Acute-Phase Protein Hemopexin Is a Negative Regulator of Th17 Response and Experimental Autoimmune Encephalomyelitis Development

Simona Rolla, Giada Ingoglia, Valentina Bardina, Lorenzo Silengo, Fiorella Altruda, Francesco Novelli and Emanuela Tolosano
J Immunol December 1, 2013, 191 (11) 5451-5459; DOI: https://doi.org/10.4049/jimmunol.1203076

Abstract

Hemopexin (Hx) is an acute-phase protein synthesized by hepatocytes in response to the proinflammatory cytokines IL-6, IL-1β, and TNF-α. Hx is the plasma protein with the highest binding affinity to heme and controls heme-iron availability in tissues and also in T lymphocytes, where it modulates their responsiveness to IFN-γ. Recent data have questioned regarding an anti-inflammatory role of Hx, a role that may be both heme-binding dependent and independent. The aim of this study was to investigate the role of Hx in the development of a T cell–mediated inflammatory autoimmune response. During experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, Hx content in serum increased and remained high. When EAE was induced in Hx knockout (Hx−/−) mice, they developed a clinically earlier and exacerbated EAE compared with wild-type mice, associated to a higher amount of CD4+-infiltrating T cells. The severe EAE developed by Hx−/− mice could be ascribed to an enhanced expansion of Th17 cells accounting for both a higher disposition of naive T cells to differentiate toward the Th17 lineage and a higher production of Th17 differentiating cytokines IL-6 and IL-23 by APCs. When purified human Hx was injected in Hx−/− mice before EAE induction, Th17 expansion, as well as disease severity, were comparable with those of wild-type mice. Taken together, these data indicate that Hx has a negative regulatory role in Th17-mediated inflammation and prospect its pharmacological use to limit the expansion of this cell subset in inflammatory and autoimmune disease.

Introduction

Hemopexin (Hx) is an acute-phase protein synthesized by hepatocytes. Systemic concentration of Hx increases several-fold in response to the proinflammatory cytokines IL-6, IL-1β, and TNF-α (1). Other than in plasma, Hx is also expressed in human and mouse brain and in the neural retina (1–5), and it can also be induced in peripheral nerves in response to injury (6–8). Moreover, Hx has been found in human cerebrospinal fluid both in physiologic and pathologic conditions (9, 10). Hx binds to free heme with high affinity (Kd <1 pM), and by scavenging heme from the bloodstream, it prevents heme-mediated endothelial and tissue damage (11). Moreover, recent works demonstrated an anti-inflammatory role for Hx through its ability to modulate the expression of proinflammatory cytokines both in a heme-dependent and -independent way (12, 13).

In humans, Hx has been chosen as a potential blood biomarker in pediatric multiple sclerosis (MS) (14) likely because of its role in the acute phase of inflammation. MS is a chronic inflammatory disease of the CNS mainly characterized by intervals of remission followed by relapse (15–17). However, acute progressive cases are documented. Onset of this disease initiates outside of the CNS through activation of CD4+ T cells by myelin-like antigenic peptides. These cells then migrate across the blood–brain barrier initiating focal inflammation. Encephalitogenic T cells that invade the CNS interact with APCs, resulting in reactivation of the T cells and activation of the APCs (18). Both IFN-γ–secreting Th1 and IL-17–secreting Th (Th17) cells have been shown to have a pathogenic role in MS (19) and experimental autoimmune encephalomyelitis (EAE), the mouse model of MS (20, 21), but an augment of circulating Th17 cells is often associated with active phases of the disease (22), indicating a key role of Th17 cells in the exacerbation of the clinical symptoms.

To investigate the role of Hx in autoimmune inflammation, we studied the course of EAE induced by immunization of Hx knockout (Hx−/−) and syngenic wild-type mice with myelin oligodendrocyte glycoprotein peptide (MOG35–55). Our data show that in the absence of Hx, the clinical symptoms of EAE are more severe and earlier and are associated to a higher number of infiltrating and circulating Th17 cells. We also found that Hx has a crucial role in the differentiation of Th17 cells, and it is able to modulate the expression levels of Th17 prodifferentiating cytokines as IL-6 and IL-23.

Materials and Methods

Mice

Hx−/− mice on the 129/Sv background (H-2b) were previously described (23). All mice were housed in our animal facility, with a 12-h dark/light cycle and access to standard laboratory chow and tap water ad libitum. Sex- and age-matched wild-type mice on the 129/Sv background were used as controls. For all experiments, 6- to 8-wk-old male mice were used. Animal studies were approved by the animal ethical committee of the University of Turin, Turin, Italy.

