Endosomal TLR3 co-receptor CLEC18A enhances host immune response to viral infection

Human C-type lectin member 18A (CLEC18A) is ubiquitously expressed in human, and highest expression levels are found in human myeloid cells and liver. In contrast, mouse CLEC18A (mCLEC18A) is only expressed in brain, kidney and heart. However, the biological functions of CLEC18A are still unclear. We have shown that a single amino acid change (S339 →R339) in CTLD domain has profound effect in their binding to polysaccharides and house dust mite allergens. In this study, we further demonstrate that CLEC18A and its mutant CLEC18A(S339R) associate with TLR3 in endosome and bind poly (I:C) specifically. Compared to TLR3 alone, binding affinity to poly (I:C) is further increased in TLR3-CLEC18A and TLR3-CLEC18A(S339R) complexes. Moreover, CLEC18A and CLEC18A(S339R) enhance the production of type I and type III interferons (IFNs), but not proinflammatory cytokines, in response to poly (I:C) or H5N1 influenza A virus (IAV) infection. Compared to wild type (WT) mice, ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice generate higher amounts of interferons and are more resistant to H5N1 IAV infection. Thus, CLEC18A is a TLR3 co-receptor, and may contribute to the differential immune responses to poly (I:C) and IAV infection between human and mouse. Ya-Lang Huang et al. report a mechanism for TLR3-mediated signaling after immune simulation and influenza virus infection by way of the co-receptor CLEC18A. This study found that a single amino acid change in CLEC18A(S339R) can enhance the production of type I and type III interferons to suppress viral replication, and increase mice survival rate after flu infection infection.

H uman C-type lectin 18 (clec18) gene cluster contains three loci clec18a, clec18b, and clec18c, and each clec18 family member encodes a polypeptide of 446 amino acids comprising a C-type lectin-like domain (CTLD) in the C terminus and a sperm-coating protein (SCP) domain (also known as CAP or TAPS domain) in the N terminus 1 . The three human Ctype lectin member 18A/B/C (CLEC18A/B/C) polypeptides are almost identical except polymorphic amino acid residues located in CTLD and SCP/CAPT/APS domains 1 . Human CLEC18A is ubiquitously expressed in human tissues with higher expression levels in myeloid cells and liver. However, the biological function of CLEC18A is still unknown, nor do we understand the impact of polymorphic amino acid residues in CTLD.
It has been shown that endosomal Toll-like receptors (TLRs) recognize diverse danger signals and nucleic acid components released from uncoated virions, and activation of endosomal TLRs induces the production interferons (IFNs) and proinflammatory cytokines to initiate inflammatory responses 2 . Endosomal TLR3 is regarded as a sentinel to RNA viruses, and activation of TLR3 leads to TRIF (TIR domain-containing adaptor-inducing IFN-β)-dependent IFN production and nuclear factor-κB (NF-κB)-dependent proinflammatory cytokine production 3 . Interestingly, the differential outcome of TLR3mediated signaling between human and mouse have been noted 4,5 . Compared to wild-type (WT) mice, TLR3 −/− mice produced lower levels of inflammatory mediators and displayed less cell infiltration after H3N2 influenza A virus (IAV) infection, despite higher virus titer being noted in the lung 6 . This observation suggests that TLR3-mediated signaling prefers to induce proinflammatory cytokine release and has a detrimental effect in IAV infection in mice. In contrast, human TLR3 is as important as RIG-I in the production of type I and type III IFNs after IAV infection 7 , and intranasal administration of TLR3 agonist reduced lung viral titers and mortality in mice 8 . These observations suggest that TLR3 activation is able to produce IFNs to protect the host from IAV infection. However, the molecular mechanism responsible for the differential TLR3-mediated signaling between human and mouse is still unknown.
