ABSTRACT

Background

   Epidemiological evidence has confirmed that periodontitis is an
   essential and independent risk factor of chronic obstructive pulmonary
   disease (COPD). Porphyromonas gingivalis, a major pathogen implicated
   in periodontitis, may make a vital contribution to COPD progression.
   However, the specific effects and molecular mechanism of the link
   between P. gingivalis and COPD are not clear.

Methods and Results

   A COPD rat model was constructed by smoke exposure combined
   intratracheal instillation of E. coli-LPS, then P. gingivalis was
   introduced into the oral cavity of COPD rats. This research observed
   that lower lung function, more severe alveolar damage and inflammation
   occurred in COPD rats with P. gingivalis group. Meanwhile, P.
   gingivalis/gingipains could colonize the lung tissues and be enriched
   in bronchoalveolar lavage fluid (BALF) of COPD rats with P. gingivalis
   group, along with alterations in lung microbiota. Proteomic analysis
   suggested that Hsp90α/MLKL-meditated necroptosis pathway was
   up-regulated in P. gingivalis-induced COPD aggravation, the detection
   of Hsp90α and MLKL in serum and lung tissue verified that Hsp90α/MLKL
   was up-regulated.

Conclusion

   These results indicate that P. gingivalis could emigrate into the
   lungs, alter lung microbiota and lead to aggravation of COPD, which
   Hsp90α/MLKL might participate in.

   KEYWORDS: Porphyromonas gingivalis, chronic obstructive pulmonary
   disease, lung microbiota, Hsp90α, MLKL, necroptosis

Introduction

   As the main cause of tooth loss, periodontitis impairs people’s quality
   of life and is an essential and independent risk factor of chronic
   obstructive pulmonary disease (COPD) [[40]1,[41]2]. COPD is a common
   and chronic respiratory disease which is characterized by irreversible
   airflow limitation due to airway and/or alveolar abnormalities [[42]3].
   Sometimes COPD patients suffer from severe exacerbations which is
   associated with significantly lower survival outcomes [[43]4,[44]5].
   Poor periodontal health, especially higher plaque scores and fewer
   teeth, is associated with the increased frequency of exacerbations in
   COPD patients [[45]6]. Some researches focused on the involvement of
   periodontal pathogens in the development of COPD [[46]7–9].
   Porphyromonas gingivalis, a major pathogens in periodontitis
   development, may play a crucial role in COPD progress [[47]10]. A
   prospective follow-up study found that COPD patients with high serum P.
   gingivalis IgG titers had significantly lower incidence and frequent of
   exacerbation than patients with normal IgG titers [[48]11]. Our
   previous studies also found a high detection rate of P. gingivalis in
   respiratory secretions of patients with COPD exacerbation, which is
   homologous to P. gingivalis in dental plaque, supported the hypothesis
   that P. gingivalis may contribute to the pathology of COPD exacerbation
   [[49]12]. However, the specific effects and molecular mechanism of the
   link between P. gingivalis and COPD are not clear.

   In healthy individuals, the lung microbiota is relatively simple and
   low in abundance that an ecological balance between lung microbiota and
   host was maintained through mechanical and immune clearance [[50]13].
   However, there is a significant disruption of the lung microbiota,
   characterized by dysbiosis in microbial composition and quantity in
   COPD [[51]14,[52]15]. Multi-omics analysis found that the altered lung
   microbiome in COPD interact with host gene expression by their
   metabolites to promote inflammation [[53]16]. The changes in airway
   microbes also shift COPD from neutrophilic to eosinophilic inflammation
   [[54]17]. Pseudomonas aeruginosa and Streptococcus pneumoniae are
   common respiratory condition pathogens that may induce acute
   exacerbation of COPD [[55]18,[56]19]. P. gingivalis was known to
   enhance the adhesion of Streptococcus pneumoniae to alveoli by
   promoting the expression of Platelet-Activating Factor Receptor through
   gingipain [[57]20]. P. gingivalis or its virulence factors can also
   cooperate with Streptococcus pneumoniae and Pseudomonas aeruginosa to
   promote alveolar epithelial cell apoptosis and aggravate respiratory
   inflammation [[58]21,[59]22]. However, no study has reported the impact
   of P. gingivalis on lung microbiota in COPD progress.

   Inflammation is the main mechanism of COPD progression, which can lead
   to small airway damage, alveolus destruction, and lung function reduced
   [[60]23]. As a cytosolic molecular chaperone, Heat shocked protein 90 α
   (Hsp90α) could regulate the phosphorylation, oligomerization and
   membrane transport of Mixed Lineage Kinase Domain Like Pseudokinase
   (MLKL) [[61]24,[62]25]. Activated MLKL can translocate to the cell
   membrane, leading to plasma membrane rupture and cell necroptosis
   [[63]26]. With the rupture of the plasma membrane, cells swell,
   chromatin shrinks, and damaged cells release a significant quantity of
   damage associated molecular patterns (DAMPs), inflammatory factors and
   cellular contents which directly causing inflammation and indirectly
   spreading inflammatory responses [[64]27–29]. Early study has found
   that Hsp90α in serum of patients with moderate-to-severe COPD are
   significantly higher than those of healthy smokers and non-smoking
   controls [[65]30]. The expression of MLKL/p-MLKL protein is increased
   in respiratory epithelial cells and macrophages of patients with severe
   COPD [[66]31]. Therefore, Hsp90α/MLKL may participate in the occurrence
   and development of COPD.

   In the present study, by constructing COPD rat model and P. gingivalis
   inoculation in COPD rats’ oral cavity, we want to investigate the
   specific effects and molecular mechanism of the link between P.
   gingivalis and COPD. So as to clarify the role of P. gingivalis in COPD
   progression and provide a novel idea on the prevention and treatment of
   COPD.

Materials and methods

Bacterial cultures

   P. gingivalis W83 (Provided by the Department of Periodontology, School
   of Stomatology, China Medical University) were cultured in fresh brain
   heart infusion (BHI) agar plate with 0.5% hemin, 0.1% menadione
   (vitamin K1),0.05% yeast extract and 5% sterile defibrinated sheep
   blood at 37°C in an anaerobic jar for 3–5 days. Monoclonal colonies in
   good growth condition (dark black and shiny) were selected to BHI
   liquid medium without sheep blood. After 16–18 h culturation, bacteria
   were collected by centrifugation at 3500 rpm for 5 min, and resuspended
   in sterile PBS. OD was measured using a UV spectrophotometer at 600 nm,
   and 1 × 10 [[67]9] CFU/mL was used as the bacterial concentration at an
   absorbance of one.

Animal model

   The Sprague-Dawley (SD) rats (180–200 g, 6–7 weeks-old) were purchased
   from Changsheng Biotechnology Co., Ltd. (Liaoning, China). After being
   fed adaptively in SPF laboratory animal room for 1 week, the rats were
   randomly divided into three groups (n = 6 per group): Control, COPD,
   COPD with P. gingivalis (COPD+P.g). The method of COPD rat modeling was
   minor modified according to some references [[68]32,[69]33]. Briefly,