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LPS reduces mortality to SeV in a dose-dependent fashion and affects viral ...
Published: 01 February 2023
FIGURE 1. LPS reduces mortality to SeV in a dose-dependent fashion and affects viral titer early during infection. ( A ) Mice were inoculated with PBS or the indicated doses of LPS 24 h before inoculation with 2 × 106 PFUs SeV. Survival was then monitored (Mantel–Cox, p = 0.0001 for all groups; n ≥ 6 per group). As a negative control, mice were treated with 3 µg LPS before inoculation with UV-SeV. ( B ) LPS- (open circle) compared with PBS-pretreated (filled circle) mice had significantly decreased lung viral titer at day 3 PI of SeV; however, by day 5 PI of SeV, there was no difference in titer. Data are presented as mean ± SEM copies of SeV. Mice numbers were the following: 3 μg LPS-UV-SeV, n = 2; 3 μg LPS, n = 17; 1 μg LPS, n = 4; 0.3 μg LPS, n = 9; 0.1 μg LPS, n = 14; 0.03 μg LPS, n = 13; PBS, n = 14. Data are pooled from three experiments. **p ≤ 0.01. FIGURE 1. LPS reduces mortality to SeV in a dose-dependent fashion and affects viral titer early during infection. (A) Mice were inoculated with PBS or the indicated doses of LPS 24 h before inoculation with 2 × 106 PFUs SeV. Survival was then monitored (Mantel–Cox, p = 0.0001 for all groups; n ≥ 6 per group). As a negative control, mice were treated with 3 µg LPS before inoculation with UV-SeV. (B) LPS- (open circle) compared with PBS-pretreated (filled circle) mice had significantly decreased lung viral titer at day 3 PI of SeV; however, by day 5 PI of SeV, there was no difference in titer. Data are presented as mean ± SEM copies of SeV. Mice numbers were the following: 3 μg LPS-UV-SeV, n = 2; 3 μg LPS, n = 17; 1 μg LPS, n = 4; 0.3 μg LPS, n = 9; 0.1 μg LPS, n = 14; 0.03 μg LPS, n = 13; PBS, n = 14. Data are pooled from three experiments. **p ≤ 0.01. More
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LPS induces robust lung recruitment of neutrophils and macrophages. Frequen...
Published: 01 February 2023
FIGURE 2. LPS induces robust lung recruitment of neutrophils and macrophages. Frequency and cell counts of lung neutrophils (PMNs, GR1+ cells; top panels), aMOs (middle panels), and iMOs (bottom panels) after administration of 0.1 µg LPS (open circle) or PBS (filled circle) i.n. at the indicated days PI of SeV (LPS/PBS given 24 h before 2 × 106 PFUs SeV). Note that day −0.75 PI of SeV is 6 h after LPS, whereas day 0 PI of SeV is 24 h after LPS; in both cases, these mice (days −0.75 and 0) did not receive SeV. n = 10 per group at 6 h, 24 h, and day 1 PI of SeV; n = 14 at days 3, 5, and 7 PI of SeV. Data are pooled from three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. FIGURE 2. LPS induces robust lung recruitment of neutrophils and macrophages. Frequency and cell counts of lung neutrophils (PMNs, GR1+ cells; top panels), aMOs (middle panels), and iMOs (bottom panels) after administration of 0.1 µg LPS (open circle) or PBS (filled circle) i.n. at the indicated days PI of SeV (LPS/PBS given 24 h before 2 × 106 PFUs SeV). Note that day −0.75 PI of SeV is 6 h after LPS, whereas day 0 PI of SeV is 24 h after LPS; in both cases, these mice (days −0.75 and 0) did not receive SeV. n = 10 per group at 6 h, 24 h, and day 1 PI of SeV; n = 14 at days 3, 5, and 7 PI of SeV. Data are pooled from three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. More
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Impact of LPS treatment on airway cytokines/chemokines. ( A ) BAL cytokines...
Published: 01 February 2023
FIGURE 3. Impact of LPS treatment on airway cytokines/chemokines. ( A ) BAL cytokines/chemokines were measured from mice treated as in ( Fig. 2 using a multiplex array. Open circles represent LPS-treated mice, whereas filled circles are PBS treated. ( B ) IFN-α, -β, and -γ mRNA expression were measured in lungs of mice treated as in (A). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n ≥ 6 for each time point for (A) and n ≥ 4 for (B). Data are pooled from three experiments. FIGURE 3. Impact of LPS treatment on airway cytokines/chemokines. (A) BAL cytokines/chemokines were measured from mice treated as in (Fig. 2 using a multiplex array. Open circles represent LPS-treated mice, whereas filled circles are PBS treated. (B) IFN-α, -β, and -γ mRNA expression were measured in lungs of mice treated as in (A). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n ≥ 6 for each time point for (A) and n ≥ 4 for (B). Data are pooled from three experiments. More
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Macrophages, not neutrophils, and type I IFN are required for LPS-mediated ...
