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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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Immunodepletion of inflammatory monocytes but not neutrophils protects the hippocampus from injury. Theiler's murine encephalomyelitis virus (TMEV)-infected mice were treated with RB6-8C5 to deplete inflammatory monocytes and neutrophils (B, E, H, K), 1A8 to deplete only neutrophils (C, F, I, L), or were treated with vehicle (A, D, G, J). Brain-infiltrating leukocytes (BILs) were collected at 18 hpi and inflammatory monocytes and neutrophils were counted by flow cytometry (A-F). RB6 treatment abrogated most of the inflammatory monocyte (B) and neutrophil response (E). 1A8 treatment abrogated the neutrophil response (F) but not the inflammatory monocyte response (C). Histological analysis of hippocampal injury (G-I) and staining for the neuron-specific marker NeuN (J-L) revealed that depletion of inflammatory monocytes and neutrophils protected the hippocampus (H) and preserved the neurons in CA1 (K) whereas depletion of only neutrophils had no effect on the injury (I, L). Scale bar in L is 500 μm and refers to G-L. M = inflammatory monocyte; N = neutrophil. The flow cytometry results are representative of 10 mice per group. The histological analyses are representative of at least five mice per group.
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Figure 9: Immunodepletion of inflammatory monocytes but not neutrophils protects the hippocampus from injury. Theiler's murine encephalomyelitis virus (TMEV)-infected mice were treated with RB6-8C5 to deplete inflammatory monocytes and neutrophils (B, E, H, K), 1A8 to deplete only neutrophils (C, F, I, L), or were treated with vehicle (A, D, G, J). Brain-infiltrating leukocytes (BILs) were collected at 18 hpi and inflammatory monocytes and neutrophils were counted by flow cytometry (A-F). RB6 treatment abrogated most of the inflammatory monocyte (B) and neutrophil response (E). 1A8 treatment abrogated the neutrophil response (F) but not the inflammatory monocyte response (C). Histological analysis of hippocampal injury (G-I) and staining for the neuron-specific marker NeuN (J-L) revealed that depletion of inflammatory monocytes and neutrophils protected the hippocampus (H) and preserved the neurons in CA1 (K) whereas depletion of only neutrophils had no effect on the injury (I, L). Scale bar in L is 500 μm and refers to G-L. M = inflammatory monocyte; N = neutrophil. The flow cytometry results are representative of 10 mice per group. The histological analyses are representative of at least five mice per group.

