WO2012016973A1 - Allergènes de contact - Google Patents

Allergènes de contact Download PDF

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WO2012016973A1
WO2012016973A1 PCT/EP2011/063271 EP2011063271W WO2012016973A1 WO 2012016973 A1 WO2012016973 A1 WO 2012016973A1 EP 2011063271 W EP2011063271 W EP 2011063271W WO 2012016973 A1 WO2012016973 A1 WO 2012016973A1
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htlr4
tlr4
tag
hmd2
sequence
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Marc Schmidt
Matthias Goebeler
Raghavan Badrinarayanan
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Transmit Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • Ni 2+ nickel
  • CHS contact hypersensitivity
  • transgenic hTLR4 expression in 77r4-deficient mice allowed efficient sensitization to Ni 2+ and elicitation of CHS.
  • Our data implicate site-specific hTLR4 inhibition as potential strategy for therapeutic intervention without affecting vital immune responses.
  • Ni 2+ nickel
  • ACD allergic contact dermatitis
  • ACD is based on a contact hypersensitivity (CHS) reaction involving antigen presentation by dendritic cells (DCs) and generation of a T lymphocyte response.
  • CHS contact hypersensitivity
  • Ni 2+ can directly trigger activation of proinflammatory intracellular signal transduction cascades that result in stimulation of the transcription factor nuclear factor- ⁇ (NF- ⁇ ) and the mitogen-activated protein (MAP) kinase p38 8 ' 9 .
  • NF- ⁇ transcription factor nuclear factor- ⁇
  • MAP mitogen-activated protein
  • the molecular mechanisms underlying generation of the proinflammatory signal are unknown.
  • Gene profiling studies revealed that Ni 2+ elicits an expression pattern reminiscent of innate immune signals, which is dominated by proinflammatory genes 10 . In most cases, the initiation of such signals relies on membrane-bound and intracellular receptors known as pattern recognition receptors (PRRs).
  • PRRs pattern recognition receptors
  • TLR Toll-like receptor
  • Ni 2+ directly activates proinflammatory intracellular signal transduction cascades, resulting in stimulation of the transcription factor NF- ⁇ (Fig. 1a).
  • NF- ⁇ transcription factor 8
  • CCL2 also known as monocyte-chemoattractant protein-1
  • IL-I interleukin 1
  • RNA interference RNA interference
  • Ni 2+ activates hTLR4, but not mTLR4
  • HEK293 cells expressing hTLR4 were transfected with either hMD2 or mMD2. Irrespective of the origin of the transfected MD2 co-receptor, hTLR4 was able to mediate Ni 2+ signals (Fig. 3b), indicating that the observed differences of the Ni 2+ responses between both species may depend on sequence variations in the TLR4 protein. We predicted that most likely non-conserved sequence motifs of hTLR4 are required for recognition of Ni 2+ signals.
  • Ni 2+ cross-links the two receptor monomers via specific histidine side chains, triggering formation of a dimer that structurally resembles the one induced by LPS 16 (Fig. 4b, Supplementary Fig. 5, Figure 11 ).
  • hTLR4 expression transfers Ni sensitivity to mouse
  • mice Animals from the resulting mouse line exhibited cell-specific expression and organ distribution of the hTLR4 transgene, typical for mTLR4 in wild- type mice and showed a mild over expression in relevant cells such as monocytes and macrophages that was comparable to previously described Tlr4 ⁇ ' ⁇ Tlr4 transgenic (TG) mice 20 (Supplementary Fig. 6, Figure 12 and unpublished data). Bone marrow-derived macrophages from Tlr4 ⁇ ' ⁇ TLR4-TG animals were responsive to LPS and Ni 2+ producing substantial amounts of TNF (Fig. 6a), IL-1 ⁇ , IL-6 and IL- 10 (Supplementary Fig. 7a, Figure 13a).
  • Ni 2+ -mediated responses such as phosphorylation of p38 MAP kinase and interferon regulatory factor (IRF) 3 and synthesis of interferon (IFN) / were only observed in cells obtained from Tlr4 ⁇ ' ⁇ TLR4-TG mice, but not from Tlr4 ⁇ ' ⁇ Tlr4-TG mice (Supplementary Fig. 7b, c, Figure 13b, c).
