EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12334 ..................................................................................................................................................................
Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis C. Yang, D. He, C. Yin & J. Tan
Abstract Neurology Department, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China
Received 12 February 2015; Accepted in revised form 9 June 2015 Correspondence to: J. Tan, The Eastern Part of Hulan Road, Xinxiang 453003, Henan Province, China. E-mail: [email protected]
Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T-cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL-17-producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC-T-cell coculture system, SiIRF4treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.
Introduction Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T-cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability [1]. Effector CD4+ T cells of Th1 and Th17 subsets are found in MS lesion and can mediate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of Th1 and Th17 cytokines, IFN-c and IL-17 is detected in MS lesions [2]. EAE can be induced by the adoptive transfer of CNS antigen-reactive Th1 and Th17 cells [3–5]. Neutralization of IL-17 by treatment with antiIL-17 antibodies has been demonstrated to reduce motor symptoms of EAE [6]. Interferon regulatory factors (IRFs), consisting of nine members, were originally identified as transcriptional regulators of type I IFNs. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function [7, 8]. Recent study demonstrated that IRF4 was
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critical for the generation of IL-17-producing Th17 cells [9]. Moreover, previous study by Magdalena Huber et al. [10] indicated that IRF4 was pivotal for differentiation of Tc17 cells in vitro and in vivo during CNS autoimmunity. However, the effect of IRF4 on EAE needs to be further investigated. In our current study, we demonstrate a crucial role for IRF4 in the development of EAE. Inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC-T-cell coculture system, SiIRF4treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression.
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346 Inhibition of IRF4 suppresses Th1 and Th17 C. Yang et al. ..................................................................................................................................................................
Materials and methods Animals. Male C57BL/6 mice, all aged 6–8 weeks, were purchase from Center of Xinxiang Medical University. Mice were maintained in a specific pathogen-free condition. All experimental procedures were examined and approved by the Institutional Animal Care and Use Committee at Xinxiang Medical University. Induction of EAE. Induction of EAE was conducted as described previously [11]. EAE was induced in C57BL/6 mice by immunization with 250 lg of MOG35-55 (myelin oligodendrocyte glycoprotein (MOG)). All peptides were dissolved in complete Freund’s adjuvant (CFA) containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra. At day 1 and 48 h after immunization, C57BL/ 6 mice were injected with 500 ng of pertussis toxin in PBS, intraperitoneally. Clinical assessment of EAE was performed after disease induction by the following criteria: 0, no disease; 1, tail paralysis; 2, hindlimb weakness or partial paralysis; 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death. A total of 100 lg of IRF4 siRNA or control siRNA was administrated via caudal vein for every 2 days from day 5 to day 15 after immunization [12]. Sequences of siRNAs were as follows: control siRNA, 50 -UAAGGCUAUGAAGA GAUACUU-30 ; and IRF4 siRNA, 50 -GGACACAC CUAUGAUGUUAUU-30 . These siRNAs were obtained from Dharmacon. For passive EAE experiments, donor mice were immunized with MOG/CFA in the same fashion as when inducing EAE, but no pertussis toxin was administered. Spleens and draining lymph nodes were collected 10 days later, single-cell suspensions were prepared, and RBCs were lysed. Cells (6 9 106 cells per ml) were cultured in RPMI1640 medium with 40 lg/ml of MOG35–55 peptide and 10 ng/ml of recombinant mouse IL-12 (R&D Systems, Houston, USA). After 3 days of culture, cells were harvested and T cells were isolated by negative selection using Dynabeads (Invitrogen, Carlsbad, CA, USA). Recipient mice were irradiated sublethally (500 cGy) and received 5 9 106 cells intravenously [13]. Histopathology. Mice were humanely euthanatized, and spinal cord was immersed in 4% paraformaldehyde for 48 h at 4 °C and paraffin-embedded. Six-micrometre-thick longitudinal sections of the spinal cord were cut to include the majority of the length of the spinal cord (from cervical to lumbar regions), containing both grey and white matters. Prior to staining, sections were deparaffinized in xylene (2 9 5 min), hydrated in graded ethanol (2 9 5 min in 100%, 5 min in 85%, 5 min 70%) to distilled water and finally rinsed in PBS. Spinal cord sections were stained with haematoxylin/eosin (Sigma, St. Louis, MO, USA) and with Luxol fast blue (American Mastertech, CA, Houston, USA) following the manufacturer’s recommendations to assess cell infiltration and demyelination, respectively. Four to five different sections
