NOD2 Mutations and Anti-Saccharomyces cerevisiae Antibodies Are Risk Factors for Crohn's Disease in African Americans

NOD2 mutations and anti-Saccharomyces cerevisiae antibodies (ASCA) are associated with Crohn’s disease (CD), ileal involvement and complicated disease behavior in whites. ASCA and the three common NOD2 mutations have not been assessed in African American (AA) adults with CD

Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6.

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.To model DILI, we immunized BALB/c, BALB/cBy, IL-6-deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI

No sex bias in baseline Treg function in BALB/c mice.

<p>(A) There was no sex bias in proliferation of T-effector cells (n = 10/group) co-cultured with naïve Tregs (Treg∶T-effector ratio 1∶1) in the presence of anti-CD3 (5 µg/mL) and anti-CD28 (2.5 µg/mL). (B) Using FACS analysis, of Tregs co-cultured as described in A, intracellular expression of inhibitory molecules PD-1(CD279, cloneJ43) was similar in female and male mice, and CTLA-4 (CD152, cloneUC10-489) was not detected in either sex (Experiments run in duplicate, n = 5–6 mice/group naive = filled histogram, stimulated = open histogram *p<0.05, **p<0.01, ***p<0.001).</p

Tregs reduce experimental anesthetic DILI in female BALB/c mice.

<p>Two weeks after the TFA-S100 immunizations, mice were given intravenous injections of 1×10⁶ freshly isolated Tregs in PBS or PBS alone (control); the mice were killed 7 days later. The degree of hepatitis was significantly reduced in mice administered Tregs compared to those administered vehicle. Data are shown as mean ± SEM., n = 8–10 mice/group, Mann-Whitney <i>U</i>, *p<0.05, **p<0.01, ***p<0.001.</p

17β-estradiol (E2) induces pro-inflammatory cytokines in naïve splenocytes from BALB/c mice.

<p>17β-estradiol (E2, 50 nM) increased IL-1β production in splenocytes cultured from naïve male BALB/c mice when compared to splenocytes cultured from naïve male BALB/c mice treated with β-cyclodextrin (BC, 5 mM) as a control (A, *p<0.05). E2 significantly increased IL-6 (B) and TNF-α (C) production in splenocytes cultured from naïve females when compared to splenocytes from naïve females treated with BC (*p<0.05). (Experiments run in duplicate, n = 5/group, Mann-Whitney <i>U</i>, * = p<0.05).</p

Anti-IL-10 antibody diminishes Tregs in TFA-S100 – immunized male and female splenocyte cultures in vitro.

<p>IL-10 blocking antibody (rat anti-mouse, 10 µg/mL), similarly diminished Tregs in female (A) and male (B) BALB/c mice isolated from TFA-S100- immunized mouse splenocytes challenged with CYP2E1 or the TFA (TFA-OVA) (Mann-Whitney <i>U</i>, *p<0.05, **p<0.01, ***p<0.001, experiments run in duplicate, n = 3 mice/group).</p

Increased T and B cells are associated with impaired splenic Treg expansion in female BALB/c mice following TFA-S100 immunizations.

<p>Splenocytes were isolated from female mice 2 weeks after TFA-S100 immunizations, stimulated with CYP2E1 (A) or TFA (B) <i>in vitro</i> (10 µg/mL) and analyzed for Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061186#pone-0061186-g006" target="_blank">Figure 6</a>. CD3+CD25 T cells and B220+ cells were significantly elevated in the same cultures from females when compared to males. (Experiments run in duplicate, n = 3 mice/group, Mann-Whitney <i>U</i>, * = p<0.05, ** = p<0.01, *** = p<0.001).</p

TFA-S100 immunizations impair splenic Treg expansion in female BALB/c mice.

<p>(A) Representative dot plot of gated CD4+ T-cells and scatter plot showing no sex differences in hepatic CD4+CD25+FoxP3+ cell (Treg) levels 3 weeks after TFA-S100 immunizations (n = 5/group). (B) Representative dot plot of gated CD4+ T-cells and scatter plot showing significantly elevated splenic Tregs in males 2 weeks after immunizations (Mann-Whitney <i>U</i>, n = 15/group *p<0.05, **p<0.01, ***p<0.001).</p

IL-6Rα antibodies diminish anesthetic DILI in female BALB/c mice.

<p>Female BALB/c mice were immunized with TFA-S100 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061186#s2" target="_blank">Methods</a> section. (A) IL-6Rα antibodies administered days 0 and 7 diminished hepatitis as demonstrated by decreased inflammation/injury scores (1.6±0.1, mean ± SE) when compared to TFA-S100 – immunized, female , BALB/c mice not treated with IL-6Rα but administered phosphate buffered saline at the same time points (2.3±0.2, mean ± SE). (B) Anti-TFA IgG and IgG2a subclass antibody levels measured in mouse sera were also decreased (p<0.05) when compared to mice immunized with TFA-S100 without blocking antibodies. (B) S100 and (C) CYP2E1 autoantibody levels were not significantly different when comparing sera of mice immunized with TFA-S100 ± anti-IL-6Rα. (Mann – Whitney <i>U</i>, n =  5/group, *p<0.05).</p