Pellino1 promoted inflammation in lung injury model of sepsis by TRAF6/ NF-κB signal pathway

This study was designed to investigate the role of Pellino1 in lung injury model of sepsis and its anti-inflammation mechanism. C57BL/6 male mice (6–7 weeks old) and Pellino1−/− male mice were subjected to laparotomy followed by extracorporeal cecum mobilization and ligation. THP-1 cells were treated with 500 ng/ml of LPS for 4 h. Both mRNA and protein expression of Pellino1 was increased at time dependence in lung tissue of lung injury model of sepsis mice. Knockout of Pellino1 attenuated lung injury and inhibited inflammation of sepsis mice. While Pellino1 protein enhanced lung injury and increased inflammation of sepsis mice. Pellino1 promoted inflammation in in vitro model of lung injury by TRAF6/ NF-κB signal pathway. TRAF6 inhibitor attenuated the effects of Pellino1 on inflammation and lung injury in mice of sepsis. Similarly, NF-κB inhibitor also suppressed the effects of Pellino1 on inflammation and lung injury in mice of sepsis. The activation of TRAF6 or induction of NF-κB attenuated the effects of Pellino1 on inflammation in in vitro model of sepsis. The inhibition of TRAF6 or suppression of NF-κB reduced the effects of Pellino1 on inflammation in in vitro model of sepsis. These results suggested that Pellino1 promoted inflammation in lung injury model of sepsis by TRAF6/ NF-κB signal pathway.


Introduction
Acute lung injury (ALI) is a clinical syndrome of acute and diffuse inflammatory lung injury caused by various internal and external pathogenic factors, which in turn causes acute respiratory failure [1]. ALI is one of the common respiratory critical illnesses. The fatality rate of ALI is as high as 40-50 % in the intensive care unit (ICU) [2]. The etiology of ALI is complex, with unclear exact pathogenesis [3]. However, it is currently believed that the excessive activation and recruitment of inflammatory cells in the lung tissue and the uncontrolled release of inflammatory factors are the root cause of ALI, independent of its specific etiology [4]. A variety of infla mmatory cytokines are involved in the occurrence and development of ALI. Among them, IL-1β and TNF-α are the two key cytokines [4]. Because they can further trigger the secretion of other pro-inflammatory cytokines, they are also called "early responsive cytokines" [4].
TRAFs are important adaptors of TNF superfamily and Toll-like/Toll/IL-1 receptor (TIR) superfamily, which play important roles in both innate immunity and acquired immunity [5]. TRAF6 is closely associated with inflammation, bone metabolism, breast development and lymph node formation, which is also involved in the pathological mechanism of immune disorders, myeloma, acute pancreatitis, prostate cancer and etc. [6,7]. Therefore, TRAF6 is expected to be a promising therapeutic target for relevant diseases [8].
The expression of Pellino1 is increased under stimulation of Toll-like receptor (TLR) ligands and inflammatory cytokines, thereby promoting inflammatory response by activating NF-κB [9]. Under IL-1 stimulation, IL receptorassociated kinase 1 phosphorylates Pellino1 protein and activates its E3 ligase activity, causing the expression of downstream signal transduction pathway, including NF-κB and inflammatory cytokines [10]. In addition, lipopolysaccharide can induce the binding between Pellino1 and receptor interacting protein 1 for subsequent ubiquitination through the TLR signal transduction pathway, thereby activating the IκB kinase-NF-κB signal transduction pathway and causing increased expression of proinflammatory cytokines [11]. This experiment investigated the role of Pellino1 lung injury model of sepsis and its anti-inflammation mechanism.

Materials and Methods
Animals model C57BL/6 male mice (6-7 weeks old) and Pellino1 −/− male mice (5-6 weeks old) were housed with free access to food and water. All aspects of the animal care and experimental protocols were approved by the Guangzhou Women and Children's Medical Center, Guangzhou Medical University Committee on Animal Care. Cecal ligation and puncture (CLP) mouse model was established for sepsis model. All mice of sepsis model were anesthetized using 50 mg/kg pentobarbital sodium (Sigma-Aldrich LLC.) and then a stump was punctured once with a 22-gauge needle to extrude a small amount of stool. The cecum was put back to the normal abdominal position and the abdomen was closed.
All mice of sham group were anesthetized using 50 mg/kg pentobarbital sodium and induced with normal saline. All Pellino1 −/− mice were anesthetized using 50 mg/kg pentobarbital sodium, and the subjected to induce sepsis model by CLP. All mice of sepsis model were anesthetized using 50 mg/kg pentobarbital sodium, subjected to laparotomy followed by extracorporeal cecum mobilization and ligation, and then injected with recombination Pellino1 protein (1 µg/mice).