per mouse were analysed under light microscopy. At least 10 randomly distributed 409 fields within the white matter of the spinal cord were captured for each section. To determine the demyelination, the white matter was outlined automatically using the wand tool. The areas covered by the Luxol fast blue stain were quantified using Image-Pro Plus software (Media Cybernetics, MD, Houston, USA). The demyelination was determined as follows: (Total white matter area-Luxol fast blue area)/Total white matter area 9 100%. Preparation and evaluation of CNS cells. Brains of mice, which were perfuse with cold PBS, were dissected and incubated in 2.5 mg/ml collagenase D for 30 min at 37 °C. Single-cell suspensions were prepared. Cells were washed in RPMI 1640 medium, and mononuclear cells were isolated using a discontinuous Percoll gradient [14]. Flow cytometry analysis. Isolated mononuclear cells from spleen, LN and CNS were cultured in 24-well plates in RPMI1640 medium supplemented with 10% FBS, 200 ng/ml phorbol myristate acetate (PMA; Sigma), 400 ng/ml ionomycin and brefeldin A (Sigma) for 4 h. The cells were harvested and stained with FITC-antihuman CD4 at 4 °C for 30 min. After washing with PBS, the cells were fixed, permeabilized and stained with APCanti-IL-17, APC-anti-IFN-c or APC-anti-Foxp3 (eBioscience, San Diego, CA, USA) at 4 °C for 30 min. The frequencies of Th17 and Th1 cells were analysed using a FACS cytometer equipped with CellQuest software (BD Pharmingen, New York, USA). Cytokine analysis. Spleens, LN cells and CNS mononuclear cells were harvested and pooled from EAE mice, and single-cell suspensions were prepared. Cells were cultured at 5 9 107 cells/well in 24-well U-bottom plates with 10 mg/ml MOG35-55 peptide in complete RPMI 1640 medium. For ELISA analysis, supernatants were harvested at 24 h of culture. The concentrations of indicated cytokines were measured by quantitative capture ELISA, according to the guidelines of the manufacturers. Adoptive transfer of DCs. DCs were isolated from the spleens of 8- to 10-week-old mice treated with SiIRF4 or control for 10 days. DCs were enriched by magnetic cell sorting using CD11c+ magnetic beads (Miltenyi, Berlin, Germany), resulting in a general purity of at least 95%. Recipient C57BL/6 mice were injected intravenously (i.v.) with 5 9 106 SiIRF4- or control-treated DCs and then immunized with MOG35–55 immediately [15]. Real-time PCR. Total RNA was extracted from cultured cells using Trizol (Invitrogen) and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in duplicate, and changes in
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C. Yang et al. Inhibition of IRF4 suppresses Th1 and Th17 347 ..................................................................................................................................................................
Figure 1 SiIRF4 ameliorates EAE development. (A) Clinical scores for mice treated with SiIRF4 after the onset of EAE; (B) At day 25 after immunization, CNSinfiltrating CD4+ and CD8+ T cells were isolated, analysed by flow cytometry and absolute numbers were calculated; (C) Indicating the demyelination rate at day 25 after immunization; (D) MOG-primed T cells from control or SiIRF4-treated mice were transferred into sublethally irradiated mice. The mice were scored for clinical signs of EAE and the mean clinical score was calculated by averaging the scores of all mice. *P < 0.05. N = 6. The experiments were repeated three times in each animal.
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relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. Western blotting. The protein levels were determined by Western blotting. Protein extracted from cells or tissue was separated on 10% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). After being blocked with 5% nonfat milk in TBS for 3 h, the membranes were incubated with indicated primary antibodies (0.2 lg/ml) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000) for 3 h. b-Actin was used as a loading control for comparison between samples. Immunoreactive bands were quantified with densitometry using Image J software (NIH, Maryland, USA). Statistical analysis. All data were presented as means SEM. ANOVA was used for statistical analysis with P < 0.05 being considered statistically significant. Data were analysed using Prism software (GraphPad Software, Inc., Beijing, China).
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Results SiIRF4 treatment ameliorates EAE development
In this study, we used a mouse model of EAE to evaluate the therapeutic effect of IRF4 inhibition with SiIRF4. Results showed that SiIRF4 treatment decreased the EAE scores and enhanced clinical recovery from EAE (Fig. 1A). Analysis of CNS-infiltrating mononuclear showed that mice treated with control PBS developed prominent inflammatory infiltration, whereas SiIRF4 treatment
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Figure 2 In vivo generation of Th17 cells and Th1 cells in EAE inhibited by SiIRF4. (A) Intracellular staining of IFN-c, IL-17 or Foxp3 in spleen cells, LN cells and CNS-infiltrating mononuclear cells. The frequencies of Th17 and Th1 cells were analysed using a FACS cytometer equipped with CellQuest software (BD Pharmingen); (B) Indicating the absolute number of IFN-c+, IL-17+ or Foxp3+ cells in CNS. *P < 0.05. N = 6.
