Anti-inflammatory Effect Of MUC1 During Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (RSV) was first characterized in 1957 and has been recognized as the most common cause of severe respiratory tract infection in young infants worldwide. RSV is the most common pathogen resulting in hospital admission in children under 5 years of age. Following RSV infection immunity is incomplete and secondary infections can occur throughout life. Despite 50 years of research there is still neither effective treatment nor any immediate prospect of a vaccine.RSV is classified in the genus Pneumovirus of the family Paramyxoviridae. The RSV genome is composed of single stranded negative sense RNA of 15,300 nucleotides encoding 11 proteins. RSV predominantly infects airway epithelial cells. These cells pave the surface of nose as well as the large and small airways. They form the first line of defense against the virus and are the site of the majority of the inflammation associated with the disease. In immuno-competent individuals the virus seldom infects other tissues. The first critical step in the infection process is entry of the virus into the cell. The airway epithelial cells are the primary target cells for RSV infection. The process can trigger activation of innate immunity, which is through the interaction between RSV G and F proteins and numerous host cell surface molecules, e.g. one or several molecules among Toll like receptor (TLR) 2,3,4,7 or 8.In mammals,12 members of the TLR family have been identified so far. TLRs are typeⅠintegral membrane glycoproteins. The extracellular N-terminal domain consists of approximately 16 to 28 leucine-rich repeats (LRRs), and each LRR consists of 20 to 30 amino acids with the conserved motif "LxxLxLxxN". The intracellular C-terminal domain is known as the Toll/IL-1 receptor (TIR) domain, which shows homology with that of the IL-1 receptor. This domain is required for the interaction and recruitment of various adaptor molecules to activate the downstream signaling pathway. The crystal structures of several TLRs with their ligand complex were recently reported. It has been shown that these complexes form heterodimers such as TLR1-TLR2, TLR4-MD2 or a homodimer such as TLR3-TLR3 after association with their respective agonist/antagonist ligands and form a horseshoe-like structure ("m" shape). It has been shown that this structure is essential for ligand binding and initiation of downstream signaling pathway. TLRs recognize various PAMPs derived from viruses, pathogenic bacteria, pathogenic fungus and parasitic-protozoa. After activation of TLRs signal transduction pathway in the airway epithelial cells by RSV infection, it can synthesize and release a variety of cytokines, e.g. IL-8, IL-6, granulocyte-macrophage colony-stimulating factor (GMCSF), transforming growth factor (TGF) and TNF-α. Among these proinflammatory cytokines, TNF-αis the most important one, no matter because it has powerful antiviral function, or it can exacerbate illness after RSV infection.TNF-αwas firstly discovered in 1975 by Dr. Loyd from Memorial Sloan-Kettering Cancer Center, New York, and its cDNA was cloned in 1984. TNF-αis primarily produced as a 212-amino acid-long typeⅡtransmembrane protein arranged in stable homotrimers. From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloprotease TNF-αconverting enzyme (TACE, also called ADAM17). The soluble 51 kilodalton (kDa) trimeric sTNF tends to dissociate at concentrations below the nanomolar range, thereby losing its bioactivity. The 17 kDa TNF protomers (185-amino acid-long) are composed of two antiparallel (3-pleated sheets with antiparallelβ-strands, forming a "jelly roll"β-structure, typical for the TNF family, but also found in viral capsid proteins. Two receptors, TNF receptor type 1 (TNFR1; CD120a; p55/60) and TNF receptor type 2 (TNFR2; CD120b; p75/80), bind to TNF. TNFR1 is expressed in most tissues, and can be fully activated by both the membrane-bound and soluble trimeric forms of TNF, whereas TNFR2 is found only in cells of the immune system, and respond to the membrane-bound form of the TNF homotrimer. As most information regarding TNF signaling is derived from TNFR1, the role of TNFR2 is likely underestimated. Upon contact with their ligand, TNF receptors also form trimers, their tips fitting into the grooves formed between TNF monomers. This binding causes a conformational change to occur in the receptor, leading to the dissociation of the inhibitory protein SODD from the intracellular death domain. This dissociation enables the adaptor protein TRADD to bind to the death domain, serving as a platform for subsequent protein binding. Following TRADD binding, three pathways can be initiated.1. Activation of NF-κB.2. Activation of the MAPK pathways.3. Induction of death signaling. The primary role of TNF is in the regulation of immune cells. TNF is also able to induce apoptotic cell death, to induce inflammation, and to inhibit tumorigenesis and viral replication. Dysregulation of TNF production has been implicated in a variety of human diseases, as well as cancer.RSV is a pneumovirus that is responsible for the majority of respiratory illness and death seen in young children. Although RSV infections typically result in nothing more than a mild upper respiratory infection,5% of