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An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization

Andrew Weaver^*, Angelika Goncalves da Silva^*, Robert K. Nuttall, Dylan R. Edwards, Steven D. Shapiro, Serge Rivest^** and V. Wee Yong^*,1

^* University of Calgary, Calgary, Alberta, Canada;
University of East Anglia, Norwich, UK;
Harvard Medical School, Boston, Massachusetts, USA; and
^** Laval University, Quebec, Canada

¹ Correspondence: University of Calgary, 3330 Hospital Dr., Calgary, Alberta T2N 4N1, Canada. E-mail: vyong{at}ucalgary.ca

SPECIFIC AIMS

The matrix metalloproteinases are implicated in multiple sclerosis (MS), where they are thought to play detrimental roles. Only a limited series of the 23 MMP members have been investigated in MS or its animal model, experimental autoimmune encephalomyelitis (EAE). The aims of this study were to examine the expression of 22 MMPs in the spinal cord of EAE mice, determine the function(s) of the most highly up-regulated MMP, and investigate the mechanisms of its activity.

PRINCIPAL FINDINGS

1. Complete spectrum of MMP alterations at peak EAE disease demonstrates a dramatic up-regulation of MMP-12
We immunized 129/SvEv mice with myelin oligodendrocyte glycoprotein (MOG, peptide 35-55). At peak disease severity, the lumbar-sacral spinal cord was removed for TaqMan real-time PCR analyses for 22 MMPs and normalized to 18S rRNA. Most MMPs were increased in EAE compared with normal animals (Fig. 1 ), but MMP-12 was the most highly expressed. There was an almost 1000-fold increase in the transcript encoding this MMP member in the spinal cord of EAE afflicted animals compared with normal ones. Some MMPs were down-regulated in EAE; these included MMP-15, -16, -17, -21, and -24. We also profiled the endogenous tissue inhibitors of metalloproteinases (TIMPs); TIMP-1 was elevated in EAE, TIMP-4 was unaltered, and TIMP-2 and -3 were reduced.

View larger version (19K): [in this window] [in a new window]   Figure 1. TaqMan real-time PCR analyses of spinal cord samples from normal controls (n of 4) and from mice at peak EAE severity (n of 6) were conducted and expressed relative to 18S rRNA levels. The normalized transcript level for each MMP in EAE was then expressed relative to controls to provide for relative fold change. The black bars denote significant increases, the empty bars reflect no change, while the gray bars denote transcripts that are down-regulated in EAE compared with normal controls. Statistical significance was determined at P < 0.05, using 2-tailed Student’s t test.

2. MMP-12 null mice have significantly worse disease outcomes than their wild-type (WT) counterparts
Since MMP-12 transcript levels were notably increased during peak disease in EAE, we addressed its involvement in EAE. We immunized MMP-12 null or WT mice with MOG and compared the resultant disease severity. Animals were followed over 73 days, during which the daily scoring was used to assess disease progress. Surprisingly, the results demonstrated that MMP-12 null mice did significantly worse than their WT counterparts. During a relapse, while the WT animals succumbed with tail and hind limb paresis/paralysis, the MMP-12 mice had forelimb paralysis in addition to tail and hind limb involvement.

Each animal’s disease burden over the entire experiment was computed by adding their daily disease score to obtain the "sum of scores" for that mouse. The MMP-12 null mice had a significantly worse sum of scores, representing a more extensive disease burden. The WT mice typically reached their peak in disease and symptoms, then remitted to the point where the animals recovered full function of all four limbs and their tail. In contrast, MMP-12 null mice achieved a higher peak disease score and for longer periods. When disease remitted in MMP-12 null mice, most animals never fully recovered function and were left with lasting impairment and weakness in tail and hind limbs.

The spinal cord histology was examined from mice at peak disease to determine the basis for the worse disease score in MMP-12 null mice. There was qualitatively more cellular infiltration and demyelination in MMP-12 null mice than with WT animals. Thus, a worse disease score in animals without MMP-12 is correlated with increased inflammation and neuropathology in the CNS.

Overall, the data implicate MMP-12 as a protective enzyme in the pathology and course of EAE.

