Molecular cloning and characterization of a novel anti-TLR9 intrabody

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.


INTRODUCTION
Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that trigger the host defense against invading pathogens by recognizing pathogen-associated molecular patterns (PAMPs) [1,2]. TLR ligands are produced by viruses, pathogenic bacteria, pathogenic fungi and parasitic eukaryotes. TLRs 1, 2, 4, 5, 6 and 10 are expressed on the cell surface, while TLRs 3,7,8,9,11,12 and 13 are expressed endosomally [3]. TLR9, which recognizes DNA, resides in the endoplasmic reticulum (ER) constitutively. Endocytosed DNA resides in early endosomes and is subsequently transported to a tubular compartment. Concurrent with the movement of DNA in cells, TLR9 redistributes from the ER to endosomes [4]. Endosomal TLRs have been implicated in autoimmune disease pathologies, such as those of rheumatoid arthritis and systemic lupus erythematosus [5,6]. Dysregulation of TLR7/TLR9 signaling has been found to promote autoimmune disease [6]. The role of TLR9 in rheumatoid arthritis is not yet fully understood. Systemic injection of CpG ODN does sensitize mice, resulting in an exaggerated arthritis if the mice are subsequently challenged with an intra-articular injection of a low dose of CpG ODN [7]. However, the pathogenesis of arthritis in TLR9 -/mice was no different to that in wild-type mice, which indicates that this receptor does not have an obvious role in this model [8]. Inhibitors of TLR signaling targeting cell surface and endosomal receptors have been developed. For example, for cell surface receptors, there are neutralizing antibodies [9,10], chaperonin 10 [11] and small-molecule antagonists [12]. Endosomal receptors can be blocked by antimalarial chloroquine and short DNA segments (immunoregulatory sequences, IRS), among others [12,13]. IRS are suppressive oligonucleotides [14,15] that are often thought to lack exclusive specificity. Effects on other TLR-family members have been shown and some inhibitory DNA segments bind signal transducer and activator of transcription 1 (STAT1) and STAT4 [16]. Furthermore, GpG-containing inhibitory sequences also interact with TLRs 3, 7 and 8 [17].
To bypass any lack of specificity, we used an intrabody with intrinsic specificity for its target [18,19]. Intrabodies can be targeted to the nucleus, mitochondria or endoplasmic reticulum (ER), where they bind their target proteins and thus persistently inhibit their function. For instance, intrabodies targeted to the ER arrest transitory target proteins preventing their further subcellular trafficking [18,19]. Intrabodies are a promising alternative to RNA-based knockdown strategies in cases of failure of the latter [20][21][22]. We previously developed an anti-TLR2 intrabody which inhibited TLR2 ligand-driven cell activation in vitro and in vivo [23]. Endosomal TLRs (including TLR9) that cannot be reached by normal antibodies due to their endosomal location are ideal targets for ER intrabodies. Here, we describe the generation and characterization of an anti-TLR9 scFv ER intrabody (αT9ib). αT9ib bound specifically to TLR9 and co-localized together with TLR9 inside the ER. As a consequence, TLR9-specific activation of RAW264.7 macrophages was inhibited. Therefore, this new intrabody appears to be a powerful tool for the inhibition of TLR9 function.