Mentions: We have previously hypothesized that the innate immune response to acute TMEV infection mediates subsequent hippocampal injury that leads to cognitive deficits [8]. In order to distinguish the role of inflammatory monocytes from neutrophils in this injury, we immunodepleted these populations and assessed hippocampal pathology (Figure 9) and cognitive performance (Figure 10). Neutrophils and inflammatory monocytes were simultaneously depleted by daily treatment from -2 dpi to +2 dpi with intraperitoneal injection of purified RB6-8C5 (500 μg/day; rat anti-Gr1). Neutrophils but not inflammatory monocytes were depleted by daily injection from -2 dpi to +2 dpi of purified 1A8 (500 μg/day; mouse anti-Ly6G). Controls received daily injections of PBS. BILs prepared at 18 hpi from control mice showed 4,898 ± 191 inflammatory monocytes and 1,515 ± 192 neutrophils (Figure 9A, D); 22.9% ± 1.0% of the BILs were CD45hi. Control mice showed robust hippocampal pathology at 7 dpi as assessed by hematoxylin and eosin (H&E) histology (Figure 9G) and immunostaining for the neuronal marker NeuN (Figure 9J). Regions of no or weak NeuN staining in the CA1 layer in Figure 9J represent neuronal dropout or pyknotic neurons. In contrast, RB6-treated mice had only 106 ± 34 inflammatory monocytes and 11 ± 3 neutrophils in the 18 hpi BILs (Figure 9B, E) and only 6.7% ± 0.4% of the BILs were CD45hi. Moreover, the hippocampus was completely preserved in these mice at 7 dpi (Figure 9H, K). The thick, multi-cell-layered NeuN staining in the CA1 layer in Figure 9K is indistinguishable from uninfected mice (data not shown). The reduction in inflammatory monocytes and neutrophils was highly significant versus control BILs (F(2,28) = 1,141.175, P < 0.001; inflammatory monocytes in control vs RB6: q(28,2) = 63.454, P < 0.001; neutrophils in control vs RB6: q(28,3) = 19.923, P < 0.001). However, while treatment with 1A8 almost completely abrogated the neutrophil response in the 18 hpi BILs (66 ± 24 neutrophils; control vs 1A8: q(28,2) = 17.917, P < 0.001) (Figure 9F) there was an actual increase in the number of inflammatory monocytes (6,251 ± 237 inflammatory monocytes; control vs 1A8: q(28,2) = 19.194, P < 0.001) (Figure 9C). The percentage of CD45hi cells in the BILs following 1A8 treatment was 23.4% ± 1.3% (control vs 1A8: q(28,2) = 1.064, P = 0.458; RB6 vs 1A8: q(28,3) = 34.188, P < 0.001). Critically, the hippocampus was robustly injured in the 1A8-treated mice (Figure 9I, L). Finally, the absence of hippocampal injury in the RB6-treated mice led to the preservation of cognitive function as assessed by Morris water maze (Figure 10). Infected, RB6-treated mice showed the same ability to learn in the maze as uninfected mice (F(2,28) = 15.508, P < 0.001; uninfected vs RB6-treated: q(28,2) = 1.089, P = 0.441) while infected, control-treated mice were completely unable to learn to navigate the maze (uninfected vs infected, control-treated: q(28,2) = 14.826, P < 0.001; RB6-treated vs control-treated: q(28,3) = 15.519, P < 0.001) [1,8]. We interpret these findings as strong support for our hypothesis that neutrophils and inflammatory monocytes are first responders to TMEV infection in the brain and that inflammatory monocytes are primarily responsible for hippocampal damage and loss of cognitive function.


