Source: International Respiratory Journal, Issue 02, 2021
Author: Liang Sicong Chen Yu
Department of Respiratory and Critical Care Medicine, Shengjing Hospital, China Medical University, Shenyang 110004
Corresponding Author: Chen Yu
Email：[email protected]
summary
Legionella infection can involve the lower respiratory tract causing Legionella pneumonia, progressing rapidly and with a high case fatality rate. Legionella enters the human body mainly parasitizes alveolar epithelial macrophages for proliferation, Legionella regulates the immune response and signaling of host cells through effector proteins, and disguises as host cell proteins to escape the killing effect, causing damage to the lungs. In this paper, we will review the research progress of the mechanism and regulatory role of lung injury caused by Legionella infection from various aspects.
Legionella is an aerobic gram-negative bacillus that is widely distributed in warm, moist environments and can grow and multiply in natural water sources, artificial cold water, hot water systems, and moist soils. Legionella was first discovered in 1976, during an outbreak of pneumonia of unknown cause during the annual meeting of veterans in Philadelphia, USA, and the causative pathogen was isolated in autopsy organizations and named Legionella. Legionella is a facultative intracellular parasite, and there are currently 58 species and 3 subspecies of Legionella found, all of which can parasitize in aquatic protozoa, and about 30 species can cause human infection. Humans are infected by inhaling contaminated aerosols. 90% of cases are caused by Legionella pneumophila[1], which can be divided into 15 serotypes. The most common pathogenic serotype in the United States and Europe is type 1, accounting for 80% of the total cases; while it is reported that only 50% of the legionnaires' disease cases in Australia and New Zealand are caused by Legionella pneumophila type 1 and 30% of Legionella long beach[2]; China currently lacks large-scale research data on the distribution of pathogenic Legionella serotypes. Legionella's ability to ingest bare DNA and, as a result, undergoes gene transfer and evolution, allowing Legionella to adapt quickly to its environment and evolve different coping strategies.
Human infection with Legionella can be divided into Legionella pneumonia and Pontiac fever according to clinical manifestations. Pontiac fever is characterized by an acute influenza syndrome with no significant lung lesions and self-healing within 3 to 5 days. Legionella pneumonia presents as acute fibrinated pyogenic pneumonia with a lobular distribution that progresses rapidly and can easily develop into fatal Legionella infection. Legionella pneumonia progresses rapidly, is limited by clinical testing methods, and has a high case fatality rate. Twenty-nine European countries collected confirmed cases of Legionnaires' disease reported from 2011 to 2015, with a case fatality rate of 9.3% [3]. The severity of Legionella infection is related to the amount of bacteria and the degree of immunity of the infected host [4]. Risk factors for Legionella infection include men, older people, smokers, people with chronic cardiopulmonary underlying disease, diabetes mellitus, malignancy, immunosuppression, and the use of tumor necrosis factor α antagonists [5]. Due to the large individual differences after Legionella infection, it is necessary to understand the lung damage caused by Legionella infection, and this paper will review the regulatory mechanism of Legionella lung damage after infection.
