Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia

https://doi.org/10.1016/j.bbi.2011.01.009Get rights and content

Abstract

Stroke is a sexually dimorphic disease with male gender considered a disadvantage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B, and CD11b+ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b+ blood cells solely in DHT-replaced animals at 24 h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96 h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.

Introduction

Male sex is considered a risk factor for stroke. Stroke occurs more frequently in men than women (Giroud et al., 1991) and outcomes after experimental stroke are worse in male animals relative to females (Herson et al., 2009). Although most studies have focused on brain outcome, it is becoming increasingly clear that stroke induces profound effects on the peripheral immune system (Dirnagl et al., 2007, Meisel et al., 2005). For example, male mice demonstrate initial activation (6–24 h) (Offner et al., 2006a) followed by suppression (96 h) of the peripheral immune system after MCAO, as evidenced by profound reduction of spleen size, splenocyte numbers and proliferative response to mitogens (Offner et al., 2006b, Vendrame et al., 2006). Of the remaining viable splenocytes, T cell abundance is curiously increased, including immunosuppressive regulatory T cells (Treg), while B lymphocytes are decreased (Liesz et al., 2009a, Offner et al., 2006b). In blood, CD11b+ cells are greatly increased (Offner et al., 2006b). These abnormalities are important, because post-stroke immunosuppression in humans and animals results in high frequency of pneumonia and septicemia (Chamorro et al., 2007, Dirnagl et al., 2007, Liesz et al., 2009a, Meisel et al., 2005). Furthermore, the importance of understanding contributors to peripheral immune suppression after stroke is highlighted by the fact that fatal infection is the most common cause of death in humans who have survived the initial stroke (Chamorro et al., 2007, Dirnagl et al., 2007, Meisel et al., 2005).

Peripheral immunosuppression is the proximate mediator of increased risk for pneumonia and septicemia post-stroke. Mice develop spontaneous pneumonia and septicemia within days after focal cerebral ischemia in parallel with the downregulation of the innate and adaptive immunity (Dirnagl et al., 2007). Animals that suffer from severe leukocyte depression show bacterial growth in blood cultures and in lung homogenates (Liesz et al., 2009a). Likewise, Prass et al. (Prass et al., 2003) observed decreased monocyte and T cell activation in animals that developed spontaneous bacterial infections after stroke. T cells are crucial in combating bacterial infection and adoptive transfer of T cells from wild-type mice but not IFNγ deficient mice inhibits stroke-induced bacterial infections demonstrating a link between cell-mediated immune response and increased susceptibility to bacterial infection.

Sex hormones modulate immune function during normal and disease-related processes (Liu et al., 2009). Testosterone, the primary circulating male hormone, and its more potent metabolite dihydrotestosterone (DHT), have well-established immunosuppressive effects in the periphery following disease and injury (Olsen and Kovacs, 1996, Palaszynski et al., 2004). Despite androgens’ well-recognized immunosuppressive potential, the importance of the intracellular androgen DHT to post-ischemic immune function has not been well-examined. We hypothesized that DHT heightens post-stroke immunosuppression and contributes in this manner to adverse outcomes in the male.

Section snippets

Animals

All experiments were conducted in accordance with the National Institutes of Health guidelines for the use of experimental animals. Age-matched sexually mature 8–10 weeks of age, male mice C57BL/6J; Charles River Laboratories, body weight 20–25 g were used in all experiments.

Castration and DHT replacement

Castration was performed in male mice 1 week before transient focal cerebral ischemia. At the time of castration, DHT was replaced via 30 day release subcutaneous pellets containing 0.5 mg DHT (Innovative Research of America,

Plasma hormone levels

As expected plasma DHT levels were lower in castrated (0.19 ± 0.09 pg/mL) (n = 12) vs in DHT-replaced mice (0.97 ± 0.32 pg/mL) (n = 18) at 96 h post-reperfusion. The plasma DHT levels from DHT-replaced animals in our study were similar to endogenous levels observed in intact naïve male mice (Uchida et al., 2009). Plasma total testosterone levels were low and similar between groups; 0.21 ± 0.013 ng/mL in castrated mice vs 0.15 ± 0.07 in DHT-replaced mice. Free testosterone levels were 0.0 ± 0.0 ng/mL in both

Discussion

These data provide the first evidence that post-ischemic peripheral immunosuppression is exacerbated by the potent androgen DHT. While MCAO reduced spleen cell density in all animals, DHT further altered surviving cell subsets. Blood-borne CD11b+ cells were also more strongly elevated in DHT-replaced animals during the early post-stroke period. DHT reduced splenocyte proliferation in response to TCR stimulation and increased splenic CD4+CD25+FoxP3+ Tregs by 96 h post-MCAO. This was in striking

Conflict of interest statement

The authors have no financial conflicts of interest.

Acknowledgments

This work was supported by NIH Grant NR003521 (PDH), AHA Grant 09POST2190040 (SD). Special thanks to Dr. Xuefang Ren for helpful discussions throughout the preparation of the manuscript.

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    S.D. and K.A. are co-first authors.

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