Fig 1: IgA-coated BEVs promote pro-inflammatory cytokine release in CD89+ cells.a IL-8 response in CD89+CD14+CD11b+ U937 monocytes exposed to uncoated or IgA-coated BEVs from B. fragilis and L. acidophilus (BEVsB/L and IgA-BEVsB/L), uncoated or IgA-coated hEVs or soluble IgA alone. Dosage of the effector samples given by total protein biomass determined by Bradford is indicated. Incubation with saline served as mock-treated control (co). (n = 10 for each data set). b IL-6 response in CD89+CD14+CD11b+ U937 monocytes exposed to uncoated or IgA-coated BEVs from B. fragilis and L. acidophilus (BEVsB/L and IgA-BEVsB/L), uncoated or IgA-coated hEVs or soluble IgA alone. Dosage of the effector samples given a total protein biomass determined Bradford is indicated. Incubation with saline served as mock-treated control (co). (n = 8 for uncoated hEVs; all other data sets n = 9). c IL-8 response in CD89-negative human intestinal organoid-derived monolayers from two healthy donors exposed to 5 µg uncoated or IgA-coated BEVs from B. fragilis and L. acidophilus (BEVsB/L and IgA-BEVsB/L). Lack of CD89 expression was confirmed by FACS (Fig. 3a). Data is presented as median ± interquartile range (n = 5 for each data set). d IL-8 (left) and IL-6 (right) response in CD14+ human monocytes isolated from three healthy donors exposed to 5 µg uncoated or IgA-coated BEVs from B. fragilis and L. acidophilus (BEVsB/L and IgA-BEVsB/L). CD89 expression was confirmed by FACS analysis (Fig. 3b). Data is presented as median ± interquartile range (n = 4 for donor 1, n = 5 for donor 2, and n = 7 for donor 3). a–d Data is presented as median ± interquartile range. Samples with no detectable signal were set to the limit of detection, which is indicated by a dotted line. Statistical difference between uncoated and IgA-coated BEVs was evaluated by two-sided Mann–Whitney U-test. Source data are provided as a Source Data file.
Fig 2: Identification of a pro-inflammatory property in EV fractions derived from UC patients.a IL-8 response in HT-29 human intestinal epithelial cells exposed to soluble effector (SEF, left) and extracellular vesicle (EV, right) fractions of the non-IBD (blue), inactive UC (yellow) and active UC (red) group. b IgA titers in soluble effector (SEF, left) and extracellular vesicle (EV, right) fractions of the non-IBD (blue), inactive UC (yellow), and active UC (red) group. c IgG titers in soluble effector (SEF, left) and extracellular vesicle (EV, right) of the non-IBD (blue), inactive UC (yellow), and active UC (red) group. d sCD89-IgA complex levels in soluble effector (SEF, left) and extracellular vesicle (EV, right) fractions of the non-IBD (blue), inactive UC (yellow), and active UC (red) group. e Representative images showing immunofluorescent stainings of human colonic biopsies derived from a non-IBD, inactive UC, and active UC patient for CD89 (red), immune cell marker CD68 (green) and total nuclei stained with DAPI (blue). Graph on the right side depicts the number of CD89+CD68+ and CD89+CD68dim/- cells per eye field for independent biopsies (non-IBD, n = 9; inactive UC, n = 10; active UC, n = 12). Scale bar = 50 µm. Data in graphs is presented as median ± interquartile range. Statistical difference against the non-IBD group was evaluated by Kruskal–Wallis with Dunn’s test. f IL-8 response in CD89+CD14+CD11b+ U937 monocytes exposed to soluble effector (SEF, left) and extracellular vesicle (EV, right) fractions of the non-IBD (blue), inactive UC (yellow) and active UC (red) group. g IL-6 response in the in CD89+CD14+CD11b+ U937 monocytes exposed to soluble effector (SEF, left) and extracellular vesicle (EV, right) of the non-IBD (blue), inactive UC (yellow) and active UC (red) group. a–d, f, g sample number as follows: non-IBD (n = 28), inactive UC (n = 30) and active UC (n = 16). Data is presented as median ± interquartile range. Samples with no detectable signal were set to the limit of detection, which is indicated by a dotted line. Statistical difference between the three groups was evaluated by Kruskal–Wallis with Dunn’s test. a–g Source data are provided as a Source Data file.