  • IRF interferon regulatory factor
  • IFN interferon
  • Tlr4 ⁇ ' ⁇ TLR4-TQ or Tlr4 ⁇ ' ⁇ Tlr4-Tg mice were sensitized to Ni 2+ (or PBS as control) and challenged with the contact allergen 1 1 days later. Twenty-four hours after elicitation, ear thickness was determined as the classical measure of a murine CHS reaction (Fig. 6b). Tlr4 ⁇ ' ⁇ TLR4-TG mice sensitized to Ni 2+ developed significant ear swelling upon elicitation with Ni while mock-sensitized animals or Tlr4 Tlr4-TG mice did not.
  • Tlr4 ⁇ ' ⁇ ⁇ /./74-transgenic mice consistently exhibited considerable leukocyte infiltration into the skin at sites of allergen challenge (Fig. 6c). By contrast, mock-treated animals did not respond.
  • hTLR4 as a crucial receptor for Ni 2+ that allows generation of a proinflammatory signal (Supplementary Fig. 8, Figure 14) and provide a novel mouse model of contact allergy to this abundant allergen.
  • Ni 2+ is the first allergen identified to directly trigger PRR signaling.
  • NF- ⁇ -, p38- and IRF3-dependent signal transduction cascades are activated that result in the expression of multiple proinflammatory genes.
  • Ni 2+ -induced proinflammatory responses are species- specific and require non-conserved histidines present in hTLR4 but not mTLR4.
  • the mechanism outlined here fundamentally differs from the recently described interaction of the major house dust mite allergen Der p 2 with TLR4 24 .
  • Der p 2 functionally mimics MD2 and critically requires the presence of LPS to trigger TLR4 signaling. It thus rather acts as sensitizer for LPS, while Ni 2+ is a direct activator of TLR4 signaling.
  • our identification of a unique Ni 2+ -responsive region in hTLR4, which is distinct from its LPS-binding domain implies possibilities to specifically interfere with Ni 2+ -induced TLR4 signaling without affecting LPS responses.
  • FIG. 1 The contact allergen Ni 2+ activates primary cells in a MyD88-hTLR4- dependent manner, (a) Fold average luciferase activation of a 6 ⁇ luciferase (luc) reporter transfected into primary human umbilical vein endothelial cells (HUVEC). Cells were stimulated for 16 h with Ni 2+ (1 .5 imM), TNF (2 ng ml "1 ) or medium as control (Ctrl), (b) Immunoblot of IL-8 expression upon stimulation with Ni 2+ , I L- 1 ⁇ and TNF in HUVEC transfected with siRNA for hMyD88 (MyD88) or a scrambled siRNA control (Scr). (c).
  • IL-8 production determined by ELISA of supernatants ⁇ left) or immunoblot of cell lysates ⁇ right) in HUVEC stimulated with Ni 2+ or LPS (E. coll 026:B6; ⁇ g ml "1 ), (d) Immunoblot of IL-8 protein expression ⁇ lower) and flow cytometric analysis of surface hTLR4 expression ⁇ upper, bold lines) in comparison to isotype staining (thin lines) in wild-type and hTLR4-hMD2-expressing HEK293 following Ni 2+ and LPS stimulation. Cells were incubated with the specified stimuli for 8 h unless indicated otherwise. Bar diagrams in (a) and (c) represent average values of three independent experiments ⁇ s.d.
  • Ni 2+ -induced gene expression requires sequence motifs present in hTLR4 but not in mTLR4.
  • TNF release determined by ELISA of primary human monocytes ⁇ left) or bone marrow macrophages from C57BI/6 wt mice ⁇ right) exposed to Ni 2+ or LPS ⁇ Salmonella minnesota R595; 1 ⁇ g ml "1 ) for 8 h.
  • Non-conserved histidines in hTLR4 provide a potential binding site for Ni 2+ .
  • (a) Protein sequence alignment of hTLR4 and mTLR4 between aa 371 -604 covering the leucine-rich repeats (LRR) 14-22 located in the C-terminal domain of the TLR4 ⁇ sheet 16 .
  • conserveed histidine residues (H) are shown underlined and in bold letters, non-conserved histidines are indicated by a box.
  • the model is based on the crystal structure of the hTLR4- hMD2 complex (PDB entry 3FXI) with LPS omitted.
  • the side chains of the histidine residues H431 , H456 and H458 (depicted as sticks, with nitrogen shown in dark grey within the ring-like structures) were repositioned with appropriate side chain rotamers, followed by addition of Ni 2+ ions (spheres) to yield a pair of metal-binding sites with a geometry similar to the one observed in an engineered serine protease/inhibitor complex 25 [PDB entry 1 SLW].
