Aggravated MRSA pneumonia secondary to influenza A virus infection is derived from decreased expression of IL‐1β

Abstract Secondary methicillin‐resistant Staphylococcus aureus (MRSA) infection is a cause of severe pneumonia with high mortality during influenza A virus (IAV) pandemics. Alveolar macrophages (AMs) mount cellular defenses against IAV and MRSA infection, which occurs via the nucleotide‐binding domain‐like receptor protein 3 (NLRP3) inflammasome. However, the activity and function of the NLRP3 inflammasome in MRSA pneumonia secondary to IAV infection remain unclear. To clarify this, we studied MRSA infection secondary to IAV both in vitro and in mouse model. The expression of the NLRP3 inflammasome was evaluated by quantitative reverse transcription polymerase chain reaction, immunofluorescence, Western blot, and enzyme‐linked immunosorbent assay. The lung pathology and the rate of weight change were observed. We found that IAV infection for 1 week activated NLRP3 inflammasome. The enhanced expression of NLRP3, caspase‐1, and cleaved caspase‐1 was associated with MRSA infection secondary to IAV, but the expression of interleukin (IL)‐1β decreased in superinfection with MRSA both in vitro and in vivo. The aggravated inflammatory pathology in MRSA pneumonia secondary to IAV infection was associated with decreased expression of IL‐1β. And increased weight loss in MRSA pneumonia secondary to IAV infection was related to decreased concentration of IL‐1β in serum. It infers that superinfection with MRSA reduces expression of IL‐1β someway, and decreased expression of IL‐1β impairs the host immunity and leads to aggravated pneumonia. These results contributed to our understanding of the detailed activity of the NLRP3 inflammasome, IL‐1β, and their relationship with aggravation of MRSA pneumonia secondary to IAV infection. Immunotherapy targeting the IL‐1β signaling pathway could be possible therapeutic strategy for secondary MRSA pneumonia.


| INTRODUCTION
The influenza virus is a member of the family Orthomyxoviridae, and can be classified into A, B, and C subtypes. Influenza A virus (IAV) infects type II epithelial cells and alveolar macrophages (AMs) in the airway and alveoli in humans, and causes acute respiratory infectious diseases including seasonal flu, bronchitis, and pneumonia. Approximately, 600 million people around the world suffer from IAV infections every year, and occasional pandemics cause greater threat to public health. 1,2 For most cases of IAV infection, the infection runs its course within 1 week, and the patient makes a full recovery. However, severe pneumonia and increased risk of mortality are sometimes caused by complications following secondary bacterial infection, which usually occurs about 1 week after IAV infection. [3][4][5][6][7] Staphylococcus aureus (S. aureus) is one of the most frequent causes of secondary infection in patients with IAV, and secondary methicillinresistant S. aureus (MRSA) pneumonia is the most lethal secondary infection. [8][9][10] Because of the high risk and severity of secondary MRSA pneumonia, antibiotic treatment has been recommended in influenza pandemic preparedness strategy. 11 However, because of the multiantibiotic resistance of MRSA, searching and applying immunotherapy for secondary MRSA pneumonia is of great necessity. 12 As the major innate immunocytes in the respiratory tract, AMs initiate immune response against infections such as IAV and MRSA via pattern recognition receptors. 13,14 These include Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs). The latter can assemble with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 to form a multiprotein complex called the inflammasome. 15 Being composed of nucleotide-binding domain-like receptor protein 3 (NLRP3), ASC, and caspase-1, the NLRP3 inflammasome plays an important role in the host response to infectious disease. The activity of NLRP3 can be suppressed by NLRP3-specific inhibitor MCC950. 16 The activation of the NLRP3 inflammasome upon infection requires two signaling pathways. 17,18 Signaling pathway 1 is the priming of transcription and synthesis of NLRP3 and pro-interleukin (IL)-1β through the TLR-MyD88-NF-κB pathway. The second signaling pathway is the formation of NLRP3 inflammasome, autocatalysis of caspase-1 into cleaved caspase-1, cleavage of pro-IL-1β into IL-1β, and secretion of IL-1β. 19,20 As an important immune signaling cytokine, IL-1β initiates downstream inflammation through multiple mechanisms, including cytokine production and neutrophil recruitment. 21 Research focused on the pathogenesis of S. aureus infection secondary to IAV has accumulated for these years. 9,14,18,[22][23][24][25][26][27][28][29] However, there is much less information available on MRSA pneumonia secondary to IAV infection. 30 Blyth et al 10 concluded that coinfection of IAV and bacteria synergistically aggravated inflammatory injury to the host. Deterioration of the lung has been observed to be enhanced by superinfection of MRSA and influenza. 22,26 However, the functions and mechanisms of the NLRP3 inflammasome in the severe MRSA pneumonia secondary to IAV infection remain unknown.
In the present study, the expression of the NLRP3 inflammasome was evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunofluorescence, Western blot, and enzymelinked immunosorbent assay (ELISA), and the lung pathology and the rate of weight change were observed. We found that enhanced expression of NLRP3 and caspase-1 during MRSA infection secondary to IAV, but the expression of IL-1β decreased in MRSA infection secondary to IAV both in vitro and in vivo. The aggravated inflammatory pathology in MRSA pneumonia secondary to IAV infection was associated with decreased expression of IL-1β. The rate of weight loss in infected mice negatively correlated with concentration of IL-1β in serum. And increased weight loss in MRSA pneumonia secondary to IAV infection was related to decreased concentration of IL-1β in serum. It infers that superinfection with MRSA reduces expression of IL-1β someway, and decreased expression of IL-1β impairs the host immunity and leads to aggravated pneumonia.