Published: 01 February 2023
FIGURE 4. Macrophages, not neutrophils, and type I IFN are required for LPS-mediated survival. ( A ) Mice were treated as in ( Fig. 2 , but given 100 µg anti-Ly6G or control IgG i.p. at the same time as LPS exposure. One day later, mice were inoculated with 2 × 106 PFUs SeV i.n., and survival was monitored. n = 14 mice in the LPS+anti-Ly6G group; n = 12 in the LPS+IgG control group; n = 2 for PBS group. Data are pooled from two experiments. ( B ) A total of 2 mg of clodronate-containing or empty (control) liposomes were given i.p., and 48 h later, mice received 0.1 µg LPS and a second i.p. dose of 0.5 mg clodronate or empty liposomes. Twenty-four hours after receiving LPS, 2 × 106 PFUs SeV was administered, and survival determined. For comparison, mice that did not receive any liposomes but were given LPS and SeV were also included (black solid line). Mantel–Cox statistical comparisons are shown on the graph. Survival was not significantly different between mice that received LPS and no other treatment and those that received LPS and empty liposomes. n = 7 for treatment with clodronate and LPS; n = 8 for treatment with empty liposome and LPS; n = 4 for LPS with SeV only group. **p ≤ 0.01, ****p ≤ 0.0001. ( C ) WT B6 or Ifnar−/− mice received 0.1 μg LPS or PBS i.n. and 24 h later were inoculated with 2 × 106 PFUs SeV or UV-SeV, and survival monitored. Ifnar−/−: n = 13 in the LPS treatment group and n = 7 in the PBS group; WT B6: n = 8 in LPS and n = 3 in PBS groups; n = 2 for UV-SeV (Ifnar−/− and WT B6) control groups. Data are pooled from two experiments. ***p ≤ 0.001. FIGURE 4. Macrophages, not neutrophils, and type I IFN are required for LPS-mediated survival. (A) Mice were treated as in (Fig. 2, but given 100 µg anti-Ly6G or control IgG i.p. at the same time as LPS exposure. One day later, mice were inoculated with 2 × 106 PFUs SeV i.n., and survival was monitored. n = 14 mice in the LPS+anti-Ly6G group; n = 12 in the LPS+IgG control group; n = 2 for PBS group. Data are pooled from two experiments. (B) A total of 2 mg of clodronate-containing or empty (control) liposomes were given i.p., and 48 h later, mice received 0.1 µg LPS and a second i.p. dose of 0.5 mg clodronate or empty liposomes. Twenty-four hours after receiving LPS, 2 × 106 PFUs SeV was administered, and survival determined. For comparison, mice that did not receive any liposomes but were given LPS and SeV were also included (black solid line). Mantel–Cox statistical comparisons are shown on the graph. Survival was not significantly different between mice that received LPS and no other treatment and those that received LPS and empty liposomes. n = 7 for treatment with clodronate and LPS; n = 8 for treatment with empty liposome and LPS; n = 4 for LPS with SeV only group. **p ≤ 0.01, ****p ≤ 0.0001. (C) WT B6 or Ifnar−/− mice received 0.1 μg LPS or PBS i.n. and 24 h later were inoculated with 2 × 106 PFUs SeV or UV-SeV, and survival monitored. Ifnar−/−: n = 13 in the LPS treatment group and n = 7 in the PBS group; WT B6: n = 8 in LPS and n = 3 in PBS groups; n = 2 for UV-SeV (Ifnar−/− and WT B6) control groups. Data are pooled from two experiments. ***p ≤ 0.001. More
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LPS-mediated survival, but not viral reduction, in SeV infection is TLR4 de...
Published: 01 February 2023
FIGURE 5. LPS-mediated survival, but not viral reduction, in SeV infection is TLR4 dependent. ( A ) WT B6 or Tlr4−/− mice were treated as in ( Fig. 2 , and survival determined. WT B6: n = 14 mice per group; Tlr4−/−: n = 6 mice per group. ****p ≤ 0.0001. Data are pooled from three experiments. ( B ) Lung SeV titers from Tlr4−/− mice treated as in (A). *p ≤ 0.05. Tlr4−/− mice were treated as in (A), and lung ( C ) aMOs and ( D ) iMOs were determined by flow cytometry at the indicated times. *p ≤ 0.05, **p ≤ 0.01. FIGURE 5. LPS-mediated survival, but not viral reduction, in SeV infection is TLR4 dependent. (A) WT B6 or Tlr4−/− mice were treated as in (Fig. 2, and survival determined. WT B6: n = 14 mice per group; Tlr4−/−: n = 6 mice per group. ****p ≤ 0.0001. Data are pooled from three experiments. (B) Lung SeV titers from Tlr4−/− mice treated as in (A). *p ≤ 0.05. Tlr4−/− mice were treated as in (A), and lung (C) aMOs and (D) iMOs were determined by flow cytometry at the indicated times. *p ≤ 0.05, **p ≤ 0.01. More
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MyD88 is required for LPS-mediated survival from SeV. <em>Myd88</em>...