  • FIG. 5 Histidines H456 and H458 in hTLR4 are required for Ni 2+ -induced proinflammatory gene expression
  • hTLR4(H456A), hTLR4(H458A) or hTLR4(H456A-H458A were stimulated with Ni + or LPS ⁇ Salmonella minnesota R595).
  • Data in (a) and (b) are representative of three independent experiments, (c) IL-6 (left) or IFN- ⁇ / ⁇ (right) cytokine production of Ni 2+ -stimulated (6h) mouse macrophages retrovirally infected to stably express wild type hTLR4, hTLR4H(456A-H458A) or empty vector as control.
  • Data represent mean values ⁇ s.d. of two independent experiments, (d) Luciferase activity of a transfected 6KB-IUC reporter in HEK293-hMD2 cells transfected with hTLR4, hTLR4(H456A-H458A), mTLR4 or mTLR4Y(454H-N456H).
  • RECTIFIED SHEET (RULE 91) ISA/EP ELISA analysis of Ni - or LPS (Salmonella minnesota R595) -induced TNF protein production by macrophages obtained from transgenic mice expressing hTLR4 [left) or mTLR4 ⁇ right) on a C57BL/10ScCr background. Data represent means of three independent experiments ⁇ s.d. (b) Ear thickness measurements of wild type mice or mice expressing transgenic mTLR4 or hTLR4 on a C57BL/10ScCr background. Mice were intracutaneously sensitized at day 0 with Ni 2+ or PBS (mock) at the abdominal wall and challenged with Ni 2+ on both ears at day 1 1 .
  • the problem of the underlying invention is to foresee a new method and a new class of substances to treat and or reduce hypersensivity (CHS) with mammals, especially humans to nickel, especially Ni 2+ .
  • CHS hypersensivity
  • Ni 2+ induces an inflammatory response via direct activation of human Toll-like receptor 4 (hTLR4).
  • hTLR4 human Toll-like receptor 4
  • mTLR4 mouse TLR4
  • mutant TLR4 proteins revealed a requirement of the non-conserved histidines 456 and 458 of hTLR4 for activation by Ni 2+ but not by the natural ligand
  • transgenic hTLR4 expression in 77r4-deficient mice allowed efficient sensitization to Ni 2+ and elicitation of CHS.
  • our Tlr4 TLR4-TG mice provide the first mouse model that allows the study of Ni 2+ -induced CHS by Ni 2+ treatment alone, which more closely resembles the human situation.
  • Such an adjuvant-free model should facilitate further research on the mechanisms of Ni 2+ -induced contact allergy without obscuring effects of additives.
  • HEK293 wild type cells or HEK293 cells stably expressing hTLR4 and/or hMD2 were cultured as described 10, 26, 27 .
  • Blood monocytes, bone marrow-derived macrophages, dendritic cells (DCs), splenic macrophages and myeloid DCs from various mouse strains were prepared, cultured and characterized by flow cytometry as earlier reported 21 ' 28"30 .
  • N1CI2 6H2O (referred to as Ni 2+ ) was purchased from Merck. Endotoxin contaminations were excluded by limulus amebocyte lysate assay (BioWhittaker) and blocking experiments using polymyxin B sulphate (Sigma). LPS from S. minnesota R595 was purchased from Alexis, LPS from E. co// 055:B5 from Sigma.
  • IRAKI -DD encoding bp 83-544 of the human IRAKI gene 31 and acting as dominant negative mutant was transfected into HUVEC using nucleofection (Amaxa ® , Lonza Cologne).
  • Expression vectors for hMD2 and mMD2 were provided by Dr. M. Muroi, National Institute of Health Sciences, Tokyo, Japan 32 .
  • Chimeric hemagglutinin (HA)-tagged TLR4 receptor genes in which coding sequences for distinct domains of hTLR4 and mTLR4 were exchanged were generously provided by A. M. Hajjar, Department of Immunology, University of Washington Medical School, Seattle, WA 15 .
  • Plasmids were transiently transfected into HEK293 cells according to standard protocols.
