1. Introduction
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that is classically divided into Crohn disease and ulcerative colitis. In IBD, which is characterized by alternative recurrence and alleviation periods, patients suffer from abdominal pain, diarrhea, bloody stools, and weight loss [
1]. It is a global disease, with increasing incidence and prevalence around the world [
2].
The etiopathogenesis of IBD has not yet been elucidated, but environmental, gut microbial, and genetic factors all play important roles [
3]. The intestinal inflammatory response in patients with IBD also involves deregulation of gut-associated lymphoid tissue (GALT) [
4]. This tissue is divided into inductive sites, which include the mesenteric lymph nodes, Peyer’s patches (in the small intestine), colonic patches, and isolated lymphoid follicles, and into effector sites, which include intraepithelial lymphocytes and lymphocytes from the
lamina propria [
5]. Dysfunction of the innate and adaptive immune responses triggers the release of numerous proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), which results in epithelial cell damage and apoptosis and alteration of the epithelial barrier [
4]. The epithelial barrier comprises epithelial cells (enterocytes), tight junction proteins, and the mucosal layer [
6] and constantly interacts with the underlying immune cells of the GALT [
7]. In IBD patients, the integrity of the epithelial barrier is compromised, as shown by increased paracellular permeability and lower levels of tight junction proteins [
8].
Although some drugs are able to modify the course of the disease and maintain remission, they are not effective in all patients and their long-term use may have adverse effects [
9]. Therefore, alternative treatment strategies for IBD are necessary. Nutritional interventions may be a good candidate. Using the
Mdr1a knockout (KO) mouse model of colitis [
10,
11], we previously showed that serum-derived bovine immunoglobulins (SBI) reduce colon permeability and the expression of oxidative markers and proinflammatory cytokines in the colonic mucosa, as well as leukocyte infiltration in the lamina propria and mesenteric lymph nodes [
12,
13]. Moreover, SBIs improve mucositis symptoms, tissue damage scores, and neutrophil and lymphocyte percentages in a rat model of mucositis [
14].
Another dietary supplement that is widely used in farm animals to enhance growth and reduce both morbidity and mortality is spray-dried porcine plasma (SDP) [
15,
16]. This supplement can modulate the intestinal immune response to inflammatory agents. For example, it reduces T lymphocyte activation, preventing the release of proinflammatory cytokines, and improves the mucosal barrier function after staphylococcal enterotoxin B challenge in rodents [
17,
18,
19]. In senescent mice, SDP supplementation reduces nonspecific basal immune activation associated with age (inflammaging) by promoting mucosal regulatory T helper (Th) lymphocytes (Treg lymphocytes) and interleukin (IL)-10 production [
18]. Moreover, SDP attenuates cognitive decline and reduces brain capillary permeability, oxidative stress, and proinflammatory cytokine expression in the brain of senescent mice [
20].
Given the ability of SBI effects reducing the severity of colitis in the
Mdr1a KO mouse model [
12,
13], we wanted to study if SDP, a compound with well-documented antioxidant and anti-inflammatory activities, can also modulate the immune response and ameliorate colitis in this animal model.
3. Discussion
The pathogenesis of IBD is still unknown. However, it is well-established that patients have a deregulated immune response involving increases in proinflammatory cytokines, which has been identified to contribute to disorders of the gastrointestinal tract [
21]. In recent years, nutraceutical compounds, which include bioactive peptides and colostrum, have been reported to have beneficial effects in IBD patients [
22]. Therefore, in the present study, we evaluated the effects of dietary supplementation with SDP on the evolution of IBD in a mouse model of spontaneous colitis.
