These authors contributed equally to this work.
CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia
Article first published online: 16 JUL 2008
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd
Volume 10, Issue 11, pages 2247–2256, November 2008
How to Cite
Lim, J. H., Ha, U.-H., Woo, C.-H., Xu, H. and Li, J.-D. (2008), CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia. Cellular Microbiology, 10: 2247–2256. doi: 10.1111/j.1462-5822.2008.01204.x
- Issue published online: 13 OCT 2008
- Article first published online: 16 JUL 2008
- Received 15 February, 2008; revised 1 July, 2008; accepted 8 July, 2008.
Bacteraemic pneumonia is a common cause of sepsis in critically ill patients today and is characterized by dysregulation of inflammation. The genetic factors predisposing to bacteraemic pneumonia are not yet fully understood. Innate immunity is pivotal for host defence against invading bacteria, and nuclear factor-kappa B (NF-κB) is central to bacteria-induced inflammation and immune responses. The deubiquitinating enzyme CYLD has been identified as a key negative regulator for NF-κB. In the present study, we investigated the role of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli inoculation, Cyld−/− mice were hypersusceptible to E. coli pneumonia with higher mortality. Innate immune response to E. coli was enhanced in Cyld−/− cells and mice. Cyld−/− cells exhibited enhanced NF-κB activation upon E. coli inoculation, and the enhanced NF-κB activation by E. coli was abolished by perturbing IκB kinase (IKK) signalling. Furthermore, IKK inhibitor rescued Cyld−/− mice from lethal infection during E. coli pneumonia along with reduced inflammation. Taken together, these data showed that CYLD acts as a crucial negative regulator for E. coli pneumonia by negatively regulating NF-κB. These findings provide novel insight into the regulation of bacteraemic pneumonia and related diseases and may help develop novel therapeutic strategies for these diseases.
The innate immune system is the first line of host defence for sensing and eliminating invading microorganisms (Cohen, 2002), and the surface epithelial cells acts as the first line of host defence against pathogenic bacteria. They recognize the invading bacteria via Toll-like receptors (TLRs). Recognition of pathogen-associated molecular patterns (PAMPs) of microorganisms by TLRs, in turn, results in activation of the cells, leading to a plethora of biological responses required for shaping both the innate and adaptive arms of the immune response (Janeway and Medzhitov, 2002). Binding of the PAMPs to TLRs activates downstream signalling pathways and results in activation of transcription factor nuclear factor-kappa B (NF-κB) (Kawai et al., 1999; 2001). The activation of NF-κB orchestrates a gene expression programme that underpins the epithelial cell-dependent immune response. Thus, NF-κB acts as a crucial regulator of inflammatory genes, including cytokines, chemokines, inflammatory enzymes, adhesion molecules and others (Barnes and Karin, 1997). Although the inflammatory response triggered by bacteria is essential for eradicating bacterial pathogen, excessive inflammatory response is clearly detrimental to the host, due to severe tissue damage (Ulevitch and Tobias, 1995; Kurt-Jones et al., 2004). To avoid overactive and detrimental inflammatory response in infectious disease, the bacteria-induced inflammatory response must be tightly regulated.
