Early Dietary HMOS reduced diabetes incidence and pancreatic insulitis in NOD-mice later in life

To determine the effects of HMOS on T1D development, four-week (Wk)-old female NOD-mice were provided with (or without) 1% HMOS containing diet from Wk4 until Wk10 of life. Clinical onset of diabetes was determined based on weekly monitored urine glucose levels. NOD-mice receiving HMOS showed delayed onset in disease development (22 ± 1.4 and 25 ± 4.5 weeks (mean ± SEM) for HMOS and control group respectively). The time of each diabetic mouse occurrence in both control and HMOS diet receiving groups were shown Fig. S1. At the end of the study, a significant reduction in the incidence of T1D was detected within NOD-mice receiving HMOS early in life (15% and 47.4% for HMOS and control group, p < 0.05 respectively, n = 19 for control, and n = 20 for HMOS group, Fig. 1A). Maximal measured blood glucose levels confirmed classification of non-diabetic and diabetic mice, since significantly higher levels were found between diabetic subgroups and non-diabetic subgroups (Fig. 1B). In addition, the mean blood glucose levels (mM) were significantly different between mouse from control and HMOS group (p < 0.05) (Fig. 1C). No changes between groups were observed in body weight over time (Fig. S1). Histological analysis of pancreatic islet inflammation (Degree of insulitis as scored on representative HE stained islets shown in Fig. 1D) revealed significantly (p < 0.001) reduced incidence of complete insulitis in HMOS-receiving NOD-mice (19%) compared to control NOD-mice (61%) (Fig. 1E). In mice receiving HMOS the mean normalized insulitis score was significantly lower (2.1 ± 0.16 (mean ± SEM) p < 0.001) than observed in control group (3.4 ± 0.16 (mean ± SEM)) (Fig. 1F). These data indicate that early HMOS supplementation can protect against T1D development.

Figure 1 Early HMOS dietary intervention protects NOD-mice from onset and development of T1D. (A) Diabetes Free percentage in control (solid line trace, n = 19) and HMOS (dash line trace, n = 20) group over time. (B) Maximal measured blood glucose levels (mM) in control (black dots, n = 9 for diabetic and n = 11 for non-diabetic) and HMOS group (white dots, n = 3 for diabetic and n = 17 for non-diabetic) were grouped according to diabetic or non-diabetic status. (C) Mean Blood glucose (mM) of control (black dots, n = 19) and HMOS group (white dots, n = 20). (D) Degree of insulitis as scored on representative HE stained islets (range 0–4). Scale bars: 50 μm. (E) Average percentage of each score in the total islets counted in the Control or HMOS group. Average of 46 islets of each mouse from Control and HMOS mice were assigned insulitis scores. White bars present No-insulitis, light grey bars present Peri-insulitis, grey bars presents Insulitis <50%, dark grey bars present Insulitis >50%, and black bars present Complete-insulitis. (F) Mean value of mean normalized insulitis scores (range 0–4) of each mouse from control and HMOS groups are shown. Data are presented as mean ± SEM, n = 19–20/group. Statistical differences between groups are depicted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 using Mann-Whitney U-test. Full size image

