Receptor Interacting Protein-2 acts as negative regulator for nontypeable Haemophilus influenzae-induced mucin MUC5AC expression

Appropriate production of mucous plays a critical role in host innate mucosal defenses against infection. However, if uncontrolled, excessive mucous may be detrimental to the host. Mucous production thus must be tightly regulated. In the present study, we investigate the role of RIP-2 in regulating nontypeable Haemophilus influenzae (NTHi)-induced up-regulation of mucin MUC5AC expression. We show that RIP-2 is a negative regulator of MUC5AC, whereas MAP kinase JNK acts as a positive regulator for MUC5AC induction by NTHi. Our studies unveil a novel role for RIP-2 in controlling mucous production in upper respiratory disease and may shed light on the identification of new therapeutic targets. Introduction Mucosal barriers in the upper respiratory tract, including the middle ear, typically aide in trapping and clearance of infectious agents as a protective mechanism for the host [1]. Mucins are a major component of mucous in these regions. Mucins are a family of high molecular weight glycoproteins secreted by goblet cells in mucosal tissues. There are more than 20 known mucins, twelve of which have been found in the respiratory tract [2]. Mucin 5AC (MUC5AC) is known to be one of the major mucins produced during upper respiratory infections, including otitis media (OM). While appropriate mucous production is protective for the host, excessive mucous production can be deleterious [1-4]. In fact, thick mucous and neutrophil nets can create a biofilmlike environment for nontypeable Haemophilus influenzae (NTHi), one of the major bacterial pathogens of OM and chronic obstructive pulmonary disease (COPD). Overproduction of mucin MUC5AC can lead to persistent, chronic infections or can worsen acute tissue injury [1, 5]. Therefore, like inflammation, mucous production must be tightly controlled. NTHi-caused upper respiratory infections, including COPD and OM, remain major health problems worldwide. COPD is the third leading cause of death in the U.S. with numbers rising each year. Hassett et al. reported the annual number of Americans affected by COPD in 2014 at over 14 million [6]. OM is the most common pediatric infectious disease in humans accounting for over 20 million physician visits and costing the U.S. upwards of 5 billion dollars annually [7, 8]. OM is characterized by mucous overproduction and inflammation in the middle ear cavity due to infection. Pediatric patients are highly susceptible to OM pathogens due to the shortened length of the Eustachian tube during development [7]. Currently, the standard treatment for both COPD and OM is systemically administered broad-spectrum antibiotics, which, leads to increased antibiotic resistance. Surgical intervention is also frequently utilized in pediatric OM patients by placing tubes to aid in drainage of the middle ear cavity. This commonplace surgical intervention and frequent use of antibiotics warrants concern for the development of drug-resistant strains of bacteria as well as the rising healthcare costs associated with both inflammatory diseases. Therefore, development of non-antibiotic therapeutic strategies is urgently needed based on a full understanding of the molecular pathogenesis of these inflammatory diseases. Receptor interacting protein-2 (RIP-2) plays a key role in activation and regulation of multiple inflammatory signaling cascades, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic receptor that detects bacterial peptidoglycans and other inflammatory stimuli [9]. Upon NOD2 detection of pathogenassociated molecular patterns (PAMPs), RIP-2 is recruited and interacts via CARD-CARD interactions. RIP-2 is a dual-specificity kinase downstream of NOD-like receptors, which undergoes autophosphorylation and subsequent polyubiquitination to drive transcription of inflammatory cytokines and chemokines [9]. RIP2 is an essential signaling adaptor, and previous studies have shown NF-κB activation is sub-optimal in its absence [10]. Previous studies have also shown that RIP-2 acts as an important adaptor molecule for ovalbumin-induced lung inflammation. However, the role of RIP-2 in bacteria-induced expression of mucin MUC5AC, a major contributor of OM and COPD pathology, remains largely unknown. In the present study, we sought to investigate the role of RIP-2 in NTHi-induced MUC5AC gene expression. Here we show that NTHi induces RIP-2 expression at mRNA and protein levels, and RIP-2 is a Correspondence to: Dr. Jian-Dong Li, Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences, Georgia State University, 100 Piedmont Avenue SE, Atlanta, GA 30303, USA, Tel: 404-413-3574; Fax: 404413-3580; E-mail: jdli@gsu.edu


