In general, due to the incomplete development of the immune system in juvenile animals, the intestines of individuals often show an allergic reaction to 7S, resulting in metabolic disorders and adverse physiological and biochemical reactions, which can have the effect of disrupting the digestion and absorption of nutrients in the intestines 12. Interestingly, a low dose of 7S can often induce a response similar to that obtained with immunostimulants 13, which is often manifested in terms of modified growth performance. For example, in turbot (Scophthalmus maximus L.), a reduction in feed utilization efficiency following ingestion of feeds containing 4–8% 7S has been observed to result in a significant reduction in specific growth rate (SGR) 14. These findings were similar to those obtained in the present study, in which we observed that hybrid grouper receiving a diet containing 8% 7S showed a significant reduction in SGR and weight gain (WG). However, whereas in turbot, the effect of ingesting of a diet containing 2% 7S did not differ significantly from that of a control diet with respect to SGR, we observed an increase in SGR in hybrid grouper fed a diet supplemented with low-dose 7S. Moreover, in turbot, it was found that the level of soybean supplantation and WG showed a first-degree relationship 15. Hybrid groupers can, however, tolerate up to 30–50% replacement of fishmeal with soybean meal in their feed, and experience no adverse effects on growth, even at soybean meal substitution levels of 10% and 30%, WG increased while did not show a significant difference 16. These observations thus indicate that hybrid grouper may be more tolerant to soybean meal than are turbot, and therefore we speculate that for turbot, the addition of 2% soybean globulin is higher than the dose used for immune enhancement.
In the present study, we found that the addition of sodium butyrate (NaB) significantly increased SGR in the bH-NaB group compared with that in the bH group, indicating that NaB can be effective in ameliorating the growth inhibition effects attributable to high doses of 7S. We also found that the feed coefficient ratio of fish in the bH and bH-NaB groups was significantly higher than that in the FM and bL groups, whereas the addition of NaB significantly reduced the FCR, indicating that NaB may enhance the growth performance by improving the feed utilization of hybrid grouper, which is similar to results obtained for grass carp 8,17. We speculate that this effect of NaB is associated with an increase intestinal antioxidant capacity and enhancement of intestinal immunity.
To further investigate the effects of 7S and NaB on the disease resistance of the hybrid grouper, we conducted a 7-day challenge test using Vibrio parahaemolyticus injected into fish at a concentration of 7.41 × 108 CFU/mL at the end of the breeding experiments. We accordingly observed the highest cumulative mortality in the bH group, thereby indicating that high doses of 7S can significantly reduce disease resistance in hybrid groupers, whereas resistance was enhanced to some extent in groupers fed a diet supplemented with NaB. Interestingly, whereas the trend in mortality in the bH group showed a consistent and rapid increase during the first 4 days post-injection, the upward trend only became apparent at day 4 post-injection in groupers fed the NaB-supplemented diet. These observations thus tend to indicate that although NaB may not be effective in ameliorating the immunosuppression caused by high doses of 7S, it can, to a certain extent, delay the onset of disease caused by V. parahaemolyticus, thereby gaining valuable time for treatment. However, given that it was unclear how this resistance is regulated, we conducted the research described in the following section.
It is widely believed that the allergic responses to specific antibody-mediated humoral immunity and T-cell-mediated cellular immunity, which induce the production of serum-specific antibodies, occur primarily in the intestinal tract of fish ingesting 7S 18. As a typical pro-inflammatory cytokine, IL-1β is primarily produced by mononuclear macrophages, and can induce the activation of neutrophils and macrophages, stimulate the release of thromboxane and platelet-activating factor, increase the permeability of epithelial and endothelial cells, and induce intestinal inflammation. In the present study, we found that levels of IL-1β in bL and bH group fish were significantly down- and upregulated, respectively. The lower levels of IL-1β in the bL group conceivably indicate that low-dose 7S can maintain normal serum immune responses by modifying IL-1β secretion, whereas the significantly elevated levels of IL-1β in the bH group, could indicate that at higher doses, 7S may promote CD4+ T-cell activation, proliferation, and differentiation by enhancing the abnormal secretion of IL-1β, thereby exacerbating the inflammatory response 19. We also observed that NaB supplementation of the grouper diet had the effect of significantly reducing serum IL-1β, thereby indicating that NaB may modulate serum immunity by inhibiting the abnormal secretion of IL-1β. In this regard, studies on rats have found that NaB can induce the overexpression of IL-1β by inhibiting the binding of p65 to the IL-1β promoter 20. We accordingly speculate that a similar mechanism operates in the hybrid grouper.
