iNOS-deficiency does not impact polyp number in APC^Min/+MSH2

<p>^−/−<b>mice.</b> Polyp count in the small intestine (A) or in the colon (B) of APC^Min/+MSH2^+/− (A^+/−M^+/−) mice and APC^Min/+MSH2^−/− (A^+/−M^−/−) mice that were iNOS proficient (N^+/−) or iNOS deficient (N^−/−).</p

Nitric oxide induces DNA mutations that are repaired by the MMR pathway.

<p>(A) Measurements of the amounts of nitric oxide in culture medium generated by various concentrations of SNAP or by macrophages that were stimulated by LPS and IFN-γ. (B) The mutant frequency at the <i>lac</i>I gene in Msh2^+/− (WT, n = 3) and Msh2^−/− (n = 3) SNAP-treated or non-treated macrophages was measured by the Big Blue mutagenesis screen. Bone marrow-derived macrophages were cultured in presence of 150 µM SNAP for 20 hrs. Genomic DNA was isolated and the mutant frequency at <i>lac</i>I gene was calculated. (C) Mutation spectrum in SNAP-treated macrophages as determined by sequencing analysis. The C:G → T:A transitions are shown. The tranversion mutations include C:G → A:T, C:G → G:C, T:A → G:C and T:A → A:T substitutions. The insertions and deletions are presents as other mutations. (D) The mutant frequency at the <i>hprt</i> gene in Msh2^+/− (WT, n = 8) and Msh2^−/− (n = 8) SNAP-treated or non-treated Pre-B cells. (E) The mutant frequency at the <i>hprt</i> gene in SW680 (n = 8) and DLD-1 (n = 8) SNAP-treated or non-treated human colorectal cancer cell lines.</p

Anti-MOG antibodies generated in WT and AID^−/− mice.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and titres of anti-MOG Ab were evaluated at day 15 (peak of disease). As expected, AID^−/− mice only made anti-MOG Ab of the IgM isotype, whereas WT mice produced anti-MOG IgG1 and IgG2c Ab (and very little IgM at this time point). The average OD for each mouse at a set concentration of serum (half-way point of the curve) is depicted, and raw OD values can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s003" target="_blank">Figure S3</a>. In this experiment, 5–6 mice per group were tested and an additional experiment tested 8–12 mice.</p

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

<div><p>Background</p><p>Clinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.</p><p>Methods</p><p>Activation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID^−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID^−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).</p><p>Conclusions</p><p>AID^−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.</p></div

Summary of EAE experiments.

<p>Summary of EAE experiments.</p

Appearance of class switched B cells in the CNS during EAE.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and spinal cords were extracted and processed at day 15 (peak of disease). B cells were identified by the expression of CD19 and B220. Class switched B cells were further identified by the absence of IgM/IgD and reported here as a percentage of the total number of B220⁺CD19⁺ B cells. Representative FACS analysis can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s002" target="_blank">Figure S2B</a>.</p

Lymph node cells from WT and AID^−/− mice produce equivalent levels of cytokines in response to immunization with rhMOG.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and draining axillary and brachial lymph nodes were harvested after 7 days post-immunization. Four million lymph node cells were plated along with 20 µg of rhMOG. Cultures were kept for 48 hours after which the supernatant was harvested and evaluated for cytokine production by ELISA. The limit of detection of the assay is 125 pg/ml for the IFNγ ELISA and 63 pg/ml for the IL-17 ELISA. Five mice per group were assessed.</p

Characteristics of the mutations within the <i>lacI</i> gene in Msh2 WT and Msh2^−/− macrophages that were SNAP-treated or untreated.

<p>Characteristics of the mutations within the <i>lacI</i> gene in Msh2 WT and Msh2^−/− macrophages that were SNAP-treated or untreated.</p

Impaired accumulation of cytokine producing CD4⁺ T cells in the CNS of AID^−/− mice.

<p>WT and AID^−/− mice were immunized with recombinant human MOG (rhMOG) and spinal cords (<b>A</b>) and brains (<b>B</b>) were extracted and processed at day 15: the peak of disease. Leukocytes were stimulated <i>ex vivo</i> with PMA/Ionomycin, and brefeldin A was added in the last 4 hours. Cells were then subjected to surface and intracellular cytokine staining. This experiment was performed twice with similar results and 6 mice per group were tested in this experiment. Representative FACS analysis can be seen in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone.0061478.s002" target="_blank">Figure S2A</a>.</p

AID^−/− mice exhibit attenuated EAE in response to rhMOG but not MOG_35-55.

<p>(<b>A</b>) WT and AID^−/− mice were immunized with MOG_35-55 in CFA and examined for clinical symptoms. A representative experiment is shown, and 2 experiments were performed with similar results (shown here are n = 6 WT and n = 5 AID^−/− mice per group). (<b>B</b>) WT and AID^−/− mice were immunized with rhMOG in CFA and examined for clinical symptoms. A representative experiment is shown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061478#pone-0061478-t001" target="_blank">Table 1</a> for all 4 experiments) with n = 11 WT and n = 7 AID^−/− mice per group. At the termination of the experiment, spinal cords were dissected and stained with H&E and counterstained with Luxol fast blue. A representative example of 6 separate mice is shown (original magnification ×200). See arrows for areas of cellular infiltrates (<b>C</b>). Cell infiltration in the spinal cord tissue was assessed as follows: meningeal infiltrate (score 1), perivascular infiltrate (score 2), parenchymal infiltrate (score 3) (<b>D</b>).</p