| Ethics statement
The experimental protocol containing euthanasia criteria was es-     combined with analgesia by intramuscular injection of ketamine (30 mg/kg weight) was performed on each mouse before blood collection and cervical dislocation. 33 The left upper lobes were used for Western blot and left lower lobes for RT-qPCR. The right lungs were used for pathology. The condition of the mice was assessed by measuring the change of body weight and the survival rate. The rate of weight change was determined using the equation: (weight at each time point − weight at zero hour)/weight at zero hour, and was analyzed between experimental groups on 1st day (24 hours), 6th day (144 hours), and 7th day (168 hours). Correlation between rate of weight change on 7th day and concentration of IL-1β in serum was investigated.   Table 1

| Statistical analysis
The experimental data were processed and analyzed with the statistical software SPSS 20.0. All results are presented as mean ± standard deviation (x ± s). Statistical significance was analyzed by unpaired t test (for 2 means) or one-way analysis of variance (for multiple groups). LSD-t or Dunnett T3 test was used for comparison between each two groups according to the homogeneity of variance.
Correlation was investigated by Pearson's product-moment correlation coefficient. Data were plotted using the GraphPad Prism software. A P value of <0.05 was considered to be statistically significant.

| Aggravated inflammatory pathology in the lung was associated with MRSA infection secondary to IAV in vivo
The pathology of the lungs confirmed that pneumonia model caused by intranasal inoculation with IAV alone or with a secondary MRSA infection was successful. Gross pathology of the lung showed congestion in the IAV group ( Figure 4B1), with more severe congestion in the lung of mice in the IAV + MRSA group ( Figure 4C1), whereas the lung of mice in the control group was normal ( Figure 4A1). Histopathology of the lungs in the control group revealed visible bronchioles and blood vessels, along with intact alveolar structure. The alveolar wall was thin, and there was no inflammatory infiltration in the alveolar cavity, nor in the interstitial substance ( Figure 4A2,A3). In the IAV group, inflammatory cell infiltration and alveolar septum thickening were observed ( Figure 4B2,B3). Whereas, more severe inflammatory damage was observed in the IAV + MRSA group. Infected lungs showed loss of alveolar architecture, infiltration of immune cells, vascular congestion, hemorrhage, bronchial obstruction, and even consolidation of lung parenchyma ( Figure 4C2,C3). These results showed that more severe inflammatory pathology of the lungs was associated with MRSA infection secondary to IAV.

| Increased weight loss of mice with secondary MRSA pneumonia was related to decreased concentration of IL-1β in serum
All of the mice in the control, IAV, and IAV + MRSA groups survived for the duration of the experiment. Weight loss was used as a marker of severity and mortality. Mice in the control group increased in body weight at a normal rate, while mice in the IAV and IAV + MRSA groups had reduced food intake and activity, as well as significant weight loss during the experiment ( Table 2). The condition of mice in the IAV + MRSA group deteriorated rapidly after coinfection with MRSA. During the 24 hours of superinfection, mice in the IAV + MRSA group lost more weight than mice in the IAV group ( Figure 5).
The rate of weight loss in the IAV and the IAV + MRSA groups on 7th day negatively correlated with concentration of IL-1β in serum on 7th day in each group, respectively. Linear trend was found from the scatter plot ( Figure 6A,B). Pearson's product-moment correlation F I G U R E 1 Expression levels of NLRP3, caspase-1, cleaved caspase-1, and IL-1β in vitro. Murine macrophages were infected with IAV for 1 week in the IAV group, with IAV for 1 week, and coinfected with MRSA for 24 hours in the IAV + MRSA group. The control group received equivalent volumes of PBS. A, Relative mRNA expression levels of NLRP3, caspase-1, and IL-1β were measured by RT-qPCR using the −ΔΔ 2 Ct method and β-actin as the reference gene. B, Concentration of IL-1β in the cell supernatant was assessed by ELISA. C and D, Relative protein expression of NLRP3, caspase-1, and cleaved caspase-1 were assessed by Western blot compared with the reference protein GAPDH. Protein level = densitometry of target protein/densitometry of GAPDH. *P < 0.05, **P < 0.01. ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IAV, influenza A virus; IL, interleukin; mRNA, messenger RNA; MRSA, methicillin resistant Staphylococcus aureus; NLRP3, nucleotidebinding domain-like receptor protein 3; PBS, phosphate-buffered saline; RT-qPCR, quantitative reverse transcription polymerase chain reaction F I G U R E 2 Immunofluorescent staining of NLRP3 in macrophages. Murine macrophages were infected with IAV for 1 week in the IAV group, with IAV for 1 week, and coinfected with MRSA for 24 hours in the IAV + MRSA group. The control group received equivalent volumes of PBS. The nucleus of murine macrophages was stained with Hoechst 33342 (blue, rank 1). NLRP3 was stained with a primary antibody and a fluorescence-conjugated secondary antibody (red, rank 2), and these were merged (rank 3). Each group was processed under the same condition. Line 1 is the control group, line 2 is the IAV group, and line 3 is the IAV + MRSA group. IAV, influenza A virus; MRSA, methicillin-resistant staphylococcus aureus; NLRP3, nucleotide-binding domain-like receptor protein 3; PBS, phosphate-buffered saline   production. They also observed that IL-1β was essential for bacterial clearance during superinfection of S. aureus and IAV. 23