Histological examination
Lung tissue samples after mice sacrificed were collected and fixed with 4 % paraformaldehyde for 24 h at room temperature. Lung tissue samples fixed with Fig. 1 Pellino1 expression in lung injuryHeat map and microarray data (a), Pellino1 mRNA (b) and protein (c and d) expressionsSham, sham control group (n = 6); 3 h-Sepsis, sepsis model after induction of 3 h group (n = 6); 6 h-Sepsis, sepsis model after induction of 6 h group (n = 6); 12 h-Sepsis, sepsis model after induction of 12 h group (n = 6); 24 h-Sepsis, sepsis model after induction of 24 h group (n = 6);. Data are presented as means ± SD using Student's t test##p < 0.01 compared with sham control group paraformaldehyde were paraffin-embedded. Lung tissue samples were cut into 5 µm sections using a paraffin slicing machine and stained with hematoxylin and eosin. Lung tissues were observed under light microscopy (magnification, × 100; BH3-MJL; Olympus Corporation, Tokyo, Japan).

Bioanalysis measurement
Processing, bronchoalveolar lavage (BAL), and myeloperoxidase (MPO, A044-1-1, Nanjing Jiancheng Bioengineering Research Institute) activity were performed as previously described literature [12]. After induction of sepsis, BAL fluid was cataloged and BAL optical density was measured at 540 nm using aliquots. BAL hemoglobin and platelet counts were measured in 1 mL BAL fluid as previously described literature [12]. BAL total protein concentration was determined by BCA Protein Assay. BAL immunoglobulin M (IgM) was determined according to the manufacturer's instructions. Pulmonary microvascular Fig. 2 Knockout Pellino1 reduced lung injury and Pellino1 protein enhanced lung injury in sepsis miceBAL neutrophil counts/mL (a), BAL protein concentrations (b), lung bacterial CFU/mL (c), circulating platelet counts (d), lung tissue by HE assay (e), BAL fluid hemoglobin (f), BAL fluid OD540 measurements at 0 h (g) and Evans blue dye measurements of lung tissue homogenates at OD620 (h) in Pellino1 −/− mice;BAL neutrophil counts/mL (i), BAL protein concentrations (j), lung bacterial CFU/mL (k), circulating platelet counts (l), lung tissue by HE assay (m), BAL fluid hemoglobin (n, BAL fluid OD540 measurements at 0 h (o) and Evans blue dye measurements of lung tissue homogenates at OD620 (p)Model, sepsis model group (n = 6); Model + Pellino1-/-, Pellino1 −/− mice of sepsis model group (n = 6); Model + Pellino1, sepsis mice model by human recombinant Pellino1 protein group (n = 6). Data are presented as means ± SD using ANOVA followed by Tukey's post hoc test##p < 0.01 compared with sepsis model group permeability was measured at 620 nm and 740 nm using the Evans blue dye extravasation technique as previously described literature [12].

Statistical analysis
Data are presented as means ± SD. A P-value of ,0.05 was considered to be significant. Student's t test or oneway analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple range tests were used for comparisons of data.