  • the composition of expressed receptors was: hTLR4, aa 27-839; mTLR4, aa 26-835; hhm, hTLR4 aa 27-616 and mTLR4 aa 614-835; mmh, mTLR4 aa26-613 and hTLR4 aa 617-839; hmh, hTLR4 aa 27-286, mTLR4 aa 286-613, and hTLR4 aa 617-839; hmm, hTLR4 aa 27-286, mTLR4 aa 286-835; hmhh, hTLR4 aa 27-286, mTLR4 aa 285-366, and hTLR4 aa
  • Mutant TLR4 receptors with single or double point mutations were generated by site-directed mutagenesis (Quick-change II site- directed mutagenesis kit , Stratagene) and verified by sequencing: hTLR4(H431A), hTLR4(H456A), hTLR4(H458A), hTLR4(H456A-H458A), hTLR4(H431 A-H456A), hTLR4(H431 A-H458A), mTLR4(Y454H-N456H).
  • Retroviral gene transfer Selected hTLR4 constructs were cloned into the pMYs- IRES-GFP retroviral vector and transiently transfected into PlatE packaging cells. Clarified and filtered supernatants containing retroviral particles were used directly to infect mouse macrophages in the presence polybrene.
  • RNA interference HUVEC were transfected with validated small interfering RNA (siRNA) from Qiagen (siRNAs against hMyD88 (SI000300909), hTLR4 (SI100151004), hMD2 (SI03246446) or scrambled siRNA) at a final concentration of 200 nM using Oligofectamine (Invitrogen Life Technologies).
  • siRNA small interfering RNA
  • HUVEC were transiently transfected with 6KB-IUC and ubiquitin-dependent Renilla luciferase as described 10 .
  • Luciferase activity was measured using the DualGlo Luciferase Assay System (Promega). ⁇ -dependent luciferase activities were normalized to the luminescence generated by the Renilla luciferase control reporter and expressed as fold stimulation.
  • cytokines cytokines Detection of cytokines.
  • Cell culture supernatants were assayed for human IL-8 or TNF and mouse IL-6 or IL-10 using ELISA kits from BD Biosciences or eBioscience.
  • ELISA kits from BD Biosciences or eBioscience.
  • murine IL-1 ⁇ macrophages lysates were analysed by ELISA (eBioscience).
  • Mouse TNF and IFN- / ⁇ were determined using bioassays as described 30 .
  • mice Generation of hTLR4 transgenic mice.
  • Mice expressing hTLR4 on a Tlr4 '/' background were constructed by pronuclear injection of fertilized eggs derived from the C57BL/10ScCr line 19 with the TLR4 BAC clone 152C16 described by and obtained from A. Poltorak and B. Beutler 35 . Bacterial vector sequences were removed before injection.
  • the BAC clone contains approximately 100 Kb of human genomic sequences beginning in the region between 14 Kb and 30 Kb upstream of the major transcriptional start site of TLR4, extending through the 12 Kb genomic region of TLR4 containing all exons and ending in a region between 41 Kb and 91 Kb downstream of the TLR4 polyA site.
  • mice Expression of the transgene in mice was monitored by RT-PCR using total RNA extracted from various organs (data not shown). Furthermore, surface expression of hTLR4 on macrophages, Mac-1 + blood leukocytes and B lymphocytes but not T lymphocytes obtained from transgenic mice was confirmed by flow cytometry. The introduction of the TLR4 transgene conferred LPS sensitivity to C57BL/10ScCr mice as verified by cytokine production (TNF, IL-6) following LPS treatment of mice in vivo or mouse macrophages in vitro and mitogenic activity after treatment of splenic B lymphocytes in vitro (data not shown).
  • TNF cytokine production
  • VogI, T. et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat. Med. 13, 1042-1049 (2007).
  • Muroi, M., Ohnishi, T. & Tanamoto, K. MD-2 a novel accessory molecule, is involved in species-specific actions of Salmonella lipid A. Infect. Immun. 70, 3546-3550 (2002).
  • Figure 7 Expression of a dominant negative mutant of IRAKI .
  • IRAKI -DD abrogates Ni 2+ -induced expression of the NF- ⁇ target gene CCL2.
  • Filled profiles represent CCL2 expression, empty profiles isotype controls. Cells have been gated on co-transfected green fluorescent protein to control for efficient transfection. Data are representative of two independent experiments.
  • Figure 8 Proinflammatory activation by Ni 2+ requires hTLR4.
