SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection

Abstract

Objectives: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms.

Materials and methods: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysM^Cre/+ :Shp2^flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2^flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysM^Cre/+ :Shp2^flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected.

Results: LysM^Cre/+ :Shp2^flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages.

Conclusions: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.

Keywords: chemo-attractants; inflammatory response; macrophage polarization; protein-tyrosine phosphatase SHP2; secondary bacterial pneumonia; type I interferon.

Figure 1

SHP2 deficiency leads to higher mortality and decreased neutrophil accumulation in mice with PR8/S aureus coinfection. (A) The lungs were harvested from the mice challenged with PBS/PBS, PR8 (200 PFU)/PBS, PBS/S.aureus (SA) (5 × 10⁷ CFU) or PR8/SA. The Shp2 mRNA expression was detected by qPCR. n = 5 mice per group. *P < .05, **P < .01. Data are representative from 3 independent experiments. (B) Following infection of influenza PR8 (200 PFU) for 5 d, mouse survival was monitored in Shp^fl/fl and LysMCre:Shp2^fl/fl mice after secondary SA (1 × 10⁷ CFU) challenge. n = 16 mice per group, **P < .01. Data are combined from 2 separate experiments. (C‐H) Shp^fl/fl and LysMCre:Shp2^fl/fl mice were infected with 200 PFU of PR8 for 5 d and then challenged with SA (5 × 10⁷ CFU) for 6 h or 24 h. Mice treated with PBS alone were used as negative controls. The bacterial load (C) in the BALF was measured. The number of total cells (D) and neutrophils (E) was counted, and the level of MPO (F) was determined in the BALF. Representative results of lung tissues by H&E staining (G) and the histogram of lung injury scores (H) were shown 24 h after secondary SA infection. Magnification for HE sections: 100×, scale bar: 100 μm. n = 4‐6 mice per group, *P < .05. Data are representative of 2 independent experiments with similar results

Figure 2

Deletion of SHP2 results in high levels of type I IFN and attenuated production of chemokines in mice with secondary S aureus infection. (A‐C) Shp^fl/fl and LysMCre:Shp2^fl/fl mice were treated with 5 × 10⁷ CFU S aureus (SA) for 6 h or 24 h, following an inoculation with 200 PFU PR8 for 5 d. The concentrations of IFN‐α (A), IFN‐β (B), KC (C) and MIP‐2 (D) in the BALF were detected by ELISA. n = 4 mice per group. (E‐H) Mice were challenged with PR8 (200 PFU) for 5 d and then treated by an instillation of SA (5 × 10⁷ CFU), with or without recombinant murine KC and MIP‐2 (1 μg each), for 6 h. The bacterial load (E) in the BALF was detected. The number of total cells (F) and neutrophils (G), as well as the level of MPO (H) in the BALF, was determined. n = 4 mice per group. *P < .05, **P < .01. Representative data of 2‐3 independent experiments are shown

Figure 3

Deletion of SHP2 suppresses inflammatory cytokine production via modulation of the macrophage phenotype in mice with secondary S aureus infection. Shp^fl/fl and LysMCre:Shp2^fl/fl mice were treated with 5 × 10⁷ CFU S aureus (SA) at 5 d after an inoculation of 200 PFU PR8 influenza. (A‐G) The relative levels of mRNA for M1‐associated genes including IL‐6, TNF‐α, IL‐12b, iNOS and M2 genes (eg, Arg1, Ym1 and Fizz1) in lung tissues were assessed by qPCR at 6 h and 24 h after secondary SA infection. n = 5 mice in each group. *P < .05. (H‐I) CD206 and Dectin‐1 expression on CD11b⁺ F4/80⁺ macrophages from the BALF was analysed by flow cytometry, and the mean fluorescence intensity (MFI) of CD206 and dectin‐1 was calculated 24 h after secondary SA infection. n = 3 mice per group. *P < .05. Representative data of 2‐3 independent experiments are shown

Figure 4

Loss of SHP2 skews macrophage differentiation to M2 phenotype and abates its phagocytic activity in response to poly(I:C) and S aureus dual stimulation. The peritoneal macrophages from Shp^fl/fl and LysMCre:Shp2^fl/fl mice were stimulated with poly(I:C) (20 μg/mL) for 24 h and then incubated with S aureus (MOI, 10) for 3, 6 and 12 h, respectively. (A‐D) Expression of M1 gene marker including IL‐6, TNF‐α, IL‐12b and iNOS was measured by qPCR. (E‐G) Expression of M2 gene markers (eg, Arg‐1, YM‐1 and Fizz1) was determined by qPCR. *P < .05, **P < .01. (H) After pre‐stimulation by poly(I:C) (20 μg/mL) for 24 h, the PMs were incubated with FITC‐labelled bacteria for 1 and 2 h. Then, the fluorescence positivity was analysed by flow cytometry. Representative traces of bacteria engulfed were shown by red (Shp^fl/fl) and blue line (LysMCre:Shp2^fl/fl), respectively. Data are representative of 3 independent experiments

Figure 5

SHP2 regulates NF‐κB and IRF3 signalling upon poly(I:C) and S aureus serial stimulation. The peritoneal macrophages from Shp^fl/fl and LysMCre:Shp2^fl/fl mice were stimulated with poly(I:C) (20 μg/mL) for 24 h and then incubated with S aureus (MOI, 10) for 15, 30, 60 and 120 min, respectively. Phosphorylated and total protein levels of JNK, p38, Erk1/2, p65 and IRF3 were detected by Western blot. Representative images (A) and quantitative analysis of blots (B) from 3 independent experiments were shown. *P < .05