Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 -/- CD4+ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.

Previously we have identified heme oxygenase 1 (Hmox1, HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 -/-CD4 + T cells to promote inflammation [8]. HO-1 is a rate-limiting intracellular enzyme that degrades heme into biliverdin and free iron and CO [9]. The reaction products have potent anti-inflammatory and antioxidative effect. Our previous study also showed that restoring function in chronically-stimulated CD4 + T cells would depend on the capacity of miR-155 to regulate HO-1 expression. HO-1 regulatory activity is centered on T cell proliferation and migration to sites of inflammation. We then questioned whether this observation could be extended to other systems where miR-155 was shown to control the activity of chronically-stimulated T cells. The regulatory role played by miR-155 on T cell-mediated chronic inflammation is well established in mouse experimental autoimmune encephalomyelitis (EAE) model [6,10]. Myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG 35-55 ) immunization upregulated the expression of miR-155 in T cells, and miR-155-deficient mice were highly resistant to EAE [6]. In addition, induction of HO-1 by metal protoporphyrins or exposure to its product CO would arrest EAE progression and Hmox1-deficient mice are more sensitive to EAE. The failure to attenuateEAE progression in Hmox1 -/mice by HO-1 activation confirmed that the protective effect requires the expression of HO-1 [11][12]. Thus, in the present study, we intended to investigate whether the resistance of miR-155 -/mice to EAE could be lifted by the inhibition of HO-1. Treatment with the HO-1 inhibitor Zn (II) Protoporphyrin IX (ZnPP) restored susceptibility to EAE in miR-155-deficient mice. Increased severity of the disease was accompanied by an enhanced T cell infiltration of the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of autoimmune T cells in EAE.

Materials and Methods Mice
Rag1 -/and miR-155 +/+ B6 congenic mice were purchased from Charles River. MiR-155 -/-B6 mice were purchased from Jackson Laboratories. All age and sex matched mice used in this study were between 6-10 weeks old and were bred in individually ventilated cages on the same rack under specific pathogen-free conditions (FELASA) at the Institute for Medical Immunology, UniversitéLibre de Bruxelles, Belgium.

EAE
EAE was induced in mice aged from 6-8 weeks old. The detailed protocols for immunization and scoring were described previously [13]. In adoptive transfer experiments, spleen CD4 + T cells from miR-155 -/mice were collected by positive selection with FITC-conjugated antimouse CD4 (BD Biosciences), followed by anti-FITC magnetic beads (Miltenyi Biotec) and sorted by magnetic sorting according to manufacturer's protocol [13]. Purified miR-155 -/-CD4 + T cells were injected i.v (4x10 6 cells/mouse) into Rag1 -/recipients. Recipient mice were subjected to EAE induction 24 hours after the transfer. In all experiments, mice were sacrificed at the peak of the diseases for analysis.

Protoporphyrin treatment
Zn (II) Protoporphyrin IX (ZnPP) and Co (III) protoporphyrin IX chloride (CoPP) (Frontier Scientific Porphyrin Products) were dissolved as previously described [8]. Solution without protoporphyrin was used as vehicle. ZnPP or CoPP was administered i.p every 2 days (200 μl/ mouse, 5 mg/kg). For the EAE experiment, the protoporphyrin injection was started at the immunization with MOG 35-55 . For miR-155 -/-CD4 + T cell adoptive transfer experiments, ZnPP injection was started at the time of the transfer.

Flow cytometry
Single cell suspensions of spleen, draining lymph node (inguinal region) and brain from EAE mice were collected at the indicated time, the detailed antibodies and methods for surface and intracellular staining and analysis were described previously [13].

Statistical analysis
Results were presented as Mean ±SEM. Mann-Whitney's U test was used to compare two groups. For multiple group comparisons, one-way ANOVA tests were performed followed up by Tukey's post-hoc multiple comparisons test. P< 0.05 was considered statistically significant.

Ethics statement
This study was carried out in strict accordance with the Royal Decree (N°2013024221) of the 29th of May 2013 on the protection of experimental animals and published on the 10th of July 2013 in the Belgium code of law. The protocol was approved by the Comité d'Ethique et du Bien-Etre Animal of the Institute for Medical Immunology of the Université Libre de Bruxelles (Permit Number: LA1500518). All surgery was performed under xylazine/ketamine anesthesia, and all efforts were made to minimize suffering.

