Sepsis, a serious clinical problem, is characterized by a systemic inflammatory response to infection and may lead to organ failure which contributes to its high mortality (1). Despite all efforts to better comprehend this pathology, little progress has been made in clinic. In recent years, several classes of pattern-recognition receptors (PRRs) have been identified as the key receptors recognizing molecular patterns of microbial pathogens including Toll-like receptors (TLRs) (2). Several reports have identified TLR signaling is intimately implicated in the hyper-inflammatory response and tissue injury during sepsis (3–5).
All TLRs, except for TLR3, signal through the common myeloid differentiation factor 88 (MyD88)-dependent pathway. TLR3 totally and TLR4 partially via a Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathway (6, 7). Activation of MyD88-dependent pathway leads to the early activation of transcription factors nuclear factor-kappa B (NF-κB) and the expression of inflammatory response-related genes, such as tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). On the other hand, activation of the TRIF-dependent pathway initiates the type 1 IFN response as well as late NF-κB activation (8). Thus, various pathogen-associated microbial patterns (PAMPs)/TLRs interactions result in NF-κB activation and consequent expression of pro-inflammatory cytokines. There is now strong evidence that many cellular signaling molecules are shared among the TLRs and contribute to signaling pathways crosstalk (9, 10). Targeting a single TLR has shown efficient protection from septic shock in preclinical models, but unfortunately they failed in recent trials in patients with severe sepsis (11).
CD14 is a 55 kD glycosylphosphatidylinositol-anchored receptor and widely expressed in cells as a cell membrane or secreted protein (12). For a long time, CD14 was known as an accessory molecule facilitating lipopolysaccharide (LPS) to bind TLR4–MD2 complexes, thereby increasing sensitivity of LPS (13). Recent advances in CD14 biology have confirmed that CD14 also recognizes a variety of other exogenous and endogenous molecular patterns and is involved in signaling through TLR2, TLR3, TLR7, and TLR9 (14–16). These studies suggested that CD14 has broad ligands and involved in TLRs-mediated inflammatory response. A phase 1 clinical trial has shown that IC14, a recombinant anti-CD14monoclonal antibody, was well tolerated in both healthy subjects and severe sepsis patients and does not increase the incidence of secondary bacterial infection (17). Another clinic study has shown that IC14 improved organ function in patients with sepsis, but no differences in mortality may be due to small sample size (18). Thus, targeting CD14 may be a novel treatment strategy for sepsis.
In the present study, we sought to determine the effect of sepsis on CD14 expression in the tissue of lung, liver, and kidney of septic mice in vivo. Macrophage is the major producer of cytokines during sepsis. Activation of TLRs in macrophages contributed to inflammatory diseases (19, 20). We stimulated peritoneal macrophages with different TLR agonists and detected the effect of TLR on CD14 expression in vitro. In addition, after identifying TLR1-9 activation except TLR3 activation increased CD14 expression, we examined the relative roles of MyD88-dependent and MyD88-independent TLR signaling by utilizing MyD88−/− and TRIF−/− mice or TRIF inhibitor. Our study suggested that MyD88 signaling was the dominant pathway regulating CD14 upregulation, which consequences with the hyper inflammation and high mortality during severe polymicrobial sepsis.
MATERIALS AND METHODS
Male C57BL/6, MyD88−/−, and TRIF−/− mice were bred at the core facility at the University of Pittsburgh. All mice were used at the age of 6 to 12 week. Animal protocols were approved by the Animal Care and Use Committee of the University of Pittsburgh. All the experiments were performed in strict adherence to the National Institutes of Health Guidelines for the Use of Laboratory Animals.
Mouse model of polymicrobial sepsis
The cecal ligation and puncture (CLP) model was used to induce polymicrobial sepsis, as described previously (21). In brief, male mice were anesthetized with sevoflurane. After general anesthesia, the cecum was mobilized, ligated, and punctured through a 22-gauge needle. The bowel was repositioned and the abdomen with sterile suture was closed. The mice were resuscitated with prewarmed sterile saline subcutaneous after CLP operation immediately. Two hours following surgery, mice received a subcutaneous injection of antibiotics (imipenem, 25 mg/kg of body weight in 1 mL saline). Mice were sacrificed and blood samples were collected in tubes containing heparin for ELISA. Left lung, liver, and kidney were harvested from mice at indicated time and frozen in liquid nitrogen immediately and then stored at −80°C until further use (Western blot assay). Right lung was harvested from mice 24 h after the CLP procedure and fixed in 4% paraformaldehyde phosphate-buffered saline (PFA) for immunohistochemistry study.
