Gut commensal Limosilactobacillus reuteri induces atypical memory-like phenotype in human dendritic cells in vitro.

Memory-like responses in innate immune cells confer nonspecific protection against secondary exposures. A number of microbial agents have been found to induce enhanced or diminished recall responses in innate cells, however, studies investigating the ability of probiotic bacteria to trigger such effects are lacking. Here, we show that priming of human monocytes with a secretome from the gut probiotic bacterium Limosilactobacillus (L.) reuteri induces a mixed secondary response phenotype in monocyte-derived dendritic cells (mo-DCs), with a strong IL-6 and IL-1ß response but low TNFα, IL-23 and IL-27 secretion. Instead, blood DC priming with L. reuteri-secretome resembles a tolerant state upon secondary exposure. A similar pattern was found in conventional and gut-like (retinoic acid exposed) DCs, although retinoic acid hampered TNFα and IL-6 production and enrichment of histone modifications in L. reuteri-secretome primed mo-DC cultures. Further, we show that the memory-like phenotype of mo-DCs, induced by priming stimuli, is important for subsequent T helper (Th) cell differentiation pathways and might determine the inflammatory nature of Th cells. We also show enhanced recall responses characterized by robust inflammatory cytokines and lactate production in the gut-like mo-DCs derived from ß-glucan primed monocytes. Such responses were accompanied with enriched histone modifications at the promoter of genes associated with a trained phenotype in myeloid cells. Altogether, we demonstrate that a gut commensal-derived secretome prompts recall responses in human DCs which differ from that induced by classical training agents such as ß-glucan. Our results could be beneficial for future therapeutic interventions where T cell responses are needed to be modulated.

Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells.

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, γδ T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-γ expression in MAIT-, γδ T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate γδ T- and NK cells was fully dependent on the presence of both monocytes and αß T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.

A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection.

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.