Activation of macrophages by Toll-like receptors (TLRs) and functionally related proteins is essential for host defense and innate immunity. TLRs recognize a wide variety of pathogen-associated molecules. Here, we demonstrate that the meningococcal outer membrane protein NhhA has immunostimulatory functions and triggers release of proinflammatory cytokines from macrophages. NhhA-induced cytokine release was found to proceed via two distinct pathways in RAW 264.7 macrophages. Interleukin-6 (IL-6) secretion was dependent on activation of TLR4 and required the TLR signaling adaptor protein MyD88. In contrast, release of tumor necrosis factor (TNF) was TLR4 and MyD88 independent. Both pathways involved NF-B-dependent gene regulation. Using a PCR-based screen, we could identify additional targets of NhhA-dependent gene activation such as the cytokines and growth factors IL-1␣, IL-1␤, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In human monocyte-derived macrophages, G-CSF, GM-CSF, and IL-6 were found to be major targets of NhhA-dependent gene regulation. NhhA induced transcription of IL-6 and G-CSF mRNA via TLR4-dependent pathways, whereas GM-CSF transcription was induced via TLR4-independent pathways. These data provide new insights into the role of NhhA in host-pathogen interaction.

Summary Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)‐COL‐DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H‐2q‐restricted DBA/1 mice to assess the preventive effect on collagen‐induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4+ T‐cell responses were reduced. Concomitant with a reduced effector T‐cell activity with lower IFNγ, IL‐13 and IL‐17A production, we observed an increase in IL‐10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up‐regulated IL‐10 and regulatory T‐cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T‐cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis.

Curative therapies against autoimmune diseases are lacking. Indeed, most of currently available treatments are only targeting symptoms. We have developed a novel strategy for a therapeutic vaccine against autoimmune diseases based on intranasal administration of a fusion protein tolerogen, which consists of a mutant, enzymatically inactive, cholera toxin A1-subunit genetically fused to disease relevant high affinity peptides and a dimer of D-fragments from protein A. The CTA1R7K-MOG/PLP-DD fusion proteins effectively reduced clinical symptoms in the experimental autoimmune encephalitis (EAE)-model of multiple sclerosis (MS). The treatment induced Tr1 cells, in the draining lymph node, which produced IL-10 and suppressed effector CD4+ T cell responses. This effect was dependent on IL-27 signalling, since treatment was ineffective in bone marrow chimeras lacking IL-27Rα within their hematopoietic compartment. scRNA-seq of dendritic cells (DC) in draining lymph nodes demonstrated distinct gene transcriptional changes of cDC1, including enhanced lipid metabolic pathways, induced by the tolerogenic fusion protein. Thus, our results with the tolerogenic fusion protein demonstrates the possibility to vaccinate and protect against disease progression by reinstating tolerance in MS and other autoimmune diseases.

We have developed a novel platform for intranasal treatment of autoimmune diseases in which ADP-ribosylation determines whether immunity or tolerance is induced. Hence, cholera toxin A1-subunit based immunomodulation through the CTA1-peptide-DD fusion protein promotes enhancement, while inactive mutants induce suppression. Whereas the target population after intranasal administration of both constructs was CD103+CD11blow DC’s, both active and inactive mutants induced strong CD4 T cell priming, but they differentially affected CD4 T cell differentiation. This way inactive mutant constructs generated regulatory CD4 T cells producing IL-10 and effectively preventing experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA), when carrying relevant peptides. Targeted DCs expressed low levels of CD80, CD86 and CD40, while, by contrast, ADP-ribosylating CTA1-peptide-DD constructs gave significantly enhanced co-stimulation. Suppression was global in that regulatory T cells following treatment were able to suppress adoptively transferred naïve CD4 T cells and could subsequently maintain tolerance. The use of specifically tolerance-inducing fusion proteins may be a way forward in the search of effective treatments against autoimmune diseases. The immunomodulating effect on the CD103+CD11blow DCs will be described and the requirement for CTA1-binding to Gsa evaluated in detail, using Cre-lox Gsa-deficient mice.

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