  • Figure 11 Structural modeling of Ni 2+ interaction with hTLR4. Modeling of the (Ni 2+ ) 2 -(hTLR4) 2 complex in ribbon representation (side view) with the putative positions of the bound Ni 2+ molecules indicated. The model is based on the crystal structure of the hTLR4-hMD2 complex (PDB entry 3FXI) with LPS omitted. Molecular modeling and graphics preparation were performed using PyMOL software.
  • FIG 12 (Supplementary Figure 6) Expression of TLR4 in transgenic Tlr4 " " TLR4- TG and Tlr4 " " Tlr4-TG mice and human peripheral blood monocytes, (a) Flow cytometry analysis of surface hTLR4 expression on human peripheral blood monocytes (CD14 hl ), murine peripheral blood monocytes (F4/80 hl ) and purified bone marrow (BM)-derived macrophages from Tlr4 " " TLR4-TG mice. Numbers in the displayed histogram overlays represent ratios of geometric mean fluorescence intensity (GMFI) obtained for the indicated or-hTLR4 stained population (black lines) and its corresponding IgG control (grey lines).
  • GMFI geometric mean fluorescence intensity
  • FIG. 13 (Supplementary Figure 7) Ni 2+ -induced activation of murine macrophages, (a-c) Bone marrow-derived macrophages obtained from transgenic mice expressing either hTLR4 (left) or mTLR4 (right) on a C57BL/10ScCr background were exposed to 1 imM Ni 2+ , 0.1 pg ml "1 LPS (Salmonella minnesota R595) or medium as indicated. Thereafter, synthesis of cytokines IL-1 ⁇ , IL-6, IL-10 (a) or IFN- / ⁇ (c) was determined by ELISA. Data represent means ⁇ s.d. of three independent experiments.
  • FIG 14 (Supplementary Figure 8) Proinflammatory signalling pathways activated by Ni 2+ .
  • an antigen-specific, T-lymphocyte-activating signal (“signal 1 ") and a proinflammatory signal (“signal 2”).
  • the latter signal is sensed by hTLR4-hMD2 resulting in the activation of NF-KB, p38 and IRF3 and subsequent expression of cytokines, adhesion molecules and type I interferons.
  • Soluble human Toll-like receptor 4 (hTLR4)-derived proteins State of the art Contact hypersensitivity (CHS) to nickel (Ni 2+ ) and cobalt (Co 2+ ) are the most important among the CHS disorders because of the high incidence of sensitized individuals, which are assumed to exceed 65 million people in Europe only for nickel hypersensitivity, and due to socio-economic reasons. CHS disorders regularly occur work-related and can lead to long-term disability in severe cases. Moreover, metals like nickel are often components of medical implants and are suspected, for example, to promote the so-called in-stent-restenose in patients with cardiovascular stents, which is a severe postoperative complication.
  • TLR4 Toll-like receptor 4
  • LPS lipopolysaccharide
  • TLR4-MD2 trap molecules that are a fusion of the extracellular domain of murine TLR4 (mTLR4), its soluble cofactor murine MD2 (mMD2) and the Fc domain of immunoglobulin G (IgG-Fc).
  • mTLR4 murine TLR4
  • mMD2 soluble cofactor murine MD2
  • IgG-Fc immunoglobulin G
  • TLR4 antagonists like TAK242 and E5565 (Eritoran).
  • TAK242 inhibits signal transduction after LPS binding to TLR4 whereas the lipid A analog E5565 directly competes binding of LPS to the TLR4/MD2 complex.
  • Clinical studies with these antagonists have been discontinued due to missing efficacy. None of these approaches to inhibit TLR4 in LPS-induced sepsis was tested so far in the context of contact hypersensitivity.
  • a main disadvantage of chelating agents is their low specificity. Moreover, in the case of systemic application (e.g. during treatment of in-stent-restenose) solubility problems may occur and the chelating agents may also unspecifically bind other bivalent ions that could disturb the function of essential ion-binding proteins such as hemoglobin.
  • the aim of the invention is to provide a method for synthesis of substances that are applicable in a method for identification of substances inhibiting TLR4 activation as a result of stimulation by contact allergens or bacterial LPS.
  • TLR4 soluble human TLR4 molecules applied in an assay that is used for identification of substances inhibiting TLR4 activity. Moreover, the molecules are applied for therapeutic intervention of TLR4-mediated contact hypersensitivity or sepsis.
  • the invention is based on the finding that also the metal cobalt (Co 2+ ) in addition to nickel is a contact allergen that activates proinflammatory gene expression, like for example IL-8, via TLR4 (Fig. 17).