Dietary HMOS influenced cytokine profile in NOD-mice

Both immune system regulation and microbiota modulation are suggested to impact T1D development12. To test the effects of dietary HMOS on systemic immunity, serum cytokines in NOD-mice were measured at endpoint. Significantly reduced concentrations of IL-17 were detected in HMOS-treated compared to control NOD-mice (p < 0.01, Fig. 2A), which was mainly present in the non-diabetic NOD-mice (p < 0.05, HMOS-Non-dia vs Control-Dia; p < 0.05, HMOS-Non-dia vs Control-Non-dia). Although no difference in the IFN-γ level between groups was found, non-diabetic mice from HMOS group showed significantly lower level than the two diabetic subgroups (p < 0.01, HMOS-Non-dia vs HMOS-Dia; p < 0.05, HMOS-Non-dia vs Control-Dia, Fig. 2B). In addition, a tendency towards an increased IL-4 level by HMOS dietary intervention was observed (p = 0.06, Fig. 2C), and significant difference was found between HMOS-Non-dia and HMOS-Dia (p < 0.01) subgroups, and HMOS-Non-dia and Control-Dia subgroups (p < 0.01). Surprisingly, TNF-α was significantly increased in HMOS-treated as compared to control NOD-mice (p < 0.01, Fig. 2C), and the non-diabetic mice from HMOS showed higher levels than those from Control group (p < 0.05). In addition, significantly decreased Leptin levels were detected in HMOS-treated NOD-mice as compared to control (p < 0.01, Fig. 2D), and the control diabetic subgroup showed higher levels than all the other three subgroups (p < 0.05, p < 0.05, p < 0.05, Control-Dia vs Control-Non-dia, HMOS-Dia, or HMOS-Non-dia, respectively).

Figure 2 Early HMOS dietary intervention changes serum cytokines profile. The concentrations of (A) IL-17, (B) IFN-γ, (C) IL-4, (D) TNF-α, and (E) Leptin were analyzed in serum of HMOS-modulated and untreated NOD-micedepicted as either total group or separated as Diabetic (Dia) and Non-diabetic (Non-dia) mice as indicated. Data are presented as Mean ± SEM, n = 17/20/group. Statistical differences between groups are depicted as *p < 0.05 and **p < 0.01using Mann-Whitney U-test. Full size image

Dietary HMOS altered the fecal and ceacal microbiota composition of NOD-mice overtime

Knowing that immune and microbiome development are correlated, we analyzed fecal microbiota compositions of NOD-mice receiving either HMOS or control diet using high-throughput 16S ribosomal RNA gene amplicon sequencing at different time points during the experiment. The fecal microbiota of both NOD-mice groups was dominated by three bacterial relative abundance (RA) phyla: Firmicutes, Bacteroidetes, and Verrucomicrobia (Fig. 3A). At baseline (Wk4), no microbial differences were detected prior to intervention (data not shown). Due to HMOS intervention, a gradual change between Firmicutes and Bacteroidetes was detected, leading to a significant increase in the RA of Firmicutes and decrease in the RA of Bacteroidetes at Wk30 (Fig. 3A). Additionally, the Firmicutes-to-Bacteroidetes (F/B) ratio, which has been negatively correlated with glycemic level and reduces overtime in T1D development17,18, indeed declined in both groups at Wk9 and Wk14. At Wk14 and Wk30, HMOS-treated NOD-mice showed significantly higher F/B ratio than the control NOD-mice (Fig. 3B). Further analysis between diabetic and non-diabetic NOD-mice, of both control and HMOS-treated groups, indicated significantly higher F/B ratio in the non-diabetic mice at Wk30 (Fig. S3). This data suggest, that HMOS intervention promoted a microbiota composition preceding prevention of T1D development and differed from untreated control NOD-mice.

Figure 3 Early HMOS dietary intervention alters fecal microbiota composition over time. Fecal samples at Wk4 (baseline), Wk9 (start intervention), Wk14 (post-intervention) and Wk30 (endpoint) were analyzed using 16 S rRNA. (A) Phylum level pie charts, organized by weekly collection time points, significance indicated either by: (#)False Discovery Rate (FDR) p < 0.05; (*)Kruskal-Wallis p < 0.05. (B) The Firmicutes-to-Bacteroidetes ratio significant differences noted in the fecal microbiota of NOD-mice receiving HMOS or control diet. (C) Relative abundance of order level's overall Segmented Filamentous Bacteria (SFB). (D) Relative abundance of families S24–7 and Lachnospiraceae in NOD-mice receiving HMOS or control diet. (E) The number of observed genera (richness) of bacteria of NOD-mice receiving HMOS dietary intervention or not. (F) Stacked column plots of the total number of rarefied sequences (11,000) of the top 90% genera. (G) Stacked column plots of relative abundance of genus SCFA-producing bacteria. (H) Relative abundance of six different bacterial genera from control and HMOS groups. Data are presented as mean ± SEM, n = 17–20/ group. Statistical differences between groups are depicted as *p < 0.05 and **p < 0.01, using Mann-Whitney U-test. Full size image

