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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 infiltrate present in the brain of mice acutely infected with Theiler's murine encephalomyelitis virus (TMEV). TMEV-infected (B-G) or sham-infected (A) mice were killed at 18 h post infection (hpi). Brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (BILs). BILs were stained with antibodies against CD45, CD11b, F4/80, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. No CD45hi cells were observed in sham-infected mice (A). In contrast, TMEV-infected mice exhibited a large population of CD45hi cells at 18 hpi. Analysis of the CD45hi cells in BILs from infected mice revealed the presence of CD11b++ cells that were also strongly positive for the Gr1 antigen (C) and CD11b+++ cells that were positive for both the Gr1 antigen (C) and the 1A8 antigen (D). In addition, a population of the Gr1+ cells was also positive for F4/80 (E); no 1A8+ cells were F4/80+ (F). Combined gating revealed that CD45hiCD11b++Gr1+ cells were also F4/80+ while CD45hiCD11b+++Gr1+ cells were F4/80-. Quantitation of neutrophils (CD45hi1A8+Gr1+CD11b+++F4/80-) and inflammatory monocytes (CD45hi1A8-Gr1+CD11b++F4/80+) in BILs from 10 individual mice revealed that about 80% of the CD45hi cells belonged to one of these populations (G). Error bars in (G) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (A-F) are representative.
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Figure 2: Flow cytometric assessment of the infiltrate present in the brain of mice acutely infected with Theiler's murine encephalomyelitis virus (TMEV). TMEV-infected (B-G) or sham-infected (A) mice were killed at 18 h post infection (hpi). Brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (BILs). BILs were stained with antibodies against CD45, CD11b, F4/80, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. No CD45hi cells were observed in sham-infected mice (A). In contrast, TMEV-infected mice exhibited a large population of CD45hi cells at 18 hpi. Analysis of the CD45hi cells in BILs from infected mice revealed the presence of CD11b++ cells that were also strongly positive for the Gr1 antigen (C) and CD11b+++ cells that were positive for both the Gr1 antigen (C) and the 1A8 antigen (D). In addition, a population of the Gr1+ cells was also positive for F4/80 (E); no 1A8+ cells were F4/80+ (F). Combined gating revealed that CD45hiCD11b++Gr1+ cells were also F4/80+ while CD45hiCD11b+++Gr1+ cells were F4/80-. Quantitation of neutrophils (CD45hi1A8+Gr1+CD11b+++F4/80-) and inflammatory monocytes (CD45hi1A8-Gr1+CD11b++F4/80+) in BILs from 10 individual mice revealed that about 80% of the CD45hi cells belonged to one of these populations (G). Error bars in (G) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (A-F) are representative.

Mentions: Given the conflicting observations that exist in the literature regarding temporal staging of innate and adaptive immune cell infiltration into the acutely infected brain [2-5,11], we sought to determine the identity of the cells observed in Figure 1. Using our established protocol for isolating a highly enriched population of leukocytes from the brain [9], we observed a robust population of CD45hi cells in TMEV-infected mice at 18 hpi (Figure 2B) that are not present in the brain of sham-infected (Figure 2A) or uninfected mice (data not shown). Further phenotyping revealed that most of the CD45hi cells in the brain-infiltrating leukocytes (BILs) were CD11b positive, albeit at two different intensity levels. We routinely distinguished CD11b++ and CD11b+++ populations (Figure 2C, D). Critically, these populations could also be distinguished by levels of surface staining with the Gr1 antibody (Figure 2C) and the 1A8 antibody (Figure 2D), such that CD11b++ cells were positive for Gr1 but negative for 1A8 and CD11b+++ cells were positive for both markers (Figure 2C, D). Likewise, the population of CD45hi cells could be distinguished by surface F4/80 expression, with some F4/80+ cells positive for Gr1 but no F4/80+ cells positive for 1A8. CD11b is a component of the αMβ2 integrin complex that is found on the surface of monocytes, granulocytes, macrophages, and natural killer cells [12]. The F4/80 antigen is a member of the epidermal growth factor 7 transmembrane family that is found on various macrophage populations including tissue resident macrophages such as microglia [13,14]. The Gr1 antigen is a mixture of Ly6C and Ly6G that is expressed on the surface of circulating monocytes and neutrophils [15]. In contrast, the 1A8 antigen is explicitly Ly6G and is only observed on neutrophils and granulocytes but not on monocytes or macrophages [15,16]. On the basis of our findings, we have established the following definitions, which are also elaborated below: CD45hiCD11b++F4/80+Gr1+1A8- cells are inflammatory monocytes; CD45hiCD11b+++F4/80-Gr1+1A8+ cells are neutrophils. We find that approximately 60% of the CD45hi cell population in the brain at 18 hpi are inflammatory monocytes and approximately 20% are neutrophils (Figure 2G).