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

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

Immunodepletion of inflammatory monocytes but not neutrophils protects the hippocampus from injury. Theiler's murine encephalomyelitis virus (TMEV)-infected mice were treated with RB6-8C5 to deplete inflammatory monocytes and neutrophils (B, E, H, K), 1A8 to deplete only neutrophils (C, F, I, L), or were treated with vehicle (A, D, G, J). Brain-infiltrating leukocytes (BILs) were collected at 18 hpi and inflammatory monocytes and neutrophils were counted by flow cytometry (A-F). RB6 treatment abrogated most of the inflammatory monocyte (B) and neutrophil response (E). 1A8 treatment abrogated the neutrophil response (F) but not the inflammatory monocyte response (C). Histological analysis of hippocampal injury (G-I) and staining for the neuron-specific marker NeuN (J-L) revealed that depletion of inflammatory monocytes and neutrophils protected the hippocampus (H) and preserved the neurons in CA1 (K) whereas depletion of only neutrophils had no effect on the injury (I, L). Scale bar in L is 500 μm and refers to G-L. M = inflammatory monocyte; N = neutrophil. The flow cytometry results are representative of 10 mice per group. The histological analyses are representative of at least five mice per group.
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Figure 9: Immunodepletion of inflammatory monocytes but not neutrophils protects the hippocampus from injury. Theiler's murine encephalomyelitis virus (TMEV)-infected mice were treated with RB6-8C5 to deplete inflammatory monocytes and neutrophils (B, E, H, K), 1A8 to deplete only neutrophils (C, F, I, L), or were treated with vehicle (A, D, G, J). Brain-infiltrating leukocytes (BILs) were collected at 18 hpi and inflammatory monocytes and neutrophils were counted by flow cytometry (A-F). RB6 treatment abrogated most of the inflammatory monocyte (B) and neutrophil response (E). 1A8 treatment abrogated the neutrophil response (F) but not the inflammatory monocyte response (C). Histological analysis of hippocampal injury (G-I) and staining for the neuron-specific marker NeuN (J-L) revealed that depletion of inflammatory monocytes and neutrophils protected the hippocampus (H) and preserved the neurons in CA1 (K) whereas depletion of only neutrophils had no effect on the injury (I, L). Scale bar in L is 500 μm and refers to G-L. M = inflammatory monocyte; N = neutrophil. The flow cytometry results are representative of 10 mice per group. The histological analyses are representative of at least five mice per group.
Mentions: We have previously hypothesized that the innate immune response to acute TMEV infection mediates subsequent hippocampal injury that leads to cognitive deficits [8]. In order to distinguish the role of inflammatory monocytes from neutrophils in this injury, we immunodepleted these populations and assessed hippocampal pathology (Figure 9) and cognitive performance (Figure 10). Neutrophils and inflammatory monocytes were simultaneously depleted by daily treatment from -2 dpi to +2 dpi with intraperitoneal injection of purified RB6-8C5 (500 μg/day; rat anti-Gr1). Neutrophils but not inflammatory monocytes were depleted by daily injection from -2 dpi to +2 dpi of purified 1A8 (500 μg/day; mouse anti-Ly6G). Controls received daily injections of PBS. BILs prepared at 18 hpi from control mice showed 4,898 ± 191 inflammatory monocytes and 1,515 ± 192 neutrophils (Figure 9A, D); 22.9% ± 1.0% of the BILs were CD45hi. Control mice showed robust hippocampal pathology at 7 dpi as assessed by hematoxylin and eosin (H&E) histology (Figure 9G) and immunostaining for the neuronal marker NeuN (Figure 9J). Regions of no or weak NeuN staining in the CA1 layer in Figure 9J represent neuronal dropout or pyknotic neurons. In contrast, RB6-treated mice had only 106 ± 34 inflammatory monocytes and 11 ± 3 neutrophils in the 18 hpi BILs (Figure 9B, E) and only 6.7% ± 0.4% of the BILs were CD45hi. Moreover, the hippocampus was completely preserved in these mice at 7 dpi (Figure 9H, K). The thick, multi-cell-layered NeuN staining in the CA1 layer in Figure 9K is indistinguishable from uninfected mice (data not shown). The reduction in inflammatory monocytes and neutrophils was highly significant versus control BILs (F(2,28) = 1,141.175, P < 0.001; inflammatory monocytes in control vs RB6: q(28,2) = 63.454, P < 0.001; neutrophils in control vs RB6: q(28,3) = 19.923, P < 0.001). However, while treatment with 1A8 almost completely abrogated the neutrophil response in the 18 hpi BILs (66 ± 24 neutrophils; control vs 1A8: q(28,2) = 17.917, P < 0.001) (Figure 9F) there was an actual increase in the number of inflammatory monocytes (6,251 ± 237 inflammatory monocytes; control vs 1A8: q(28,2) = 19.194, P < 0.001) (Figure 9C). The percentage of CD45hi cells in the BILs following 1A8 treatment was 23.4% ± 1.3% (control vs 1A8: q(28,2) = 1.064, P = 0.458; RB6 vs 1A8: q(28,3) = 34.188, P < 0.001). Critically, the hippocampus was robustly injured in the 1A8-treated mice (Figure 9I, L). Finally, the absence of hippocampal injury in the RB6-treated mice led to the preservation of cognitive function as assessed by Morris water maze (Figure 10). Infected, RB6-treated mice showed the same ability to learn in the maze as uninfected mice (F(2,28) = 15.508, P < 0.001; uninfected vs RB6-treated: q(28,2) = 1.089, P = 0.441) while infected, control-treated mice were completely unable to learn to navigate the maze (uninfected vs infected, control-treated: q(28,2) = 14.826, P < 0.001; RB6-treated vs control-treated: q(28,3) = 15.519, P < 0.001) [1,8]. We interpret these findings as strong support for our hypothesis that neutrophils and inflammatory monocytes are first responders to TMEV infection in the brain and that inflammatory monocytes are primarily responsible for hippocampal damage and loss of cognitive function.

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