Globally, pneumonia is a severe public health problem with a large disease burden and a major cause of mortality and morbidity. The disease burden caused by pneumonia affects more children worldwide than other diseases (O’Brien et al., 2019). Older patients have a high risk of death afflicted by pneumonia (Quinton and Mizgerd, 2015). Some of the bacterial and viral infections causing acute pneumonia or sepsis result in serious inflammatory damage to the lungs, thereby leading to the progression of acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), especially in critically ill patients (Dai et al., 2018). Multidrug-resistant microbes (“superbugs”), such as Streptococcus pneumoniae, group A Streptococcus, Klebsiella pneumoniae, Staphylococcus aureus, Mycoplasma pneumoniae, and Pseudomonas aeruginosa can cause severe pneumonia and were the main inducers of acute pathogenic factors (Ravi Kumar et al., 2018).

P. aeruginosa, a common opportunistic pathogen, can cause life-threatening respiratory infections and is responsible for hospital-acquired infections and ventilator-associated pneumonia (VAP). The high adaptability and an increasing number of multidrug-resistant P. aeruginosa constitute a threat for those who suffer from Chronic Obstructive Pulmonary Disease (COPD) or Cystic Fibrosis (CF; Aloush et al., 2006). Unfortunately, because of its intrinsic and extrinsic drug resistance and the capacity of P. aeruginosa to form biofilms, a P. aeruginosa infection is notoriously difficult to treat with antibiotics (Moore and Flaws, 2011). Multidrug-resistant pathogen-induced death is estimated to reach 10 million by 2050, and thereby exceeding deaths caused by cancer and diabetes combined worldwide. Thus, the development of alternative therapeutic methods is critical (O’Neill, 2016). The QS system is a cell-to-cell system that enables microbe populations to change their behavior based on population density. The QS system of P. aeruginosa plays a key role in coordinating various activities, including biofilm formation and the release of virulence factors. The QS system of P. aeruginosa is mainly regulated by four QS network subsystems, including lasI/lasR, rhlI/rhlR, PQS, and IQS systems (Lee and Zhang, 2015).

P. aeruginosa-induced pneumonia expresses a myriad of virulence factors, including flagella, pili, lipopolysaccharides (LPS), elastase, alkaline phosphatase, exotoxin A, as well as components of the type III secretion system (T3SS; Sadikot et al., 2005). In addition, the ability of P. aeruginosa to form biofilms is conducive to establishing infections in VAP and CF patients and is difficult to eradicate (Costerton et al., 1999). P. aeruginosa-induced pneumoniae is a complex process. Several surface-associated elements, including flagella, fimbriae, and LPS, contact host respiratory epithelia through a number of aggregated pilis (Gellatly and Hancock, 2013). Once contact with host epithelia has occurred, T3SS can be activated. P. aeruginosa T3SS is a major determinant of virulence, and its expression is frequently associated with acute invasive infections. Moreover, P. aeruginosa T3SS has been linked to increased mortality in infected patients (Hauser, 2009). Elastase (lasB lasA) has been shown to directly injure lung tissue through disruption of epithelial tight junctions and basal membranes. Elastase may increase the recruitment of neutrophils into the airways, which can result in serious inflammation (Kipnis et al., 2006). Pyocyanin induces direct damage to the respiratory tract as epithelial necrosis, and slowing tracheal mucociliary transport results from damaged ciliary movement (Lau et al., 2004). Virulence factors, such as type III secretory proteins, QS systems, and LPS, activate the host immune response. Therefore, the activation of macrophages, neutrophilic granulocytes, and T cells induces the secretion of cytokines, chemotactic factors, and another inflammatory mediator, thus leading to lung injury and mortality (Driscoll et al., 2007).

For thousands of years, tea originating from China has gained the world’s taste. It has become the daily health drink for many individuals. In general, these health benefits result from the phenolic compounds that are present in green tea, particularly catechins. Numerous studies have demonstrated the diverse activities of Epigallocatechin-3-gallate (EGCG), including antioxidant, antibacterial, antiviral, antitumor and anti-inflammatory activities. In our previous study, we demonstrated that EGCG significantly acted against the expression of P. aeruginosa QS-regulated virulence in vitro, and significantly increased the survival rate of Caenorhabditis elegans infected with P. aeruginosa (Hao et al., 2021). QS quenching, called anti-virulence therapy, has been considered a suitable strategy to settle the multidrug resistance problem (Clatworthy et al., 2007). Taken together, our study provides additional support for drinking tea as an effective method against bacterial infections. These findings can pave the way for the use of EGCG as a therapeutic medicine to treat bacterial infections in the lungs.