Since polymorphic amino acid residues were found in the CTLD domain and SCP domain among members of CLEC18 family, and distinct glycan-binding affinity in vitro was found between the CTLD domain of CLEC18A(S339) and CLEC18A-1(R339) 1 , we are interested to understand the differential functions of S339 versus R339 in host immunity in vivo. To address this question, we generate CLEC18A(S339R) mutant, which is identical to CLEC18A except amino acid 339 (S→R). Here we reported that CLEC18A and CLEC18A(S339R) associate with TLR3 and bind poly (I:C) directly. To understand their roles in TLR3-mediated signaling, we generated ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice to investigate their functions in poly (I:C) stimulation and IAV infection. We found that macrophages and alveolar epithelial cells (AECs) from ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice produce higher amounts of IFNs (type I and type III) and IFNstimulated genes (ISGs) after incubation with poly (I:C) or H5N1 IAV. Furthermore, ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice are more resistant to H5N1 infection with higher survival rates than WT littermates (CLEC18A(S339R) > CLEC18A > WT). These observations suggest that CLEC18A and CLEC18A(S339R) act as TLR3 co-receptors in the endosome, and may contribute to the differential immune responses to TLR3-mediated signaling between human and mouse.

Results
Expression of mouse endogenous CLEC18A and generation of ROSA-CLEC18A/ CLEC18A(S339R) knock-in mice. While human CLEC18A is ubiquitously expressed and highest expression levels were noted in immune cells and liver 1 , mouse CLEC18A (mCLEC18A) messenger RNA (mRNA) is only expressed in brain, kidney, and heart (Fig. 1a). To confirm the expression of mCLEC18A, we generated human anti-mCLEC18A monoclonal antibody (mAb) to detect mCLEC18A in mouse tissues (lanes 1-8, Fig. 1b). We harvested cell lysates from 293T cells transfected with vector alone (lane 9, Fig. 1b) or Flagtagged full-length mCLEC18A (lane 10, Fig. 1b), followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before blotting to polyvinylidene difluoride (PVDF) paper. The blots were incubated with anti-mCLEC18A mAb (lanes 9 and 10, Fig. 1b) and anti-Flag mAb (lanes 9 and 10, Fig. 1c), respectively, to determine the expression of mCLEC18A. We found that anti-mCLEC18A mAb is able to detect endogenous mCLEC18 in the brain (lane 1, Fig. 1b) and 293T cells transfected with full-length mCLEC18A (lane 10, Fig. 1b), while the expression levels of mCLEC18A in heart and kidney (lanes 3 and 7, Fig. 1b) were too low to be detected under the same condition. We further examined the expression of endogenous mCLE18A by flow cytometry (Supplementary Fig. 1). Even we can detect 293T cells transfected with full-length mCLEC18A complementary DNA (cDNA) ( Supplementary  Fig. 1d), we cannot detect mCLEC18A in mouse peripheral blood, bone marrow, and splenocytes. These observations further confirmed that the tissue distribution of mCLEC18A is distinct from the human counterpart.
To understand the functions of CLEC18A-mediated immune responses in vivo, we generated ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice to express human CLEC18A and CLEC18A(S339R), respectively, to address this question. We found that CLEC18A and CLEC18A(S339R) were expressed ubiquitously, and the highest expression level was found in the brain, bone marrow, and splenocytes in ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice (Fig. 1d). The expression of human CLEC18A and CLEC18A(S339R) in ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice tissues is similar to what we observed in human tissues 1 , although the expression levels are lower than that in human liver and kidney. Western blot analysis further confirmed the expression of CLEC18A and CLEC18A (S339R) in mouse bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) (Fig. 1e).