TNF-α is a further key cytokine that plays role in mediating intestinal inflammatory processes 21, and has been shown to exacerbate the progression of enteritis by inducing the release of pro-inflammatory factors 22. Consequently, determining changes in the levels of TNF-α is important with respect to monitoring inflammatory responses in the body. We found that a low dose of 7S significantly reduced serum TNF-α levels in the hybrid grouper, whereas in contrast, a high dose had the effect of promoting the secretion of TNF-α. Similarly, in turbot, it has been found that supplementing feed with 4–8% 7S increased the expression of TNF-α, which ultimately inhibited growth. These observations indicate that 7S has a regulatory effect on TNF-α secretion in both hybrid grouper and other fish. Indeed, it has previously been found that the percentage of CD4+ lymphocyte sub-populations in mice increases linearly with increasing 7S levels, and thus we hypothesized that the elevation of TNF-α in the high-dose 7S group may be associated with an increase in CD4+ lymphocyte sub-populations in groupers 23. We also found that supplementation of the grouper diet with NaB had the effect of reducing serum TNF-α to levels comparable with those observed in FM group fish, which we suspect could be attributable to the fact that NaB can inhibit TNF-α secretion by down-regulating LITAF expression 24. This effect may also be associated with the fact that NaB inhibits mast cell activation and suppresses the release of mast cell inflammatory mediators.
Similarly to TNF-α, the type II interferon IFN-γ play roles in immune and inflammatory responses. 7S is treated by APC cells and the 7S soymetide is exposed, which binds to B- or T-cell-transformed immunoglobulins 25. On the basis of the findings of the present study, it would appear that both low and high doses of 7S can promote the secretion of IFN-γ, and we accordingly speculate that this may be closely related to the activation of T cells by 7S as an antigenic protein. This assumption tends to be supported by our observations indicating that dietary supplementation with NaB was associated with a significant elevation in the levels of serum IFN-γ. NaB was also found to inhibit certain IFN-γ functions by inhibiting the downstream signaling of IFN-γ and thus the production of IFN-γ-induced factors 26. We accordingly conjecture that, similar to humans, NaB can reduce the production of toxic substances in groupers by blocking the induction of IFN-γ on inflammatory cells. To examine this possibility, we conducted the experiments described in the following section.
Nitric oxide (NO), a free radical gas produced from l-arginine via the catalytic activity of nitric oxide synthase (NOS), plays an important role in host defense and inflammatory responses, and can interact with cytokines such as TNF-α and IFN-γ, thereby influencing the course of inflammatory responses 27–29. Currently, however, there is a degree of controversy as to the mechanism whereby NO is regulated in the intestines 27,30. In the present study, we found that NOS showed an elevated trend in the bL group and NO was significantly elevated in the fish in this group. A similar pattern was also observed in groupers in the bH group. Notably in this regard, we also detected significant increases and decreases in the levels of IFN-γ and TNF-α in the bL group, respectively, the former of which can enhance NO secretion by stimulating macrophages and inducing the production of nitric oxide synthase 31. However, given that TNF-α showed an increase and decrease in the bL and bH groups, respectively, we were unable to determine whether TNF-α is involved in the regulation of NO production. Nevertheless, we speculate that either TNF-α is not involved at low doses of 7S but is involved at high doses, or, alternatively, that TNF-α does not play a role in regulating the macrophage production of NO in the hybrid grouper. In addition, we observed that dietary supplementation with NaB maintained the significant upregulated production of IFN-γ, and resulted in the recovery of TNF-α to FM group levels. Correspondingly, NOS levels were significantly reduced, and NO showed a non-significant downward trend. Thus, taking into consideration the results obtained for both IFN-γ and TNF-α, we are more inclined to believe that NaB downregulates NOS activity by inhibiting the release of TNF-α 32, thereby reducing NO levels. The ONOO− is produced by the rapid binding of NO to oxygen radicals, which may be an important factor in cell damage, energy consumption, and cell death 33. We found that levels of ONOO− in the bL group were significantly downregulated, which contrasts with the elevated levels of NO in this group. Our observations tended to indicate that both IFN-γ and IL-1β and TNF-α are involved in the regulation of the NO-binding peroxide-generating step of ONOO− production. And from the analysis just presented, TNF-α and IL-1β are very important in the regulation of ONOO− generation34, particularly at high doses of 7S. We also found that NaB supplementation significantly reduced NOS levels, which may be related to the inhibition of NOS promoter-dependent transcriptional activity by NaB 35. Excess ONOO− can adversely affect tissues primarily by damaging DNA, inactivating enzymes, and degrading mitochondria, and thus the downregulation of ONOO− in the bH-NaB group may be related to the fact that NaB can enhance the provision of energy to cells 36,37, thereby preventing the generation of ONOO− and thus preventing its negative effects.
