Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain
© Howe et al; licensee BioMed Central Ltd. 2012
Received: 15 August 2011
Accepted: 9 March 2012
Published: 9 March 2012
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.
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.
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.
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.
Viral infection of the central nervous system (CNS) may induce clinically relevant outcomes that range from coma, paralysis, and death to persistent cognitive impairment, seizures, and epilepsy . Many viral infections of the CNS are acute, with viral clearance mediated by the adaptive arm of the immune system. However, the relationship between the delayed adaptive antigen-specific T and B cell-mediated response that eventually controls and eliminates the viral pathogen and the rapid but largely non-specific innate immune system response is poorly understood. Indeed, different viruses and different hosts exhibit disparate relationships between the innate and adaptive response to infection. For example, McGavern and colleagues have shown in mice infected with lymphocytic choriomeningitis virus that pathogenic neutrophils and inflammatory monocytes are recruited to the brain by antiviral CD8+ T cells. Depletion of the CD8+ T cell response reduced the neutrophil and inflammatory monocyte burden in the CNS and delayed pathogenesis . In contrast, Bergmann and colleagues observed that neutrophils and inflammatory monocytes were the first leukocytes to infiltrate the brain in mice infected with the non-lethal neurotropic JHM strain of mouse hepatitis virus (JHMV) , and depletion or blockade of monocytes impaired subsequent T cell infiltration into the brain parenchyma . Lane and colleagues also observed a very early neutrophil response to CNS infection with JHMV, and in contrast to the Bergmann et al. findings implicating monocytes, they found that blocking neutrophil entry into the CNS resulted in impairment of the subsequent T cell response . Finally, Lokensgard and colleagues observed an early neutrophil and inflammatory monocyte response in the brain following infection with herpes simplex virus 1 and this response preceded lymphocyte infiltration by a week . Overall, the common denominator in all of these studies was the ensuing neuropathology triggered by infiltrating neutrophils and inflammatory monocytes.
Our previous experience with the Theiler's murine encephalomyelitis virus (TMEV) model of acute picornavirus infection of the CNS  indicated that brain pathology and the functional sequelae of such injury occur as a result of an early, preadaptive immune response. Indeed, hippocampal pyramidal neurons were clearly injured at 1 day after infection . We have hypothesized that neutrophils and inflammatory monocytes, as part of an early wave of first responders to infection, are responsible for hippocampal injury and loss of memory function observed in TMEV-infected mice, but the relative contribution of each population to this injury was unclear. In the present study we phenotyped the brain-infiltrating leukocyte population at several acute time points after infection with TMEV. We found that neutrophils and inflammatory monocytes were present in the brain within 12 h of infection, indicating that in this model system infiltration of such innate effectors is a hyperacute response. Furthermore, we found that the absence of an inflammatory monocyte response but not the absence of a neutrophil response resulted in neuroprotection and cognitive preservation.
At 5 to 8 weeks of age, mice were infected by intracranial injection of 2 × 105 plaque-forming units (PFU) of the Daniel's strain of the Theiler's murine encephalomyelitis virus (TMEV) in 10 μL RPMI (the media used to grow the virus) . When relevant, sham-infected mice received intracranial injection of 10 μL virus-free RPMI.
C57BL/6/J (no. 000664) mice aged 4 to 6 weeks were acquired from The Jackson Laboratories (Bar Harbor, ME, USA). Upon arrival, mice were acclimatized for at least 1 week prior to use. Breeding pairs of LysM:eGFP mice  were kindly provided by Dr. David Sacks (National Institutes of Health/National Institute of Allergy and Infectious Diseases) and bred in house. Mice were group housed in the Mayo Clinic College of Medicine research vivarium under conventional conditions with ad libitum access to food and water. Sex was mixed for all experiments. All animal experiments conformed to the National Institutes of Health and Mayo Clinic Institutional Animal Care and Use Committee guidelines.
Histology and immunostaining
Following intraperitoneal injection of a terminal dose of pentobarbital (100 mg/kg), mice were perfused via intracardiac puncture with 50 mL of 4% paraformaldehyde in phosphate-buffered saline (PBS). For paraffin sections, the brain was postfixed in 4% paraformaldehyde at 4°C for 24 h and then blocked via coronal cuts at the level of the optic chiasm and infundibulum. Tissue blocks were embedded in paraffin, sectioned at 5 microns, mounted on charged slides, rehydrated, and stained with hematoxylin and eosin. For vibratome sections, the brain was postfixed in 4% paraformaldehyde at 4°C for 6 h and then blocked to isolate the hippocampal field. Tissue blocks were embedded in agar and sectioned at 80 microns. Free floating sections were blocked in PBS plus 10% normal donkey serum for 1 h, incubated overnight at 4°C with primary antibodies diluted 1:100 in block, washed, incubated with fluorescently tagged secondary antibody diluted 1:200 in PBS, washed, and mounted on gelatin-subbed slides. CD45 was detected with clone 30-F11 (BD Biosciences, San Jose, CA, USA). CD11b was detected with clone M1/70 (BD Biosciences). Ly6C/G was detected with clone Gr1, RB6-8C5 (BD Biosciences). Ly6G was detected with clone 1A8 (BD Biosciences).
