Neha M. Sahasrabudhe

The impact of dietary fibers on dendritic cell responses IN VITRO is dependent on the differential effects of the fibers on intestinal epithelial cells

SCOPE In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied. METHODS AND RESULTS The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This was different when DCs and fibers were co-cultured together with supernatants from human epithelial cells (Caco spent medium). Caco spent medium enhanced the production of IL-12, IL-1Ra, IL-6, IL-8, TNF-α, MCP-1 (monocyte chemotactic protein), and MIP-1α but this was strongly attenuated by the dietary fibers. This attenuating effect on proinflammatory cytokines was dependent on the interaction of the fibers with Toll-like receptors as it was reduced by Pepinh-myd88. The interaction of galacto-oligosaccharides, chicory inulin, wheat arabinoxylan, barley β-glucan with epithelial cells and DCs led to changes in the production of the Th1 cytokines in autologous T cells, while chicory inulin, and barley β-glucan reduced the Th2 cytokine IL-6. The Treg-promoting cytokine IL-10 was induced by galacto-oligosaccharides whereas chicory inulin decreased the IL-10 production. CONCLUSIONS Our results suggest that dietary fibers can modulate the host immune system not only by the recognized mechanism of effects on microbiota but also by direct interaction with the consumers mucosa. This modulation is dietary fiber type dependent.

DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1

In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP), and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid, and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

Arabinoxylan activates Dectin-1 and modulates particulate β-glucan-induced Dectin-1 activation

SCOPE Arabinoxylan is one of the most commonly consumed dietary fiber. Immunomodulation by arabinoxylan is documented but the mechanisms by which these immune-effects are accomplished are unknown. METHODS AND RESULTS By applying reporter cell lines for Toll-like receptors (TLRs) and Dectin-1, we demonstrated that arabinoxylan interacts with Dectin-1 receptors and not with TLRs. Arabinoxylan activates Dectin-1 to a similar magnitude as soluble β-glucans. Soluble β-glucans are known to inhibit the particulate β-glucan-induced activation of Dectin-1. As arabinoxylan is also soluble, the inhibiting capacity of arabinoxylan on particulate β-glucan-activated Dectin-1 cell lines was studied. It was found that this inhibition was similar to that of soluble β-glucan and was caused predominantly by inhibition of the Dectin-1A transcript variant. The Dectin-1 inhibitory function of arabinoxylan was further confirmed in human dendritic cells that demonstrated reduced production of IL-10 and TNF-α. The production of the antifungal cytokines IL-4 and IL-23 were increased in dendritic cells stimulated with arabinoxylan and particulate β-glucan. In contrast to soluble β-glucan, arabinoxylan did not enhance production of IL-10, TNF-α, and IL-23. CONCLUSION Arabinoxylan activates Dectin-1 and supports antifungal immune responses in human dendritic cells. The mode of action of arabinoxylan is similar but not identical to that of soluble β-glucans.

β2→1-Fructans Modulate the Immune System In Vivo in a Microbiota-Dependent and -Independent Fashion

It has been shown in vitro that only specific dietary fibers contribute to immunity, but studies in vivo are not conclusive. Here, we investigated degree of polymerization (DP) dependent effects of β2→1-fructans on immunity via microbiota-dependent and -independent effects. To this end, conventional or germ-free mice received short- or long-chain β2→1-fructan for 5 days. Immune cell populations in the spleen, mesenteric lymph nodes (MLNs), and Peyer’s patches (PPs) were analyzed with flow cytometry, genome-wide gene expression in the ileum was measured with microarray, and gut microbiota composition was analyzed with 16S rRNA sequencing of fecal samples. We found that β2→1-fructans modulated immunity by both microbiota and microbiota-independent effects. Moreover, effects were dependent on the chain-length of the β2→1-fructans type polymer. Both short- and long-chain β2→1-fructans enhanced T-helper 1 cells in PPs, whereas only short-chain β2→1-fructans increased regulatory T cells and CD11b−CD103− dendritic cells (DCs) in the MLN. A common feature after short- and long-chain β2→1-fructan treatment was enhanced 2-alpha-l-fucosyltransferase 2 expression and other IL-22-dependent genes in the ileum of conventional mice. These effects were not associated with shifts in gut microbiota composition, or altered production of short-chain fatty acids. Both short- and long-chain β2→1-fructans also induced immune effects in germ-free animals, demonstrating direct effect independent from the gut microbiota. Also, these effects were dependent on the chain-length of the β2→1-fructans. Short-chain β2→1-fructan induced lower CD80 expression by CD11b−CD103− DCs in PPs, whereas long-chain β2→1-fructan specifically modulated B cell responses in germ-free mice. In conclusion, support of immunity is determined by the chemical structure of β2→1-fructans and is partially microbiota independent.