Published: 01 February 2023
FIGURE 6. MyD88 is required for LPS-mediated survival from SeV. Myd88−/− mice were treated with 0.1 µg LPS (dotted line) or PBS (solid line) 24 h before i.n. inoculation with 2 × 105 PFUs SeV, and survival was then determined. n = 12 mice in the LPS-treated group and n = 10 in the PBS group. Data are pooled from two experiments. FIGURE 6. MyD88 is required for LPS-mediated survival from SeV. Myd88−/− mice were treated with 0.1 µg LPS (dotted line) or PBS (solid line) 24 h before i.n. inoculation with 2 × 105 PFUs SeV, and survival was then determined. n = 12 mice in the LPS-treated group and n = 10 in the PBS group. Data are pooled from two experiments. More
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Oral immunization with the Yptb1(pYA5199) vaccine confers T cell–mediated c...
Published: 01 February 2023
FIGURE 1. Oral immunization with the Yptb1(pYA5199) vaccine confers T cell–mediated comprehensive protection against pneumonic Y. pestis infection. ( A ) On the 42nd day after immunization, mice (n = 15, mixed males and females) were i.n. challenged with 5 × 103 CFUs (50 LD50) of Y. pestis KIM6+(pCD1Ap). Survival was recorded for 14 dpi. ( B ) The bacterial burden in the lung, spleen, and liver at 2 dpi. ( C ) Ab responses. Serum anti-YPL Ab titers in immunized mice at 14 and 28 dpv (left); the ratios of IgG2a/IgG1 and IgG2b/IgG1 to YPL (right). ( D ) Representative flow plots showing the frequency of lung CD4+ and CD8+ T cells and the respective cells producing IFN-γ, TNF-α, or IL-17A in mice (n = 6 females). ( E ) At dpv 42, lung single cells obtained from Yptb1(pYA5199) and sham mice were in vitro stimulated for 72 h with YPL. The absolute numbers of lung CD4+ and CD8+ T cells and ( F ) lung T cells expressing IFN-γ, TNF-α, or IL-17A were determined by flow cytometer. ( G ) Serum transfer. Naive mice (n = 10, females) were i.p. injected with different volumes of serum collected from sham and Yptb1(pYA5199)-immunized mice at 42 dpv. Twenty-four hours postinjection, recipient mice were i.n. challenged with 10 LD50 of Y. pestis. ( H ) Lung T cell transfer. Naive mice treated with a single dose of irradiation (5 Gy) were i.v. administered CD4+ and/or CD8+ T cells (2 × 106 cells/mouse) isolated from sham or Yptb1(pYA5199)-immunized mice at 42 dpv. At 24 h postadministration, mice (n = 10, females) were i.n. infected with 10 LD50 of Y. pestis, and survival was monitored for 15 d. ( I ) T cell depletion. Mice (n = 10, females) were depleted of CD4+ and/or CD8+ T cells by i.p. administration of anti-CD4, anti-CD8, or anti-CD4 plus anti-CD8 mAbs (500 µg/each mouse in 200 µl) and then i.n. challenged with 50 LD50 of Y. pestis. Each symbol in the individual bar graph represents a data point obtained from an individual mouse. Each experiment was performed two times with identical conditions. Data obtained from experiments were pooled and analyzed and are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 1. Oral immunization with the Yptb1(pYA5199) vaccine confers T cell–mediated comprehensive protection against pneumonic Y. pestis infection. (A) On the 42nd day after immunization, mice (n = 15, mixed males and females) were i.n. challenged with 5 × 103 CFUs (50 LD50) of Y. pestis KIM6+(pCD1Ap). Survival was recorded for 14 dpi. (B) The bacterial burden in the lung, spleen, and liver at 2 dpi. (C) Ab responses. Serum anti-YPL Ab titers in immunized mice at 14 and 28 dpv (left); the ratios of IgG2a/IgG1 and IgG2b/IgG1 to YPL (right). (D) Representative flow plots showing the frequency of lung CD4+ and CD8+ T cells and the respective cells producing IFN-γ, TNF-α, or IL-17A in mice (n = 6 females). (E) At dpv 42, lung single cells obtained from Yptb1(pYA5199) and sham mice were in vitro stimulated for 72 h with YPL. The absolute numbers of lung CD4+ and CD8+ T cells and (F) lung T cells expressing IFN-γ, TNF-α, or IL-17A were determined by flow cytometer. (G) Serum transfer. Naive mice (n = 10, females) were i.p. injected with different volumes of serum collected from sham and Yptb1(pYA5199)-immunized mice at 42 dpv. Twenty-four hours postinjection, recipient mice were i.n. challenged with 10 LD50 of Y. pestis. (H) Lung T cell transfer. Naive mice treated with a single dose of irradiation (5 Gy) were i.v. administered CD4+ and/or CD8+ T cells (2 × 106 cells/mouse) isolated from sham or Yptb1(pYA5199)-immunized mice at 42 dpv. At 24 h postadministration, mice (n = 10, females) were i.n. infected with 10 LD50 of Y. pestis, and survival was monitored for 15 d. (I) T cell depletion. Mice (n = 10, females) were depleted of CD4+ and/or CD8+ T cells by i.p. administration of anti-CD4, anti-CD8, or anti-CD4 plus anti-CD8 mAbs (500 µg/each mouse in 200 µl) and then i.n. challenged with 50 LD50 of Y. pestis. Each symbol in the individual bar graph represents a data point obtained from an individual mouse. Each experiment was performed two times with identical conditions. Data obtained from experiments were pooled and analyzed and are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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Lung CD4<sup>+</sup> and CD8<sup>+</sup> T<sub>RM</sub> cells are activated...
Published: 01 February 2023
FIGURE 2. Lung CD4+ and CD8+ TRM cells are activated by oral Yptb1(pYA5199) immunization and expand after pulmonary Y. pestis infection. Mice were i.v. injected via the tail vein with 5 µg of FITC-conjugated anti-CD45.2 mAbs diluted in 200 µl of sterile PBS to distinguish lung TRM cells from circulating T cells. Five minutes postinjection, mice were euthanized to isolate lung single cells (n = 5 females). Lung cells isolated from Yptb1(pYA5199)-immunized and sham mice were in vitro stimulated with PMA + ionomycin. ( A ) Representative flow plots of lung CD4+ and CD8+ TRM cells producing IFN-γ, TNF-α, and/or IL-17A in immunized and sham mice at 42 dpv. Lung TRM cells were gated based on CD45 (i.v. injection), CD4+/CD8+, CD44+, and CD69+, as well as intracellular cytokines (IFN-γ+, TNF-α+, and IL-17A+), in the flow cytometry protocol (top). Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A (bottom). ( B ) Representative flow plots of lung CD4+ and CD8+ TRM cells in mice at 2 dpi (top). Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A (bottom) (n = 10 females). ( C ) Kinetic analysis of lung CD4+ and CD8+ TRM cells after pulmonary Y. pestis infection (n = 10 females). Data obtained from a minimum of two experiments are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 2. Lung CD4+ and CD8+ TRM cells are activated by oral Yptb1(pYA5199) immunization and expand after pulmonary Y. pestis infection. Mice were i.v. injected via the tail vein with 5 µg of FITC-conjugated anti-CD45.2 mAbs diluted in 200 µl of sterile PBS to distinguish lung TRM cells from circulating T cells. Five minutes postinjection, mice were euthanized to isolate lung single cells (n = 5 females). Lung cells isolated from Yptb1(pYA5199)-immunized and sham mice were in vitro stimulated with PMA + ionomycin. (A) Representative flow plots of lung CD4+ and CD8+ TRM cells producing IFN-γ, TNF-α, and/or IL-17A in immunized and sham mice at 42 dpv. Lung TRM cells were gated based on CD45− (i.v. injection), CD4+/CD8+, CD44+, and CD69+, as well as intracellular cytokines (IFN-γ+, TNF-α+, and IL-17A+), in the flow cytometry protocol (top). Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A (bottom). (B) Representative flow plots of lung CD4+ and CD8+ TRM cells in mice at 2 dpi (top). Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A (bottom) (n = 10 females). (C) Kinetic analysis of lung CD4+ and CD8+ TRM cells after pulmonary Y. pestis infection (n = 10 females). Data obtained from a minimum of two experiments are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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CD4<sup>+</sup> and CD8<sup>+</sup> T<sub>RM</sub> cells established in the...