EAE in mice

Hx−/− and wild-type mice were immunized s.c. with 200 μg MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; Sigma, St. Louis, MO) emulsified in CFA containing 400 μg Mycobacterium tuberculosis H37Ra (Sigma). At days 0 and at day 2 postimmunization, 200 ng pertussis toxin (from Bordetella pertussis; Sigma) was administered i.p. Clinical score was assessed daily, in a blinded manner, on a scale of 0–5: 0, no disease; 1, limp tail; 2, hind-limb weakness; 3, hind-limb paralysis; 4, hind-limb and forelimb paralysis; 5, moribund/dead state. The onset of the disease refers to clinical score equal to 1.

In rescue experiments, purified human Hx (Athens Research, Athens, GA) was injected i.p. in Hx−/− mice at day 0 of EAE at a dose of 2 mg/animal.

Tissue histology and immunohistochemistry

Hx−/− and wild-type mice were sacrificed at day 28 after induction of EAE and perfused with 35 ml ice-cold PBS through the left ventricle. Spinal cords were isolated and fixed in 4% paraformaldehyde for 24 h, dehydrated in PBS–30% sucrose (Sigma) for 5 d, embedded in optimal cryopreserving tissue compound (OCT; Miles Laboratories, Elkhart, IN), snap-frozen, and sectioned transversally (7 μm/section). Sections were stained with H&E. High-resolution images of sections were captured at ×100 magnification using an Olympus BH-2 microscope, coupled to an Olympus camera. The total cell infiltration (inflammatory index) was assessed as follows: each spinal cord section was subdivided into 16 areas, and each subdivided area displaying lymphocyte infiltration was assigned a score of 1; thus, each animal had a potential maximum score of 16. Four sections of the lumbar spinal cord per animal were analyzed. The presence of CD4+ cells was evaluated by immunofluorescence: sections were incubated 1 h at room temperature with rat anti-mouse CD4-FITC diluted 1:100 (Biolegend, San Diego, CA); Hoechst (Invitrogen, Carlsbad, CA) was used to stain the nuclei. High-resolution images of sections were captured at 200× magnification on a Zeiss fluorescence microscope (Zeiss, Thornwood, NY) with Zeiss ApoTome image visualization system. The number of CD4+ infiltrated cells in four consecutive sections of the lumbar spinal cord of four wild-type and four Hx−/− mice was blindly assessed. Evaluation of demyelization was performed by immunohistochemistry. Sections were incubated overnight with a rat anti-mouse myelin basic protein (MBP) Ab (Abcam, Cambridge, U.K.), diluted 1:100, followed by biotin-conjugated secondary Ab. The sections were visualized on an Olympus BH-2 microscope, coupled to an Olympus camera. As a control of the primary Ab specificity, serial sections were always processed in parallel with the secondary Ab alone.

Sera analysis

Serum was collected from the tail vein at day 0 and at days 2, 4, 7, 14, and 28 after immunization. One microliter of serum was separated on 10% NaDodSO4-PAGE and analyzed by Western blotting using an anti-Hx Ab (mAb 3D6/E12) (1). An anti-mouse IgG Ab (Abcam) was used as control. Alternatively, red Ponceau staining was used as loading control. Membranes were then incubated with 1:5000 HRP-conjugated anti-mouse IgG (Abcam) and revealed by ECL on a ChemiDoc system (Bio-Rad Laboratories, Segrate, Italy). Quantification of proteins was performed by densitometry analysis. IL-6 content in the sera was measured by ELISA kit (Biolegend).

Lymphocyte isolation

To isolate spinal cord and brain infiltrates, we sacrificed Hx−/− and wild-type mice at days 14 or 28 after induction of EAE, and we performed perfusion with 35 ml ice-cold PBS through the left ventricle. Spinal cords and brains were dissected out and digested with 2.5 mg/ml collagenase D (Roche, Mannheim, Germany) for 45 min at 37°C. The tissue was homogenized in cold PBS, and the tissue suspensions were passed through a 70-μm cell strainer to obtain a single-cell suspension and then centrifuged. Cells were resuspended in 37% Percoll (GE Healthcare Europe GmbH, Milan, Italy) and overlaid with 70% Percoll. After centrifugation at 1000 × g for 25 min, the cell monolayer at the 70–37% interphase was collected, washed in RPMI 1640 (Invitrogen), and used for flow cytometry. Spleens and lymph nodes (LNs) were collected from mice at day 0 and at days 7 and 14 of EAE, harvested, and single-cell suspensions were prepared by passage through a 70-μm cell strainer. Blood from Hx−/− and wild-type mice at days 7, 14, 21, and 28 after EAE induction was collected in heparinized tubes, and PBMCs were isolated by Ficoll (EuroClone, Pero, Italy) gradient.