  • the activity of TLR4 induced by cobalt and subsequent expression of proinflammatory genes is mediated by sequences that are
  • RECTIFIED SHEET (RULE 91) ISA/EP specific for human TLR4 and that are not found in TLR4 of mouse (Fig. 18a, 18b).
  • Co 2+ -induced gene expression requires cofactor MD2 but occurs independently of the species origin of MD2 (Fig. 18c, 18d). Both species exhibit different susceptibilities to Co 2+ -induced proinflammatory responses that are determined by sequence specific variances in the genes encoding human and mouse TLR4 (Fig. 19).
  • the histidine residues H456 and H458 in human TLR4 (hTLR4) are required for Co 2+ -induced proinflammatory gene expression (Fig. 20).
  • s-hTLR4 alone is sufficient for inhibition of TLR4 activity inducing proinflammatory gene expression in response to contact with the metals nickel or cobalt. Therefore s- hTLR4 is applicable for therapy of metal-induced contact allergy. However, in combination with its cofactor MD2, s-hTLR4 is applicable for inhibition of LPS- induced proinflammatory gene expression following TLR4 activation, since the binding sites of LPS and metals at TLR4 are different and activation by LPS needs the presence of MD2 in contrast to activation by metals.
  • s-hTLR4 soluble human TLR4
  • sequence ID 6 site-directed mutagenesis is applied.
  • the nucleotide sequence coding for s-hTLR4 according to sequence ID 2 consists of the leader sequence of the murine Ig ⁇ immunoglobulin chain with the nucleotide sequence for a hemagglutinin tag (HA-tag) fused to its 3 ' end for later purification of synthesized protein and of the sequence coding for the extracellular domain (amino acids 1 to 628) of human TLR4 (hTLR4 according to sequence IDs 1 and 5; Figure 15) fused downstream of the HA-tag in accordance with the reading frame and with the glutamine residue (Q) at position 628 exchanged with a stop codon (Q628->Stop628; Figure 16; Seq. IDs 2 and 6).
  • forward and reverse primers according to sequence IDs 3 and 4 are applied that hybridize with the hTLR4 sequence according to sequence ID 1. This trun
  • ISA/EP hTLR4 sequence misses the coding sequence for the transmembrane domain of wildtype hTLR4, resulting in secretion of s-hTLR4 protein, for example into the supernatant of cultured cells expressing s-hTLR4 (Fig. 21a).
  • the complete s- hTLR4 sequence is cloned by known methods into an appropriate plasmid vector that is transfected by common techniques into an appropriate human cell line like for example HEK-293, which in one embodiment has no endogenous expression of the TLR4 cofactor human MD2 (hMD2, according to sequence IDs 7 (nt sequence) and 8 (aa sequence)) allowing hMD2-free purification of s-hTLR4 (Fig. 21a, right side).
  • the cell line for example HEK-293, is stably expressing hMD2 (HEK-293-hMD2) in order to allow later purification of s-hTLR4/hMD2 heterodimer complexes (Fig. 21a, left side).
  • s-hTLR4 is expressed by the transfected cells during incubation of the cell culture in appropriate cell culture medium at 37°C and 5% CO2 in an incubator for 24 hours and secreted into the cell culture supernatant.
  • the produced s-hTLR4 protein exhibits the histidine residues that are crucial for binding of metal ions like nickel (Ni 2+ ) or cobalt (Co 2+ ) at positions 431 , 456, and 458, as well as binding sites for its cofactor hMD2.
  • the s-hTLR4 protein or the s-hTLR4/hMD2 heterodimer complex is purified from cell culture supernatants via affinity chromatography using the HA-tag.
  • the HA-tag is exchanged with another peptide tag that is applicable for protein purification, like for example Isopeptag, BCCP, Myc-tag, Calmodulin-tag, FLAG-tag, His-tag, Maltose binding protein-tag, Nus-tag, Glutathione-S-transferase-tag, Green fluorescent protein-tag, Thioredoxin-tag, S-tag, Softag 1 , Softag 3, Strep-tag, SBP-tag, Ty tag.
  • Appropriate tags are known to the experts.
  • a nucleotide sequence coding for a protease restriction site is placed between the peptide tag and the TLR4/1-628 coding sequence with the aim to be able to cut the peptide tag from the TLR4/1-628 peptide by use of the corresponding protease enzyme after the purification procedure using the tag.
