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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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Flow cytometric assessment of the timecourse of neutrophil and inflammatory monocyte infiltration during acute infection. Brain-infiltrating leukocytes (BILs) were prepared from uninfected (A-C) mice or from mice infected with Theiler's murine encephalomyelitis virus (TMEV) for 12 h (D-F), 18 h (G-I), or 24 h (J-L). Cells were stained with antibodies against CD45, CD11b, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. Essentially no Gr1+ (B) or 1A8+ cells (C) were observed in uninfected mice and the forward scatter (FSC) vs side scatter (SSC) plot showed a clear absence of the larger, more granular cells present at later time points (A). In contrast, TMEV-infected mice exhibited both CD11b++Gr1+ inflammatory monocytes and CD11b+++1A8+ neutrophils as early as 12 hpi (D-E), with inflammatory monocytes outnumbering neutrophils (E, F). Both of these populations increased in number by 18 hpi (G-I). Neutrophils continued to increase in the infiltrate by 24 hpi (K, L) while inflammatory monocytes began to decrease at this time point (K, L). The granular profile of neutrophils was also clearly present by FSC and SSC at 24 hpi (J). Results are representative of more than 20 mice at each timepoint.
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Figure 7: Flow cytometric assessment of the timecourse of neutrophil and inflammatory monocyte infiltration during acute infection. Brain-infiltrating leukocytes (BILs) were prepared from uninfected (A-C) mice or from mice infected with Theiler's murine encephalomyelitis virus (TMEV) for 12 h (D-F), 18 h (G-I), or 24 h (J-L). Cells were stained with antibodies against CD45, CD11b, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. Essentially no Gr1+ (B) or 1A8+ cells (C) were observed in uninfected mice and the forward scatter (FSC) vs side scatter (SSC) plot showed a clear absence of the larger, more granular cells present at later time points (A). In contrast, TMEV-infected mice exhibited both CD11b++Gr1+ inflammatory monocytes and CD11b+++1A8+ neutrophils as early as 12 hpi (D-E), with inflammatory monocytes outnumbering neutrophils (E, F). Both of these populations increased in number by 18 hpi (G-I). Neutrophils continued to increase in the infiltrate by 24 hpi (K, L) while inflammatory monocytes began to decrease at this time point (K, L). The granular profile of neutrophils was also clearly present by FSC and SSC at 24 hpi (J). Results are representative of more than 20 mice at each timepoint.

Mentions: BILs were prepared from uninfected mice and at 12, 18, and 24 hpi (Figure 7) in order to determine how rapidly neutrophils and inflammatory monocytes enter the brain. We found a robust population of inflammatory monocytes present at 12 hpi (Figure 7E, F), suggesting that the cells had started infiltrating even earlier. Analysis of BILs at 6 hpi showed a small but inconsistent population of inflammatory monocytes (data not shown), indicating that infiltration began in earnest between 6 and 12 hpi. A small population of neutrophils was present in the BILs by 12 hpi (Figure 7E, F). By 18 hpi the density of both neutrophils and inflammatory monocytes had increased (Figure 7H, I). By 24 hpi the number of neutrophils continued to increase while the number of inflammatory monocytes decreased relative to 18 hpi (Figure 7K, L). Cell counts and relative percent of each cell population are provided in Table 1.


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

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

Flow cytometric assessment of the timecourse of neutrophil and inflammatory monocyte infiltration during acute infection. Brain-infiltrating leukocytes (BILs) were prepared from uninfected (A-C) mice or from mice infected with Theiler's murine encephalomyelitis virus (TMEV) for 12 h (D-F), 18 h (G-I), or 24 h (J-L). Cells were stained with antibodies against CD45, CD11b, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. Essentially no Gr1+ (B) or 1A8+ cells (C) were observed in uninfected mice and the forward scatter (FSC) vs side scatter (SSC) plot showed a clear absence of the larger, more granular cells present at later time points (A). In contrast, TMEV-infected mice exhibited both CD11b++Gr1+ inflammatory monocytes and CD11b+++1A8+ neutrophils as early as 12 hpi (D-E), with inflammatory monocytes outnumbering neutrophils (E, F). Both of these populations increased in number by 18 hpi (G-I). Neutrophils continued to increase in the infiltrate by 24 hpi (K, L) while inflammatory monocytes began to decrease at this time point (K, L). The granular profile of neutrophils was also clearly present by FSC and SSC at 24 hpi (J). Results are representative of more than 20 mice at each timepoint.
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Figure 7: Flow cytometric assessment of the timecourse of neutrophil and inflammatory monocyte infiltration during acute infection. Brain-infiltrating leukocytes (BILs) were prepared from uninfected (A-C) mice or from mice infected with Theiler's murine encephalomyelitis virus (TMEV) for 12 h (D-F), 18 h (G-I), or 24 h (J-L). Cells were stained with antibodies against CD45, CD11b, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. Essentially no Gr1+ (B) or 1A8+ cells (C) were observed in uninfected mice and the forward scatter (FSC) vs side scatter (SSC) plot showed a clear absence of the larger, more granular cells present at later time points (A). In contrast, TMEV-infected mice exhibited both CD11b++Gr1+ inflammatory monocytes and CD11b+++1A8+ neutrophils as early as 12 hpi (D-E), with inflammatory monocytes outnumbering neutrophils (E, F). Both of these populations increased in number by 18 hpi (G-I). Neutrophils continued to increase in the infiltrate by 24 hpi (K, L) while inflammatory monocytes began to decrease at this time point (K, L). The granular profile of neutrophils was also clearly present by FSC and SSC at 24 hpi (J). Results are representative of more than 20 mice at each timepoint.
Mentions: BILs were prepared from uninfected mice and at 12, 18, and 24 hpi (Figure 7) in order to determine how rapidly neutrophils and inflammatory monocytes enter the brain. We found a robust population of inflammatory monocytes present at 12 hpi (Figure 7E, F), suggesting that the cells had started infiltrating even earlier. Analysis of BILs at 6 hpi showed a small but inconsistent population of inflammatory monocytes (data not shown), indicating that infiltration began in earnest between 6 and 12 hpi. A small population of neutrophils was present in the BILs by 12 hpi (Figure 7E, F). By 18 hpi the density of both neutrophils and inflammatory monocytes had increased (Figure 7H, I). By 24 hpi the number of neutrophils continued to increase while the number of inflammatory monocytes decreased relative to 18 hpi (Figure 7K, L). Cell counts and relative percent of each cell population are provided in Table 1.

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