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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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Characterization of the Ly6 antigen series on brain-infiltrating leukocytes (BILs) and peripheral blood leukocytes (PBLs). Flow cytometric analysis of PBLs (A, C, E, G, I, K, M, O) and BILs (B, D, F, H, J, L, N, P) collected from the same mouse 18 h after infection with Theiler's murine encephalomyelitis virus (TMEV) revealed the presence of readily distinguishable neutrophil populations in both cell preparations. However, inflammatory monocytes were only detected in the BILs. Consistent with the analyses performed above, the CD45hiCD11b++F4/80+ population of inflammatory monocytes was positive for Gr1 (Ly6C/G), 7/4 (Ly6B), and AL-21 (Ly6C) but negative for 1A8 (Ly6G). The CD45hiCD11b+++F4/80- neutrophil population was positive for all four Ly6 series antigens. Of note, CD45hiCD11b++F4/80+ inflammatory monocytes in the BILs expressed higher levels of surface Ly6C/G (B, D) and Ly6B (J, L) as compared to CD45hiCD11b+++F4/80- neutrophils and expressed 10-fold higher levels of the AL-21 antigen (N, P). The CD45+CD11b+ macrophage population in the blood was negative for all Ly6 series antigens. Results are representative of five mice.
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Figure 4: Characterization of the Ly6 antigen series on brain-infiltrating leukocytes (BILs) and peripheral blood leukocytes (PBLs). Flow cytometric analysis of PBLs (A, C, E, G, I, K, M, O) and BILs (B, D, F, H, J, L, N, P) collected from the same mouse 18 h after infection with Theiler's murine encephalomyelitis virus (TMEV) revealed the presence of readily distinguishable neutrophil populations in both cell preparations. However, inflammatory monocytes were only detected in the BILs. Consistent with the analyses performed above, the CD45hiCD11b++F4/80+ population of inflammatory monocytes was positive for Gr1 (Ly6C/G), 7/4 (Ly6B), and AL-21 (Ly6C) but negative for 1A8 (Ly6G). The CD45hiCD11b+++F4/80- neutrophil population was positive for all four Ly6 series antigens. Of note, CD45hiCD11b++F4/80+ inflammatory monocytes in the BILs expressed higher levels of surface Ly6C/G (B, D) and Ly6B (J, L) as compared to CD45hiCD11b+++F4/80- neutrophils and expressed 10-fold higher levels of the AL-21 antigen (N, P). The CD45+CD11b+ macrophage population in the blood was negative for all Ly6 series antigens. Results are representative of five mice.

Mentions: Confusion exists in the literature regarding the use of Ly6 antigens as markers of neutrophils and inflammatory monocytes. Historically, Gr1 staining of the Ly6C/G heteroantigen was used to define neutrophils. More recently, it has become clear that Gr1 marks both neutrophils and a population of inflammatory monocytes. The availability of the 1A8 marker, which only recognizes Ly6G and only labels neutrophils, now allows for better resolution of these populations [15,19]. In order to further validate our phenotype definitions, we also examined the Ly6 series in blood at 18 hpi and compared the profile to BILs from the same animal (Figure 4). In general, the staining for Gr1 (Ly6C/G) (Figure 4A-D) and 1A8 (Ly6G) (Figure 4E-H) was higher on the brain-infiltrating cells as compared to the same populations in the blood. Of note, brain-infiltrating inflammatory monocytes exhibited elevated Gr1 staining compared to brain-infiltrating neutrophils (Figure 4B). We were unable to identify a population of inflammatory monocytes in the blood in the infected mice, suggesting either that these cells are not circulating at this time or that so many have been recruited to the brain that the blood levels are reduced to below detection (Figure 4A, E, I, M). Surface 1A8 labeling was increased on neutrophils that had entered the brain (Figure 4F) as compared to circulating neutrophils (Figure 4E), suggesting that this molecule may be upregulated as neutrophils home to target tissues. Finally, inflammatory monocytes exhibited higher surface levels of 7/4 (Ly6B-specific) and AL-21 (Ly6C-specific) staining as compared to neutrophils in the brain (Figure 4J, L, N, P). Based on these observations, in addition to differential surface CD11b, Gr1, and 1A8 staining, we can further distinguish brain-infiltrating monocytes from brain-infiltrating neutrophils on the basis of surface expression of the 7/4 and AL-21 antigens. Brain-resident microglia/macrophages were consistently negative for all Ly6 antigens (not shown).


