Fig 2: Anti-coliticeffect of capsaicin in mice and its inhibition on Ca2+signaling in IEC-6 cells pretreated with TNF-α.A and B, summary data showing the time course of body weight and mouse colon length after different treatments without (Ctrl) or with capsaicin (intragastrically (10 mg/kg) once per day for 7 days), DSS, or DSS + Cap (n = 5). C and D, summary data showing the time courses of stool score and MPO after different treatments as in A and B. E, histological examination and immunohistological analysis showing colonic micro-structure (the upper panels) and TRPV4 immunostaining (the lower panels) after different treatments as in A and B. The scale bar represents 100 μm for each image. F and G, summary data showing histological score and TRPV4 protein level after different treatments as in A and B. H and I, summary tracings of [Ca2+]cyt time course in response to GSK (10 nM) in IEC-6 cells without (n = 22) or with (n = 28) TNF-α (40 ng/ml) pretreatment. J–M, summary tracings of [Ca2+]cyt time course showing the inhibitory effect of Cap at different doses on GSK-induced [Ca2+]cyt signaling in IEC-6 cells pretreated with TNF-α. N, summary data showing the inhibitory effect of Cap at different doses on peaks of GSK-increased [Ca2+]cyt signaling described as in H–M. The data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 were performed by Student’s t test. [Ca2+]cyt, cytosolic Ca2+ concentrations; DSS, dextran sodium sulfate; IECs, intestinal epithelial cells; MPO, myeloperoxidase; TRPV, transient receptor potential vanilloid.

Fig 3: Myeloperoxidase is functionally expressed in primary GECs and activity increases in response to ATP and P. gingivalis infection. (A) Immunoperoxidase staining of MPO in H2O2 (100 μM) and ATP (3 mM) cells at 40x magnification performed for visual detection. Image J was used to quantify the relative staining intensity and compared to untreated. N = 9, **p < 0.01. (B) Quantitative ELISA measurement of MPO expression in primary GECs displayed as pg/mL in H2O2, ATP pre-treated, and P. gingivalis (MOI100) infected cells. N = 3, *p < 0.05 as compared to untreated. (C) MPO activity assay during ATP pre-treatment and P. gingivalis (MOI100) infection in primary GECs calculated based on the standard curve of activity tested empirically (see Methods Section). N = 3, *p < 0.05 as compared to untreated.

Fig 4: NET killing assay and fluorescent staining for NETs in infected mouse lungs. (A) HL60 immortalized promyeloid cells were differentiated for 5 to 6 days, seeded in 24-well plates, and activated with 25 nM phorbol myristate acetate, after which 20 μM cytochalasin D was added to prevent killing via phagocytosis. Bacteria were added at an MOI of 10. Bacterial killing was expressed as a percentage of the counts obtained from control wells. Data are means and SEM (n = 3). All statistical comparisons were to the matched treatment of the parent strain. ****, P < 0.00005. (B) Immunofluorescent staining and confocal imaging of infected mouse lung sections. Nuclei were stained with DAPI (blue), bacteria were stained with anti-NTHi rabbit polyclonal sera and secondary Alexa Fluor 488 antibody conjugate (green), and NETs were stained using anti-histone H3 citrulline R2, R8, and R17 (red) and propidium iodide (cyan). These confocal images are representative images of mouse lungs 48 h postinfection. Images were taken at ×90 magnification. Bars, 10 μm. (C) MPO activity in lung homogenate. Data are means and SD (n = 12 to 18). Statistical significance was assessed by the Mann-Whitney nonparametric t test.

Fig 5: TNFAIP6+ MSCs have enhanced anti-inflammatory activities in the mouse model of acute inflammation. A Serum level of IL-6, TNF-α, IFN-γ, and IL-1β was determined after 24 h post-LPS stimulation via ELISA (n = 8). B The total cell number in BAL was determined after 24 h post-LPS stimulation by flow cytometry (n = 8). C The neutrophil number in BAL was determined as CD45+CD11b+Ly-6G+Ly-6Cmed after 24 h post-LPS stimulation by flow cytometry (n = 8). D The MPO activity was quantified after 24 h post-LPS stimulation (n = 8). E The CD45+ cells in the lung were measured after 7 days post-LPS stimulation flow cytometry (n = 8). F Representative figures for HE staining of lung tissues after 7 days post-LPS stimulation. * indicates P < 0.05. TNFAIP6 tumor necrosis factor alpha-induced protein 6; IL-6 interleukin 6; TNF-α tumor necrosis factor alpha; IFN-γ interferon gamma; IL-1β interleukin 1 beta; BAL bronchoalveolar lavage; and MPO myeloperoxidase