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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2014 Oct 31;111(45):16053–16058. doi: 10.1073/pnas.1418966111

Interleukin 10 modulation of pathogenic Th17 cells during fatal alphavirus encephalomyelitis

Kirsten A Kulcsar 1, Victoria K Baxter 1, Ivorlyne P Greene 1,1, Diane E Griffin 1,2
PMCID: PMC4234572  PMID: 25362048

Significance

Mosquito-borne alphaviruses are important causes of epidemic encephalomyelitis. The immune response plays an important role in disease; however, immune-mediated mechanisms of pathogenesis and regulation are not understood. In this study, we determined that a pathogenic Th17 response occurs during fatal alphavirus encephalitis. Furthermore, the regulatory cytokine, interleukin 10, plays an important role in modulating the pathogenic Th17 response. In the absence of interleukin 10, the Th17 response is increased in magnitude and displays a more pathogenic phenotype, resulting in accelerated disease progression. These findings are important for understanding the pathogenesis of virus infections in the central nervous system and the identification of therapeutic interventions that focus on immune modulation in the central nervous system.

Keywords: GM-CSF, Sindbis virus, immunopathology, viral encephalitis, interleukin-10

Abstract

Mosquito-borne alphaviruses are important causes of epidemic encephalomyelitis. Neuronal cell death during fatal alphavirus encephalomyelitis is immune-mediated; however, the types of cells involved and their regulation have not been determined. We show that the virus-induced inflammatory response was accompanied by production of the regulatory cytokine IL-10, and in the absence of IL-10, paralytic disease occurred earlier and mice died faster. To determine the reason for accelerated disease in the absence of IL-10, immune responses in the CNS of IL-10−/− and wild-type (WT) mice were compared. There were no differences in the amounts of brain inflammation or peak virus replication; however, IL-10−/− animals had accelerated and increased infiltration of CD4+IL-17A+ and CD4+IL-17A+IFNγ+ cells compared with WT animals. Th17 cells infiltrating the brain demonstrated a pathogenic phenotype with the expression of the transcription factor, Tbet, and the production of granzyme B, IL-22, and GM-CSF, with greater production of GM-CSF in IL-10−/− mice. Therefore, in fatal alphavirus encephalomyelitis, pathogenic Th17 cells enter the CNS at the onset of neurologic disease and, in the absence of IL-10, appear earlier, develop into Th1/Th17 cells more often, and have greater production of GM-CSF. This study demonstrates a role for pathogenic Th17 cells in fatal viral encephalitis.

Encephalitic arthropod-borne viruses are important causes of morbidity and mortality worldwide (1, 2). In the Americas, the mosquito-borne alphaviruses infect neurons and cause outbreaks of encephalomyelitis with high mortality in horses and humans. In addition, Chikungunya virus, a newly emerging and rapidly spreading old-world alphavirus, can also cause neurologic disease (3–5). The immune-mediated inflammatory response in the central nervous system (CNS) is necessary for virus clearance, but can also cause neuronal damage. Several lines of evidence suggest that fatal alphavirus encephalomyelitis is mediated by the immune response to virus infection rather than virus infection per se (6, 7), but the components of the immune response involved in causing neuronal death and mechanisms for regulating this immunopathology have not been identified.

We have used a well-characterized model of fatal alphavirus encephalomyelitis to identify immune contributors to fatal disease. Neuroadapted Sindbis virus (NSV) infects neurons in the brain and spinal cord with a particular tropism for hippocampal and motor neurons and causes fatal paralytic disease in adult C57BL/6 mice (8–10). Infected neurons die during this disease process, but neuronal death is caused by the immune response to infection rather than by damage from virus replication (7, 10). Mice can be protected from fatal disease if the CNS inflammatory response is inhibited, despite the fact that this results in a failure of virus clearance (7, 11). Studies of immune-deficient mice have implicated T cells in this immunopathologic process (6, 9), but the types of T cells involved, their pathologic function, and the regulation of this response during fatal disease are not known.

Results

IL-10 Deficiency Accelerates the Course of Fatal Encephalomyelitis.

Interleukin (IL) 10 is a critical regulatory cytokine produced by many types of cells that determines the balance between inflammation and immune regulation by influencing antigen presentation, T-cell differentiation, cytokine production, and intensity of inflammation (12, 13). During fatal CNS infection induced by intranasal inoculation of 4–6-wk-old C57BL/6J mice with 105 pfu NSV, Il10 mRNA was increased in both the brain and spinal cord (P < 0.0001; Fig. 1A). To determine the importance of IL-10 production in the CNS during the induction of fatal encephalomyelitis, we compared the disease course of NSV-infected wild-type (WT) C57BL/6J mice to that of IL-10–deficient C57BL/6J mice (B6.129P2-Il10tm1Cgn/J; IL-10−/−). Disease was accelerated in IL-10−/− mice with more rapid onset of paralysis (P < 0.0001; Fig. 1C) and earlier death (mean day of death was 8 d compared with 10 d, P < 0.0001; Fig. 1D). Accelerated disease progression occurred during the time of Il10 mRNA increase in WT mice, 5–7 d after infection (Fig. 1 A and C). IL-10 deficiency did not affect virus replication or peak virus titers, although titers at day 7 suggest that virus clearance was delayed (Fig. 1B).

Fig. 1.

Fig. 1.

