University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Veterinary and Biomedical Science Veterinary and Biomedical Sciences, Department of 2015 Association of Autophagy in the Cell Death Mediated by Dihydrotestosterone in Autoreactive T Cells Independent of Antigenic Stimulation Ting Jia University of Nebraska-Lincoln, [email protected] Annandurai Anandhan University of Nebraska-Lincoln, [email protected] Chandirasegaran Massilamany University of Nebraska-Lincoln, [email protected] Rajkumar A. Rajasekaran University of Nebraska-Lincoln, [email protected] Rodrigo Franco University of Nebraska-Lincoln, [email protected] SeFoe nelloxtw pa thige fors aaddndition addal aitutionhorsal works at: http://digitalcommons.unl.edu/vetscipapers Part of the Biochemistry, Biophysics, and Structural Biology Commons, Cell and Developmental Biology Commons, Immunology and Infectious Disease Commons, Medical Sciences Commons, Veterinary Microbiology and Immunobiology Commons, and the Veterinary Pathology and Pathobiology Commons Jia, Ting; Anandhan, Annandurai; Massilamany, Chandirasegaran; Rajasekaran, Rajkumar A.; Franco, Rodrigo; and Reddy, Jay, "Association of Autophagy in the Cell Death Mediated by Dihydrotestosterone in Autoreactive T Cells Independent of Antigenic Stimulation" (2015). Papers in Veterinary and Biomedical Science. 183. http://digitalcommons.unl.edu/vetscipapers/183 This Article is brought to you for free and open access by the Veterinary and Biomedical Sciences, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Veterinary and Biomedical Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Ting Jia, Annandurai Anandhan, Chandirasegaran Massilamany, Rajkumar A. Rajasekaran, Rodrigo Franco, and Jay Reddy This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/vetscipapers/183 Published in Journal of Neuroimmune Pharmacology 10 (2015), pp. 620–634; doi: 10.1007/s11481-015-9633-x Copyright © 2015 Springer Science+Business Media New York. Used by permission. Submitted April 29, 2015; accepted September 15, 2015; published online September 29, 2015. digitalcommons.unl.edudigitalcommons.unl.edu Association of Autophagy in the Cell Death Mediated by Dihydrotestosterone in Autoreactive T Cells Independent of Antigenic Stimulation Ting Jia, Annandurai Anandhan, Chandirasegaran Massilamany, Rajkumar A. Rajasekaran, Rodrigo Franco, and Jay Reddy School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln Corresponding author — Jay Reddy, Room 202, Bldg. VBS, Lincoln, NE 68583, USA; [email protected] Abstract Introduction Gender disparity is well documented in the mouse model Multiple sclerosis (MS) is thought to be an autoimmune of experimental autoimmune encephalomyelitis (EAE) in- disease, but the antigens that induce it are unknown (Hae- duced with proteolipid protein (PLP) 139–151, in which fe- gert and Marrosu 1994). Although men are less likely than male, but not male, SJL mice show a chronic relapsing-re- women to develop MS, they tend to develop a more severe, mitting paralysis. Furthermore, dihydrotestosterone (DHT) chronic form of the disease (Compston et al. 2006). Disease has been shown to ameliorate the severity of EAE, but the severity in female MS patients is reduced during pregnancy, underlying mechanisms of its protective effects are unclear. but the symptoms of MS return to their pre-pregnant state Using major histocompatibility complex (MHC) class II dex- within 6 months post-delivery (Confavreux et al. 1998). Fur- tramers for PLP 139-151, we tested the hypothesis that DHT thermore, reduced severity of MS during pregnancy is corre- selectively modulates the expansion and functionalities of lated with higher estriol levels, and oral estriol treatment of antigen-specific T cells. Unexpectedly, we noted that DHT in- relapsing-remitting MS patients reduces the number of en- duced cell death in antigen-specific, autoreactive T cells, but hancing lesions seen on magnetic resonance imaging (Soldan the effects were not selective, because both proliferating and et al. 2003). Such an alteration in the disease course, although non-proliferating cells were equally affected independent attributed to elevated amounts of T helper (Th) 2 cytokines of antigenic stimulation. Furthermore, DHT-exposed PLP during pregnancy, may be due to additional factors. 