Results
The resistance of miR-155 -/mice to EAE was lifted by HO-1 inhibition We have previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 -/-CD4 + T cells to promote inflammation [8]. We then tested whether this observation could be extended to other systems where miR-155 was shown to control the activity of chronically-stimulated T cells. The regulatory role played by miR-155 on T cell-mediated chronic inflammation is well established in mouse experimental autoimmune encephalomyelitis (EAE) model [6,10]. Thus, experimental protocols were designed to identify a possible role for miR-155 in HO-1-mediated regulation of T cell activity in EAE. Both miR-155 +/+ and miR-155 -/mice were immunized with MOG 35-55 peptide. As anticipated, miR-155 -/mice displayed a lower peak of disease severity, in contrast, miR-155 +/+ mice exhibited a more pronounced pathology (mean maximal score 2.17±0.38 vs 0.33±0.11, p<0.05 by two tailed Mann-Whitney U test). In order to determine whether HO-1 was responsible for the incapacity of miR-155-deficient mice to develop EAE, we injected repeatedly HO-1 inhibitor ZnPP in MOG 35-55 peptide-immunized miR-155 -/mice and compared with animals injected with vehicle for the development of EAE. As shown in Fig 1, HO-1 inhibition worsened significantly the clinical signs of EAE in miR-155 -/mice (mean maximal score 1.16±0.27 vs 0.33±0.11, p<0.05 by two tailed Mann-Whitney U test). This observation was consistent with a role for miR-155 in repressing HO-1-mediated regulatory process in the development of EAE. In agreement with what has been published previously [11], induction of HO-1 expression and function by CoPP administration reversed paralysis (mean maximal score 2.17±0.38 vs 1.00±0.34, p<0.05 by two tailed Mann-Whitney U test). (Fig 1). On the contrary, injecting HO-1 inhibitor to miR-155 +/+ mice did not enhance disease pathology (S1 Fig). Thus, worsened disease caused by HO-1 inhibition was only observed in miR-155 -/mice.

Inhibition of HO-1 promote T cell infiltrated into the brain
The pathogenesis of EAE initiates with immune cell infiltration into the brain [6]. Mice from the indicated groups were sacrificed and single cell suspensions were isolated from spleen, draining lymph nodes (LN) and brain on day 20 after immunization. The cellular content of each organ was characterized by flow cytometry. As depicted in Fig 2, the cellularity in brain was significant altered among the indicated four groups, whereas cellularity in spleen and lymph node were comparable. As expected, compared with miR-155 +/+ mice, the lack of miR-155 expression caused an important drop in the number of B220 + cells, CD3 + T cells and CD11b + myeloid cells present within the brain. Inhibition of HO-1 by ZnPP, however, restored brain cellular infiltrates in miR-155 -/mice to numbers comparable to those observed in wild type counterparts, again supporting a role for HO-1 in regulating the inflammation of the central nervous system (CNS) in the absence of miR-155. However, induce HO-1 expression by CoPP treatment in miR-155 +/+ mice failed to modify the cellularity in the brain.

HO-1 inhibition promotes Th1 and Th17 responses in CNS during EAE in miR-155 -/mice
During EAE, myelin peptide-specific Th1 and Th17 effector cells in CNS are responsible for disease pathology [15][16]. We then examined spleen, draining lymph node and brain for the presence of Th1 and Th17 cells during EAE. Results presented in Fig 3 show that both the numbers of Th1 and Th17 cells present in the lymph node or brain of miR-155 -/mice treated with ZnPP were very similar to those in vehicle-treated miR-155 +/+ mice. This contrasted sharply with the modest numbers of Th1 and Th17 cells present in spleen, lymph node or brain of miR-155 -/mice treated with vehicle only. On contrary, the total numbers of these inflammatory T cell populations in the LN and brain were similar in miR-155 +/+ treated with vehicle versus miR-155 +/+ mice treated with CoPP. Altogether, these observations reinforced the idea that miR-155 promotes T cell-mediated inflammation by targeting HO-1.

T cell intrinsic role for miR-155 in the development of inflammatory T cells during EAE
To test whether the failure of miR-155 -/-T cells to mediate EAE could be corrected by the inhibition of HO-1, we adoptively transferred 4x10 6 purified naïve miR-155 -/-CD4 + T cells into Rag1 -/congenic recipients and induced EAE 24 hours later. One group of mice were treated with ZnPP, the second one received only the vehicle. MiR-155 +/+ CD4 + T cells transferred group was used as positive control. Mice receiving ZnPP had a substantially more severe and accelerated disease course compared to vehicle-treated mice (Fig 4A). At the peak of the diseases, mice from the three groups were sacrificed and cells from brain were separated. Consistently, increased numbers of cells were observed in the brain of mice that received ZnPP (Fig 4B).
We also examined spleen, draining lymph node and brain for the presence of Th1 and Th17 CD4 + T cells in the indicated groups. Results presented in Fig 5A showed that the percentage of Th1 cells in the spleen, draining lymph node or brain of miR-155 -/mice treated with ZnPP were very similar to those in vehicle-treated mice. However, the absolute number of Th1 cells in the spleen and brain of the ZnPP treated mouse was significantly increased than the vehicle treated mouse (Fig 5B). Altogether, these observations reinforced the idea that miR-155 can promote T cell-mediated inflammation by targeting HO-1 expression in T cells.