Flow cytometry

Spinal cord and brain mononuclear cells, LNs cells, spleen cells (SPCs), and PBMCs were cultured in 48-well plates (1 × 106 cells/well) and activated with 50 ng/ml PMA (Sigma), 500 ng/ml Ionomycin (Sigma), and 10 ng/ml brefeldin A (Sigma) for 18 h. Cells were then harvested, washed in PBS-1% BSA, and labeled on the surface with FITC anti-mouse CD4 mAb (Biolegend). Cells were then washed and fixed in 4% paraformaldehyde for 15 min at room temperature; after washing, cells were permeabilized with 5% saponin (Sigma) in PBS and labeled intracellularly with PerCP anti-mouse IL-17 (Biolegend) or PE anti-mouse IFN-γ mAb (Biolegend), 20 min at 4°C. Stained cells were acquired on a FACSCalibur and analyzed with CellQuest (BD Biosciences, Erembodegem, Belgium). Each plot represents the results from 50,000 events. To depict cytokine release in response to MOG35–55, we cultured SPCs isolated at day 0 and at days 7 and 14 postimmunization with or without MOG35–55 (10 μg/ml) in 24-well plates (4 × 106 cells/well) for 10 d. After 3 d, IL-2 (1 ng/ml) and IL-23 (10 ng/ml) were added to the cultures and replaced every 3 d. At day 9, cells were activated with PMA, Ionomycin, and brefeldin A for 18 h to perform intracellular staining as previously described. The percentage of MOG35–55–specific Th17 and Th1 cells was measured by flow cytometry subtracting the percentage of cells cultured in absence of MOG35–55. For the measurement of cytokine production, cells were cultured with MOG35–55 peptide for 48 h and supernatants tested by using an ELISA kit (Biolegend).

Th17 cell differentiation

Spleens were collected from Hx−/− and wild-type untreated mice, and single-cell suspensions were prepared by passage through a cell strainer (BD Biosciences). CD4+ naive cells were positively isolated by anti-CD4 and anti-CD62L microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and 2 × 106 CD4+ naive cells were cultured in 24-well plates, coated with 2 μg/ml anti-CD3 mAb and 2 μg/ml anti-CD28 (BD Biosciences) in the presence of Th17 prodifferentiating cytokines, 50 ng/ml IL-6 (Peprotech, Rocky Hill, NJ), 5 ng/ml TGF-β (Peprotech), and 10 ng/ml IL-23 (R&D Systems, Abingdon, U.K.), with or without IFN-γ blocking Ab. Supernatants were collected after 48 h and IL-17 production was measured by using an ELISA kit (Biolegend). Lymphocytes of the negative fraction were used as memory cells and cultured with or without 10 μg/ml IL-23 plus IFN-γ blocking Ab.

Cell proliferation

Spleen cells were harvested and single-cell suspensions were prepared by passage through a cell strainer (BD Biosciences, Erembodegem, Belgium). Splenocytes were cultured in triplicate in round-bottom 96-well plates in the absence or presence of different concentrations of MOG35–55 peptide (0, 1, 10, 100 μg/ml). After 48 h, the cultures were pulsed with 1 μCi [3H]thymidine (TdR) (Amersham, Milan, Italy). After 18 h, the cells were harvested and [3H]TdR uptake was evaluated by TopCount Microplate reader (Packard Instrument, Wellesley, MA).

Quantitative real-time PCR analysis

Macrophages and dendritic cells were separated from spleens collected at day 0 and at day 7 after EAE induction using anti-F4/80 and anti-CD11c microbeads, respectively, following the manufacturer’s instructions (Miltenyi Biotec). Total RNA was extracted from cells using RNeasy mini kit (Qiagen, Milan, Italy). cDNA was prepared from 1 μg total RNA using Promega M-MLV Retro Transcriptase (Promega Italia, Milan, Italy). Quantitative real-time (qRT)-PCR was performed on a 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Primers and probes were designed using the ProbeFinder software (http://www.roche-applied-science.com). All results were normalized to 18S mRNA.

Statistical analyses

Statistical analyses were performed with GraphPad Prism (GraphPad Software, San Diego, CA) by using one-way or two-way ANOVA, or the nonparametric two-tailed Mann–Whitney U test. A p value <0.05 was considered significant.

