Influence of Porphyra-derived polysaccharides and oligosaccharides on attenuating food allergy and modulating enteric microflora in mice

ABSTRACT
 Recently, there have been reports on the immunomodulatory properties of polysaccharides (PP), oligosaccharides (PO), and mixtures of PO/PP derived from Porphyra. To explore their potential in alleviating allergic responses and enteric dysbiosis, this study delved deeper into their effects using a murine model of food allergy. Daily administration of Porphyra samples mitigated allergic diarrhea, enteritis, and allergen-specific IgE production. Moreover, Porphyra samples exerted a significant reduction in the secretion of IL-2, IL-4, IFN-γ, and IL-17A, along with an elevation in the IL-10/IL-4 ratio in splenocytes upon allergen stimulation. Similarly, expression of IL-4 and IFN-γ in the duodenum was suppressed. Notably, allergic mice displayed a distinct fecal microflora profile compared to that of normal mice. Interestingly, PP and PO treatments exhibited different effects on improving enteric dysbiosis. These findings collectively suggest the potential of PP and PO as functional food candidates for the management of allergic enteritis and dysbiosis.


Introduction
The prevalence of food allergy, an immune disorder resulting from the consumption of food allergens, continues to rise, affecting approximately 3-10% of adults and 8% of children (Messina & Venter, 2020).One potential cause of food allergy is the failure of oral tolerance to food allergens (Vickery et al., 2011).Upon crossing the epithelial barrier, food allergens are recognised by dendritic cells, which subsequently present them to naïve T cells (Renz et al., 2018;Vickery et al., 2011).This interaction elicits the differentiation of naïve T helper (Th) cells into IL-4-secreting Th2 cells, promoting IgE secretion by B cells (Anvari et al., 2019).The allergen-specific IgE binds to the highaffinity immunoglobulin ε receptor on mast cells (Anvari et al., 2019;Kumar et al., 2012).When the gastrointestinal tract is exposed to the same allergen, IgE crosslinking induces mast cell degranulation and the secretion of inflammatory factors, leading to enteritis (Kumar et al., 2012;Ray, 2006;Vickery et al., 2011).Currently, there are no effective treatments for food allergy, highlighting the urgent need for the development of novel therapeutic and preventive strategies.
The composition of enteric microflora plays a crucial role in intestinal immunity and modulation of allergic responses.Various factors, including dietary choices, have been identified to influence the composition of enteric microflora (Hooper et al., 2012).Studies have shown that enteric dysbiosis can trigger and exacerbate intestinal inflammation and even lead to sepsis caused by opportunistic pathogens, primarily due to the disruption of the intestinal barrier (Gill et al., 2006;Huang et al., 2020).Therefore, maintaining a balanced enteric microflora is crucial for the development of immune tolerance.It has been proposed that preventing enteric dysbiosis could maintain the balance between Th1 and Th2 immune responses and preserve an adequate number of regulatory T (Treg) cells in the intestinal-associated lymphoid tissue.Additionally, treatment with probiotics has been shown to inhibit intestinal inflammation, stimulate Treg cell proliferation, and enhance the secretion of IL-10 (Atarashi et al., 2013;Rodriguez et al., 2012).
Dietary indigestible carbohydrates, such as oligosaccharides and polysaccharides, possess the unique ability to selectively enhance the growth or activity of beneficial enteric probiotics (Davani-Davari et al., 2019).In the intestinal tract, Bacteroides and Firmicutes are the predominant bacterial phyla.Bacteroides, in particular, has the capability to metabolise and utilise the polysaccharides present in the intestine (Cockburn & Koropatkin, 2016).Previous research suggests that natural polysaccharides can promote the growth of Bacteroidetes, reduce the abundance of Proteobacteria, and increase the population of microorganisms that produce short-chain fatty acids in the intestine.Consequently, polysaccharides contribute to maintaining a balanced enteric microflora and creating an unfavourable environment for opportunistic pathogens (Di et al., 2018).
Porphyra sp., a type of red seaweed commonly consumed in East Asia, serves as both an economic crop and a food source.It contains various bioactive components, especially polysaccharides, which constitute 20-40% of the dry weight of Porphyra (Bhatia et al., 2013;Cian et al., 2012;Mu & Wu, 2011;Wang et al., 2008).These polysaccharides share a structural similarity to agarose and are characterised by the presence of β-D -galactose, 6-O-methyl-β-D -galactose, α-L -galactose, and α-L -galactose-6-sulphate (Cian et al., 2015).Numerous studies have highlighted the diverse bioactivities of Porphyra polysaccharides.For instance, Shi et al. demonstrated that sulphated polysaccharides extracted from P. haitanensis can mitigate allergic reaction in mice (Shi et al., 2015).Oligosaccharides derived from the hydrolysis of Porphyra polysaccharides have also exhibited bioactivities.Osumi et al. used bacterial enzymes to hydrolyse P. yezoensis-derived polysaccharides, resulting in oligosaccharides that showed hypocholesterolemic potential (Osumi et al., 2002).Wu et al. employed Aeromonas salmonicida MAEF108 enzymes to hydrolyse Porphyra polysaccharides and obtain Porphyra oligosaccharides with antioxidant activity (S.-C.Wu et al., 2005).Our recent study has provided evidence supporting the effects of PP, PO, and their mixtures on regulating antigen-specific humoral responses and splenic cytokine production, highlighting their potential protective role against allergen-induced immune responses (Wei et al., 2022).
To assess the potential use of PP, PO, and their mixtures in managing allergy, the objective of this study was to investigate their anti-allergic effects in a murine model of food allergy.Furthermore, the profile of immune response and gut microflora was illustrated to unravel the mechanisms underlying the actions of PP and PO.