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IL-10 is important in regulating disease progression independent of virus replication. WT and IL-10−/− mice were infected intranasally with 105 pfu NSV. (A) Il10 mRNA expression measured by quantitative real-time PCR in the brains (filled square) and spinal cords (open square) of NSV-infected WT mice. Ct values were normalized to GAPDH. Ct values and fold change were calculated relative to uninfected controls (ΔΔCt). Data are pooled from two independent experiments and represent the mean ± SEM of 4–6 mice at each time point; *P < 0.05, ***P < 0.001, ****P < 0.0001. (B) Virus titers were determined using brain homogenates prepared from WT (filled square, solid line) and IL-10−/− (open circle, dashed line) mice. Data are pooled from three independent experiments and presented as the mean ± SEM of 6–9 mice at each time point; **P < 0.01. (C) Signs of disease in WT and IL-10−/− mice infected with NSV were monitored daily. The clinical score scale was as follows: 0, no symptoms; 1, abnormal hind-limb and tail posture, ruffled fur, and/or hunched back; 2, unilateral hind-limb paralysis; 3, bilateral hind-limb paralysis and/or moribund; 4, dead. Data are pooled from three independent experiments and presented as the mean ± SEM; WT, n = 27 and IL-10−/−, n = 36; ****P < 0.0001. (D) Survival was assessed using a Kaplan–Meier curve and log-rank test. The mean day of death was 10 for WT mice and 8 for IL-10−/− mice. Data are pooled from three independent experiments; WT, n = 35 and IL-10−/−, n = 37; ****P < 0.0001.

To identify the reason for accelerated disease in the absence of IL-10, we first assessed the magnitude of the CNS inflammatory response by isolating and counting cells infiltrating the brain (Fig. 2A) and by scoring hematoxylin and eosin (H&E)-stained coronal brain sections for inflammation (Fig. 2 B and C). The numbers of infiltrating cells and amount of inflammation increased through 7 d after infection, but these parameters were not different between WT and IL-10−/− mice. To determine whether the relative proportions of myeloid and lymphoid cells contributing to the inflammatory process differed, we used flow cytometry for identification of cells isolated from the brain (Fig. 2 D–O). There were no differences in numbers or percentages of microglia (Fig. 2 D and E), natural killer (NK) cells (Fig. 2 J and K), CD4+ T cells (Fig. 2 L and M), or CD8+ T cells (Fig. 2 N and O). WT mice had more monocyte/macrophages (P < 0.05; Fig. 2F), and IL-10−/− mice had more neutrophils (P < 0.01; Fig. 2 H and I) at 7 d after infection.

Fig. 2.

Fig. 2.

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The inflammatory response to NSV infection is similar in WT and IL-10−/− mice. WT (filled square or bar, solid line) and IL-10−/− (open circle or bar, dashed line) mice were infected with NSV and inflammation assessed. (A) The average number of live cells isolated from the brains (n = 6–10, pooled) of mice after infection. Data represent the mean ± SEM from four separate experiments. (B and C) Brains were collected from uninfected and NSV-infected mice at 3, 5, and 7 d after infection. (B) Extent of inflammation in coded H&E-stained coronal sections scored on a scale from 0 to 3, with an extra point given to sections that had excessive parenchymal cellularity. Data are presented as the mean ± SEM of three WT (black bars) and three IL-10−/− (open bars) mice. (C) Representative H&E-stained coronal sections of uninfected mice and infected mice 7 d after infection (20×). (D–O) Flow cytometric analysis of isolated cells pooled from the brains (n = 6–10) of WT or IL-10−/− mice without infection and 3, 5, and 7 d after infection. The absolute number of cells per brain and frequency (percentage of live cells) of microglia (D and E), macrophages/monocytes (F and G), neutrophils (H and I), NK cells (J and K), CD4+ T cells (L and M), and CD8+ T cells (N and O) were determined. The data represent the mean ± SEM from two to four independent experiments; *P < 0.05, **P < 0.01.

To determine whether increased neutrophil infiltration into the CNS was responsible for accelerated disease in IL-10−/− mice, neutrophils were depleted using antibody to Ly6G or control antibody given at the time of infection and 4 d after infection (14). Depletion of circulating neutrophils was confirmed to be greater than 90% by blood smears (Fig. S1A) and greater than 65% in the brain (Fig. S1 B and C). The presence or absence of neutrophils did not alter the disease course in IL-10−/− mice (Fig. S1 D and E). Therefore, neither differences in numbers nor overall types of CNS inflammatory cells explained accelerated disease in the absence of IL-10.

As both CD8+ and CD4+ T cells have been implicated in immunopathologic processes during NSV infection (6), we next assessed the possibility that the absence of IL-10 led to functional differences in these cell populations even though overall numbers were similar. Flow cytometry and intracellular cytokine staining were used to measure production of the effector molecules TNFα, IFNγ, and granzyme B by CD8+ T cells isolated from the brain at 5 d (Fig. 3 A–G) and 7 d (Fig. 3 H–N) after infection. At 5 d after infection, the percentage of CD8+ T cells expressing granzyme B was lower in IL-10−/− mice than WT mice (68.8% vs. 86.4%, P = 0.0247; Fig. 3B), whereas the number and percentage of CD8+ T cells producing IFNγ (Fig. 3 D and E) and TNFα (Fig. 3 F and G) were similar. A decrease in CD8+ T cells producing granzyme B in IL-10−/− mice would be predicted to decrease, rather than increase, cytotoxicity, but could slow virus clearance (Fig. 1B) (15, 16). At 7 d after infection, no differences were observed between WT and IL-10−/− mice in the frequency or number of CD8+ T cells expressing IFNγ, TNFα, or granzyme B (Fig. 3 H–N). Analysis of fluorescence intensity indicated that amounts of granzyme B per CD8+ T cell were similar in WT and IL-10−/− mice (Fig. 3 A and H). Therefore, differences in numbers or function of CD8+ T cells did not explain accelerated disease in IL-10−/− mice.

Fig. 3.

Fig. 3.