CLEC18A enhances IFN production and ISG expression after influenza virus infection. As mouse BMDMs do not express endogenous CLEC18A, we compared the responses of BMDMs isolated from WT, ROSA-CLEC18A, and ROSA-CLEC18A (S339R) mice to various TLR ligands, respectively, followed by examining the expression of type I IFNs (IFN-α and IFN-β) and proinflammatory cytokines. Among the TLR ligands tested, high molecular weight (HMW) poly (I:C) enhanced the expression of IFN-α, IFN-β, IFN-γ-inducible protein 10 (IP-10), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Interestingly, the expression levels of IFN-α, IFN-β, and IP-10 were further upregulated in ROSA-CLEC18A and ROSA-CLEC18A(S339R) BMDMs (Fig. 2a). Similar to poly (I:C) stimulation, higher expression levels of IFN-α and IFN-β were found in ROSA-CLEC18A and ROSA-CLEC18A(S339R) BMDMs stimulated with RNA viruses, including H5N1 IAV and dengue virus (DV) (Fig. 2b). In contrast, other TLR ligand has no obvious effect to upregulate cytokine production ( Supplementary Fig. 2). We further knocked down the endogenous CLEC18 in human macrophage to confirm its role in TLR3-mediated signaling. We found that knockdown of CLEC18 significantly reduced poly (I:C) and virus-, but not LPS-, induced IFN production ( Supplementary  Fig. 3). To further examine the role of CLEC18A and CLEC18A (S339R) in TLR3-mediated signaling, human HT1080 cells were transfected by small interfering RNAs (siRNAs) to knock down endogenous CLEC18A. After recovery overnight, the transfected cells were transfected with pMACS.Kk-HA(C)-hCLEC18A and pMACS.Kk-HA(C)-hCLEC18A(S339R) to express CLEC18A and CLEC18A(S339R), respectively. We found that overexpression of CLEC18A and CLEC18A(S339R) enhanced the expression of IFN-α and IP-10 after poly (I:C) stimulation ( Supplementary  Fig. 4). These observations further confirm that CLEC18A is able to enhance TLR3-mediated signaling. Moreover, we examined the expression of ISGs critical in viral infection 9,10 . While Ch25h and Mx1 were upregulated, the expression of ISG15 was similar in both ROSA-CLEC18A and ROSA-CLEC18A(S339R) BMDMs post H5N1 IAV and DV infections (Fig. 2c). In addition, virus titer and the expression of matrix protein (M) and nucleoprotein (NP) were downregulated in ROSA-CLEC18A and CLEC18A (S339R) BMDMs (Fig. 2d). It is interesting to note that CLEC18A (S339R) is more potent than CLEC18A to enhance IFN production and suppress virus replication in all the assays.
In addition to type I IFNs, we further investigated the expression of type III IFN, which plays a critical role in antiinfluenza virus infection 11 , in AECs after H5N1 IAV infection.
Compared to WT mice, both type I IFNs (IFNα4 and IFNβ) and type III IFN (IFNλ2/3) were upregulated, while the expression of TNF-α and IL-6 was similar in AECs isolated from ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice (Fig. 3a). Moreover, the expression of M and NP genes was suppressed in AECs expressing human CLEC18A and CLEC18A(S339R) (Fig. 3b). Similar to what we observed in macrophages, CLEC18A(S339R) is more potent than CLEC18A to induce IFN expression and suppress virus replication in virus-infected AECs, suggesting that the polymorphic amino acid residue [S 339 →R 339 ] in CTLD domain has a profound effect to upregulate IFN production and suppress H5N1 IAV replication.
We further generated CLEC18A deletion mutants to identify minimal domain responsible for interaction with TLR3. We found that all the CLEC18 deletion mutants interact TLR3 efficiently except CLEC18 Δ28-292 a.a. (Fig. 6h-k). Therefore, we conclude that CTLD domain is dispensable for CLEC18A-TLR3 interaction, and TLR3 interacts with all the rest parts of CLEC18A, including CAP-, EGF-and EGK-like domains.
CLEC18A and CLEC18A(S339R) protect host from H5N1 IAV-induced inflammation and lethality. We further examined the effect of CLEC18A and CLEC18A(S339R) in host defense against H5N1 IAV infection. To address this question, mice were inoculated with H5N1 IAV (1500 PFU/mouse) via intranasal route to observe body weight change and survival rate for 21 days. We found that CLEC18A(S339R) is more potent than CLEC18A to protect mice from H5N1 IAV-induced body weight loss (Fig. 8a) and lethality (Fig. 8b). Compared to WT mice, less cell infiltration and pulmonary inflammation were noted in ROSA- CLEC18A and ROSA-CLEC18A(S339R) mice after H5N1 IAV infection (Fig. 8c). Moreover, virus titer was suppressed dramatically in CLEC18A(S339R) mice (Fig. 8d), and the expression of viral M and NP mRNAs is downregulated in ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice at day 7 post infection (Fig. 8e). We further investigate the cytokine amount in bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assay (ELISA) (Fig. 8f). At days 4 and 7 post infection, higher levels of IFN-α, IFN-β, and IP-10 were noted in ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice. Moreover, a higher level of IFNλ2/3 was observed on day 4, but not on day 7 post infection, while the expression of proinflammatory cytokines (TNF-α, CCL2, and CCL5) was similar in WT, ROSA-CLEC18A, and ROSA-CLEC18A(S339R) mice (Fig. 8f). These observations indicated that CLEC18A and CLEC18A(S339R) are able to upregulate the expression of type I and type III IFNs to suppress H5N1 IAV replication in vivo.