Materials and methods
2.1.Chemicals, reagents, detection kits and Porphyra-derived polysaccharides (PP) and oligosaccharides (PO) Chemicals and reagents were procured from Sigma Chemical (St.Louis, MO, USA), unless specified otherwise.Cell culture reagents were obtained from GE Healthcare Life Sciences (Marlborough, MA, USA).ELISA kits for cytokine and immunoglobulin (Ig) measurements were sourced from eBioscience, Inc. (San Diego, CA, USA).The necessary reagents and antibodies for immunohistochemical (IHC) staining were acquired from Vector Laboratories, Inc. (Newark, CA, USA), and Thermo Fisher Scientific Inc. (Waltham, MA, USA).The source of dried Porphyra sp. and methods for PP and PO preparation were the same as described in the previous study (Wei et al., 2022).In addition, the characteristics of PP and PO were shown in the previous study (Wei et al., 2022).

Food allergy model and design of animal experiment
Female BALB/c mice at 5 weeks of age were procured from the National Laboratory Animal Center of Taiwan.The mice were housed in the laboratory animal facility of the National Taiwan Ocean University (NTOU) and allowed free access to water and food for 1 week prior to the conduction of experiment.As shown in Figure 1(A), mice were divided into following groups (5 mice per group): the naïve group (NA), the vehicle group (VH), and the treatment groups.In the VH group, mice received a daily oral gavage of 0.1 mL phosphate-buffered saline (PBS).In the treatment groups, mice were administered a daily oral gavage of 150 mg/kg body weight of PP, PO, or PO/PP mixtures (PO:PP = 2:1, 1:1, and 1:2; referred to as O2P1, O1P1, and O1P2, respectively) dissolved in 0.1 mL PBS.The dosage regimen was according to the results in a previous study (Wei et al., 2022).
The protocol and methods for the induction of food allergy were the same as that reported in the previous study (Huang et al., 2020).Except for the mice in the NA group, all other mice were sensitised intraperitoneally with ovalbumin (OVA) and alum as adjuvant on day 4 and day 18.From day 32 to day 40, the sensitised mice were subjected to repeated challenges with OVA via oral gavage every other day.On the days of sensitisation and challenge, treatment of Porphyra samples was at least 4 h prior to the sensitisation or challenge.The severity of allergic diarrhea, occurring 30-60 min after the challenge, was assessed by scoring the fecal form on a scale of 0-3: 0 for normal feces, 1 for soft feces, 2 for sloppy feces, and 3 for diarrhea.On day 41, the mice were euthanized, and the duodenal tissues, which showed obvious histopathological changes in the employed model as described in the previous studies, were collected for histopathological examination (Huang et al., 2009(Huang et al., , 2010(Huang et al., , 2017)).The protocol and methods of immunohistochemical (IHC) staining for IL-4 and IFN-γ detection were the same as that reported in the previous study (Huang et al., 2010).Hematoxylin and Eosin (H & E)-stained sections and IHC-stained sections were observed by an upright microscope (Olympus BX53) and analysed using ImageJ software to determine histopathological changes, the crypt/villus ratio, and the area ratios of IL-4 and IFN-γ positive signals to the total tissue area, following previously described methods (Huang et al., Figure 1.Protocol of the animal experiment, diarrhea scores and histopathological examination of duodenal tissues.(A) BALB/c mice were divided into the naïve group (NA), the vehicle group (VH), and the treatment groups (5 mice per group).In the VH group, mice received a daily oral gavage of 0.1 mL PBS.In the treatment groups, mice were administered a daily oral gavage of 150 mg/kg body weight of PP, PO, or PO/PP mixtures (PO:PP = 2:1, 1:1, and 1:2; referred to as O2P1, O1P1, and O1P2, respectively) dissolved in 0.1 mL PBS.Except for the mice in the NA group, all other mice were sensitised with ovalbumin (OVA) on day 4 and day 18.From day 32 to day 40, the sensitised mice were subjected to repeated challenges with OVA via oral gavage every other day.On day 41, the mice were euthanized, and the samples of feces, spleen and duodenal tissues were isolated for further experiments.(B) The severity of allergic diarrhea, occurring 30-60 min after the challenge, was assessed by scoring the fecal form on a scale of 0-3: 0 for normal feces, 1 for soft feces, 2 for sloppy feces, and 3 for diarrhea.(C) Representative images of H&E-stained duodenal sections are shown, highlighting villus edema (marked by red arrows) and crypt hyperplasia (marked by yellow arrows).(D) The crypt/villus ratio was quantified using ImageJ software.Data are presented as mean ± SEM (n = 5) and represent three independent experiments.Statistical analysis was performed to compare the different groups, with significance indicated by the symbols # (p < 0.05 compared to the NA group) and *(p < 0.05 compared to the VH group).2009,2010,2017).Spleen samples were individually isolated to prepare splenocyte suspensions, and fresh fecal samples were collected after the final challenge for nextgeneration sequencing (NGS) analysis of 16S rRNA gene, as described in previous studies (Huang et al., 2020).

Treatment of splenocytes and measurement of cytokine and Ig
Splenocytes prepared from the spleen isolated from each mouse were seeded into 24-well culture plates at a concentration of 5 × 10 6 cells/mL.These cells were then treated with OVA for a period of 24-72 h.After the designated treatment time, the supernatants from the culture plates were collected.The levels of IFN-γ, IL-2, IL-4, IL-10, and IL-17A were measured using ELISA.Furthermore, the levels of total IgG, IgE, OVAspecific IgG, and OVA-specific IgE in the serum samples were also measured using ELISA.The ELISA protocol was conducted following the instructions provided by the supplier.

Statistical analysis
Comparison between groups were conducted by use of ANOVA followed by a Student's t-test (SigmaPlot V14, Systat Software Inc.).The differences were considered significant at p < 0.05.

