Background• In humans,10 members of the toll-likereceptor(TLR)family of proteins recognize differentpathogen-associated molecular patterns(PAMPs)throughtheir luminal leucine-rich repeats.• They localize on cell surface (TLR1, 2, 4, 5,6and11) or inendosomes (TLR3, 7,8and9)• TLRs are essential in the early events of innate immunityas well as in the development of robust adaptive immuneresponses.• Microbial products, such as LPS and DNA, trigger signalingcascades
• The recognition of PAMPs by TLRs ultimately leads toNF-κ B and AP-1 activation and the production of manypro-inflammatory cytokines, such as TNF-α and IL -6.Additionally, type 1 interferon s are induced through thephosphorylation of IRF3 and IRF7. Thus, TLRs areimportant in the early innate immune responses againstpathogens. These initial mediators and the activation ofantigen presenting cells (APCs)will also impact theensuing adaptive immunity.
Tollip Structure• TLR signals are regulated by molecules such as thesuppressor of cytokine signaling1 (SOCS1)and Toll-Interacting protein(Tollip). it is an inhibitory adaptorprotein within Toll-like receptors (TLR). The TLR pathwayis a part of the innate immune system that recognizesstructurally conserved molecular patterns of microbialpathogens, leading to an inflammatory immune response.
Cont.• A TBD (Tom1-binding domain) and a CUE (coupling ofubiquitin to endoplasmic reticulum degradation)domain, located on the N-and C-terminal regionsrespectively, confer a potential for multiple proteininteractions Finally, a C2 (internal protein kinase Cconserved region 2) domain binds phosphoinositidesand is responsible for the intracellular trafficking ofthe protein to the endocytic pathway.
Experimental Evidence for Tollipfunction• While experiments on deficient mice suggested thatTollip was needed for maximal cytokine productionin response to low doses of TLR agonists, moststudies imply a negative regulatory role for Tollip invarious signaling pathways.• A high throughput shRNA screen identified Tollip asa potential regulator of MHC class II (MHC II)trafficking. Up to now, only two E3 ubiquitin ligaseshave been shown to modify MHC class II molecules.These are the membrane-associated RING-CH(MARCH) 1
MARCH function• MARCH1 is mostly expressed in the spleen and morespecifically in follicular B cells. Comparably to the class IItrans-activator (CIITA), which is the master regulator ofMHC II gene transcription, MARCH1 appears to be themaster regulator of MHC II expression where MARCHproteins add ubiquitin to target lysines in substrateproteins, thereby signaling their vesicular transportbetween membrane compartments. MARCH1 down-regulates the surface expression of majorhistocompatibility complex (MHC)class II molecules andother glycoproteins by directing them to the lateendosomal/ lysosomal compartment
Hypotheses of the study• Hypothesized that Tollip has regulatory rolein the trafficking of MHC II molecules.
Materials and Methods• AntibodiesL243(HLA-DR), CerCLIP.1(CLIP/HLA-DR complexes), BU45 (human invariantchain)• ReagentsPoly(I:C) Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic analog ofdouble-stranded RNA (dsRNA)• Cell lines and miceC57BL/6 (B6), M1K-O mice (knock out mice), Xid mice (Btk knockout)• Plasmids and constructs• Transfections• Flow cytometry• Immunoprecipitation and western-blot analysis
Cont.• Bioluminescence resonance energy transfer (BRET)experiment• Microscopy• Luciferase assay• siRNA• Real-time quantitative PCR
Results/ DiscussionFig. 1. The response to poly(I:C) and LPS is impaired in the Ii KO and M1 KO mice. (A) Splenocytes fromC57BL/6, Ii KO and Xid mice were isolated and treated ex vivo for 24 h with LPS prior to RNA extractionand qPCR analysis of TNFα mRNA expression. (B) Splenocytes from C57BL/6, Ii KO and M1 KO mice wereisolated and treated ex vivo for 24 h with either LPS or poly(I:C) prior to RNA extraction and qPCR analysisof TNFα mRNA expression. Expression is illustrated as fold level compared to the value of untreatedC57BL/6 cells, which was set at 1. Data is representative of at least two different experiments.
Fig. 2. HLA-DR interacts with TLR3 in live cells. (A) HEK 293E CIITA cells were transfected withTLR3-flag. 48 h post-transfection, cells were lysed and immunoprecipitated with a flag specificantibody and blotted for HLA-DRα or HLA-DMβ. Asterisks represent the antibodies. (B) HEK 293Tcells were transfected with HLA-DR–Rluc and increasing amounts of TLR3-EYFP. The BRET ratiowas calculated by dividing the fluorescence with substrate, subtracted from the fluorescencewithout substrate, by the luminescence. Error bars represent standard deviation obtained fortwo different transfections.