The incidence of sepsis/septic shock in the USA ranges from 400 000 to 750 000 cases per year, and mortality due to sepsis is approximately 30% (Angus et al., 2001; Cohen, 2002). Moreover, the incidence of septic shock is worryingly on the raise, in part as a result of the growing number of subjects immunocompromised because of medical interventions such as organ transplantation or chemotherapy for cancer, or simply because in a globally ageing population the number of people with weaker immune systems has expanded (Edgeworth et al., 1999; Martino et al., 1999). Moreover, treatment of septic shock relies largely on intravenous administration of antibiotics, but under some circumstances these conventional therapies may promote further release of endotoxins from the cell envelope of killed bacteria, exacerbating septic shock itself (Byl et al., 2001). For this reason, the quest for novel methods able to resolve the symptoms is actively pursued. Pneumonia is a common cause of sepsis in critically ill patients today (Marrie et al., 1998). Especially, bacteraemic pneumonia is a highly specified subgroup of pneumonia that is potentially life-threatening and is characterized by dysregulation of inflammation following primarily bacterial infection. Escherichia coli is the second most common cause of bacteraemic pneumonia, and pneumonia due to E. coli has a reported mortality of up to 70% and often results in death because of sepsis and septic shock (Packham and Sorrell, 1981; Angus et al., 2001). Despite tremendous effort put into improving survival, bacteraemic pneumonia by E. coli remains a major cause of morbidity and mortality in patients hospitalized in intensive care units worldwide (Diekema et al., 1999; Angus et al., 2001; Bochud and Calandra, 2003).
Cylindromatosis (CYLD) was originally identified as a tumour suppressor, loss of which causes a benign human syndrome CYLD (Bignell et al., 2000). Recent studies have indicated that CYLD, a member of the deubiquitinating enzyme family that specifically digests polyubiquitin chains, deubiquitinates TRAF2, TRAF6 and TRAF7 and acts as a negative regulator for activation of NF-κB by tumour necrosis factor receptor (TNFR) and TLR (Brummelkamp et al., 2003; Kovalenko et al., 2003; Trompouki et al., 2003; Yoshida et al., 2005; Lim et al., 2007a). CYLD also negatively regulates activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK (Reiley et al., 2004; 2007; Yoshida et al., 2005; Zhang et al., 2006; Lim et al., 2007a,b; Sakai et al., 2007) both in vitro and in vivo. In addition, CYLD negatively regulates antiviral receptor TLR7 upon bacterial infection, thus playing an important role not only in the bacterial infection, but also in the viral infection (Sakai et al., 2007). Moreover, the expression of CYLD is itself under the control of NF-κB (Jono et al., 2004; Yoshida et al., 2005), suggesting that CYLD is involved in a negative feedback regulation of NF-κB activation and NF-κB-dependent gene expression.
Given the fact that NF-κB is crucial for regulating host immune and inflammatory response in bacteria infection, particularly in Gram-negative bacterial pneumonia and CYLD is an important regulator of NF-κB in bacterial infection, it is logical to hypothesize that CYLD may act as a negative regulator for innate immure response against invading bacteria during E. coli pneumonia. In the present study, we show that CYLD indeed acts as a crucial negative regulator for lethal infection in E. coli pneumonia. These studies bring new insights into the regulation of E. coli pneumonia and related diseases such as severe sepsis and septic shock and may open up new avenues for treating these diseases.
Cyld−/− mice are hypersusceptible to E. coli pneumonia
Escherichia coli is the second most common cause of bacteraemic pneumonia. Although LPS, E. coli endotoxin, has been commonly used to study the underlying molecular mechanisms of the pathogenesis of this disease, study using live bacteria offers many advantages to present natural bacteraemic pneumonia, sepsis and septic shock in human patient. Moreover, animal models of live E. coli pneumonia have been successfully established and used by many research groups to study these diseases (Mizgerd et al., 2000; Ong et al., 2003; Jeyaseelan et al., 2005). In order to investigate the role of CYLD in E. coli pneumonia and related diseases, mouse model of E. coli pneumonia was induced as previously reported (Ong et al., 2003). Wild-type (WT) and Cyld−/− mice were intratracheally (i.t.) inoculated with either lethal dose [1 × 107 colony-forming units (cfu) per mouse] or sublethal dose (5 × 106 cfu per mouse) of E. coli, and survival was monitored for 6 days after E. coli inoculation. In lethal dose of E. coli (1 × 107 cfu per mouse) inoculation, WT mice started to die within 1 day after inoculation and resulted in 80% of mortality in 6 days. However, Cyld−/− mice inoculated with E. coli exhibited 90% lethality within 1 day, and all E. coli-inoculated Cyld−/− mice died within 3 days (100% lethality in 3 days) (Fig. 1A). Moreover, in sublethal dose inoculation (5 × 106 cfu per mouse) experiments, no death was found in WT mice by the end of the experiments. In contrast, E. coli-inoculated Cyld−/− mice started to die within 1 day, and 70% of E. coli-inoculated Cyld−/− mice died within 4 days (70% mortality in 4 days) (Fig. 1B).