The gastrointestinal tract can develop different inflammatory pathologies, with changes in epithelial permeability leading to altered mucosal functions [
23]. Here,
Mdr1a KO mice showed an increase in the histopathological index, indicating intestinal deterioration. In addition, KO mice had higher epithelial permeability and lower abundance of tight junction proteins than WT animals, which is consistent with the alterations in epithelial permeability and in tight junction structure observed in patients with ulcerative colitis and Crohn disease [
8]. Although SDP supplementation did not improve the histopathological index, it prevented the increase in crypt epithelial permeability and the reduction in E-cadherin abundance observed in
Mdr1a KO animals. This effect is similar to that observed in the same model of colitis after supplementation with SBI, which is an immunoglobulin concentrate [
12]. Previous work has also shown that SDP can prevent increases in small intestine permeability during acute intestinal inflammation induced by an enterotoxin [
17].
IBD patients show lower abundance of goblet cells and changes in mucin expression and secretion [
24], which reduce the thickness of the mucus layer [
25] and thereby compromise barrier function. In addition to mucins, goblet cells also synthesize trefoil factors, which are secretory proteins that stabilize the mucus layer [
26] and facilitate intestinal epithelial restitution [
27]. Our
Mdr1a KO mice showed a reduction in the number of goblet cells in the colon, as well as in the expression of
Muc2 and
Tff3, which would contribute to an increase in the epithelial permeability of the colon [
28]. Indeed, it has been described that a reduction in goblet cells and
Muc2 would be associated with an increase in pathogenic bacteria in the mucus layer and a greater penetration of these microorganisms into the mucosa [
29]. SDP supplementation did not improve goblet cell abundance in KO mice but did attenuate the decrease in
Muc2 expression and maintained
Tff3 expression at similar levels to those of healthy animals. Therefore, SDP helps to maintain barrier integrity and prevent the entry of pathogenic microorganisms that could eventually perpetuate inflammation in the colonic mucosa.
Both MUC1 and MUC4 are membrane-bound mucins that are elevated in patients with IBD during the inactive phase of the disease, suggesting a crucial role of these mucins in the development of the condition [
30]. In addition, aberrant expression of
Muc1 is involved in intestinal barrier dysfunction during inflammation, with overexpression of
Muc1 correlated with increased intestinal permeability [
31]. On the other hand, neutrophil infiltration is associated with increased
Muc4 expression [
32]. SDP supplementation reduced the overexpression of
Muc1 and
Muc4 observed in KO mice, suggesting that SDP-supplemented mice had lower immune activity and an enhanced colon barrier.
Mdr1a KO mice showed an increase in immune cell recruitment in the
lamina propria and in the intraepithelial compartment, as observed in IBD patients [
33]. This infiltration is triggered by monocytes, neutrophils, and Th lymphocytes [
34]. The infiltration is accompanied by chemokines and cytokines that promote the recruitment and activation of these cells, leading to unrestrained accumulation of activated immune cells that impair mucosal homeostasis and perpetuate the inflammatory response [
35]. We found that SDP supplementation reduced the accumulation of activated monocytes and neutrophils in the colonic mucosa, which is consistent with reduced production of MIP-1β and MCP-1. This effect is notable because these chemokines are important mediators for the recruitment and accumulation of neutrophils and macrophages in colitis models [
36]. The SDP diet also reduced the percentage of activated Th cells in the
lamina propria, diminishing an immune subset that plays a main role in initiating and shaping IBD pathogenesis [
37]. The decline in Th cell activation led to a reduction in proinflammatory cytokines, such as IL-2 and IFN-γ. This is noteworthy because activated Th lymphocytes are also characterized by alterations in cytokine production, which result in a disturbed balance between pro- and anti-inflammatory cytokines [
38,
39]. Along this line, similar effects of SDP supplementation have been observed in other rodent models of jejunum and pulmonary acute inflammation [
40,
41,
42].