Segmented filamentous bacteria (SFB) have been positively linked with diabetes protection in NOD-mice19. HMOS-treated NOD-mice indicated higher RA of SFB Clostridiales compared to control NOD-mice (Fig. 3C), which lead to a significant difference at Wk30 (p < 0.05). At the taxonomic family level, the RA of S24–7, a dominate bacteria originating from the phylum Bacteroidetes20, was significantly reduced in the HMOS-treated NOD-mice at Wk14 and Wk30; whereas the RA of Lachnospiraceae, a member of Clostridium cluster XIVa that produces butyrate21, was significantly higher in HMOS-treated NOD-mice, when compared to the control mice at Wk30 (Fig. 3D). Additionally, bacterial richness was significantly higher in HMOS-treated NOD-mice compared with control NOD-mice at Wk30 (p < 0.01, Fig. 3E).

The longitudinal study model of RA genera taxa that indicated significance between untreated control and HMOS-treated NOD-mice fecal samples are depicted in (Fig. 3F). Interestingly, we detected increased RA of SCFAs-producing bacteria in HMOS non-diabetic mice (13% at Wk14, and 12% at Wk30) when compared to HMOS diabetic mice (2% at Wk14, and 2.5% at Wk30) (Fig. 3G). Overall, diabetic mice showed reduced SCFA-producer taxa compared to non-diabetic mice at Wk14 and Wk30 in both groups of NOD-mice. It is noteworthy that within those genera, HMOS-treated NOD-mice displayed more abundant Akkermansia at Wk30, which is a mucin-degrader in the gut22. Besides, the RA of [Ruminococcus] (Wk14), Ruminococcus (Wk9 and Wk30), Oscillospira (Wk30), unclassified genus of Lachnospiraceae (Wk14 and 30), and Coprococcus (Wk9) were observed significantly higher in HMOS treated mice than in control mice (Fig. 3H).

Dietary HMOS maintained alpha and beta diversity of the fecal microbiota in NOD-mice

Low diversity of gut microbiota, i.e. number, abundance, and distribution of bacteria has been linked to increased risk of T1D18,23. Therefore, we analyzed alpha and beta diversity of fecal microbiota in NOD-mice at different time points. At taxonomic level of genus, no significant changes in alpha diversity indices24 were detected in HMOS-treated NOD-mice overtime (Fig. 4B–D), except for bacterial richness (ANOVA; p < 0.01) (Fig. 4A). Particularly, within the HMOS-treated group, significantly higher bacterial richness at Wk30 was detected in non-diabetic mice than in the other disease and time comparisons (Fig. 4A). Additionally, bacterial richness was significantly higher in HMOS-treated NOD-mice compared with control NOD-mice at Wk30 (p < 0.01, Fig. 3E). In contrast, significant loss of diversity in the Shannon (ANOVA; p < 0.01), Simpson (Kruskal-Wallis; p < 0.01), and evenness (ANOVA; p < 0.001) indices were detected in control NOD-mice (Fig. 4B–D). Finally, Wk14 and Wk30 fecal taxon-specific differences were observed within the context of overall microbial community analyses (ANOSIM) that revealed significant differences between the diabetic and non-diabetic NOD-mice in both groups, at the genus level (Fig. 4E). Our finding that NOD mice lose their bacterial diversity over time in T1D development, are consistent with previous studies demonstrating that the level of bacterial diversity diminished overtime in diabetic children18 and HMOS dietary intervention seems to prevent NOD-mice from losing their bacterial diversity. This observation was supported by the Bray-Curtis dissimilarity index indicating greater variability in microbial community HMOS non-diabetic mice at Wk30. (Fig. S4).