To evaluate the physiological changes during E. coli pneumonia, body weight and rectal body temperature, two major physiological indicators of the severity of disease, were measured prior to and every 24 h after 5 × 106 cfu of E. coli inoculation for 6 days. Upon E. coli inoculation, WT mice showed a slight body weight loss compared with control inoculated WT mice, and became fully recovered within 4 days. However, the body weight loss was significantly enhanced in E. coli-inoculated Cyld−/− mice compared with E. coli-inoculated WT mice, and failed to recover by the end of the experiments (Fig. 1C). In addition, severe hypothermia was also found in E. coli-inoculated Cyld−/− mice, but not in WT mice (Fig. 1D). Taken together, these data indicate that CYLD deficiency results in hypersusceptibility to E. coli pneumonia in vivo.
E. coli-induced innate immune response is enhanced in lungs of Cyld−/− mice in vivo
Because CYLD is critical for the survival during lethal E. coli pneumonia and dysregulated innate immune response is known to contribute significantly to the mortality in bacteraemic pneumonia, the effects of CYLD deficiency on the innate immune response to E. coli pneumonia were investigated. WT and Cyld−/− mice were i.t. inoculated with 5 × 106 cfu of E. coli, and pathological changes and mRNA expressions of innate immune mediators such as IL-1β and IL-6 were measured by histopathological analysis and real-time quantitative PCR (Q-PCR) analysis respectively. Upon i.t. inoculation of E. coli, polymorphonuclear leukocytes (PMN) infiltration and migration into the perivascular, peribronchial and alveolar spaces were observed in lungs of WT mice, and E. coli-inoculated Cyld−/− mice exhibited greater enhancement of PMN infiltration and migration compared with WT mice (Fig. 2A and B). Consistent with the histopathological findings in lung tissues of E. coli-inoculated mice, mRNA expressions of IL-1β and IL-6 from the lungs of E. coli-inoculated mice were enhanced in Cyld−/− mice (Fig. 2C). Collectively, these results suggest that CYLD is a crucial negative regulator of innate immune response to E. coli in vivo.
CYLD deficiency results in enhanced NF-κB activation upon E. coli inoculation in Cyld−/− cells in vitro
Given the fact that innate immune response to E. coli is enhanced in Cyld−/− mice and CYLD is a key regulator of NF-κB signalling, we investigated whether CYLD deficiency enhances innate immure response to E. coli in Cyld−/− mice by enhancing NF-κB activity. Mouse embryonic fibroblasts (MEFs) from WT and Cyld−/− mice were treated with E. coli at the concentration of 100 multiplicity of infection (moi), and the levels of phosphorylation and subsequent degradation of IκBα, an important regulator of NF-κB activation, were then measured by immunoblot analysis. Upon E. coli inoculation, cells from Cyld−/− mice showed accelerated and enhanced phosphorylation and degradation of IκBα compared with those from WT mice (Fig. 3A). Furthermore, E. coli-induced NF-κB promoter activity was greatly enhanced in Cyld−/− cells (Fig. 3B) along with enhanced upregulation of NF-κB-dependent genes IL-1β and IL-6 (Fig. 3C). Thus, these findings suggest that CYLD acts as a critical regulator of innate immune response to E. coli through inhibition of NF-κB activation in vitro.