As previously mentioned, the strong oxidizing properties of ONOO− and its interaction with cytokines such as TNF-α and IL-1β can induce cellular damage and thus disrupt the normal morphology of tissues. To better visualize the effects of NO and ONOO− on the intestinal tract of hybrid grouper, we prepared histomorphological sections of the hindgut, which is particularly susceptible to 7S-related disturbance (Periodic acid Schiff and Alcian Blue staining, AB-PAS). Intestinal development in fish can often reflect digestive absorption, and in this regard, plica height is typically used as an indicator of intestinal health. However, we would argue that plica height alone does not truly reflect the status of intestinal health. Therefore, as an alternative, we used the ratio of intestinal diameter to plica height (Id/Ph), which we believe to be a more reliable indicator of intestinal integrity. The larger this ratio, the greater is the space occupied by folds in the intestines, and thus the greater is the efficiency of absorption 17. On the basis of the findings of the present study, we established that a high dose of 7S can significantly inhibited the development of plicae in the distal intestine of hybrid grouper, thereby reducing the absorptive area of the intestinal fold and thus the efficiency with which feed nutrients are absorbed. However, supplementing the grouper diet with NaB reversed the detrimental effects of high-dose 7, resulting in a reduction in the Id/Ph ratio, and significantly enhancing intestinal development. Similar results have also been obtained for the yellow drum (Nibea albiflora, Richardson) 38.
The mucus secreted by the mucous cells of fish contains non-specific immunochemical substances, including lysozyme, transfer factors, chitin, and complementary substances, that are resistant to pathogenic microorganisms 39. On the basis of AB-PAS staining, mucous cells can be classified into four types: I (pure red), II (pure blue), III (purple reddish), and IV (blue purple). From Fig. 2, it can be seen that most of the mucous cells in the distal intestine of hybrid grouper are type II cells. As an acidic mucous cell, the acidic mucus secreted by type II mucous cells plays an important role in regulating the transport of proteins and their residues, lubricating coarse feed, and increasing the viscosity of mucus and immune protection 40. In the present study, we observed that the numbers of type II mucus cells in the intestines of bL and bH-NaB group fish were not significantly different from those in the FM group, whereas in contrast, numbers were significantly reduced in the bH group, thereby indicating that a high dose of 7S can significantly inhibit the formation of type II mucus cells and thus result in the reduced secretion of antimicrobial active substances. Conversely, supplementing the grouper diet with NaB was found to effectively increase the abundance of type II mucous cells, thereby enhancing the ability of the distal intestine of hybrid groupers to resist pathogenic microorganisms, and thus protecting the distal intestine.
Subsequent to the ingestion of antigenic proteins in feed, most is converted to amino acids and other small molecules. However, owing to the high molecular weight of 7S, a small fraction is not readily broken down and directly enters the lymphatic system via the intestinal mucosal spaces to induce an immune response 41. When the antigen re-enters the body from the intestines, it will cause intestinal mucosa damage and edema, leading to inflammation. In this regard, although numerous studies have examined the effects of antigenic proteins on the intestines of fish, few have investigated the root cause of the inflammatory effects.