Introduction
Mucosal barriers in the upper respiratory tract, including the middle ear, typically aide in trapping and clearance of infectious agents as a protective mechanism for the host [1]. Mucins are a major component of mucous in these regions. Mucins are a family of high molecular weight glycoproteins secreted by goblet cells in mucosal tissues. There are more than 20 known mucins, twelve of which have been found in the respiratory tract [2]. Mucin 5AC (MUC5AC) is known to be one of the major mucins produced during upper respiratory infections, including otitis media (OM). While appropriate mucous production is protective for the host, excessive mucous production can be deleterious [1][2][3][4]. In fact, thick mucous and neutrophil nets can create a biofilmlike environment for nontypeable Haemophilus influenzae (NTHi), one of the major bacterial pathogens of OM and chronic obstructive pulmonary disease (COPD). Overproduction of mucin MUC5AC can lead to persistent, chronic infections or can worsen acute tissue injury [1,5]. Therefore, like inflammation, mucous production must be tightly controlled.
NTHi-caused upper respiratory infections, including COPD and OM, remain major health problems worldwide. COPD is the third leading cause of death in the U.S. with numbers rising each year. Hassett et al. reported the annual number of Americans affected by COPD in 2014 at over 14 million [6]. OM is the most common pediatric infectious disease in humans accounting for over 20 million physician visits and costing the U.S. upwards of 5 billion dollars annually [7,8]. OM is characterized by mucous overproduction and inflammation in the middle ear cavity due to infection. Pediatric patients are highly susceptible to OM pathogens due to the shortened length of the Eustachian tube during development [7]. Currently, the standard treatment for both COPD and OM is systemically administered broad-spectrum antibiotics, which, leads to increased antibiotic resistance. Surgical intervention is also frequently utilized in pediatric OM patients by placing tubes to aid in drainage of the middle ear cavity. This commonplace surgical intervention and frequent use of antibiotics warrants concern for the development of drug-resistant strains of bacteria as well as the rising healthcare costs associated with both inflammatory diseases. Therefore, development of non-antibiotic therapeutic strategies is urgently needed based on a full understanding of the molecular pathogenesis of these inflammatory diseases.
Receptor interacting protein-2 (RIP-2) plays a key role in activation and regulation of multiple inflammatory signaling cascades, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic receptor that detects bacterial peptidoglycans and other inflammatory stimuli [9]. Upon NOD2 detection of pathogenassociated molecular patterns (PAMPs), RIP-2 is recruited and interacts via CARD-CARD interactions. RIP-2 is a dual-specificity kinase downstream of NOD-like receptors, which undergoes autophosphorylation and subsequent polyubiquitination to drive transcription of inflammatory cytokines and chemokines [9]. RIP-2 is an essential signaling adaptor, and previous studies have shown NF-κB activation is sub-optimal in its absence [10]. Previous studies have also shown that RIP-2 acts as an important adaptor molecule for ovalbumin-induced lung inflammation. However, the role of RIP-2 in bacteria-induced expression of mucin MUC5AC, a major contributor of OM and COPD pathology, remains largely unknown.
In the present study, we sought to investigate the role of RIP-2 in NTHi-induced MUC5AC gene expression. Here we show that NTHi induces RIP-2 expression at mRNA and protein levels, and RIP-2 is a (PVDF) membranes. The membrane was blocked with a solution of Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk. The membrane was then incubated in a 1:2,000 dilution of a primary antibody in 5% bovine serum albumin-TBS-T. After three washes in TBS-T, the membrane was incubated with 1:5,000 dilution of the corresponding secondary antibody in 5% non-fat dry milk-TBS-T. Respective proteins were visualized by using Amersham ECL Prime Reagent (GE Healthcare Life Science).