  • Appropriate sequences encoding protease restriction sites are also known to the experts.
  • the MD2 binding sites within the hTLR4/1-628 part of s- hTLR4 is deleted or destroyed by insertion of applicable point mutations.
  • the purified s-hTLR4 is applied for an assay that is used for identification of substances or molecules that are able to inhibit the activity of hTLR4. Moreover, the assay is used for investigation of the mode of action of these inhibiting substances or molecules.
  • the substances are labeled with an appropriate fluorescent dye like for example Acridine orange, Acridine yellow, Alexa Fluor, 7- Aminoactinomycin D, 8-Anilinonaphthalene-1 -sulfonate, ATTO dyes, Auramine- rhodamine stain, Benzanthrone, Bimane, 9,10-Bis(phenylethynyl)anthracene, 5,12- Bis(phenylethynyl)naphthacene, Blacklight paint, Brainbow, Calcein, Carboxyfluorescein, Carboxyfluorescein diacetate succinimidyl ester, Carboxyfluorescein succinimidyl ester, 1 -Chloro-9,1 0-bis(phenylethynyl)anthracene, 2-Chloro-9,10-bis(phenylethynyl)anthracene, 2-Chloro-9,10-diphen
  • Binding of the substances to s-hTLR4 is monitored afterwards by fluorescence-based methods like for example microplate readers or fluorescence microscopes that are known to the experts.
  • s-hTLR4 proteins are immobilized to an ELISA microtiter plate by common methods and then incubated with the fluorescence- labeled substances. Substances not bound to the immobilized s-hTLR4 are removed by washing procedures, and fluorescence of complexes consisting of s-hTLR4 and the labeled substance is monitored.
  • a competition ELISA is used for identification of hMD2- competing substances.
  • purified s-hTLR4 is immobilized by common techniques on an ELISA plate and incubated with fluorescently labeled hMD2 molecules and at the same time with one of the substances that shall be tested. In the presence of hMD2-competing substances, that bind with a higher affinity to the immobilized s-hTLR4 than hMD2, no fluorescence is detectable.
  • purified hMD2 cofactor molecules are immobilized together with s-hTLR4 on the same ELISA plate with both building a molecule complex (s-hTLR4/hMD2). Afterwards, the plate is incubated with fluorescently labeled LPS in parallel with the single substances of interest to be tested for competition. After washing procedures that remove non-bound molecules, no fluorescence is detectable when the substance instead of the fluorescently labeled LPS is bound to the s-hTLR4/hMD2 complexes.
  • purified hMD2 is immobilized on the microtiter plate and afterwards incubated with fluorescently labeled purified s-hTLR4 and the substance of interest.
  • FIG. 15 Schematic presentation of human Toll-like receptor 4 (hTLR4) with Ig- kappa chain and an HA-tag fused to the N-terminus of hTLR4. Histidine residues crucial for binding of nickel or cobalt are labelled as H456/H458, and the transmembrane domain is named TM-Domain.
  • hTLR4 Toll-like receptor 4
  • FIG 16 Schematic presentation of human Toll-like receptor 4 (hTLR4) with Ig- kappa chain and an HA-tag fused to the N-terminus of hTLR4. Histidine residues crucial for binding of nickel or cobalt are labelled as H456/H458, and Q628 * designates the amino acid residue that is replaced by a stop codon leading to a truncated soluble hTLR4 protein (s-hTLR4) missing the transmembrane domain and the C-terminal part of hTLR4.
  • Figure 17 The contact allergen cobalt (Co ) activates proinflammatory gene expression via hTLR4.
  • FIG. 18 Co 2+ -induced gene expression requires sequence motifs present in hTLR4 but not in mTLR4.
  • Co 2+ -induced proinflammatory gene expression requires MD2 but occurs independently of the species origin of MD2.
  • FIG. 19 Sequence specific variances in the genes for human and mouse TLR4 determine the different susceptibilities of both species to Co 2+ -induced proinflammatory responses (a) Analysis of luciferase activity of a transfected 6x ⁇ - luc reporter or (b) IL-8 release of HEK293-hMD2 cells transfected with human TLR4 (hTLR4), hTLR4 bearing inactivating alanin replacements at the putative metal- binding histidines (hTLR4-H456A-H458A), murine TLR4 (mTLR4), or a "humanized" mTLR4 mutant containing histidine substitutions at the corresponding positions in the mouse sequence (mTLR4-Y454H-N456H), respectively.