Results

Hx is upregulated during EAE

To understand whether Hx was modulated during EAE in mice, we first analyzed its expression in the liver of wild-type mice before and at day 7 after the immunization with MOG35–55 peptide. Hx mRNA levels increased in wild-type liver at day 7 postinjection (Fig. 1A). Moreover, we analyzed Hx in the serum of wild-type mice collected before and at days 2, 4, 7, 14, and 28 after MOG35–55 peptide immunization. As shown in Fig. 1B and 1C, Hx levels increased by day 2 after immunization and remained high until day 14. In addition, Hx expression was also upregulated in the CNS of wild-type mice with EAE (Supplemental Fig. 1). Thus, Hx is induced during murine EAE.

FIGURE 1.
FIGURE 1.

Hx is upregulated during EAE. (A) qRT-PCR analysis of Hx mRNA level in the liver of wild-type mice before and at day 7 postimmunization. Data represent mean ± SEM. **p < 0.01, n = 5 mice per group. (B) Representative Western blot of Hx level in the serum of two wild-type mice during EAE. IgG level was used as loading control. (C) Serum levels of Hx were quantified by densitometry. The results were representative of three independent experiments. Data represent the mean ± SEM. **p < 0.01. AU, Arbitrary units; RQ, relative quantity.

Loss of Hx exacerbates EAE disease

To investigate whether Hx modulates the autoimmune response, we examined the effects of Hx deficiency on the development of EAE. Disease progression was analyzed in wild-type and Hx−/− mice at different time points after immunization with MOG35–55 peptide. In wild-type mice, the initial signs of EAE were observed 11.6 ± 1.9 d after immunization (Fig. 2A, 2B). In contrast, in Hx−/− mice, the onset of EAE occurred on day 7.7 ± 1.3 (Fig. 2A, 2B). Moreover, 28 d after immunization, Hx−/− mice exhibited a higher EAE score compared with wild-type mice (3.30 + 0.60 versus 2.40 + 0.43; Fig. 2C). Consistent with these observations, Hx−/− mice displayed a massive inflammatory infiltration in the CNS compared with wild-type mice (Fig. 2D). Zones of demyelization in CNS, detectable where inflammatory infiltrates occurred, were more extended in Hx−/− mice than in wild-type mice (Fig. 2E). Among inflammatory cells, the number of CD4+ T cells was significantly higher in the CNS of Hx−/− mice than in that of wild-type animals (Fig. 3A). To better characterize CNS-infiltrated CD4 T cells, we isolated spinal cord and brain mononuclear cells from wild-type and Hx−/− mice at days 14 and 28 postimmunization, activated polyclonally with PMA, Ionomycin, and brefeldin A, and stained with an anti-CD4 Ab and anti–IL-17 or anti–INF-γ Abs. As shown in Fig. 3B–D, the number of CD4+ T cells producing IL-17 (Th17) in the spinal cord, as well as in the brain, was significantly increased in Hx−/− mice as compared with wild-type animals at both days 14 and 28, whereas that of CD4+ T cells producing IFN-γ (Th1) was comparable. Taken together, these results indicate that the loss of Hx results in a more severe EAE associated to a higher number of Th17-infiltrating cells in the CNS.

FIGURE 2.
FIGURE 2.

Loss of Hx exacerbates EAE disease in vivo. (A) Clinical scores of EAE in Hx−/− and wild-type mice, expressed as cumulative disease score over 28 d. Data represent mean ± SD of a representative experiment. ***p < 0.001, n = 4 for each group. (B) Day of the onset (score = 1) and (C) clinical score at day 28 postimmunization. Data represent mean ± SEM of 10 mice per each genotype pooled from 3 independent experiments. **p < 0.01, ***p < 0.001. (D) Representative sections of the spinal cord of a wild-type mouse and an Hx−/− mouse at day 28 postimmunization stained with H&E showing the massive inflammatory infiltration in Hx−/− animal. Inflammatory index reported on the right was calculated as reported in Materials and Methods. Data represent mean ± SEM. *p < 0.05, n = 4 mice per each genotype. (E) Representative sections of the spinal cord of a wild-type mouse and an Hx−/− mouse at day 28 postimmunization processed by immunohistochemistry with an anti-MBP Ab. (D and E) Original magnifications ×4 (left), ×20 (right).

FIGURE 3.
FIGURE 3.

