Nur77 deficiency leads to systemic inflammation in elderly mice

Recent studies suggest that Nur77 deficiency in mice enhances atherosclerosis [3, 4] and LPS-induced sepsis [15], suggesting that Nur77 is implicated in inflammation. To further evaluate the potential role of Nur77 in inflammation in vivo, Nur77^−/− mice were observed in this study. We compared splenic differences in 2-month-old Nur77^−/− mice and wild-type (WT) mice and found no significant differences in the size and weight of their spleens (Fig. 1a). Interestingly, we found that 8-month-old Nur77^−/− mice were more prone to develop splenomegaly (Fig. 1b, left) and increased spleen weight (Fig. 1b, right). Thus, these observations indicated that Nur77 deficiency was associated with an increased inflammatory pathology in elderly mice. Our results were consistent with a recent report that Nur77^−/− mice displayed hyperplasia in the jejunum and colon, and increased crypt depth and villus length of the jejunum and colon [16].

Our investigation further showed that Nur77 deficiency in elderly mice led to exacerbation of inflammatory pathology as characterized by increased inflammatory cell infiltration and inflammatory cytokines production. As shown in Fig. 1c, Nur77^−/− mice had more severe inflammatory cell infiltration in the liver, lung, spleen and kidney, and more hyperplasia of fibrous tissue in the lungs. The elderly Nur77-deficient mice also exhibited increased susceptibility to glomerulonephritis with enlargement of the kidney glomeruli, which can be linked to systemic disorders or certain infections [17, 18]. Additionally, the spleens of elderly Nur77^−/− mice showed expansion of red pulp and decreased white pulp than WT mice (Fig. 1c). Combined, these phenotypical findings suggest a progression of systemic inflammation in Nur77^−/− elderly mice.

Changes in inflammatory cytokines expression are known to readily occur in systemic inflammation [19]. Unsurprisingly, the mRNA expression of pro-inflammatory cytokines, including Tnfα and Il6, was higher in the liver and spleen tissues (Fig. 2a) from Nur77^−/− mice than those from Nur77^+/+ mice. The concentration of IL-6 in serum of 8-month-old Nur77^−/− mice was also elevated (Fig. 2b). There were no differences in the gene expression of Tnfα and Il6 in 2-month-old mice in the two groups (Additional file 1: Figure S1a). A recent study found that mice lacking all Nr4a receptors including Nur77 had impaired regulatory T cell (Treg) development and resulted in systemic lethal autoimmunity [20], suggesting a potential role of Nr4a receptors in autoimmune disease. Nur77 has also been suggested to be implicated in Treg differentiation and self-reactive T cells selection [21, 22]. Herein, we also investigated whether Nur77 deficiency in mice was involved in the pathogenesis of autoimmune disease. Analysis of serum immunoglobulin showed increased IgG1 and IgE in elderly Nur77^−/− mice than in WT mice (Fig. 2c). However, we did not observe any differences in the Ig levels in younger 2-month-old mice in the two groups (Additional file 1: Figure S1b). Together, these data indicate that elderly Nur77^−/− mice developed systemic inflammation and autoimmunity, supporting a role of Nur77 in inflammation and immunity. Our results are consistent with previous reports that Nur77^−/− mice showed increased susceptibility to atherosclerosis [4, 14] and restenosis [10], and demonstrate that Nur77 has functional roles in protecting mice from inflammatory and autoimmune diseases.

Accumulating studies have shown that polarization of macrophages occurs in response to inflammatory diseases [23]. Two opposite and competing macrophage phenotypes, M1 (pro-inflammatory) and M2 (anti-inflammatory), have been defined and identified in a range of physiologic and pathological processes [23]. Several genes, including CXCL11, INDO, and MRC1, have been shown to discriminate between M1 and M2 macrophages [24]. M1 phenotype is typically associated with an increased CXCL11 and INDO expression but associated with a decreased MRC1 expression [24]. In this study, we isolated peritoneal macrophages from elderly 8-month-old Nur77^+/+ and Nur77^−/− mice, and found that Nur77 deficiency in mice significantly enhanced Cxcl11 and Indo mRNA expression, but reduced Mrc1 expression (Fig. 3a), indicating that the absence of Nur77 in macrophages led to enhanced polarization of macrophages toward a pro-inflammatory M1 phenotype. M1-like macrophages are also generally considered to produce numerous pro-inflammatory mediators such as TNFα, IL-12, and IL-6 [25]. Similar phenomena were also observed in our current study. As shown in Fig. 3b, Nur77 deficiency in peritoneal macrophages markedly enhanced the expression of Tnfα and Il6, indicating that Nur77 plays an important role in regulation of macrophage phenotype and functions. These results are strongly supported by a recent report that Nur77 deletion polarizes macrophages toward an inflammatory phenotype and increases inflammatory cytokines production in atherosclerosis [14]. Together with our observations that increased inflammatory cells infiltration and inflammatory cytokines production in elderly Nur77^−/− mice (Figs. 1 and 2), thus, we speculate that Nur77-deficiency skews the macrophage phenotype to M1 subset, and subsequently leads to the development of systemic inflammation in elderly mice.