1 Legionella infection macrophage mechanism
Legionella has evolved in the environment the ability to survive in protozoan cells, a trait that also allows Legionella to survive and replicate in mammalian macrophages, which play a crucial role in the initial stages of host infection with Legionella, determining whether Legionnaires' disease occurs or not. After Legionella reaches the respiratory tract, it first enters the cell due to the phagocytosis of alveolar macrophages, and the body's complement system can enhance the role of Legionella into the cell, and its main outer membrane protein can directly activate complement C3, increasing its success rate of being engulfed by macrophages [6]. Once the bacteria enter the cell, they produce a membrane-encased well-defined vesicle called Legionella-containing vacuole (LCV), which is a complex process that requires the assistance of the type 4 secretion system (T4SS) to form LCV. Current studies have shown that effector proteins from the Arf[7], Rab[8], Ran[9] and Rap[10] families are associated with LCV formation and legionella replication. Due to the action of T4SS, Legionella releases more than 300 effector proteins into host cells[11], which are released into the cytoplasm of host cells, where the endoplasmic reticulum and mitochondria of the recruiting cells wrap around the LCV and become structures similar to the rough-faced endoplasmic reticulum, after which the LCV is tightly and continuously together with the endoplasmic reticulum [12]. SidM (DrrA) mediates Rab1 activation, thereby mediating endoplasmic reticulum and membrane-derived vesicles to LCV recruitment[13]. Phosphatidyl inositol plays an important role in the formation of LCV. In live-cell imaging using Legionella pneumophila infection with Platycos pneumophila infection, it was observed that After being engulfed by cells, Legionella acquired phosphatidylinositol-3,4,5-triphosphate and phosphatidyl inositol-4-phosphate from the cell membrane after being engulfed by Legionella pneumophila, a process completed within 60 s after the bacteria were engulfed and did not require T4SS action; after 60 s, the phagosome was recruited abundant phosphatidyl inositol-3-phosphate; 2 h after infection, under the action of T4SS, The phosphatidylinositol-3-phosphate on the surface of the LCV gradually sheds, slowly recruiting phosphatidylinositol-4-phosphate, and the volume within the LCV begins to expand, surrounded by the endoplasmic reticulum; until 8 h after infection, the phosphatidylinositol-4-phosphate is undergoing stable accumulation, and the phosphatidyl inositol conversion is the decisive step in the LCV maturation process, regulated by T4SS, and the Icm-deficient mutants cannot be converted, thus being quickly degraded in host cells [14]. Legionella converts phosphatidylinositol through the following pathways: (1) effector proteins directly bind to membrane surface phospholipids (SidC, SidM, AnkX, LidA, RidL, SetA, LtpM) ;(2) Effector proteins act as bacterial phosphositol phosphatase (SidF, SidP), phosphositositol kinase (LepB, LegA5), phospholipase (VipD, PlcC, LpdA); (3) Host cells are recruited by effector proteins for phosphoinol phospholipase or kinase (RalF, SidM)[15]. Like other intracellular parasites, Icm/Dot's effector proteins develop the ability to mimic host proteins and interfere with the signaling and exchange pathways of host cells[16], preventing LCVs from binding to intracellular lysosomes, so that LCVs are not destroyed by lysosomes within macrophages, making them a relatively safe environment for Legionella to replicate internally. The replication of Legionella in vivo can be divided into 2 stages, the replication period and the exponential period. Legionella in the replicating phase is present in LCV without flagella, and the replication phase can last for 16 to 20 h, during which time LCV utilizes nutrients within the phagocyte cytoplasm itself, and the effector protein AnkB increases the content of free amino acids required by Legionella by inducing proteasome hydrolysis of host proteins [17]. Other studies have shown that the Lgt family works synergistically with the SidE family, releasing amino acids from host cells for Legionella utilization [18]. In order to maintain legionella replication levels within cells and prevent premature termination of the replication cycle, effector protein SdhA maintains LCV membrane stability and prevents disruption of LLV membranes within host cells [19]. In the later stages of infection, the outer membrane of LCV is gradually degraded, and Legionella is scattered in the cytoplasm of the host cell and on the outer membrane of different organelles[20], forming an elastic vesicle-like mature intracellular structure, and when the nutrients in the vesicle are exhausted, Legionella releases virulence factors that rupture the host cell. Legionella at this time, with flagella, is highly motility and pathogenic, escapes from the ruptured host cell, is engulfed by new macrophages, and opens the next round of infection.
2 Regulatory mechanisms within macrophages after Legionella infection
Whether Legionella develops into a serious infectious disease after infecting a human body is related to whether it can replicate in large quantities within cells. The use of legionella infection with mice can not replicate the legionella pneumonia model, the use of legionella infection of macrophages in mice, found that legionella can not complete the replication and survival of the cells, this is because the mice in the early stage of infection that effectively cleared the pathogen in the cells and prevented the occurrence of further infection. Legionella enters macrophages and replicates inside the vesicles, autophagy is an important part of immunity, cells recognize and engulf pathogens after initiating autophagy programs, limiting the replication of pathogens internally, but legionella has been found to regulate the autophagy of host cells through a variety of effector proteins. Choy et al. [21] found that the effector protein RavZ inhibits autophagy by dissociating Atg8/LC3 from phosphatidylethanolamine. The effector protein LpSpI acts on the synthesis of host cell sphingosine to reduce autophagy [16]. In the late autophagy stages, synaptic fusion protein 17 on autophagosomes mediates autophagosome fusion with lysosomals, and Legionella effector protein Lpg1137 binds and lyses synaptic fusion protein 17, preventing early steps in autophagy and apoptosis [22]. In recent years, it has been found that the Icm/Dot system inhibits intracellular signaling and regulates host cell immunity by inducing monounitination of the E2 enzyme UBE2N by substrate MavC (Lpg2147).
