Role of testosterone in regulating induction of TNF-α in rat spleen via ERK signaling pathway

Highlights

• Testosterone withdrawal is associated with strong inflammation.

• Orchidectomy triggers NO production and LPS-evoked TNF-α secretion ex vivo.

• p-ERK protein expression was markedly enhanced on orchidectomized rats.

• Orchidectomy resulted in splenomegaly and promoted splenocyte proliferation rate.

• Testosterone attenuated inflammatory molecules secretion in vitro.

Abstract

Spleen is a pivotal organ for regulating immune homeostasis. It has been shown that testosterone diminishes secretion of various inflammatory molecules under multiple conditions. However, the mechanisms of action of endogenous testosterone affecting immune responses in the spleen remain unknown. The aim of the present study was to evaluate the immune functions of the spleen in response to testosterone withdrawal after orchidectomy, and the impact of splenocytes on the bacterial endotoxin lipopolysaccharide (LPS)-induced secretion of inflammatory molecules. Male rats were divided into 3 groups, i.e. intact, orchidectomized (Orch) and orchidectomized plus replacement of testosterone propionate (TP) (Orch + TP). The Orch and Orch + TP rats underwent bilateral orchidectomy one week before TP replacement (2 mg/kg body weight) or sesame oil in intact rats as controls for seven days. Orch resulted in a significant increase of spleen weight and basal secretion of nitric oxide (NO) from splenocytes. Additionally, LPS up-regulated cell proliferation and the secretion of tumor necrosis factor-alpha (TNF-α) in splenocytes of Orch rats. Orch further up-regulated phosphorylation of extracellular signal-regulated kinases. Interestingly, the plasma corticosterone concentration in the Orch group was higher than that in the intact and Orch + TP groups. Deficiency of testosterone-elevated TNF-α and NO secretion in response to LPS were confirmed in the rat splenocytes. Testosterone also significantly attenuated LPS-elicited release of TNF-α and NO in a dose-dependent manner. However, testosterone did not suppress splenic blastogenesis at doses in the 10⁻¹⁰–10⁻⁷ M concentration range. In this context, testosterone might have a protective role against inflammatory responses in the spleen. The present study provides evidence to indicate that testosterone might modulate the immune system.

Introduction

Testosterone, synthesized and secreted mainly by the Leydig cells of the testes [1], is responsible for maintaining spermatogenesis and secondary sexual characteristics [2], [3] such as increased bone and muscle mass [4], and sex drive in the male. Recently, there is an increasing interest in the immuno-endocrine system, including the relationship between testosterone and chronic inflammatory diseases. Low levels of testosterone have been linked to the increase of cardiovascular risk factor [5], mortality [6], and diabetes mellitus [7], [8]. These observations raised the possibility that testosterone exerts immunomodulatory effects.

On stimulation by the bacterial endotoxin lipopolysaccharide (LPS) [9], immune cells secrete pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 [10], [11] and/or other inflammatory molecules. In turn, these cytokines induce other host immune responses associated with inflammatory diseases, including sepsis [12], asthma [13], rheumatoid arthritis and inflammatory bowel disease [14]. TNF-α is a key mediator of inflammation. Recently TNF-α has been attributed a major role in the development of pathological processes in the promotion of expression of genes associated with inflammation [15] and in leukocyte extravasation [16]. Intriguingly, regardless of whether it is a physiological consequence of aging or a surgical intervention, low levels of testosterone have been associated with increased levels of TNF-α [11] or other inflammatory mediators [17], [18]. These findings, taken together with the observation of cytokine attenuations by testosterone, suggest that testosterone may exert potent anti-inflammatory effects.

Toll-like receptor 4 (TLR-4) recognizes specific patterns of microbial conserved components such as LPS, and activates mitogen-activated protein kinases, including extracellular signal regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and the p38 subfamilies consequently leading to the release of pro-inflammatory cytokines, such as TNF-α and IL-1β [19], [20]. Several experimental studies have documented that testosterone not only decreases the synthesis of pro-inflammatory cytokines [11], [21], [22], but also promotes the secretion of anti-inflammatory mediators such as IL-10 [23]. On the other hand, it has been shown that testosterone could abrogate LPS-induced cell proliferation [21] and down-regulate TLR-4 expression in macrophages [11] and non-immune cell types such as prostate smooth muscle cells [21]. Nevertheless, few works have studied the mechanism of the effects of testosterone on the signaling downstream of TLR-4. Moreover, little is known about the impact of immune responses in the spleen caused by testosterone withdrawal. The mechanisms underlying alterations of TLR-4 downstream proteins by testosterone deficiency are also unclear. Accordingly, the present study was carried out to determine the effects of testosterone depletion via orchidectomy on the secretion of TNF-α and TLR-4 in the downstream signaling pathway in the rat spleen.