The expression of Pellino1 in lung injury
To examine the changes of gene expression in lung tissue of lung injury model of sepsis mice, we first measured Pellino1 expression. As a result, the expression of Pellino1 was enhanced in lung tissue of lung injury . Data are presented as means ± SD using Student's t test##p < 0.01 compared with sepsis model group ( Fig. 1a and b). Both mRNA and protein expression of was increased at time dependence in lung tissue of lung injury model of sepsis mice (Fig. 1 c-e).
Knockout of Pellino1 reduced lung injury and Pellino1 protein enhanced lung injury in sepsis mice We further examined the function of Pellino1 in lung injury in sepsis mice. Consequently, we found that the inhibition of BAL neutrophil, BAL protein concentrations, lung bacterial CFU, BAL fluid hemoglobin (Hgb) concentration, lung injury, BAL fluid OD540 and lung tissue homogenates at OD620, and circulating platelet counts were increased in lung injury in Pellino1 −/− mice of sepsis ( Fig. 2a and h).
Recombinant Pellino1 protein was used to induce lung injury in sepsis mice. As expected, recombinant Pellino1 protein increased BAL neutrophil, BAL protein concentrations, lung bacterial CFU, BAL fluid Hgb concentration, lung injury, BAL fluid OD540 and lung tissue homogenates at OD620, and inhibited circulating platelet counts in lung injury of sepsis mice ( Fig. 2I and P).

Pellino1 promoted inflammation both in vivo and vitro model of lung injury by TRAF6/ NF-κB
To define the mechanism of Pellino1 in lung injury, we analyzed the gene expression of Pellino1-regulated inflammation gene in lung injury. As a result, the expression of TRAF6/ NF-κB was up-regulated and TRAF6/ NF-κB may be a target spot of Pellino1 in vitro by over-expression of Pellino1 ( Fig. 3a and d). Pellino1 was pulled down by TRAF6 in a mutual pulldown experiment using anti-Flag antibody (Fig. 3e). Over-expression of Pellino1 increased the expression of TRAF6 in vitro (Fig. 3f).
Moreover, over-expression of Pellino1 induced the protein expression of Pellino1, TRAF6 and NF-κB in vitro of lung injury (Fig. 4a, b, d and f). Downregulation of Pellino1 suppressed the protein expression Fig. 6 The regulation of TRAF6 participated in the effects of Pellino1 in mice model of lung injury modelTRAF6 and NF-κB protein expressions (a, b, d), TNF-α level (c), lung tissue by HE assay (e), IL-6 levels, IL-1β and IL-18 levels (f, g, and h), BAL neutrophil counts/mL (i), BAL protein concentrations (j), lung bacterial CFU/mL (k), circulating platelet counts (l), BAL fluid hemoglobin (m), BAL fluid OD540 measurements at 0 h (n) and Evans blue dye measurements of lung tissue homogenates at OD620 (o)Model, sepsis model group (n = 6); Model + Pellino1, sepsis mice model by human recombinant Pellino1 protein group (n = 6); Model + Pellino1 + C25-140, sepsis mice model by human recombinant Pellino1 protein and TRAF6 inhibitor group (n = 6). Data are presented as means ± SD using ANOVA followed by Tukey's post hoc test##i < 0.01 compared with sepsis model group; **p < 0.01 compared with sepsis model by human recombinant Pellino1 protein group of Pellino1, TRAF6 and NF-κB in vitro of lung injury (Fig. 4b c, 4e, 4g). Over-expression of Pellino1 increased the levels of TNF-α, IL-6, IL-1β and IL-18 in vitro ( Fig. 4h and k). While down-regulation of Pellino1 reduced the levels of TNF-α, IL-6, IL-1β and IL-18 in vitro ( Fig. 4l and o).
Next, we found that Pellino1 protein could induced the protein expression of Pellino1, TRAF6 and NF-κB and increased the levels of TNF-α, IL-6, IL-1β and IL-18 in mice of sepsis ( Fig. 5a and g). Pellino1 −/− mice suppressed the protein expression of Pellino1, TRAF6 and NF-κB and inhibited the levels of TNF-α, IL-6, IL-1β and IL-18 in mice of sepsis (Fig. 5h n).