Influence of PP, PO and PO/PP mixtures on alleviating allergic diarrhea and enteritis in mice
In our previous study, it was found that the dosage of 150 mg/kg of PP, PO, and PO/ PP mixtures had a modulatory effect on antigen-specific immune responses (Wei et al., 2022).Therefore, the same dosage of 150 mg/kg was employed to investigate and compare the anti-allergic effects of PP, PO, and PO/PP mixtures.During the experiment, no abnormal behaviour or activity were observed in any of the mice, revealing that the employed dosage did not cause any obvious adverse effects on the mice.The diarrhea scores of mice in the VH group increased with each challenge.However, scores of mice in PP, PO, and PO/PP mixtures groups were statistically lower than those in the VH group at the 4th and 5th challenges, indicating that Porphyra samples could reduce the severity of allergic diarrhea (Figure 1(B)).Furthermore, the effects of PP, PO, and PO/PP mixtures on improving allergic enteritis were assessed through histopathological examination.In the VH group, significant tissue damage, villus edema, and crypt hyperplasia were observed (Figure 1(C)).However, the treatment groups showed milder histopathological changes compared to the VH group (Figure 1(C)).The ratio of crypt depth to villus height is a commonly used index for assessing gut health (Pluske et al., 1996).Treatment with Porphyra samples resulted in a reduced crypt/villus ratio compared to the VH control group (Figure 1(D)), indicating the protective effects of PP, PO, and PO/PP mixtures against allergic enteritis.

Impact of PP, PO and PO/PP mixtures on serum antibodies production
The impact of Porphyra samples on IgG production was investigated to understand their effects on humoral immunity.The levels of total IgG were comparable between the VH group and treatment groups (Figure 2(A)).This results reveals that PP, PO, and PO/PP mixtures had limited influence on the production of total IgG, indicating that their effects on humoral immunity for general pathogen protection were minimal (Vidarsson et al., 2014).Furthermore, the levels of total IgE, another type of antibody, were not significantly altered by the treatment of Porphyra samples (Figure 2(B)).Since allergen-specific IgE and IgG play crucial roles in the initiation and inhibition of allergeninduced mast cell degranulation through the cross-linking of IgE receptors, the levels of OVA-specific IgE and IgG were specifically investigated (Finkelman, 2007).Compared to that in the NA group, the increased level of OVA-specific IgE in the VH group indicated a successful induction of type I hypersensitivity (Figure 2(D)).Compared to the VH group, the levels of OVA-specific IgE were down-regulated, while the levels of OVA-specific IgG were slightly up-regulated in mice treated with Porphyra samples (Figure 2(C,D)).These results indicate that the modulatory effects of Porphyra samples on humoral immunity were allergen-specific, specifically affecting the production of allergen-specific IgE in response to OVA.In line with the results of the current study, Persiyanova et al. have demonstrated that polysaccharides derived from the brown algae Fucus evanescens could raise the level of OVA-specific IgG (Persiyanova et al., 2020).As the binding of allergen-specific IgG to allergen blocks the binding of IgE and allergen (Finkelman, 2007), the up-regulation of OVA-specific IgG is suggested as one of the potential manifestations of effective treatment.