Construction of an adenoviral vector encoding αT9ib, production of the recombinant virus and infection in vitro
An adenoviral vector to carry a bicistronic expression cassette driving expression of the ER intrabody αT9ib and the reporter gene eGFP was constructed via two subcloning steps. In the first step, the coding sequence of the anti-TLR9 ER intrabody, including the ER signal peptide, myc-tag and ER retention signal, was amplified via PCR from the pCMV/myc/ER vector carrying the cds of αT9ib using the primers TLR9-ERRS-BACK-phosph: 5'-ATGGGATGGAGCTGTATCATCCTC-3' and TLR9-ERRS-FOR-phosph: 5'-CTACAGCTCGTCCTTCTCGCTTGC-3'. The amplicon was ligated into the SmaI linearized vector pGEM/IRES/EGFP [27] containing the IRES sequence of the polio virus followed by the reporter gene eGFP. After transformation in E. coli DH5α, positive clones (pGEM/αT9ib/IRES/EGFP) containing the bicistronic expression cassette of the αT9ib gene and the reporter gene eGFP served as templates in the second step to derive AdVαT9ib particles and infect RAW264.7 cells [23]. RAW264.7 cells were infected with recombinant adenovirus at a multiplicity of infection (moi) of 50 for AdVGFP, AdVαT2ib and AdVαT9ib. The adenoviral vector contains the genome of a replication-deficient adenovirus type 5 subgenus C in which the E1 and E3 region is deleted [27].
NF-κB-dependent luciferase assay in HEK293 cells overexpressing specific TLRs and αT9ib 3 x 10 4 human embryonic kidney (HEK293) fibroblastoid cells were seeded in each well of a 96-well cell culture plate and transfected with plasmids directing constitutive expression of mTLR9 or other TLRs, αT9ib or control intrabody (anti-VEGFR 2 intrabody scFv A7, αVR-ib [25]) and Renilla luciferase, as well as NF-κB-dependent expression of firefly luciferase. Challenge with TLR agonists and the assay itself were performed as described [23].
Visualization of intracellular accumulation of TNFα by flow cytometry RAW264.7 macrophages were co-transfected with expression plasmids for eGFP and αT9ib or control plasmid pCMV/myc/ER. After 4 days, the cells were challenged with TLR agonists and intracellular flow cytometry was performed according to the method described in [23]. Intracellular TNFα in GFP-positive cells was detected with allophycocyanin-labeled rat anti-mouse TNFα antibody (BD Biosciences, clone MP6-XT22). Flow cytometry analyses were performed using a FACS-Calibur equipped with CellQuest software (Becton Dickinson).

ELISA
TNFα concentration was determined using a mouse TNFα ELISA Kit (BD Biosciences, BD OptEIA) according to the manufacturer's instructions.

Construction of the anti-TLR9 scFv intrabody-coding sequence
Amplification of a DNA fragment encoding the variable domain of the heavy chain (VH) of the hybridoma 5G5 resulted in a 386-bp PCR product. The corresponding variable domain of the light chain (VL) encompassed 329 bp. The linker-encoding fragment VH-(G 4 S) 3 -VL encompassed 95 bp. PCR assembly of VH, VL and the synthetic linker-encoding fragments resulted in a 754-bp construct (Fig. 1A). Ligation of the anti-TLR9 scFv DNA into the plasmid pCMV/myc/ER resulted in an anti-TLR9 intrabody (αT9ib) construct comprising a myc-tag and the ER retention sequence fused to the 3' terminus of the anti-TLR9 scFv sequence (Fig. 1B).
Co-localization of αT9ib and TLR9 inside the ER compartment and intracellular binding of αT9ib to murine TLR9 Co-localization of αT9ib and mTLR9 was visualized by immunofluorescence microscopy ( Fig. 2A). Both resided within a lattice structure identical with the ER compartment. Co-staining of αT9ib, mTLR9 and ER resident marker calnexin indicated localization of αT9ib and mTLR9 inside the ER (upper panel). Co-transfection with cherry-CD63 (endosomal marker) expression plasmid showed no localization of αT9ib and mTLR9 inside the endosomal compartment (lower panel). Specific intracellular binding of mTLR9 and αT9ib was further verified by co-immunoprecipitation performed with anti-HA antibody and with anti-myc antibody (Fig. 2B). Lack of immunoprecipitation of mTLR9 with the control intrabodies αT2ib and αVR-ib in lysates of HEK293 cells expressing respective protein pairs demonstrated αT9ib specificity for its cognate antigen.

Functionality of αT9ib upon transient overexpression in HEK293 cells
In order to analyze αT9ib function, NF-κB-driven reporter gene activity was determined in HEK293 cells overexpressing both mTLR9 and αT9ib upon TLR9 specific challenge. Co-transfection of αT9ib and mTLR9 expression plasmid DNA effectively inhibited TLR9 activity (Fig. 3A). By contrast, cellular activation through either TLR4 or TLR3 was not influenced by co-expression of αT9ib. Moreover, another ER intrabody (αVR-ib) recognizing the vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) did not block TLR9 activation. Immunofluorescence staining of HEK293 cells co-transfected with αT9ib and mTLR9 expression plasmids demonstrates co-expression of both proteins in almost every transfected cell (Fig. 3B).