In this study, it was demonstrated that EGCG protects mice against P. aeruginosa-induced lung damage by inhibiting the virulence controlled by QS systems. P. aeruginosa is a major cause of acute nosocomial infections and pneumonia (Kizny Gordon et al., 2017). Nosocomial bacteremia pneumonia is associated with significant mortality, morbidity, length of hospital stays and cost. In addition, despite important medical advances, there have been no improvements in the treatment of P. aeruginosa infections over the past two decades (Melsen et al., 2013; Jain et al., 2015). The mechanisms by which P. aeruginosa resists antibiotics include intrinsic, acquired and adaptive resistance, such as efflux pumps, antibiotic-inactivating enzymes, impermeable outer membrane proteins, horizontal transfer of resistance genes or mutational changes, and adaptive resistance refers to the formation of biofilms (Livermore, 2002; Breidenstein et al., 2011). P. aeruginosa is resistant to diverse types of antibiotics, including quinolones, aminoglycosides, and β-lactams (Hancock and Speert, 2000). Due to the increasing frequency of antibiotic resistance of P. aeruginosa, novel therapeutic approaches to treat P. aeruginosa infections are of utmost importance. Furthermore, novel treatments may be used alone or in combination with conventional therapies, such as the inhibition of QS and bacterial lectins, the use of iron chelation, phage therapy, vaccine strategies, as well as the use of nanoparticles, antimicrobial peptides and electrochemical scaffolds (Chatterjee et al., 2016). In our previous study, the minimal inhibitory concentration (MIC) of EGCG against P. aeruginosa was confirmed to be 512 μg/ml (Hao et al., 2021). However, EGCG reduced virulence phenotypes, such as biofilm, protease, elastase activity, swimming, and swarming motility at concentrations where no growth inhibition was observed. In the present study, we aimed to explore the anti-infection ability of EGCG based on the QS mechanism in a mouse model of P. aeruginosa infection. Our results indicated that EGCG significantly alleviated P. aeruginosa-induced pulmonary edema, decreased the bacterial load and the level of proinflammatory cytokines in the lung, and enhanced the survival rates of mice. Moreover, EGCG significantly reduced the expression of QS-regulated virulence factors in vivo. Taken together, these findings showed that EGCG has the ability to mitigate the release of virulence and may alleviate inflammation-caused damage in vivo.

EGCG is one of the richest ingredients in green tea-derived polyphenols, and its biological activities have been extensively studied (Jigisha et al., 2012; Steinmann et al., 2013). EGCG can inhibit infections by reducing biofilm formation and toxin release in bacteria (Zhao et al., 2021). However, few studies have evaluated the clinical effect of EGCG in animal models. In several previous studies, the anti-inflammation effect of EGCG was demonstrated. Lee et al. demonstrated that EGCG protected against TNF-α-mediated lung inflammation by down-regulation of oxidative stress and expression of intercellular adhesion molecule (ICAM)-1 in A549 cells or human pulmonary alveolar epithelial cells (HPAEpiCs) as well as in mouse lungs (Lee et al., 2013). In a recent study, the effect of green tea (GTE) and EGCG on macrophage polarization was evaluated in vitro and it was determined whether the treatment could ameliorate inflammatory responses in vivo. Results showed that GTE and EGCG decreased M1-macrophages and increased Treg cells in bone marrow to inhibit inflammation. EGCG and GTE prevent LPS-induced inflammatory damage contributing to restore the immune system homeostasis through increasing M2-macrophages, N2-neutrophils and Tregs in the spleen and blood (Azambuja et al., 2022). The activation of M2 macrophages, which polarized by Th2 cytokines such as IL-4 and IL-13, can enhance the effect anti-inflammatory and immunoregulatory. Consequently, increasing the release of anti-inflammatory cytokines IL-10 and TGF-β (Shapouri-Moghaddam et al., 2018). Wang et al. (2019) demonstrated that pretreatment with EGCG attenuated LPS-induced ALI as manifested by fewer pathological changes in pulmonary edema and the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the lung, serum, and BALF. The protective mechanism was associated with suppressing the TLR-4/NF-kB-p65 pathway that mediates inflammation.