The major histocompatibility complex (MHC) processes foreign antigens, generating antigenic peptide-MHC molecular complexes, which are present in T cells and play an important role in inducing immune responses 42. In this process, MHC II and MHC I present exogenous and endogenous antigens, respectively, and in the present study, we focused on the mechanisms associated with the former. In mammals, MHC II encapsulates foreign antigens within vesicular primary endosomes in response to an antigen signal. The principal genes involved in the MHC II antigen presentation process are GILT, AEP, and CTSB 43,44, which function cooperatively to transport the primary endosomes to lysosomes for hydrolysis and antigen degradation. Thereafter, the complexes enter the Golgi apparatus for processing, and are thereby converted to antigenic peptide-MHC II molecular complexes, which activate CD4 T cells and produce corresponding immune responses 45. In this regard, rather than binding directly to the promoter of the MHC II gene, CIITA has been shown to act by controlling MHC II gene transcription via a synergistic interaction with the essential transcription factors RFX5, CREB, and NFY 46,47. In the present study, we found that the addition of a low dose of 7S promoted the synergistic association between CIITA and CREB1 and NFY to initiate the transcription of MHC II, thus presenting the foreign 7S antigenic protein (Fig. 7(a)). In contrast, we detected no significant changes in RFX5, and thus MHC I was not upregulated. Studies in patients with type III naked lymphocyte syndrome (BLS) have shown that the RFX complex is required for CIITA-mediated MHC I activity 47, and it is speculated that this is also the case in hybrid grouper. In response to receiving the antigenic signal from 7S, MHC II forms an intranuclear body regulated by GILT, AEP, and CTSB, and is subsequent to processing within lysosomes and the Golgi system to form an antigenic peptide-MHC II molecular complex, which then transmits the signal to CD4 T cells to activate the immune response. In the distal intestine of hybrid grouper, AEP does not appear to be involved in transduction to nucleosomes, but rather promotes the transport of nucleosomes to lysosomes by upregulating GILT and downregulating CTSB, as has also been observed in mice 48. However, the involvement of AEP appears to be related to the type of antigen detected by an organism, as AEP preferentially deals with viral antigens, as opposed to macromolecular protein antigens 49. CD4 T cells are activated upon receiving signals from the molecular complex, which in turn secrete cytokines with immunomodulatory properties. In combination with the results obtained from our examination of serum-related immunity, these findings indicate that CD4 T cells and macrophages can enhance intestinal immunity in hybrid groupers by inhibiting the secretion of the pro-inflammatory factors IL-1β and TNF-α and promoting secretion of the anti-inflammatory factor IFN-γ.
The development of intestinal inflammation is generally accompanied by the abnormal expression of constituents of the PI3K/Akt/mTOR pathway 50, the regulation of which by anti-nutritional factor-induced intestinal inflammation is currently incompletely understood. PI3K is a heterodimer composed of the regulatory subunit p85 and the catalytic subunit p110, the activities of which provide a signal for Akt, using PIP3 as a second messenger 51. mTOR is an atypical serine/threonine protein kinase comprising two complexes, mTOR C1 and mTOR C2. The former consists of three core components, namely, mTOR, Raptor, and mLST8. mTOR is the catalytic subunit of the complex, whereas mLST8 is associated with the catalytic domain of mTOR C1, which stabilizes kinase activation, although is not the catalytic subunit of mTOR C1. mLST8 however, is associated with the catalytic domain of mTOR C1 that stabilizes kinase activation. Rictor does not seem to affect mTOR C1 activity 52. mTOR C2 consists of three core components, namely, mTOR, Rictor, and mLST8, among which mTOR is a component of the catalytic subunit of the complex, as in mTOR C1. mTOR is a rapamycin-insensitive partner that can interact with Protor, although the physiological function of this interaction remains to be determined. mLST8 is a complex catalytic subunit of mTOR C2 53.
Akt also receives signals from mTOR C2, thereby promoting the expression of the core protein Rheb via the TSC1/TSC2 complex, which regulates mTOR C1 54. On the basis of the low-dose 7S results obtained in the present study, we established that in response to this treatment, Akt is not regulated by mTOR C2 or PI3K, and does not promote Rheb expression via TSC2, thereby disrupting the regulation of mTOR C1. Moreover, in mTOR C1, TEL2 promotes the expression of 4E-BP2, although EIF4E is unaffected, whereas in mTOR C2, TEL2, and Sin1 are likely to be the key components responsible for the upward modulation of RhoA. Furthermore, although the expression of TSC1 is elevated, in combination with Rheb, the regulation of mTOR C1 does not appear to be controlled by Akt. Given the aforementioned observations, we speculate that activation of the PI3K/Akt/mTOR pathway in response to low doses of 7S is superficial and does not have a fundamental effect on key downstream proteins, with CD4 T-cell activation playing a major role.