Effects of PP, PO and PO/PP mixtures on regulating splenic and duodenal cytokine production
To investigate the effects of Porphyra samples on regulating allergen-induced Th subset activation, the production of IL-2, IFN-γ, IL-17A, IL-4 and IL-10, representing the activation of Th1, Th17, and Th2 and Treg subsets, by OVA-stimulated splenocytes was measured.OVA sensitisation and challenge led to an increase in the secretion of these cytokines (Figure 3(A-E)), indicating the activation of Th1, Th17, and Th2 subsets.Treatment of Porphyra samples mitigated the OVA-induced activation of Th1, Th17, and Th2 subsets to varying degrees, as evidenced by the decreased levels of these cytokines (Figure 3(A-E)).IL-10 is predominantly generated by Th2 cells or Treg cells in splenocytes.To understand the impact of Porphyra samples on the activation of Treg cells, the production of TGF-β, another regulatory cytokine, was measured.However, the level of TGF-β was too low to detect by ELISA.Instead, the ratio of IL-10/IL-4 was calculated to evaluate the source of IL-10.Treatment with Porphyra samples significantly increased the IL-10/IL-4 ratio (Figure 3(F)), suggesting a potential upregulation of Treg immunity as a mechanism for the anti-allergic effects of Porphyra samples.However, further studies will be required to address this issue.Since systemic (splenic) and mucosal (duodenal) responses may differ, the duodenal expression of IL-4 and IFN-γ were measured to elucidate the intestinal Th1/Th2 immune balance.Consistent with the results of splenic cytokine production, OVA sensitisation and challenge elevated the duodenal expression of IL-4 and IFN-γ.However, treatment with Porphyra samples suppressed duodenal expression of IL-4 and IFN-γ (Figure 4), suggesting the influence of Porphyra samples in alleviating allergen-induced Th1 and Th2 immune responses.
Although both IL-10 and TGF-β are classified as regulatory cytokines secreted from Foxp3 + Treg cells, it is primarily IL-10, not TGF-β, that inhibits mast cell activation and intestinal inflammatory responses in murine models of food allergy (Burrello et al., 2018;Huang et al., 2010).This phenomenon was also observed in allergic mice treated with Porphyra samples in this study.Furthermore, the balance between effector Th cell subsets plays an essential role in controlling inflammation and allergy (Chaplin, 2010).Cytokine production is a common indicator for Th subset activation, with each subset producing specific cytokines.Indeed, IL-2 is involved in clonal expansion of lymphocytes.IFN-γ and IL-17A contribute to different inflammatory responses in certain immune disorders.IL-4 induces IgE isotype switching and subsequent type I hypersensitivity.IL-10 serves to restrict the immune response of the host and preserve immune balance (Chaplin, 2010).Based on the results of the current study, the anti- allergic effects of Porphyra samples were closely related to the attenuation of allergenspecific Th subset activation through the upregulation of IL-10 production.

