Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.

BACKGROUND: Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence. RESULTS: Coexpression of alphaT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with alphaT2ib indicated interaction of alphaT2ib with its cognate antigen within cells. alphaT2ib inhibited NF-kappaB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding alphaT2ib into HEK293 cells demonstrated high efficiency of the TLR2-alphaT2ib interaction. The alphaT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-alphaT2ib. Transduction with AdValphaT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFalpha mRNA accumulation, as well as TNFalpha translation and release by macrophages were largely abrogated upon transduction of alphaT2ib. alphaT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdValphaT2ib. Systemic transduction applying AdValphaT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. CONCLUSION: The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of alphaT2ib in microbial or viral infections.

Experimental models of acute infection and Toll-like receptor driven septic shock.

Mainly Gram-negative and Gram-positive bacterial infections, but also other infections such as with fungal or viral pathogens, can cause the life-threatening clinical condition of septic shock. Transgression of the host immune response from a local level limited to the pathogen's place of entry to the systemic level is recognised as a major mode of action leading to sepsis. This view has been established upon demonstration of the capacity of specific pathogen-associated molecular patterns (PAMPs) to elicit symptoms of septic shock upon systemic administration. Immune stimulatory PAMPs are agonists of soluble, cytoplasmic, as well as/or cell membrane-anchored and/or -spanning pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). However, reflection of pathogen-host crosstalk triggering sepsis pathogenesis upon an infection by a host response to challenge with an isolated PAMP is incomplete. Therefore, an experimental model more reflective of pathogen-host interaction requires experimental host confrontation with a specific pathogen in its viable form resulting in a collective stimulation of a variety of specific PRRs. This chapter describes methods to analyse innate pathogen sensing by the host on both a cellular and systemic level.

Human but not murine toll-like receptor 2 discriminates between tri-palmitoylated and tri-lauroylated peptides.

Toll-like receptors (TLRs) mediate activation of the immune system upon challenge with microbial agonists, components of disintegrating cells of the body, or metabolic intermediates of lipidic nature. Comparison of murine (m) and human (h) TLR2 primary sequences revealed 65% of identical residues within the extracellular domains in contrast to 84% in the intracellular domains. Comparative analysis of TLR2-driven cell activation by various TLR2 agonists showed that the tri-lauroylated lipopeptide analog (Lau(3)CSK(4)) is recognized efficiently through mTLR2 but not hTLR2. Genetically complemented human embryonic kidney 293 cells and murine TLR2(-/-) embryonic fibroblasts, as well as human and murine macrophage cells, were used for this analysis. In contrast to cellular activation, which depended on blockable access of the TLR2-ligand to TLR2, cellular uptake of Lau(3)CSK(4) and tri-palmitoylated peptide (P(3)CSK(4)) was independent of TLR2. A low-conserved region spanning from leucine-rich repeat (LRR) motif 7 to 10 was found to control TLR2 species-specific cell activation. Exchange of mLRR8 for hLRR8 in mTLR2 abrogated mTLR2-typical cell activation upon cellular challenge with Lau(3)CSK(4) but not P(3)CSK(4), implicating mLRR8 as a central element of Lau(3)CSK(4) recognition. The point mutation L112P within LRR3 abrogated hTLR2-dependent recognition of lipopeptides but merely attenuated mTLR2 function, whereas deletion of the N-terminal third of each LRR-rich domain (LRRs 1 to 7) had the opposite effect on P(3)CSK(4) recognition. Despite similar domain structure of both TLR2 molecules, species-specific properties thus exist. Our results imply distinct susceptibilities of humans and mice to challenge with specific TLR2 ligands.