EAE in Hx−/− mice is associated with an increased infiltration of Th17 cells. (A) Representative sections of the spinal cord of a wild-type mouse and an Hx−/− mouse at day 28 postimmunization processed by IFL with an anti–CD4-FITC Ab. The number of CD4+ cells, counted as reported in Materials and Methods (right panel). Original magnification ×200. Data represent mean ± SEM. *p < 0.05, n = 4 mice per each genotype. (B) Th17 and Th1 cells infiltrating the spinal cord and the brain of wild-type and Hx−/− mice at day 14 of the disease, detected by flow cytometry. Data represent mean ± SEM. *p < 0.05, ***p < 0.001, n = 4 mice per genotype. (C) Th17 and Th1 cells infiltrating the spinal cord and the brain of wild-type and Hx−/− mice at day 28 of the disease, detected by flow cytometry. Data represent mean ± SEM. *p < 0.05, **p < 0.01, n = 6 mice per genotype. (D) Representative dot plots are shown: numbers in the quadrants indicate the percentage of cytokine-secreting cells gated on CD4+ cells. The mean clinical score of mice used in the analyses is reported in (B) and (C).

Loss of Hx results in an expansion of Th17 cells

Because we observed a difference in the amount of Th17 cells infiltrating the CNS between Hx−/− and wild-type mice, the expansion of Th17 and Th1 cells during EAE in LN cells, SPCs, and PMBCs was assessed. LN cells, SPCs, and PMBCs were isolated from wild-type and Hx−/− mice at days 7 and 14 postimmunization, activated polyclonally with PMA, Ionomycin, and brefeldin A, and then stained intracellularly to detect the production of IL-17 and INF-γ. At day 7 of the disease, corresponding to the disease onset in Hx−/− mice, Hx−/− mice showed a higher percentage of Th17 cells in LNs and spleen compared with wild-type mice (Fig. 4A, 4B). By contrast, at day 14, the percentage of Th17 cells was higher in LNs and spleen of the wild-type counterpart. A significantly higher percentage of Th17 cells was also observed in PBMCs of Hx−/− mice compared with wild-type mice (Fig. 4C). No difference in the percentage of Th1 cells in LNs, spleen, and PBMCs between Hx−/− and wild-type mice was detected (Fig. 4A–C).

FIGURE 4.
FIGURE 4.

Loss of Hx results in an expansion of Th17 cell population. Ex vivo analysis of Th17 and Th1 cells in LN cells (A) and SPCs (B) of wild-type and Hx−/− mice at days 7 and 14 postimmunization and in PMBCs during the disease (C). Cells from wild-type (n = 5) and Hx−/− (n = 5) mice were stimulated in vitro with PMA and Ionomycin for 18 h, and IL-17 and IFN-γ quantified by flow cytometry analysis. Data represent mean ± SEM. *p < 0.05, ***p < 0.001. (D) Flow cytometry analysis of Th17 and Th1 cells and ELISA assay for IL-17 and INF-γ after in vitro stimulation with MOG35–55 peptide of SPCs isolated from wild-type and Hx−/− mice before and at days 7 and 14 postimmunization. Data represent mean ± SEM. *p < 0.05, **p < 0.01. (E) [3H]TdR uptake of SPCs isolated from wild-type and Hx−/− mice, and cultured in the absence or presence of different concentrations of MOG35–55 peptide. Results are expressed as the Δcpm.

Then the response of lymphocytes after in vitro stimulation with MOG35–55 was investigated. SPCs were isolated at day 0 and at days 7 and 14 after immunization, cultured for 10 d in the presence of 10 μg/ml MOG35–55 peptide, and then checked for the cytokine profile. As shown in Fig. 4D, SPCs isolated from Hx−/− mice at days 7 and 14 showed a higher percentage of MOG35–55–specific Th17 cells compared with wild-type cells. By contrast, SPCs from wild-type mice displayed a higher percentage of MOG35–55–specific Th1 cells compared with cells from Hx −/− mice. Dosage of IL-17 in the supernatants of cells cultured for 48 h with MOG35–55 peptide confirmed the difference between SPLs isolated from Hx−/− and wild-type mice (Fig. 4D). The percentage of MOG35–55–specific cells producing both IL-17 and IFN-γ was very low but significantly higher in Hx−/− mice than in wild-type controls (Supplemental Fig. 2). Analysis of T cell proliferation in the presence of different concentrations of MOG35–55 peptide demonstrated that cells isolated from the spleen of wild-type and Hx−/− mice had the same proliferative potential (Fig. 4E). These results indicate that Hx deficiency results in an unbalance between Th17 and Th1 cell populations, favoring Th17 differentiation.