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

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

Characterization of the Ly6 antigen series on brain-infiltrating leukocytes (BILs) and peripheral blood leukocytes (PBLs). Flow cytometric analysis of PBLs (A, C, E, G, I, K, M, O) and BILs (B, D, F, H, J, L, N, P) collected from the same mouse 18 h after infection with Theiler's murine encephalomyelitis virus (TMEV) revealed the presence of readily distinguishable neutrophil populations in both cell preparations. However, inflammatory monocytes were only detected in the BILs. Consistent with the analyses performed above, the CD45hiCD11b++F4/80+ population of inflammatory monocytes was positive for Gr1 (Ly6C/G), 7/4 (Ly6B), and AL-21 (Ly6C) but negative for 1A8 (Ly6G). The CD45hiCD11b+++F4/80- neutrophil population was positive for all four Ly6 series antigens. Of note, CD45hiCD11b++F4/80+ inflammatory monocytes in the BILs expressed higher levels of surface Ly6C/G (B, D) and Ly6B (J, L) as compared to CD45hiCD11b+++F4/80- neutrophils and expressed 10-fold higher levels of the AL-21 antigen (N, P). The CD45+CD11b+ macrophage population in the blood was negative for all Ly6 series antigens. Results are representative of five mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368782&req=5

Figure 4: Characterization of the Ly6 antigen series on brain-infiltrating leukocytes (BILs) and peripheral blood leukocytes (PBLs). Flow cytometric analysis of PBLs (A, C, E, G, I, K, M, O) and BILs (B, D, F, H, J, L, N, P) collected from the same mouse 18 h after infection with Theiler's murine encephalomyelitis virus (TMEV) revealed the presence of readily distinguishable neutrophil populations in both cell preparations. However, inflammatory monocytes were only detected in the BILs. Consistent with the analyses performed above, the CD45hiCD11b++F4/80+ population of inflammatory monocytes was positive for Gr1 (Ly6C/G), 7/4 (Ly6B), and AL-21 (Ly6C) but negative for 1A8 (Ly6G). The CD45hiCD11b+++F4/80- neutrophil population was positive for all four Ly6 series antigens. Of note, CD45hiCD11b++F4/80+ inflammatory monocytes in the BILs expressed higher levels of surface Ly6C/G (B, D) and Ly6B (J, L) as compared to CD45hiCD11b+++F4/80- neutrophils and expressed 10-fold higher levels of the AL-21 antigen (N, P). The CD45+CD11b+ macrophage population in the blood was negative for all Ly6 series antigens. Results are representative of five mice.
Mentions: Confusion exists in the literature regarding the use of Ly6 antigens as markers of neutrophils and inflammatory monocytes. Historically, Gr1 staining of the Ly6C/G heteroantigen was used to define neutrophils. More recently, it has become clear that Gr1 marks both neutrophils and a population of inflammatory monocytes. The availability of the 1A8 marker, which only recognizes Ly6G and only labels neutrophils, now allows for better resolution of these populations [15,19]. In order to further validate our phenotype definitions, we also examined the Ly6 series in blood at 18 hpi and compared the profile to BILs from the same animal (Figure 4). In general, the staining for Gr1 (Ly6C/G) (Figure 4A-D) and 1A8 (Ly6G) (Figure 4E-H) was higher on the brain-infiltrating cells as compared to the same populations in the blood. Of note, brain-infiltrating inflammatory monocytes exhibited elevated Gr1 staining compared to brain-infiltrating neutrophils (Figure 4B). We were unable to identify a population of inflammatory monocytes in the blood in the infected mice, suggesting either that these cells are not circulating at this time or that so many have been recruited to the brain that the blood levels are reduced to below detection (Figure 4A, E, I, M). Surface 1A8 labeling was increased on neutrophils that had entered the brain (Figure 4F) as compared to circulating neutrophils (Figure 4E), suggesting that this molecule may be upregulated as neutrophils home to target tissues. Finally, inflammatory monocytes exhibited higher surface levels of 7/4 (Ly6B-specific) and AL-21 (Ly6C-specific) staining as compared to neutrophils in the brain (Figure 4J, L, N, P). Based on these observations, in addition to differential surface CD11b, Gr1, and 1A8 staining, we can further distinguish brain-infiltrating monocytes from brain-infiltrating neutrophils on the basis of surface expression of the 7/4 and AL-21 antigens. Brain-resident microglia/macrophages were consistently negative for all Ly6 antigens (not shown).

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