Deoxynivalenol, T-2 toxin, and zearalenone, major Fusarium mycotoxins, contaminate human food on a global level. Exposure to these mycotoxins during pregnancy can lead to abnormalities in neonatal development. Therefore, the aim of this study was to investigate the effects of Fusarium mycotoxins on human placental epithelial cells. As an in vitro model of placental barrier, BeWo cells were exposed to different concentrations of deoxynivalenol, zearalenone or T-2 toxin. Cytotoxicity, effects on barrier integrity, paracellular permeability along with mRNA and protein expression and localization of junctional proteins after exposure were evaluated. Induction of proinflammatory responses was determined by measuring cytokine production. Increasing mycotoxin concentrations affect BeWo cell viability, and T-2 toxin was more toxic compared to other mycotoxins. Deoxynivalenol and T-2 toxin caused significant barrier disruption, altered protein and mRNA expression of junctional proteins, and induced irregular cellular distribution. Although the effects of zearalenone on barrier integrity were less prominent, all tested mycotoxins were able to induce inflammation as measured by IL-6 release. Overall, Fusarium mycotoxins disrupt the barrier of BeWo cells by altering the expression and structure of junctional proteins and trigger proinflammatory responses. These changes in placental barrier may disturb the maternal–fetal interaction and adversely affect fetal development.

Deoxynivalenol (DON), a highly prevalent mycotoxin food contaminant, is known to have immunotoxic effects. In the current study, the potential of dietary interventions with specific mixtures of trans-galactosyl-oligosaccharides (TOS) to alleviate these effects were assessed in a murine influenza vaccination model. Vaccine-specific immune responses were measured in C57Bl/6JOlaHsd mice fed diets containing DON, TOS or a combination, starting 2 weeks before the first vaccination. The direct effects of TOS and its main oligosaccharide, 3′-galactosyl-lactose (3′-GL), on DON-induced damage were studied in Caco-2 cells, as an in vitro model of the intestinal epithelial barrier. Exposure to DON significantly reduced vaccine-specific immune responses and the percentages of Tbet⁺ Th1 cells and B cells in the spleen. DON significantly altered epithelial structure and integrity in the ileum and reduced the SCFA levels in the cecum. Adding TOS into DON-containing diets significantly improved vaccine-specific immune responses, restored the immune cell balance in the spleen and increased SCFA concentrations in the cecum. Incubating Caco-2 cells with TOS and 3′-GL in vitro further confirmed their protective effects against DON-induced barrier disruption, supporting immune modulation. Overall, dietary intervention with TOS can attenuate the adverse effects of DON on Th1-mediated immune responses and gut homeostasis. These beneficial properties might be linked to the high levels of 3′-GL in TOS.

Early-life nutrition significantly impacts vaccination efficacy in infants, whose immune response to vaccines is weaker compared to adults. This study investigated vaccination efficacy in female C57Bl/6JOlaHsd mice (6 weeks old) fed diets with 0.7% galacto-oligosaccharides (GOS)/long-chain fructo-oligosaccharides (lcFOS) (9:1), 0.3% human milk oligosaccharides (HMOS), or a combination (GFH) for 14 days prior to and during vaccination. Delayed-type hypersensitivity (DTH) was measured by assessing ear swelling following an intradermal challenge. Influvac-specific IgG1 and IgG2a levels were assessed using ELISAs, while splenic T and B lymphocytes were analyzed for frequency and activation via flow cytometry. Additionally, cytokine production was evaluated using murine splenocytes co-cultured with influenza-loaded dendritic cells. Mice on the GFH diet showed a significantly enhanced DTH response (p < 0.05), increased serological IgG1 levels, and a significant rise in memory B lymphocytes (CD27+ B220+ CD19+). GFH-fed mice also exhibited more activated splenic Th1 cells (CD69+ CXCR3+ CD4+) and higher IFN-γ production after ex vivo restimulation (p < 0.05). These findings suggest that GOS/lcFOS and HMOS, particularly in combination, enhance vaccine responses by improving memory B cells, IgG production, and Th1 cell activation, supporting the potential use of these prebiotics in infant formula for better early-life immune development.

Figure: Background: Siglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni . However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. Results: Here we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1 ⁺ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis -binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans -interactions with ligands. Conclusion: In conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.

Cancer vaccines' efficacy to stimulate T cell responses in patients is currently sub-optimal, and this can be potentially overcome by the delivery of antigens directly to antigen-presenting cells (APCs). To achieve this, single domain antibodies (nanobodies, Nbs) specific for receptors on APCs can be conjugated to liposomes to deliver cancer antigens. Here, we generated three CD169-specific Nb liposomes containing 3 different Nb clones (1B5, 1C1, and 2C2) and one CLEC9A-specific Nb liposome. Blocking experiments with CD169-specific antibodies indicated distinct binding epitopes on CD169 for 2C2 compared with 1B5 and 1C1. Both 2C2 and 1C1 Nb liposomes showed stronger binding and uptake by CD169-overexpressing cell lines and monocyte derived dendritic cells compared to 1B5 liposomes. Analysis of primary blood-derived APCs demonstrated strong uptake of 2C2 and 1C1 Nb liposomes by Axl+ Siglec-6+ DCs, classical monocytes, and DC3s, whereas CLEC9A-specific Nb liposomes were taken up by CLEC9A-expressing XCR1+ DC1s. In contrast, when gp100-specific T cell activation was evaluated using gp100-containing CD169-specific Nb liposomes and moDCs, only 1B5 Nb liposomes led to an enhanced T cell response. Using enriched blood DCs, we also observed an increased T cell activation by gp100-containing CLEC9A-specific Nb liposomes. To conclude, CD169- and CLEC9A-specific Nb liposomes are specifically taken up by primary CD169- or CLEC9A-expressing human APCs, and, depending on the Nb, can lead to a higher T cell activation. These results highlight the potential of Nb liposomes to improve vaccination strategies for immunotherapy.

Dendritic cells (DCs) are important orchestrators of immune responses. Their development in the bone marrow is controlled by transcription factors, but epigenetic mechanisms remain poorly understood. DOT1L is emerging as a key epigenetic regulator in immune cells. By mapping DOT1L-mediated histone H3K79 methylation in canonical DC subsets, we observed that DOT1L modified common as well as DC subset-specific genes. In vitro- or in vivo- induced deletion of Dot1l followed by in vitro cell culture resulted in a decrease in myeloid progenitors and plasmacytoid DCs (pDCs) and an increase in cDC2s, while cDC1s remained unchanged. In vitro generated Dot1l- KO DCs were unable to produce IFNα upon stimulation. Moreover, transcriptomes of Dot1l -KO DC subsets exhibited enrichment of antigen presentation pathways and MHC class II surface levels were upregulated in pDCs. Mechanistically, inhibition of DOT1L linked the observed effects to its methyltransferase activity. Together, our data indicate that in DCs DOT1L differentially affects the development of canonical subsets and suppresses antigen presentation pathways.