139- 151-specific T cells did not show any shift in cytokine To determine the mechanisms of gender differences in production; rather, frequencies of cytokine-producing PLP- susceptibility to central nervous system (CNS) autoimmu- specific T cells were significantly reduced, irrespective of T nity, mouse models of experimental autoimmune encepha- helper (Th) 1, Th2, and Th17 subsets of cytokines. By evalu- lomyelitis (EAE) are commonly employed. EAE can be in- ating cell death and autophagy pathways, we provide evi- duced by immunizing susceptible mouse strains with the dence for the induction of autophagy to be associated with CNS myelin antigens, which include myelin proteolipid cell death caused by DHT. Taken together, the data provide protein (PLP), myelin basic protein (MBP), and myelin ol- new insights into the role of DHT and indicate that cell death igodendrocyte glycoprotein (MOG), or their corresponding and autophagy contribute to the therapeutic effects of andro- immunogenic peptide fragments PLP 139-151, MBP 89-101/ gens in autoreactive T cells. MBP Ac1-11, and MOG 35-55 in complete Freund’s adju- Keywords: DHT, Myelin proteolipid protein, MHC class II vant (Miller et al. 2007; Constantinescu et al. 2011; McCar- dextramers, Autoreactive T cells, Apoptosis, Autophagy thy et al. 2012). While gender bias in susceptibility to EAE 620 Autophagy in DHT-induced cell death in autoreactive T cells 621 has been noted with both PLP and MBP antigens, EAE in- equally affected, independent of antigenic stimulation, but duced with PLP 139-151 has been commonly used, because with no shift in the production of cytokines. it results in manifestations of the disease phenotypes that are distinct by gender: only females develop chronic relaps- ing-remitting paralysis (Bebo et al. 1996). However, clinical Materials and Methods relapses could be induced in males by orchidectomy, point- ing to the pivotal role androgens play in the regulation of Mice CNS autoimmunity (Bebo et al. 1998). Consistent with this notion, the therapeutic efficacy of androgens has been tested Five-to-six-week-old male and female SJL/J (H-2s) mice were in EAE, with the result that both dihydrotestosterone (DHT) procured from the Jackson Laboratory (Bar Harbor, ME). and dihydroepiandrosterone have been shown to ameliorate The mice were maintained according to the animal proto- the severity of EAE when administered either during the in- col guidelines of the University of Nebraska–Lincoln, Lin- duction or effector phase of the disease (Dalal et al. 1997; coln, NE. Du et al. 2001; Palaszynski et al. 2004). Additionally, female mice treated with DHT experience less severe EAE accom- Peptide Synthesis and Immunization Procedures panied by enhanced interleukin (IL)-10 production (Dalal et al. 1997). These observations suggest that androgens act on PLP 139-151 (HSLGKWLGHPDKF) and neuraminidase T cells and may mediate anti-inflammatory effects. In sup- (NASE) 101–120 (EALVRQGLAKVAYVYKPNNT) were syn- port of this hypothesis, expression of an androgen recep- thesized on 9-fluorenylmethyloxycarbonyl chemistry (Neo- tor has been demonstrated in splenocytes and thymocytes peptide, Cambridge, MA). All peptides were HPLC-purified in mice and humans (Kovacs and Olsen 1987; Benten et al. (>90 %), identity was confirmed by mass spectroscopy, and 1999; Benten et al. 2002). the peptides were dissolved in 1 × phosphate-buffered sa- Furthermore, using the EAE adoptive transfer protocol, line (PBS) prior to use. To generate primary T cell cultures we and others have demonstrated that PLP 139-151-reactive for PLP 139-151, SJL mice were immunized subcutaneously T cells generated in male SJL mice induce EAE in females, (s.c.) with PLP 139-151 emulsified in complete Freund’s adju- but severity is low and the clinical signs of EAE take a long vant in multiple sites in the inguinal flank and sternal regions time to appear. On the contrary, cells from female mice in- (100 μg/ mouse) (Miller et al. 2007; Massilamany et al. 2010). duce EAE in males with a disease severity comparable to its severity in female recipients of female cells (Bebo et al. T Cell Proliferative Assay 1999; Massilamany et al. 2011b). These data suggest that male hormones play an important role in controlling au- Single-cell suspensions were prepared from the draining toimmune responses. Because PLP 139-151-induced EAE lymph nodes, hereafter termed lymph node cells (LNCs), is classically a CD4 T cell-mediated disease, any immuno- from PLP-immunized mice on day 10 post-immunization modulatory effects exerted from the use of androgens are or from naïve mice, and the cell pellets were resuspended in expected to influence mainly this subset of T cells. Consis- 1 × ammonium chloride potassium buffer (Lonza, Walkers- tent with this notion, DHT treatment of myelin-reactive T ville, MD) to lyse erythrocytes as described (Massilamany cells has led to deviation of immune response from a pro- et al. 2011a). After washing, cell pellets were resuspended in inflammatory