These results were similar to the data obtained in our study; we demonstrated that EGCG alleviates lung damage, including pathological injury and pulmonary edema in P. aeruginosa-induced pulmonary infection. Moreover, EGCG decreased the P. aeruginosa load and infection mortality in the lung. EGCG significantly inhibited the expression of TNF-α, IL-1β, IL-6, and IL-17 in the lung but increased the expression of anti-inflammatory cytokines IL-4 and IL-10. We speculate that EGCG may improve the immunity ability through promoting the polarization of M2 macrophages. Hence EGCG increased the level of anti-inflammatory cytokines IL-4 and IL-10. Further work needs to be performed to identify the underlying mechanisms involved. The ELISA results showed that the protein expression levels of TNF-α and IL-17 decreased in a dose-dependent manner, compared with PAO 1 group. Therefore EGCG can reduce P. aeruginosa-induced inflammation in the lung. Liu et al. demonstrated that tea polyphenols increased the survival rate of Caenorhabditis elegans against K. pneumonia infection to 73.3 and 82.2% (Liu et al., 2020). Previous results showed that microencapsulation of EGCG exhibited therapeutic outcomes by resolution of inflammation in tuberculosis bacteria-infected lungs and a significant reduction in bacterial burden (Sharma et al., 2020). Many studies have suggested that EGCG and other polyphenol compounds could be potential antivirulence agents for pulmonary infection (Sriram et al., 2009; Ling et al., 2012). The potent anti-inflammatory activities add further appeal to the medicinal use of EGCG or other green tea polyphenol-rich products.

P. aeruginosa can express a plethora of virulence factors that facilitate invasion and damage host tissues (Crousilles et al., 2015), and are controlled by complex, intersecting regulatory circuits and multiple signaling systems (Nadal Jimenez et al., 2012; Balasubramanian et al., 2013). Among these, QS regulates virulence factors (proteases, elastase, rhamnolipid, exotoxins, and pyocyanin) and plays an important role in acute P. aeruginosa infections (Hauser, 2009; Lee and Zhang, 2015). QS participates in biofilm formation, which plays an important role during chronic infections and antibiotic resistance. In recent years, antimicrobial agents with great potential have been studied and are now known as antivirulence therapies (Hauser, 2009). This intervention targets virulence factors or virulence regulatory pathways that will not result in inhibition of bacterial growth or bacterial cell death but block their pathogenicity (Hauser, 2009). Consequently, the emergence of drug-resistant strains is decreasing. Interference with QS-mediated signaling and alleviation of virulence contribute to clearance of infecting bacteria by host defenses (Saeki et al., 2020). Our results confirmed the anti-QS ability of EGCG against P. aeruginosa in vivo. EGCG significantly inhibited the expression of QS-relevant genes, including lasI, lasR, rhlI, rhlR, pqsA, pqsR, phzA, phzH, phzM, phzS, lasA, lasB, rhlA, and rhlC. Moreover, EGCG had a better capacity to inhibit virulence than the positive CIP group. According to the gene expression results, EGCG displayed an excellent ability to inhibit proinflammatory cytokines. At 80 mg/kg, EGCG significantly increased anti-inflammatory cytokines IL-10 and IL-4, which was not observed in the CIP group. We found that the expression of lasR/lasI and rhlR/rhlI systems, the leading virulence factor during P. aeruginosa infection, was significantly inhibited at 80 mg/kg. Pyocyanin, a green color product of P. aeruginosa, can interfere with host oxidative stress responses by increasing intracellular levels of reactive oxygen species (Lau et al., 2004). Our results show that the inhibition ability of EGCG on the expression of QS-regulated virulence factors was better than that of CIP, especially in the QS systems lasR/lasI, rhlR/rhlI and pqsR/pqsA. Thus, these results suggested that EGCG not only alleviates inflammation but also reduces the damage of P. aeruginosa infection by targeting QS system-regulated virulence, thereby resulting in a higher protective effect than CIP. EGCG is a great potential QS inhibitor against infection that not only reduces the release of virulence factors in vitro but also effectively inhibits the expression of virulence factors in vivo.

In conclusion, our results provide proof that EGCG alone can ameliorate acute P. aeruginosa infection in the lungs by QS and inflammation inhibition. The in vivo results indicate that the QS inhibitor, EGCG,s may serve as a potential approach in treating bacterial infections. EGCG, a rich ingredient of tea, is indispensable to human life and may play an important role in preventing bacterial infections.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Animal Ethical Committee of Sichuan Agricultural University.

HT and SH contributed to conceptualization, methodology, validation, and investigation. LZ and FS contributed to formal analysis and investigation. YL, QW, HG, CL, JL, and ZZ contributed to writing—original draft and resources. GY contributed to writing—review and editing, supervision, resources, and funding acquisition. All authors contributed to the article and approved the submitted version.

This work was supported by International Science and Technology Cooperation Program of Sichuan: 2020YFH0143; Traditional Chinese Veterinary Medicine Laboratory of the National Ethnic Affairs Commission [2020] 03.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.