Increased spleen volume may occur in obesity, and can be considered a stable marker of inflammation, as well as of changes in the activation of splenic immune activity. In this regard, the expressions of pro inflammatory cytokines, such as, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) appear to be augmented in obesity 1, while interleukin-10 (IL-10), an anti-inflammatory cytokine synthesized in the marginal zone of the spleen (B cells), appears to be reduced 2. Thus, the bidirectional interaction between obesity and immune response disorders indicates association with splenic activity, a relationship that has only recently been recognized 3.

Obesity is also associated with vitamin deficiency, in particular, reduced vitamin D (VD) plasma status has been observed in obese subjects 4. Insufficient plasma levels of VD are responsible for disturbances in immune responses, with consequent effects on splenic immune activity 5. However, the exact mechanisms involved in the relationship between obesity and VD deficiency are unknown, as both conditions have direct effects on immune responses 6. Athletes who are VD deficient and aim to improve their physical performance, as well as, invigorate their immune responses, can be benefited with VD supplementation 7 associated with exercises. As previously shown, VD-deficient-rats that received intraperitoneally VD supplementation, presented muscle protein metabolism and muscle mass improving 8, what could be positive to the exercise practice and recovery. Furthermore, VD was effective on modulating the exercise-induced muscle damage and inflammation in rats 9. Exercise decreases the adipose tissue content and metabolic abnormalities present in obesity 610 these effects may be due to sympathetic activation and the action of catecholamines (adrenaline and noradrenaline) 1. Interestingly, the catecholamines have significant effects on immune activity, including on splenic immune response modulation 11.

In a hypothalamic model of obesity, induced by neonatal treatment with monosodium L-glutamate (MSG), neuroendocrine abnormalities such as, excessive adiposity, hyperinsulinemia, insulin resistance (IR), imbalance of autonomic activity, dyslipidemia, glucose intolerance, cardiovascular complications and hyperleptinemia 1213 are associated with changes in immune function 14, including splenic dysfunction 1516. Interestingly, recent studies have demonstrated that VD, when administered intraperitoneally in MSG obese rats, improved immune responses and metabolic abnormalities 1718. Moreover, splenectomy in MSG-obese rats improves IR and reduces adiposity 16. Thus, in the present study, we evaluated the effects of chronic VD supplementation associated with swimming training on morphological aspects of the spleen in lean and MSG obese rodents.

All experimental procedures were conducted with male newborn Wistar rats; animal protocols were approved by the Committee on Ethics in Animal Experimentation (CEUA/November,15/2015) of the State University of West of Parana. During lactation and growth, rats were maintained in adequate conditions, under controlled lighting (8:00 - 20:00h) and temperature (22 ± 2°C), with free access to standard chow and water, according to the guidelines of the National Council for Control of Animal Experiments (CONCEA).

Neonatal MSG administration induces hypothalamic lesions, resulting in dysfunctions in the autonomic nervous system (ANS) and endocrine abnormalities, promoting obesity without any alteration in food intake 2223. In the present study, as previously demonstrated in the literature 1723, MSG-treated rats demonstrated higher WAT accumulation and reduced BW, in relation to CON lean rats.

The rats treated with MSG present an altered inflammatory profile and IR 2023. The role of the spleen in this disorder is exemplified by the fact that splenectomy in MSG-treated rats improves obesity and IR, without modifying plasma inflammatory cytokines 16. About the spleen, is a secondary lymphoid organ responsible for immune surveillance against blood-circulating pathogens and the WP is a splenic region rich in lymphocytes T and B 25. Thus, we observed that the spleens of MSG-treated rats demonstrated a reduced weight, accordingly to previously findings 2526. Additionally, were verified alterations in splenic morphology in spleen of MSG-rats, such as increased WP area, reduced occupation of GC and augmented thickness of LN, in relation to the spleen of CON lean rats. However, other studies have shown that the MSG-treated rats had atrophy of the WP, absence of GC and abundance of hemosiderophages in the spleen, these data are different from our findings2627. Moreover, these authors also observed high presence of cell elements of hematopoiesis, especially megakaryocytes in the red pulp (RP) and large congested blood vessels with thick walls in spleen of MSG-treated rats 2627. In addition, was found a decrease in CD3+ T-lymphocyte number in the splenic tissue of MSG-treated rats, besides an unclear differentiation between the RP and WP zones, and a reduction in LN size 27. In contrast, in the present study we observed augmented thickness of LN in MSG-treated rats, in relation to spleens from CON-lean rats.