348 Inhibition of IRF4 suppresses Th1 and Th17 C. Yang et al. .................................................................................................................................................................. A
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showed decreased CD4+ and CD8+ leucocytes infiltration and significantly alleviated demyelination (Fig. 1B–C). Next, we transferred T cells from MOG-immunized control or SiIRF4-treated mice into wild-type recipients. Mice that received SiIRF4-treated T cells displayed lower EAE scores and better clinical recovery from EAE (Fig. 1D). SiIRF4 inhibits Th1 and Th17 cell differentiation in EAE
We examined the presence of IL-17 (Th17), interferon-c (IFN-c) (Th1) or Foxp3 (Treg)-producing CD4+ T cells during EAE in lymph nodes, spleen and the CNS. Mice were harvested on day 25 after immunization with MOG35-55. Intracellular cytokine staining showed that the frequencies of both Th1 and Th17 cells in the splenocytes, lymph nodes cells and CNS mononuclear cells were lower in SiIRF4-treated mice than that in controls (Fig. 2A). The frequencies of Foxp3+ Tregs in the splenocytes, lymph nodes cells and CNS mononuclear cells were much higher in SiIRF4-treated mice than that in controls (Fig. 2A). The absolute numbers of Th1 and Th17 cells in CNS were also markedly smaller in SiIRF4-treated mice than that in controls (Fig. 2B). And there was no significant difference in the number of Treg infiltration in CNS between the two groups (Fig. 2B). CD4+ T cells in control-treated mice underwent cell divisions in vitro with MOG35-55, whereas CD4+ T cells in SiIRF4-treated mice had a reduced proliferation (Fig. 3A). In parallel, CCK8 assays via splenocytes produced similar differences (Fig. 3B). Supernatants were harvested at 24 h of culture and analysed by
Figure 3 SiIRF4 affects inflammatory T cell development in vitro during EAE. (A) To investigate the effect of SiIRF4 on T cell proliferation in vitro, the splenocytes harvested from mice 25 days after EAE induction were labelled with carboxyfluorescein succinimidyl amino ester (CFSE). CFSE loss by CD4+ proliferating cells from the two groups was assayed by flow cytometry after restimulation with MOG35-55 (10 mg/ml) for 72 h. The average frequency of CD4+ proliferating cells is presented; (B) The harvested splenocytes was also restimulated with MOG35-55 (10 mg/ml) for 72 h, and added CCK-8 solution incubated at 37 °C for the final 4 h. Cell proliferation was evaluated by stimulation index and the average frequency is presented; (C) Indicates the production of IFN-c and IL-17A by splenocytes, LN cells and CNS-infiltrating mononuclear cells from control and SiIRF4treated EAE mice. *P < 0.05. N = 6.
ELISA. SiIRF4-treated mice showed decreased production of both IL-17A and IFN-c compared to their controls (Fig. 3C). SiIRF4 attenuates EAE via a direct effect on DC
Myeloid-derived suppressive cells (MDSCs) are a heterogeneous group of myeloid cells comprised of hematopoietic progenitor cells and precursors of macrophages, granulocytes and dendritic cells (DCs) [16]. Recently, the close relationship between IRF4 and DCs has been demonstrated [17, 18]. Therefore, we tested whether SiIRF4 regulates the expression of Th1- and Th17polarizing cytokine by DCs. For this, we cultured CD4+ T cells with CD11c+ DCs from control- and SiIRF4treated mice and analysed cytokine expression. In our DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells (Fig. 4A). To investigate whether SiIRF4-induced myeloid DCs could modulate clinical responses in vivo, we isolated CD11c+ DCs from SiIRF4-treated mice, adoptively transferred them into recipient mice and then immunized with MOG35-55. These CD11c+ DCs ameliorated EAE development in recipient mice (Fig. 4B–C). SiIRF4 alters cytokine expression in DCs
DCs are a major source of different cytokines important for initiation and regulation of an immune response. Therefore, we analysed the cytokines expression by Q-PCR. As
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Figure 4 SiIRF4 attenuates EAE via a direct effect on DC. (A) CD4+ T cells isolated from C57BL/6 mice were cultured with CD11c+ DCs derived from control and SiIRF4-treated mice on day 25 after EAE induction. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IFN-c and IL-17; (B) CD11c+ cells from SiIRF4-treated donor mice attenuates EAE. 5 9 106 purified splenic CD11c+ cells from mice treated with SiIRF4 or control were injected i.v. into recipient C57BL/6 mice and then immunized with MOG35–55. (C) At day 25 after immunization, CNS-infiltrating CD4+ and CD8+ T cells were isolated and analysed by flow cytometry and absolute numbers were calculated. *P < 0.05. N = 6.