For the survival study, mice were monitored every 12 h for up to 3 days following Sham or CLP procedure.
Peritoneal macrophages were isolated from C57BL/6 or MyD88−/− mice, 3 d after i.p. injection of 2 mL of 4% thioglycollate and plated in RPMI1640 medium containing 10% FBS, 50 U/mL penicillin G sodium and streptomycin sulfate and maintained overnight and then washed away floaters with PBS and incubated in fresh medium. On the next day, the adherent macrophages were incubated in serum-free medium RPMI1640 at 37°C for 2 h and then stimulated with TLR agonist (tlrl-kit1mw, Invivogen, San Diego, Calif) for indicated time in Opti-MEM medium (Gibco, Carlsbad, Calif).
Flow cytometry analysis
Peritoneal macrophages were cultured as described above and treated for 16 h as indicated with medium alone, 200 ng/mL Pam3CSK4 (TLR1 agonist), 5 × 107 HKLM (TLR2 agonist), 10 μg/mL PolyI:C (TLR3 agonist), 10 ng/mL LPS (TLR4 agonist), 100 ng/mL FLA-ST (TLR5 agonist), 20 ng/mL FLS-1 (TLR6 agonist), 1 μg/mL SSRNA40 (TLR7 agonist), 5 μM ODN1826 (TLR9 agonist). Following treatment, surface expression of CD14 was determined by flow cytometry. Dead cells were stained by a Live/Dead Fixable dead cell stain kit (Invitrogen). Cells were washed in cold PBS and then stained for 30 min on ice with FITC anti-mouse CD14 (123307, Biolegend). Stained cells were washed twice prior to flow cytometry analysis. Data were collected on a LSR II (BD Biosciences, Franklin Lakes, NJ) and the data were analyzed with FlowJo software. Mean fluorescent intensity (MFI) was determined by the geometric mean of the fluorescence of gated macrophages.
CD14 antibody was obtained from BD Pharmingen (553738, 1:1,000). In brief, lung, liver, and kidney tissue were homogenized in lysis buffer with protease inhibitor cocktails. Macrophages isolated from WT mice were stimulated with TLR1–9 agonist or pretreated with resveratrol (22) at different concentrations (0–20 μM) and then exposed to LPS (10 ng/mL) for indicated time. Macrophages isolated from Myd88−/− mice were treated with LPS (10 ng/mL and 100 ng/mL) or other TLR ligands, including Pam3CSK4, HKLM, FLS-1, and SSRNA40 for 16 h. Macrophages were lysed in lysis buffer with protease inhibitor cocktails on ice for 15 min. Both tissue and cells lysates were centrifuged at 15,000 g for 15 min at 4°C and supernatants were collected. 80 μg of tissue lysates and 30 μg of cells lysates were separated by SDS-PAGE gel and transferred to Reinforced NC membrane. The membranes were blocked with 5% skim milk in 0.1% Tween 20/TBS buffer and then incubated with CD14 antibody overnight. Simultaneously, for normalized comparisons, anti-β-actin antibody (1:2,000) was used as internal controls. Blots were probed with horse radish peroxidase (HRP)-conjugated anti-rat IgG or anti-mouse IgG for 1 h at room temperature. Bands were visualized using Super Signal West Pico Chemiluminescent Substrate. The levels of target protein and control proteins were analyzed using Image J software and further statistical analyses were performed.
The fresh lung tissue specimens were fixed in 4% PFA, dehydrated and embedded in paraffin. The samples were cut into 5 μm sections and placed onto slides. Endogenous peroxidase was quenched by H2O2. Mouse monoclonal CD14 antibody (ab182032, 1:500 diluted) was added on the sections as the primary antibody. The sections were incubated with polymer for 30 min and developed in DAB solution for 5 min. Then sections were counterstained with hematoxylin and mounted with cover slip.
Serum was collected after CLP at indicated time. Measurement of TNF-α or IL-6 concentration was carried out using TNF-α /IL-6 ELISA kit (R&D Systems, Minneapolis, Minn) following the manufacturer's protocol.
All data in the figures are expressed as mean ± SEM. Data were analyzed through Student t test or one-way analysis of variance (AOVAN) followed by LSD post-hoc test to compare the mean values. Differences in survival were determined using the log-rank test. A value of P < 0.05 was considered to be statistically significant.