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CD8+ T-cell effector molecule expression is similar in the brains of WT and IL-10−/− mice during NSV infection. Isolated cells were pooled (n = 6–10) from the brains of WT and IL-10−/− mice at 5 d (A–G) and 7 d (H–N) after infection; stimulated with PMA and ionomycin in the presence of brefeldin A to assess IFNγ, TNFα, and granzyme B production; and analyzed by flow cytometry. Contour plots show the gating of CD8+ T cells that produce IFNγ, TNFα, and granzyme B at 5 d (A) and 7 d (H) after infection and are representative of three independent experiments. Histograms show the fluorescence intensity of granzyme B expression in CD8+ T cells isolated from the brains of WT (black) and IL-10−/− (orange) mice relative to the isotype control (gray filled). The frequency of CD8+ T cells that produce IFNγ (B and I), TNFα (D and K), and granzyme B (F and M) calculated as the mean ± SEM from three independent experiments. The number of CD8+ T cells that produce IFNγ (C and J), TNFα (E and L), and granzyme B (G and N) was calculated as the mean ± SEM from three independent experiments; *P < 0.05.

IL-10 Deficiency Increases the Numbers of Th17 Cells in the CNS.

CD4+ T cells can differentiate into several functional groups defined by transcription factor expression and cytokine production. Because both Th1 and Th17 subsets have been implicated in autoimmune disease in the CNS (17), we used flow cytometry and intracellular cytokine staining to analyze brain CD4+ cells for production of signature cytokines IFNγ (Th1) and IL-17A (Th17) (Fig. 4). At 5 d after infection, when signs of neurologic disease appear in IL-10−/− mice (Fig. 1C), the IFNγ-producing CD4+ population was similar (Fig. 4 A–C), but there was a fivefold increase in the frequency (2.97% vs. 14.62%, P = 0.0063; Fig. 4D) and in the number (6.4 × 102 vs. 5.2 × 103 cells per brain, P = 0.0153; Fig. 4E) of IL-17–producing CD4+ T cells in IL-10−/− mice compared with WT mice. At 7 d after infection, when neurologic signs of disease become prominent in WT mice, the numbers of IL-17–producing as well as IFNγ-producing CD4+ T cells increased in WT mice, but the percentage (0.41% vs. 1.94%, P = 0.0120; Fig. S2 A and D) and numbers (1.2 × 103 vs. 5.4 × 103 cells per brain, P = 0.0508; Fig. S2E) of Th17 cells remained higher in IL-10−/− mice. In the spinal cord, Th17, but not Th1, cells were also more prevalent in IL-10−/− mice than WT mice (P = 0.0420; Fig. S2 F–J).

Fig. 4.

Fig. 4.

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The Th17 response is increased in the brains of IL-10−/− mice compared with WT mice at the onset of clinical disease. Mononuclear cells were isolated from the brains (n = 6–10) of WT and IL-10−/− mice at 5 d after NSV infection and stimulated with PMA and ionomycin in the presence of brefeldin A to assess the production of IFNγ (Th1) and IL-17A (Th17) in CD4+ T cells. (A) Contour plots show the gating for IFNγ+ and IL-17A+ cells within the brain CD4+ T-cell population. Plots are representative of three independent experiments. (B–E) Quantification of the frequency of CD4+ T cells that express IFNγ (B) and IL-17A (D), as well as the number of CD4+IFNγ+ (C) and CD4+IL-17A+ (E) T cells in the brains of WT (black) and IL-10−/− (white) mice. Data are shown as the mean ± SEM from three independent experiments; *P < 0.05, **P < 0.01. (F–I) Analysis of Il17a (F and G) and Ccl20 (H and I) mRNAs in the brains (F and H) and spinal cords (G and I) of WT (filled square, solid line) and IL-10−/− (open circle, dashed line) mice during NSV infection. Gene Ct values were normalized to Gapdh, and fold change was calculated relative to uninfected controls (ΔΔCt). Data are pooled from two independent experiments and presented as the mean ± SEM from four to six mice at each time point; *P < 0.05, **P < 0.01, ***P < 0.001.

To assess the effect of IL-10 deficiency on Th17-related gene expression in the CNS, we compared the levels of mRNAs for Il17a and for the Th17-specific chemokine Ccl20 (18) in the brains and spinal cords of WT and IL-10−/− mice (Fig. 4 K–N). Il17a mRNA levels were higher in IL-10−/− compared with WT mice in the brain (P = 0.0144; Fig. 4K) and spinal cord (P = 0.0095; Fig. 4L). Ccl20 mRNA levels were higher in IL-10−/− mice in the spinal cord (P = 0.0126; Fig. 4N), but not the brain (Fig. 4M).

Therefore, both Th1 and Th17 cells began infiltrating the CNS of WT mice coincident with the onset of paralysis and were present during fatal NSV infection. In the absence of IL-10, accelerated disease correlated with an increase in the Th17, but not the Th1, response.

Th17 Cells in the CNS of Mice with Fatal Viral Encephalomyelitis Have a Pathogenic Phenotype That Is Enhanced in the Absence of IL-10.

Th17 cells are polyfunctional, have multiple phenotypes, and can play both detrimental and beneficial roles in disease pathogenesis (19–21). To determine the phenotype of the Th17 cells entering the brain at the onset of neurological disease, CD4+IL-17A+ cells from the brain at 5 d after NSV infection were examined for production of granzyme B, IL-22, and GM-CSF, cytokines associated with a pathogenic phenotype in experimental autoimmune encephalomyelitis (EAE) (19–21) (Fig. 5). Th17 cells from both WT and IL-10−/− mice expressed all three cytokines. The proportions of Th17 cells producing granzyme B (23.0% vs. 10.8%, P = 0.028; Fig. 5 A and E) and IL-22 (41.5% vs. 22.6%, P = 0.0104; Fig. 5 A and G) were greater in WT than IL-10−/− mice, but there were more granzyme B+ (9.5 × 102 vs. 9.0 × 101, P = 0.0328; Fig. 5F) and IL-22+ (6.5 × 102 vs. 7.9 × 101, P = 0.0369; Fig. 5H) Th17 cells in the brains of IL-10−/− compared with WT mice. Analysis of fluorescence intensity indicated that the amount of granzyme B expressed by each positive Th17 cell was similar (Fig. 5A).