Loss of Hx affects both the differentiative potential of CD4+ T cells and the expression of Th17-polarizing cytokines

Data reported in the previous section demonstrated an expansion of Th17 cell population in Hx−/− mice subjected to EAE. This might be because of a higher predisposition of naive CD4+ T cells of Hx−/− mice to differentiate toward Th17 lineage and/or to altered extrinsic factors promoting Th17 differentiation. To discriminate between these two possibilities, we evaluated the Th17 differentiative potential of CD4+ naive and memory T cells isolated from Hx−/− mice. As shown in Fig. 5A, naive CD4 T cells isolated from Hx−/− mice and activated in vitro with anti-CD3/CD28 mAbs were able to produce an enhanced amount of IL-17 compared with that produced by naive CD4+ cells from wild-type mice. This amount of IL-17 was further enhanced in the presence of Th17 polarizing cytokines alone or admixed with anti–IFN-γ neutralizing mAb, and it was higher compared with that produced by naive CD4+ from wild-type mice. By contrast, memory CD4 T cells from wild-type and Hx−/− mice produced similar amounts of IL-17 when stimulated with anti-CD3/CD28 mAb alone, whereas memory CD4 T cells from Hx−/− mice displayed a striking increase of IL-17 production compared with memory CD4 T cells from wild-type mice when activated in the “Th17 polarizing condition,” namely, IL-23 and anti–IFN-γ neutralizing mAb (Fig. 5B).

FIGURE 5.
FIGURE 5.

Loss of Hx results in enhanced differentiative potential toward Th17 lineage, as well as in an enhanced production of Th17-polarizing cytokines. (A) IL-17 production in cultures of CD4 naive T cells isolated from untreated wild-type and Hx−/− mice, and stimulated with polarizing cytokines (IL-6, TGF-β, and IL-23) alone or in combination with anti–IFN-γ mAb. Data represent mean ± SEM. *p < 0.05, **p < 0.01. (B) IL-17 production in cultures of CD4 memory T cells isolated from untreated wild-type and Hx−/− mice, and stimulated with IL-23 plus anti–IFN-γ mAb. Data represent mean ± SEM. ***p < 0.001. (C and D) qRT-PCR analysis of IL-6, IL-23, and IL-12 mRNA level in macrophages (C) and dendritic cells (D) isolated from the spleen of wild-type and Hx−/− mice before and at day 7 postimmunization. Results were representative of three independent experiments. Data represent mean ± SEM. *p < 0.05, **p < 0.01, n = 4 mice/genotype for each experimental point. (E). IL-6 serum levels in wild-type and Hx−/− mice before and at days 7 and 14 postimmunization. IL-6 (pg/ml) was measured by ELISA assay. Data represent the mean ± SEM. (F) qRT-PCR analysis of IL-6 and IL-23 mRNA level in the brain of wild-type and Hx−/− mice at day 28 postimmunization. Data represent mean ± SEM. *p < 0.05, n = 4 mice per genotype.

Then we evaluated the expression of the cytokines that drive Th17 differentiation (IL-6 and IL-23) or Th1 differentiation (IL-12) in macrophages (F4/80+ cells) and dendritic cells (CD11c+ cells) of Hx−/− and wild-type mice with EAE. IL-6 and IL-23 were strongly induced during disease in macrophages and dendritic cells isolated from both wild-type and Hx−/− mice, but this induction was significant higher in cells from Hx−/− animals (Fig. 5C, 5D). IL-12 expression did not change during disease and was comparable between Hx−/− and wild-type mice (Fig. 5C, 5D). Consistent with these data, the amount of IL-6 in the sera was higher in Hx−/− mice than in wild-type controls (Fig. 5E). Finally, in the brain of Hx−/− mice, IL-6 and, to a lesser extent, IL-23 mRNA were significantly higher than in that of wild-type animals at day 28 postimmunization (Fig. 5F). These results indicate that both intrinsic and extrinsic factors may contribute to the expansion of Th17 cells in Hx−/− mice.

Injection of purified Hx in Hx−/− mice rescues the disease severity and the expansion of Th17 cells

To further assess the potential protective role of Hx in the development of EAE, we injected a single dose of purified human Hx in Hx−/− mice at day 0 of EAE and evaluated disease progression and Th17 expansion. As shown in Fig. 6A, exogenous Hx was detectable by Western blotting in the serum of Hx−/− mice only until day 2 of EAE.

FIGURE 6.
FIGURE 6.