Flagella protein is the key to activating immunity after Legionella infects the body. After infecting the macrophages of mice with Legionella, the macrophages detect and recognize the flagella protein by Nlrc4 and Naip5, forming The Nap5/NLR4 inflammatory body, further activating caspase-1 within the cell, leading to the release of IL-1β and IL-18, initiating the death procedure of the cells. Toll-like receptor 5 (TLR5) also recognizes flagellins, activating the nuclear transcription factor κB pathway leading to cell death. Experiments such as Akhter [24] observed that this process is accompanied by the rupture of DNA in the nucleus, so this process is cytostatic death, limiting the replication and spread of Legionella in vivo. A / J mice in infection with Legionella can form a model similar to human Legionella pneumonia, the reason is that the Naip5 gene expression level defects in this kind of mice, can not cause cell recognition of flagellin, allowing Legionella to replicate in large quantities within the cell, experiments showed that the motility of flagella has no effect on the toxicity of Legionella, after knocking out the fla gene, Legionella flagella can not move, but can make Legionella evade the recognition mechanism of macrophages, in non-A / J mice macrophages for replication [25]。 Unlike mice, human macrophages allow Legionella to replicate in large quantities, which may stem from the fact that humans express only 1 NAIP gene (hNAIP)[26], which also activates the capase-1 pathway to restrict legionella growth by recognizing flagella proteins[27] and recognizes Prgl, a needle-like protein in the bacterium T3SS[28], but not as strongly as Nap5.
In addition to flagellins, legionella lipopolysaccharide (LPS) and dsDNA can also activate the formation of inflammatory bodies within cells to limit their survival within cells. Normally, human recognition of bacterial LPS is carried out by TLR4, but because of the long fatty acid chain of Legionella lipid A, which affects the recognition of TLR4, Legionella LPS is carried out by TLR2 [29]. LPS activates caspase-11 (caspase-4 in humans), a process that triggers the formation of holes and leads to the secretion of IL-1α [30]. Caspase-11/caspase-4 simultaneously promotes the binding of lysosomals to LCV to inhibit replication of pathogens [31]. But the mechanism by which LPS leaks into the host cytoplasm and activates caspase-11/caspase-4 remains unclear. Legionella dsDNA also activates the inflammatory body AIM2 within macrophages, which subsequently activates the caspase-1 pathway, leading to cytochromia [32].
3 Immunomodulatory mechanisms of the body after Legionella infection
After entering the human body, Legionella first activates the innate immune response, which produces a clearing effect on it. Innate immune cells such as monocytes, neutrophils, and natural killer cells all play an important role in this process. These cells recognize Legionella and infected macrophages through pattern recognition receptors and exert immune effects to clear them. Brown et al. [33] isolated and quantified lung inflammatory cells in mice infected with Legionella pneumonia, and the results suggested that after 1 day of infection, alveolar macrophages accounted for a small fraction of the total inflammatory cells, and by day 2, only 1% of the total number of inflammatory phagocytes, and 97% were monocyte-derived cells (MC) and neutrophils. Macrophages release IL-1α after engulfing Legionella, causing alveolar epithelial cells to secrete chemokines that induce neutrophils to recruit large numbers to the site of infection [34]. Another study also confirmed that 72 h after infection with Legionella pneumophila, mice had a massive outbreak of pro-inflammatory cytokines, with large numbers of neutrophils and monocytes pouring into the lungs [35].
