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γ-Conglutin (γ-C) from lupin seeds has been identified as a potent allergen with cross reactivity to peanuts. Here, we investigated how γ-C affected the response in bone marrow-derived dendritic cells (DCs) to bacterial stimuli. γ-C enhanced L. acidophilus NCFM (LaNCFM)-induced IL-12, IL-10, and IL-23 dose-dependently. In contrast, together with E. coli Nissle or LPS, γ-C reduced the production of IL-12 but not of IL-23 and IL-10. Enzyme-hydrolyzed γ-C also enhanced LaNCFM-induced IL-12 and IL-23 production. All preparations induced ROS production in the DCs. The mannose receptor ligands mannan and dextran and the clathrin inhibitor monodansylcadaverine partly inhibited the endocytosis of γ-C. Kunitz trypsin inhibitor and the scavenger receptor ligand polyG also enhanced LaNCFM-induced IL-12, indicating the involvement of receptors other than C-type lectin receptors. The endocytosis of labeled γ-C increased dose-dependently by addition of unlabeled γ-C, which coincided with γ-C's tendency to aggregate. Taken together, γ-C aggregation affects endocytosis and affects the cytokine production induced by gram-positive and gram-negative bacteria differently. We suggest that γ-C is taken up by the same mechanism as other food proteins but due to aggregation is present in higher concentration in the DCs. This could influence the resulting T-cell response in a microbial stimuli-dependent way.

Stilbenoids are anti-inflammatory and antioxidant compounds, with resveratrol being the most investigated molecule in this class. However, the actions of most other stilbenoids are much less studied. This study compares five monomeric (resveratrol, piceatannol, pterostilbene, pinostilbene, and trimethoxy-resveratrol) and two dimeric (dehydro-δ-viniferin and trans -δ-viniferin) stilbenoids for their capability to modulate the production of bacteria-induced cytokines (IL-12, IL-10, and TNF-α), as well as lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), in murine bone marrow-derived dendritic cells. All monomeric species showed dose-dependent inhibition of E. coli -induced IL-12 and TNF-α, whereas only resveratrol and piceatannol inhibited IL-10 production. All monomers, except trimethoxy-resveratrol, inhibited L. acidophilus -induced IL-12, IL-10, and TNF-α production. The dimer dehydro-δ-viniferin remarkably enhanced L. acidophilus -induced IL-12 production. The contrasting effect of resveratrol and dehydro-δ-viniferin on IL-12 production was due, at least in part, to a divergent inactivation of the mitogen-activated protein kinases by the two stilbenoids. Despite having moderate to high total antioxidant activity, dehydro-δ-viniferin was a weak inhibitor of LPS-induced ROS formation. Conversely, resveratrol and piceatannol potently inhibited LPS-induced ROS formation. Methylated monomers showed a decreased antioxidant capacity compared to resveratrol, also depending on the methylation site. In summary, the immune-modulating effect of the stilbenoids depends on both specific structural features of tested compounds and the stimulating bacteria.

Methicillin resistant Staphylococcus aureus (MRSA) has developed resistance to most β-lactam antibiotics leaving few treatment options against infections with MRSA. Through mannose receptors, mannan potentiates IL-12 production induced by Gram-positive bacteria, a cytokine crucial in the clearance of S. aureus infection. We investigated the IL-12 potentiating effect of mannan pre-treatment of bone marrow-derived dendritic cells prior to stimulation with clinical MRSA strains. Mannan almost doubled IL-12 as well as IFN-β production in response to USA300, also when USA300 was treated with the β-lactam cefoxitin. The MRSA-induced IL-12 production was dependent on bacterial uptake and reactive oxygen species (ROS). Mannan alone induced ROS production, and in combination with USA300, the ROS produced corresponded to the sum induced by mannan and USA300. Addition of a monoclonal antibody against the mannose receptor likewise enhanced USA300-induced IL-12 and induced ROS production. Mannan addition further increased the endocytosis as well as the rate of endosomal killing of bacteria. Pre-treatment with soluble β-glucans also induced ROS and potentiated the USA300-induced IL-12 indicating that other C-type receptors may play a similar role. In the presence of the pro-inflammatory mediators, GM-CSF or IFN-γ, the mannan-enhanced IL-12 production increased further. The USA300-induced and the mannan-facilitated enhanced IFN-β and IL-12 showed same dependency on MAPK c-Jun N-terminal kinase signaling, suggesting that mannan enhances the signals already induced by the bacteria, rather than changing them. We suggest that the C-type lectin-induced ROS production is a key factor in the IFN-β and IL-12 potentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eld, Johnsen, Nielsen, Jørgensen, Lindstrøm-Svendsen, Baldry, Ingmer and Frøkiær.)

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of β-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that β-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)