Obesity is associated with an imbalance between pro and anti-inflammatory cytokines 1. IL-10 is an important anti-inflammatory cytokine, where the spleen is responsible for approximately 30% of circulating IL-10 levels 2. Both subcutaneously-administered MSG and oral MSG administration appear to induce a reduction in plasma IL-10, suggesting that this agent is able to provoke splenic hypo-function and atrophy 1428. However, is important to recognize that subcutaneous MSG administration induces hormonal alterations, such as, absence of the growth hormone releasing hormone (GHRH) and growth hormone (GH), as well as high cortisol levels 29. As mentioned by Dorshkind and Horseman (2000) 30, the immunomodulatory properties of GH result from its ability to counteract the negative effects mediated by stress-induced glucocorticoids upon the immune system. In addition, severe somatotrophy deficiency of Ghrh−/− mice essentially affects the spleen and the B compartment of the adaptive immune system, inducing reduced spleen size amongst other effects 31. Taken together, these data show that neonatal subcutaneous MSG administration can exert, direct and indirect immunotoxic effects on the spleen.

The effects of VD on the immune system are well established 32, with VD deficiency playing a role in several immunological diseases 33. Additionally, VD supplementation appears to improve glucose tolerance, insulin sensibility and dyslipidemia in obese subjects 18. We have recently confirmed that oral VD supplementation is able to improve IR and dyslipidemia in MSG-obese rats without affecting adiposity 34. However, in CON lean animals, VD appeared to elevate fat aggregation, especially in relation to exercised rats. The impact of VD on WAT has demonstrated contradictory results. For example, in high fat diet (HFD) obese mice, VD supplementation reduced WAT, but in isolated 3T3-L1 adipocytes, VD has been reported to stimulate adipogenesis 3536. Moreover, VDR-/- mice have reduced WAT content 37. In contrast, exercise reduced WAT in lean rats; an effect that was not observed in MSG, suggesting that obese rats present a lower lipolytic response to exercise. Accordingly, neonatal MSG treatment has been shown to alter lipolysis in some fat depots 38.

Interestingly, we observed that VD supplementation promoted an augmentation in the WP area, as well as in the GC area, in spleens from CON lean rats, without altering histological aspects in spleens of MSG-obese rats. VD supplementation can elevate secretion of IL-2 and the proliferation index of T lymphocytes in the spleens of immunosuppressant mice 39 and, in HFD-obese mice, VD supplementation improves spleen morphology, inducing increased WP, with proliferation of LN and augmented immune activity in the spleen 33. These, data suggest that VD supplementation was unable to attenuate or revert the splenic histological abnormalities induced by neonatal MSG treatment. However, changes, induced by VD, in spleen functionality cannot be disregarded. Jin et al. (2018) demonstrated that intraperitoneally-administered VD induced the infiltration of Treg cells in secondary immune organs, such as the spleen and lymphatic nodes 40. Moreover, as mentioned, MSG-obese rats do not produce GH, but present excessive cortisol levels, events that could prevent the effects of VD on the spleen. Further studies are necessary to clarify these hypotheses in the MSG-obese model.

The role of exercise in metabolism is well established; exercise improves glucose and lipid homeostasis 10 and can modulate the immunological system 40. Whether exercise has positive or negative effects on immune responses depends on the intensity, frequency and duration of exercise training 4142. Exercise stimulates the sympathetic nervous system (SNS) by inducing the release of catecholamines (adrenaline and noradrenaline) from the adrenal glands, and stimulates the direct action of noradrenaline in tissues innervated by post-ganglionic sympathetic neurons 424344. Importantly, the spleen is richly innervated by the SNS and noradrenaline (NE) exerts important immunomodulatory effects in splenocytes 10.

