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 (LysMCre/+:Shp2flox/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 (Shp2flox/flox). We infused additional KC and MIP‐2 to examine the reconstitution of antibacterial immune response in LysMCre/+:Shp2flox/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 LysMCre/+:Shp2flox/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.

methicillin-resistant strains, became the predominant superinfecting pathogens and caused fulminant pneumonia with fatal clinical outcomes following influenza infection. 5,7 It has been reported that the pulmonary host innate defence against secondary bacterial infection can be impaired by the preceding influenza challenge through multiple mechanisms. 8,9 Viral infection was shown to suppress function of neutrophils, leading to impaired phagocytosis and the attenuated generation of intracellular reactive oxygen species in response to subsequent bacterial infection. 6,10,11 Type I interferons (IFNs), including IFN-α and IFN-β, were typically induced during viral infection and played an indispensable role in host defence. 12 However, type I IFNs have been reported to facilitate post-influenza bacterial infection by reducing the production of chemo-attractants such as keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, which are critical for recruiting neutrophils to the site of infection. [13][14][15] A recent study showed signal transducer of activation and transcription (STAT) 1, a vital transcription factor in IFN signalling, was detrimental during influenza and MRSA superinfection by suppressing type 17 immune response. STAT1 deletion resulted in increased neutrophils in lungs and decreased bacterial burden upon superinfection. 16 In addition to neutrophils, macrophages also participate in host defence by phagocytosing and killing invasive bacteria in the lungs. 17,18 Macrophages can be phenotypically polarized towards classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages), both of which are involved in a variety of inflammatory diseases. 19  Src homology 2 domain-containing phosphatase (SHP2) is a member of non-receptor protein-tyrosine phosphatases subfamily and ubiquitously expressed in the cytoplasm. 21 SHP2 has been shown to regulate cellular functions in multiple lung diseases, including carcinoma, pulmonary fibrosis, chronic airway diseases and lung infection. [22][23][24][25] SHP2 can be activated by respiratory syncytial virus (RSV) and contributed to host antiviral activity. 26 It also has been identified to be critical for clearance of Haemophilus influenzae by skewing macrophage phenotypic differentiation. 25 In addition, SHP2 deficiency in myeloid cells alleviated pulmonary inflammation in acute lung injury. 27 Moreover, SHP2 was found to disrupt IL-10-STAT3 signalling and its dependent anti-inflammatory response in human and mouse macrophages in the context of colonic inflammation. 28 However, to date, it remains unclear whether SHP2 is associated with susceptibility to the post-influenza bacterial infection.
In the present study, we established a murine model of post-influenza S aureus pneumonia to investigate the mechanisms involved in the impaired host antibacterial response following primary influenza challenge. Here, we demonstrat that mice with SHP2 deficiency are more susceptible to secondary S aureus infection. Moreover, such enhanced susceptibility is associated with the overproduction of type I IFNs and M2-biased macrophage differentiation.

| Mice
Shp2 flox/flox and LysM Cre/+ mice on the C57BL/6 background were crossed with each other to generate conditional Shp2 knockout mice as previously described. 23

| Establishment of a mouse model of postinfluenza S aureus pneumonia
The influenza virus PR8 strain was propagated in Madine Darby canine kidney (MDCK) cells and stored in aliquots at −80°C. Virus titers were determined using plaque assay on MDCK cells. In specific, 200 μL of the viral stock was serially diluted and incubated on MDCK monolayers at 37°C for 2 hours. After the incubation, cells were overlaid with viral growth medium (including MEM, NaHCO 3, 10% BSA, 1% DEAE Dextran, 1 μg/mL TPCK trypsin and 2% agarose) as described before and incubated for 72 hours at 34°C in a 5% CO 2 atmosphere. 13 The cells were fixed by 4% formaldehyde and stained with 1% (wt/vol) crystal violet to determine virus titers by counting the number of plaques. The wells containing of 30-100 plaques were suitable for counting, and the virus titers was calculated by the following formula: virus titers (plaque-forming units [PFU]/ mL) = plaques × dilution × 5. The clinically isolated S aureus strain presenting multilocus sequence type ST15 and agr type II was cultured and counted as previously described. 30,31 In brief, S aureus was

| Quantification of PR8 virus in the lungs
The mice were sacrificed at 5 days after influenza infection. The whole lung was removed and homogenized in 1 mL PBS by mechanical grinding. After three cycles of freeze/thaw to release the virus, the supernatants of lung homogenates were collected for viral titration by plaque assay mentioned above.