Fig. 5.

Fig. 5.

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Th17 cells found in the brains of NSV-infected WT and IL-10−/− mice have a pathogenic phenotype. (A) Mononuclear cells were isolated and pooled from brains (n = 6–10) of WT and IL-10−/− mice 5 d after infection and stimulated with PMA and ionomycin in the presence of brefeldin A to assess the production of GM-CSF, granzyme B, and IL-22 by Th17 cells (CD4+IL-17A+). (A) Contour plots show the gating for GM-CSF, granzyme B, and IL-22 production within the CD4+ T-cell population. Histograms show the fluorescence intensity of GM-CSF and granzyme B expression in Th17 cells isolated from the brains of WT (black) and IL-10−/− (orange) mice relative to the isotype control (gray, filled). Contour plots and histograms are representative of three independent experiments. (B–H) The frequencies and numbers of CD4+IL-17A+ T cells producing GM-CSF (B and C), granzyme B (E and F), and IL-22 (G and H) in NSV-infected WT (black) and IL-10−/− (white) mice. (D) The mean fluorescence intensity of CD4+IL-17A+GM-CSF+ T cells in the brains of WT (black) and IL-10−/− (white) mice. Data represent the mean ± SEM from three independent experiments; *P < 0.05.

Greater differences were observed for Th17 cell production of GM-CSF. In IL-10−/− mice, both the proportion (23.8% vs. 15.7%, P = 0.0173; Fig. 5 A and B) and numbers (1.1 × 103 vs. 4.1 × 101, P = 0.0268; Fig. 5C) of Th17 cells producing GM-CSF were higher than in WT mice. Additionally, the Th17 cells in the brains of IL-10−/− mice had higher mean fluorescence intensities for GM-CSF than those in the brains of WT mice, indicating a higher level of GM-CSF production per cell (P = 0.0179; Fig. 5 A and D). Together, these data show that the Th17 cells in the CNS during fatal NSV infection have a pathogenic phenotype and that, in the absence of IL-10, the pathogenic Th17 response is amplified and GM-CSF production increased. A definitive role for a particular Th17 product could not be identified because neither treatment with neutralizing antibody to IL-17 nor GM-CSF improved the outcome in IL-10−/− or WT mice compared with control antibody (Fig. S3 A–H).

Th17 cells develop independently of Tbet and IFNγ, but in tissue can acquire expression of these signature Th1 factors along with an increase in pathogenicity (22). An important pathogenic role is suggested by the target organ presence of IL-17A+IFNγ+ Th1/Th17 cells during EAE and autoimmune colitis (21) and by the fact that mice deficient in the transcription factor Tbet are protected from development of EAE (23, 24). Because pathogenic Th17 cells can develop into multifunctional pathogenic Th1/17 cells, we examined brain CD4+ T cells for expression of Tbet and the Th17 transcription factor, RORγt, in conjunction with the production of IL-17A and IFNγ (Fig. 6). WT mice had mostly Th1 cells (IFNγ+Tbet+) and Th17 cells (IL-17A+RORγt+) with very few Th1/Th17 cells (IFNγ+IL-17A+) (Fig. 6 A and B). In the absence of IL-10, however, in addition to an increase in the proportion of Th17 cells (8.98% vs. 2.53%, P = 0.0003), there was also an increase in the Th1/Th17 population (4.28% vs. 0.46%, P = 0.0262; Fig. 6 A and B).

Fig. 6.

Fig. 6.

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Th17 cells in the brains of NSV-infected WT and IL-10−/− mice express Tbet and RORγt and develop into Th1/Th17 cells in the absence of IL-10. Mononuclear cells isolated and pooled from the brains (n = 6–10) of WT and IL-10−/− mice 5 d after infection were stimulated with PMA and ionomycin in the presence of brefeldin A to assess the production of IFNγ and IL-17A by CD4+ T cells. (A) Contour plots showing IFNγ and IL-17A production within the CD4+ T-cell population. Plots are representative of three independent experiments. (B) Quantification of the IFNγ+ (Th1), IL-17A+ (Th17), and IFNγ+IL-17A+ (Th1/Th17) CD4+ T cells in the brains of WT (black) and IL-10−/− (white) mice presented as the mean ± SEM from three independent experiments; *P < 0.05, ***P < 0.001. (C) Tbet and RORγt transcription factor expression in IFNγ+ (Th1), IL-17A+ (Th17), and IFNγ+IL-17A+ (Th1/Th17) CD4+ T cells. (D) Histograms show Tbet and RORγt expression in WT (black) and IL-10 KO (orange) relative to an isotype control (gray, filled). Contour plots and histograms are representative of three independent experiments.

The IFNγ+ cells in the brains of WT and IL-10−/− mice expressed Tbet, with little expression of RORγt, consistent with classic Th1 cells (Fig. 6 C and D, Top). The IL-17A+ cells expressed RORγt, the canonical Th17 lineage transcription factor, as well as Tbet, a marker of pathogenicity (Fig. 6 C and D, Middle). The IFNγ+IL-17A+ cells in the brains of IL-10−/− mice also expressed RORγt and Tbet (Fig. 6 C and D, Bottom). These data show that populations of pathogenic Th17 cells are present in the CNS at the onset of neurological disease in WT and IL-10−/− mice. The increased presence of Th1/Th17 cells in the absence of IL-10 suggests that these cells are regulated by IL-10 and may contribute to the accelerated disease progression.

