Toll‐like receptor 4: A promising therapeutic target for pneumonia caused by Gram‐negative bacteria

Abstract Gram‐negative bacteria (GNB) emerge as important pathogens causing pulmonary infection, which can develop into sepsis due to bacterial resistance to antibiotics. GNB pneumonia poses a huge social and economic burden all over the world. During GNB infection in the lung, Toll‐like receptor 4 (TLR4) can form a complex with MD2 and CD14 after recognizing lipopolysaccharide of GNB, initiate the MyD88‐ and TRIF‐dependent signalling pathways and stimulate host non‐specific immune response. In this review, we summarize recent progress in our understanding of the role of TLR4 in GNB pneumonia. The latest experimental results, especially in TLR4 knockout animals, suggest a promising potential of targeting TLR4 signalling pathway for the treatment of GNB pneumonia. Furthermore, we highlight the benefits of Traditional Chinese Medicine as novel candidates for the therapy of GNB pneumonia due to the modulation of TLR4 signalling pathway. Finally, we discuss the promise and challenge in the development of TLR4‐based drugs for GNB pneumonia.


| INTRODUC TI ON
Streptococcus pneumoniae is a main cause of pneumonia, followed by other Gram-positive bacteria such as Staphylococcus aureus and Bacillus anthracis. However, recent epidemiological study showed increasing incidence of pneumonia caused by Gram-negative bacteria (GNB), and the most common is Pseudomonas aeruginosa, followed by Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, Bordetella pertussis and Moraxella catarrhalis. Pneumonia caused by GNB is difficult to treat due to their antibiotic-resistant characteristic. For example, P aeruginosa showed the resistance to most antimicrobials including ceftazidime, meropenem and piperacillin/tazobactam. K pneumoniae showed high rate of beta-lactam resistance, including resistance to third-generation cephalosporins and carbapenems. 1 Moreover, pneumonia can lead to sepsis in immunocompromised hosts, which remains one of the major causes of death. The incidence and mortality rate of sepsis keep rising worldwide, especially in low-and middle-income countries. 2 Toll-like receptor 4 (TLR4) can identify exogenous pathogens by binding to lipopolysaccharide (LPS) of GNB, stimulate the production of antimicrobial peptides and induce the nonspecific immune responses such as the activation of nuclear factor-kB (NF-kB) pathway in the macrophage. 3 The activation of TLR4   4 In the new era of antibiotic-resistant bacteria, 5 it is necessary to explore TCM for the treatment of GNB pneumonia based on the pivot role of TLR4 in infectious pneumonia.

| G NB PNEUMONIA
Antibiotic-resistant GNB infections become the leading causes of death caused by infectious pneumonia. Especially, uncontrolled inflammatory response to GNB infection is associated with high morbidity and mortality, which can turn pneumonia into sepsis due to the antibiotic resistance.
Pneumonia resulting from GNB is a leading cause of mortality and morbidity with a rise in the prevalence of early-onset ventilator-associated and community-acquired pneumonia. It was reported that bacterial antibiotic resistance could cause more than 25 000 deaths every year in Europe. 6 In China, the prevalence of infectious diseases has been increasing yearly. From 2014 to 2016, the number of bacteria isolated from clinical cases, especially antibiotic-resistant GNB, has kept increasing ( Figure 1). [7][8][9] Given the emergence of antibiotic-resistant bacteria, it is urgent to develop new strategies to treat pneumonia by antibiotic-resistant GNB. 10-12

| LPS AC TIVATE S TLR4 S I G NALLING
Toll-like receptor 4 plays a crucial role in mediating innate immune responses to infections in pneumonia, especially to GNB infection.

| Structure of tlr4
Toll-like receptor 4 is the first identified member of TLR family. 15 TLR4 is a transmembrane protein characterized by an extracellular domain containing leucine-rich repeats (LRRs) where the MD-2 molecule is associated, and a cytoplasmic tail harbouring a conserved region known as Toll/IL-1 receptor (TIR) domain. 16 The extracellular domain is responsible for ligand binding, receptor dimerization and initiation of intracellular signalling, whereas the intracellular domain shares a significant sequence and structural homology with the interleukin-1 receptor (IL-1R) family. 16

| Recognition of lps by tlr4
The complex crystal structure helps explain why LPS structural properties are ideal for TLR4 signalling activation. LPS has six lipid chains, five of which are completely submerged inside the pocket in MD-2, whereas the sixth chain is exposed to the sur- TcpB, a TIR domain-containing protein produced by Brucella melitensis, can interact with MAL, MyD88 and TLR4. However, no interaction is observed between TcpB and TRAM or IRAK-2, demonstrating that MAL-dependent signalling pathway is the specific target for TcpB. Furthermore, competition assays demonstrated that TcpB can disrupt TLR4-MAL interaction, but could not interfere with MAL-MyD88 interaction. 21 These results provide new insights into the mechanism of immunomodulation of TLR signalling by bacterial TIR domain-containing protein at the structural level.
TcpB mimics MAL properties to bind to phosphoinositides in the plasma membrane through its N-terminal domain and competes F I G U R E 2 LPS/TLR4 signalling pathway. LPS binds to LBP and forms a complex with MD-2/TLR4. Intracellular LPS/TLR4 signalling can be transduced through either MyD88-dependent or MyD88-independent TRIF/ TRAM pathways, which mediate the transactivation of pro-inflammatory cytokine with MAL for the interaction with MyD88. 16 However, Sengupta et al demonstrated that TcpB interacts with MAL but not with MyD88, and TcpB does not interfere with the interaction between MAL and MyD88. 22 Site-directed mutagenesis analysis showed that residues Glu 183 , Ser 244 and Arg 288 in the TIR domain are required for MyD88 homo-dimerization, and mutations on these residues suppress the recruitment of IRAK1/4 and NF-B activation. Importantly, overexpression of MyD88 mini-proteins comprising the Glu183 residue competes with the homo-dimerization of endogenous MyD88 protein and impairs TLR signalling in immune cells. 23  Interestingly, a study showed that LPS pre-conditioning redirected TLR4 singling via TRIF-IRF3 pathway but not MyD88 pathway. 27 The suppression of NF-κB activity is repressed in LPS pre-conditioning mice, whereas the production of pro-inflammatory cytokine does not vary, suggesting that other signalling cascades are involved in the production of pro-inflammatory cytokine.