Mice and animal experiments
C57BL/6 and RIP-2 -/mice (7-8 weeks old) were purchased from the Jackson Laboratory. Anesthetized mice were inoculated with NTHi at a concentration of 1 × 10 7 CFU per mouse or saline as control [2]. The inoculated mice were sacrificed at 6 and 12 h postinoculation. Eardrums of mice were inspected for signs of middle ear inflammation. Dissected mouse middle ears were then subjected to total RNA extraction and histologic analyses. All animal experiments were approved by the Institutional Animal Care and Use Committee at Georgia State University.

Histology
As previously described [2], formalin-fixed paraffin-embedded mouse middle ear tissues were sectioned (4 µm), and then stained with hematoxylin and eosin (H&E) to visualize inflammatory responses and pathological changes in the middle ear. Images of stained cells and tissue sections were recorded with light microscopy system (Carl Zeiss).

Statistical analyses
All experiments were repeated in at least three independent experiments. Data are shown as mean ± SD of n determinations. Statistical analysis was assessed with two-tailed unpaired Student t-test; p < 0.05 was considered statistically significant.

NTHi induces RIP-2 in human epithelial cells in vitro and in mouse middle ear in vivo
RIP-2 is a dual specificity kinase known to modulate inflammatory signaling pathways. Previous studies have shown the role of RIP-2 to be complex in various diseases. For example, RIP-2 deficient mice were shown to be resistant to developing arthritis and autoimmunemediated cephalomyelitis associated with a multiple sclerosis model [9,19,20]. Paradoxically, dysregulation of the NOD2/RIP-2 pathway has been implicated in dysregulation of cell signaling pathways during viral infection [9,21]. To investigate the role of RIP-2 in NTHi-induced mucin expression in our experimental models, we first examined whether NTHi induces RIP-2 in vitro. We treated human epithelial cells with NTHi and measured RIP-2 mRNA expression by performing quantitative PCR (Q-PCR) analysis. NTHi induced RIP-2 expression at mRNA level in human middle ear epithelial HMEEC-1 cells ( Figure  1A) and human bronchial epithelial BEAS-2B cells ( Figure 1B). We also confirmed a time-dependent induction of RIP-2 at the protein level ( Figure 1C). Consistent with in vitro findings, NTHi-induced RIP-2 mRNA expression was also confirmed in the middle ear of mice by q-PCR analysis ( Figure 1D). Collectively, these results that NTHi induces expression of RIP-2 in vitro and in vivo.

RIP-2 acts as a negative regulator for NTHi-induced MUC5AC expression
On the basis that mucous overproduction is a hallmark of NTHi-infections and NTHi markedly induce RIP-2 expression, negative regulator of MUC5AC expression, whereas c-Jun N-terminal kinase (JNK) is a positive regulator for MUC5AC induction. Thus, our study provides the direct evidence for the first time of the negative regulation of MUC5AC induction by NTHi via RIP-2 signaling pathway and may lead to the development of new therapeutic strategies for suppressing MUC5AC overproduction.

Bacterial strains and culture conditions
Clinical isolate of NTHi 12 was used in this study. NTHi were grown as previously described [12][13][14][15][16]. For in vitro experiments, NTHi was resuspended in PBS and used at a multiplicity of infection (MOI) of 50. For in vivo experiments, NTHi was resuspended in isotonic saline and used at a concentration of 1 x 10 7 CFU per mouse.

Real-time quantitative RT-PCR analysis
Total RNA extraction and RT-qPCR were performed as previously described [2][3][4][12][13][14][15][16][17]. The relative quantities of mRNAs were determined by using the comparative Ct method and were normalized by using human cyclophilin for in vitro or mouse glyceraldehyde-3phosphate dehydrogenase (GAPDH) for in vivo as endogenous controls. The primer sequences of human MUC5AC, mouse MUC5AC, human cyclophilin and mouse GAPDH were described previously [2,18]. The primer sequences of RIP-2 are as follows: human RIP-2 (

Plasmids and transfections
RIP-2 siRNA (SR305775) and control siRNA (SR30004) were purchased from Origene. All transient transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturers' instructions. For experiments with inhibitors, the transfected cells were pretreated with or without chemical inhibitors for 1 h followed by 5 h of incubation with NTHi.