Impact of PP, PO and PO/PP mixtures on enteric microflora
In this study, the profile of enteric microflora was investigated using 16S rRNA gene sequencing.The main bacterial classes identified in the enteric microflora were Bacteroidia, Bacilli, and Clostridia.Compared to that in mice of the NA group.The relative abundance of Bacilli and Clostridia was decreased, while the relative abundance of Bacteroidia was increased in the VH group (Figure 5(A)).However, treatment with PP and PO resulted in different impact on the enteric microflora.PP treatment decreased the relative abundance of Bacteroidia, and the predominant bacterial class was Bacilli.On the other hand, PO treatment decreased the relative abundance of Bacteroidia and increased the relative abundance of Clostridia, Lachnospiraceae, Actinobacteria, and The microbial diversity and relative abundance of fecal bacterial classes in each group are represented using pie charts.These charts provide a snapshot of the composition and distribution of different bacterial classes in the fecal samples from each experimental group.(B) Linear discriminant analysis (LDA) effect size (LEfSe) graphics were generated to analyse the differences between the groups of mice.The horizontal bars in the graphics represent the effect size, while the length of the bar corresponds to the log 10 transformed LDA score, indicated by vertical dotted lines.The colour scheme distinguishes between mice in the VH group (red) and mice in the PO or PP groups (green).To identify taxa of bacteria that exhibited statistically significant changes in their relative abundance (p < 0.05), the taxonomic names of these bacteria are labelled alongside the horizontal lines in the LEfSe graphics.A threshold of 2.0 on the logarithmic LDA score was applied to determine discriminative features.
Coriobacteriia (Figure 5(A)).To further analyse the specific bacteria affected by PP and PO treatment, LEfSe analysis was performed.It was found that the relative abundance of Bifidobacterium was 4.8-fold higher in the PP group compared to that in the VH group (Figure 5(B)).The relative abundance of Clostridia, Lachnospiraceae, Actinobacteria, and Coriobacteriia was at least 3.6-fold higher in the PO group compared to that in the VH group (Figure 5(B)).These findings indicate that PP and PO exerted distinct effects on modulating enteric dysbiosis, which could be attributed to their different molecular weights and sulphate contents (Wei et al., 2022).
Previous studies have shown that Bifidobacterium has beneficial effects in ameliorating food allergy by modulating the populations and activities of Th subsets and Treg cells (Gu et al., 2023;Liu et al., 2018).Clostridia has been reported to limit allergen exposure, suppress sensitisation, and then prevent against the development of food allergy (Stefka et al., 2014).The role of Lachnospiraceae and Actinobacteria in the development or prevention of food allergy is still debated.While some studies have observed a higher prevalence of Lachnospiraceae in children with egg and milk allergy, others have demonstrated a potential protective association with milk allergy (Berni Canani et al., 2016;Fazlollahi et al., 2018;Feehley et al., 2019).Actinobacteria abundances have been found to be both higher and lower in allergic populations compared to healthy individuals in different studies (Dethlefsen et al., 2007;Ling et al., 2014).Limited information is available regarding the relationship between Coriobacteriia and food allergy, but recent studies suggest a potential negative effect of Coriobacteriia on allergic rhinitis and asthma (Jin et al., 2023;Zheng et al., 2022).Polysaccharides and oligosaccharides are known to be metabolised by enteric microbes, and their metabolites are important for maintaining intestinal barrier integrity and immune tolerance (Cheng et al., 2022;Iribarren et al., 2021;Wu et al., 2023).Since the fecal microflora profile differed between PP-and PO-treated mice, further metabolome analysis would be necessary to understand how PP and PO influence the enteric microflora and how the affected microbes modulate the immune system and ameliorate allergic responses.

