Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK-MAPK and NF-κB Signal Pathways through TLR4

Abstract

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α using qPCR and ELISA. Blocking signal-regulated JNK-MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK-MAPK and RelA(p65) pathways.

Keywords: Haemophilus parasuis; PotD; polyamine; pro-inflammatory cytokines; signal pathway.

Figure 1

Purification of rPotD protein. Lane 1: Ultrasonic-lysed crude protein of pET-potD-BL21(DE3); lane 2: purified rPotD protein (dialyzed, ultra-filtrated); lane 3: purified rPotD protein (without being ultra-filtrated); lane 4: low concentration of elution buffer; M: protein molecular mass standard (Novoprotein).

Figure 2

Determination of specific IgG titers in serum samples collected from each mouse after receiving three rounds of immunization. IgG titers were determined using indirect ELISA coated with purified recombinant PotD protein. The titer was defined as the absorbance of the highest dilution at 450 nm which was at least 0.1 above that of the background wells in which serum samples were replaced with PBS. Short vertical lines indicate value below detection limit. These experiments were repeated four times. Each error bar represents one standard deviation from the mean.

Figure 3

Immunofluorescent (IF) assay for the level of CD positive T-cells in spleen tissues. Spleen tissues were harvested from mice in different groups after 7 dpi, and used for FFPE treatment, pathological slice preparation, and IF assay for the levels of immunocyte CD3⁺/CD4⁺/CD8⁺. (A) IF analysis and H&E reference points (100×). (B) IF analysis (725×) and statistical analysis of IOD of T lymphocyte CD3⁺/CD4⁺/CD8⁺. Relative values of IOD_R were calculated by the ratio of the IOD of CY3-fluorescence intensity (IOD_C) and DAPI (IOD_D).

Figure 3

Immunofluorescent (IF) assay for the level of CD positive T-cells in spleen tissues. Spleen tissues were harvested from mice in different groups after 7 dpi, and used for FFPE treatment, pathological slice preparation, and IF assay for the levels of immunocyte CD3⁺/CD4⁺/CD8⁺. (A) IF analysis and H&E reference points (100×). (B) IF analysis (725×) and statistical analysis of IOD of T lymphocyte CD3⁺/CD4⁺/CD8⁺. Relative values of IOD_R were calculated by the ratio of the IOD of CY3-fluorescence intensity (IOD_C) and DAPI (IOD_D).

Figure 4

Lymphocyte Proliferation Assay. Two weeks after the booster immunization, the splenocytes (1 × 10⁶ cells/well) from immunized groups and mock group were cultured with 10 μg/well of proteins and ConA (positive control) for 72 h at 37 °C with 5% CO₂. Levels of lymphocyte proliferation in each group were detected using an MTS method and the absorbance at 490 nm (A_490nm) was measured using a Varioskan Flash (Thermo Scientific). (A) Microexamination of spleen lymphocyte proliferation in the mice immunized with rPotD+adjuvant. (B) Spleen lymphocyte proliferation analysis. These experiments were repeated three times. Each error bar represents one standard deviation from the mean.

Figure 5

The concentrations of (A) IL-1, (B) IL-4, and (C) IFN-γ in cultured primary spleen cells of mice. Production in cultured spleen cells of mice injected with proteins plus MONTANIDE™ GEL 01 adjuvant or the adjuvant control (negative control). Results are expressed in picograms per milliliter. The asterisk * indicates significance at a p < 0.05 level. These experiments were repeated three times. Each error bar represents one standard deviation from the mean.

Figure 5

The concentrations of (A) IL-1, (B) IL-4, and (C) IFN-γ in cultured primary spleen cells of mice. Production in cultured spleen cells of mice injected with proteins plus MONTANIDE™ GEL 01 adjuvant or the adjuvant control (negative control). Results are expressed in picograms per milliliter. The asterisk * indicates significance at a p < 0.05 level. These experiments were repeated three times. Each error bar represents one standard deviation from the mean.

Figure 6

The survival rate of mice challenged with a lethal dose (1.4 × 10⁹ CFU/mouse) of H. parasuis SH0165. (A) In active immunization, PBS/rPotD-alone/adjuvant-alone groups triggered 100% (10/10) death in mice model within 48 h post-injection, while rPotD+adjuvant group engendered only 30% (3/10) of death within a 72 h-period observation. (B) In passive immunization, all the mice receiving antisera elicited by PBS/ rPotD alone/adjuvant alone died after the challenge with H. parasuis SH0165 within 36 h. One half of mice (5/10) immunized passively with antisera from rPotD mixed with adjuvant were protected from the lethal H. parasuis challenge. Mock group indicates mice that were neither vaccinated nor challenged with SH0165, while PBS group indicates mice receiving both PBS inoculation and bacteria injection. All assays were performed at least two times independently. Survival data were recorded every 12 h for a period of 72 h and plotted using GraphPad Prism v. 8.0.2.

Figure 7

Histopathologic analysis of mice lungs (200×). Lung tissues were harvested from mice in different groups after 72 h dpi, and used for histological examination (H&E) staining and histopathological assay. Mock group (Mock): no visible pathological damage. rPotD-adjuvant group: thickening alveolar walls and inflammatory cells infiltration (black arrow); expanded alveoli, thinner alveolar walls (yellow arrow); broken alveolar walls (red arrow). Adjuvant-alone group: necrosis, karyopycnosis and hyperchromatism and fragmentation of the epithelial cells in the alveolar walls (black arrow); inflammatory cells infiltration (red arrow); dilated capillaries congested with red blood cells (yellow arrow); local hemorrhage (blue arrow).