Published: 01 February 2023
FIGURE 3. CD4+ and CD8+ TRM cells established in the lungs of FTY720-treated Yptb1(pYA5199)-immunized mice offer protection against pulmonary Y. pestis infection. ( A ) Scheme of the immunization, FTY720 treatment, TRM cell analysis, and Y. pestis infection. The lung single-cell isolated Yptb1(pYA5199)+FTY720-immunized and sham mice were in vitro stimulated with PMA + ionomycin. ( B ) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in FTY720-treated Yptb1(pYA5199)-immunized mice at 42 dpv (n = 5 females). ( C ) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in FTY720-treated Yptb1(pYA5199)-immunized mice at 2 dpi. ( D ) On 42 dpv, FTY720-treated Yptb1(pYA5199)-immunized mice (n = 15, mixed males and females) were i.n. challenged with 50 LD50 of Y. pestis. Survival was recorded for 15 d. ( E ) The bacterial burden was evaluated in the lung, spleen, and liver at 2 dpi (n = 6, mixed males and females). ( F ) Scheme of the FTY720 treatment 10 d before oral Yptb1(pYA5199) immunization and until the end of the experiment. ( G ) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells in pre- and post-FTY720–treated Yptb1(pYA5199)-immunized mice at 42 dpv. ( H ) On 42 dpv, mice (n = 5, mixed males and females) were i.n. challenged with 50 LD50 of Y. pestis. ( I ) Adoptive transfer of FACS-sorted lung circulating (CD45+) T cells or TRM cells (CD45) from Yptb1(pYA5199)-immunized mice. Sorted cells were injected into irradiated (5 Gy) naive mice via the intratracheal route. At 24 h after administration, recipient mice (n = 10 females) were i.n. challenged with 10 LD50 of Y. pestis. In the bar plots, each symbol represents a data point obtained from an individual mouse. Data are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 3. CD4+ and CD8+ TRM cells established in the lungs of FTY720-treated Yptb1(pYA5199)-immunized mice offer protection against pulmonary Y. pestis infection. (A) Scheme of the immunization, FTY720 treatment, TRM cell analysis, and Y. pestis infection. The lung single-cell isolated Yptb1(pYA5199)+FTY720-immunized and sham mice were in vitro stimulated with PMA + ionomycin. (B) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in FTY720-treated Yptb1(pYA5199)-immunized mice at 42 dpv (n = 5 females). (C) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in FTY720-treated Yptb1(pYA5199)-immunized mice at 2 dpi. (D) On 42 dpv, FTY720-treated Yptb1(pYA5199)-immunized mice (n = 15, mixed males and females) were i.n. challenged with 50 LD50 of Y. pestis. Survival was recorded for 15 d. (E) The bacterial burden was evaluated in the lung, spleen, and liver at 2 dpi (n = 6, mixed males and females). (F) Scheme of the FTY720 treatment 10 d before oral Yptb1(pYA5199) immunization and until the end of the experiment. (G) Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells in pre- and post-FTY720–treated Yptb1(pYA5199)-immunized mice at 42 dpv. (H) On 42 dpv, mice (n = 5, mixed males and females) were i.n. challenged with 50 LD50 of Y. pestis. (I) Adoptive transfer of FACS-sorted lung circulating (CD45+) T cells or TRM cells (CD45−) from Yptb1(pYA5199)-immunized mice. Sorted cells were injected into irradiated (5 Gy) naive mice via the intratracheal route. At 24 h after administration, recipient mice (n = 10 females) were i.n. challenged with 10 LD50 of Y. pestis. In the bar plots, each symbol represents a data point obtained from an individual mouse. Data are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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Local depletion of lung resident CD4<sup>+</sup> and/or CD8<sup>+</sup> T c...