Particulate β-glucans synergistically activate TLR4 and Dectin-1 in human dendritic cells

SCOPE The major receptor for β(1-3)-glucans on immune cells is considered to be Dectin-1 receptor. Particulate β-glucans induce stronger immune responses than soluble β-glucans by clustering of Dectin-1 receptors. Here, it was hypothesized that activation of other pattern recognition receptors such as Toll-like receptor 4 (TLR4) can also contribute to enhanced activity of immune cells after exposure to particulate β-glucans. METHODS AND RESULTS To test this hypothesis, reporter cell lines were designed expressing TLR4 with either Dectin-1A or Dectin-1B, that is, one of the two transcript variants of human Dectin-1 receptors. Enhanced NF-κB activation was observed after stimulation with particulate β-glucans in both Dectin-1A-TLR4 and the Dectin-1B-TLR4 cell lines. This was different with soluble β-glucans, which enhanced activation in Dectin-1A-TLR4 cell lines but not in Dectin-1B-TLR4 cells. The synergistic activation of TLR4 and Dectin-1 by particulate β-glucans was confirmed in human dendritic cells. The effects of particulate β-glucan induced TLR4 binding were regulatory as blocking TLR4 enhanced pro-inflammatory cytokine IL-23, IL-4, IL-6, and TNF-α production. CONCLUSION These results suggest that TLR4 and Dectin-1 are synergistically activated by particulate β-glucans, wherein TLR4 activates an immune regulatory pathway in human dendritic cells. Our data suggest that β-glucan is an immune regulatory ligand for TLR4.

Dietary Fiber Pectin Directly Blocks Toll-Like Receptor 2–1 and Prevents Doxorubicin-Induced Ileitis

Dietary carbohydrate fibers are known to prevent immunological diseases common in Western countries such as allergy and asthma but the underlying mechanisms are largely unknown. Until now beneficial effects of dietary fibers are mainly attributed to fermentation products of the fibers such as anti-inflammatory short-chain fatty acids (SCFAs). Here, we found and present a new mechanism by which dietary fibers can be anti-inflammatory: a commonly consumed fiber, pectin, blocks innate immune receptors. We show that pectin binds and inhibits, toll-like receptor 2 (TLR2) and specifically inhibits the proinflammatory TLR2–TLR1 pathway while the tolerogenic TLR2–TLR6 pathway remains unaltered. This effect is most pronounced with pectins having a low degree of methyl esterification (DM). Low-DM pectin interacts with TLR2 through electrostatic forces between non-esterified galacturonic acids on the pectin and positive charges on the TLR2 ectodomain, as confirmed by testing pectin binding on mutated TLR2. The anti-inflammatory effect of low-DM pectins was first studied in human dendritic cells and mouse macrophages in vitro and was subsequently tested in vivo in TLR2-dependent ileitis in a mouse model. In these mice, ileitis was prevented by pectin administration. Protective effects were shown to be TLR2–TLR1 dependent and independent of the SCFAs produced by the gut microbiota. These data suggest that low-DM pectins as a source of dietary fiber can reduce inflammation through direct interaction with TLR2–TLR1 receptors.