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Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain.

Howe CL, Lafrance-Corey RG, Sundsbak RS, Lafrance SJ - J Neuroinflammation (2012)

Bottom Line: Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus.Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain.These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. howe@mayo.edu

ABSTRACT

Background: Neuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus.

Methods: The identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the Theiler's murine encephalomyelitis virus. Behavioral consequences of immune cell depletion were assessed by Morris water maze.

Results: Inflammatory monocytes, defined as CD45hiCD11b++F4/80+Gr1+1A8-, and neutrophils, defined as CD45hiCD11b+++F4/80-Gr1+1A8+, were found in the brain at 12 h after infection. Flow cytometry of brain-infiltrating leukocytes collected from LysM: GFP reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. Microscopy of sections from infected LysM:GFP mice showed that infiltrating cells were concentrated in the hippocampal formation. Immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. Immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. Immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the Morris water maze.

Conclusions: Neutrophils and inflammatory monocytes rapidly and robustly responded to Theiler's virus infection by infiltrating the brain. Inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. Depletion of inflammatory monocytes and neutrophils with the Gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

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Visualization of the green flourescent protein (GFP)+ infiltrate in the brain of Theiler's murine encephalomyelitis virus (TMEV)-infected LysM:eGFP mice. TMEV-infected LysM:eGFP reporter mice were killed by intracardiac perfusion of 4% paraformaldehyde 18 h after infection. Vibratome sections were prepared and analyzed by epifluorescence microscopy. The distribution of GFP+ neutrophils and inflammatory monocytes at low magnification (A, B) matches the distribution of inflammatory infiltrate observed in Figure 1. Higher magnification of a cross-section through the alveus and corpus callosum (C) and at the hippocampal fissure (D) shows the presence of both GFPhi and GFPmid cells with a variety of morphologies consistent with migratory behavior. Comparison of the epifluorescent signal (E) and the brightfield image (F) of a cross-section of the hippocampus confirms the presence of numerous GFP+ neutrophils and inflammatory monocytes at the hippocampal fissure (HF), in the alveus (alv), and proximal to the pyramidal neurons of CA1. Cg = cingulum bundle; DG = dentate gyrus. Green represents GFP in (A-E), blue in (C-D) shows 4',6-diamidino-2-phenylindole (DAPI). Results are representative of eight mice.
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Figure 6: Visualization of the green flourescent protein (GFP)+ infiltrate in the brain of Theiler's murine encephalomyelitis virus (TMEV)-infected LysM:eGFP mice. TMEV-infected LysM:eGFP reporter mice were killed by intracardiac perfusion of 4% paraformaldehyde 18 h after infection. Vibratome sections were prepared and analyzed by epifluorescence microscopy. The distribution of GFP+ neutrophils and inflammatory monocytes at low magnification (A, B) matches the distribution of inflammatory infiltrate observed in Figure 1. Higher magnification of a cross-section through the alveus and corpus callosum (C) and at the hippocampal fissure (D) shows the presence of both GFPhi and GFPmid cells with a variety of morphologies consistent with migratory behavior. Comparison of the epifluorescent signal (E) and the brightfield image (F) of a cross-section of the hippocampus confirms the presence of numerous GFP+ neutrophils and inflammatory monocytes at the hippocampal fissure (HF), in the alveus (alv), and proximal to the pyramidal neurons of CA1. Cg = cingulum bundle; DG = dentate gyrus. Green represents GFP in (A-E), blue in (C-D) shows 4',6-diamidino-2-phenylindole (DAPI). Results are representative of eight mice.

Mentions: We further confirmed the presence of neutrophils and inflammatory monocytes in the brain following infection using LysM:GFP reporter mice. These animals express enhanced green fluorescent protein (GFP) at the lysozyme M locus, yielding GFPhi neutrophils and GFPmid monocytes [10]. No other cells express GFP in these mice, making them an excellent tool for tracking neutrophils and inflammatory monocytes. We observed robust and clearly distinguished populations of GFPhi and GFPmid cells in the BILs at 18 hpi (Figure 5B). Gating into GFPhi, GFPmid, and GFPneg populations confirmed that our CD11b+++Gr1+1A8+ neutrophil population was almost exclusively GFPhi (Figure 5D, G), while our CD11b++Gr1+1A8- inflammatory monocyte population was almost exclusively GFPmid (Figure 5E, H). The GFPneg population in the 18 hpi BILs exhibited a CD11b+Gr1-1A8- immunophenotype consistent with microglia (resident macrophages) (Figure 5F, I). The clear distinction between GFP+ and GFP- cells in the BILs population was further exploited by assessing the physical location of infiltrating GFP+ cells in vibratome sections of brain from 18 hpi mice (Figure 6). We found that the majority of GFP+ cells were located in proximity to the hippocampal formation (Figure 6A), with high concentrations of labeled cells just superior and lateral to the hippocampus proper. Higher magnification also showed dense clusters of GFP+ cells in the corpus callosum and alveus (Figure 6B, C, E) and in the hippocampal fissure (Figure 6D, E). This suggests that the white matter tracts overlying the hippocampus and the vasculature-rich hippocampal fissure are primary sites for infiltration of neutrophils and inflammatory monocytes. These observations are consistent with the histological analyses shown in Figure 1 and further support a model in which neutrophils and inflammatory monocytes are rapid responders to brain infection. Of note, the robust localization of these cells to the hippocampal formation is consistent with our previously published observations that the hippocampus is the primary site of pathology following TMEV infection and that this damage is not a direct result of the virus but is rather a bystander phenomenon [8].


Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain.