In the present study, CON lean-exercised rats presented augmented LN size, without change in spleen weight. Previous studies have demonstrated that exercise in rodents and humans 4546 can reduce spleen weight, via an effect that appears to be dependent on exercise intensity 45. Using a similar exercise training protocol as that applied herein, Shimojo et al. (2019) recently demonstrated that swimming training reduces TNF-alpha production in the spleens of C57BL/6J male mice during endotoxemia 47. Similar results were obtained by Chen et al. (2016) 48. In this study, exercise dampened the secretion of inflammatory mediators, through partial inhibition of TLR4 and p-NF-κB, and activation of PI3K/p-Akt expression in the spleen. Moderate physical training can also abolish the effects of nutritional programming on splenic morphometry in adult rats 49. In contrast, in MSG-treated rats, we observed that swimming training did not modify histological aspects of the spleen. Some studies have demonstrated that positive effects of exercise in immune system are dependent upon HPA activation 50. Neonatal MSG treatment promotes disruption in the HPA axis, potential diminishing the benefits of exercise on the spleen.

Several nutritional strategies have been associated with exercise training, with the aim of improving physical performance or accelerating weight loss 5152. Moreover, prolonged exercise and heavy training are associated with depressed immune function; a condition improved by VD supplementation 5153. In the present study, the association of VD plus swimming training did not change body weight or WAT mass in either CON lean or MSG-obese rats. Aly et al. (2016) reported that regular moderate exercise training improves the effects of VD by enhancing vitamin D receptor (VDR) responsivity 53. Moreover, Carillho et al. (2013) showed that, in obese or overweight humans, VD supplementation plus exercise elevates muscle potency, in association with an improved VD status and a reduction in waist circumference 54. However, as demonstrated by Van den Heuvel et al. (2013), high intensity exercise exerts a greater impact on plasma VD levels 55. The association of VD supplementation with physical exercise improves metabolic conditions in patients with MS, by maximizing the effects of exercise and should be recommended 54.

To date, no study has investigated the effect of the combination of exercise and VD on the spleen. We demonstrate, for the first time, that there was no additive effect of the association of exercise and VD supplementation on morphological aspects of spleen. In the spleens of CON lean rats, the isolated effects of exercise or VD were maintained. It should be emphasized that both exercise and VD augmented the thickness of the LN, but this effect was not further exacerbated in VD-exercised rats, suggesting that pathways by which these effects are mediated may be similar. In contrast, while VD elevated the area of WP and the size of GC, exercise reduced the GC area as well as GC occupation, suggesting an antagonist impact on this parameter. Recently, forced swimming training was demonstrated to reduce WP and increase apoptotic index in splenocytes 55. It is probable that immunological effects regulated by exercise are determined by SNS activity effects on the spleen, while the action of VD could be, at least in part, dependent upon VDR activation and modulation of other hormonal pathways, such as the action of glucocorticoids 53545657.

In summary, our data show that neonatal MSG treatment reduces spleen size, in association with an increase in WP area and LN thickness and a reduction in the GC region. Neither exercise nor VD supplementation were able to restore histological aspects in the spleens of hypothalamic obese rats. In contrast, in the spleens of control lean rats, isolated effects of exercise and VD were observed. Thus, both interventions augmented LN thickness, but VD alone elevated WP and GC area, while swimming training reduced GC area, indicating that different splenic pathways were modulated by exercise and by VD supplementation.

We wish to thank the Endocrinological Physiology and Metabolism Laboratory (LAFEM) research group, as well as to CAPES by scholarship of MSc thesis of Zoé Maria Guareschi and to the Laboratory of Tissue Biology and Reproduction of UNIOESTE.