Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE.

<p>B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).</p

PSEN1 cKO mice are susceptible to EAE.

<p>WT and PSEN1 cKO mice were immunized with MOG35-55 to induce active EAE. A. Clinical course of EAE. B. Peak clinical score of EAE mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. Symbols indicate the percentage of cells from individual mice. Open circles indicate results from WT and filled squares indicate PSEN1 cKO mice. Error bars represent SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).</p

Impact of GSI on Th1 effector differentiation.

<p>A-C. Effects of GSI on the generation of encephalitogenic Th1 T cells. T cells from MBP1-11 TCR transgenic mice were activated <i>in vitro</i> with MBP ac1-11 peptide and IL-12. Cell cultures were stimulated in the presence of DMSO or GSI. 8x10⁶ T cells were transferred into B10.Pl recipients and mice were graded for the development of EAE. A. Mean EAE disease course. B. Peak clinical score. C. Cumulative EAE score. D-F. Effects of GSI on Th1 polarization with IL-12. Splenocytes were stimulated <i>in vitro</i> with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 alone with IL-12. IFNγ and Tbet expression was measured by intracellular flow cytometry at 72 hours using gates for live CD4+ T cell events. E. Percentages of IFNγ expressing CD4+ T cells. F. Percentages of T cells expressing Tbet. Results shown are representative of at least two experiments. Open circles indicate DMSO treatment. Filled squares indicate GSI. Symbols represent individual mice (panels B and C) or replicate cultures (panels E and F). The numbers in FACS plots indicate cell percentages within each quadrant. Asterisks indicate statistically significant differences between groups (* p<0.05, ** p<0.01 and ***p <0.001).</p

Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis

<div><p>Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. <i>In vivo</i> GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. <i>In vitro</i>, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses <i>in vivo</i> and potently regulate T effector differentiation <i>in vitro</i>, however, they are dispensable for EAE.</p></div

GSI treatment inhibits effector differentiation, activation and proliferation <i>in vitro</i>.

<p>A-E. Effects of GSI on Th1 differentiation. Splenocytes were stimulated <i>in vitro</i> with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 and either DMSO or GSI. A-C. IFNγ and Tbet expression were determined by intracellular staining and flow cytometry. A. Representative flow cytometry plots showing IFNγ and Tbet expression. B. IFNγ expression was measured by intracellular flow cytometry. C. Tbet expression was measured by intracellular flow cytometry. D and E. Expression analysis of IL12Rβ1 and IL12Rβ2. Total cellular RNA was isolated from each culture and target gene expression determined by real time PCR. D. Expression of IL12Rβ1. E. Expression of IL12Rβ2. F-J. Effects of GSI on T cell activation. Splenocytes from 2D2 TCR transgenic mice were activated <i>in vitro</i> with MOG35-55 peptide in the presence of DMSO or GSI. At 72 hours of stimulation, flow cytometry was used to measure expression of the activation markers CD25, CD44 and CD69. F. Representative flow cytometry plots showing CD25 and CD44 expression. G. The percentage of T cells expressing CD25. H. Expression of CD44 by T cells. I. The percentages of T cells expressing CD69. J. Quantitation of CD25 expression on activated T cells (CD4 and CD44 gated events). K-N Effects of GSI on T cell proliferation. Splenocytes were labeled with Cell-trace and stimulated for 96 hours with antibodies to CD3 and CD28 in the presence of DMSO or DBZ. Th1 differentiation was promoted by the addition of anti-IL-4 alone (neutral), or in combination with IL-2. K. Proliferation and intracellular IFNγ staining were detected by flow cytometry. L and M. Division index and Proliferation index were calculated using gates to measure the percentages of T cells within each cell division. O. Expression of IFNγ by T cells that had undergone 4 or more cell divisions. P. Expression of CD25 by T cells cultured in the absence or presence of IL-2. Flow cytometry plots are gated on live CD4+ T cells. Results shown are representative of at least two experiments. The numbers in FACS plots indicate cell percentages within each quadrant. Individual symbols indicate results from replicate wells. Open circles indicate treatment with DMSO, filled squares indicate GSI treatment. Error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).</p

Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on the clinical course of experimental autoimmune encephalomyelitis (EAE).

<p>A single intravenous (i.v.) injection of PEG minocycline-liposomes given after disease onset at day 15 post-immunization resulted in significant amelioration of clinical disease for eight days compared to a single injection of PBS when given shortly after onset of clinical disease (A). This effect was not sustained (A). In subsequent experiments, PEG-liposomes were administered every five days. Treatment with i.v. PEG minocycline-liposomes initiated after disease onset at day 15 post-immunization and administered every five days was as effective in ameliorating clinical EAE as treatment with daily intraperitoneal (i.p.) injections of minocycline (B). In contrast, i.v. injections of empty PEG-liposome every five days, or minocycline i.p. injections every five days had no detectable effect on the clinical course of EAE (B). The time of treatment initiation is indicated by a red arrow.</p

Effects of PEG minocycline-liposomes on the expression of matrix metalloproteinase (MMP)-9 in splenocytes and CNS-infiltrating leukocytes.