Discussion
It has been demonstrated that human macrophages and DCs only produce type I IFNs, but not TNF and IL-6, after TLR3 ligand stimulation. In contrast, mouse macrophages produce both type I IFNs and proinflammatory cytokine after poly (I:C) stimulation 4 . However, the underlying mechanism of the differential responses between human and mouse cell after poly (I:C) stimulation is still unknown. In this work, we demonstrated that human CLEC18A and CLEC18A(S339R) associate with TLR3, and act as coreceptor to bind poly (I:C). Interestingly, enhanced expression of type I and type III IFNs was noted in macrophages and epithelial cells isolated from ROSA-CLEC18A and CLEC18A(S339R) mice, respectively.
It is surprising to find that poly (I:C) binds to CLEC18A (K D = 2.10E − 08) and CLEC18A(S339R) (K D = 5.59E − 09) with high affinity. Both CLEC18A and CLEC18A(S339R) are typical C-type lectins with "QPD," "WND," and "WIGL" motifs. The presence of QPD motif is in accord with its preferential binding to galactan 1 , while the "WND" motif is shown to promote binding to galactose and N-acetylgalactosomine 16 . Furthermore, the characteristic "WIGL" motif is highly conserved in canonical CTLD sequences 16 . Thus, CLEC18A is the first canonical C-type lectin that can bind poly (I:C) and acts as TLR3 co-receptor to enhance anti-viral immunity.
It is interesting to note that a single amino acid change [S 339 →R 339 ] in CTLD domain has a potent effect to enhance type I and type III IFN productions and anti-viral immunity. Compared to BMDM-CLEC18A, BMDM-CLEC18A(S339R) expressed higher levels of IFNs (Fig. 2b) and ISGs (Fig. 2c) with lower level of viral NP gene (Fig. 2d) expression after H5N1 IAV infection. Moreover, higher expression of IFN-β with a lower level of viral M gene was found in lung epithelial cells isolated from ROSA-CLEC18A(S339R) mice (Fig. 3b). This observation is in accord with a stronger induction of p-TBK1 and p-IRF3 in CLEC18A(S339R) (Fig. 5c). Furthermore, higher levels of type III IFN (Fig. 8f) with lower levels of virus titer and less lung inflammation were found in ROSA-CLEC18A(S339R) mice (Fig. 8c, d). The differential responses between CLEC18A and CLEC18A(S339R) alleles are in accord with a higher survival rate of ROSA-CLEC18A(S339R) mice after H5N1 IAV infection (Fig. 8b). Thus, the polymorphic amino acid residue S339R in the CTLD of CLEC18A seems contributing significantly in host immunity to H5N1 infection. Further study is needed to confirm the role of CLEC18A(S339R) allele in host defense against various microbial infections in the future.
In this work, we demonstrated that both CLEC18A and CLEC18A(S339R) bind to poly (I:C) and enhances poly (I:C)induced IFNs production via forming a heterocomplex with TLR3. The higher affinity of CLEC18A(S339R) than CLEC18A may be attributed to the positive charge of R 339 located in the CLEC18A(S339R) CTLD, thereby increasing its binding to negatively charged poly (I:C). Moreover, ROSA-CLEC18A (S339R) mice produce higher amount of type I IFNs than ROSA-CLEC18A mice, and are more resistant to H5N1 IAV infection. These observations suggest that ROSA-CLEC18A(S339R) allele may be beneficial to host upon RNA virus infection. This speculation is supported by our observation that DV induced higher amount of IFNs in ROSA-CLEC18A(S339R) than ROSA-CLEC18A and WT mice. Among the species we examined 1 , Takifugu rubripes CLEC18A contains N corresponding to S/R 339 in CTLD, while mice also contain "S" in CTLD domain. In addition to amino acid residue 339, another polymorphic amino acid residue was found at 421 in CTLD domain [(D 421 (CLE-C18A)→N 421 (CLEC18C)]. The D 421 →N 421 mutation converts the "WND" motif into WNN, and thus may attenuate their binding to galactose and N-acetylgalactosomine 16 , and even other glycan and non-glycan ligands. In addition to CTLD domain, polymorphic amino acid residues located in N-terminal SCP/ CAP domain was also noted 1 . Thus, CLEC18 is a highly polymorphic C-type lectin, and large-scale human population is necessary to understand whether another polymorphic amino acid residue exists in positions 339 and 421, and investigate the association of CLEC18A polymorphic amino acid residues in host defense against virus, bacteria, and other microbes in the future.