The regulation of TRAF6 was involved in the effects of Pellino1 in lung injury model
We continued to examine the role of TRAF6 in the effects of Pellino1 in lung injury model. TRAF6 inhibitor (C25-140) could suppress the protein expression of TRAF6 and NF-κB, reduced lung injury and inhibited the levels of TNF-α, IL-6, IL-1β and IL-18 in mice treated with Pellino1 protein (Fig. 6a and h). TRAF6 inhibitor reduced BAL neutrophil, BAL protein concentrations, lung bacterial CFU, BAL fluid Hgbconcentration, lung injury, BAL fluid OD540 and lung tissue homogenates at OD620, and increased circulating platelet counts in lung injury of mice treated with Pellino1 protein (Fig. 6i and o).
SITRAF6 plasmid suppressed the protein expression of TRAF6 and NF-κB in vitro following over-expression of Pellino1 (Fig. 7a, b and e). While TRAF6 plasmid induced the protein expression of TRAF6 and NF-κB in vitro following down-regulation of Pellino1 (Fig. 7 ce). SITRAF6 plasmid reduced the levels of TNF-α, IL-6, IL-1β and IL-18 in vitro following over-expression of Pellino1 ( Fig. 7f and i). TRAF6 plasmid promoted the Fig. 7 The regulation of TRAF6 participated in the effects of Pellino1 in vitro modelSITRAF6 plasmid suppressed TRAF6 and NF-κB protein expressions in vitro model following over-expression of Pellino1 (a, b, e). TRAF6 plasmid induced TRAF6 and NF-κB protein expressions in vitro model following down-regulation of Pellino1 (c, d, e). SITRAF6 plasmid reduced TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following overexpression of Pellino1 (f, g, h, i). TRAF6 plasmid promoted TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following down-regulation of Pellino1 (j, k, l, m)Negative, negative group (n = 3); Pellino1, over-expression of Pellino1 group; siPellino1, down-regulation of Pellino1 group (n = 3); Pellino1 + siTRAF6, over-expression of Pellino1 and down-regulation of TRAF6 group (n = 3); siPellino1 + TRAF6, down-regulation of Pellino1 and over-expression of TRAF6 group (n = 3). Data are presented as means ± SD using ANOVA followed by Tukey's post hoc test##p < 0.01 compared with negative group; **p < 0.01 compared with over-expression of Pellino1 group or down-regulation of Pellino1 group levels of TNF-α, IL-6, IL-1β and IL-18 in vitro following down-regulation of Pellino1 (Fig. 7j m).

The regulation of NF-κB was involved in the effects of Pellino1 in lung injury model
We further investigated the role of NF-κB n the effects of Pellino1 in lung injury model. NF-κB inhibitor (JSH-23) suppressed the protein expression of NF-κB, reduced lung injury and inhibited the levels of TNF-α, IL-6, IL-1β and IL-18 in mice treated with Pellino1 protein (Fig. 8a and f). NF-κB inhibitor decreased BAL neutrophil, BAL protein concentrations, lung bacterial CFU, BAL fluid Hgb concentration, lung injury, BAL fluid OD540 and lung tissue homogenates at OD620, and increased circulating platelet counts in lung injury of mice treated with Pellino1 protein (Fig. 8g n).