3. Mechanisms of MMP-12 in EAE: MMP-12 is involved in T effector cell polarization
We killed animals at a point of divergence between WT and MMP-12 null mice to evaluate possible differences in cytokine expression by splenocytes and lymphoid node cells (LNCs) in both groups. A proliferation assay first revealed there was no significant difference between splenocytes and LNCs from MMP-12 null and WT mice after stimulation with MOG in vitro, indicating that the precursor frequency of MOG antigen-specific cells was similar in both groups. When supernatants from cultured cells were assayed by ELISA for a representative Th1 cytokine, interferon- (IFN-), both the 10 and 20 µg/mL MOG-induced IFN- levels were significantly greater in the MMP-12 null samples than in WT cells.

To evaluate Th cell polarization further, RNA was extracted from splenocytes and LNCs of mice at the point of divergence of disease and examined for changes in IFN- and interleukin-5 (IL-5, a representative Th2 cytokine) using real-time PCR. MMP-12 null cells upon MOG antigen restimulation in vitro displayed a 14-fold increase in IFN- levels relative to the unstimulated cells whereas IL-5 was decreased (Fig. 2 A, B). We then used the ratio of IFN- to IL-5 for each individual mouse, which was further averaged across each group. Such analyses highlight that MMP-12 null splenocytes were significantly skewed toward a Th1 phenotype (P<0.001, 2-tailed unpaired t test) (Fig. 2C ). In contrast, WT splenocytes were biased toward a Th2 polarizing condition (Fig. 2D ), and this ratio was significantly different from 1 (P<0.05, 2-tailed unpaired t test vs. unstimulated WT cells). The modest ratio of Th2 to Th1 of 2 for the WT cells presumably reflects a disease stage in which the WT mice were just beginning to remit and where Th2 cytokines were just beginning to increase over those of Th1. In studies by others, EAE is highly Th1 polarized in the initial stages of disease while Th2 cytokines predominate during the remission phase.

View larger version (21K): [in this window] [in a new window]   Figure 2. An increase in Th1 bias is evident in MMP-12 null lymphoid cells. A, B) Each bar is the relative expression of the cytokine of interest in MOG stimulated vs. unstimulated condition, for either null or WT cells. C) The Th1/Th2 ratio is obtained by analyzing the individual interferon- to IL-5 level for that mouse and the group mean ± SE is then displayed; D) the opposite ratio. The level of statistical significance denotes comparison between WT and null cells. E, F) MMP-12 null mice have significantly higher expression of T-bet and lower expression of GATA-3, a condition of transcriptional regulation that favors a Th1 bias. All bars represent n of 4 samples. **P< 0.01, ***P< 0.001.

Collectively, these data indicate that the mechanism by which MMP-12 is protective in EAE is accounted for in part by altering a Th1 to a Th2 milieu.

We addressed possible mechanisms that could underlie the differences in the T effector cell polarization between WT and MMP-12 null mice. We examined the expression of the transcriptional regulators for Th1 and Th2 promotion, which are T-bet and GATA-3, respectively. Real-time PCR analyses of the lymphoid cells showed that the level of T-bet transcripts was significantly increased in MMP-12 null cells after MOG stimulation (P<0.001, 2-tailed unpaired t test, compared with unstimulated state) (Fig. 2E ). In contrast, there was a significant decrease in GATA-3 expression (Fig. 2F ) after MOG stimulation in null cells (P<0.01 compared with unstimulated state).

Overall, the combined data from cytokine and transcription factor analyses indicate that at the point of disease divergence, WT T cells are beginning to show a slight Th2 bias and mice subsequently remit. In contrast, MMP-12 null animals at this same score have increased T-bet and reduced GATA-3 expression, favoring a T effector phenotype that is highly polarized toward a Th1 type; in concordance, animals continue to worsen clinically. Therefore, the data indicate that despite similar clinical deficits at this point in the disease, WT and MMP-12 null mice have profoundly divergent T effector cell responses driving disease pathology, thus linking an MMP to T cell polarization for the first time (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate for the first time that a highly up-regulated MMP in EAE, MMP-12, is protective against the disease. The results provide a new link between two important regulators of inflammation, that of MMPs and Th1/Th2 polarization. Further studies are warranted to define precisely how MMP-12 alters T-bet and GATA-3 expression to modulate T cell polarization, thereby affecting disease outcome.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2030fje; doi: 10.1096/fj.04-2030fje

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