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Figure 5 SiIRF4 alters cytokine expression in DCs. CD11c+ DCs were isolated from SiIRF4- or control-treated naive (unimmunized) mice, and the expression of TNF-a, IL-1b, IL-6, IL-12/23 (p40), IL-10 and CD80 was analysed by Q-PCR (A) and Western blotting (B). *P < 0.05. N = 6.
shown in Fig. 5, CD11c+ DCs from SiIRF4-treated naive (unimmunized) mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. We also found that SiIRF4 treatment resulted in significant reduction in the expression of CD80 (Fig. 5).
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Discussion Our current study demonstrates a crucial role for IRF4 in the development of EAE. Inhibition of IRF4 with SiIRF4 decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our
350 Inhibition of IRF4 suppresses Th1 and Th17 C. Yang et al. .................................................................................................................................................................. DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. CD4+ Th cells play an important role in the adaptive immune system by activating and directing other immune cells. There are at least four distinct Th cell subsets (Th1, Th2, Th17 and regulatory T cells), which have been demonstrated to control immune response [19]. Both Th1 and Th17 cells have been shown in clinical and preclinical studies to be critically involved in the pathogenesis of autoimmune diseases including MS, rheumatoid arthritis and inflammatory bowel disease, whereas Th2 cells are implicated in asthma and allergy when aberrantly stimulated [20, 21]. IFN-c-producing Th1 and IL-17-producing Th17 cells are key proinflammatory mediators of cellular immunity that underlie crucial events during development of EAE. Both MS and EAE are thought to be initiated by myelin-reactive CD4+ T cells that produce IFN-c and IL-17 [22–24]. The Th1 lineage of cytokine can help Th17 cells invade the brain and spinal cord, thus triggering EAE [25, 26]. The high percentage of Th17 cells has an impact on the inflammation in the brain and the severity of disease [27]. Treg cells have been demonstrated to be capable of controlling CNS autoimmunity in several EAE models. Several studies described that the transfer of CD25+ Treg ameliorated EAE symptoms [28–30]. This study showed that the attenuation of EAE in SiIRF4-treated mice is associated with decreased expression of IL-17 and IFN-c and increased expression of Foxp3 in the peripheral lymphoid organs and CNS. The need for parenchymal APC is based on the fundamental immunological principle of reactivation for CD4+ T-cell effector function within the target tissue. A role for DC in directing T-cell transit from the perivascular space in postcapillary venules has been proposed [31, 32]. Recently, DCs have been demonstrated as a therapeutic target in MS and EAE [33]. CD11c+ DCs have been shown to be sufficient to initiate this autoimmune demyelinating disorder [28]. Furthermore, antigen presentation by myeloid DCs has been implicated in driving progression of relapsing EAE [34]. In our DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Furthermore, we isolated CD11c+ DCs from SiIRF4-treated mice, adoptively transferred them into recipient mice and then immunized with MOG35-55. The results showed that these CD11c+ DCs ameliorated EAE in recipient mice. All of these indicated that the effect of SiIRF4 on Th1 and Th17 cell differentiation may be mediated via modulation of DCs.
Cytokines secreted by DCs are critical messengers that control T-cell activation and differentiation. CD11c+ DCs from SiIRF4-treated mice displayed significantly reduced production of the pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. These proinflammatory cytokines have been shown to promote CNS tissue damage in EAE [35, 36]. IL-1b is required for Th17 differentiation and in mice; it synergizes with IL-6 and IL-23 in induction of Th17 cells [37]. In addition to its potential direct toxicity, TNF-a perpetuates the inflammatory process through recruitment of cells [38]. IL-10 is an anti-inflammatory cytokine with paracrine and autocrine effects [39]. We conclude that SiIRF4 reduces the Tcell stimulatory capacity of DCs, which has been shown to be necessary for the survival of encephalitogenic T cells in the effector phase of the immune response [31]. In summary, our study provides evidence that SiIRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE. Inhibition of IRF4 with SiIRF4 decreases EAE scores and leucocyte infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. And this effect was via a direct regulation of DCs. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in immunoregulation, IRF4 may be an effective target in attenuating MS.