Figure 4 Early HMOS dietary intervention changes the alpha diversity and beta diversity of fecal microbiota of the non-diabetic and diabetic mice within each group at four collection points. (A) richness. (B) Shannon’s index. (C) Simpson’s index. (D) evenness. Alpha diversity indices data are represented as mean ± SEM, n = 17–20/ group, *p < 0.05, **p < 0.01, ***p < 0.001, using one-way ANOVA test. (E) Inter-group analysis of similarity (ANOSIM) (Beta diversity). Global R comparison was based on ANOSIM performed within the software R package, as described in the text. P-values were calculated based on a permutational analysis, employing 9,999 permutations. p < 0.05, n = 17–20/group. CD: Control Diabetic; CND: Control Non-diabetic; HD: HMOS Diabetic; HND: HMOS Non-diabetic. Full size image

In addition, functional predictions using PICRUSt analysis indicated that the RA of a majority of reference gene pathways trended different in fecal samples collected from HMOS-treated and control NOD-mice (data not shown). Of note, the RA of three pathways trended higher in HMOS-treated non-diabetic compared to control non-diabetic mice, at Wk14 and Wk30, specifically the carbohydrate digestion and absorption pathway which is associated to SCFAs-producing bacteria (Fig. S5). Overall results indicate and point to specific microbial changes, as well as diversity playing a role in protection of T1D in the NOD-mice receiving early HMOS diet.

Figure 5 Early HMOS dietary intervention increases production of SCFAs in the fecal samples which are correlated to severity of insulitis. (A) Concentrations of total SCFAs (sum of acetic acid, propionic acid, and butyric acid), (B) acetic acid, (C) propionic acid, and (D) butyric acid shown as in total or diabetic (Dia) and Non-diabetic (Non-dia) of control and HMOS group, fecal samples were collected from Wk9. Data are presented as mean ± SEM, n = 17–20/ group. Statistical differences between groups are depicted as *p < 0.05 and **p < 0.01using Mann-Whitney U-test. (E) and (F) Significant correlations between acetic acid and butyric acid with insulitis score, Spearman correlation analysis was used, r- and p-value included respectively. Full size image

Dietary HMOS increased fecal SCFAs levels in NOD-mice during dietary intervention

Changes in microbial ecology promoted us to assess SCFAs levels in feces and cecum content by NMR spectroscopy. Within fecal samples from HMOS-treated NOD-mice, a significantly increased total fecal SCFAs (p < 0.0001) concentration was detected compared to control group (at Wk9, which is during the HMOS provision) (Fig. 5A). Specifically, acetic acid (p < 0.0001), propionic acid (p < 0.01), and butyric acid (p < 0.05) were all significantly higher in fecal samples of NOD-mice receiving HMOS− compared to control diets. More importantly, we found that all samples with higher levels of SCFAs were predominantly present in the HMOS-receiving-non-diabetic mice (Fig. 5A–D). In addition, HMOS-treated NOD-mice had relatively higher total SCFAs concentrations detected in cecum content as compared to control mice (Fig. S6). No differences in SCFAs were detected between the groups at Wk14 and Wk30, which are 4 to 20 weeks post HMOS intervention respectively. Consistently with previous study in which SCFAs (specifically, acetate and butyrate) have been demonstrated to directly protect against pancreatic islet inflammation7 and diabetes incidence8, our correlation analysis between individual SCFAs and insulitis score showed a significantly inverse correlation of acetic acid (spearman correlation R-0.64 and p < 0.01 for HMOS, and R-0.36 with p = 0.12 for control group) (Fig. 5B) and butyric acid (R-0.50 and p < 0.05 for HMOS, and R-0.39 and p = 0.09 for control group) (Fig. 5C) with individual insulitis scores. Together, these results indicate that HMOS mediated protection against development of autoimmune T1D might be established through interaction between microbial derived metabolites SCFA (acetate, propionate, and butyrate) post intervention and immune development.

