Oral administration of oat beta-glucan preparations of different molecular weight results in regulation of genes connected with immune response in peripheral blood of rats with LPS-induced enteritis

Among the genes up-regulated in the peripheral blood of LPS-challenged rats, we found mRNA encoding four granzymes: A, B, C, and K (Gzma, Gzmb, Gzmc, and Gzmk, respectively) and a granzyme-like protein 1 (LOC102553861) (Supplementary Table 1). Granzymes belong to the family of serine proteases expressed by cytotoxic T lymphocytes (Tc) and natural killer (NK) cells. These proteases serve to protect an organism against viral infections and cellular transformation [20]. They are known to induce apoptotic cell death via the granule exocytosis pathway [21]. However, an increasing body of evidence indicates that granzymes may also have other functions in inflammation, i.e., granzymes A, B, and K may be involved in the production, release and/or processing of proinflammatory cytokines [22]. Studies on mice have shown that granzyme A has a direct role in the inflammatory response to LPS [23], whereas the extracellular form of this protein activates primary human monocytes to release TNF-α, IL-1β, IL-6, and IL-8 [24]. Similarly, granzyme B has been shown to enhance LPS-induced TNF-α release from monocytes in vitro; however, it was not able to exert this effect when administered alone [25]. The recombinant murine granzyme K induced the release of IL-1β from peritoneal mouse macrophages, and this effect was observed after priming the cells with LPS, suggesting an additive or synergistic effect of granzymes and bacterial compounds [26]. The role of granzyme C is less clear, although its expression was detected in activated CD4 + and CD8 + T lymphocytes, as well as in NK cells stimulated with IL-15 [27]. The transcriptomic analysis performed in our study also confirmed that intravenous administration of LPS to rats caused a systemic inflammatory response. Interestingly, a comparison of the gene expression profile of the LPS-G1 vs. LPS-G0 groups revealed a 2.35-fold decrease in the expression of gene encoding the granzyme C-like protein (RGD1560650), pointing to potential antiinflammatory activity of G1 beta-glucan in the LPS-challenged rats supplemented with this glucose polymer (Supplementary Table 2). Even more pronounced decrease (12.594-fold) in expression was noted in the case of Serp2 gene in rats from LPS-G1 group. The same gene was 3.388 up-regulated in LPS-challenged rats fed the control diet (LPS-G0) (Table 3, Supplementary Tables 1 and 2). Serp2 encodes stress-associated endoplasmic reticulum protein family member 2, belonging to the family of serine protease inhibitors, termed serpins, which are key regulators of numerous biological pathways involved in initiation of inflammation, coagulation, angiogenesis, apoptosis, extracellular matrix composition, and complement activation responses [28]. The exact function of Serp2 expressed by mammalian species has not been fully elucidated so far; however, Messud-Petit and co-workers [29] showed that Serp2 specifically binds to IL-1β-converting enzyme (ICE), and can prevent the onset of inflammation by reducing the levels of the active proinflammatory cytokine IL-1β. Therefore, the weakening of the immune response is linked to Serp2 gene function. To our knowledge, this is the first report showing a strong effect of beta-glucans on Serp2 gene expression, strongly supporting the immunomodulatory activities of these polysaccharides.