Discussion
Due to the rapid onset and severe condition of ALI, there is still a lack of effective therapeutic methods for ALI despite accumulative therapeutic approaches for clinically critically ill patients in recent years [13]. Sepsis-related ALI is still the clinical syndrome with the highest mortality rate in ICU [14]. Therefore, we found that Pellino1 mRNA and protein expressions were increase at time dependence in lung tissue of lung injury model of sepsis mice [15,16]. Recombination Pellino1 protein enhanced lung injury in sepsis mice by induction release of inflammation factor. Hughes et al. suggest that Fig. 8 The regulation of NF-κB participated in the effects of Pellino1 in mice model of lung injury modelNF-κB protein expressions (a, b), TNF-α, IL-6 levels, IL-1β and IL-18 levels (c, d, e, and f), lung tissue by HE assay (g), BAL neutrophil counts/mL (h), BAL protein concentrations (i), lung bacterial CFU/mL (j), circulating platelet counts (k), BAL fluid hemoglobin (l), BAL fluid OD540 measurements at 0 h (m) and Evans blue dye measurements of lung tissue homogenates at OD620 (n)Model, sepsis model group (n = 6); Model + Pellino1, sepsis mice model by human recombinant Pellino1 protein group (n = 6); Model + Pellino1 + JSH-23, sepsis mice model by human recombinant Pellino1 protein and NF-κB inhibitor group (n = 6). Data are presented as means ± SD using ANOVA followed by Tukey's post hoc test##p < 0.01 compared with sepsis model group; **p < 0.01 compared with sepsis model by human recombinant Pellino1 protein group Pellino-1 regulated immune responses in models of inflammatory lung disease [17]. Taken together, our findings demonstrated that Pellino1 is an important regulator in lung injury of sepsis, and therefore it may serve as a novel therapeutic target for lung injury of sepsis.
TRAF6 is a member of the TNF receptor family. A large number of studies have confirmed that TRAF6 plays an important role in innate immunity and acquired immunity, cell apoptosis, stress response, and inflammation [18]. Studies have also found that the expression of TRAR6 is first decreased and then increased in lung tissues in mice with ALI [7,19,20]. In addition, TLR4-TRAF6 pathway is considered to be involved in inflammation-related lung injury. Moreover, TRAF6 also plays an important role in the process of ALI and inflammation [18]. We observed that Pellino1 promoted inflammation in vivo and vitro model of lung injury by TRAF6/ NF-κB signal pathway. Strickson et al. identified that Pellino1 partially restored IL-1 signaling by TRAF6 in primary macrophages [21]. Moreover, these results indicated Pellino1 regulate inflammation may participate in lung injury of sepsis.
Accumulative studies have confirmed that the activation of NF-kB plays an important role in the occurrence and development of many critical illnesses, and the activation of NF-kB is also associated with a variety of inflammatory factors [22]. As a pleiotropic regulatory factor, NF-kB is at the core of inflammation and anti-inflammation [23]. Therefore, how to selectively regulate the expression of certain target genes is particularly critical for inflammatory reaction of sepsis [24]. However, the anti-inflammatory and immune effects of NF-kB inhibitors detected so far are nonspecific for inflammatory factors of sepsis [25]. Excessive inhibition of NF-kB activation would destroy the dynamic balance between pro-inflammatory and antiinflammatory factors, which would decrease the body's defense capabilities and further aggravate disease progression [26]. Similar to this study, we found that the activation of TRAF6 or induction of NF-κB reduced the effects of Pellino1 on inflammation in vitro model of sepsis. The inhibition of TRAF6 or suppression of Fig. 9 The regulation of NF-κB participated in the effects of Pellino1 in lung injury modelSiNF-κB plasmid suppressed NF-κB protein expressions in vitro model following over-expression of Pellino1 (a, c). NF-κB plasmid induced TRAF6 and NF-κB protein expressions in vitro model following down-regulation of Pellino1 (b, c). SiNF-κB plasmid reduced TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following over-expression of Pellino1 (d, e, f, g). NF-κB plasmid promoted TNF-α, IL-6, IL-1β and IL-18 levels in vitro model following down-regulation of Pellino1 (h, i, j, k)Negative, negative group (n = 3); Pellino1, over-expression of Pellino1 group; siPellino1, down-regulation of Pellino1 group (n = 3); Pellino1 + siNF-κB, overexpression of Pellino1 and down-regulation of NF-κB group (n = 3); siPellino1 + NF-κB, down-regulation of Pellino1 and over-expression of NF-κB group (n = 3). Data are presented as means ± SD using ANOVA followed by Tukey's post hoc test##p < 0.01 compared with negative group; **p < 0.01 compared with over-expression of Pellino1 group or down-regulation of Pellino1 group NF-κB reduced the effects of Pellino1 on inflammation in vitro model of sepsis. Wang et al. suggest that Pel-lino1 regulates neuropathic pain through the regulation of MAPK/NF-κB signaling in the spinal cord [27]. Furthermore, we found that Pellino1 triggered TRAF6/ NF-κB signaling and enhanced inflammation in lung injury of sepsis. Collectively, these results suggest that Pellino1 may be an important regulator of inflammation in lung injury of sepsis by TRAF6/ NF-κB signaling.

Conclusions
We demonstrated that Pellino1 mRNA and protein expressions were increase in lung tissue of lung injury of sepsis and enhanced lung injury in sepsis mice by induction TRAF6/ NF-κB signaling. Our data suggest that Pel-lino1 may be an ideal target for the alleviation of inflammation in lung injury of sepsis.