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 infiltrate present in the brain of mice acutely infected with Theiler's murine encephalomyelitis virus (TMEV). TMEV-infected (B-G) or sham-infected (A) mice were killed at 18 h post infection (hpi). Brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (BILs). BILs were stained with antibodies against CD45, CD11b, F4/80, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. No CD45hi cells were observed in sham-infected mice (A). In contrast, TMEV-infected mice exhibited a large population of CD45hi cells at 18 hpi. Analysis of the CD45hi cells in BILs from infected mice revealed the presence of CD11b++ cells that were also strongly positive for the Gr1 antigen (C) and CD11b+++ cells that were positive for both the Gr1 antigen (C) and the 1A8 antigen (D). In addition, a population of the Gr1+ cells was also positive for F4/80 (E); no 1A8+ cells were F4/80+ (F). Combined gating revealed that CD45hiCD11b++Gr1+ cells were also F4/80+ while CD45hiCD11b+++Gr1+ cells were F4/80-. Quantitation of neutrophils (CD45hi1A8+Gr1+CD11b+++F4/80-) and inflammatory monocytes (CD45hi1A8-Gr1+CD11b++F4/80+) in BILs from 10 individual mice revealed that about 80% of the CD45hi cells belonged to one of these populations (G). Error bars in (G) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (A-F) are representative.
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Figure 2: Flow cytometric assessment of the infiltrate present in the brain of mice acutely infected with Theiler's murine encephalomyelitis virus (TMEV). TMEV-infected (B-G) or sham-infected (A) mice were killed at 18 h post infection (hpi). Brain was collected fresh and processed for the isolation of brain-infiltrating leukocytes (BILs). BILs were stained with antibodies against CD45, CD11b, F4/80, Gr1 (Ly6C/G) and 1A8 (Ly6G) and analyzed by flow cytometry. No CD45hi cells were observed in sham-infected mice (A). In contrast, TMEV-infected mice exhibited a large population of CD45hi cells at 18 hpi. Analysis of the CD45hi cells in BILs from infected mice revealed the presence of CD11b++ cells that were also strongly positive for the Gr1 antigen (C) and CD11b+++ cells that were positive for both the Gr1 antigen (C) and the 1A8 antigen (D). In addition, a population of the Gr1+ cells was also positive for F4/80 (E); no 1A8+ cells were F4/80+ (F). Combined gating revealed that CD45hiCD11b++Gr1+ cells were also F4/80+ while CD45hiCD11b+++Gr1+ cells were F4/80-. Quantitation of neutrophils (CD45hi1A8+Gr1+CD11b+++F4/80-) and inflammatory monocytes (CD45hi1A8-Gr1+CD11b++F4/80+) in BILs from 10 individual mice revealed that about 80% of the CD45hi cells belonged to one of these populations (G). Error bars in (G) represent 95% confidence intervals; black circles represent individual animals; 10 mice per group were analyzed and the flow plots in (A-F) are representative.
Mentions: Given the conflicting observations that exist in the literature regarding temporal staging of innate and adaptive immune cell infiltration into the acutely infected brain [2-5,11], we sought to determine the identity of the cells observed in Figure 1. Using our established protocol for isolating a highly enriched population of leukocytes from the brain [9], we observed a robust population of CD45hi cells in TMEV-infected mice at 18 hpi (Figure 2B) that are not present in the brain of sham-infected (Figure 2A) or uninfected mice (data not shown). Further phenotyping revealed that most of the CD45hi cells in the brain-infiltrating leukocytes (BILs) were CD11b positive, albeit at two different intensity levels. We routinely distinguished CD11b++ and CD11b+++ populations (Figure 2C, D). Critically, these populations could also be distinguished by levels of surface staining with the Gr1 antibody (Figure 2C) and the 1A8 antibody (Figure 2D), such that CD11b++ cells were positive for Gr1 but negative for 1A8 and CD11b+++ cells were positive for both markers (Figure 2C, D). Likewise, the population of CD45hi cells could be distinguished by surface F4/80 expression, with some F4/80+ cells positive for Gr1 but no F4/80+ cells positive for 1A8. CD11b is a component of the αMβ2 integrin complex that is found on the surface of monocytes, granulocytes, macrophages, and natural killer cells [12]. The F4/80 antigen is a member of the epidermal growth factor 7 transmembrane family that is found on various macrophage populations including tissue resident macrophages such as microglia [13,14]. The Gr1 antigen is a mixture of Ly6C and Ly6G that is expressed on the surface of circulating monocytes and neutrophils [15]. In contrast, the 1A8 antigen is explicitly Ly6G and is only observed on neutrophils and granulocytes but not on monocytes or macrophages [15,16]. On the basis of our findings, we have established the following definitions, which are also elaborated below: CD45hiCD11b++F4/80+Gr1+1A8- cells are inflammatory monocytes; CD45hiCD11b+++F4/80-Gr1+1A8+ cells are neutrophils. We find that approximately 60% of the CD45hi cell population in the brain at 18 hpi are inflammatory monocytes and approximately 20% are neutrophils (Figure 2G).

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