Cyld−/− mice exhibited enhanced innate immune response to E. coli through enhanced NF-κB activation in vivo
To further confirm whether enhanced innate immune response to E. coli infection in Cyld−/− mice is due to the enhanced NF-κB activation in vivo, we investigated the effects of IκB kinase (IKK) inhibitor Wedelolactone on innate immune response in vivo. We first evaluated the inhibitory effect of Wedelolactone on E. coli-induced NF-κB activation in human alveolar epithelial cells A549. A549 cells were transiently transfected with NF-κB luciferase reporter gene followed by treatment with E. coli at the concentrations of 10 and 100 moi with or without pre-treatment of Wedelolactone. E. coli-induced NF-κB activation was measured as relative luciferase activity. NF-κB luciferase activity was induced upon E. coli treatment, and pre-treatment of Wedelolactone markedly reduced E. coli-induced NF-κB activation (Fig. 4A). Subsequent mRNA expression of NF-κB-dependent gene IL-1β also was significantly inhibited by pre-treatment of Wedelolactone in a dose-dependent manner (Fig. 4B). Next we examined the effect of inhibition of IKKs on the innate immune response to E. coli in WT and Cyld−/− mice. WT and Cyld−/− mice were i.t. inoculated with 5 × 106 cfu of E. coli with or without pre-inoculation of Wedelolactone 2 h prior to E. coli inoculation [10 mg kg−1, intraperitoneally (i.p.)], and mRNA expression of IL-1β was measured from the lungs of E. coli- and control-inoculated mice 6 h after E. coli inoculation. E. coli-induced mRNA expression of IL-1β was significantly inhibited by inhibition of IKKs in the lungs of both WT and Cyld−/− mice (Fig. 4C). Consistent with these results, systemic production of macrophage-inflammatory protein-2 (MIP-2) protein was also significantly inhibited by pre-treatment of Wedelolactone (Fig. 4D). Moreover, histopathological analysis showed that inhibition of NF-κB activation by pre-treatment of Wedelolactone markedly reduced inflammation in lungs of E. coli-inoculated Cyld−/− mice along with reduced PMN recruitment into lung (Fig. 4E). Together, these data suggest that Cyld−/− mice exhibited enhanced innate immune response to E. coli through enhanced NF-κB activation in vivo.
Inhibition of NF-κB activation rescued Cyld−/− mice from lethal E. coli pneumonia
As inhibition of NF-κB activation by inhibiting IKKs abolished enhanced innate immune response to E. coli infection in Cyld−/− mice, we investigated whether inhibition of NF-κB by inhibition of IKKs can rescue the Cyld−/− mice from lethal E. coli pneumonia in vivo. Cyld−/− mice were first i.t. inoculated with 1 × 107 cfu or 5 × 106 cfu of E. coli with or without pre-inoculation of Wedelolactone (10 mg kg−1, i.p.) 2 h prior to E. coli inoculation. Survival was monitored for 6 days after inoculation. At higher dose of E. coli inoculation (1 × 107 cfu per mouse), Cyld−/− mice inoculated with E. coli without Wedelolactone started to die within 1 day after inoculation and resulted in 100% of mortality within 3 days. In contrast, none of the Wedelolactone-pre-inoculated Cyld−/− mice died during E. coli pneumonia (100% survival) (Fig. 5A). Similar results were also observed in low-cfu inoculation groups (Fig. 5B). Consistent with these results, body weight loss and hypothermia during E. coli pneumonia were also significantly attenuated by inhibition of IKKs in Cyld−/− mice (Fig. 5C and D). Taken together, these results indicate that CYLD deficiency resulted in hypersusceptibility to E. coli during lethal infection via enhanced NF-κB activation, thereby providing in vivo evidence for the negative role of CYLD in regulating bacteraemic pneumonia.