Among the 533 genes regulated in the blood of rats from the LPS-G1 group in comparison with the LPS-G0 group, several other transcripts were directly connected with the host immune response. Among the 275 up-regulated genes listed in Supplementary Table 2, special attention should be paid to those encoding: TNF receptor superfamily member 5 (CD40); immunoglobulin lambda-like polypeptide 1 (Igll1); immunoglobulin delta heavy chain secreted form (IgD) mRNA; Fc fragment of IgE, low affinity II receptor for (CD23) (Fcer2); TNF receptor-associated factor 3 (Traf3) and NLR family, pyrin domain-containing 1B protein (Nlrp1b). Products of these genes are involved in the signaling pathways induced in response to inflammatory processes, such as the TLR signaling pathway and the NLR signaling pathway (Table 5). Currently, increasing number of data demonstrate that beta-glucans are able to regulate both innate and adaptive immunity. Macrophages and dendritic cells are considered the main target cells of beta-glucans, although neutrophils, B cells, T cells, and natural killer cells are also known to be activated by these polysaccharides [30]. Beta-glucans, which cannot directly penetrate cell membrane due to their large molecular size [31], may act as pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface receptors called pattern recognition receptors (PRRs) [32]. Among PRRs that are best known to bind beta-glucans are dectin-1 and the Toll-like receptors (TLRs). Binding of beta-glucans to dectin-1 and TLRs induces intracellular signaling pathways which play an important role in the innate immune response [33, 34]. Beta-glucan-activated signaling cascades eventually lead to the release of cytokines, including IL-12, IL-6, TNF-α, and IL-10 [35].

Our study revealed that CD40 was among the genes connected with TLR-induced signaling pathways, which were up-regulated in LPS-G1 group when compared to LPS-G0 group. CD40 protein is a member of the TNF receptor (TNFR) superfamily recognizing CD40L molecule (Supplementary Table 2). CD40L is produced by both Th1 and Th2 helper T cells, as well as by mast cells, basophils, and eosinophils [36]. Signals induced by CD40L expressed in Th1 and Th2 cells are known to be required for macrophage and B cell activation, respectively [37]. The production of IgE by B cells is induced by CD40-stimulated ERK kinase activation transduced via serine/threonine protein kinase Tpl2 [37]. Interestingly, our study also demonstrated increased expression of other genes connected with TLR signaling and B cell activity. Genes encoding TNF receptor-associated factor 3 (Traf3), and immunoglobulins: IgD, Igll1, and the Fc fragment of IgE (Fcer2) were up-regulated in the LPS-G1 group when compared with the LPS-challenged group (LPS-G0). These results may indicate that dietary supplementation with high-molecular-weight oat beta-glucan in animals with an LPS-induced inflammatory state resulted in a modulation of the immune response of the organism, at least partially mediated by molecules involved in the TLR signaling pathway and B lymphocytes activation. In fact, our previous report on the effect of low- and high-molecular-weight oat beta-glucans on the inflammatory and oxidative stress status in the colons of rats with LPS-induced enteritis also demonstrated that G1 beta-glucan caused a significant increase in B cells within the population of intraepithelial lymphocytes [4]. At the same time, we observed a significant decrease in the levels of proinflammatory cytokines in the colon tissue lysates from rats with enteritis fed the G1 beta-glucan-enriched diet [4].

In addition to the changes in the expression of genes connected with the TLR signaling pathway, we also noted up-regulated levels of Nlrp1b mRNA (NLR family, pyrin domain-containing 1B), which is involved in the NLR signaling pathway (Supplementary Table 2). In animals, the NLR protein family is a class of innate immune molecules comprising PRRs localized in the cellular cytoplasm to recognize microbial products. Among these proteins, NOD1 and NOD2 are known to detect bacterial cell wall components [38]. The activation of NLR proteins results in inflammatory responses mediated by NFκB, MAPK or caspase-1 activation, accompanied by subsequent secretion of proinflammatory cytokines [38]. Other proteins belonging to the NLR family are involved in the formation of inflammasomes in cells. Inflammasomes are macromolecular scaffolds that are composed of an NLR, the adaptor protein ASC, and caspase-1, which form in the cytosol following NLR activation in response to specific microbe- and/or damage-associated molecular patterns [39]. As the sensor component of the inflammasomes, NLRP1 plays a crucial role in innate immunity and inflammation. In response to pathogens and other damage-associated signals, NLP1 initiates the formation of the inflammasome polymeric complex [40]. Furthermore, NLRP1 has been shown to be important in attenuating gastro-intestinal inflammation and tumorigenesis [39]. It was, therefore, interesting to observe that supplementing the diet of LPS-challenged rats with G1 beta-glucan elevated the expression of Nlrp1, providing another argument to the hypothesis that beta-glucans have protective properties against inflammation. It should be noted, however, that the increase in Nlrp1 expression was detected only in the case of the G1 beta-glucan, but not in the case of the low-molecular-weight G2 beta-glucan.