  • Stable HEK293-hMD2 cells were transfected with HA-tagged hTLR4, HA-mTLR4 or HA-hTLR4 (H456A-H458A) and exposed to Co 2+ , LPS (1 ⁇ g ml "1 ) or medium (ctrl) for 8 h.
  • (a) Flow cytometric staining of intracellular IL- 8 protein expressed by the positively transfected cell population. Transfection of the indicated constructs was monitored by HA-immunostaining and gating on the HA- positive population. Representative overlays of IL-8 staining (black lines) and isotype- staining (grey lines) of three independent experiments are shown.
  • Figure 21 (a) Western analysis of the indicated HA-tagged hTLR4 proteins in supernatants and total lysates from transfected HEK293-hMD2 cells (left) or HEK293 wild-type cells (right), respectively. Data demonstrate hMD2-independent release of truncated HA-hTLR4-Q628 * into the supernatant. Lysates and supernatants were taken 48h after transfection with the indicated hTLR4 constructs, (b) IL-8 ELISA of HEK293 hTLR4/hMD2 cells incubated with undiluted supernatants produced by HEK wt cells transfected with the indicated constructs.

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Abstract

La présente invention concerne un nouveau procédé et une nouvelle classe de substances pour traiter et/ou réduire l'hypersensibilité de contact (CHS) des mammifères, particulièrement les humains, au nickel, particulièrement Ni2+. Etonnamment, il a été découvert que l'inhibition de hTLR4 spécifique d'un site est une stratégie potentielle pour l'intervention thérapeutique sans affecter les réponses immunitaires vitales. Nous démontrons ici que Ni2+ induit une réponse inflammatoire via une activation directe du récepteur 4 Toll-like humain (hTLR4). De façon remarquable, l'activation de TLR4 induite par Ni2+ est spécifique d'une espèce puisque TLR4 de souris (mTLR4) est incapable de produire cette réponse. Des études avec des protéines TLR4 mutantes ont révélé que les histidines 456 et 458 de hTLR4 doivent ne pas être conservées pour obtenir une activation par Ni2+ mais pas par le ligand naturel LPS. En conséquence, l'expression de hTLR4 transgénique dans des souris déficientes en Tlr4 a permis une sensibilisation efficace à Ni2+ et la provocation d'une CHS. Les allergies au nickel (Ni2+) sont la cause la plus fréquente d'hypersensibilité de contact (CHS) dans les pays industrialisés. A la fois un signal spécifique des lymphocytes T et un signal pro-inflammatoire sont nécessaires pour un développement efficace de la CHS. Nous démontrons ici que Ni2+ induit une réponse inflammatoire via une activation directe du récepteur 4 Toll-like humain (hTLR4). De façon remarquable, l'activation de TLR4 induite par Ni2+ est spécifique d'une espèce puisque TLR4 de souris (mTLR4) est incapable de produire cette réponse. Des études avec des protéines TLR4 mutantes ont révélé que les histidines 456 et 458 de hTLR4 doivent ne pas être conservées pour obtenir une activation par Ni2+ mais pas par le ligand naturel LPS. En conséquence, l'expression de hTLR4 transgénique dans des souris déficientes en Tlr4 a permis une sensibilisation efficace à Ni2+ et la provocation d'une CHS. Nos données impliquent une inhibition de hTLR4 spécifique du site comme stratégie potentielle pour une intervention thérapeutique sans affecter les réponses immunitaires vitales. En outre, nous montrons que la réponse inflammatoire liée à l'hypersensibilité via l'activation directe du récepteur 4 Toll-like humain (hTLR4) est non seulement induite par le nickel mais également par le cobalt (Co2+). De plus, l'invention décrit étonnamment la synthèse de molécules hTLR4 tronquées (s-hTLR4) auxquelles il manque le domaine transmembranaire et la partie C-terminale de la protéine qui sont utilisables dans un procédé d'identification de substances pour traiter ou réduire l'hypersensibilité de contact(CHS) ou une septicémie chez des mammifères par inhibition de l'activation de hTLR4.
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US10030067B2 (en) * 2013-03-15 2018-07-24 University Of Florida Research Foundation, Incorporated Compounds for treating neurodegenerative proteinopathies
AU2014228886B2 (en) * 2013-03-15 2018-11-29 University Of Florida Research Foundation Compounds for treating neurodegenerative proteinopathies
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