In response to supplementation with a high dose of 7S (Fig. 7(b)), MHC I synthesis is also upregulated via RFX5 activation, and a comparison with the response to low doses indicates that RFX5 is essential for MHC I regulation 47. Subsequent to the formation of nuclear endosomes, it appears that GILT and AEP, although not GILT and CTSB, are involved in regulation. This observation indicates that the expression of AEP may be dependent not to the type of antigen, but rather the dose of antigen. In this case, following the activation of CD4 T cells by the antigenic peptide-MHC II molecular complex, the secretion of IFN-γ remains at high levels, and there are also significant increases in the expression of the pro-inflammatory factors IL-1β and TNF-α, thereby contributing to the development of intestinal inflammation. Interestingly in this regard, we also detected an upregulation of endogenous antigen presentation by MHC I, which we suspect may be associated with the elevated levels of body endogenous antigens induced by high doses of 7S. Likewise, we found that the PI3K/Akt/mTOR pathway was also fully activated in response to stimulation with high doses of 7S. Commencing with PI3K, which activates Akt by upregulating 3-PDK1, we found that IKKα was also upregulated, and although we did not examine the role played by NF-kB in the present study, we speculate that Akt may induce the transfer of NF-κB to the nucleus via IKKα. With respect to mTOR C1, the components depressor, Raptor, and mLST8 are activated, resulting in an mTOR C1-induced upregulation of 4E-BP1 and 4E-BP2, which in turn results in an increase in EIF4E secretion. Moreover, p70 S6K also increased the level of EIF4B via mTOR C1. Upregulation of eIF-4E is often considered a marker of early colon cancer, and it has also been reported that 4E-BP1 and 4E-BP2 limit the anti-inflammatory response of macrophages by inhibiting IL-10 55. Thus, high doses of 7S-induced intestinal inflammation in the hybrid grouper may be associated with elevated levels of 4E-BP1 and 4E-BP2 and a suppression of the anti-inflammatory effects of macrophages. In addition, the expression of TSC1 is elevated, thereby in turn promoting the expression of Rheb, which relays signals from Akt to mTOR C1. Furthermore, with respect to mTOR C2, mLST8, depressor, and RICTOR are also activated, thereby elevating the levels of SGK1, which is involved extensively in the regulation of inflammation, and not only induces cardiac inflammation by activating the NLRP3 inflammasome 56 but can also exacerbate inflammation by stimulating NF-kB 57.
As a common feed additive used to supplement the diets of livestock, poultry, and aquatic animals, NaB can be hydrolyzed in the gastrointestinal tract to form butyric acid, which provides a source of energy to the intestinal epithelial cells, and contributes to the repair of damaged intestinal mucosa and strengthening intestinal immunity. However, the mechanisms whereby NaB influences intestinal repair have yet to be determined. In the present study, we found that supplementation of feed containing a high dose of 7S with appropriate levels of NaB facilitated the transcription of MHC I and MHC II via the upregulated expression of CREB1 and downregulation of NFY, and that MHC II expression was maintained at a high level (Figure(c)). These observation thus tend to indicate that dietary supplementation with NaB has the effect of enhancing the efficiency of antigenic protein presentation and processing in hybrid groupers. In addition, a concomitant reduction in the expression of MHC I may be indicative of a reduction in the severity of the inflammatory response compared with that seen in groupers receiving a high dose of 7S in the absence of NaB 58. In addition GILT and AEP are inhibited during the development of nucleosomes, which subsequently migrate to the lysosomes and Golgi apparatus. There is also a reduction in expression of the pro-inflammatory factors IL-1β and TNF-α, thereby indicating a weakening of the inflammatory response. Although PI3K expression was also upregulated, the expression of 3-PDK1 and Akt remained at levels similar to those observed in groupers receiving a high dose of 7S. However, we found that the key transcription factor IKKα, which is transduced by Akt to NF-kB, was suppressed. On the basis of these observations, we thus speculate that NaB supplementation could suppress the activation of NF-κB 59, although we did not verify this assumption in the present study. With regards to the mTOR C1 complex, we noted an inhibition of mLST8 and Raptor, which in turn downregulated the expression and secretion of 4E-BP1 and EIF4B, and although the expression of p70 S6K was maintained at a high level, the formation of the downstream product EIF4B was inhibited.
Collectively, these findings indicate that the protective effect of NaB on the hindgut of hybrid grouper is closely associated with small molecule proteins downstream of mTOR C1, such as EIF4E. Although TSC1 showed high expression, that of Rheb was significantly reduced, and taken together with the results obtained for groupers receiving feed supplemented with high and low doses of 7S, we speculate that there are probably alternative mechanisms regulating the pathway from Akt to mTOR C1. With respect to mTOR C2, we established that the downregulated expression of mLST8 and Sin1 and upregulation of PRR5 promoted an increase in the expression of RhoA and PKC, and that SGK1 concomitantly remained at high levels. RhoA and PKC are generally considered to function as signal molecules associated with the inflammatory response, and are accordingly also commonly used as targets to suppress inflammation 60,61. Thus, NaB does not appear to alleviate intestinal inflammation via mTOR C2, but rather exacerbates the progression of intestinal inflammation.