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. 2016 Feb 22:13:46.
doi: 10.1186/s12974-016-0513-y.

Sustained TNF production by central nervous system infiltrating macrophages promotes progressive autoimmune encephalomyelitis

Alice Valentin-Torres  1 , Carine Savarin  2 , David R Hinton  3 , Timothy W Phares  4 , Cornelia C Bergmann  5 , Stephen A Stohlman  6
Affiliations

Sustained TNF production by central nervous system infiltrating macrophages promotes progressive autoimmune encephalomyelitis

Alice Valentin-Torres et al. J Neuroinflammation. .

Abstract

Background: Tumor necrosis factor (TNF) has pleiotropic functions during both the demyelinating autoimmune disease multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). How TNF regulates disability during progressive disease remains unresolved. Using a progressive EAE model characterized by sustained TNF and increasing morbidity, this study evaluates the role of unregulated TNF in exacerbating central nervous system (CNS) pathology and inflammation.

Methods: Progressive MS was mimicked by myelin oligodendrocyte glycoprotein (MOG) peptide immunization of mice expressing a dominant negative IFN-γ receptor alpha chain under the human glial fibrillary acidic protein promoter (GFAPγR1∆). Diseased GFAPγR1∆ mice were treated with anti-TNF or control monoclonal antibody during acute disease to monitor therapeutic effects on sustained disability, demyelination, CNS inflammation, and blood brain barrier (BBB) permeability.

Results: TNF was specifically sustained in infiltrating macrophages. Anti-TNF treatment decreased established clinical disability and mortality rate within 7 days. Control of disease progression was associated with a decline in myelin loss and leukocyte infiltration, as well as macrophage activation. In addition to mitigating CNS inflammation, TNF neutralization restored BBB integrity and enhanced CNS anti-inflammatory responses.

Conclusions: Sustained TNF production by infiltrating macrophages associated with progressive EAE exacerbates disease severity by promoting inflammation and disruption of BBB integrity, thereby counteracting establishment of an anti-inflammatory environment required for disease remission.