Published: 01 February 2023
FIGURE 4. Local depletion of lung resident CD4+ and/or CD8+ T cells in Yptb1(pYA5199)-immunized mice impairs protection against pneumonic plague. ( A ) Scheme of immunization, FTY720 treatment, T cell depletion, and survival against pneumonic plague infection. ( B ) Survival study in the lung T cell–depleted mice. The Yptb1(pYA5199)+FTY720-immunized mice (n = 6 females) were depleted of CD4+ and/or CD8+ T cells at the lung mucosal site by i.n. administration of 200 µg of anti-CD4, anti-CD8, or both mAbs and then i.n. challenged with 50 LD50 of Y. pestis. ( C ) Lung Y. pestis burden at 2 dpi (n = 5 females). ( D ) Representative flow plots showing the percentages of neutrophils and AMs in the BAL fluid at 2 dpi. ( E and F ) The number of neutrophils (E) and AMs (F) in the BAL fluid of control and T cell–depleted mice (n = 6 females) at 2 dpi. ( G ) Analysis of cytokines and chemokines in the BAL fluid samples collected at 2 dpi. Each symbol represents a data point obtained from an individual mouse. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 4. Local depletion of lung resident CD4+ and/or CD8+ T cells in Yptb1(pYA5199)-immunized mice impairs protection against pneumonic plague. (A) Scheme of immunization, FTY720 treatment, T cell depletion, and survival against pneumonic plague infection. (B) Survival study in the lung T cell–depleted mice. The Yptb1(pYA5199)+FTY720-immunized mice (n = 6 females) were depleted of CD4+ and/or CD8+ T cells at the lung mucosal site by i.n. administration of 200 µg of anti-CD4, anti-CD8, or both mAbs and then i.n. challenged with 50 LD50 of Y. pestis. (C) Lung Y. pestis burden at 2 dpi (n = 5 females). (D) Representative flow plots showing the percentages of neutrophils and AMs in the BAL fluid at 2 dpi. (E and F) The number of neutrophils (E) and AMs (F) in the BAL fluid of control and T cell–depleted mice (n = 6 females) at 2 dpi. (G) Analysis of cytokines and chemokines in the BAL fluid samples collected at 2 dpi. Each symbol represents a data point obtained from an individual mouse. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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IFN-γ and IL-17A play a critical role in protection against pulmonary <ital
Published: 01 February 2023
FIGURE 5. IFN-γ and IL-17A play a critical role in protection against pulmonary Y. pestis challenge. ( A ) Scheme of neutralization of IFN-γ, IL-17A, or both in Yptb1(pYA5199)-immunized mice. Mice administrated with PBS (sham) were used as control. ( B ) Survival study with cytokine neutralization. The Yptb1(pYA5199)-immunized mice (n = 10 females) were i.p. injected with 200 µg of anti–IFN-γ, anti–IL-17A, or both mAbs and then i.n. challenged with 50 LD50 of Y. pestis. ( C ) Lung Y. pestis burden in the respective group of mice at 2 dpi. ( D and E ) The number of neutrophils (D) and AMs (E) in the BAL fluid. ( F and G ) The absolute number of lung neutrophils (F) and AMs (G) in the lungs of mice (n = 6 females) treated with anti–IFN-γ, anti–IL-17A, anti–IFN-γ/IL-17A, or isotype control Abs at 2 dpi. ( H and I ) The number of CD4+ (H) and CD8+ TRM (I) cells in the lungs of mice (n = 6 females) treated with the respective Abs at 2 dpi. ( J ) Analysis of cytokines and chemokines in the BAL fluid of mice (n = 6 females) treated with the respective Abs at 2 dpi. ( K ) Representative H&E-stained lung sections of oral Yptb1(pYA5199)-immunized mice treated with anti–IFN-γ, anti–IL-17A, anti–IFN-γ/IL-17A, or isotype control Abs, or sham mice collected at 2 dpi. A black arrowhead indicates a reduced alveolar lacunar space; a red arrowhead indicates a lung lesion or hemorrhage. Each symbol represents a data point obtained from an individual mouse. Data are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 5. IFN-γ and IL-17A play a critical role in protection against pulmonary Y. pestis challenge. (A) Scheme of neutralization of IFN-γ, IL-17A, or both in Yptb1(pYA5199)-immunized mice. Mice administrated with PBS (sham) were used as control. (B) Survival study with cytokine neutralization. The Yptb1(pYA5199)-immunized mice (n = 10 females) were i.p. injected with 200 µg of anti–IFN-γ, anti–IL-17A, or both mAbs and then i.n. challenged with 50 LD50 of Y. pestis. (C) Lung Y. pestis burden in the respective group of mice at 2 dpi. (D and E) The number of neutrophils (D) and AMs (E) in the BAL fluid. (F and G) The absolute number of lung neutrophils (F) and AMs (G) in the lungs of mice (n = 6 females) treated with anti–IFN-γ, anti–IL-17A, anti–IFN-γ/IL-17A, or isotype control Abs at 2 dpi. (H and I) The number of CD4+ (H) and CD8+ TRM (I) cells in the lungs of mice (n = 6 females) treated with the respective Abs at 2 dpi. (J) Analysis of cytokines and chemokines in the BAL fluid of mice (n = 6 females) treated with the respective Abs at 2 dpi. (K) Representative H&amp;E-stained lung sections of oral Yptb1(pYA5199)-immunized mice treated with anti–IFN-γ, anti–IL-17A, anti–IFN-γ/IL-17A, or isotype control Abs, or sham mice collected at 2 dpi. A black arrowhead indicates a reduced alveolar lacunar space; a red arrowhead indicates a lung lesion or hemorrhage. Each symbol represents a data point obtained from an individual mouse. Data are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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Lung T<sub>RM</sub> cells activated by oral Yptb1(pYA5199) immunization con...