CD14 expression increased in multiple organs in a time-dependent manner during CLP sepsis
CLP, a relevant model of polymicrobial sepsis, was used to detect the expression characteristic of CD14 during severe sepsis. Lung, liver, and kidney were isolated at various time intervals up to 24 h after CLP surgery. CD14 protein levels were assessed by Western blot. As shown in Figure 1A, CD14 levels increased as early as 2 h with further increases to 24 h. Immunohistochemistry of CD14 confirmed higher expression at 24 h in the lung of septic mice (Fig. 1B). The liver and kidney showed similar expression patterns with detectable increases at 8 h after CLP followed by dramatic increases at 24 h (Fig. 1, C and D). This result demonstrates that CD14 expression increases across multiple organs in polymicrobial sepsis over the 24 h period.
The effect of various TLR agonists on CD14 expression in macrophages
CD14 expression is inducible in macrophages (23). Macrophages rapidly response to the presence of pathogens through the detection of PAMP molecules through TLR (6). To determine if specific TLRs were involved in the regulation of CD14 expression, peritoneal macrophages were exposed to ligands for TLR1–9. CD14 expression levels were measured at 4 and 16 h, respectively. The Western blot analysis showed that CD14 expression in peritoneal macrophages increased in a time-dependent manner after all TLR1–9 agonists stimulation with the exception of the TLR3 agonist PolyI:C (Fig. 2A). Cell membrane CD14 expression was corresponded to Western blot analysis (Fig. 2B). These results together indicated that all TLR agonists tested, except of PolyI:C, increased CD14 expression.
CLP failed to increase CD14 expression in MyD88−/− mice but not in TRIF−/−mice
All TLRs, but not TLR3, signal through the MyD88-dependent pathway. TLR3 signals via exclusively TRIF-dependent pathway. In contrast, TLR4 signals through both MyD88 and TRIF (6, 7). To examine which adaptor protein contributed to sepsis-induced CD14 expression, we measured the expression levels of CD14 in the lung, liver, and kidney of WT, MyD88−/−, and TRIF−/−mice after CLP by Western blot. As shown in Figure 3A–C, CLP-induced CD14 expression was abolished in MyD88−/− mice but not in TRIF−/−mice in all three organs.
LPS increased CD14 expression via MyD88-dependent but TRIF-independent pathway in macrophages
To further determine the role of MyD88 in CD14 upregulation, we analyzed the protein expression level of CD14 in LPS-stimulated macrophages isolated from WT and MyD88−/−mice. As shown in Figure 4, MyD88-deficient macrophages showed a much lower level of CD14 compared with WT macrophages after LPS stimulation (Fig. 4A). In addition, other TLR ligands such as Pam3CSK4, HKLM, FLS-1, and SSRNA40 induced CD14 expression that was also significantly decreased in macrophages isolated from MyD88 KO mice (Fig. 1B). To further confirm TRIF is not required for CD14 upregulation, we used TRIF inhibitor to suppress TRIF signaling (22). As shown in Figure 4C, resveratrol pretreatment increased CD14 expression in a dose-dependent manner in macrophages after LPS stimulation.
The impact of MyD88 or TRIF deletion on CLP-induced mortality and cytokine production
Studies have focused on the role of MyD88 and TRIF in sepsis but the results are conflicting. We compared survival rates and circulating cytokines release following CLP in WT, MyD88−/−, and TRIF−/− mouse. As expected, 80% of mice in the WT group were dead by 3 d (Fig. 5A). However, only 30% of MyD88−/− mice perished by day 3. In contrast to the WT and MyD88−/− mice, all of the TRIF−/− mice died by day 2. These results indicate that MyD88 signaling contributes to mortality in this model of severe sepsis, in comparison TRIF exerts protective function.
Excessive elevation of pro-inflammatory cytokines in circulation is a major contributor to remote organ injury after CLP (24). IL-6 and TNF-α levels were assessed as a measure of systemic inflammation over the initial 24 h. As illustrated in Figure 5B and C, compared with control mice, which had undetectable levels of IL-6 and TNF-α, sepsis increased both cytokines levels in the serum. MyD88 deletion resulted in much lower IL-6 and TNF-α levels compared with TRIF deletion had no such effect at same time point. Taken together, these results point to the center role of MyD88, but not TRIF signaling in regulating systemic inflammation and CD14 expression in CLP-induced sepsis.