Discussion

T-cell–mediated damage drives pathogenesis of fatal alphavirus encephalomyelitis. We identified Th1 and pathogenic Th17 cells in the CNS at the onset of neurological disease. IL-10 expression increased in the CNS after infection and was identified as a critical regulator of the Th17 and Th1/Th17 response. In the absence of IL-10, paralytic disease was accelerated concomitant with increased infiltration of pathogenic Th17 and Th1/Th17 cells into the CNS. Pathogenic Th17 cells expressed effector cytokines IL-17A, IL-22, granzyme B, and GM-CSF and transcription factors RORγt and Tbet, with a marked increase in production of GM-CSF in the absence of IL-10.

The role of IL-10 regulation of the immune response during viral encephalitis has received limited analysis. IL-10 has a protective role during acute coronavirus and flavivirus encephalitis in mice, but the mechanisms by which outcome is improved were not identified (25–27). Although IL-10 deficiency can promote an overall increase in inflammation (28–30), this did not occur in alphavirus-induced encephalomyelitis. Rather, IL-10 deficiency facilitated a selective increase in the proportion of CD4+ T cells in the inflammatory response that were Th17 cells.

IL-10 directly inhibits both differentiation and proliferation of Th17 cells (31–33), and IL-10 deficiency leads to an increase in Th17 cells in mice infected with leishmania (34), influenza virus (35), and respiratory syncytial virus (RSV) (36). During pulmonary infection with influenza virus, an increase in Th17 cells is associated with a better outcome, whereas for infection with RSV and leishmania, the disease was more severe. In none of these studies was the function or cytokine profile of the Th17 cells characterized to provide insight into how IL-10 modulated Th17-associated disease. During NSV infection of the CNS, IL-10 deficiency also led to more severe disease and an increase in Th17 cells. Functional characterization of the Th17 cells revealed a pathogenic phenotype with production of granzyme B, IL-22, and GM-CSF.

The effector function(s) of Th17 cells that cause neurologic disease are not clear, but multiple factors may contribute. IL-17 production is reported to be both protective and damaging (17, 37–40). IL-17 can induce neuronal cell death in vitro (41), and EAE is attenuated in mice lacking IL-17 (42). Neutralization of IL-17 did not improve outcome after NSV infection, but a role for IL-17 in neuronal damage cannot be excluded because of unknown CNS penetration of the antibody. Granzyme B induces neurotoxicity by activation of protease-activated G protein-coupled receptors on neurons (43), and both granzyme B and IL-22 contribute to blood–brain barrier disruption (44).

GM-CSF is a potent mediator of Th17 cell damage during EAE (45). GM-CSF activates microglia, enhances myeloid cell recruitment, and promotes Th17 cell differentiation (46–48). The greatest influence of IL-10 deficiency on Th17 function was increased production of GM-CSF on both a population and single-cell basis. Thus, although a neutralizing antibody did not improve the outcome, GM-CSF cannot be excluded as a contributor to immune-mediated damage in viral encephalomyelitis.

Together, these studies have characterized the functional features of Th17 cells that develop in the absence of IL-10 and have identified pathogenic Th17 and Th1/17 cells as participants in the immunopathologic process leading to fatal viral encephalomyelitis. This understanding of the pathogenesis of acute virus-induced neurologic disease will aid in identifying therapeutic interventions that focus on immune modulation as well as antiviral drugs.

Methods and Materials

Animals and Infection.

C57BL/6J WT and B6.129P2-Il10tm1Cgn/J (C57BL/6J IL-10−/−) (49) mice were purchased from Jackson Laboratories and bred in house. The NSV strain of Sindbis virus (50) was grown and assayed by plaque formation in BHK cells. For infection, sex-matched 4–6-wk-old mice were inoculated intranasally with 105 pfu NSV in 20 μL HBSS. For assessment of morbidity and mortality, mice were monitored daily. The scoring system used was as follows: 0, no signs of disease; 1, abnormal hind-limb and tail posture, ruffled fur, and/or hunched back; 2, unilateral hind-limb paralysis; 3, bilateral hind-limb paralysis or full-body paralysis; 4, dead. For tissue collection, mice were anesthetized with isoflurane and bled by cardiac puncture. Mice were perfused with ice-cold PBS, and brains and spinal cords were collected and used fresh or snap frozen and stored at −80 °C. All experiments were performed according to guidelines approved by the Johns Hopkins University Institutional Animal Care and Use Committee.

Virus Assays.

Tissue was homogenized in cold PBS to make 10% (wt/vol) brain and spinal cord homogenates and was clarified by centrifugation. Infectious virus was assayed by plaque formation on BHK-21 cells. Data are plotted as the mean of the log10 value of plaque forming units ± SEM. For statistical purposes, samples in which no virus was detected at a 1:10 dilution were assigned a value of 0.85, halfway between the limit of detection and 0.

Histology.

Uninfected and NSV-infected mice at 3, 5, and 7 d after infection were perfused with ice-cold PBS followed by 4% (wt/vol) paraformaldehyde. Brains were removed and cut into 2-mm coronal slices with an adult Mouse Brain Slicer (Zivic Instruments) that were postfixed in 4% (wt/vol) paraformaldehyde for 24 h at 4 °C. Slices were washed in PBS and embedded in paraffin. Coronal brain sections (10 μm) were stained with H&E, coded, and scored blindly as previously described (51) using a 0–3 scale: 0, no detectable inflammation; 1, one to two small inflammatory foci per section; 2, moderate inflammatory foci in up to 50% of 10× fields; 3, moderate to large inflammatory foci in greater than 50% of 10× fields. An additional point was added for excessive parenchymal cellularity, allowing for a maximal score of 4.