<p>The expression levels of MMP-9 were significantly down-regulated on day 20 after active induction of experimental autoimmune encephalomyelitis (EAE) in splenocytes of mice following treatment with daily intraperitoneal (i.p.) injections of minocycline, and after intravenous (i.v.) injections of PEG minocycline-liposomes every 5 days compared to experimental animals that received no treatment, that were treated with i.v. injections of empty PEG-liposome every five days, or with minocycline i.p. injections every five days, as shown by ELISA (A). The proteolytic activity of MMP-9 measured by zymography was also significantly diminished in mice treated daily with i.p. injections of minocycline and i.v. injections of PEG minocycline-liposomes every five days (B). MMP-9 protein expression in CNS mononuclear cells was not found to be significantly different between experimental groups (C).</p

Immunization with human (h)AQP4_281-300 leads to an expansion of antigen-specific CD4⁺ T cells <i>in vivo</i>, and an Ig isotype switch in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice.

<p>(A) Following immunization with human (h)AQP4_281-300, an expansion of antigen-specific CD4⁺ T helper cells was detected by tetramer staining of lymph node cells. The fluorescent signal of <i>HLA-DRB1*03</i>:<i>01</i>-loaded tetramers minus the fluorescent signal of empty <i>HLA-DRB1*03</i>:<i>01</i> tetramers is shown. CD4⁺ T helper cells provide soluble mediators that drive B cell differentiation immunoglobulin (Ig) class switching. To determine whether hAQP4_281-300-reactive CD4⁺ T cells are capable of causing IgM to IgG isotype switching in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice, the concentration of Ig against hAQP4_281-300, mAQP4284-299, or with whole-length hAQP4 protein in serum of immunized mice was quantified longitudinally. Since the NMO-IgG is a human IgG1 isotype, both, the murine IgG2a and IgG2b isotype were examined as they have similar properties with regard to complement binding and the Fcγ receptor. A switch from IgM to IgG2b was detected in mice immunized with hAQP4_281-300 peptide with regard to (B) antibody responses against hAQP4_281-300 and (C) whole-length AQP4 protein. An Ig isotype switch from IgM to IgG2b was also detectable in mice immunized with whole-length AQP4 protein with regard to (D) antibody responses against hAQP4_281-300 and (E) whole-length AQP4 protein.</p

Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

<i>HLA-DRB1*03</i>:<i>01</i> transgenic mice are disease resistant to active immunization with human aquaporin 4 (hAQP4), and adoptive transfer of hAQP4-specific T cells.

<p>(A) <i>HLA-DRB1*03</i>:<i>01</i> mice were actively immunized with proteolipid protein (PLP)_91-110 (100 μg/100 μl/mouse; positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152720#pone.0152720.ref025" target="_blank">25</a>]), or varying AQP4 antigens*(whole-length hAQP4 protein, hAQP4_281-300, murine (m)AQP4_281-300, hAQP4_281-300 with a Quil-A Incomplete Freund Adjuvant (IFA) booster on day 14 post-immunization, mAQP4_281-300 with a Quil-A IFA booster on day 14 post immunization, and hAQP4_281-300 plus mAQP4_281-300) emulsified in Complete Freund Adjuvant (CFA). Immunization with a positive control proteolipid protein (PLP)_91-110, a dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> led to typical EAE. (B) Lymph node cells taken from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 or mAQP4_281-300 were restimulated for three days and passively transferred into <i>HLA-DRB1*03</i>:<i>01</i> mice. None of these experimental approaches resulted in clinical disease. (C) Paraffin sections were stained with haematoxlin eosin (H&E) and luxol fast blue (LFB). Representative sections of the spinal cords from PLP_91-110 and hAQP4_281-300 immunized mice are shown. On histopathological examination there were no visible signs of cellular infiltration, inflammation, or demyelination within the brain and spinal cord in any experimental paradigms other than in active immunization with PLP_91-110, the dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> that led to typical EAE (spinal cord shown; inflammatory infiltrates and areas of demyelination are indicated by black arrows). (D) Fifteen days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with PLP_91-110 or hAQP4_281-300, pupillary reflex was measured via a mouse pupillometry. Mice actively immunized with hAQP4_281-300 and the control antigen PLP_91-110 did not show altered pupillary responses.</p