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Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis C. Yang, D. He, C. Yin & J. Tan
Abstract Neurology Department, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China
Received 12 February 2015; Accepted in revised form 9 June 2015 Correspondence to: J. Tan, The Eastern Part of Hulan Road, Xinxiang 453003, Henan Province, China. E-mail: [email protected]
Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T-cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL-17-producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC-T-cell coculture system, SiIRF4treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.
Introduction Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T-cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability [1]. Effector CD4+ T cells of Th1 and Th17 subsets are found in MS lesion and can mediate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of Th1 and Th17 cytokines, IFN-c and IL-17 is detected in MS lesions [2]. EAE can be induced by the adoptive transfer of CNS antigen-reactive Th1 and Th17 cells [3–5]. Neutralization of IL-17 by treatment with antiIL-17 antibodies has been demonstrated to reduce motor symptoms of EAE [6]. Interferon regulatory factors (IRFs), consisting of nine members, were originally identified as transcriptional regulators of type I IFNs. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function [7, 8]. Recent study demonstrated that IRF4 was
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critical for the generation of IL-17-producing Th17 cells [9]. Moreover, previous study by Magdalena Huber et al. [10] indicated that IRF4 was pivotal for differentiation of Tc17 cells in vitro and in vivo during CNS autoimmunity. However, the effect of IRF4 on EAE needs to be further investigated. In our current study, we demonstrate a crucial role for IRF4 in the development of EAE. Inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC-T-cell coculture system, SiIRF4treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression.
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Materials and methods Animals. Male C57BL/6 mice, all aged 6–8 weeks, were purchase from Center of Xinxiang Medical University. Mice were maintained in a specific pathogen-free condition. All experimental procedures were examined and approved by the Institutional Animal Care and Use Committee at Xinxiang Medical University. Induction of EAE. Induction of EAE was conducted as described previously [11]. EAE was induced in C57BL/6 mice by immunization with 250 lg of MOG35-55 (myelin oligodendrocyte glycoprotein (MOG)). All peptides were dissolved in complete Freund’s adjuvant (CFA) containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra. At day 1 and 48 h after immunization, C57BL/ 6 mice were injected with 500 ng of pertussis toxin in PBS, intraperitoneally. Clinical assessment of EAE was performed after disease induction by the following criteria: 0, no disease; 1, tail paralysis; 2, hindlimb weakness or partial paralysis; 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death. A total of 100 lg of IRF4 siRNA or control siRNA was administrated via caudal vein for every 2 days from day 5 to day 15 after immunization [12]. Sequences of siRNAs were as follows: control siRNA, 50 -UAAGGCUAUGAAGA GAUACUU-30 ; and IRF4 siRNA, 50 -GGACACAC CUAUGAUGUUAUU-30 . These siRNAs were obtained from Dharmacon. For passive EAE experiments, donor mice were immunized with MOG/CFA in the same fashion as when inducing EAE, but no pertussis toxin was administered. Spleens and draining lymph nodes were collected 10 days later, single-cell suspensions were prepared, and RBCs were lysed. Cells (6 9 106 cells per ml) were cultured in RPMI1640 medium with 40 lg/ml of MOG35–55 peptide and 10 ng/ml of recombinant mouse IL-12 (R&D Systems, Houston, USA). After 3 days of culture, cells were harvested and T cells were isolated by negative selection using Dynabeads (Invitrogen, Carlsbad, CA, USA). Recipient mice were irradiated sublethally (500 cGy) and received 5 9 106 cells intravenously [13]. Histopathology. Mice were humanely euthanatized, and spinal cord was immersed in 4% paraformaldehyde for 48 h at 4 °C and paraffin-embedded. Six-micrometre-thick longitudinal sections of the spinal cord were cut to include the majority of the length of the spinal cord (from cervical to lumbar regions), containing both grey and white matters. Prior to staining, sections were deparaffinized in xylene (2 9 5 min), hydrated in graded ethanol (2 9 5 min in 100%, 5 min in 85%, 5 min 70%) to distilled water and finally rinsed in PBS. Spinal cord sections were stained with haematoxylin/eosin (Sigma, St. Louis, MO, USA) and with Luxol fast blue (American Mastertech, CA, Houston, USA) following the manufacturer’s recommendations to assess cell infiltration and demyelination, respectively. Four to five different sections
