Immune cells from human blood and murine spleen express mRNA of the prolactin receptor short isoform 1b and S3 respectively, without being affected by stress

MARÍA TAMARA MORENO SOSA; JUAN PABLO MACKERN OVERTI

Mendoza

Simposio; II International Symposium on Translational Medicine; 2019

Secretaría de Posgrado, RR II y Extensión Facultad de Ciencias Médicas - UNCuyo

Autoimmune diseases are multifactorial diseases in which environmental factors, hormonal status and genetics are involved. However the precise contribution of psychological stress exposure to autoimmune aetiology remains unclear. Furthermore, the role of psychological stress exposure in worsening spontaneous experimental T cell autoimmunity is still understudied.The stress response is triggered by the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which induces the corticotropin-releasing factor - adrenocorticotropic hormone -cortisol cascade which may impact in several tissues including the immune system. Additionally, adrenal epinephrine and pituitary PRL are also secreted in response to stress. PRL displays many physiological functions including immune modulation, mammary gland development and different effects on the central nervous system including stress responses. All functions of PRL are mediated by PRL receptors (PRL-R). PRL-R exists as long and short isoforms produced by alternative splicing of the intracellular cytoplasmic domain of the gene being the long and short the activating and inhibitory isoforms respectively. It is known that B cells treated with estrogen increase the expression of PRL-Rs transcripts which in turn may promote autoimmunity. Although PRL secretion in response to stress are well described, there is no information about changes in PRL-R mRNA expression in immune cells as well as whether stress would enhance autoimmunity. Our aim was to investigate whether PRL-Rs mRNA expression are expressed in immune cells and whether chronic stress enhances development of T cell mediated autoimmunity increasing PRL-Rlong mRNA expression. To answer these question, we evaluate the presence of mRNA PRL-Rlong and short isoforms in human and murine immune cells. Additionally, we used an in vivo approach consisting in Type 1 Diabetes susceptible NOD and Balb/c mice being exposed to chronic unpredictable stress (CUS) consisting in one of five different stressors (restraint, isolation, forced swim, change light-dark cycle and tilting home cage at 45° inclination for 24 hours) randomly chosen daily for 3 months. Following stress, immune cell infiltration in pancreas were evaluated by flow cytometry and histology. Using specific primers for human and murine isoforms we evaluated gene expression by Real Time PCR. PCR products were visualized by agarose gel electrophoresis stained with SYBRGold. We found that murine as well as human leukocytes express PRL-RLong and also PRL-RShort. These results demonstrate that immune cells express both prolactin receptor isoforms. To our knowledge this is the first report showing that human leukocytes express PRL-RShort. It has been reported that stress induces several behavioral alterations including locomotor activity. To determine whether autoimmune susceptible host showed an altered behavior compared to a control strain Balb/c and NOD mice were exposed to CUS for three months and locomotor activity were evaluated by the open field test prior and post experimental procedure. The increased mean of locomotor activity in both, NOD and Balb/c CUS group compared to NOD and Balb/c untreated groups reflected by the average number of fields entered in the open field test suggest that stress modifies behavior in both strains. Interestingly, mice strains respond to stress by different behavior. NOD CUS mice displayed motor activity than Balb/c CUS mice. Additionally, stressed NOD mice gained less weight at the end of the protocol than untreated NOD mice. The finding that CUS differentially altered behavior between NOD and Balb/c mice indicates that these strains may have a differential stress responses. To evaluate whether this disparity in the behavior response to CUS treatment is due to differential HPA axis activity between both strains, we determined corticosterone levels in serum samples collected at the end of the study. Interestingly, despite NOD and Balb/c mice displayed different behavior in response to stress, this condition induce the production of similar levels of corticosterone in both, NOD and Balb/c, strains compared to untreated mice (Figure 1). To assess whether stress worsens autoimmunity we evaluated the infiltration of immune cells within the pancreas after CUS treatment. We determined the frequency of total leukocytes by flow cytometry assays staining pancreatic cell suspensions derived from untreated and CUS groups of NOD and Balb/c mice with anti-CD45 monoclonal antibodies. We found that CUS treatment enhanced the frequency of CD45+ cells only in NOD mice compared to UT NOD group and both, untreated and CUS, Balb/c groups (Figure 2 A and B). To test whether the CD45+ cells increase in pancreas after stress was due to higher infiltration of T cells we evaluated the presence of CD4+ and CD8+ T cells by staining pancreatic cell suspensions with anti-CD4 and -CD8 monoclonal antibodies. As shown in Figure 2, CD4+ T cells from NOD CUS treated mice are increased compared to UT group while CD8+ T cells were unaffected (Figure 2 C to F). In contrast to NOD mice, Balb/c mice showed no differences in CD45+ nor CD4+ T cell infiltration in pancreas after stress treatment suggesting no autoimmunity development (Figure 2 A, C and E). Thus, leukocyte infiltration of the pancreas in autoimmune susceptible NOD mice could be modulated by chronic stress. To assess where is located the stress-induced infiltration in target organ we conducted histological studies of the pancreas. Surprisingly, CUS increased mononuclear cell infiltration in pancreas interlobular areas of NOD mice (Figure 3 A and B). In contrast, pancreatic islets areas displayed similar amount of infiltration in untreated and CUS NOD mice groups (Figure 3 C and D). As expected, both Balb/c mice groups, untreated and CUS, did not displayed pancreatic infiltration. These results suggest that stress strengthen T cell recruitment to specific areas of target organ in autoimmune susceptible mice.To determine whether immune cells from autoimmune susceptible NOD mice are differentially modulated by stress to be more sensitive to the stress-response hormone prolactin, we evaluate the expression of the activating PRL-R Long and the short isoforms in spleen after CUS procedure. Interestingly, we found no changes in mRNA expression in either PRL-Rs isoforms. Although, these results suggest that chronic psychological stress enhances T cell mediated autoimmunity progression in NOD mice, whether PRL-receptors are involved in the disease progression remains unknown. None the less, our data indicate that chronic stress may be a potential aetiological agent of autoimmune diseases.