A comparison of the peripheral blood transcriptomic profile between the LPS-G2 and LPS-G0 groups revealed the smallest number of differentially expressed genes (Table 2). These findings are in line with the other results obtained in the course of our studies on the role of dietary supplementation with beta-glucans in rats with LPS-induced enteritis, which all showed that low-molecular-weight beta-glucan (G2) was the less potent modulator of the immune response [4, 8]. In general, large molecular weight beta-glucans are regarded more biologically active, whereas short beta-glucans with molecular weight below 5000–10,000 Da were shown to be inactive [41]. Furthermore, soluble beta-glucans appear to be stronger immunostimulators than insoluble ones [30]. Our observations confirm these findings, as the high-molecular-weight beta-(1 → 3)/(1 → 4)-linked oat d-glucan showed better antiinflammatory and antioxidative potential in colon and peripheral blood of rats challenged with LPS [4, 8]. This is at least partially connected with its physical property, i.e., viscosity and solubility, in the intestine, where the organism is first exposed to beta-glucans action.

Although, in our present study, a substantially lower number of genes connected with immune response were regulated under the influence of low-molecular-weight oat beta-glucan (G2), it is worth noting that IL34 mRNA showed increased expression in the LPS-G2 group when compared to LPS-G0 group (Supplementary Table 3). Interleukin 34 (IL-34) promotes the proliferation, survival, and differentiation of monocytes and macrophages. It is also known to stimulate the release of proinflammatory chemokines, and thereby plays an important role in innate immunity and in inflammatory processes [42]. Foucher et al. [43] have shown that IL-34 induces the differentiation of monocytes into resident macrophages (MΨ). Their results have demonstrated that IL-34-stimulated macrophages have potent immunosuppressive properties. Upon LPS stimulation, those MΨ macrophages treated with IL-34 produced high levels of antiinflammatory IL-10 and low levels of proinflammatory IL-12 [43]. To our knowledge, our report is the first showing a possible relationship between beta-glucans and IL-34 expression; however, Sarinho et al. [44] demonstrated that subcutaneous beta-(1 → 3)-d-glucan injections applied to children with mild-to-moderate persistent asthma resulted in the patients having increased serum IL-10 levels. Those authors suggested that beta-glucans may be used to modulate allergic sensitisation and may be beneficial in restoring Th2 cells function.

A comparison of the gene expression profile between the LPS-G2 and LPS-G0 groups also revealed a 2.7-fold down-regulation of the prostaglandin E receptor 3 (PTGER3) mRNA levels in the blood of the LPS-challenged rats supplemented with G2 beta-glucan (Supplementary Table 3). This receptor is involved in the prostaglandin E2 signaling pathway (Table 6). So far, the majority of data suggest that prostaglandin E receptor (EP3) mostly inhibits immune responses. In an OVA-induced asthma model, inflammation was more pronounced in EP3-deficient mice than in wild-type mice, and an EP3 agonist suppressed inflammation [45]. EP3 stimulation also resulted in the inhibition of skin inflammation in a contact hypersensitivity model [46]. Further studies are necessary to determine the possible relationship between the molecular activity of beta-glucans and decreased PTGER3 expression.

