Fig 1: Shed Plasmodium berghei T-cell immunomodulatory protein (PbTIP) in host circulation exhibited binding on macrophages and reduced their inflammatory responses while upregulating the Th2-type/anti-inflammatory responses in vitro. (A) Brief incubation of recombinant PbTIP (10 µg/ml) with RAW 264.7 cells followed by immunostaining against the protein revealed its binding on the cell surface (top left panels). The shed PbTIP in malaria-infected mouse serum also displayed binding on RAW 264.7 cells in vitro (middle left panel). No binding on RAW 264.7 cells was observed with healthy mouse serum (right control panel). Parasite blood-stage lysate (obtained from parasite free–thaw lysis) containing various fragments of PbTIP also exhibited binding on RAW 264.7 cells (bottom left panel). Images were taken at ×200 magnification on a Zeiss Axio imager M2 microscope. DAPI was used to stain the nuclei of cells. Scale bar, 10 µm. (B) Recombinant PbTIP binding upon incubation with murine peritoneal macrophages and RAW 264.7 cells. Images were taken at a higher magnification to unveil surface binding of PbTIP. Scale bar, 10 µm (top panel) and 5 µm. Glutathione S-transferase (GST) protein was used as a negative control for binding. (C) PbTIP surface binding reduced the ability of macrophages to mount inflammatory responses upon lipopolysaccharide (LPS) stimulation: (i) IL-1ß was reduced by sixfold; (ii) TNF-a was reduced by 15-fold; (iii) IFN-? was reduced by 2.6-fold; (iv) IL-12p40 was reduced by threefold. (D) The levels of anti-inflammatory cytokines such as TGF-ß and IL-10, however, increased by five and ninefold, respectively, with longer exposure to PbTIP (20 h). The experiment was repeated more than three times, and C t values obtained from quantitative PCR (qPCR) were used to calculate the relative fold change using the 2-?? C t method. Statistical significance values (p-values) of the transcript fold change between the LPS-stimulated and LPS+PbTIP RAW 264.7 cells were given for various cytokine genes: for IL-1ß, TNF-a, IFN-?, and IL12p40, 0.0032, 0.008, 0.01, and 0.04, respectively. For the anti-inflammatory cytokines, such as IL-10 and TGF-ß, the p-values were 0.0002 and 0.004, respectively. Student’s t-test was performed to calculate the statistical significance of the data. *, ** and *** signifies p values in the range of 0.05 to 0.01, 0.005 to 0.001 and 0.0005 to 0.0001, respectively.
Fig 2: DMXAA treatment enhanced the infiltration of CD3+ T cells and macrophages in the tumor. (A) Representative IHC and phenotype images of the control and DMXAA-treated tumor-bearing mice. T cells are marked as yellow (upper) or red (lower). (B) Flow cytometry analysis of the macrophages and (C) T cells. The gating strategy is shown in the left panel; the numbers of M1, M2, and T cells of treatment group were higher than those in the control group. The percentages of M1, M2, and T cells are presented in a bar graph (right panel). (D) The IFN-? and IL-12p40 concentrations in BAL fluid analyzed using an ELISA assay. The results are expressed as mean±SD. *p<0.05. ns, not significant; IFN, interferon; IL, interleukin; IHC, immunohistochemistry; BAL; Bronchoalveolar lavage; ELISA, enzyme-linked immunosorbent assay.
Fig 3: NTB-DCs vaccine significantly increased the antibody and cellular immune responses. (A) Six-week-old C57BL/6J APCMin/+ mice were given AOM for 4 weeks and administered F. nucleatum for 8 weeks. At week 20, the mice were immunized twice a week with DCs control, TBI-DCs, BNE-DCs vaccine and NTB-DCs vaccine for 4 weeks. (B) TBI and NTB adjuvant increased the antibody responses. Mice (n = 10) were immunized DCs control, TBI-DCs, BNE-DCs vaccine and NTB-DCs vaccine. At week 24, mice sera were harvested, IgG and IgG subgroups were detected by ELISAs. (C) TBI and NTB adjuvant significantly increased the cellular immune response. Splenocytes of immunized mice (n=10) were stimulated with antigen for 3 days. The levels of IFN-?, interleukin-4 (IL-4), IL-12p40 and IL-17A in supernatant were determined using the corresponding ELISA kit. (D) ELISPOT analysis on IFN-? and IL-17A spot-forming cells among splenocytes. The results are shown as the means ± SD. *P <0.05; **P < 0.01; ***P < 0.001.
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