per mouse were analysed under light microscopy. At least 10 randomly distributed 409 fields within the white matter of the spinal cord were captured for each section. To determine the demyelination, the white matter was outlined automatically using the wand tool. The areas covered by the Luxol fast blue stain were quantified using Image-Pro Plus software (Media Cybernetics, MD, Houston, USA). The demyelination was determined as follows: (Total white matter area-Luxol fast blue area)/Total white matter area 9 100%. Preparation and evaluation of CNS cells. Brains of mice, which were perfuse with cold PBS, were dissected and incubated in 2.5 mg/ml collagenase D for 30 min at 37 °C. Single-cell suspensions were prepared. Cells were washed in RPMI 1640 medium, and mononuclear cells were isolated using a discontinuous Percoll gradient [14]. Flow cytometry analysis. Isolated mononuclear cells from spleen, LN and CNS were cultured in 24-well plates in RPMI1640 medium supplemented with 10% FBS, 200 ng/ml phorbol myristate acetate (PMA; Sigma), 400 ng/ml ionomycin and brefeldin A (Sigma) for 4 h. The cells were harvested and stained with FITC-antihuman CD4 at 4 °C for 30 min. After washing with PBS, the cells were fixed, permeabilized and stained with APCanti-IL-17, APC-anti-IFN-c or APC-anti-Foxp3 (eBioscience, San Diego, CA, USA) at 4 °C for 30 min. The frequencies of Th17 and Th1 cells were analysed using a FACS cytometer equipped with CellQuest software (BD Pharmingen, New York, USA). Cytokine analysis. Spleens, LN cells and CNS mononuclear cells were harvested and pooled from EAE mice, and single-cell suspensions were prepared. Cells were cultured at 5 9 107 cells/well in 24-well U-bottom plates with 10 mg/ml MOG35-55 peptide in complete RPMI 1640 medium. For ELISA analysis, supernatants were harvested at 24 h of culture. The concentrations of indicated cytokines were measured by quantitative capture ELISA, according to the guidelines of the manufacturers. Adoptive transfer of DCs. DCs were isolated from the spleens of 8- to 10-week-old mice treated with SiIRF4 or control for 10 days. DCs were enriched by magnetic cell sorting using CD11c+ magnetic beads (Miltenyi, Berlin, Germany), resulting in a general purity of at least 95%. Recipient C57BL/6 mice were injected intravenously (i.v.) with 5 9 106 SiIRF4- or control-treated DCs and then immunized with MOG35–55 immediately [15]. Real-time PCR. Total RNA was extracted from cultured cells using Trizol (Invitrogen) and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in duplicate, and changes in
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C. Yang et al. Inhibition of IRF4 suppresses Th1 and Th17 347 ..................................................................................................................................................................
Figure 1 SiIRF4 ameliorates EAE development. (A) Clinical scores for mice treated with SiIRF4 after the onset of EAE; (B) At day 25 after immunization, CNSinfiltrating CD4+ and CD8+ T cells were isolated, analysed by flow cytometry and absolute numbers were calculated; (C) Indicating the demyelination rate at day 25 after immunization; (D) MOG-primed T cells from control or SiIRF4-treated mice were transferred into sublethally irradiated mice. The mice were scored for clinical signs of EAE and the mean clinical score was calculated by averaging the scores of all mice. *P < 0.05. N = 6. The experiments were repeated three times in each animal.
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relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. Western blotting. The protein levels were determined by Western blotting. Protein extracted from cells or tissue was separated on 10% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). After being blocked with 5% nonfat milk in TBS for 3 h, the membranes were incubated with indicated primary antibodies (0.2 lg/ml) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000) for 3 h. b-Actin was used as a loading control for comparison between samples. Immunoreactive bands were quantified with densitometry using Image J software (NIH, Maryland, USA). Statistical analysis. All data were presented as means SEM. ANOVA was used for statistical analysis with P < 0.05 being considered statistically significant. Data were analysed using Prism software (GraphPad Software, Inc., Beijing, China).
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Results SiIRF4 treatment ameliorates EAE development
In this study, we used a mouse model of EAE to evaluate the therapeutic effect of IRF4 inhibition with SiIRF4. Results showed that SiIRF4 treatment decreased the EAE scores and enhanced clinical recovery from EAE (Fig. 1A). Analysis of CNS-infiltrating mononuclear showed that mice treated with control PBS developed prominent inflammatory infiltration, whereas SiIRF4 treatment
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Figure 2 In vivo generation of Th17 cells and Th1 cells in EAE inhibited by SiIRF4. (A) Intracellular staining of IFN-c, IL-17 or Foxp3 in spleen cells, LN cells and CNS-infiltrating mononuclear cells. The frequencies of Th17 and Th1 cells were analysed using a FACS cytometer equipped with CellQuest software (BD Pharmingen); (B) Indicating the absolute number of IFN-c+, IL-17+ or Foxp3+ cells in CNS. *P < 0.05. N = 6.