infected children fall prey to more severe lower respiratory tract infections and bronchiolitis.Severe lower respiratory disease results in as many as 130,000 pediatric hospitalizations annually in the U.S..Similarly, RSV has been linked to high mortality in the institutionalized elderly and the immunocompromised, particularly bone marrow transplant recipients. Furthermore, it has been proven that exposure to RSV infection early in life can lead to an increased susceptibility to suffer from recurrent allergic wheezing and asthma. These strains on public health make the development of an effective RSV vaccine a high priority.Unfortunately,the realization of this goal has been thwarted by the legacy of failed vaccine trials in the 1960s with a formalin-inactivated, alum-precipitated RSV preparation (FI-RSV). The FI-RSV vaccine caused more severe illness, increased rates of hospitalization, and some mortality. Anyway, it is proved that TNF-αis the primary perpetrator of T-cell-mediated lung injury after RSV infection. In this condition, there must be some mechanisms to inhibit excess secretion of TNF-α, which can exacerbate illness after RSV infection. Our previous data shows that TNF-αcan induce mucin 1, cell surface associated (MUC1) in epithelial cells, which is a kind of anti-inflammatory molecular. Interestingly, our recent data shows that MUC1 can inhibit TNF-αsecretion through suppression of TLRs signaling. We can easily image that a negative feedback regulation circle exists after RSV infection in airway to prevent exacerbated lung damage. The negative feedback regulation circle is, RSV infection in airway can induce TNF-αproduced by airway epithelial cells through the recognization and interaction between RSV and TLRs, up-regulated TNF-αcan interact with TNFR1 and up-regulate MUC1 expression. Inducible MUC1 can inhibit TNF-αproduction through blocking the signal pathway of TLRs, and prevent aggravating lung illness after RSV infection, finally.MUC1 is a member of the mucin family and encodes a membrane bound, glycosylated phosphoprotein. The protein is anchored to the apical surface of many epithelia by a transmembrane domain, with the degree of glycosylation varying with cell type. It contains extracellular domain, transmembrane domain and cytoplasmic tail. Extracellular domain also includes a 20 amino-acid-long variable number tandem repeat (VNTR) domain, with the number of repeats varying from 20 to 120 in different individuals and domain found in sea urchin sperm protein, enterokinase, agrin (SEA). MUC1 cytoplasmic tail has several motifs, which serve the interaction between phosphatidylinositol 3 kinase (PI-3K), growth factor receptor-bound protein 2 (GRB2) and so on. The protein serves a protective function by binding to pathogens and also functions in cell signaling transduction.In order to prove that MUC1 has the anti-inflammatory role after RSV infection, we designed the following experiments. The experiments contain three steps: First step, identify whether RSV can induce MUC1 expression and TNF-αrelease after infection.Confluent A549 cells were treated for 24 h with RSV (MOI=0,1.0 or 5.0) after serum starve 24 h. Total RNA was analyzed for the levels of MUCl mRNA by using real-time RT-PCR, and data were normalized to GAPDH mRNA levels.300μl supernatant was examined by ELISA for TNF-αrelease level. The results show that RSV can induce MUCl expression and TNF-αrelease.Second step, time course study of induction of MUC1 and TNF-αby RSV in A549 cells.Confluent A549 cells were treated with RSV (MOI=0 or 5.0) for 6,12,24,48 and 72 h after serum starve for 24 h.300μl supernatant was examined by ELISA for TNF-αexpression level. And equal protein aliquots of cell lysate were examined by ELISA, too. The induction of MUC1 expression is as early as 12 h after RSV infection, and it reaches the peak at 24 h after infection and keeps to 48 h, then the MUC1 expression level begins to go down at 72 h. TNF-αrelease increases at 6 h after RSV infection, even earlier (data not shown). And it reaches the peak at 24 h after infection, and then the level goes down at 48 h after infection, of which the time points are earlier than that of induction of MUC1 expression. It is just illustrated that RSV infection firstly increase the production of TNF-αof epithelial cells, then TNF-αinduce the expression of MUC1 in auto-secrete manner, at the end, increased MUC1 protein suppresses the TNF-αrelease, which is like a negative feedback regulation circle to reduce lung damage caused by excess TNF-α.Third step, the experiments of using sTNFR1, over-expression MUC1 and knocking-down MUC1 prove that MUC1 is an anti-inflammatory molecular during RSV infection.After we used sTNFR1 to block TNF-αfunction, we can see the decrease of MUC1 expression and increase of TNF-αrelease 24 h after RSV infection. This means that RSV induces MUC1 expression through TNF-α—TNFR1 axis. Over expressed MUC1 by transfecting pcDNA3.1-MUC1 into A549 cells can suppress the TNF-αrelease 24 h after RSV infection. And down-regulated MUC1 by transfecting MUC1-siRNA into A549 cells enhances the TNF-αrelease 48 h after RSV infection, when the TNF-αlevel should go down. In sum, MUC1 is an anti-inflammatory molecular during RSV infection by negative feedback regulation circle mechanism.