Th17 cells play an important role in host defense against extracellular pathogens but are also associated with the development of inflammatory responses such as those seen in IBD [
43]. Here,
Mdr1a KO mice showed a considerable increase in IL-17 release in the colonic mucosa, indicating a clear bias toward the Th17 immune response. SDP decreased the concentration of IL-17 in the colonic mucosa. This effect may be clinically relevant because IL-17 induces the release of chemokines and other chemoattractants from epithelial and endothelial cells that promote the inflammatory response through the recruitment of neutrophils [
44]. Therefore, the reduced IL-17 release observed in KO mice supplemented with SDP might explain the lower activation of neutrophils and their reduced infiltration into the colonic mucosa. Similar effects of SDP supplementation were observed in mice with acute lung inflammation induced by lipopolysaccharide (LPS), with a reduction in the activation of neutrophils and their infiltration into lung tissue [
42,
45].
Immune activation and proinflammatory cytokine release can disassemble tight junction proteins, increasing epithelial permeability [
46]. This can trigger and perpetuate local inflammation in IBD [
47]. Furthermore, both
Tnf-α and
Ifn-γ induce
Inos expression, which also has deleterious effects on intestinal integrity [
48]. In general, our results showed that SDP supplementation reduced the immune response during colitis development. A similar pattern was also observed with SBI supplementation in
Mdr1a KO mice [
13].
The intestinal mucosa is normally maintained in a state of controlled inflammation in which there is a balance between protective immune responses and tolerance to self-antigen and commensal bacteria [
49]. Dendritic cells participate in the preservation of Th intestinal tolerance through the activation and maintenance of Treg cells [
50], which in turn control immune responses in the gut by inhibiting the proliferation and effector functions of other T cells. Inflammation in IBD may be caused by an alteration in the balance between Treg and proinflammatory activated Th cells [
51]. Our KO mice showed a reduced proportion of dendritic cells and activated dendritic cells, which correlates well with the reduction in the Treg population. In our experiments, SDP supplementation increased the percentage of dendritic cells and their activation in the lamina propria of the KO mice, as well as the proportion of Treg lymphocytes in the colonic mucosa. SDP supplementation also reduced the ratio between activated Th lymphocytes and Treg lymphocytes, indicating that SDP restores the balance between these lymphocyte populations. A similar response pattern has been observed in the Staphylococcal enterotoxin B model of mild intestinal inflammation [
19] and in acute lung inflammation induced by LPS [
45]. In both cases, the challenge increased the activated Th/Treg ratio while SDP restored the pre-challenge ratio. The importance of these effects lies in the role of Treg in the suppression of Th effector cells through the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β [
52].
SDP supplementation did not modify colon TGF-β concentration but did promote IL-10 release. The lack of a TGF-β response was unexpected because, in other inflammation models, such as intestinal and genitourinary acute inflammation models, SDP induces the secretion of TGF-β [
41,
53]. However, in acute lung inflammation, only mature TGF-β (and not total TGF-β) is increased by SDP [
42].
On the other hand, SDP supplementation increased the release of IL-10 during chronic inflammation. These results are consistent with the acute intestinal inflammation induced by staphylococcal enterotoxin B [
19], with the acute lung inflammation provoked by LPS [
42], and with the uterine inflammation induced by stress [
53], in which SDP supplementation increases the expression of this anti-inflammatory cytokine. In a very different condition, such as in a mouse model of senescence, SDP increases the brain concentration of IL-10, suggesting its participation in the regulation of immune responses in nonmucosal tissues [
20]. All of these effects of SDP on IL-10 are remarkable because this cytokine plays an important anti-inflammatory role in restricting and suppressing the inflammatory responses, thus minimizing tissue damage in response to microbial challenges [
54].
Besides the effect of SDP on the immune system itself, it can also act through changes in the intestinal microbiota. In this regard, Moretó et al. [
55] showed that supplementation with SDP increases the proportion of bacterial families that improve intestinal barrier function and are well-known mediators of anti-inflammatory and tolerogenic responses, such as the Lactobacillaceae family. In fact, different species of the genus
Lactobacillus have been shown to be effective in promoting the expression of
Il-10 [
22] or in reducing the expression of adhesion molecules that promote leukocyte recruitment in an experimental colitis model [
56].