Injection of purified Hx in Hx−/− mice rescues the disease severity and the expansion of Th17 cells. (A) Representative Western blot of Hx in the serum of three Hx−/− mice at different times of EAE. Ponceau red staining was used as loading control. (B) Clinical scores of EAE in wild-type, Hx−/−, and Hx-injected Hx−/− mice, expressed as cumulative disease score over 28 d. Data represent mean ± SD of a representative experiment. *p < 0.05, ***p < 0.001, #p < 0.05, ###p < 0.001; n = 4 for each group. (C) Ex vivo analysis of Th17 and Th1 cells in PMBCs of wild-type, Hx−/−, and Hx-injected Hx−/− mice at days 0, 7, 14, 21, and 28 postimmunization. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ##p < 0.01. (D) Th17 and Th1 cells infiltrating the spinal cord and the brain of wild-type, Hx−/−, and Hx-injected Hx−/− mice at day 28 of the disease, detected by flow cytometry. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n = 4 mice per genotype. (E) qRT-PCR analysis of IL-6 and IL-23 mRNA level in the brain of wild-type, Hx−/−, and Hx-injected Hx−/− mice at day 28 postimmunization. Data represent mean ± SEM. *p < 0.05, **p < 0.01, n = 4 mice per genotype.

A single dose of Hx was able to rescue the EAE severity in Hx−/− mice (Fig. 6B). The amount of circulating Th17 cells (Fig. 6C) during disease progression as well as the amount of infiltrated Th17 cells in the spinal cord and in the brain (Fig. 6D) at day 28 of EAE were significantly reduced in Hx-injected Hx−/− mice and were comparable with those of wild-type animals. As expected, the amount of circulating (Fig. 6C) and infiltrated (Fig. 6D) Th1 cells were not affected by Hx injection.

As shown in Fig. 6E, the expression of IL-6 and IL-23 in the CNS at day 28 of EAE was reduced in Hx-injected Hx−/− mice compared with untreated Hx−/− animals and was similar to that of wild-types.

These data indicate that a single dose of Hx in Hx−/− mice was able to limit Th17 cell expansion, thus controlling EAE severity.

Discussion

In this study, we demonstrated that Hx affects the severity of EAE by limiting the expansion of the Th17 cell population. This conclusion comes from the observation that Hx−/− mice developed a more severe and earlier EAE than wild-type animals, associated with a higher amount of circulating and infiltrating Th17 cells autoreactive against myelin. A direct effect of Hx in controlling EAE development is further demonstrated by the “rescue experiment” in which Hx administration in Hx−/− mice before EAE induction has been shown to limit Th17 cell expansion and attenuate disease severity.

Both Th1 and Th17 cells have been defined as effector T cell subsets involved in the pathogenesis of the EAE, but Th17 cells are considered to be more pathogenic than Th1 cells (24). Autoantigen-specific Th17 cells have been shown to break and transmigrate across the blood–brain barrier, and in the CNS induce inflammation by neutrophil recruitment (20) and killing of neurons (25). Thus, the more severe EAE developed by Hx−/− mice may be ascribed to the higher amount of Th17 cells infiltrating the CNS and circulating in the periphery of Hx−/−mice compared with wild-type controls.

The Th17-driven EAE developed by Hx−/− mice is due to both intrinsic and extrinsic factors. We showed that CD4+ T cells isolated from Hx−/− mice polarize more efficiently toward Th17 lineage than cells isolated from wild-types controls. The absence of Hx seems to predispose naive CD4 cells to differentiate into Th17 lineage as CD4+ naive cells from Hx−/−mice produce an enhanced amount of IL-17 compared with the same cells from wild-type mice. By contrast, activated memory CD4 cells produce an equal amount of IL-17 in Hx−/− and wild-type mice, and this is increased in memory CD4 T cells from Hx−/− mice only when activated in the presence of IL-23 and in the absence of IFN-γ. These data suggest that the absence of Hx favors the differentiation of naive CD4+ cells toward Th17 lineage and enhances the stabilization and expansion of memory Th17 T cells by IL-23, particularly when IFN-γ is neutralized.

The enhanced potential of CD4+ naive T cells to differentiate toward Th17 lineage might be because of the reduced sensitivity to IFN-γ of lymphocytes of Hx−/− mice as we previously demonstrated that, in the absence of Hx, T cells displayed a reduced IFN-γ–STAT1–dependent phosphorylation (26). IFN-γ signaling plays a key role in Th17 differentiation and function (27). It was already demonstrated that the neutralization of IFN-γ effect by a specific mAb to IFN-γ or to the IFN-γR1 chain was required for the massive development of activated T lymphocytes cultured in the presence of IL-23 into Th17 cells (28). The Hx-mediated control of CD4+ T cell responsiveness to IFN-γ might be related to the Hx ability to modulate iron availability within cells (1), thus affecting the expression of Transferrin receptor that has already been shown to regulate INFγ-R2 expression at the plasma membrane (29, 30). Because Transferrin receptor expression was lower in T lymphocytes from Hx−/− mice both in normal condition (26) and after EAE induction (data not shown), developing T cells from MOG35–55–immunized Hx−/− mice showed refractoriness to IFN-γ and were more prone to polarize into Th17 cells.