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Figures

Fig. 1
Fig. 1
TNF is primarily produced by Iba1+ cells during EAE. a Confocal Z-stack images showing TNF in white matter of spinal cords from WT and GFAP∆R1γ during acute and chronic EAE. Magnification ×100. Bar graph depicts TNF+ area calculated as an average of three fields per group. b TNF mRNA expression in FACS sorted astrocytes and microglia from GFAP-GFP/GFAP∆R1γ during acute (d19 p.i.) and chronic EAE (d30 p.i.). c Confocal Z-stack images (magnification ×100) with orthogonal views illustrating TNF co-staining with Iba1 in spinal cord white matter from WT and GFAP∆R1γ during acute and chronic EAE. Data are representative of three mice per group. P values determined by Student’s t test
Fig. 2
Fig. 2
TNF secretion is sustained in infiltrating macrophages during progressive EAE. TNF production by macrophages and microglia determined by intracellular staining during a acute (d19 p.i.) and b chronic (d30 p.i.) EAE in WT (black line) or GFAP∆R1γ (red line) mice. Numbers in histograms depict the percentage of TNF producing WT cells (black) or GFAP∆R1γ cells (red). Dotted line represents isotype control. Bar graphs show mean fluorescence intensity (MFI) of TNF staining in macrophages and microglia from WT and GFAP∆R1γ mice during acute and chronic disease. Data represent the mean values and ± SEM of three independent experiments with six mice per group per experiment. P values determined by Student’s t test
Fig. 3
Fig. 3
Anti-TNF treatment ameliorates progressive EAE. EAE was induced in WT and GFAP∆R1γ mice. At the peak of acute disease (d19 p.i.), WT and GFAP∆R1γ were divided into two groups with equal clinical scores. WT + αTNF and GFAP∆R1γ + αTNF received i.p. injections of anti-TNF mAb (0.5 mg), whereas WT and GFAP∆R1γ received equal amounts of isotype mAb control. Mice were treated every 2 days for a total of four dosages. a Clinical scores of αTNF treated and control cohorts pre and 10 days post treatment b Kinetics of clinical recovery and c percent survival in anti-TNF treated GFAP∆R1γ mice relative to control treated GFAP∆R1γ and WT mice. Data represent the mean values and ± SEM of four separate experiments with five mice per group per experiment. P values are determined by Student’s t test. d Percentage area of demyelination in spinal cord white matter calculated by analysis of transverse sections at six separate levels per mouse. Data represent the mean ± SEM of seven to nine individual mice per group. Statistical differences determined by two-tailed unpaired t test with *p < 0.05
Fig. 4
Fig. 4
Anti-TNF treatment decreases inflammation during progressive EAE. a Total numbers of CD45hi inflammatory cells in the CNS at d30 p.i. of WT and GFAPγR1∆ mice treated with α-TNF or α-βgal mAb. b Frequencies of CD8+ and CD4+ T cells as well as CD11b+ myeloid cells within CD45hi infiltrating cells in the CNS of the three mouse groups as depicted. c Total numbers of Th1 (IFN-γ-producing) and Th17 (IL-17 producing) CD4+ T cells derived from the CNS (d30 p.i.) assessed after PMA and ionomycin stimulation. d IFN-γ and IL-17 in supernatants of splenocytes from immunized mice (d30 p.i.) stimulated with MOG35–55 peptide as determined by ELISA. Data represent the mean values and ± SEM of three separate experiments with five mice per group per experiment. P values determined by Student’s t test
Fig. 5
Fig. 5
TNF blockade restores BBB integrity during progressive EAE. a BBB integrity in brains and spinal cords determined by sodium fluorescein permeability assay determined at day 25 p.i. in WT and GFAPγR1∆ mice treated with α-TNF or control mAb. Data is represented as fold increase over naïve values with six mice per group. b Confocal images (Z-stack, magnification ×40) of spinal cords stained with laminin (green), IgG (red), and DAPI (blue) at d30 p.i. Images representative of two individual mice per group. P values determined by Student’s t test
Fig. 6
Fig. 6
TNF neutralization promotes anti-inflammatory responses. a MHC class II expression on macrophages and microglia derived from WT and GFAPγR1∆ mice treated with α-TNF or control mAb determined by flow cytometry at d30p.i. b IL-10 levels in the CNS of mouse groups as depicted determined by ELISA (D30p.i.). Foxp3 (c) and IL-27p28 (d) mRNA expression in the CNS determined by RT-PCR. Data represent the mean values and ± SEM of three separate experiments with five mice per group per experiment. P values determined by Student’s t test
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References

    1. McCoy MK, Tansey MG. TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation. 2008;5:45. doi: 10.1186/1742-2094-5-45. - DOI - PMC - PubMed
    1. Selmaj K, Raine CS, Cannella B, Brosnan CF. Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J Clin Invest. 1991;87:949–54. doi: 10.1172/JCI115102. - DOI - PMC - PubMed
    1. Spuler S, Yousry T, Scheller A, Voltz R, Holler E, Hartmann M, Wick M, Hohlfeld R. Multiple sclerosis: prospective analysis of TNF-alpha and 55 kDa TNF receptor in CSF and serum in correlation with clinical and MRI activity. J Neuroimmunol. 1996;66:57–64. doi: 10.1016/0165-5728(96)00020-3. - DOI - PubMed
    1. Begum F, Zhu W, Cortes C, MacNeil B, Namaka M. Elevation of tumor necrosis factor alpha in dorsal root ganglia and spinal cord is associated with neuroimmune modulation of pain in an animal model of multiple sclerosis. J Neuroimmune Pharmacol. 2013;8:677–90. doi: 10.1007/s11481-013-9449-5. - DOI - PubMed
    1. Probert L, Akassoglou K, Pasparakis M, Kontogeorgos G, Kollias G. Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha. Proc Natl Acad Sci U S A. 1995;92:11294–8. doi: 10.1073/pnas.92.24.11294. - DOI - PMC - PubMed

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