Fig 3: IgA-coated BEVs and EV fractions from UC patients promote a CD89-dependent pro-inflammatory cytokine release in mouse bone marrow derived cells (BMDC).a IL-6 and TNF-α response in bone marrow derived cells (BMDC) from CD89wt/wt (littermates, LM) and CD89tg/wt (CD89-transgenic, CD89) mice exposed to uncoated or IgA-coated BEVs from B. fragilis and L. acidophilus (BEVsB/L and IgA-BEVsB/L) as well as soluble IgA alone. Dosage of the effector samples given by total protein biomass determined by Bradford is indicated. Incubation with saline served as mock-treated control (co). (n = 10 for each data set). b IL-6 and TNF-α response in bone marrow derived cells (BMDC) from CD89tg/wt (CD89-transgenic, CD89) mice exposed to 1 µg IgA-coated BEVs from B. fragilis and L. acidophilus (IgA-BEVsB/L) in the absence (-) or presence (+) of the Syk inhibitor R406. (n = 6 for each data set). c IL-6 (left) and TNF-α (right) response in bone marrow derived cells (BMDC) from CD89wt/wt (littermates, LM) and CD89tg/wt (CD89-transgenic, CD89) mice exposed to EV fractions derived from the non-IBD (blue) and active UC (red) group (seven randomly chosen representatives). (n = 7 for each data set). d IL-6 and TNF-α response in bone marrow derived cells (BMDC) from CD89tg/wt (CD89-transgenic, CD89) mice exposed to 1 µg EV fractions derived from three representative active UC patients (UC1, UC2, or UC3) in the absence (-) or presence (+) of the Syk inhibitor R406. (n = 4 for each data set). a–d Data is presented as median ± interquartile range. Samples with no detectable signal were set to the limit of detection, which is indicated by a dotted line. Statistical difference in panel a was evaluated by Kruskal–Wallis (comparison of groups receiving the same BEVsB/L dosage) with Dunn’s test. Statistical differences in all other panels [comparison of (B)EV samples with and without inhibitor or non-IBD and active UC groups] were evaluated by two-sided Mann–Whitney U-test. Source data are provided as a Source Data file.
Fig 4: P. micra induces inflammation and impairs the osteogenic capacity of PDLSCs. (A) IHC staining of rat periodontal ligament showing the expression levels of ALPL, Col 1 and Osx in the control group and P. micra-infected group. Scale bars, 20 μm. (B) ALP staining and ARS staining of PDLSCs infected by P. micra. Scale bars, 2 mm and 200 μm. (C) Quantitative results of ALP staining in (B). (D) Quantitative results of ARS staining in (B). (E–F) Validation of osteogenesis-related markers in PDLSCs during persistent P. micra infection by Western blot. GAPDH was set as control. (G) qRT-PCR analysis was performed to validate IL-6 and IL-8 mRNA levels of PDLSCs infected with P. micra. (H) Quantification of IL-6 and IL-8 levels in the supernatant of PDLSCs infected with P. micra. (I) Western blot revealed NF-κB and ERK1/2 signalling in PDLSCs at 0–2 h after co-culture with P. micra (MOI = 100). Data are presented as the mean ± SD of three independent experiments (n = 3) in (A, C-D, F–I). P values were determined by two-tailed unpaired Student's t-test in (A) and one-way ANOVA test in (C-D, F–I). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.
Fig 5: P. micra attaches to PDLSCs via its surface protein TmpC. (A) Representative SEM images of P. micra attaching to PDLSCs in the first hour of infection. Scale bars, 3 μm. (B) Representative TEM images of P. micra attaching to and invading PDLSCs in the first hour of infection. The adhered P. micra are zoomed with red box. The invaded P. micra are zoomed with blue box. Scale bars, 2 μm. (C) Representative fluorescence images of P. micra attaching to and invading PDLSCs. Scale bars, 10 μm. (D) Schematic representation of biotin pull-down. (E) P. micra surface protein TmpC pulled down by biotinylated surface protein of PDLSCs. (F) Schematic representation of the insertional inactivation of TmpC in P. micra. DNA fragment containing ermB gene was constructed and transformed into P. micra wild type (P. micra WT) for homologous recombination. (G) PCR of insertion mutants of P. micra. Bacterial chromosomal DNA was used as template for the PCR and different primers indicated in (F) were used to validate ermB insertion. (H) ALP and ARS staining of PDLSCs infected by P. micra WT or P. micra ΔtmpC. Scale bars, 400 μm. (I) Quantitative results of ALP staining in (H). (J) Quantitative results of ARS staining in (H). (K) Western blot revealed altered protein expression of Col 1, ALPL, Runx2 and BSP in PDLSCs infected by P. micra WT or P. micra ΔtmpC. GAPDH was set as control. (L) Western blot showing the activation of NF-κB and ERK1/2 signalling pathways in PDLSCs treated with TmpC protein. GAPDH was set as control. (M) qPCR analysis showing altered IL-6 and IL-8 mRNA levels in PDLSCs treated with TmpC. Data are presented as the mean ± SD of three independent experiments (n = 3) in (I–M). P values were determined by one-way ANOVA test in (I–M). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.
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