It has been previously shown that CYLD is a negative regulator of NF-κB and the NF-κB-dependent innate immune response to bacterial pathogens (Lim et al., 2007a; Sakai et al., 2007). However, it is still unclear if CYLD plays a critical role in host survival in E. coli pneumonia, which is characterized by profound inflammation. In this report, we have investigated the role of CYLD in regulation of innate immune responses to E. coli infection. We show that CYLD is a critical negative regulator for the innate immune response to E. coli via inhibition of NF-κB. The substantial increase in mortality after E. coli infection in Cyld−/− mice reveals the importance of CYLD in host defence. Taken together, our studies provide direct evidence that CYLD acts as a crucial suppressor for the inflammatory responses and thereby protects against lethal infection of E. coli. Our results also raise the possibility that variations in the Cyld gene may potentially represent an important susceptibility factor for E. coli pneumonia.
The airway epithelial surface plays a key role in host defence response in lung diseases including bacterial infections by recognizing highly conserved bacterial PAMPs. Recognition of PAMPs results in activation of host cells, leading to a plethora of biological responses required for shaping both the innate and adaptive arms of the immune response (Janeway and Medzhitov, 2002). Binding of the bacterial components to TLRs activates downstream signalling pathways, and results in activation of transcription factor, NF-κB (Kawai et al., 1999; 2001), which is known as a major regulator of inflammatory genes including cytokines, chemokines, inflammatory enzymes, adhesion molecules and others (Barnes and Karin, 1997). A critical balance between activation and subsequent deactivation of the NF-κB is of particular importance in the host survival. Although activation of the NF-κB signalling pathway is critical for mounting an immune response to eliminate invading pathogens, negative regulation of the NF-κB signalling pathways is equally important for preventing prolonged and detrimental inflammatory response and the resultant tissue damage (Pinsky, 2004).
We previously reported that CYLD deficiency protects mice from Streptococcus pneumoniae-induced alveolar haemorrhage and lethality, thus acting as a negative regulator for host survival in lethal S. pneumoniae infections by negatively regulating MKK3-p38 MAPK signalling independently of NF-κB activation (Lim et al., 2007b). Of particular interest in the current study is the in vivo evidence that, in infections mainly characterized by inflammation rather than structural damage of local tissues, CYLD actually acts as an important host survival factor by deactivating NF-κB pathway. With the exception of the report showing that CYLD downregulation was found in patients with chronic intestine inflammation (Costello et al., 2005), to date there has been no available report for increased susceptibility to infectious disease or enhanced mortality in bacteraemic pneumonia and related diseases in CYLD patients. One of the major reasons is likely because the CYLD was just recently identified and most of the CYLD studies originally focused on skin tumour. Although a more complete understanding of immunological functions of CYLD in bacteraemic pneumonia and other inflammatory diseases await the informational study on the CYLD patients, it is possible that the primary role of CYLD is to protect the host from over-reactive inflammatory response by terminating the bacteria-induced NF-κB activation during inflammation and infection. Thus, the present report together with previous studies suggests that CYLD plays a diverse role in regulating a variety of pathological processes. Identification of the critical role for CYLD in the pathogenesis of E. coli pneumonia may lead to the development of novel therapeutic strategy for the management of patients with bacteraemic pneumonia, sepsis and septic shock.
Cell and cell culture
Human alveolar epithelial cell A549 was maintained in F-12K medium (Invitrogen Life Technologies) as described previously (Sakai et al., 2007). MEF cells were obtained from the day 13 embryos and maintained in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies) as described previously (Lim et al., 2007b). Both cell culture mediums were supplemented with 10% fetal bovine serum (Invitrogen Life Technologies), 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin. All cells were cultured in a 5% saturated CO2 atmosphere at 37°C.
Bacteria and bacteria culture
Escherichia coli was purchased from American Tissue Culture Collection (ATCC; ATCC 25922) and grown on chocolate agar plates and in brain–heart infusion broth at 37°C in a humidified 5% CO2 water-jacketed incubator. For in vitro cell experiments, E. coli was inoculated into cell culture medium at the concentration of 10 or 100 moi. For in vivo animal experiments, mid-log-phase E. coli obtained from 6 h of incubation were prepared at the concentration of 2 × 108 cfu ml−1 in saline by centrifugation followed by washing with sterile saline.
