An inappropriately mounted or dysregulated immune response can cause considerable morbidity and mortality in a number of diseases. One such example is sepsis, which is among the most common causes of death in the United States, with over 750,000 cases presenting annually, of which more than one-quarter are fatal (36
). Excessive inflammation is the hallmark of a number of related infectious pathologies as well, including sepsis, acute respiratory distress syndrome (ARDS) and multiple organ failure (37
). LPS derived from bacterial sources can contribute to these diseases, and does so by interacting with MD-2 and TLR4. To circumvent an overactivated host immune response to LPS, it is imperative that TLR4 signal transduction be tightly regulated, but the precise molecular mechanisms by which this is accomplished are only partly understood.
Here we further elucidate the complexities involved in averting a prolonged and dysregulated immune response to LPS by the identification of a naturally occurring alternatively spliced isoform of human MD-2, which we have termed MD-2s. We report that human MD-2s is generated by skipping exon 2 of the MD-2
gene, which leads to an in-frame deletion of 30 amino acids spanning positions 39–69, and one amino acid substitution (D38G). Under similar conditions and using primers specific to the murine MD-2
gene, we could not detect a corresponding murine splice variant. A prior study reported an alternatively spliced version of murine MD2 (MD-2B) (21
), which lacks the first 54 bases of exon 3 and downregulates LPS signaling. However, there are no data provided as to whether it is secreted or inducible and the human relevance is unknown (see for comparison between MD-2s and MD-2B).
Comparison of the MD-2 splice variants, MD-2s and MD-2B.
Our results identify MD-2s as an important negative regulatory component of the TLR4 signaling pathway. The mRNA expression profile of MD-2s in human tissues revealed that it is ubiquitously expressed, suggesting that this isoform may perform a widespread role in modulating TLR4-mediated responses. Previous studies have shown that IFN-γ exerts anti-inflammatory responses by inducing specific secreted inhibitors, examples include the IL-1 receptor antagonist (IL-1Ra) (38
) and IL-18 binding protein (IL-18BP) (39
). Both molecules suppress the activity of IL-1 and IL-18 respectively. Many LPS-inducible negative regulators have also been identified, such as smTLR4 and MyD88s. We determined that MD-2s is upregulated in response to IFN-γ, IL-6 and LPS, indicating that MD-2s may be a key component involved in the negative regulation of TLR4 signaling.
Similar to full-length MD-2, MD-2s is a secreted glycoprotein. However, MD-2s overexpression failed to trigger NF-κB activation and IL-8 secretion following LPS treatment, indicating the importance of exon 2 for MD-2 function. Importantly, we also observed that MD-2s negatively regulated both NF-κB activation and IL-8 secretion following LPS stimulation, suggesting that MD-2s may be used as a therapeutic or preventative agent for modulating endotoxemia. Whereas the murine isoform, MD-2B, inhibited TLR4 from being expressed on the cell surface (21
), MD-2s is anchored to the cell surface of TLR4 expressing cells, and both proteins localize together on the cell membrane. We also determined that MD-2s and TLR4 immunoprecipitated together. This may have been predicted, given that MD-2s retains most of the residues reported to be essential in mediating a MD-2-TLR4 interaction, with the exception of I66 and R68 (31
). In addition, MD-2s immunoprecipitated with LPS. This is consistent with a study demonstrating that a 15 residue peptide fragment of MD-2, encompassing the F126 loop (positions 119–132), still binds LPS (41
). Additional studies have verified that residues within this fragment of MD-2 are essential for LPS binding (34
). This region is preserved in MD-2s and most likely confers the ability of MD-2s to associate with LPS. Furthermore, although the hydrophobic pocket of MD2-s is predicted to be disrupted, with the exception of K58 all the MD-2 residues that have been shown to be involved in the main dimerization interface of the TLR4-MD-2-LPS complex, are preserved in MD-2s. Whilst it appears that these residues are sufficient to form an effective interaction between MD-2s and LPS, the binding affinity is most likely affected.
Several studies have shown that negative regulators can control TLR signal transduction by inhibiting the formation of active signaling complexes, including the recently identified splice variant TAG (20
), IRF-4 (43
), RP105 (44
) and the IL-1Ra (45
). Based on our results, we propose that MD-2s functionally modulates TLR4 signaling by inhibiting the formation of an active MD-2-TLR4 signaling complex. In addition, it is possible that MD-2s may behave like a decoy co-receptor by binding LPS and TLR4 to form a non-functional complex that does not activate NF-κB, thereby negatively regulating signaling.
Collectively our results define an important mechanistic role for MD-2s in modulating the LPS-TLR4 signal transduction pathway at the initial phase of activation. MD-2s therefore represents a prospective target for pharmacological intervention and development of new therapeutic and preventive strategies for sepsis and other diseases resulting from an over-exuberant MD2-TLR4-induced immune response.