulted in lesion-specific recruitment of large numbers of T cells in contrast to very limited T-cell infiltration in TOVA-recipient and -naïve perforant pathway-deafferented mice. By double immunofluorescence and confocal microscopy, infiltration with TMBP but not TOVA enhanced the microglial response to axonal transection and microglial phagocytosis of myelin debris associated with the degenerating axons. Because myelin antigen-specific immune responses may provoke protective immunity, increased phagocytosis of myelin debris might enhance regeneration after a neural antigen-specific T cell-mediated immune response in multiple sclerosis.