Howe CL, Lafrance-Corey RG, Sundsbak RS, Lafrance SJ - J Neuroinflammation (2012)

Visualization of the green flourescent protein (GFP)+ infiltrate in the brain of Theiler's murine encephalomyelitis virus (TMEV)-infected LysM:eGFP mice. TMEV-infected LysM:eGFP reporter mice were killed by intracardiac perfusion of 4% paraformaldehyde 18 h after infection. Vibratome sections were prepared and analyzed by epifluorescence microscopy. The distribution of GFP+ neutrophils and inflammatory monocytes at low magnification (A, B) matches the distribution of inflammatory infiltrate observed in Figure 1. Higher magnification of a cross-section through the alveus and corpus callosum (C) and at the hippocampal fissure (D) shows the presence of both GFPhi and GFPmid cells with a variety of morphologies consistent with migratory behavior. Comparison of the epifluorescent signal (E) and the brightfield image (F) of a cross-section of the hippocampus confirms the presence of numerous GFP+ neutrophils and inflammatory monocytes at the hippocampal fissure (HF), in the alveus (alv), and proximal to the pyramidal neurons of CA1. Cg = cingulum bundle; DG = dentate gyrus. Green represents GFP in (A-E), blue in (C-D) shows 4',6-diamidino-2-phenylindole (DAPI). Results are representative of eight mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Visualization of the green flourescent protein (GFP)+ infiltrate in the brain of Theiler's murine encephalomyelitis virus (TMEV)-infected LysM:eGFP mice. TMEV-infected LysM:eGFP reporter mice were killed by intracardiac perfusion of 4% paraformaldehyde 18 h after infection. Vibratome sections were prepared and analyzed by epifluorescence microscopy. The distribution of GFP+ neutrophils and inflammatory monocytes at low magnification (A, B) matches the distribution of inflammatory infiltrate observed in Figure 1. Higher magnification of a cross-section through the alveus and corpus callosum (C) and at the hippocampal fissure (D) shows the presence of both GFPhi and GFPmid cells with a variety of morphologies consistent with migratory behavior. Comparison of the epifluorescent signal (E) and the brightfield image (F) of a cross-section of the hippocampus confirms the presence of numerous GFP+ neutrophils and inflammatory monocytes at the hippocampal fissure (HF), in the alveus (alv), and proximal to the pyramidal neurons of CA1. Cg = cingulum bundle; DG = dentate gyrus. Green represents GFP in (A-E), blue in (C-D) shows 4',6-diamidino-2-phenylindole (DAPI). Results are representative of eight mice.
Mentions: We further confirmed the presence of neutrophils and inflammatory monocytes in the brain following infection using LysM:GFP reporter mice. These animals express enhanced green fluorescent protein (GFP) at the lysozyme M locus, yielding GFPhi neutrophils and GFPmid monocytes [10]. No other cells express GFP in these mice, making them an excellent tool for tracking neutrophils and inflammatory monocytes. We observed robust and clearly distinguished populations of GFPhi and GFPmid cells in the BILs at 18 hpi (Figure 5B). Gating into GFPhi, GFPmid, and GFPneg populations confirmed that our CD11b+++Gr1+1A8+ neutrophil population was almost exclusively GFPhi (Figure 5D, G), while our CD11b++Gr1+1A8- inflammatory monocyte population was almost exclusively GFPmid (Figure 5E, H). The GFPneg population in the 18 hpi BILs exhibited a CD11b+Gr1-1A8- immunophenotype consistent with microglia (resident macrophages) (Figure 5F, I). The clear distinction between GFP+ and GFP- cells in the BILs population was further exploited by assessing the physical location of infiltrating GFP+ cells in vibratome sections of brain from 18 hpi mice (Figure 6). We found that the majority of GFP+ cells were located in proximity to the hippocampal formation (Figure 6A), with high concentrations of labeled cells just superior and lateral to the hippocampus proper. Higher magnification also showed dense clusters of GFP+ cells in the corpus callosum and alveus (Figure 6B, C, E) and in the hippocampal fissure (Figure 6D, E). This suggests that the white matter tracts overlying the hippocampus and the vasculature-rich hippocampal fissure are primary sites for infiltration of neutrophils and inflammatory monocytes. These observations are consistent with the histological analyses shown in Figure 1 and further support a model in which neutrophils and inflammatory monocytes are rapid responders to brain infection. Of note, the robust localization of these cells to the hippocampal formation is consistent with our previously published observations that the hippocampus is the primary site of pathology following TMEV infection and that this damage is not a direct result of the virus but is rather a bystander phenomenon [8].

Bottom Line: Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus.Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain.These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. howe@mayo.edu

ABSTRACT

Background: Neuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus.

Methods: The identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the Theiler's murine encephalomyelitis virus. Behavioral consequences of immune cell depletion were assessed by Morris water maze.

Results: Inflammatory monocytes, defined as CD45hiCD11b++F4/80+Gr1+1A8-, and neutrophils, defined as CD45hiCD11b+++F4/80-Gr1+1A8+, were found in the brain at 12 h after infection. Flow cytometry of brain-infiltrating leukocytes collected from LysM: GFP reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. Microscopy of sections from infected LysM:GFP mice showed that infiltrating cells were concentrated in the hippocampal formation. Immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. Immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. Immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the Morris water maze.

Conclusions: Neutrophils and inflammatory monocytes rapidly and robustly responded to Theiler's virus infection by infiltrating the brain. Inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. Depletion of inflammatory monocytes and neutrophils with the Gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

Show MeSH
Related in: MedlinePlus