Among genes commonly regulated in both groups supplemented with beta-glucans (LPS-G1 and LPS-G2), two genes particularly have drawn our attention: Mtus1 (microtubule associated tumor suppressor 1) which was significantly up-regulated, and Ube2C (ubiquitin-conjugating enzyme E2C) which was significantly down-regulated by both beta-glucans (Table 4). Data in the literature show that the expression of Mtus1 is down-regulated in a wide range of cancers: colon tumors, breast cancer, ovarian cancer, head and neck squamous cell carcinoma, as well as prostate cancer cell lines [47‐50]. Transfections of HUVEC cells with Mtus1 siRNA resulted in significantly increased cell proliferation, showing the important antimitotic properties of this gene [50]. On the other hand, Ube2c high expression has been shown to be positively correlated with unfavorable prognosis in various cancer types of: breast, bladder, lung, and thyroid gland [51‐55]. UBE2C is an integral component of the ubiquitin proteasome system required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit [51]. Cells overexpressing Ube2c ignore the mitotic spindle checkpoint signals and lose genomic stability, which is a hallmark of cancer [55]. In our study, the expression of Ube2c was over fivefold down-regulated in LPS-G1 group and over threefold down-regulated in LPS-G2 group when compared with LPS-G0 group (Table 4). So far, there are no data showing a direct relationship between beta-glucans and the aforementioned genes; however, the anticancer properties of beta-glucans have been widely suggested. The effect of beta-(1 → 3),(1 → 4)-d-glucan of high molecular weight in the form of a highly purified water extract was tested in vitro on human skin melanoma HTB-140 cells [56]. The results showed a concentration-dependent increase in apoptosis induction manifested by augmented activation of caspase-3/-7 and an increased number of cells positively stained with annexin V-FITC. The same compound of low molecular weight was used to test antitumor capacity in the following cancer cells: Me45, A431, and normal HaCaT and murine macrophages [57]. The study showed that beta-(1 → 3),(1 → 4)-d-glucan significantly decreased the viability of cancer cells in a dose- and time-dependent manner, while the polysaccharide was non-toxic for normal cells. The effect of both high- and low-molecular-weight beta-(1 → 3),(1 → 4)-d-glucans was also studied on human lung adenocarcinoma cells, human multidrug-resistant small cell lung cancer cells, and normal human keratinocytes, showing an induction of oxidative stress and the cytotoxic properties of beta-glucan preparations in cancer cells but not normal keratinocytes [58]. Furthermore, research on beta-glucans isolated from other sources (yeast and fungi) also provides the evidence of the anticancer properties of these compounds. Beta-(1 → 3)-d-glucan from Saccharomyces cerevisiae has been shown to exert antitumor effects in S180 tumor-bearing mice without toxicity in normal mouse cells. Treatment with beta-(1 → 3)-d-glucan resulted in an increased rate of tumor inhibition in a dose-dependent manner, and significantly potentiated immune responses in mice [59]. In another study, Maitake D-Fraction extracted from Grifola frondosa (Maitake mushroom) decreased the size of lung, liver, and breast tumors in > 60% of patients when it was combined with chemotherapy in a two-arm controlled study compared with chemotherapy alone [60]. In a prospective clinical trial of the short-term immune effects of oral beta-glucan in patients with advanced breast cancer, 23 female patients with advanced breast cancer were compared with a control group of 16 healthy females [61]. Beta-(1 → 3),(1 → 6)-d-glucan was taken daily by oral administration and blood samples were collected on days 0 and 15 of the trial. The results showed that, despite a relatively low initial white cell count, oral beta-glucan stimulated the proliferation and activation of peripheral blood monocytes in the patients with advanced breast cancer. In the present study, the up-regulation of Mtus1 and significant down-regulation of Ube2c in the peripheral blood of the LPS-challenged rats supplemented with beta-glucans may support the hypothesis concerning the potential anticancer effects of these polysaccharides.

