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. 2009 Aug;175(2):661-7.
doi: 10.2353/ajpath.2009.081093. Epub 2009 Jul 16.

The complement inhibitor FUT-175 suppresses T cell autoreactivity in experimental autoimmune encephalomyelitis

Qing Li  1 , Kristine Nacion, Hong Bu, Feng Lin
Affiliations

The complement inhibitor FUT-175 suppresses T cell autoreactivity in experimental autoimmune encephalomyelitis

Qing Li et al. Am J Pathol. 2009 Aug.

Abstract

Several recent studies have shown that interacting antigen presenting cells and/or T cells produced complement activation products C5a and C3a, are integrally involved in T-cell activation, and promote the generation of myelin oligodendrocyte glycoprotein (MOG(35-55))-specific interferon-gamma and interleukin-17-producing T cells in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. In this study, we tested whether FUT-175, a clinical pharmaceutical that has been shown to inhibit the formation of C3/C5 convertases, can attenuate myelin-specific T-cell responses, as well as disease severity in experimental autoimmune encephalomyelitis. In vitro, FUT-175 inhibited local C5a/C3a production by antigen presenting cell-T-cell complexes and attenuated MOG(35-55)-specific Th1 and Th17 responses with little nonspecific cytotoxicity. In vivo administration of FUT-175 delayed experimental autoimmune encephalomyelitis disease onset, lowered clinical scores, decreased central nervous system inflammation, and reduced demyelination. The FUT-175-treated mice exhibited decreased numbers of MOG(35-55)-specific interferon-gamma- and interleukin-17-producing T cells. In addition, results from the FUT-175 treatment of naive recipients of adoptively transferred splenocytes from MOG(35-55)-immunized mice suggested that the effect of FUT-175 was on MOG-specific cellular responses and not on anti-MOG antibodies. These results argue that complement regulators, which inhibit C5a/C3a production, may have therapeutic efficacy in multiple sclerosis and in other clinical conditions in which T cells drive disease pathogenesis.

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Figures

Figure 1
Figure 1
FUT-175 inhibits local C5a and C3a productions by APC-T cell partners during antigen specific T cell activation. A: C5a and C3a detections in concentrated culture supernatants from co-cultured bone marrow-derived dendritic cells, OT-II CD4+ T cells, 3 μg/ml OVA323–339 peptide and different concentrations of FUT-175. Recombinant mouse C5a was used as positive control (rC5a). B: C5a and C3a detected in A were semiquantified using the software package MetaMorph 7.5 (Molecular Devices, Downington, PA).
Figure 2
Figure 2
FUT-175 inhibits MOG35–55-specific T cell responses in vitro: 6 × 105 spleen cells from MOG35–55 immunized mice were aliquoted into each well of a 96 well ELISPOT plate coated with IFNγ- or IL-17-capturing mAb. Then 5 μg/ml MOG35–55 peptide, together with increasing concentrations of FUT-175, was added into the plate and the mixtures were incubated for 24 hours at 37°C, 5% CO2. After developing, numbers of IFNγ (A) and IL-17 (B) producing cells were counted using an ImmunoSPOT Imaging system (Cleveland, OH). C: FUT-175 has little nonspecific cytotoxicity after 72 hours incubation at a concentration as high as 400 μg/ml: 2 × 105 splenocytes from mice with EAE were incubated with 5 μg/ml MOG35–55 peptide and different concentrations of FUT-175 (0, 25,100, 400 μg/ml) at 37°C, 5% CO2. At different time points (0, 24, 48, 72 hours), cell viabilities were assessed by adding 20 μl of indicator dye resazurin from the CellTiter-Blue Cell Viability Assay kit (Promega, MI) and incubating for another 1 hour. After this, fluorescence 560Ex/590Em were measured using a fluorescence plate reader (Molecular Devices, Downington, PA).
Figure 3
Figure 3
FUT-175 treatment protects mice from EAE. A: Clinical scores of mice receiving FUT-175 or PBS treatments one day after immunization. Mice were treated with 10 mg/kg FUT-175 or equal volume of PBS i.p. every day 1 day after MOG35–55 immunization. Clinical scores were monitored daily and average scores are shown. (n = 18, P < 0.05) For histological analysis, cervical portion of spinal cords were collected from PBS treated (B,×40 and D, ×200) and FUT-175 treated mice (C, ×40 and E, ×200) 25 days after immunization and processed for H&E staining or Luxol Fast Blue staining (G, H). The average infiltrated cell numbers in a 10,000 μM2 dorsal area were analyzed by the software package MetaMorph 7.5 (Molecular Devices, Downington, PA) and compared (F). The average myelin areas in a 50,000 μmol/L2 white matter area were quantified using the same software package and compared between the PBS- and FUT-175-treated mice (I). *P < 0.05.
Figure 4
Figure 4
Mice treated with FUT-175 exhibit suppressed MOG35–55-specific T cell responses in vivo. FUT-175-treated mice (n = 18) and control mice (n = 20) were sacrificed 25 days after immunization and T cell responses against MOG35–55 in each group were assayed by IFNγ and IL-17 ELISPOT assays using 6 × 105 spleen cells incubated with different concentrations of MOG35–55 at 37°C and 5% CO2 for 24 hours (*P < 0.05).
Figure 5
Figure 5
Effects of FUT-175 treatments on coagulation and systemic complement activation. A: FUT-175 treatments prolonged coagulation time. aPPT were analyzed on an automatic coagulation analyzer using sodium citrate treated plasma samples collected from PBS and 10 mg/kg FUT-175-treated mice (n = 10). B: FUT-175 treatments inhibited systemic complement activation. Deposited C3b levels on EshA (classical pathway) or Zymosan (alternative pathway) were quantitated by flow cytometry using serum samples collected from PBS- and 10 mg/kg FUT-175-treated mice *P < 0.05.
Figure 6
Figure 6
FUT-175 treatments suppress EAE induced by adoptive transferred MOG35–55-reactive T cells in naïve recipient mice. A: After i.v. injection of the in vitro-amplified MOG35–55 reactive T cells, naïve recipient mice were randomly divided into two groups (n = 5 in each group). One group received FUT-175 treatments every day (10 mg/kg) while the other group received same volume of PBS. Disease severities were scored daily and average scores are presented. B: Twelve days after adoptive transfer, mice were sacrificed and T cell responses against MOG35–55 were measured and compared between the groups by IFNγ and IL-17 ELISPOT assays. The experiments were repeated with similar results (not shown). *P < 0.05.
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