Fig. 1. Assembly and primary sequence of human/murine TLR9 cross-reactive intrabody.
A -PCR amplified variable domain encoding sequences of mAb 5G5, the linker DNA, and the product of the assembly PCR. The amplification products were separated using agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Molecular weights 386 and 329 bp respectively correspond to the expected size of the variable domains of the immunoglobulin G (IgG) heavy (VH) and light chain (VL), 95 bp to the amplified linker DNA fragment (L) and 754 bp to the expected apparent size of the product of scFv assembly PCR. B -Primary sequence of human/murine TLR9-crossreactive intrabody. The coding (lower line) and amino acid (upper line; ###: stop codon) sequences of αT9ib are shown, including the ER signal peptide, the myc-epitope, and the ER retention sequence, which are all shown in green. The complementarity-determining regions (CDR1-CDR3) of the variable domains of the heavy and light chain are printed in bold red. The synthetic linker (shown in bold blue italic letters), localized between the VH and VL domains, was introduced by assembly PCR. SalI and NotI restriction sites were used to clone the anti-TLR9 scFv fragment into pCMV/myc/ER. Fig. 2. Subcellular co-localization and co-immunoprecipitation of αT9ib and murine TLR9 upon transfection. A -Immunofluorescence analysis by laser scanning confocal microscopy of fixed and permeabilized HEK293 cells transiently transfected with the mTLR9-myc expression plasmid, HA-tagged αT9ib and cherry-CD63 (endosomal marker). Expression of mTLR9-myc was visualized using FITC-labeled goat anti-myc antibody. Expression of HA-tagged αT9ib was detected using rabbit anti-HA antibody and Cy5-labeled goat anti-rabbit antibody. The expression of calnexin was visualized using anti-calnexin antibody and Cy3-conjugated goat anti-mouse antibody. Scale bar: 10 m. B -For immunoprecipitation, 1 x 10 6 HEK293 cells were co-transfected with HA-tagged mTLR9 expression plasmid together with myc-tagged αT2ib expression plasmid, myctagged αVR-ib expression plasmid, or myc-tagged αT9ib expression plasmid. After 72 h, the cells were lysed and incubated overnight with rabbit anti-HA-agarose-conjugated antibody (IP αHA, left) or anti-myc antibody (IP αmyc, right) for 1 h, followed by incubation with Protein G PLUS Agarose overnight. The precipitates were analyzed using 10% SDS-PAGE and probed either with mouse anti-myc and goat anti-mouse PO-labeled antibodies to visualize ib-myc (lower panel) or with rabbit anti-HA and goat anti-rabbit PO-labeled antibodies to detect TLR9-HA (upper panel). Non-precipitated cell lysates that were single-transfected with HA-tagged mTLR9 expression plasmid or myc-tagged αT9ib expression plasmid were used as positive controls. Lysate with HA-tagged mTLR9 was used as a negative control for detection with anti-myc antibody and vice versa.