Brain-infiltrating leukocyte preparation
Our published protocol was followed with slight modification . Briefly, homogenized brain material was centrifuged through a 30% Percoll gradient at 7,800 g ave for 30 minutes without braking. The washed and strained cell suspension was then centrifuged on a 1.100 g/mL Percoll layer for 20 minutes at 800 g ave. The interface containing neutrophils and inflammatory monocytes was collected, washed, and used for flow cytometry.
Flow cytometry buffer contained 1% bovine serum albumin and 0.02% sodium azide in PBS. Blocking buffer contained flow cytometry buffer, supernatant from the 2.4G2 hybridoma (Fc block; anti-CD16/32; American Type Culture Collection, Manassas, VA, USA no. HB-197), and fetal bovine serum at a ratio of 10:5:1. After isolation, cells were blocked at 4°C for 30 minutes. Primary antibodies were used at 1:200 and incubated for 30 minutes at 4°C. Stained cells were washed three times in flow cytometry buffer and fixed in 2% paraformaldehyde prior to flow cytometric analysis on a BD FACSCalibur (BD Biosciences). Files were analyzed offline using FlowJo 7.5 (Windows version; Tree Star, Inc., Ashland, OR, USA). CD45 was detected with clone 30-F11 (BD Biosciences no. 557235). CD11b was detected with clone M1/70 (BD Biosciences no. 553312). F4/80 was detected with clone BM8 (eBiosciences no. 53-4801-82). Ly6C/G was detected with clone Gr1, RB6-8C5 (BD Biosciences no. 553129). Ly6G was detected with clone 1A8 (BD Biosciences No. 551467). Ly6B was detected with clone 7/4 (Caltag no. RM6504). Ly6C was detected with clone AL-21 (BD Biosciences no. 560595).
Morris water maze
Cognitive performance was assessed beginning at 14 days postinfection using our previously published methodology .
All graphs show mean ± 95% confidence intervals. Cell counts in the depletion experiments were assessed by one-way analysis of variance (ANOVA). Morris water maze performance was assessed by two-way ANOVA. Pairwise analyses, when appropriate, used the Student-Newman-Keuls method. All tests utilized α = 0.05, β = 0.2.
Results and discussion
Immune cells rapidly infiltrate the brain of TMEV-infected mice but not sham-infected mice
Brain infiltrating cells are enriched in neutrophils and inflammatory monocytes
Neutrophils and inflammatory monocytes enter the brain within 12 h of infection
Quantitation of brain infiltrating inflammatory monocytes and neutrophils (brain-infiltrating leukocytes (BILs)) at 12, 18, and 24 h post infection (hpi)
Number of inflammatory monocytes
Number of neutrophils
Inflammatory monocytes as percentage of CD45hi BILs
Neutrophils as percentage of CD45hi BILs
17 ± 11
3 ± 3
1,151 ± 270
106 ± 40
60.6 ± 28.9
5.7 ± 3.5
4,299 ± 381
1,228 ± 127
57.5 ± 8.9
16.4 ± 2.8
3,226 ± 250
1,645 ± 231
51.5 ± 7.7
26.3 ± 5.6
Inflammatory monocytes injure the hippocampus
In contrast to other viral models in which neutrophils apparently do not infiltrate the CNS until recruited by other immune populations such as lymphocytes , our findings suggest that neutrophils are one of the earliest responders in the TMEV model. As in other viral model systems [4–6], we predict that these cells will serve to prime the way for the adaptive response. In addition, our time course findings suggest that inflammatory monocytes precede neutrophils into the brain and the 1A8 depletion experiments show that inflammatory monocytes are competent to enter the CNS in the absence of neutrophils. Indeed, in the absence of neutrophils there was a significant increase in the number of inflammatory monocytes in the brain at 18 hpi, suggesting that neutrophils may exert a regulatory effect that slows or reverses inflammatory monocyte accumulation in the brain. The depletion experiments also show that inflammatory monocytes are required for the loss of CA1 pyramidal neurons that occurs in the first few days of infection  while neutrophils appear to be dispensable for this injury and the downstream consequences. Ongoing experiments will assess the impact of inflammatory monocytes versus neutrophils in the recruitment of the adaptive immune system and eventual control of the virus. Likewise, ongoing experiments in chemokine receptor knockout hosts will determine the relative impact of different chemotactic pathways in the separate and integrated neutrophil and inflammatory monocyte responses.