Discussion
Immunization with MOG 35-55 induces a chronic form of EAE in C57BL/6 mice, mostly mediated by CD4 + T cells [17]. More recently, the regulatory role played by miR-155 on T cellmediated inflammation was established in EAE model [6]. Moreover, induction of HO-1 expression was also shown to suppress T cell-mediated neuro-inflammation and improve EAE in C57BL/6 mice immunized with MOG 35-55 [11]. Interestingly, the same study demonstrated that blocking HO-1 by ZnPP treatment in miR-155 +/+ mice had no effect on EAE induction. Since we have previously shown that HO-1 is a target of miR-155, this situation could be attributed to the absence of HO-1 expression in chronically-stimulated miR-155-sufficient T cells. We confirmed that in vivo induction of HO-1 by repeated injection of HO-1 activator, CoPP, attenuated clinical signs of EAE in normal miR-155 +/+ mice. ZnPP treatment worsened the clinical signs of EAE in miR-155 -/mice. This observation is consistent with a role for miR-155 in repressing HO-1-mediated regulation of T cells and in promoting the development of EAE. Inhibition of HO-1 by ZnPP restored brain cellular infiltrates in miR-155 -/mice, again supporting a role for HO-1 in regulating the inflammation of the central nerve system in the absence of miR-155. Though protoporphyrin treatment could restore antigen-driven expansion and tissue migration of chronical-stimulated miR-155 -/-T cells, it did not improve the capacity of T cells to produce effector cytokines, such as IFN-γ [8]. In the adoptive transfer model, the percentage of Th1 cell was similar regardless of ZnPP treatment. However, the absolute number of Th1 cells was increased in ZnPP-treated group and the increased severity of the disease in miR-155 -/animals treated with the HO-1 inhibitor was caused by an enhanced T cell infiltration into the brain. Thus, miR-155-dependent regulation of HO-1 function primarily would appear to control T cell proliferation and migration to sites of inflammation. Interestingly, it has been well demonstrated that mitochondrial reactive oxygen species (mROS) signaling is required in T cells for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 production [18]. Since lack of HO-1 induces mitochondrial oxidative stress and ROS production [19], one could hypothesize that miR-155-mediated control of HO-1 expression in T cells would be essential for IL-2-dependent proliferation and expansion following activation by antigen. This hypothesis is reinforced by the observation that HO-1 inhibition in T cells stimulates IL-2 production [8,20]. We observed that, though ZnPP treatment induced EAE in miR-155 -/treated mice, it did not restore the clinical disease to wild-type levels. It is well established that microRNA usually targets the expression of many genes and that their overall effect on biological systems results from the regulation of multiple gene expression. Since HO-1 has been identified as one of miR-155 targets, we can hypothesize that treatment by ZnPP in our experiment does not regulate the anti-inflammatory function of other miR-155 targets and this situation could explain why HO-1 inhibition alone does not restore EAE to wild-type levels in miR-155 -/mice. Other miR-155 targets that have been identified are Socs1, Smad and Ifngr [21][22][23]. They all have been characterized for their anti-inflammatory function. Whether inhibition of their activity in conjunction with that of HO-1 could restore EAE in miR-155-deficient animals remain to be explored.
In conclusion, our work has confirmed the importance of miR-155 in T cells for inhibiting HO-1 expression and promoting T cell-driven inflammation leading to autoimmune disease. It remains, however, to evaluate precisely the mechanism by which HO-1 deletion promotes T cell-mediated EAE in miR-155 -/mice. This could help to explore novel and effective therapies against auto-immune diseases.
Supporting Information S1 Fig. Inhibition of HO-1 had no effect on miR-155 +/+ mice in EAE. EAE was induced in miR-155 +/+ mice that received ZnPP or vehicle alone. Disease severity was regularly scored based on clinical symptoms (n = 7-10). Data are shown as Mean ± SEM. Data are representative of two independent experiments. (TIF)