Fig 1: Bp–specific nasal CD4+ TRM cells protect IL-17 KO mice against nasal colonization and induce recruitment of neutrophils.a Schematic of the transfer protocol. C57BL/6 mice were infected nasally (i.n.) with 106 CFU of B1917GR. CD4+ TRM cells were purified from noses of infected C57BL/6 mice 14 d.p.c. A total of 104 CD45iv− CD45+ CD44+ CD69+ CD4+ TRM cells were transferred intravenously (i.v.) to IL-17 KO mice 10 h.p.c. and 7 d.p.c. (blue arrows). Control IL-17 KO and C57BL/6 mice received PBS (black arrows). CFU in the nasal tissues was counted at indicated time points post challenge. Nasal cells were collected 14 d.p.c. and analyzed by flow cytometry (FACS). b C57BL/6 (in gray), IL-17 KO (KO, in purple), and transferred IL-17 KO mice (KOt, in blue) were infected with 105 CFU of B1917GR, and CFU numbers were counted in the noses at indicated time points. c Representative graphs showing neutrophil (CD45iv− CD45+ Ly-6G+) recruitment in the nose of C57BL/6 (left panels), IL-17 KO (KO, middle panels), and transferred IL-17 KO mice (KOt, right panel) mice 14 days after infection with 105 CFU of B1917GR. Neutrophil recruitments of non-infected and infected mice are depicted in the upper (gray) and lower (red) panels, respectively. Numbers indicate percentages of events in each square. d C57BL/6 (in gray), IL-17 KO (KO, in purple), and transferred IL-17 KO mice (KOt, in blue) mice were infected with 105 CFU of B1917GR (open bars) or left uninfected (gray bars), and absolute numbers of neutrophils per nose were measured 14 d.p.c. e Representative graphs showing neutrophil (CD45+ Ly-6G+) recruitment in the nose of BALB/c mice vaccinated with aPV (middle panels) or wPV (right panels) or left unvaccinated (left panels) 7 days (7 d.p.c.) and 28 days (28 d.p.c.) after infection with 106 CFU of B1917GR. Numbers indicate percentages of events in each square. f Absolute numbers of neutrophils in the noses of BALB/c mice immunized with aPV (in yellow) or wPV (in blue), or left un-immunized (in gray) at indicated time points after challenge with 106 CFU of B1917GR. Results shown are geometric means ± SD. n = 3–5. Ordinary one-way ANOVA (b, d) were performed to compare KO mice to KOt (*, b, d) or to C57BL/6 mice (#, b) and to evaluate the induction of neutrophils in infected C57BL/6 (*, d). Kruskal–Wallis tests were performed to compare aPV (#) and wPV (*) immunized BALB/c mice to control mice (f). *,#P < 0.05, **,##p < 0.001, ***p < 0.001, and ****p < 0.0001. Only significant differences are indicated.
Fig 2: Scavenging of isoLG reduces splenic myeloid and lymphoid expansion in a mouse model of SLE.Spleens and cells were isolated at the time of sacrifice from 32-week-old B6.SLE123 mice. Single-cell suspensions were prepared from freshly isolated mouse tissue via enzymatic digestion and mechanical dissociation. Live cell singlets were analyzed. (A) Representative spleens revealing a reduction in spleen size from B6.SLE123 mice treated with 2-HOBA. Quantitation of (B) live cells and (C) CD45+ cells. (D) Representative FACS plots for CD3+ T cells. Quantitation of (E) CD3+ T cells, (F) CD4+ T cells, (G) CD8+ T cells, (H) DCs, and (I) CD19+ B cells. Data were analyzed using 1-way ANOVA (n = 6–9, *P < 0.05, **P < 0.01, ***P < 0.001).
Fig 3: USNB lyses cells and causes immunogenic cell death. C57BL/6 mice implanted with RM1 cells were left untreated or were treated with anti-PD1, USNB or USNB+anti-PD1. One day (A) and 4 days (B) after treatment, the absolute numbers of leukocytes (CD45+), DCs (CD11c+), monocytes (CD11b+), NK cells (49b+) and macrophages (CD68+) in tumor tissue were analyzed by flow cytometry. RM1-OVA cells were treated with USNB, US or NB in vitro, and the supernatants were collected and analyzed. (C, D) Western blot of tumor antigen OVA levels and immunogenic cell death marker levels in the supernatants, respectively. (E) ATP concentration in the supernatants of RM1 cells in the different treatment groups. (F, G) Transmission electron microscopy images of RM1 cells obtained immediately after treatment with US, NB or USNB. The blue arrow indicates the fragmented Golgi apparatus; the yellow arrow indicates swollen mitochondria; and the red arrow indicates fragmented nuclei. The scale bars in the global and magnified images represent 5 and 1 µm, respectively. (H) Flow cytometry analysis of PD1, CD69, CD25, TNF-α and IFN-γ expression levels in CD8+ T cells after ex vivo coculture assay. Gene ontology analysis of differentially expressed genes in tumor masses between (I) USNB and untreated, (J) USNB+anti-PD1 and USNB, (K) USNB+anti-PD1 and anti-PD1, (L) USNB +anti-PD1 and untreated samples. The bars represent the mean±SEM (n=3 per group in E). The p value was calculated by unpaired t-test (A, B, E). (A, B) The data are representative of two independent experiments with at least five mice per group. (C–H) The data are from three independent experiments, and representative results are shown. IFN-γ, interferon gamma; UT, untreated; NB, nanobubble; NK, natural killer; TME, tumor microenvironment; TNF-α, tumor necrosis factor alpha; US, ultrasound; USNB, ultrasound-stimulated nanobubble.
Fig 4: Morphology of serially transplanted cervical cancer PDXs.A) representative example of an H&E stained cervical squamous cell carcinoma sample showing morphology of the tumour biopsy, primary, secondary and tertiary PDXs. B) Typical examples of negative staining for anti-human CD45 staining (second column), and anti-mouse CD45 (third column). Insets show examples of CD45 positive staining in human cervix biopsies and mouse kidney Scale bars 50 µm.
Fig 5: Microglia become proinflammatory 96 h after reperfusion injury stroke. (a) C57Bl/6J mice underwent sham surgery, 1‐h tMCAO or pMCAO and were culled after 96 h. The brain was separated into left (contralateral) and right (ipsilateral) hemispheres and underwent flow cytometric analysis to quantify live, unstimulated CD45+7‐AAD− leukocytes. Microglia were defined as CD45midCD11b+. (b) Proportion of microglia from live CD45+ cells. (c) Numbers of microglia normalized to the corresponding cerebral hemispheric weight. Percentage of microglia positive for (native) intracellular (d) pro‐IL‐1β, (e) TNF‐α and (f) Arg1 were assessed. Data are shown as mean ± s.e.m.: (b, c) n = 3 or 15, (d–f) n = 4 per group of one to three independent experiments. Data in b, d and e were analyzed by the unpaired t‐test per normality. *P < 0.05, **P < 0.01, ****P < 0.0001. 7‐AAD, 7‐aminoactinomycin D; Arg1, arginase 1; CL, contralateral; IL, ipsilateral; IL‐1β, interleukin‐1β; pMCAO, permanent middle cerebral artery occlusion; tMCAO, temporary middle cerebral artery occlusion; TNF, tumor necrosis factor. [Colour figure can be viewed at wileyonlinelibrary.com]
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