Neutrophils play an important role in the early stages of scavenging Legionella in vivo, but the number of neutrophils decreases rapidly 48 h after infection [36]. On the one hand, neutrophils directly engulf Legionella through the action of reactive oxygen species, and on the other hand, the production of tumor necrosis factor (TNF) binds to the tumor necrosis factor receptor (TNFR) on the surface of macrophages, so that macrophage lysosomes bind to LCV to inhibit the replication of Legionella [37].
After being recruited to the foci of infection, monocytes differentiate into MCs in situ, producing IL-12 to promote bacterial clearance, and then stimulate natural killer cells and local memory T cells, natural killer T cells, and γδT cells to produce interferon γ (interferon-γ, IFN-γ), enhancing mc killing of bacteria. After infection progresses to the third day, the number of MC is significantly increased, becoming the main force for the removal of pathogens, and the MC kills the bacteria directly through the stimulation of IFN-γ[38], which stays in the lungs longer than neutrophils, and can reactivate antigen-specific T cells and induce cytokine secretion [39].
Natural killer cells are the main source of IFN-γ produced after infection with Legionella, which is recognized by TLR after infection with Legionella, and activates natural killer cells through the MyD88-dependent pathway to produce a large number of IFN-γ [40], mediating the defense effect against Legionella. Mice lacking MyD88 signaling are unable to control legionella replication in vivo and can cause the spread of bacteria to the liver and spleen [41]. Neutrophils activate caspase-1 and release IL-18 during the clearance of Legionella, which also plays an important role in the activation of natural killer cells [42].
Although macrophages have a limited immune role in the innate immune process caused by Legionella infection, and infected phagocytes produce low levels of TNF and IL-12 due to the action of Legionella effector proteins, experiments such as Copenhaver [43] have demonstrated that macrophages (bystander cells) that do not engulf Legionella infection can play a strong synergistic role with infected macrophages during Legionella infection. Infected macrophages produce IL-1α and IL-1β acting on the IL-1R of the spectator cell, causing the bystander cells to release IL-6, TNF and IL-12 in large quantities, thereby producing an immune effect on Legionella.
Due to the decisive role of innate immunity during Legionella infection, there are currently few studies of adaptive immunity during Legionella infection. Seven days before infection, CD4+ T cells and CTL cells enter lung tissue to participate in infection clearance, and mice that consume CD4+ T cells and CD8+ T cells using drugs die rapidly after infection with Legionella [44]. Legionella antigen-specific T cells are found in mediastinal lymph nodes after infection for 3 days, and on day 6, CD4+ T cells differentiate into Th1 and Th17 cells infiltrating into the lungs to secrete IFN-γ, IL-1, and IL-17 to assist in innate immunity [45].
B cells are also involved in adaptive immunity to Legionella infection, and LPS, membrane proteins, and heat shock proteins can stimulate B cells to produce specific IgG and IgA antibodies after Legionella infection [46]. Studies have shown that 7 to 10 days after infection, with the help of CD4 + T cells, the lungs produce a large number of memory B cells. However, it is doubtful whether specific antibodies against Legionella have a protective effect in vivo, and subcutaneous injection of Legionella heat shock protein 60 in mice can lead to the production of IgG, which acts as a protective effect in the first 48 hours of infection, but does not play a role in guinea pigs [40]. In another case, mice were immunized using recombinant type A flagellin (rFLA) and then a lethal dose of Legionella pneumophila intravenously, and 60 percent of the rFLA group was used, suggesting that rFLA could elicit an immune response in mice [47], but whether further applications can be tested remains to be tested.
4 Principles of lung damage caused by Legionella pneumonia
The clinical symptoms of Legionella pneumonia lack specificity, but are often accompanied by extrapulmonary manifestations such as gastrointestinal symptoms and central nervous system symptoms. The pathology of the acute phase of Legionella infection is fibrinous purulent pneumonia and acute diffuse alveolar injury, often scattered in small abscesses, later lung exudates and hyaluronic membrane mechanization and interstitial fibrosis. Pulmonary vascular muscular arteries often present with non-necrotizing vasculitis changes, and about one-third of patients involve the pleura with serous, serous fibrinous, or purulent pleurisy changes [48]. Modeling Legionella pneumonia in mice found interstitial inflammation, massive cell infiltration, granuloma formation, and perivascular lymphocyte ligation in mouse lung tissue, and severe alveolar wall necrosis 48 h after infection [49].