Other than the intrinsic higher capability of CD4 T cells from Hx−/− mice to differentiate toward Th17 lineage, our data indicate that also extrinsic factors may contribute to Th17 cell expansion in Hx−/− mice. We showed that macrophages and dendritic cells isolated from Hx−/− mice with EAE produced higher amounts of IL-6 and IL-23 than cells isolated from wild-type animals. IL-6 is a key stimulator for Th17 differentiation and, accordingly, IL-6–deficient mice are protected from EAE (31). IL-23 produced by APCs stabilizes the Th17 phenotype by promoting secretion of IL-17A, IL-17F, and IL-22, and by helping Th17 cells to acquire effector function (32). Of note, mice deficient for IL-23 are completely resistant to EAE induction (33). The increase of IL-6 and IL-23 in Hx−/− mice could be related to the Hx ability to modulate cytokine production (12). It has already been shown that Hx was able to downregulate the secretion of IL-6 and TNF-α from macrophages in response to LPS in a manner that is distinct from its function as a heme scavenger (12). This might occur by limiting TLR4- and TLR2-induced cytokine production. TLR signals, induced by pathogen-associated molecular patterns contained in the CFA, are necessary for the induction of EAE and influence the CD4 Th cell–dependent inflammation that causes EAE (34). Absence of TLR4 exacerbates EAE and is associated with increased expression of IL-6 and IL-23 in splenic dendritic cells and increased frequency of Th17 cells (35). By contrast, deficiency of TLR2 in CD4+ T cells substantially impaired Th17 response in vivo and the pathogenesis of EAE (36). In this article, we show that Hx is able to modulate not only the secretion of IL-6, but also that of IL-23, an effect similar to that obtained by TLR4 blockade (35), confirming the emerging hypothesis that Hx may modulate the response to TLR4 and TLR2 ligands, thus playing a role in acute and chronic inflammatory diseases (12, 13). The elucidation of the mechanisms by which Hx could modulate TLR activity needs more studies.

Hx was reported to be produced in the CNS by ependymal cells, neurons, and glial cells (1, 2, 4, 5). In the CNS, Hx controls iron accumulation in oligodendrocytes and affects their differentiation (4, 37). Accordingly, the expression of the MBP along with the density of myelinated fibers in the basal ganglia and in the motor and somatosensory cortex of Hx−/− mice was strongly reduced compared with wild-type controls (37). Thus, it is possible that the exacerbated demyelination we observed in Hx−/− mice with EAE was due not only to massive Th17 cell infiltration, but also to the impairment of Hx-deficient oligodendrocytes to respond to exogenous insults. This conclusion is consistent with data showing that iron deposition in the brain contributes to MS pathogenesis (38). Thus, Hx would control, on one hand, systemic signals driving Th17 differentiation and, on the other hand, local signals in CNS affecting oligodendrocyte function.

Hx has been reported to be induced in pediatric MS (14). Our data on Hx−/− mice with EAE may give an explanation to such induction. Hx upregulation is expected to contribute to the inflammatory response thus controlling the severity of the disease. Moreover, by controlling T cell responsiveness to IFN-γ, Hx induction might limit Th17 cell differentiation. Results obtained by injecting purified Hx in Hx−/− mice strongly support these conclusions. Finally, the previously reported observation that Hx expression in CNS controls oligodendrocyte function suggests that all of these factors may contribute to disease establishment and progression. Thus, Hx administration may be beneficial in clinical settings, such as autoimmune and inflammatory diseases, characterized by dysregulation of T cell differentiation.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • ↵1 S.R. and G.I. equally contributed to this work.

  • ↵2 F.N. and E.T. equally contributed to this work.

  • This work was supported by grants from the Federazione Italiana Sclerosi Multipla (2007/R/10 and 2009/R19), Ministero dell’Istruzione, dell’Università e della Ricerca, Progetti di Rilevante Interesse Nazionale; Regione Piemonte: Ricerca Industriale e Sviluppo Precompetitivo, Ricerca Industriale “Converging Technologies”, Progetti Strategici su Tematiche di Interesse Regionale o Sovra Regionale, and Progetti di Ricerca Sanitaria Finalizzata.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    EAE
    experimental autoimmune encephalomyelitis
    Hx
    hemopexin
    Hx−/−
    Hx knockout
    LN
    lymph node
    MBP
    myelin basic protein
    MOG
    myelin oligodendrocyte glycoprotein
    MS
    multiple sclerosis
    qRT
    quantitative real-time
    SPC
    spleen cell
    TdR
    thymidine.

  • Received November 6, 2012.
  • Accepted September 20, 2013.

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