Figure 8

Viable nalidixic acid-resistant colonies were counted for H. parasuis isolated from PotD-immunized (A) or non-treated (B) SPF mice at 12 h postinjection. One hundred microliters per diluted lung tissue homogenate were plated on TSA++ supplemented with 200 µL nalidixic acid (100 mg/mL). Plates were incubated at 37 °C overnight, and visible bacterial colonies were counted manually. Graph represents mean ± SD for 4 mice/group for each treatment (* indicates a significant difference (p < 0.05) between groups of immune treatment using paired t-test, and plus sign indicates mean value in each group.).

Figure 9

ELISA detection of the levels of pro-inflammatory cytokines induced by rPotD. Induction of cytokines in Raw 264.7 macrophages by rPotD stimulation. Raw 264.7 macrophages were incubated with 15 and 30 µg/mL rPotD, and 200 ng/mL LPS (positive control) for 12 h, as well as single culture media (negative control). Protein levels of (A) IL-1β, (B) IL-6, and (C) TNF-α in the culture supernatants were determined by ELISA. All assays were performed at least three times independently. Each error bar represents one standard deviation from the mean.

Figure 9

ELISA detection of the levels of pro-inflammatory cytokines induced by rPotD. Induction of cytokines in Raw 264.7 macrophages by rPotD stimulation. Raw 264.7 macrophages were incubated with 15 and 30 µg/mL rPotD, and 200 ng/mL LPS (positive control) for 12 h, as well as single culture media (negative control). Protein levels of (A) IL-1β, (B) IL-6, and (C) TNF-α in the culture supernatants were determined by ELISA. All assays were performed at least three times independently. Each error bar represents one standard deviation from the mean.

Figure 10

Analysis of the recognition receptor of PotD. Anti-TLR2 and anti-TLR4 monoclonal antibodies were used to block their corresponding Toll-like receptor or both TLR-2/4. Protein levels of (A) IL-1β, (B) IL-6, and (C) TNF-α in the culture supernatants were determined by ELISA. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 10

Analysis of the recognition receptor of PotD. Anti-TLR2 and anti-TLR4 monoclonal antibodies were used to block their corresponding Toll-like receptor or both TLR-2/4. Protein levels of (A) IL-1β, (B) IL-6, and (C) TNF-α in the culture supernatants were determined by ELISA. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 11

MTT Analysis of cytotoxicities of signaling pathway inhibitors (inhibitory rate curve). Cytotoxicities of signaling pathway inhibitors SB203580 (p38-MAPK inhibitor), U0126 (Erk1/2 inhibitor), SP600125 (JNK inhibitor), and PDTC (NF-κB inhibitor) were determined as an inhibition ratio, which is equal to (1 − (A − A_blank)/(A_c − A_blank)), in which A represents the A_570nm of experimental groups (inhibitor agent-treated); A_blank represents background wavelength in blank wells (no cell and no agent); A_c represents A_570nm in control groups, where cells were only treated with MTT agent, but not any inhibitor agent. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 12

Western Blotting (WB) detection of the signaling molecules involved in cytokine secretion. Raw 264.7 macrophage was treated with 15 μg/mL of purified rPotD for 12 h, and used for detection of signaling molecule in MAPK and NK–κB signaling pathways. (A) Western blot analysis. (B) gray intensity. (C) Ratios between phosphorylated activated signals. C1–C3 represent three parallel mock groups, while P1–P3 represent three parallel Hp–rPotD challenged groups. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 12

Western Blotting (WB) detection of the signaling molecules involved in cytokine secretion. Raw 264.7 macrophage was treated with 15 μg/mL of purified rPotD for 12 h, and used for detection of signaling molecule in MAPK and NK–κB signaling pathways. (A) Western blot analysis. (B) gray intensity. (C) Ratios between phosphorylated activated signals. C1–C3 represent three parallel mock groups, while P1–P3 represent three parallel Hp–rPotD challenged groups. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 13

Effects of inhibitors on the pro-inflammatory cytokine secretion induced by PotD based on ELISA. Signaling pathway inhibitors SB203580 (p38–MAPK inhibitor), U0126 (Erk1/2 inhibitor), SP600125 (JNK inhibitor), and PDTC (NF–κB inhibitor) were used to block their corresponding MAPK and NF–κB signal pathways. Protein levels of IL–1β (A), IL–6 (B), TNF–α (C) in the culture supernatants were determined by ELISA. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.

Figure 13

Effects of inhibitors on the pro-inflammatory cytokine secretion induced by PotD based on ELISA. Signaling pathway inhibitors SB203580 (p38–MAPK inhibitor), U0126 (Erk1/2 inhibitor), SP600125 (JNK inhibitor), and PDTC (NF–κB inhibitor) were used to block their corresponding MAPK and NF–κB signal pathways. Protein levels of IL–1β (A), IL–6 (B), TNF–α (C) in the culture supernatants were determined by ELISA. These experiments were repeated 3 times. Each error bar represents one standard deviation from the mean.