Figure 6 HMOS, HMOS + Butyrate, and HMOS + Acetate induce direct immunomodulatory effects on murine bone marrow derived DCs phenotype and cytokines microenvironment in vitro. (A) Representative histograms of maturation, co-stimulatory, and inhibitory markers expression on DCs and (B) MFI of given markers expression were shown. (C) Representative histograms of migratory markers expression on DCs and (D) median florescence of intensity of given migratory markers were shown. DCs were treated by medium, HMOS, HMOS + Butyrate, or HMOS + Acetate with (black lines) or without (grey lines) LPS activation for 24 h. Filled histograms represent isotype controlled mAb staining. Gating strategy shown in Fig. S7. (E) Concentrations of IL-10, IL-12p70, and IL-6 by medium- (black bars), HMOS− (white bars), HMOS + Butyrate- (dark grey bars), HMOS + Acetate-DC (grey bars. Data are presented as mean ± SEM, n = 3–4 for DCs activation experiment. Statistical differences between groups are depicted as *p < 0.05, **p < 0.01 and ***p < 0.01 using Mann-Whitney U-test. Full size image

HMOS and SCFAs induce tolerogenic DC phenotype in vitro

Intestinal produced SCFAs are known to exert their effects in different organs25, indeed both acetate and butyrate protect NOD-mice from developing T1D via limiting the autoimmune T cells and promoting functional regulatory T cells8. Moreover, specific HMOS, such as 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), and Lacto-N-neotetraose (LNnT) have been detected within systemic circulation26,27, which allows direct interactions with DCs, the pivotal immune regulators that can drive T-cell priming and differentiation. A subpopulation of DCs with tolerogenic phenotypes and functions are suggested to control effector and regulatory mechanisms relevant to pathology of autoimmune diseases such as T1D28. Both SCFAs and synthetic oligosaccharides can induce DCs with regulatory phenotypes via distinct mechanisms29,30. Therefore, HMOS alone and/or HMOS combined with butyrate or acetate were tested on murine bone marrow derived immature DCs (iDCs) for 24 h after which Th1-type of DCs were induced by adding LPS, DC-phenotype and T-cell induction capacity were subsequently analyzed using flow cytometry. Exposure of iDCs to physiological concentrations of HMOS, HMOS + butyrate, or HMOS + acetate induced a semi-mature phenotype compared to untreated control characterized by induction (MFI) of MHC-II and co-stimulatory molecules CD86, CD80, and CD40 expression, while increased expression of inhibitory molecules PD-L1 and OX40-L were detected (Fig. 6A,B). LPS alone enhanced MFI of MHCII, CD86, CD80, and CD40 on iDCs, but addition of LPS did not change HMOS, HMOS + butyrate, and HMOS + acetate modulated DC-phenotypes (Fig. 6A,B). Interestingly, the expression levels (MFI) of PD-L1 and OX40-L on HMOS− and HMOS + acetate-DC were significantly increased and stable in the presence of LPS as compared to untreated iDCs, pointing to the potential of inducing Tregs31 and skewing immune responses32. It is worth mentioning that, the observed effects of HMOS are not due to the potential LPS contamination since elimination of LPS by polymyxin B, which is an effective LPS inhibitor, does not influence the HMOS effects (Fig. S8).

CC-chemokine receptor 7 (CCR7) essentially contributes to immunity by guiding cells to and within lymphoid organs33. HMOS− and HMOS + acetate-DCs expressed higher levels of CCR7 compared with untreated DCs; LPS up-regulated CCR7 expression in HMOS-DCs. The expression of CXCR3 could only be detected on HMOS-DCs; LPS increased CXCR3 expression on all the three types of DCs (Fig. 6C,D), suggesting increased potential DCs to reach the inflammatory site in the pancreas33. This correlated to significant increased release of IL-10 by HMOS−, HMOS + butyrate- and HMOS + acetate-DC compared to untreated DCs, which were reduced upon LPS stimulation. Moreover, IL-12p70, and IL-6 release of HMOS, HMOS + butyrate, and HMOS + acetate treated DCs in the presence of LPS was significantly lower than control LPS-DCs (Fig. 6E), collectively showing a direct modulation of DCs by HMOS and SCFAs.