Published: 01 February 2023
FIGURE 6. Lung TRM cells activated by oral Yptb1(pYA5199) immunization confer long-lasting protection. ( A ) Scheme of the immunization, immune parameter analysis, and challenge in the long-term animal study. ( B ) Kinetic analysis of lung CD4+ and CD8+ TRM cells in Yptb1(pYA5199)-immunized mice treated with or without FTY720 at 30, 60, 90, and 120 dpv (n = 6 females). ( C ) Mice were i.v. injected via the tail vein with 5 µg of FITC-conjugated anti-CD45.2 mAbs diluted in 200 µl of sterile PBS to distinguish lung TRM cells from circulating T cells. Five minutes postinjection, mice were euthanized to isolate lung single cells (n = 5 females). Lung cells isolated from Yptb1(pYA5199), Yptb1(pYA5199)+FTY720-immunized, and sham mice were in vitro stimulated with PMA + ionomycin. Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in Yptb1(pYA5199)-immunized mice treated with or without FTY720 and control mice was performed at 2 dpi. ( D ) On the 120th day after immunization, mice (n = 10, equal number of males and females) were i.n. challenged with 50 LD50 of Y. pestis. ( E ) The bacterial burden was evaluated in the lung, liver, and spleen of mice (n = 6, mixed males and females) at 2 dpi. Data obtained from experiments are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. FIGURE 6. Lung TRM cells activated by oral Yptb1(pYA5199) immunization confer long-lasting protection. (A) Scheme of the immunization, immune parameter analysis, and challenge in the long-term animal study. (B) Kinetic analysis of lung CD4+ and CD8+ TRM cells in Yptb1(pYA5199)-immunized mice treated with or without FTY720 at 30, 60, 90, and 120 dpv (n = 6 females). (C) Mice were i.v. injected via the tail vein with 5 µg of FITC-conjugated anti-CD45.2 mAbs diluted in 200 µl of sterile PBS to distinguish lung TRM cells from circulating T cells. Five minutes postinjection, mice were euthanized to isolate lung single cells (n = 5 females). Lung cells isolated from Yptb1(pYA5199), Yptb1(pYA5199)+FTY720-immunized, and sham mice were in vitro stimulated with PMA + ionomycin. Quantitative analysis of the number of lung CD4+ or CD8+ TRM cells and corresponding cells producing IFN-γ, TNF-α, and/or IL-17A in Yptb1(pYA5199)-immunized mice treated with or without FTY720 and control mice was performed at 2 dpi. (D) On the 120th day after immunization, mice (n = 10, equal number of males and females) were i.n. challenged with 50 LD50 of Y. pestis. (E) The bacterial burden was evaluated in the lung, liver, and spleen of mice (n = 6, mixed males and females) at 2 dpi. Data obtained from experiments are presented as the mean ± SD. The statistical analysis is described in the Materials and Methods. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. More
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eCIRP increases the expression of CD112 in neutrophils. ( A ) A total of 3 ...
Published: 01 February 2023
FIGURE 1. eCIRP increases the expression of CD112 in neutrophils. ( A ) A total of 3 × 106 BMDNs were sorted by negative selection using a magnetic column and treated with rmCIRP (1 μg/ml) for 2 h, and mRNA levels of CD112 were assessed by real-time PCR. ( B ) A total of 2 × 106 BMDNs were treated with rmCIRP at different time points, and the protein levels of CD112 were assessed by Western blotting. ( C ) Purified 1 × 106 BMDNs were treated with rmCIRP at different doses and time points, and CD112 protein expression on the surface of BMDNs was assessed using flow cytometry. A representative gating strategy is shown in PBS-treated sample. Total BMDNs were gated for the single granulocyte population based on FSC and SSC. Single granulocytes were subsequently gated for the Ly6G-AF488-positive cell population and further gated into those staining for CD112-BV421-positive cells in the PBS group. (C and E ) Representative histograms of CD112-BV421-positive neutrophil population and ( D and F ) their corresponding bar diagrams representing the mean frequency of CD112 expression are shown at different doses and different time points. n = 5–10 samples/group obtained from at least four mice. Experiments were performed two or three times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test for (A) mRNA and (B–F) by one-way ANOVA and Student-Newman-Keuls method for protein expression. *p < 0.05 versus PBS. FIGURE 1. eCIRP increases the expression of CD112 in neutrophils. (A) A total of 3 × 106 BMDNs were sorted by negative selection using a magnetic column and treated with rmCIRP (1 μg/ml) for 2 h, and mRNA levels of CD112 were assessed by real-time PCR. (B) A total of 2 × 106 BMDNs were treated with rmCIRP at different time points, and the protein levels of CD112 were assessed by Western blotting. (C) Purified 1 × 106 BMDNs were treated with rmCIRP at different doses and time points, and CD112 protein expression on the surface of BMDNs was assessed using flow cytometry. A representative gating strategy is shown in PBS-treated sample. Total BMDNs were gated for the single granulocyte population based on FSC and SSC. Single granulocytes were subsequently gated for the Ly6G-AF488-positive cell population and further gated into those staining for CD112-BV421-positive cells in the PBS group. (C and E) Representative histograms of CD112-BV421-positive neutrophil population and (D and F) their corresponding bar diagrams representing the mean frequency of CD112 expression are shown at different doses and different time points. n = 5–10 samples/group obtained from at least four mice. Experiments were performed two or three times, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test for (A) mRNA and (B–F) by one-way ANOVA and Student-Newman-Keuls method for protein expression. *p &lt; 0.05 versus PBS. More
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eCIRP induces CD112 expression on neutrophils through the TLR4 pathway. ( A...