| THE ROLE OF TLR4 IN PNEUMONIA
It was reported that TLR4 expression at mRNA and protein levels significantly increased at around 2 to 6 hours after intracerebral haemorrhage, peaked at day 3, declined at day 5, while remained elevated compared with baseline even on day 7. 28 In another study, exposure to LPS can enhance TLR4 mRNA expression after 1 hour in autologous human alveolar macrophages and monocytes, with a subsequent decrease in TLR4 mRNA level after 24 hours, 29  It was reported that TLR4/MyD88-triggered myeloid-derived suppressor cells (MDSCs) help resolve inflammation during bacterial pneumonia. 33 The cytoadherence of M pneumoniae induces inflammatory responses in macrophages through TLR4, and the response can be inhibited by TLR4 inhibitor VIPER. 34 These results were confirmed in TLR2 and TLR4 double-knockout (DKO) mice. 34 In addition, TLR2/4 DKO mice were more susceptible to K pneumoniae infection than single TLR2 KO or TLR4 KO mice, suggesting that TLR2 and TLR4 play cooperative role in lung innate immune responses during K pneumoniae infection. 35 However, B pseudomallei LPS signalling occurs solely through TLR4 in murine, whereas TLR2 plays an additional role in human. 36 The patients who develop post-operative pneumonia show a trend of significant reduction in TLR4 expression compared with those without pneumonia. Similarly, TLR4-deficient mice had impaired survival with higher bacterial loads and diminished production of inflammatory mediators, indicating that TLR4 signalling is required to induce a protective immune response to common GNB. 37 Based on these reports, the dual role of TLR4 in infectious pneumonia seems to be managed by the inhibitory or stimulatory factors for a balance. Nevertheless, TLR4 remains a potential target for inhibiting undesired inflammatory responses.

| TCM TARG E TING TLR4 A S P OTENTIAL DRUG FOR PNEUMONIA
Due to the challenge from antibiotic-resistant bacteria, it is very urgent for us to explore new treatment of pneumonia with TCM which will not cause antibiotic resistance. At present, various promising synthetic and plant-derived strategies are being tested. Houttuynia can affect TLR4 expression directly or indirectly. 38 Emodin inhibits influenza viral pneumonia, by inhibiting IAV-induced activation of TLR4, MAPK and NF-kB pathways and activating Nrf2 signalling. 39 Ugonin M is a unique flavonoid isolated from Helminthostachys zeylanica, and it suppresses the production of pro-inflammatory molecules such as nitric oxide, IL-1, TNF-α and IL-6. Moreover, Ugonin M inhibits not only NF-κB and MAPK activation but also TLR4 protein expression. 4

| VALIDATI ON OF TLR4 A S A PROMIS ING THER APEUTI C TARG E T FOR G NB PNEUMONIA
In recent years, with the development of genomics, structure biology and bioinformatics, increasing evidence validates that TLR4 is crucially involved in the pathogenesis of GNB pneumonia and emerges as a promising therapeutic target for GNB pneumonia.
A meta-analysis showed that TLR4 A299G polymorphism was significantly associated with the susceptibility to pneumonia. 43

| CON CLUS I ON S AND PER S PEC TIVE S
Based on all the above discussion, it is undoubted that TLR4 plays an important role in GNB pneumonia. TLR4 signalling pathway involved in lung inflammatory injury is composed of ligands, TLR4 itself, receptor and its downstream pathways, including adaptor proteins MyD88 and TRIF and transcription factors such as NF-κB. 48,49 Therefore, TLR4 ligands and receptors are all promising targets for developing effective treatment of pneumonia. A variety of TLR4 antagonists has been identified (summarized in Table 1). Future studies are needed to investigate the interaction between TLR4 and these molecules in the initiation and development of GNB pneumonia.
Traditional Chinese Medicine has emerged as a novel approach to control GNB pneumonia due to the advantages of not causing antibiotic resistance. Recent studies have shown that TCM could target TLR4 signalling to achieve the efficacy to ameliorate or even eradicate GNB pneumonia. Therefore, it is important to explore TCM to develop new therapeutic intervention for pneumonia. 50 However, it is important to notice that TLR4 signalling is involved in a variety of physiological and pathological processes in human body, and thus, the specificity of TLR4-targeted therapy may be the biggest challenge in the clinical. Recent advances in crystal structure analysis of TLR4 and the development of better drug design tools may help address the challenge.
In summary, GNB pneumonia remains a serious threat to human health for the coming years. Appropriate screening and optimization of TLR4 agonists and antagonists, especially from TCM, would provide future therapeutics for GNB pneumonia.

ACK N OWLED G EM ENTS
We apologize to the researchers in the field whose publications are not referenced due to space limitation.

CO N FLI C T O F I NTE R E S T S
All authors report no potential conflicts.

AUTH O R CO NTR I B UTI O N
Junying Ding wrote the manuscript and Qingquan Liu proof the manuscript.