Mononuclear Cell Isolation.

Single-cell suspensions were made from brain and spinal cord tissue homogenized in RPMI + 1% FBS, 1 mg/mL collagenase (Roche), and 0.1 mg/mL DNase (Roche) using the GentleMACS system (Miltenyi). The homogenate was incubated (37 °C, 15 min), homogenized again, incubated again, and filtered (70 μm). Myelin debris and red blood cells were removed by centrifugation on a 30/70% percoll gradient for 30 min at 4 °C. Mononuclear cells at the interface were collected, resuspended in PBS + 2 mM EDTA, and live cells counted using trypan blue exclusion.

Flow Cytometry.

Approximately 1–2 × 106 cells were stained with the violet Live/Dead Fixable Cell Stain Kit (Invitrogen) in PBS + 2 mM EDTA, blocked with rat anti-mouse CD16/CD32 (BD Pharmingen), diluted in PBS + 2 mM EDTA + 0.5% BSA, surface-stained for 25 min on ice, fixed, and resuspended in 200 μL PBS + 2 mM EDTA + 0.5% BSA. All antibodies were from BD Pharmingen or eBioscience: CD45 (clone 30-F11), CD11b (clone M1-70), Ly6G (clone 1A8), Ly6C (clone HK1.4), CD3 (clone 17A2), CD4 (clone RM4-5), CD8 (clone 53–6.7), and NK1.1 (clone PK136). Cell types were defined as follows: microglia (CD45loCD11b+Ly6G−Ly6C−), macrophages/monocytes (CD45hiCD11b+Ly6G−Ly6C+), neutrophils (CD45+CD11b+Ly6G+Ly6Cint), NK cells (CD45+CD3−NK1.1+), T cells (CD3+), CD4 T cells (CD3+CD4+), and CD8 T cells (CD3+CD8+).

For intracellular cytokine staining, 2–3 × 106 cells were stimulated in RPMI + 1% FBS containing 50 ng/mL of phorbol-12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin in the presence of GolgiPlug-brefeldin A (BD Pharmingen) for 4 h. After surface staining, cells were fixed and permeabilized using either the CytoFix/CytoPerm kit (BD Pharmingen) or the Foxp3 Transcription Factor Fix/Perm kit (eBioscience). Intracellular markers were stained for 25 min on ice and resuspended in 200 μL of PBS + 2 mM EDTA + 0.5% BSA. All antibodies were from BD Pharmingen or eBioscience: IFNγ (clone XMG1.2), IL-17a (clone eBio17B7), TNFα (clone MP6-XT22), granzyme B (clone NGZB), Tbet (clone ebio4B10), RORγt (clone B2D, eBioscience), GM-CSF (clone MP1-22E9), granzyme B (clone NGZB), and IL-22 (clone 1H8PWSR). Data were acquired with a BD FACS Canto II using FACS Diva software (version 6.0) and analyzed using FlowJo 8.8.7 (TreeStar Inc.).

Gene Expression Analysis Using Real-Time PCR.

RNA was isolated from frozen tissue using the RNeasy Lipid Mini RNA Isolation Kit (Qiagen). RNA was quantified using a nanodrop spectrophotometer, and cDNA was prepared with the High Capacity cDNA Reverse Transcription Kit (Life Technologies) using 0.5–2.5 μg RNA. Quantitative real-time PCR was performed using 2.5 μL cDNA, TaqMan gene expression arrays (Il10, Il17a, and Ccl20), and 2× Universal PCR Mastermix (Applied Biosystems). Gapdh mRNA levels were determined using the rodent primer and probe set (Applied Biosystems). All reactions were run on the Applied Biosystems 7500 real-time PCR machine with the following conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min for 50 cycles. Transcript levels were determined by normalizing the target gene Ct value to the Ct value of Gapdh. This normalized value was used to calculate the fold change relative to the average of the uninfected control (ΔΔCt method).

Neutrophil Depletion.

Mice were administered 0.5 mg rat anti-Ly6G antibody (clone 1A8, BioXcell) or the rat IgG2a isotype control (clone 3A2, BioXcell) intraperitoneally in 200 μL PBS at the time of infection and 4 d after infection. To document neutrophil depletion, blood smears were examined for the percentage of white blood cells that were neutrophils. To document depletion in the CNS, brains collected 7 d after infection were analyzed by flow cytometry.

Cytokine Neutralization.

To neutralize IL-17a, mice were treated with 250 μg of mouse anti–IL-17a (clone 17F3, BioXcell) or the mouse IgG1 isotype control (clone MOPC-21, BioXcell) antibody intraperitoneally in a volume of 200 μL (diluted in PBS) at the time of infection and again at 3 and 6 d after infection. To neutralize GM-CSF, mice were treated with 250 μg of rat anti–GM-CSF (clone MP1-22E9, BioXcell) or the rat IgG2a isotype control (clone 3A2, BioXcell) antibody intraperitoneally in a volume of 200 μL (dlluted in PBS) at 2, 4, 6, and 8 d after infection.

Statistical Analysis.

Data from two to four independent experiments or at least three mice per group were used. Survival was compared using Kaplan–Meier survival curves (log rank test). Differences during the course of infection in a single group were determined using one-way ANOVA and Dunn posttests. Differences between groups during the course of infection were determined using two-way ANOVA and Bonferroni posttests. Differences between groups at a single time point were determined using an unpaired, two-tailed Student t test with a 95% confidence interval. All statistical analyses used Prism 5 (GraphPad).

