Combined regulation of pro-inflammatory cytokines production by STAT3 and STAT5 in a model of B. pertussis infection of alveolar macrophages

Abstract

Bordetella pertussis is a highly contagious respiratory pathogen responsible for whooping-cough or pertussis. Despite high vaccination coverage worldwide, this gram-negative bacterium continues to spread among the population. B. pertussis is transmitted by aerosol droplets from an infected individual to a new host and will colonize its upper respiratory tract. Alveolar macrophages (AMs) are effector cells of the innate immune system that phagocytose B. pertussis and secrete both pro-inflammatory and antimicrobial mediators in the lungs. However, understanding their role in B. pertussis pathogenesis at the molecular level is hampered by the limited number of primary AMs that can be collected in vivo. In order to decipher the regulation of innate response induced by B. pertussis infection, we used for the first time self-renewing, non-transformed cells, called Max Planck Institute (MPI) cells, which are phenotypically and functionally very close to pulmonary AMs. Using optimized infection conditions, we characterized the entry and the clearance of B. pertussis within MPI macrophages. We showed that under these conditions, MPI cells exhibit a pro-inflammatory phenotype with the production of TNF, IL-1β, IL-6 and MIP-2α, similarly to primary AMs purified from broncho-alveolar fluids of mice. In addition, we explored the yet uncharacterized role of the signal transduction activator of transcription (STAT) proteins family in the innate immune response to B. pertussis infection and showed for the first time the parallel regulation of pro-inflammatory cytokines by STAT3 and STAT5 in MPI macrophages infected by B. pertussis. Altogether, this work highlights the interest of using MPI cells for experiments optimization and preliminary data acquisition to understand B. pertussis interaction with AMs, and thus significantly reduce the number of animals to be sacrificed.

Keywords: Bordetella pertussis; STAT proteins; alveolar macrophages; cytokines; infection.

Figure 1

TNF production by MPI cells infected with B. pertussis. (A) TNF production 24h after infection measured by ELISA from cell culture supernatant of MPI cells incubated for 0,5h with different MOI of B. pertussis. (B) MPI cell activity assessed using MTT assay, 24h after infection of MPI cells with B. pertussis for 0,5h. (C) TNF production 24h post-infection measured by ELISA from cell culture supernatant of MPI cells incubated with B. pertussis for 0.5, 1 or 2h with a MOI of 50. Lines represent the median of triplicates or quadruplicates. Statistical test used: Kruskal-Wallis followed by a Conover’s test for multiple comparisons. Data are representatives of two independent experiments. *: p-value <0,05, **: p-value <0,01.

Figure 2

Internalization of B. pertussis by the MPI cells. (A) Electron micrograph of a cross-sectionned MPI cell incubated for 2h with B. pertussis at a MOI of 50. B. pertussis is entrapped by cytoplasmic protrusions synthesized by the cell. Scale bar, 500 nm. (B) Representative Flow cytometry chart of MPI cells infected for 1h with CFSE-stained-B. pertussis at a MOI of 50.

Figure 3

Impact of B. pertussis internalization on TNF response by MPI cells. (A) Number of colony-forming unit (CFU) internalized by MPI cells pre-treated or not with cytochalasin D (CytD) and incubated with B. pertussis for 1h at a MOI of 50. (B) TNF production 24h after B. pertussis infection measured by ELISA from cell culture supernatant of MPI cells pre-treated or not with Cyt D and incubated with B. pertussis for 1h with a MOI of 50. Lines represent the median of quadruplicates. Statistical test used: Mann-Whitney. Data are representatives of 2 experiments. *: p-value < 0,05.

Figure 4

Intracellular clearance of B. pertussis by MPI cells. (A) Electron micrograph of a cross-sectioned MPI cells after 4h of incubation with B. pertussis at a MOI of 50, showing the fusion of a B. pertussis-containing phagosome with a lysosome. Scale bar, 500 nm. (B) Kinetics of intracellular survival of B. pertussis inside MPI cells after 1h of contact between cells and bacteria at a MOI of 50. The end of contact time corresponds to T0. Colony Forming Units of B. pertussis from infected-macrophages lysates were counted after 5 days of culture on BG agar plates. The percentage of intracellular survival was calculated with the ratio of (CFU_{Time of interest}/CFU_T0)*100. The bars represent the mean of quadruplicates. Data are representatives of 3 independent experiments.

Figure 5

Cytokine responses of MPI cells and primary murine alveolar macrophages infected with B. pertussis. MPI cells and AM were incubated for 1h with B. pertussis at a MOI of 50. 24h post-infection, production of TNF (A), IL-1β (B), IL-6 (C), MIP-2α (D) and IL-22 (E) was measured by the Luminex technology. Lines represent the median of five biological replicates and the graph is representative of 2 experiments. Statistical test used: Mann-Whitney. *: p-value < 0,05.

Figure 6

Inhibition of STAT5 phosphorylation in MPI cells cultured without GM-CSF and infected or not with B. pertussis. (A) Phospho-STAT5 (p-STAT5), STAT5 and β-actin levels detected by immunoblot 4h post-infection in lysates of MPI cells incubated for 1h with B. pertussis at a MOI of 50. The end of contact time corresponds to T0. Non-infected cells were added as control. NI: Non-infected cells, BP: B. pertussis infected-cells. (B) Fold-protein expression of STAT5 and p-STAT5 in B. pertussis-infected MPI cells cultured in the presence or not of GM-CSF. Bars represent the median of 3 independent experiments.

Figure 7

B. pertussis clearance in MPI cells with and without GM-CSF. Kinetics of intracellular survival of B. pertussis inside MPI cells. MPI cells were incubated for 1h with B. pertussis at a MOI of 50. The end of contact time corresponds to T0. Bars represent the mean of quadruplicates with the standard deviation (SD). Data are representatives of 3 independent experiments. ***: p-value <0,001.

Figure 8

Cytokine expression and secretion by B. pertussis-infected MPI cells. MPI cells were incubated for 1h with B. pertussis at a MOI of 50. The end of contact time corresponds to T0. (A) Expression of tnf, il-1b, il-6 and mip-2a transcripts in MPI cells cultured in medium containing or not GM-CSF, 6h post-infection. Bars represent the median of triplicates (B) Cytokines production measured by ELISA in the supernatants of B. pertussis-infected cells 24h post-infection. Lines represent the median of quadruplicates. Statistical test used: Mann-Whitney. Data are representatives of 3 independent experiments. *: p-value < 0,05.

Figure 9

Inhibition of Tyr705 STAT3 phosphorylation, but not Ser727 STAT3, in MPI cells cultured without GM-CSF and infected or not with B. pertussis. MPI cells were incubated for 1h with B.pertussis at a MOI of 50. The end of contact time corresponds to T0. (A, B) Immunoblot of MPI cell lysates infected with B. pertussis 4h post-infection, using specific antibodies to detect (A) p-STAT3 (Tyr705), (B) p-STAT3 (Ser727), (A, B) STAT3 and β-actin. NI, Non-infected cells; BP, B. pertussis infected-cells. (C) Fold-protein expression of p-STAT3 (Tyr705), p-STAT3 (Ser727), STAT3. Results represent the median of 3 independent experiments.