Conclusion
This study provides valuable insights into the anti-allergic effects of PP and PO.The distinct effects observed between PP and PO on attenuating inflammation, modulating the production of allergen-specific antibodies and cytokines and enteric microflora could be attributed to their differences in molecular weight and chemical composition.These findings open up possibilities for the development of functional foods containing Porphyra-derived oligosaccharides and polysaccharides as therapeutic options for managing allergic enteritis and dysbiosis.

Figure 2 .
Figure 2. The production of antibodies in serum of mice.Serum sample was collected from each mouse, and the concentrations of immunoglobulins, including (A) total IgG, (B) total IgE, (C) OVAspecific IgG, and (D) OVA-specific IgE, were measured.Data are presented as mean ± SEM (n = 5) and represent three independent experiments.Statistical analysis was conducted to compare the different groups, and the symbols # (p < 0.05 compared to the NA group) and *(p < 0.05 compared to the VH group) were used to indicate significant differences.

Figure 3 .
Figure 3. Cytokine production from OVA-stimulated splenocytes.Splenocyte suspensions prepared from mice in each experimental group were cultured in the presence of OVA.The levels of (A) IL-2, (B) IFN-γ, (C) IL-17A, (D) IL-4 and (E) IL-10 in the supernatants were measured, and the ratio of (F) IL-10/IL-4 was calculated.Data are expressed as mean ± SEM (n = 5) and represent three independent experiments.Statistical analysis was performed to compare the different groups, and the symbols # (p < 0.05 compared to the NA group) and *(p < 0.05 compared to the VH group) were used to denote significant differences.

Figure 4 .
Figure 4.The expression of IL-4 and IFN-γ in the duodenal tissues of mice.Representative images of the IHC-stained sections are presented in (A, C), where the brown signals indicate the presence of IL-4 or IFN-γ.(B, D) The area occupied by IL-4 and IFN-γ positive signals was measured, and the ratio of positive area to the total tissue area was calculated using ImageJ software.Data are expressed as mean ± SEM (n = 5) and represent three independent experiments.Statistical analysis was conducted to compare the different groups, and the symbols # (p < 0.05 compared to the NA group) and *(p < 0.05 compared to the VH group) were used to indicate significant differences.

Figure 5 .
Figure5.Full-length 16S rRNA gene analysis of fecal microbiota.(A) The microbial diversity and relative abundance of fecal bacterial classes in each group are represented using pie charts.These charts provide a snapshot of the composition and distribution of different bacterial classes in the fecal samples from each experimental group.(B) Linear discriminant analysis (LDA) effect size (LEfSe) graphics were generated to analyse the differences between the groups of mice.The horizontal bars in the graphics represent the effect size, while the length of the bar corresponds to the log 10 transformed LDA score, indicated by vertical dotted lines.The colour scheme distinguishes between mice in the VH group (red) and mice in the PO or PP groups (green).To identify taxa of bacteria that exhibited statistically significant changes in their relative abundance (p < 0.05), the taxonomic names of these bacteria are labelled alongside the horizontal lines in the LEfSe graphics.A threshold of 2.0 on the logarithmic LDA score was applied to determine discriminative features.