Transfection and luciferase assay
Transient transfection of NF-κB luciferase reporter gene was conducted using TransIT-LT1 reagent (Mirus) following the manufacturer's instruction. Trasnfected cells were pre-treated with IKK inhibitor Wedelolactone or vehicle for 2 h, followed by E. coli inoculation. The luciferase assay was conducted in triplicate and luciferase activity was normalized with respect to β-galactosidase activity.
Real-time Q-PCR analysis
Quantitative PCR analysis of human IL-1β, TNF-α and IL-8 and mouse IL-1β, IL-6, TNF-α and MIP-2 was conducted as follows. Total RNA was isolated from cells and lung tissues of mice using TRIzol (Invitrogen Life Technologies) following the manufacturer's instruction. Reverse transcriptase reaction was conducted using TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer's instruction. PCR amplification was performed with SYBR green universal master mix (Applied Biosystems). Reactions were amplified and quantified using an ABI 7500 sequence detector and manufacturer's software (Applied Biosystems). The relative quantity of human and mouse mRNA was obtained using the comparative threshold cycle (CT) method and was normalized using pre-developed TaqMan assay reagent human cyclophilin and mouse GAPDH as an endogenous control for human mRNA and mouse mRNA respectively.
Mice and animal experiments
C57BL/6 mice, background mouse strain for targeting of Cyld gene, were purchased from National Cancer Institute (NIH, USA) and used as WT control mice for Cyld−/− mice. Cyld−/− mice were reported previously (Zhang et al., 2006; Lim et al., 2007a,b). E. coli-induced septic shock model was established as described previously with slight modification (Karzai et al., 1999; 2003; Li et al., 2008) as follows. For the mouse model of E. coli pneumonia-induced septic shock, WT and Cyld−/− mice were i.t. inoculated with 5 × 106 or 1 × 107 cfu of E. coli in 50 μl of saline, and saline as control. Mortality was recorded every 12 h after inoculation for 6 days. Body weight and body temperature were measured prior to inoculation and every 24 h after inoculation for 6 days.
For mRNA expression of inflammatory mediators, WT and Cyld−/− mice were i.t. inoculated with 5 × 106 cfu of E. coli, and mice were sacrificed 6 h after inoculation with overdose injection of sodium pentobarbital. Total RNA was extracted from the lungs and mRNA expression was measured by Q-PCR analysis as described above. Blood was collected from the vena cava (one part of 3.2% sodium citrate and nine parts of blood), and concentration of MIP-2 was measured from the plasma with ELISA assay kit (R&D systems, USA).
For the histopathological analysis, E. coli-inoculated WT and Cyld−/− mice were sacrificed 24 h after inoculation with overdose injection of sodium pentobarbital. Lung tissues were fixed with 10% buffered formaldehyde overnight with rocking, embedded in paraffin and sectioned at 5 μm thickness. Sections were then stained with haematoxylin and eosin (H&E) to visualize inflammatory response and pathological changes in the lung. H&E-stained lung sections were then evaluated by light microscopy using Axiovert 40 CFL (Carl Zeiss), and images were recorded with an AxioCam MRC (Carl Zeiss).
For perturbing of NF-κB signalling, Cyld−/− mice were i.p. inoculated with 10 mg kg−1 IKK inhibitor Wedelolactone followed by i.t. inoculation of E. coli. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at University of Rochester.
Differences in survival between WT and Cyld−/− mice after E. coli inoculation with or without Wedelolactone were determined by Kaplan–Meier analysis using SPSS 14.0 software (SPSS). All in vivo survival rate data were evaluated by log-lank test and statistical significance was accepted at a value of P < 0.05. All other in vivo and in vitro data were evaluated by Student's t-test.
This work was supported by grants from National Institute of Health DC004562 and DC005843 (to J.-D. Li). We thank members of Dr Li's laboratory for helpful discussion and suggestions.
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