| Cell counting in BALF
BALF was collected and centrifuged for 10 min at 300 g. The supernatants were stored at −80°C until detection, and the erythrocytes of the cell pellets were removed using lysis buffer (STEMCELL, Vancouver, Canada). After total cell counting, approximately 2 × 10 5 cells were loaded onto a slide by cytospin and stained with Giemsa stain to count the number of neutrophils.

| Histopathological examination
The whole lung was fixed in a 4% paraformaldehyde neutral buffer solution and embedded with paraffin. The samples were sliced into 4 μm sections for haematoxylin and eosin (H&E) staining.
Morphometric analysis was conducted under an optical photomicroscope (Olympus). Lung injury in the specimen was evaluated blindly and graded from 0 (normal) to 4 (severe) following four items: interstitial inflammation, neutrophil infiltration, congestion and oedema.
The score was calculated by adding the individual scores for each item. Lung injury score of each mouse was calculated as the mean of four lung sections. 32

| Phagocytosis assay
The S aureus was labelled to assess the phagocytic capability of macrophages. 35

| Statistical analysis
All data analyses were performed using GraphPad Prism version 5 (GraphPad Software, Inc.). Quantitative data are expressed as mean ± SEM, and significance in differences was determined with student t test or one-way ANOVA with Bonferroni's post hoc analysis where appropriate. Mouse survival rates were analysed with log-rank test. A threshold of P < .05 was considered statistically significant.

| SHP2 deficiency leads to higher mortality and impaired bacterial clearance in mice with postinfluenza S aureus pneumonia
SHP2 has been reported to be activated and involved in host defence against infection. 25,26 Interestingly, we found that the level of SHP2 expression was upregulated by either influenza or S aureus infection and was much higher in mice with post-influenza S aureus pneumonia ( Figure 1A and Figure S1A). Then, we challenged con-

| Deletion of SHP2 results in enhanced induction of type I IFN and attenuated production of chemokines in mice with secondary S aureus infection
During influenza infection, the induction of type I IFNs has been shown to be closely related to secondary bacterial pneumonia. 13,14 The current study revealed that SHP2 deletion led to a high level

| Deletion of SHP2 suppresses inflammatory cytokines via modulating macrophage phenotype in mice upon secondary S aureus infection
In addition to neutrophils, macrophages also participate in the host immune response to post-influenza bacterial infection. Chen et al found that M2 macrophages led to a hypersusceptibility to secondary bacterial infection. 36 Our data showed that compared to the control mice, Shp2 knockout mice displayed significantly lower levels of

| Loss of SHP2 skews macrophage differentiation in response to poly(I:C) and S aureus co-stimulation
The mechanisms by which SHP2 regulates the inflammation re-

| SHP2 is required for the phosphorylation of NF-κB and IRF3 in response to poly(I:C) and S aureus dual stimulation
It has been well established that NF-κB p65 can activate inflam- Therefore, SHP2 holds a discrepancy role in regulating NF-κBdependent inflammatory cytokines expression and IRF3-dependent interferon production.