Supplementary Material

Supplementary File
pnas.201418966SI.pdf (329.2KB, pdf)

Acknowledgments

We thank Tricia Niles for help with flow cytometry, Damien Chopy for help with early experiments, and Drs. Alan Scott and Jay Bream for helpful advice. This work was supported by Grants F31 NS076223 (to K.A.K.), T32 OD011089 (to V.K.B.), T32 AI007247 (to I.P.G.), and R01 NS087539 (to D.E.G.) from the National Institutes of Health.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1418966111/-/DCSupplemental.

References

  • 1.Hollidge BS, González-Scarano F, Soldan SS. Arboviral encephalitides: Transmission, emergence, and pathogenesis. J Neuroimmune Pharmacol. 2010;5(3):428–442. doi: 10.1007/s11481-010-9234-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Go YY, Balasuriya UBR, Lee C-K. Zoonotic encephalitides caused by arboviruses: Transmission and epidemiology of alphaviruses and flaviviruses. Clin Exp Vaccine Res. 2014;3(1):58–77. doi: 10.7774/cevr.2014.3.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Chusri S, Siripaitoon P, Hirunpat S, Silpapojakul K. Case reports of neuro-Chikungunya in southern Thailand. Am J Trop Med Hyg. 2011;85(2):386–389. doi: 10.4269/ajtmh.2011.10-0725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Staples JE, Breiman RF, Powers AM. Chikungunya fever: An epidemiological review of a re-emerging infectious disease. Clin Infect Dis. 2009;49(6):942–948. doi: 10.1086/605496. [DOI] [PubMed] [Google Scholar]
  • 5.Nelson J, Waggoner JJ, Sahoo MK, Grant PM, Pinsky BA. Encephalitis caused by Chikungunya virus in a traveler from the Kingdom of Tonga. J Clin Microbiol. 2014;52(9):3459–3461. doi: 10.1128/JCM.01288-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Rowell JF, Griffin DE. Contribution of T cells to mortality in neurovirulent Sindbis virus encephalomyelitis. J Neuroimmunol. 2002;127(1-2):106–114. doi: 10.1016/s0165-5728(02)00108-x. [DOI] [PubMed] [Google Scholar]
  • 7.Greene IP, Lee E-Y, Prow N, Ngwang B, Griffin DE. Protection from fatal viral encephalomyelitis: AMPA receptor antagonists have a direct effect on the inflammatory response to infection. Proc Natl Acad Sci USA. 2008;105(9):3575–3580. doi: 10.1073/pnas.0712390105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Jackson AC, Moench TR, Trapp BD, Griffin DE. Basis of neurovirulence in Sindbis virus encephalomyelitis of mice. Lab Invest. 1988;58(5):503–509. [PubMed] [Google Scholar]
  • 9.Kimura T, Griffin DE. Extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted Sindbis virus. Virology. 2003;311(1):28–39. doi: 10.1016/s0042-6822(03)00110-7. [DOI] [PubMed] [Google Scholar]
  • 10.Nargi-Aizenman JL, Griffin DE. Sindbis virus-induced neuronal death is both necrotic and apoptotic and is ameliorated by N-methyl-D-aspartate receptor antagonists. J Virol. 2001;75(15):7114–7121. doi: 10.1128/JVI.75.15.7114-7121.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Nargi-Aizenman JL, et al. Glutamate receptor antagonists protect from virus-induced neural degeneration. Ann Neurol. 2004;55(4):541–549. doi: 10.1002/ana.20033. [DOI] [PubMed] [Google Scholar]
  • 12.Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. doi: 10.1146/annurev.immunol.19.1.683. [DOI] [PubMed] [Google Scholar]
  • 13.Couper KN, Blount DG, Riley EM. IL-10: The master regulator of immunity to infection. J Immunol. 2008;180(9):5771–5777. doi: 10.4049/jimmunol.180.9.5771. [DOI] [PubMed] [Google Scholar]
  • 14.Garraud K, et al. Differential role of the interleukin-17 axis and neutrophils in resolution of inhalational anthrax. Infect Immun. 2012;80(1):131–142. doi: 10.1128/IAI.05988-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Haile Y, et al. Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity. J Immunol. 2011;187(9):4861–4872. doi: 10.4049/jimmunol.1100943. [DOI] [PubMed] [Google Scholar]
  • 16.Wang T, et al. Granzyme B mediates neurotoxicity through a G-protein-coupled receptor. FASEB J. 2006;20(8):1209–1211. doi: 10.1096/fj.05-5022fje. [DOI] [PubMed] [Google Scholar]
  • 17.Petermann F, Korn T. Cytokines and effector T cell subsets causing autoimmune CNS disease. FEBS Lett. 2011;585(23):3747–3757. doi: 10.1016/j.febslet.2011.03.064. [DOI] [PubMed] [Google Scholar]
  • 18.Yamazaki T, et al. CCR6 regulates the migration of inflammatory and regulatory T cells. J Immunol. 2008;181(12):8391–8401. doi: 10.4049/jimmunol.181.12.8391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lee Y, et al. Induction and molecular signature of pathogenic TH17 cells. Nat Immunol. 2012;13(10):991–999. doi: 10.1038/ni.2416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol. 2009;27:485–517. doi: 10.1146/annurev.immunol.021908.132710. [DOI] [PubMed] [Google Scholar]
  • 21.Peters A, Lee Y, Kuchroo VK. The many faces of Th17 cells. Curr Opin Immunol. 2011;23(6):702–706. doi: 10.1016/j.coi.2011.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hirota K, et al. Fate mapping of IL-17-producing T cells in inflammatory responses. Nat Immunol. 2011;12(3):255–263. doi: 10.1038/ni.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Bettelli E, et al. Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis. J Exp Med. 2004;200(1):79–87. doi: 10.1084/jem.20031819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yang Y, et al. T-bet is essential for encephalitogenicity of both Th1 and Th17 cells. J Exp Med. 2009;206(7):1549–1564. doi: 10.1084/jem.20082584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Trandem K, et al. Virally expressed interleukin-10 ameliorates acute encephalomyelitis and chronic demyelination in coronavirus-infected mice. J Virol. 2011;85(14):6822–6831. doi: 10.1128/JVI.00510-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hayasaka D, et al. TNF-α acts as an immunoregulator in the mouse brain by reducing the incidence of severe disease following Japanese encephalitis virus infection. PLoS ONE. 2013;8(8):e71643. doi: 10.1371/journal.pone.0071643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Tun MMN, et al. Protective role of TNF-α, IL-10 and IL-2 in mice infected with the Oshima strain of Tick-borne encephalitis virus. Sci Rep. 2014;4:5344. doi: 10.1038/srep05344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lin MT, Hinton DR, Parra B, Stohlman SA, van der Veen RC. The role of IL-10 in mouse hepatitis virus-induced demyelinating encephalomyelitis. Virology. 1998;245(2):270–280. doi: 10.1006/viro.1998.9170. [DOI] [PubMed] [Google Scholar]