In previous studies, we found that human CLEC18 is detectable in culture supernatant 1 and human serum 17,18 . However, we cannot detect human CLEC18A/CLEC18A(S339R) in the ROSA-CLEC18 knock-in mouse serum. It may be attributed to lower expression of human CLEC18A/CLEC18A(S339R) in the knockin mice, because the expression levels of CLEC18 in human immune cells and liver 1 are much higher than what we observe in ROSA-CLEC18A/CLEC18A(S339R) mice (Fig. 1a-c). Even though the functions of soluble CLEC18 are still unknown, CLEC18 serum level correlates with the stage of hepatitis B virus (HBV) infection and is a potential biomarker to predict HBeAg loss and seroconversion in CHB patients under nucleos(t)ide analog therapy 17 . Moreover, higher CLEC18 serum level positively correlated with viral loads 18 in HCV patients. These observations suggest that CLEC18 may also participate in the pathogenesis of liver inflammation caused by hepatitis B virus and HCV. Our observation that CLEC18A acts as a TLR3 coreceptor is the first step to explore its potential role in innate immunity against viral infection, and more studies are necessary to understand how CLEC18A contributes to host innate immunity against viral infections in the future.
Isolation of primary mouse alveolar epithelial type II cells. C57BL/6 mice were sacrificed by an overdose of isoflurane. The inferior vena cava was cut and lungs were perfused with PBS via the right ventricle until it turned white in color. A small nick was made into the exposed trachea to insert a shortened 20 G angiocatheter that was fixed and a total volume of 3 ml of sterile dispase was injected (BD Biosciences), followed by injection of 500 μl of sterile 1% low-melting agarose in PBS (Lonza). After a few minutes of incubation, the lungs were removed and placed into a tube containing 1 ml of dispase for 45 min (25°C). Lungs were then transferred into a culture dish containing Dulbecco's modified Eagle's medium (DMEM)/10 mM HEPES/100 U/ml DNaseI (Sigma)/antibiotics for 5 min, and the tissue was carefully dissected from the airways and large vessels. The single cell was successively suspended through a 100 and 40 μm strainers. The cell was centrifuged at 300 × g for 10 min at 4°C, then the cell was resuspended with RBC lysis buffer for 5 min to lyse the presence of RBCs, and finally, it was directly added to 10 ml DMEM supplemented with 10% FCS/HEPES and antibiotics. The cell was centrifuged and the resuspended cell was placed on prewashed 100-mm tissue culture plates that had been coated for 24-48 h at 4°C with 42 μg CD45 and 16 μg CD32 (BD Pharmingen). After incubation for 2 h at 37°C, type II cells were gently collected from the plate and filtered by 40 μm strainers to suspend the single cell. AECs were plated into 6-well cell culture plates at 1.5 × 10 6 /well, followed by detecting prosurfactant protein C (Abcam, ab40879), the specific marker for alveolar epithelial type II cells, by flow cytometry to determine the purity.
Transfection of macrophages and HT1080 cells by CLEC18 siRNA and CLEC18 expression vectors. Human macrophages were seeded at 7 × 10 4 cells/well in 96well microtitration plates. After incubation at 37°C overnight, cells were transfected with 100 nM siRNA in 2.5 μl HiPerFect, and further incubated at 37°C for 48 h before harvesting for the functional assay. Alternatively, HT1080 cells were seeded in 12-well microplate (1.2 × 10 5 cells/well) and incubated at 37°C overnight, followed by incubation with 10 nM siRNA in 6 μl HiPerFect at 37°C for 48 h to knock down endogenous CLEC18. The siRNA-transfected HT1080 cell cells were then seeded in 12-well microplate (1.2 × 10 5 cells/well) and incubated at 37°C overnight, followed by transfection with 1 μg plasmids in 4 μl Turbofect transfection reagent (Thermo Scientific™, catalog number R0532), and further incubated at 37°C for 24 h before functional assay.