Published: 01 February 2023
FIGURE 2. eCIRP induces CD112 expression on neutrophils through the TLR4 pathway. ( A and B ) Purified BMDNs from WT or TLR4−/− mice were treated with 1 μg/ml of rmCIRP for 2 h, and CD112 expression on the BMDNs was assessed using flow cytometry. ( C and D ) BMDNs were pretreated with 10 μg/ml of anti-TLR4-neutralizing Ab or isotype IgG Ab for 30 min and then stimulated with 1 μg/ml of rmCIRP for 2 h. (A and C) Representative histograms indicating CD112-BV421-positive neutrophils using the previous gating and (B and D) the corresponding bar diagrams of the frequency of CD112+ neutrophils are shown. n = 4–8 samples/group obtained from at least two mice. Experiments were performed twice. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls test. *p < 0.05 versus PBS; #p < 0.05 versus WT CIRP or isotype IgG Ab. FIGURE 2. eCIRP induces CD112 expression on neutrophils through the TLR4 pathway. (A and B) Purified BMDNs from WT or TLR4−/− mice were treated with 1 μg/ml of rmCIRP for 2 h, and CD112 expression on the BMDNs was assessed using flow cytometry. (C and D) BMDNs were pretreated with 10 μg/ml of anti-TLR4-neutralizing Ab or isotype IgG Ab for 30 min and then stimulated with 1 μg/ml of rmCIRP for 2 h. (A and C) Representative histograms indicating CD112-BV421-positive neutrophils using the previous gating and (B and D) the corresponding bar diagrams of the frequency of CD112+ neutrophils are shown. n = 4–8 samples/group obtained from at least two mice. Experiments were performed twice. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls test. *p &lt; 0.05 versus PBS; #p &lt; 0.05 versus WT CIRP or isotype IgG Ab. More
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In vivo administration of rmCIRP increases frequencies of CD112<sup>+</sup>...
Published: 01 February 2023
FIGURE 3. In vivo administration of rmCIRP increases frequencies of CD112+ neutrophils in blood and spleen. After 4 h of rmCIRP i.p. injection, the blood and spleen were collected. ( A ) A representative sample shows the flow cytometry gating strategy in blood. Blood cells were gated for the single granulocytes based on FSC and SSC characteristics and then gated into the Ly6G-AF488- and CD112-BV421-positive cell population. Representative histograms of CD112 expression on neutrophils are shown. ( B ) Flow cytometric analysis representing the frequencies of CD112+ neutrophils in blood is shown. ( C ) Representative samples providing flow cytometry gating strategy and ( D ) their flow cytometric analysis of CD112+ neutrophils in spleen are shown. n = 7 samples/group. Experiments were performed twice, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test. *p < 0.05 versus vehicle. FIGURE 3. In vivo administration of rmCIRP increases frequencies of CD112+ neutrophils in blood and spleen. After 4 h of rmCIRP i.p. injection, the blood and spleen were collected. (A) A representative sample shows the flow cytometry gating strategy in blood. Blood cells were gated for the single granulocytes based on FSC and SSC characteristics and then gated into the Ly6G-AF488- and CD112-BV421-positive cell population. Representative histograms of CD112 expression on neutrophils are shown. (B) Flow cytometric analysis representing the frequencies of CD112+ neutrophils in blood is shown. (C) Representative samples providing flow cytometry gating strategy and (D) their flow cytometric analysis of CD112+ neutrophils in spleen are shown. n = 7 samples/group. Experiments were performed twice, and all data were used for analysis. Data are expressed as mean ± SEM and compared by Student t test. *p &lt; 0.05 versus vehicle. More