Secondary bacterial pneumonia following influenza infection is
often severe with high mortality. In 1979, Jakab et al reported that influenza A increased susceptibility to S aureus infection. 41 In the 2009 H1N1 pandemic, the aetiological importance of S aureus as a cause of post-influenza pneumonia had been further recognized among fatal cases. 42,43 However, the underlying molecular mechanisms for secondary bacterial pneumonia remain to be further illustrated. In the present study, we demonstrate that tyrosine phos- However, antiviral IFN pathways may potentiate secondary bacterial infection. It has been reported that influenza-induced type I IFN inhibited Th17 immunity and increased susceptibility to secondary S aureus pneumonia. 45 Nakamura et al reported that an enhanced type I IFN response was associated with decreased production of the chemokine CCL2, which impaired the recruitment of macrophages and bacterial clearance in mice co-infected with influenza virus and S pneumonia. 46 Moreover, other studies have demonstrated that type I IFNs induced by viral infection led to insufficient elimination of bacteria by inhibiting the production of chemokines (eg, KC and MIP-2) responsible for neutrophil recruitment into the lungs. 13,15 Conversely, the overexpression of KC in the transgenic mice displayed a beneficial effect on bacterial clearance, which was related to a vigorous migration of neutrophils in the lung. 47 Interestingly, intratracheal inoculation of KC and MIP-2 could effectively recover Macrophages exhibit remarkable plasticity during the maturation process and can be differentiated into either M1 or M2 phenotype.
M1 macrophages produce abundant pro-inflammatory mediators and promote bactericidal activity, whereas M2 macrophages are associated with the resolution of inflammation and persistence of bacteria. 49 A previous study reported that M2 macrophages were in an activated state during influenza infection and induced an impaired host innate immune response at the early antibacterial process. 36 Gopal et al claimed that STAT2 deficiency increased accumulation of M1, M2 and M1/M2 co-expressing macrophages by influenza-MRSA superinfection, which was associated with increased bacterial clearance. 50 SHP2-mediated regulation in macrophages has been shown to restrain the IL-4-induced M2 phenotype in pulmonary fibrosis. 23 The antibacterial M1 macrophages in Haemophilus influenzae pneumonia were preferentially activated via SHP2-dependent NF-κB p65 signalling. 25 In addition, disruption of SHP2 in monocyte/macrophages was found to alleviate neutrophil recruitment and inflammation in LPS-induced acute lung injury, 27 and conditional knockout of Shp2 in the lung epithelia was shown to reduce pulmonary inflammation in cigarette smoke-exposed mice. 24 These studies supported the role of SHP2 for driving macrophages towards an M1 phenotype and enhancing the inflammatory response. In contrast, Guo et al reported that SHP2 negatively regulated NLRP3 activation and decrease overproduction of pro-inflammatory cytokines including IL-1β and IL-18 in macrophages. 51 Therefore, the role of SHP2 in the innate immunity appears to be diverse dependent on different immune cells and disease models.
In the current study, we revealed that SHP2 deletion increased the expression of M2-associated markers and decreased the levels of M1 markers in both primary macrophages and the lungs upon secondary bacterial infection. And the altered macrophage phenotype induced by SHP2 deficiency contributed to poor host antibacterial immunity in the coinfection model.
NF-κB is activated in response to various stimuli or stresses.
NF-κB p65 was reported to regulate the expression of the pro-inflammatory and antibacterial genes in macrophages. [52][53][54] We previously demonstrated that infection with S aureus induced phosphorylation of NF-κB p65 in macrophages, which shifted the cells into M1 phenotype and promoted the antibacterial response. 38 In this study, we further revealed that SHP2 could drive M1 macrophage polarization and amplify the inflammatory response by activation of NF-κB p65 in response to dual stimulation. IRF3, a member of IRF family, is the key transcription factor involved in the activation of IFN-α/β expression. 55 An et al reported that SHP2 negatively regulated TLR3/ TLR4 induced TIR-domain-containing adapter-inducing IFN-β (TRIF)dependent type I IFN production. 34 In addition, Park et al recently identified SHP2 as a negative regulator of TLR2-induced IFN-β production. 56 Consistent with these findings, our data demonstrated IRF3 to be further activated upon dual stimulation with poly(I:C) and S aureus in vitro, which could be responsible for the increased IFN production in SHP2-deficient macrophages.
In summary, this study dissects the molecular mechanisms associated with post-influenza bacterial infection. We identify SHP2 as one of key factors required to mount a vigorous immune response to a secondary bacterial infection. Moreover, SHP2 regulates negatively the production of type I IFNs and induces the M1-biased macrophage differentiation required for a protective antibacterial inflammatory response. Our findings highlight the importance of innate immunity for restricting secondary bacterial infection in the lungs following influenza infection.

ACK N OWLED G EM ENTS
We thank Dr G. Feng (University of California, San Diego, CA) for providing the Shp2 flox/flox mice. This work was supported by grants from National Natural Science Foundation of China (81770008, 81570005, and 81570004).

CO N FLI C T O F I NTE R E S T
The authors declare they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.