Western blot
Western blot procedures were previously described [2][3][4][12][13][14][15][16][17]. Western blots were performed using whole-cell extracts, separated on 8-10 % SDS-PAGE gels, and transferred to polyvinylidene difluoride we hypothesized that RIP-2 may play a critical role in regulating mucin production [22]. To determine the role of RIP-2 in MUC5AC induction, we utilized small interfering RNA (siRNA) specific for RIP-2 in human epithelial cells. We first evaluated the efficiency of RIP-2 knockdown. As shown in Figure 2A, RIP-2 siRNA efficiently depleted RIP-2 expression. Next, we evaluated the effect of siRNA RIP-2 on MUC5AC induction. Interestingly, siRIP-2 markedly enhanced NTHi-induced MUC5AC up-regulation in HMEEC-1 cells ( Figure  2B). Moreover, we determined if NTHi also induces effusion in the middle ear of the mouse otitis media models. As shown in Figure 2C, increased effusion was observed in RIP-2 deficient mice compared with WT control mice as early as 12 hours post-infection. Together, our data indicate that RIP-2 is a negative regulator for NTHi-induced MUC5AC expression.

JNK acts as a positive regulator for NTHi-induced MUC5AC up-regulation
MAP kinase JNK has been shown to play a critical role in mediating bacteria-induced inflammatory responses [4,23]. Thus, we sought to determine the role of JNK in NTHi-induced MUC5AC expression. We first confirmed the activation of JNK by NTHi. NTHi induced potent activation of JNK in a time-dependent manner in human epithelial cells ( Figure 3A). We next evaluated the effects of JNK specific inhibitor (SP600125) on NTHi-induced MUC5AC expression. As shown in Figure 3B, inhibition of JNK suppressed NTHi-induced MUC5AC expression, thereby indicating a critical role of JNK as a positive regulator for NTHi-induced MUC5AC expression. The role of JNK in regulating bacteria-induced host mucosal immune responses is highly complex and may act differently depending on the pathogen as well as on the host genes. The positive regulation of MUC5AC expression by JNK may be due to its role in mediating the activation of the signaling pathways including the transcription factors leading to the transcriptional up-regulation of MUC5AC expression. We previously reported that JNK negatively regulates MUC5AC expression via targeting negative activator protein-1 (AP-1) site in Streptococcus pneumoniae infection [4]. Thus, it is possible that JNK positively regulates NTHi-induced MUC5AC expression through the AP-1 site in the promoter region of MUC5AC gene. Future studies are needed to confirm this hypothesis by using AP-1 site mutants of MUC5AC-Luc luciferase reporter promoter constructs.

JNK acts as a negative regulator for NTHi-induced RIP-2 expression
Having demonstrating the positive role of JNK in mediating the up-regulation of mucin MUC5AC by NTHi, we next sought to explore if JNK may also mediate up-regulation of MUC5AC by suppressing the expression of RIP-2, the negative regulator for NTHi-induced MUC5AC expression. Interestingly, inhibition of JNK using JNK specific inhibitor enhanced NTHi-induced expression of RIP-2 ( Figure 4). This exciting result provides direct evidence to unveil the complex molecular mechanism by which JNK mediates NTHi-induced mucin MUC5AC expression likely via up-regulation of MUC5AC transcription and also via inhibition of the induction of negative regulator RIP-2 expression ( Figure 5).
Taken together, our studies bring new insights into the complex regulatory mechanism underlying bacteria-induced up-regulation of mucous and may be helpful for developing new therapeutic strategies for suppressing mucous overproduction in upper respiratory infectious diseases. Future studies are needed for further investigating the precise molecular mechanisms underlying tight regulation of mucous overproduction.  Copyright: ©2017 Bohn AO. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.