348 Inhibition of IRF4 suppresses Th1 and Th17 C. Yang et al. .................................................................................................................................................................. A
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showed decreased CD4+ and CD8+ leucocytes infiltration and significantly alleviated demyelination (Fig. 1B–C). Next, we transferred T cells from MOG-immunized control or SiIRF4-treated mice into wild-type recipients. Mice that received SiIRF4-treated T cells displayed lower EAE scores and better clinical recovery from EAE (Fig. 1D). SiIRF4 inhibits Th1 and Th17 cell differentiation in EAE
We examined the presence of IL-17 (Th17), interferon-c (IFN-c) (Th1) or Foxp3 (Treg)-producing CD4+ T cells during EAE in lymph nodes, spleen and the CNS. Mice were harvested on day 25 after immunization with MOG35-55. Intracellular cytokine staining showed that the frequencies of both Th1 and Th17 cells in the splenocytes, lymph nodes cells and CNS mononuclear cells were lower in SiIRF4-treated mice than that in controls (Fig. 2A). The frequencies of Foxp3+ Tregs in the splenocytes, lymph nodes cells and CNS mononuclear cells were much higher in SiIRF4-treated mice than that in controls (Fig. 2A). The absolute numbers of Th1 and Th17 cells in CNS were also markedly smaller in SiIRF4-treated mice than that in controls (Fig. 2B). And there was no significant difference in the number of Treg infiltration in CNS between the two groups (Fig. 2B). CD4+ T cells in control-treated mice underwent cell divisions in vitro with MOG35-55, whereas CD4+ T cells in SiIRF4-treated mice had a reduced proliferation (Fig. 3A). In parallel, CCK8 assays via splenocytes produced similar differences (Fig. 3B). Supernatants were harvested at 24 h of culture and analysed by
Figure 3 SiIRF4 affects inflammatory T cell development in vitro during EAE. (A) To investigate the effect of SiIRF4 on T cell proliferation in vitro, the splenocytes harvested from mice 25 days after EAE induction were labelled with carboxyfluorescein succinimidyl amino ester (CFSE). CFSE loss by CD4+ proliferating cells from the two groups was assayed by flow cytometry after restimulation with MOG35-55 (10 mg/ml) for 72 h. The average frequency of CD4+ proliferating cells is presented; (B) The harvested splenocytes was also restimulated with MOG35-55 (10 mg/ml) for 72 h, and added CCK-8 solution incubated at 37 °C for the final 4 h. Cell proliferation was evaluated by stimulation index and the average frequency is presented; (C) Indicates the production of IFN-c and IL-17A by splenocytes, LN cells and CNS-infiltrating mononuclear cells from control and SiIRF4treated EAE mice. *P < 0.05. N = 6.
ELISA. SiIRF4-treated mice showed decreased production of both IL-17A and IFN-c compared to their controls (Fig. 3C). SiIRF4 attenuates EAE via a direct effect on DC
Myeloid-derived suppressive cells (MDSCs) are a heterogeneous group of myeloid cells comprised of hematopoietic progenitor cells and precursors of macrophages, granulocytes and dendritic cells (DCs) [16]. Recently, the close relationship between IRF4 and DCs has been demonstrated [17, 18]. Therefore, we tested whether SiIRF4 regulates the expression of Th1- and Th17polarizing cytokine by DCs. For this, we cultured CD4+ T cells with CD11c+ DCs from control- and SiIRF4treated mice and analysed cytokine expression. In our DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells (Fig. 4A). To investigate whether SiIRF4-induced myeloid DCs could modulate clinical responses in vivo, we isolated CD11c+ DCs from SiIRF4-treated mice, adoptively transferred them into recipient mice and then immunized with MOG35-55. These CD11c+ DCs ameliorated EAE development in recipient mice (Fig. 4B–C). SiIRF4 alters cytokine expression in DCs
DCs are a major source of different cytokines important for initiation and regulation of an immune response. Therefore, we analysed the cytokines expression by Q-PCR. As
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Figure 4 SiIRF4 attenuates EAE via a direct effect on DC. (A) CD4+ T cells isolated from C57BL/6 mice were cultured with CD11c+ DCs derived from control and SiIRF4-treated mice on day 25 after EAE induction. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IFN-c and IL-17; (B) CD11c+ cells from SiIRF4-treated donor mice attenuates EAE. 5 9 106 purified splenic CD11c+ cells from mice treated with SiIRF4 or control were injected i.v. into recipient C57BL/6 mice and then immunized with MOG35–55. (C) At day 25 after immunization, CNS-infiltrating CD4+ and CD8+ T cells were isolated and analysed by flow cytometry and absolute numbers were calculated. *P < 0.05. N = 6.