HMOS and SCFAs modulate BMDC functions in vitro

Knowing that HMOS−, HMOS + acetate-, and HMOS + butyrate modulated DC phenotype, we assessed effect on effector CD4 + T cells activation (i.e Th-1 and Th17-cells) and Treg differentiation. Upon co-culture of HMOS− and HMOS + acetate-modulated DCs with purified naïve splenic CD4 + T-cells significantly higher percentages of Tregs were detected than when co-cultured with untreated DCs (p < 0.01, p < 0.05, respectively), whilst HMOS + butyrate-DC did not change proportion of Tregs compared with untreated iDC (Fig. 7A,B).

Figure 7 HMOS−, HMOS + butyrate-, and HMOS + acetate-modulated DCs limit effector T cells and promote functional Tregs differentiation. (A) Co-cultured splenic naïve T cells were analyzed by flow cytometry, gating strategy shown in Fig. S9. Representative plots of Foxp3 + CD25 + (Treg), Tbet + CD69 + (Th1), and RORγT + CCR6 + (Th17) CD4 T cells primed by different DCs are shown. (B) Percentage of Treg, Th1, and Th17 cells of CD4 cells primed by medium- (black bars), HMOS− (white bars), HMOS + butyrate- (dark grey bars), HMOS + Acetate-DC (grey bars). (C) IL-10, IFN-γ, and IL-6 levels in the supernatant at day 6 from co-culture. (D) Representative plots and (E) the degree of responder T cell proliferation after co-culturing with Tregs primed by different DCs, gating strategy is shown in Fig. S10. CFSE-labeled naïve CD4 + T cells as responder cells were co-cultured with Tregs primed by different DCs at ratio of 1:1 for 4 days, CD3/CD28 beads were used to activate the responder T cells. Proliferation of FITC-positive cells was analyzed by flow cytometry and suppressive functionality was determined by comparing the dividing (Div) CD4 + T cells. Data are presented as mean ± SEM, 4 independent allogenic stimulation assays and 3 independent suppressive assays were performed. Statistical differences between groups are depicted as *p < 0.05, **p < 0.01 and ***p < 0.001 using Mann-Whitney U-test. Full size image

In addition, a significant increase of Tbet + CD69 + population (Th1) was induced by HMOS modulated DCs compared with untreated DCs (p < 0.05) (Fig. 7A,B). Upon LPS stimulation, all these three types of DCs, particularly HMOS + acetate-modulated DCs (p < 0.01), significantly decreased the induction of Th1-cells. Unexpectedly, HMOS-DC primed significantly higher percentage of RORγT + CCR6 + (Th17) cells compared with untreated DCs (p < 0.05), whereas DCs stimulated by addition of HMOS and butyrate exhibited relative lower percentage of Th17 cells than untreated DCs (Fig. 7A,B). The addition of LPS in the iDCs was effective in the induction of Th17-cells, however, in the presence of HMOS + butyrate, this effect was significantly reduced (p < 0.01, Fig. 7A,B), which is in line with the reduced cytokine responses (IL-17) detected in vivo. The T-cells priming by different DCs was further supported by their cytokine profiles (Fig. 7C). Splenic naïve CD4 + T cells co-cultured with HMOS, and HMOS + butyrate modulated DCs released significantly higher levels of IL-10 compared with those co-cultured with untreated iDCs (p < 0.05). Besides, LPS stimulated DCs induced higher secretion of IFN-γ and IL-6 by the co-cultured cells, and HMOS, HMOS + butyrate, and HMOS + acetate modulated DCs significantly dampened the release of IFN-γ (p < 0.05, p < 0.05, p < 0.05, respectively), and IL-6 (p < 0.01, p < 0.01, p < 0.01, respectively) upon LPS stimulation (Fig. 7C). More importantly, all the Tregs primed by HMOS−, HMOS + butyrate-, HMOS + acetate-modulated DCs displayed a significant higher suppressive capacity in effector T cells compared with untreated iDCs (p < 0.05, p < 0.05, p < 0.05, Fig. 7D,E). These data collectively support that the induction of tDCs by HMOS and microbial derived metabolites may be involved in the prevention of diabetes as observed in NOD-mice receiving HMOS.