In the course of the immune response, the release of Legionella's own virulence factors, cytokines, and white blood cell products causes the occurrence of acute inflammation. Legionella has antigens typical of gram-negative bacteria, including LPS, flagella protein, heat shock protein, outer membrane protein, etc., and can spread lymphoidally and hematogenously to extrapulmonary organs, causing multisystem symptoms. Usually LPS is the main cause of pulmonary fiber exudation, but Legionella LPS is low endotoxin and hypopyrogenic, compared to other gram-negative bacteria, the fatty acid chain of Legionella LPS is too long, using Legionella's LPS to treat cells, the concentration of LPS required by cells to produce inflammatory factors is 1 000 times that of highly pathogenic E. coli [50], and Legionella LPS can not be combined with the LPS receptor CD14 of monocytes, so it can be recognized by TLR2 in humans, However, it manifests as low toxicity. PilY1 is a protein exposed to the surface of Legionella and is highly homologous to PilY1 on the surface of Pseudomonas aeruginosa.[51] Hoppe et al. [52] found that it is closely related to the virulence of Legionella, which can effectively increase the adhesion of Legionella to the cell surface, enhance the motility of flagella and the formation of biofilms. Increased capillary permeability in the lungs after Legionella infection, damage to the junction between vascular endothelial cells and vascular endothelial cells, and inflammation lead to the formation of pulmonary edema [53].
The body's innate immunity produces inflammatory cytokine storms and inflammatory cell infiltration when it clears Legionella [54]. Neutrophils, etc. undergo incomplete phagocytosis and disruption of lysosomal leakage into tissues [55]. The lysosome contains acid hydrolases, neutral proteases and lysozymes, which can degrade extracellular components such as collagen fibers, basement membranes, cellulose, elastin and other extracellular components, and play an important role in the tissue destruction of purulent inflammation. Intrinsic immunity causes tissue damage during the removal of pathogens, and regulatory T cells suppress the damaging inflammatory response by secreting IL-10 and transforming growth factor β [56].
During the establishment of a mouse Legionella pneumonia model, a significant increase in the number of infiltrative macrophages was observed after 6 d of infection, and the formation of granulomatous tissue around the blood vessels was aggravated. Friedman et al. [57] believe that the secretion of IL-2, TNF, etc. after infection induces the immune response of Th1 cells, activates macrophages in the late stage of immunity, and causes mechanized pneumonia in the late stage of infection. However, the specific mechanism of pulmonary fibrosis caused by Legionella infection is still unclear. Elevated hiF-1α levels during Mycobacterium tuberculosis infection have been found to be associated with the formation of granulation tissue [58], but further research is needed as to whether this regulatory form is also present in Legionella infection.
5 Other regulatory mechanisms in Legionella infection
There are individual differences in the performance of Legionella after infection with the human body, and some healthy people do not develop severe pneumonia after infection with Legionella, but manifest as self-limiting Pontiac fever. Current clinical studies suggest that patients with risk factors are more susceptible to severe Legionnaires' disease. A survey of Legionnaires' disease in Europe from 2011 to 2015 showed a higher incidence of Legionnaires' disease in Europe and an increase in incidence with age, which may be directly related to smoking habits in older men [3]. Smoking is thought to increase lung sensitivity to bacterial infections and affect macrophage function [59], but the specific mechanism of effect remains unclear.
The severity of Legionella pneumonia depends explicitly on the amount of bacteria inhaled by the host and the host's own immune status, and patients with a weakened immune status are more susceptible to severe Legionnaires' disease [60]. Cytomegalovirus infection, chemotherapy for malignancy, and immunosuppressants after organ transplantation are all high risk factors for legionella infection, and treatment must be extended or combined therapy when anti-infective drugs are administered [61]. In one mouse trial, immunosuppressive states due to sepsis were susceptible to secondary Legionella infection and were more susceptible to blood transport to the spleen and liver to form abscesses, which was associated with decreased proliferation of CD4+ T cells [62]. Lanternier et al. [63] confirmed that the prevalence of Legionella pneumonia in patients clinically treated with TNF-α inhibitors was significantly increased. Deficiency of TNF and TNFR can seriously affect the course of Legionella pneumonia [64], and legionella infection in TNFR knockout mice is more severe and has a higher mortality rate than in wild-type mice, and TNFR deficiency leads to neutrophil recruitment to the lungs and prevents bacteria from being removed [65]. The case fatality rate may be as high as 19 percent in patients with Legionella pneumonia in immune compromise and 2.5 to 6 percent in patients with nonimpressed Legionella pneumonia [66]. Therefore, for patients with immune compromise, a combination of anti-infective drugs is currently recommended for a course of at least 3 weeks. Because Legionella is an intracellular pathogen, its response time to anti-infective drugs is long, and too short treatment regimens may lead to recurrence of Legionella pneumonia [4].
