Molecular Structural Characterizations And Innate Immune Functions Of The DNA-Sensing Receptors In Mouse

The innate immunity is the earliest line that hosts defense against pathogenic infection. The detection of pathogen associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) activates a series of signaling cascades triggering the secretion of I type IFNs, inflammatory factors and chemokines, and then the innate immunity of host comes into play leading to the elimination of invading pathogens ultimately. The DNA generated during viral infection and replication is one of important PAMPs. Hence, the host could defense against the infection of DNA viruses is the key that how to recognize viral DNA by the PRRs. In the present study, five mouse DNA recognition receptors including Toll-like receptor 8 (TLR8), Toll-like receptor 9 (TLR9), DNA-dependent activator of IFN-regulatory factors (DAI), absent in melanoma 2 (AIM2), and leucine-rich repeat in Flightless-1 interaction protein 1 (LRRFIP1) were cloned, and then the genetic evolution and structural characterization were analysed. Furthermore, functions of the five receptors lay the foundation for further uncovering the mechanisms of the interaction between host and pathogene, the molecular mechanisms of immune response against viral infections, and the mechanisms of immune evasion of pathogene. Progress in this study are as follows:1. Five DNA recognition receptor genes of mouse were cloned and identified.The mouse TLR8 (mTLR8) and mouse TLR9 (mTLR9) were cloned from mouse peripheral blood lymphocytes and the mouse DAI (mDAI), the mouse AIM2 (mAIM2) and the mouse LRRFIP1 (mLRRFIP1) were cloned from the mouse splenic lymphocytes by RT-PCR. The sequence analysis demonstrated that the ORF of cloned mTLR8 gene is 3099 bp, encoding 1032 amino acids; the ORF of cloned mTLR9 gene is 3099 bp, encoding 1032 amino acids; the ORF of cloned mDAI gene is 1236 bp, encoding 411 amino acids; the ORF of cloned mAIM2 gene is 1065 bp, encoding 354 amino acids; the ORF of cloned mLRRFIP1 gene is 2190 bp, encoding 729 amino acids.2. The molecular structural characterizations and genetic evolution of the five DNA recognition receptors were predicted and analysed. The structures of the cloned genes were predicted by the bioinformatics softwares. The results showed that mTLR8 and mTLR9 contain LRRs ectodomain, transmembrane domain and TIR cytoplasmic domain, and the LRRs display a space structure like a solenoid coil; mDAI contains two Z-DNA domains (Za and Zβ), D3 domain and signaling transduction domain; mAIM2 contains HIN-200 domain and PYD domain; mLRRFI1l contains two coiled-coil domains and forms a dimer. The genetic evolution analysis demonstrated that mTLR8, mTLR9, mDAI, mAIM2 and mLRRFIP1 gene are rather close to other mammals respectively, indicating that these sequences are relative conserved. These results demonstrated that the cloned genes possess the molecular structural characterizations of PRRs.3. Various promoters of effector molecules in innate immunity signaling were cloned and the report systems were constructed. The mouse IFN-a2, IFN-a4, IFN-β and TNF-α promoter sequences were cloned from mouse genome by PCR. The sequencing results showed that these amplified promoter sequences were consistent with reference sequences respectively. Moreover, these sequences were inserted into the plasmid pGL-4.10, and the mouse IFN-a2, IFN-a4, IFN-β and TNF-a promoter luciferase reporting recombinant plasmids were constructed. Meanwhile, the eukaryotic expression recombinant plasmids of five DNA recognition receptors were constructed and the signaling report systems were verified, which provide mareials and platform for monitoring the mouse PRRs in recognizing in innate immunity.4. The characteristic of expression level in different tissues of mTLR8 mRNA was analysed and the Sanction in recognizing was studied preliminary. The mRNA expression profile of mTLR8 mRNA in different tissues was analyzed by real-time quantitative RT-PCR. The results showed that mTLR8 mRNA is expressed at detectable level in all the monitored tissues, including PBMCs, heart, liver, spleen, lung, kidney, small intestine, and muscle. Notably, expression was most abundant in the lung, followed by the heart and spleen. The results of mTLR8 in recognizing showed that the over-expressing of mTLR8 induced NF-κB activation and TNF-a production in mTLR8-transfected HEK293 cells. However, a combination of poly (dT) ODN plus the R848 and A/T-rich DNA could not activate mTLR8. The results indicated that mTLR8 has the particularity in molecular structure, expression and function.5. The functions of three DNA recognition receptors of mouse were analysed which lay solid foundation for further studying the signaling of DNA PRRs. The functions of mDAI, mAIM2 and mLRRFIP1 in recognizing were studied by the established report systems of signaling in innate immunity respectively. The results showed that A/T-rich or C/G-rich DNA could activate mDAI, mAIM2 and mLRRFIP1, and induce the transcription of corresponding transcription factors and the secretion of cytokines, which indicating that mDAI, mAIM2 and mLRRFIP1 may play a vital role in innate immune responses against DNA virus infections.