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Figure 5 SiIRF4 alters cytokine expression in DCs. CD11c+ DCs were isolated from SiIRF4- or control-treated naive (unimmunized) mice, and the expression of TNF-a, IL-1b, IL-6, IL-12/23 (p40), IL-10 and CD80 was analysed by Q-PCR (A) and Western blotting (B). *P < 0.05. N = 6.
shown in Fig. 5, CD11c+ DCs from SiIRF4-treated naive (unimmunized) mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. We also found that SiIRF4 treatment resulted in significant reduction in the expression of CD80 (Fig. 5).
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Discussion Our current study demonstrates a crucial role for IRF4 in the development of EAE. Inhibition of IRF4 with SiIRF4 decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our
350 Inhibition of IRF4 suppresses Th1 and Th17 C. Yang et al. .................................................................................................................................................................. DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Next, we adoptively transfer CD11c+ DCs from SiIRF4-treated mice into recipient mice and found that these CD11c+ DCs ameliorated EAE. Furthermore, CD11c+ DCs from SiIRF4-treated naive mice exhibited significantly reduced expression of pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. CD4+ Th cells play an important role in the adaptive immune system by activating and directing other immune cells. There are at least four distinct Th cell subsets (Th1, Th2, Th17 and regulatory T cells), which have been demonstrated to control immune response [19]. Both Th1 and Th17 cells have been shown in clinical and preclinical studies to be critically involved in the pathogenesis of autoimmune diseases including MS, rheumatoid arthritis and inflammatory bowel disease, whereas Th2 cells are implicated in asthma and allergy when aberrantly stimulated [20, 21]. IFN-c-producing Th1 and IL-17-producing Th17 cells are key proinflammatory mediators of cellular immunity that underlie crucial events during development of EAE. Both MS and EAE are thought to be initiated by myelin-reactive CD4+ T cells that produce IFN-c and IL-17 [22–24]. The Th1 lineage of cytokine can help Th17 cells invade the brain and spinal cord, thus triggering EAE [25, 26]. The high percentage of Th17 cells has an impact on the inflammation in the brain and the severity of disease [27]. Treg cells have been demonstrated to be capable of controlling CNS autoimmunity in several EAE models. Several studies described that the transfer of CD25+ Treg ameliorated EAE symptoms [28–30]. This study showed that the attenuation of EAE in SiIRF4-treated mice is associated with decreased expression of IL-17 and IFN-c and increased expression of Foxp3 in the peripheral lymphoid organs and CNS. The need for parenchymal APC is based on the fundamental immunological principle of reactivation for CD4+ T-cell effector function within the target tissue. A role for DC in directing T-cell transit from the perivascular space in postcapillary venules has been proposed [31, 32]. Recently, DCs have been demonstrated as a therapeutic target in MS and EAE [33]. CD11c+ DCs have been shown to be sufficient to initiate this autoimmune demyelinating disorder [28]. Furthermore, antigen presentation by myeloid DCs has been implicated in driving progression of relapsing EAE [34]. In our DC-T-cell coculture system, SiIRF4-treated DCs resulted in significantly less IFN-c and IL-17 production from T cells. Furthermore, we isolated CD11c+ DCs from SiIRF4-treated mice, adoptively transferred them into recipient mice and then immunized with MOG35-55. The results showed that these CD11c+ DCs ameliorated EAE in recipient mice. All of these indicated that the effect of SiIRF4 on Th1 and Th17 cell differentiation may be mediated via modulation of DCs.
Cytokines secreted by DCs are critical messengers that control T-cell activation and differentiation. CD11c+ DCs from SiIRF4-treated mice displayed significantly reduced production of the pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-12/IL-23 (p40), and a corresponding increase in anti-inflammatory IL-10 expression. These proinflammatory cytokines have been shown to promote CNS tissue damage in EAE [35, 36]. IL-1b is required for Th17 differentiation and in mice; it synergizes with IL-6 and IL-23 in induction of Th17 cells [37]. In addition to its potential direct toxicity, TNF-a perpetuates the inflammatory process through recruitment of cells [38]. IL-10 is an anti-inflammatory cytokine with paracrine and autocrine effects [39]. We conclude that SiIRF4 reduces the Tcell stimulatory capacity of DCs, which has been shown to be necessary for the survival of encephalitogenic T cells in the effector phase of the immune response [31]. In summary, our study provides evidence that SiIRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE. Inhibition of IRF4 with SiIRF4 decreases EAE scores and leucocyte infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. And this effect was via a direct regulation of DCs. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in immunoregulation, IRF4 may be an effective target in attenuating MS.
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