MMP-independent role of TIMP-1 at the blood brain barrier during viral encephalomyelitis

Abstract

Infection of the CNS (central nervous system) with a sublethal neurotropic coronavirus (JHMV) induces a vigorous inflammatory response. CD4⁺ and CD8⁺ T cells are essential to control infectious virus but at the cost of tissue damage. An enigma in understanding the contribution of T cell subsets in pathogenesis resides in their distinct migration pattern across the BBB (blood brain barrier). CD4⁺ T cells transiently accumulate within the perivascular space, whereas CD8⁺ T cells migrate directly into the CNS parenchyma. As MMPs (matrix metalloproteinases) facilitate migration across the glia limitans, specific expression of the TIMP (tissue inhibitor of MMPs)-1 by CD4⁺ T cells present in the perivascular cuffs suggested that TIMP-1 is responsible for stalling CD4⁺ T cell migration into the CNS parenchyma. Using TIMP-1 deficient mice, the present data demonstrate an increase rather than a decrease in CD4⁺ T cell accumulation within the perivascular space during JHMV infection. Whereas virus control was not affected by perivascular retention of CD4⁺ T cells, disease severity was decreased and associated with reduced IFNγ (interferon γ) production. Moreover, decreased CD4⁺ T cell recruitment into the CNS parenchyma of TIMP-1 deficient mice was not associated with impaired T cell recruiting chemokines or MMP expression, and no compensation by other TIMP molecules was identified. These data suggest an MMP-independent role of TIMP-1 in regulating CD4⁺ T cell access into the CNS parenchyma during acute JHMV encephalitis.

Figure 1. Disease severity is decreased in TIMP-1^−/− compared to WT mice, despite similar virus clearance and extent of demyelination

(A) Clinical symptoms were monitored daily in WT and TIMP-1^−/− mice. Data represent the mean of seven experiments with 15–20 mice per experiment, **P<0.01 and ***P<0.001. (B) Virus replication in brains of WT and TIMP-1^−/− mice analyzed by plaque assay. Data represent the average±S.E.M. of six individual mice per group per time point combined from two experiments. (C) Percentage of demyelination in spinal cord white matter of WT and TIMP-1^−/− mice between days 7 and 14 p.i. Data represent the mean±S.E.M. of at least three mice per group per time point. (D) IFNγ mRNA expression analyzed by real-time PCR in naïve, JHMV infected WT and TIMP-1^−/− mice. Data represent the mean±S.E.M. of three individuals per group per time point.

Figure 2. TIMP-1 deficiency does not alter CNS inflammation

Numbers of total inflammatory leukocytes (CD45^hi), neutrophils (ly6G⁺), monocytes (F4/80⁺), CD8⁺ and CD4⁺ T cells per brain in WT and TIMP-1^−/− infected mice. Data represent the mean±S.E.M. of 12 mice per group per time point combined from four separate experiments (n=3 per time point and group per experiment).

Figure 3. CD4⁺ T cells are retained in the perivascular space of TIMP-1^−/− mice

(A) CD4⁺ T cell perivascular cuffs in WT and TIMP-1^−/− mice at day 7 p.i. analyzed by immunofluorescent microscopy. The two basement membranes of the BBB stained with laminin (red), delimit the perivascular space. CD4⁺ T cells appear in green. Scale bar, 25 μm. (B) Total number of CD4⁺ T cells and their distribution in the perivascular space versus parenchyma analyzed in WT and TIMP-1^−/− mice at day 7 and 10 p.i. Data represent the mean±S.E.M. of six mice per group combined from two separate experiments (with n=3 for each individual experiment).

Figure 4. Chemokine expression in infected WT and TIMP-1^−/− mice

CCL5 and CXCL10 mRNA, measured by real-time PCR, and protein, measured by ELISA, in naïve or infected WT and TIMP-1^−/− mice. Data represent the mean±S.E.M. of three to seven individual mice per group per time point.

Figure 5. TIMPs and MMPs do not compensate for TIMP-1 deficiency during JHMV infection

Expression of TIMP-1, -2, -3 and -4 mRNA (A), and MMP-3 and -12 mRNA (B), analyzed by real-time PCR in naïve and JHMV infected WT and TIMP-1^−/− mice. Transcript levels depict the average±S.E.M. of three individual brains per group per time point representative of two separate experiments. (C) MMP9 activity determined by zymography of CNS mononuclear cells isolated from infected WT and TIMP-1^−/− mice at days 3 and 7 p.i. Data are representative of two experiments.