Our findings indicate that neutrophils and inflammatory monocytes rapidly and robustly respond to TMEV infection by infiltrating the brain. We hypothesize that these effector cells, and inflammatory monocytes in particular, may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing CNS pathology associated with acute viral infection.
This work was supported by grant R01 NS64571 from the NIH/NINDS and by a kind gift from Don and Fran Herdrich.
- Buenz EJ, Rodriguez M, Howe CL: Disrupted spatial memory is a consequence of picornavirus infection. Neurobiol Dis 2006, 24:266–273.View ArticlePubMedGoogle Scholar
- Kim JV, Kang SS, Dustin ML, McGavern DB: Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis. Nature 2009, 457:191–195.View ArticlePubMedGoogle Scholar
- Bergmann CC, Lane TE, Stohlman SA: Coronavirus infection of the central nervous system: host-virus stand-off. Nat Rev Microbiol 2006, 4:121–132.View ArticlePubMedGoogle Scholar
- Savarin C, Stohlman SA, Atkinson R, Ransohoff RM, Bergmann CC: Monocytes regulate T cell migration through the glia limitans during acute viral encephalitis. J Virol 2010, 84:4878–4888.View ArticlePubMedPubMed CentralGoogle Scholar
- Hosking MP, Liu L, Ransohoff RM, Lane TE: A protective role for ELR + chemokines during acute viral encephalomyelitis. PLoS Pathog 2009, 5:e1000648.View ArticlePubMedPubMed CentralGoogle Scholar
- Marques CP, Cheeran MC, Palmquist JM, Hu S, Urban SL, Lokensgard JR: Prolonged microglial cell activation and lymphocyte infiltration following experimental herpes encephalitis. J Immunol 2008, 181:6417–6426.View ArticlePubMedPubMed CentralGoogle Scholar
- Buenz EJ, Howe CL: Picornaviruses and cell death. Trends Microbiol 2006, 14:28–36.View ArticlePubMedGoogle Scholar
- Buenz EJ, Sauer BM, Lafrance-Corey RG, Deb C, Denic A, German CL, Howe CL: Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection. Am J Pathol 2009, 175:668–684.View ArticlePubMedPubMed CentralGoogle Scholar
- Lafrance-Corey RG, Howe CL: Isolation of brain-infiltrating leukocytes. J Vis Exp 2011, 52:2747.Google Scholar
- Faust N, Varas F, Kelly LM, Heck S, Graf T: Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 2000, 96:719–726.PubMedGoogle Scholar
- Kang SS, McGavern DB: Inflammation on the mind: visualizing immunity in the central nervous system. Curr Top Microbiol Immunol 2009, 334:227–263.PubMedGoogle Scholar
- Abram CL, Lowell CA: The ins and outs of leukocyte integrin signaling. Annu Rev Immunol 2009, 27:339–362.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin HH, Stacey M, Stein-Streilein J, Gordon S: F4/80: the macrophage-specific adhesion-GPCR and its role in immunoregulation. Adv Exp Med Biol 2010, 706:149–156.View ArticlePubMedGoogle Scholar
- van den Berg TK, Kraal G: A function for the macrophage F4/80 molecule in tolerance induction. Trends Immunol 2005, 26:506–509.View ArticlePubMedGoogle Scholar
- Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE: Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 2008, 83:64–70.View ArticlePubMedGoogle Scholar
- Nagendra S, Schlueter AJ: Absence of cross-reactivity between murine Ly-6C and Ly-6G. Cytometry A 2004, 58:195–200.View ArticlePubMedGoogle Scholar
- Ford AL, Goodsall AL, Hickey WF, Sedgwick JD: Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol 1995, 154:4309–4321.PubMedGoogle Scholar
- Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA, Leenen PJ: Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol 2004, 172:4410–4417.View ArticlePubMedGoogle Scholar
- Mallya M, Campbell RD, Aguado B: Characterization of the five novel Ly-6 superfamily members encoded in the MHC, and detection of cells expressing their potential ligands. Protein Sci 2006, 15:2244–2256.View ArticlePubMedPubMed CentralGoogle Scholar
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