Patients with clinically found Legionella infection can have multiple infections with other bacteria at the same time, resulting in treatment failure. Mizrahi et al. [67] used second-generation sequencing technology to analyze the sputum of patients diagnosed with Legionella infection with pathogen genes, and other pathogens such as Streptococcus pneumoniae and Acinetobacter were detected, and the composition of the co-infected bacteria was different according to the proportion of Legionella detected. Differences in susceptibility to Legionella have been suggested to be associated with differences in the composition of the pulmonary microbiota [68]. A study of respiratory specimen pathogen sequence detection during treatment in patients with Legionella pneumonia showed that the diversity of lung microorganisms in patients with incurable Legionella pneumonia decreased significantly compared with healthy patients, and sequencing showed that patients were not sensitive to treatment because Legionella developed resistance genes, which may be due to changes in catecholamine levels and inflammatory factors in the lung bubbles during lung infection that favored the growth of specific flora, including streptococcus, increasing the time and difficulty of treatment for Legionella pneumonia [69]. ]。 However, the specific mechanism still needs to be further studied and explained.
Studies in animal models have shown that Legionella behaves very differently in different mouse strains infected, depending on the different gene expressions of legionella flagella recognition receptors in mice. Therefore, some teams have speculated whether the individual differential performance of humans infected with Legionella is also due to genetic diversity. Hawn et al. [70] conducted a genomic analysis of patients in an outbreak of Legionella pneumonia in the Netherlands suggested that humans with TLR5 stop code heterozygotes were more likely to develop severe Legionella pneumonia. Different strains of Legionella also differ in the performance of infection with mammalian macrophages [71], and the survival results of Legionella pneumophila strains of different serotypes (clinical strains, environmental strains) in the same mouse-derived macrophages are abnormal, and the diversity of Legionella genes is also one of the factors leading to different outcomes of host infection. A recent study sequenced Legionella genes for potentially effective drug targets, analyzing 4,358 proteins of Legionella, 18 of which were selected as specific drug targets, and 11 of which were suitable for the development of narrow-spectrum antibiotics, but drug development needs to be further studied [72].
Infections with Legionella in humans are usually due to inhalation of aerosols contaminated with Legionella, but Legionella is a facultative endocytositial parasitic bacterium that can parasitize aquatic protozoa in environmental water, and parasitism in amoeba may lead to increased virulence of Legionella. Brieland et al. [73] found that inhaled elevated levels of inflammatory factors released by lung tissues of mice containing Legionella amoeba were introduced compared to mice infected with Legionella alone. Legionella growing inside Amoeba is more virulent than Legionella growing on culture media and exhibits greater replication in macrophages .[74] Other studies have shown that mutant strains of Legionella that have lost their virulence are restored to the lungs of mice when inoculated with amoeba [75]. Although rare, there have been clinical reports of lung amoeba infection in immunosuppressed patients [76], and this co-infection requires vigilance.
6 Summary and Outlook
The high mortality rate of Legionella pneumonia and the presence of underlying diseases and immunosuppression further increase the chance of Legionella infection, but the specific causes and interactions are unclear. Fully understanding and understanding the causes of lung damage caused by Legionella and its regulatory mechanism are of great help to find factors that can improve Legionella lung damage in the study, and it is hoped that through the study of this, the clinical difficulties in the treatment of Legionella pneumonia can be solved, and new ideas for the treatment of Legionella pneumonia can be proposed.
Conflicts of Interest All authors declare that there is no conflict of interest
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