Sepsis, characterized by a systemic bacterial infection, is a life-threatening condition that can lead to multi-organ failure and death in intensive care unit patients (1). TLRs are a family of innate immune recognition receptors that detect PAMPs and play a key role in initiating inflammatory responses and contribute to pathogenesis of sepsis (3–5). Infectious and/or inflammatory diseases induce CD14 expression. Previously, studies indicated that CD14 is involved in TLR2, 3, 4, 7, and 9 signaling and enhances TLRs activation induced inflammatory response (14–16). Inhibition of CD14 either via an anti-antibody or by gene deficiency has been shown to protect against infection in different animal models (25, 26). Recent studies indicated that soluble CD14 subtype (presepsin) has diagnostic and prognostic value for sepsis (27, 28). Therefore, regulation of CD14 is crucial in TLR-mediated immune responses and related disease progresses. Previously, the study indicated that CD14 mRNA level was significantly increased in the lung, liver, and kidney from CLP mice (29). In the present study, we found that polymicrobial sepsis-induced TLRs activation increased CD14 protein expression in the lung, liver, and kidney in a time-dependent manner. During infection, macrophages are the first line of defense. However, macrophages and TLRs over activation induced overwhelming inflammatory response and contributed to tissue injury in inflammatory diseases (19, 20). CD14 increased TLRs induced inflammation (13–16). To detect whether TLR activation increased CD14 expression in turn, we stimulated peritoneal macrophages with different TLR agonist and observed that TLR1–9 agonist exception of the TLR3 agonist significantly increased CD14 expression in vitro.
TLRs induce inflammatory reactions by the activation of signaling pathways mediated by MyD88 and TRIF. MyD88 is the central adapter protein for signal transduction of all TLRs, except for TLR3. TLR4 partially and TLR3 completely induces TRIF-dependent pathway (6, 7). Many studies have focused on the role of MyD88 and TRIF in response to sepsis but shown conflicting results. Using the CLP model (30) or a similar model (31), studies have established MyD88 deficiency markedly decreased inflammatory cytokines and improved the survival rate in mice. However, some other studies have shown that MyD88-deficient mice were more susceptible to E coli challenge (32) and had a higher mortality rate in the CLP model of sepsis (33). Consistent with Feng results (30), our in vivo study showed that MyD88 deficiency decreased inflammatory cytokines release and improved survival after CLP. We also observed that sepsis-induced CD14 expression was almost abolished in the tissue of MyD88−/− mice as well as in LPS-stimulated macrophages. These data suggested MyD88's regulation of inflammatory responses was mediated through CD14 expression. In this regard, impaired CD14 expression in MyD88 deficiency might be involved in the downregulation of inflammatory responses under such a condition. The previous study has shown that the mortality rate and immune response is highly dependent on the severity of CLP. Therefore, the mild model of CLP was most likely the reason for the different results in the Peck-Palmer study (33). With a more severe model of sepsis, we showed a protective effect in MyD88-deficient mice.
Similarly, the role of TRIF signaling in sepsis was often inconsistent. In a less severe sepsis mode induced by E colichallenge, Cuenca et al. (34) demonstrated that loss of TRIF in neonates markedly increases mortality that was associated with systemic bacteremia and exaggerated late inflammatory response. In contrast to the neonates, TRIF deficiency in adult mice has no effect on survival rate compared with WT mice (35). Feng et al. (30) also found that TRIF signaling played no major role in the development of organ dysfunction and mortality in severe polymicrobial sepsis (30). But in endotoxin shock, TRIF deficiency significantly increased the survival rate. In the current study, we found that TRIF deficiency played no major role in inflammatory cytokines release and reduced the survival rate in severe polymicrobial sepsis. CLP increased CD14 expression in the tissue of TRIF−/− mice was similar to WT mice. Intriguingly, pretreated WT macrophages with TRIF inhibitor did not suppress LPS-induced CD14 expression as well. Previous studies have established that TLRs such as TLR2, TLR4, and TLR9 all contributed to the pathogenesis of polymicrobial sepsis (3–5). Loss of TRIF only impaired TLR4 activation. The increased CD14 involved in other TLRs activation and enforced inflammation may substitute TRIF pathway in polymicrobial sepsis. These results may explain why in TRIF−/− mice we did not detect significantly decreased inflammatory responses in polymicrobial sepsis but Feng did in endotoxemia (30). Furthermore, TRIF signaling is required for proper function of phagocytosis as well as intracellular killing during Gram-negative bacterial infection (35). TRIF deficiency caused higher mortality in polymicrobial sepsis may be due to systemic toxicity and increased bacteremia.
In summary, the present study suggested that Myd88-dependent canonical TLR activation increased CD14 expression and CD14 engages in TLRs recognition and increases inflammation as a novel positive feedback loop. Clinical sepsis is a more complex disease, where multiple inflammatory mediators and pathways are likely to be hyperactivated. MyD88-CD14 signaling should be considered in future therapeutic options designed to treat polymicrobial sepsis.
The authors thank Rongqian Wu for help in the discussion of this study.
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