  • 29.Cheeran MC-J, Mutnal MB, Hu S, Armien A, Lokensgard JR. Reduced lymphocyte infiltration during cytomegalovirus brain infection of interleukin-10-deficient mice. J Neurovirol. 2009;15(4):334–342. doi: 10.1080/13550280903062797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Marques CP, et al. Interleukin-10 attenuates production of HSV-induced inflammatory mediators by human microglia. Glia. 2004;47(4):358–366. doi: 10.1002/glia.20045. [DOI] [PubMed] [Google Scholar]
  • 31.Huber S, et al. Th17 cells express interleukin-10 receptor and are controlled by Foxp3⁻ and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner. Immunity. 2011;34(4):554–565. doi: 10.1016/j.immuni.2011.01.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chaudhry A, et al. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity. 2011;34(4):566–578. doi: 10.1016/j.immuni.2011.03.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.McGeachy MJ, et al. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8(12):1390–1397. doi: 10.1038/ni1539. [DOI] [PubMed] [Google Scholar]
  • 34.Gonzalez-Lombana C, et al. IL-17 mediates immunopathology in the absence of IL-10 following Leishmania major infection. PLoS Pathog. 2013;9(3):e1003243. doi: 10.1371/journal.ppat.1003243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.McKinstry KK, et al. IL-10 deficiency unleashes an influenza-specific Th17 response and enhances survival against high-dose challenge. J Immunol. 2009;182(12):7353–7363. doi: 10.4049/jimmunol.0900657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Weiss KA, Christiaansen AF, Fulton RB, Meyerholz DK, Varga SM. Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection. J Immunol. 2011;187(6):3145–3154. doi: 10.4049/jimmunol.1100764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Crowe CR, et al. Critical role of IL-17RA in immunopathology of influenza infection. J Immunol. 2009;183(8):5301–5310. doi: 10.4049/jimmunol.0900995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mukherjee S, et al. IL-17-induced pulmonary pathogenesis during respiratory viral infection and exacerbation of allergic disease. Am J Pathol. 2011;179(1):248–258. doi: 10.1016/j.ajpath.2011.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Suryawanshi A, et al. Role of IL-17 and Th17 cells in herpes simplex virus-induced corneal immunopathology. J Immunol. 2011;187(4):1919–1930. doi: 10.4049/jimmunol.1100736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Savarin C, et al. IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils. J Neuroinflammation. 2012;9:104. doi: 10.1186/1742-2094-9-104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wang D-D, et al. IL-17 potentiates neuronal injury induced by oxygen-glucose deprivation and affects neuronal IL-17 receptor expression. J Neuroimmunol. 2009;212(1-2):17–25. doi: 10.1016/j.jneuroim.2009.04.007. [DOI] [PubMed] [Google Scholar]
  • 42.Komiyama Y, et al. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J Immunol. 2006;177(1):566–573. doi: 10.4049/jimmunol.177.1.566. [DOI] [PubMed] [Google Scholar]
  • 43.Wang T, et al. Granzyme B-induced neurotoxicity is mediated via activation of PAR-1 receptor and Kv1.3 channel. PLoS ONE. 2012;7(8):e43950. doi: 10.1371/journal.pone.0043950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kebir H, et al. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med. 2007;13(10):1173–1175. doi: 10.1038/nm1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.McQualter JL, et al. Granulocyte macrophage colony-stimulating factor: A new putative therapeutic target in multiple sclerosis. J Exp Med. 2001;194(7):873–882. doi: 10.1084/jem.194.7.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Codarri L, Greter M, Becher B. Communication between pathogenic T cells and myeloid cells in neuroinflammatory disease. Trends Immunol. 2013;34(3):114–119. doi: 10.1016/j.it.2012.09.007. [DOI] [PubMed] [Google Scholar]
  • 47.El-Behi M, et al. The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF. Nat Immunol. 2011;12(6):568–575. doi: 10.1038/ni.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.van Nieuwenhuijze A, et al. GM-CSF as a therapeutic target in inflammatory diseases. Mol Immunol. 2013;56(4):675–682. doi: 10.1016/j.molimm.2013.05.002. [DOI] [PubMed] [Google Scholar]
  • 49.Kühn R, Löhler J, Rennick D, Rajewsky K, Müller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell. 1993;75(2):263–274. doi: 10.1016/0092-8674(93)80068-p. [DOI] [PubMed] [Google Scholar]
  • 50.Griffin DE, Johnson RT. Role of the immune response in recovery from Sindbis virus encephalitis in mice. J Immunol. 1977;118(3):1070–1075. [PubMed] [Google Scholar]
  • 51.Rowell JF, Griffin DE. The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells. J Immunol. 1999;162(3):1624–1632. [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary File
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