Inhibition of TLR9-specific signal transduction in RAW264.7 macrophages through αT9ib expression
To demonstrate that αT9ib inhibits TLR9-specific signaling in macrophages, the αT9ib expression plasmid DNA was co-transfected with an eGFP expression plasmid into RAW264.7 macrophages that were challenged with either LPS, Fig. 3. Inhibition of TLR9 activity upon transient overexpression of αT9ib and murine TLR9 in HEK293 cells and inhibition of intracellular accumulation of TNFα in RAW264.7 macrophages transfected with αT9ib. A -Assay of NF-κB-driven reporter gene activation in HEK293 cells transiently co-expressing mTLR9, mTLR4 or hTLR3 and αT9ib or αVR-ib. The cells were respectively stimulated with CpG DNA, LPS or Poly I:C. The results are presented as the mean ± SD for one representative experiment out of 3 independent iterations. B -HEK293 cells were co-transfected with αT9ib and HA-tagged mTLR9 and stained for immunofluorescence with mouse anti-myc antibody and Cy3labeled goat anti-mouse antibody or rabbit anti-HA antibody and FITC-labeled goat antirabbit antibody. C -RAW264.7 macrophages were co-transfected with eGFP expression plasmid and pCMV/myc/ER vector or eGFP expression plasmid and αT9ib expression plasmid or αT2ib expression plasmid as a control. Four days after transfection, the cells were stimulated with LPS (100 ng/ml), Pam 3 CSK 4 (100 ng/ml), CpG-oligonucleotide 1826 (1 µM), CpG-oligonucleotide 1668 (1 µM) or R848 (10 µg/ml). The cells were fixed and permeabilized, and the intracellular TNFα was analyzed in GFP-positive cells using flow cytometry with allophycocyanin-labeled rat anti-mouse TNFα.
Pam 3 CSK 4 , CpG ODN 1826, CpG ODN 1668, or R848. TNFα production was determined by intracellular flow cytometry in GFP-positive intrabodyexpressing cells (Fig. 3C). αT9ib inhibited intracellular accumulation of TNFα upon ODN 1826 or 1668 challenge, whereas TLR2-, TLR4-and TLR7-specific activation induced by Pam 3 CSK 4 , LPS or R848 was not affected. To increase the transduction efficiency of αT9ib in macrophages, we constructed an adenoviral vector and generated AdVαT9ib particles. Myc-tag-specific immunoblot analysis of RAW264.7 cell lysates upon infection with AdVαT9ib or AdVαT2ib revealed an apparent size of adenovirally transduced αT9ib of approximately 30 kDa (Fig. 4A). Simultaneous expression of αT9ib and bicistronic eGFP was demonstrated by immunofluorescence microscopy (data not shown). Furthermore, eGFP expression was analyzed in RAW264.7 cells by flow cytometry. Macrophages were transduced equally effectively with the adenoviral construct AdVαT9ib and the control AdVGFP (Fig. 4B). Infection rates ranged from 70-80%. After 10 days, a maximum of eGFP-expressing cells was observable and the amount of living cells ranged from 80-90%. The recombinant AdVαT9ib particles were applied to demonstrate that αT9ib inhibits TNFα secretion from RAW264.7 macrophages upon challenge with CpG DNA (Fig. 4C). Non-infected cells and AdVGFP-and AdVαT9ib-infected cells responded almost equally to TLR2-or TLR4-specific challenge. The cells released TNFα in substantial amounts compared to the controls. In AdVGFPinfected cells, the release of TNFα was reduced to some extent upon challenge with CpG DNA. By contrast, cells that were infected with AdVαT9ib were mostly unable to respond to a TLR9-specific challenge.

DISCUSSION
Toll-like receptors play a central role in the development and sustainment of chronic inflammatory diseases [28]. Specific research focuses on finding TLR antagonists as novel therapeutics, for example against systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease and rheumatoid arthritis, in which the immune system is inappropriately overactive [12]. To inhibit intracellular TLRs, which cannot be targeted by classical mAbs, antimalarial drugs and short DNA sequences are used, but they often lack sufficient specificity [12,16,17]. By contrast, ER-retained intrabodies do act specifically [18] and are capable of persistently blocking receptor functions if the intrabody gene is stably expressed upon retroviral or lentiviral virus infection [29,30]. Here, we developed an ER intrabody (αT9ib) for the specific knockdown of the intracellularly localized TLR9. To the best of our knowledge, this is the first description of an ER-targeting intrabody that inhibits the function of an intracellularly localized receptor. αT9ib was constructed from the variable domains of mAb 5G5 recognizing both human and murine TLR9 [24]. Functional analysis of αT9ib demonstrated efficient and highly specific inhibition of TLR9 signaling in HEK293 cells overexpressing mTLR9 and in RAW264.7 macrophages (Figs 3 and 4C) due to the retention of TLR9 inside the ER by αT9ib. Complex formation of both molecules inside the ER was clearly demonstrated (Fig. 2A). For efficient transduction of RAW264.7 macrophages and for future experiments with mice, we subcloned the αT9ib expression cassette into an adenoviral vector that drives bicistronic expression of eGFP and αT9ib. Cellular release of TNFα from AdVαT9ib-infected RAW264.7 macrophages upon TLR9specific challenge with CpG ODN 1668 and 1826 was predominantly blocked to a level similar to the background level of unstimulated cells. Interestingly, AdVGFP-infected cells also showed a decreased release of TNFα. Referring to this, it has been demonstrated that adenoviruses may induce TLR9-dependent TNFα secretion in macrophages [31]. This might lead to a lower potential of AdVGFP-infected cells to respond to a TLR9-specific challenge, although a higher release of TNFα in unstimulated cells would be expected in this case. Other possibilities would include the influence of eGFP overexpression on TLR9 signaling. Our data show for the first time the highly specific and efficient neutralization of nascent TLR9 in the ER by an ER intrabody. Therefore, αT9ib represents a very promising experimental tool to study TLR9-driven cell activation. For example, αT9ib might be used to study the effect of TLR9 signaling on the function of specific immune cells. We have started to test the capability of αT9ib to interrupt the inflammatory reactions leading to chronic inflammatory diseases in appropriate experimental mouse models.