Detection of human and mice CLEC18 by Western blot or flow cytometry. For Western blotting analysis, cells (1 × 10 6 ) were lysed by RIPA buffer, followed by fractionation on 12% SDS-PAGE before blotting onto PVDF membrane. Membranes were probed with anti-hCLEC18 mAb (clone 3A9E6 (1)) or anti-mCLEC18 mAb (home-made antibody C8G), followed by incubation with peroxidaseconjugated goat anti-mouse polyclonal antibody (Millipore, AP181P) or donkey anti-human polyclonal antibody (Jackson ImmunoResearch, 709-035-149). Blots were developed by the Immobilon TM Western Chemiluminescent using horseradish peroxidase as the substrate (Millipore TM ). For flow cytometry analysis, primary cells were incubated with phycoerythrin (PE)-conjugated anti-mouse CD4 antibody (BioLegend, 100408) Inoculation of virus. Murine and human macrophages were infected with IAVs at a multiplicity of infection (MOI) = 2. After 1 h adsorption, cells were washed once with PBS, followed by incubation with culture medium (RPMI supplemented with 10% fetal bovine serum) for 12 h or 24 h. WT or ROSA-CLEC18 knock-in mice (9-12 weeks old) were anesthetized by intraperitoneal injection of ketamine and xylazine, followed by intranasal inoculation with H5N1 (HA,NA) virus 20 (1.5 × 10 3 PFU) in 20 μl sterile PBS. Survival rate and body weight loss were monitored until day 21 post infection.
RNA extraction and real-time PCR. Total RNA was extracted by TRIzol reagent (Invitrogen™) according to the vendor's instruction. First-strand cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific). PCR reaction was performed by LightCycler® 480 System (Roche Applied Science). All primers are listed in Supplementary Table I. Quantification of viral M and NP gene. To determine M and NP expression in infected mice, samples were harvested from infected mice for RNA extraction Fig. 6 Mapping of TLR3 and CLEC18A/CLEC18A(S339R) interaction domain. a Scheme of human TLR3 deletion mutants. b-g The 293T cells (3 × 10 6 ) were transfected with pMACS-Kk-HA-hCLEC18A/CLEC18A(S339R) (7.5 μg) and pFlag-CMV1-hTLR3 (full-length and deletion mutants) (7.5 μg) for 48 h, followed by immunoprecipitation using anti-Flag mAb. Immunoprecipitates were fractionated on SDS-PAGE and blotted to PVDF membranes before probing with anti-HA mAb and anti-Flag mAb, respectively. Blots were further incubated with peroxidase-conjugated anti-mouse IgG antibody and developed by ECL Kit. h-k The 293T cells (3 × 10 6 ) were transfected with pCMV-Tag4A-hCLEC18A (full-length and deletion mutants) and pUNO1-TLR3-HA for 48 h, followed by immunoprecipitation using anti-Flag mAb. Immunoprecipitates were fractionated on SDS-PAGE before blotting to PVDF membranes. Blots were probed with anti-HA mAb and anti-Flag mAb, respectively, followed by incubation with peroxidase-conjugate anti-mouse IgG antibody and developed by ECL Kit. Iso isotype control antibody, IP immunoprecipitation, WB western blot. Red arrows: CLEC18 or TLR3; V: vector only; [+]: Flag-tagged CLEC18A and deletion mutants were co-transfected with HA-tagged TLR3 expression vector (TLR3-HA). All WB experiments have been repeated at least three times.
using the TriRNA Pure Kit (Geneaid, Catalog No.TRP100), and first-strand cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific) as per the vendor's instruction. Quantitative real-time PCR analysis was performed using the LightCycler® 480 System (Roche Applied Science).
Determination of cytokine levels by ELISA. The levels of cytokines in culture supernatant and BALF were determined by ELISA Kits according to the vendor's instruction. All samples were stored at −80°C before assay.