BACKGROUND:Alterations in the intestinal microbiota contribute to the pathogenesis of various cardiovascular disorders, but how they affect the development of Kawasaki disease (KD) an acute pediatric vasculitis, remains unclear. METHODS:We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to assess the contribution of the intestinal microbiota to the development of vascular inflammation. We evaluated the severity of vasculitis in microbiota-depleted mice. 16S rRNA gene sequencing was used to characterize the fecal microbiome composition of LCWE-injected mice. Some groups of mice were orally treated with selected live or pasteurized bacteria, short-chain fatty acids, or Amuc_1100, the Toll-like receptor 2 signaling outer membrane protein from Akkermansia muciniphila, and their impact on vasculitis development was assessed. RESULTS:We report that depleting the gut microbiota reduces the development of cardiovascular inflammation in a murine model mimicking KD vasculitis. The development of cardiovascular lesions was associated with alterations in the intestinal microbiota composition and, notably, a decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Oral supplementation with either of these live or pasteurized individual bacteria or with short-chain fatty acids produced by them attenuated cardiovascular inflammation, as reflected by decreased local immune cell infiltrations. Treatment with Amuc_1100 also reduced the severity of vascular inflammation. CONCLUSIONS:This study reveals an underappreciated gut microbiota-cardiovascular inflammation axis in KD vasculitis pathogenesis and identifies specific intestinal commensals that regulate vasculitis in mice by producing metabolites or via extracellular proteins capable of enhancing and supporting gut barrier function.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

Changes in intestinal microbiota composition and function are associated with development of cardiovascular disease, but the role of these alterations remains unclear in Kawasaki Disease (KD), an acute pediatric vasculitis that targets coronary arteries. Using a murine model of KD vasculitis, we observed reduced development of cardiovascular lesions in germ-free and antibiotic-treated mice. KD vasculitis in mice was associated with alterations in the gut microbiota composition and decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii . Supplementation with live or pasteurized A. muciniphila or F. prausnitzii attenuated the severity of KD cardiovascular inflammation. Oral administration of the short-chain fatty acids acetate or propionate, both produced by A. muciniphila, or butyrate, produced by F. prausnitzii , reduced the development of vascular inflammation. Furthermore, treatment with Amuc_1100, a purified protein isolated from the outer membrane of A. muciniphila , decreased the severity of murine KD vasculitis. Beneficial effects of either pasteurized A. muciniphila or Amuc_1100 on murine KD vasculitis development were associated with improvement of gut barrier function. These results reveal an underappreciated gut microbiota-cardiovascular inflammation axis during murine KD vasculitis. Our findings may incite the development of novel diagnostic tools and therapeutic strategies that modulate the intestinal microbiota composition and function for KD patients.

Alterations of gut microbiota composition and function influence the development of cardiovascular disease, but the role of these aberrations remains poorly understood in Kawasaki Disease (KD), an acute pediatric vasculitis that targets coronary arteries. Using a murine model of KD vasculitis, we found that germ-free and antibiotic-treated mice displayed reduced inflammation and cardiovascular lesions. Development of KD vasculitis in mice was associated with alterations in the composition of the intestinal microbiota, specifically decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Supplementation with live or pasteurized A. muciniphila or F. prausnitzii attenuated the severity of KD cardiovascular inflammation. Oral administration of the short-chain fatty acids acetate or butyrate, which are produced by A. muciniphila and F. prausnitzii respectively, or treatment with Amuc_1100, a purified protein isolated from the outer membrane of A. muciniphila, ameliorated the severity of KD cardiovascular lesions. Reduced development of KD vasculitis in mice receiving either pasteurized A. muciniphila or Amuc_1100 was associated with improvements of gut barrier function. These results reveal an underappreciated gut microbiota-cardiovascular inflammation axis during murine KD vasculitis. Our findings may stimulate the development of novel diagnostic tools and therapeutic strategies that modulate the intestinal microbiota composition and function for KD patients. Research is supported by the NIH grants R01AI072726 to M.A. and R01HL139766 to M.N.R.

Rationale: NLRP3 (NLR family pyrin domain containing 3) activation and IL-1β (interleukin-1β) production are implicated in Kawasaki disease (KD) pathogenesis; however, a detailed and complete characterization of the molecular networks and cellular subsets involved in the development of cardiovascular lesions is still lacking. Objective: Here, in a murine model of KD vasculitis, we used single-cell RNA sequencing and spatial transcriptomics to determine the cellular landscape of inflamed vascular tissues. Methods and Results: We observe infiltrations of innate and adaptive immune cells in murine KD cardiovascular lesions, associated with increased expression of Nlrp3 and Il1b . Monocytes, macrophages, and dendritic cells were the main sources of IL-1β, whereas fibroblasts and vascular smooth muscle cells (VSMCs) expressed high levels of IL-1 receptor. VSMCs type 1 surrounding the inflamed coronary artery undergo a phenotype switch to become VSMCs type 2, which are characterized by gene expression changes associated with decreased contraction and enhanced migration and proliferation. Genetic inhibition of IL-1β signaling on VSMCs efficiently attenuated the VSMCs type 2 phenotypic switch and the development of cardiovascular lesions during murine KD vasculitis. In addition, pharmacological inhibition of NLRP3 prevented the development of cardiovascular inflammation. Conclusions: Our studies unravel the cellular diversity involved in IL-1β production and signaling in murine KD cardiovascular lesions and provide the rationale for therapeutic strategies targeting NLRP3 to inhibit cardiovascular lesions associated with KD.

More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross- reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope-derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergenspecific Ab responses were subsequently boosted with molecules containing the B cell epitope-derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

BACKGROUND:Vaccines consisting of allergen-derived peptides lacking IgE reactivity and allergen-specific T cell epitopes bound to allergen-unrelated carrier molecules have been suggested as candidates for allergen-specific immunotherapy. OBJECTIVE:To study whether prophylactic and therapeutic vaccination with carrier-bound peptides from the major birch pollen allergen Bet v 1 lacking allergen-specific T cell epitopes has influence on Bet v 1-specific T cell responses. METHODS:Three Bet v 1-derived peptides, devoid of Bet v 1-specific T cell epitopes, were coupled to KLH and adsorbed to aluminium hydroxide to obtain a Bet v 1-specific allergy vaccine. Groups of BALB/c mice were immunized with the peptide vaccine before or after sensitization to Bet v 1. Bet v 1- and peptide-specific antibody responses were analysed by ELISA. T cell and cytokine responses to Bet v 1, KLH, and the peptides were studied in proliferation assays. The effects of peptide-specific and allergen-specific antibodies on T cell responses and allergic lung inflammation were studied using specific antibodies. RESULTS:Prophylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides induced a Bet v 1-specific IgG antibody response without priming/boosting of Bet v 1-specific T cells. Prophylactic and therapeutic vaccination of mice with the peptide vaccine induced Bet v 1-specific antibodies which suppressed Bet v 1-specific T cell responses and allergic lung inflammation. CONCLUSION AND CLINICAL RELEVANCE:Vaccination with carrier-bound allergen-derived peptides lacking allergen-specific T cell epitopes induces allergen-specific IgG antibodies which suppress allergen-specific T cell responses and allergic lung inflammation.