The microarray analyses conducted in the present study also revealed that the administration of oat beta-glucans in the diet resulted in increased expression of Atg10 transcript, which belongs to the family of autophagy-related genes (ATGs) (Table 4). In addition, a comparison between the LPS-G1 and LPS-G0 experimental groups showed a significant up-regulation of another member of the ATG family, Atg13 (Supplementary Table 2). Autophagy is a homeostatic process of lysosomal degradation observed in all eukaryotic organisms. It is based on the formation of double-membrane vesicles, termed autophagosomes, which engulf long-lived cytoplasmic proteins, damaged organelles and even invasive pathogens, and transport these cargos to the lysosomes in which the cargo is degraded by the hydrolytic enzymes [62]. Autophagy is constitutively active in cells at a basal level, providing a quality control mechanism for proteins and organelles to protect cells from the adverse consequences of aggregated or misfolded proteins, as well as damaged organelles, such as mitochondria, which could induce cell death or degenerative diseases. On the other hand, autophagy is regarded as an essential prosurvival catabolic pathway induced by a wide variety of stresses, including nutrient deprivation, hypoxia, oxidative stress, growth factor withdrawal, and intracellular infections. The process is strictly regulated by several signaling pathways, and remains under the control of over 30 ATGs and their protein products. Recently, an increasing number of reports have described the role of autophagy in adaptive immunity [63], as well as in the endogenous pathway of antigen MHC II presentation [64]. Both differentially expressed autophagic genes, Atg10 and Atg13, encode proteins involved in the process of autophagy induction in cells. Currently, some reports describe a possible relationship between beta-glucans and autophagic activity in cells. Studies have shown that the recognition of beta-glucan by dectin-1 could trigger the formation of phagosomes that specifically recruit LC3, which is a protein associated with the membranes of autophagosomes, and, thus, regarded as a reliable marker of autophagy induction in cells [65]. Dectin-1 signaling was entirely sufficient to trigger LC3-associated phagocytosis of fungal beta-glucan particles [66]. Although there are no data describing the possible regulation of ATG expression by beta-glucans, our findings suggest that these polysaccharides may play a role in autophagy induction, and the connection between this process and immunomodulation should be further explored.

Finally, among the genes that were differentially expressed in the peripheral blood of the rats supplemented with beta-glucans, quite a substantial number of transcripts represented proteins involved in ion channel formation or regulation in cell membranes, e.g., Kcng2, Kcnc4, Scn7a, Nkain4, Camk1g (in LPS-G1 vs. LPS-G0 analysis); Kcnmb2, Kcnj12 (in G2-LPS vs. LPS analysis) (for a gene description see Supplementary Tables 2 and 3). These genes encode proteins of potassium, sodium, and calcium voltage channels, and interestingly, they were all down-regulated in the beta-glucan-treated groups (Supplementary Tables 2 and 3). Some reports describe a relationship between the activity of ion-voltage channels and LPS. Gerth et al. [67] exposed macrophages to LPS to study the early cellular events associated with LPS-mediated macrophage activation. They used the whole-cell patch clamp technique to analyze ion channels and measure the membrane currents in relation to the translocation of the NFκB transcription factor to the nucleus. Exogenous LPS was shown to increase the voltage-dependent outward current, whereas the voltage-dependent inward current was unaffected [67]. In parallel, the authors found that exogenous LPS led to the nuclear localization of the p65 subunit of NFκB, proving the activation of this transcription factor, which is a major process in macrophage activation, as NFκB induces the expression of a number of genes encoding many inflammatory mediators [67]. Thus, it is possible that the down-regulation of genes encoding potassium and sodium channel proteins in the LPS-G1 and LPS-G2 experimental groups may also be connected with the antiinflammatory functions of beta-glucans and the attenuation of the inflammatory state induced by LPS injection. Furthermore, Schmid-Antomarchi et al. [68] demonstrated that the level of activity of NADPH oxidase is dependent on the plasmic membrane potential, and suggested a link between the generation of reactive oxygen intermediates and the opening of the K(Ca) channels. Our previous studies showed that the effect of beta-glucans was connected with their antioxidative activities, as rats with LPS-induced enteritis that were supplemented with beta-glucans had an increased concentration of antioxidative potential markers (TAS, SOD, GR, and GPx activity; TBARS concentration) in the blood, spleen, colon tissue, stomach, and liver [4, 8‐10]. Taken all together, these findings may suggest that the decreased expression of genes connected with ion-voltage channel activity is linked with the antiinflammatory and antioxidative activity of beta-glucans observed in our in vivo experimental model.