C) FRET experiment performed in HeLa cells 48 h after transfection with TLR3-EYFP and HLA-DRα–EGFP2/β. One stack of living cells was observed by confocal microscopy. The dotted square shows thebleached area. The signal intensity for the bleached region was quantified for pre- and post-bleach. Thesignals were normalized for the ones of the corresponding regions prior to the beach and plotted in a barchart. (D) Luciferase assay of HeLa or HeLa HLA-DR1 cells transfected or not with TLR3 and the NF-κB-luciferase reporter plasmid. The cells were stimulated for 5 h with poly(I:C) prior to the addition ofluciferine. Error bars represent standard deviation obtained for two different transfections. Data is
Fig. 3. Tollip knockdown increases HLA-DR expression. (A) HeLa-CIITA-DO cells were transfected with control or TOLLIP-specificsiRNAs, and cultured for 48 h at 37 °C. The bar chart represents the mRNA expression of Tollip for cells transfected with specific orcontrol siRNA. (B) The cells were stained and analyzed by flow cytometry for cell surface and total expression of HLA-DR (L243 Ab).The mean fluorescence values (MFV) were plotted to account for variations in the levels of HLA-DR. Error bars represent standarddeviation obtained for two different transfections. (C) Cells were stained for cell surface expression of CLIP (CerCLIP) and totalexpression of invariant chain (BU45). The mean fluorescence values (MFV) were plotted to account for variations in the levels ofCLIP and invariant chain Error bars represent standard deviation obtained for two different transfections. (D) HeLa-DR1 and HeLa-DR1 TM/TM cells were transfected with control or Tollip-specific siRNAs, and cultured for 48 h in 37 °C. Cells were stained for cellsurface expression of HLA-DR (L243 Ab). The mean fluorescence values (MFV) were plotted to account for variations in the levels ofHLA-DR expression. Error bars represent standard deviation obtained for two different transfections.
Fig. 4. Tollip blocks MARCH1-mediated down-regulation of HLA-DR. (A) HEK 293E CIITA cellswere transfected with GFP-Tollip, EYFP andMARCH1 or GFP-Tollip and MARCH1. Cellswere stained for cell surface expression of HLA-DR and Tfr. Bar charts represent the meanfluorescence intensity of EYFP or GFP positivecells. (B) HeLa CIITA cells were transfected withGFP-Tollip, EYFP and MARCH1 or GFP-Tollipand MARCH1. Cells were stained for cell surfaceexpression of HLA-DR. The bar chart representsthe mean fluorescence intensity of EYFP or GFPpositive cells. (C) Cells were lysed and blottedfor HLA-DRα and HLA-DRβ. The intensity of thebands was quantified and divided by the one ofcells transfected with the YFP control. Resultsare represented as a bar chart. Data isrepresentative of at least two differentexperiments.
Fig. 5. Tollip reduces the expression ofMARCH1. (A) HeLa CIITA and HEK 293E CIITAcells were transfected with EYFP-MARCH1, GFP-Tollip or an empty vector(mock). Cell lysates were blotted for actin(asterisk) and MARCH1. The intensity of thebands was quantified, normalized to actin anddivided by the one of cells transfected withMARCH1. Results are represented as a barchart. (B) HeLa CIITA cells were transfectedwith EYFP-MARCH1, GFP-Tollip or an emptyvector (mock). Cell lysates were blotted forTollip. The intensity of the bands was quantifiedand the value obtained for cells expressingTollip alone was set to 1. Results arerepresented as a bar chart. (C) HeLa CIITA orHEK 293E CIITA cells were transfected withEYFP-MARCH1 (left panel) of EYFP-MARCH1K-0 (right panel) with or without GFP-Tollip, GFP-SOCS1 and EYFP. Cells werestained for cell surface MHC II and analysed byflow cytometry. The mean fluorescence valuesfor MHC II in cells expressing Tollip and EYFPwas set to 1. Data is representative of a leasttwo different experiments.
Figure(6)Fig. 6. Tollip interacts with MHC II. HeLa cells were transfected with MARCH1and/or GFP-Tollip and/or empty vector. Samples were immunoprecipitatedwith a HLA-DR-specific antibody and blotted for (A) Tollip and (B) DRα. Theasterisk indicates the position of the immunoprecipitating mouse antibody lightchain recognized by the goat secondary antibody. Data is representative of atleast two different experiments.
Summery• Results showed that Ii-deficiency impairs TLRresponses are in line with a functional role of MHC IImolecules in innate immunity. Morespecifically, given the well-described endosomalsorting signals of Ii, this data supports the assertionthat the intracellular pool of MHC II molecules isneeded for efficient LPS response However, theywere not able to rule out that the effect of Ii may bedue to the lower trafficking of MHC II moleculesfrom the ER in the C57BL/6 background.
Recommendation and Conclusion• The preliminary observations in this study presentedan interplay between MARCH1 and Tollip warrant amore in-depth mechanistic characterization of themolecular interactions taking place betweenTLRs, MHC II, Tollip, MARCH1 and the endocyticmachinery. Also, future studies should address morein depth the effect of Tollip on MHC II trafficking inthe absence of MARCH1 as It will be extremelyinteresting to test the impact of Ii deficiency onother mice backgrounds where surface MHC II, atleast quantitatively, appears normal.
Reference• M.-C. Bourgeois-Daigneault, A. M.Pezeshki, T. Galbas, M. Houde, M.Baril, K. Früh, A. Amrani, S. Ishido, D. Lamarre, J. Thibodeau, Tollip-induced down-regulation of MARCH1, Results in Immunology, Volume3, 2013, Pages 17-25