Bryonolic Acid Transcriptional Control of Anti-inflammatory and Antioxidant Genes in Macrophages in Vitro and in Vivo

Bryonolic acid (BA) (<b>1</b>) is a naturally occurring triterpenoid with pleiotropic properties. This study characterizes the mechanisms mediating the anti-inflammatory and antioxidant activities of BA and validates the utility of BA as a tool to explore the relationships between triterpenoid structure and activity. BA reduces the inflammatory mediator NO by suppressing the expression of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 macrophage cells. In addition, BA robustly induces the antioxidant protein heme oxygenase-1 (HO-1) in vitro and in vivo in an Nrf2-dependent manner. Further analyses of Nrf2 target genes reveal selectivity for the timing and level of gene induction by BA in treated macrophages with distinct patterns for Nrf2-regulated antioxidant genes. Additionally, the distinct expression profile of BA on Nrf2 target genes relative to oleanolic acid suggests the importance of the triterpenoid scaffold in dictating the pleiotropic effects exerted by these molecules

Disruption of <i>Smad4</i> Expression in T Cells Leads to IgA Nephropathy-Like Manifestations

<div><p>The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (<i>Smad4^{co/co;Lck-cre}</i>). Loss of <i>Smad4</i> expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that <i>Smad4^{co/co;Lck-cre}</i> mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the <i>β</i>1, 4-galactosyltransferases (<i>β</i>4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced <i>β</i>4GalT2 and <i>β</i>4GalT4 mRNA levels in B cells. These findings indicate that <i>Smad4^{co/co;Lck-cre}</i> mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype.</p></div

Degree of glomerular deposition in kidneys.

<p>Degree of glomerular deposition in kidneys.</p

Deletion of <i>Smad4</i> in T cell induced predominant IgA deposition in glomeruli and proteinuria.

<p>(A) Immunofluorecence studies showed glomerular deposition of predominantly IgA, with lesser amounts of IgG and IgM deposition in <i>Smad4^{co/co;Lck-cre}</i> mice compared with WT mice at 3 months of age. C3 deposition was occasionally observed in both mesangial areas and Bowman's capsule in <i>Smad4^{co/co;Lck-cre}</i> mice, but was restricted to Bowman's capsule in WT mice. PAS staining showed no significant difference between WT and <i>Smad4^{co/co;Lck-cre}</i> mice. Original magnification; ×400 (B) Electron microscopic overview (left panels) and detail of podocytic foot processes (right panels) for <i>Smad4^{co/co;Lck-cre}</i> (bottom panels) and WT (top panels) mice. Podocyte foot process effacement was partially observed in glomeruli from <i>Smad4^{co/co;Lck-cre}</i> mice compared to WT mice. Scale bars: 5 µm (left panels); 0.5 µm (right panels). (C) Increased albumin creatinine ratio (ACR) was observed in urines of <i>Smad4^{co/co;Lck-cre}</i> mice (n = 9) compared with WT mice (n = 9) (0.043±0.005 vs 0.083±0.007). (D) There is no significant difference in the urinary hemoglobin between WT (n = 10) and <i>Smad4^{co/co;Lck-cre}</i> mice (n = 10) (14.7± 1.94 vs 21.2±2.36). Data are expressed as the mean±SEM; P values were calculated with Student's <i>t</i> test. ***P< 0.001</p

Loss of Smad4 signaling in T cells induces increased Th2 cytokine production by T cells.

<p>The levels of (A) IL-4 and (C) IL-13 in T cell supernatant were significantly increased in <i>Smad4^{co/co;Lck-cre}</i> mice (n = 10) compared with WT mice (n = 10). <i>Smad4^{co/co;Lck-cre}</i> mice tended to have higher (B) IL-5 levels than WT mice (P = 0.066). Data are expressed as the mean±SEM; P values were calculated with Student's <i>t</i> test.*P<0.05</p

Loss of Smad4 signaling in T cells induces high serum levels of IgA.

<p>The serum levels of IgA, IgM, IgG1, and IgG2a were determined using ELISA. (A) Markedly increased serum levels of IgA in <i>Smad4^{co/co;Lck-cre}</i> mice (n = 8) compared with WT mice (n = 8) (1773±294.3 vs 545.2±71.39). The results from serum levels of (B) IgM, (C) IgG1, and (D) IgG2a showed no significant differences between the two groups. Data are expressed as the mean±SEM; P values were calculated with Student's <i>t</i> test. ***P< 0.001</p

Characteristics of circulating IgA in mice lacking Smad4 in T-cells are similar to those in patients with IgA nephropathy.

<p>(A and B) Aberrant glycosylation of circulating IgA from <i>Smad4^{co/co;Lck-cre}</i> mice (n = 6, black square) and WT mice (n = 6, black triangle) was examined by lectin ELISA. Both (A) sialic acid and (B) galactose on circulating IgA from the mutants were lower than those from controls. Data are expressed as the ratios of specific lectin-binding IgA to total IgA. (C and D) <i>Smad4^{co/co;Lck-cre}</i> mice (n = 6, black square) exhibit an increased in both (C) IgG1-IgA complexes and (D) IgG2a-IgA complexes in the circulation, compared with WT mice (n = 6, black triangle). Data are expressed as mean±SEM; P values were calculated with <i>t</i> test or Bonferroni post test. ***P<0.001, **P<0.01, and *P<0.05 (E) The predominance of polymeric IgA was observed in serum from <i>Smad4^{co/co;Lck-cre}</i> mice, but not in WT mice, as determined by Western blot.</p

Levels of Th2 cytokines in serum of <i>Smad4^{co/co;Lck-cre}</i> mice.

<p>The levels of IL-4, IL-5, and IL-13 in the serum from <i>Smad4^{co/co;Lck-cre}</i> (n = 7) and WT (n = 7) mice are were determined by ELISA. No significant differences were observed between the two groups. Data are expressed as the mean±SEM; P values were calculated with Student's <i>t</i> test.</p

RT-PCR analysis of <i>β</i>4GalT mRNAs in <i>Smad4^{co/co;Lck-cre}</i> mice.

<p>mRNA was isolated from purified B-cells (>95% CD19+), and RT-PCR was performed using primers specific for <i>β</i>4GalT1 to 7, and GAPDH. <i>β</i>4GalT 2 and 4 mRNA expression in <i>Smad4^{co/co;Lck-cre}</i> mice (n = 4, black column) were significantly reduced relative to controls (n = 4, white column). All samples were normalized to GAPDH levels. Data are expressed as mean±SEM; P values were calculated with <i>t</i> test. *P<0.05</p

PCR Primers.

<p>βGalT, β1,4 galactosyltransferase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.</p