Expressions Of TLR2and TLR4on Peripheral Blood Mononuclear Cells Frompatients With Alzheimer’s Disease

Background:Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia in the elderly, with a prevalence of over35million worldwide[1]. It is characterized by global cognitive decline and the accumulation of plaques containing the amyloid beta protein (Aβ), neurofibrillary tangles, activated microglia and astrocytes, and degenerating neurons. The causes of AD are very complex, considerable evidence has supported the notion that the innate immune response and resulting neuroinflammation may play a vital role in the pathogenesis of AD were increased.Toll-like receptors (TLRs) are transmembrane pattern recognition receptors that initiate signals in response to diverse pathogen-associated molecular patterns (PAMPs) in the innate immune system[2,3]A number of studies have reported an association between TLRs and AD, especially TLR2, that play a key role in inflammatory neurodegeneration binding the highly hydrophobic amyloid peptides or lipopolysaccharide (LPS). Moreover, TLR2are involved in the microglia-mediated inflammatory response, Aβ plaque formation and Aβ clearance in Alzheimer’s disease (AD). Our previous studies have shown that variants in the TLR2genes are associated with the risk of AD.In view of the functionality of the variants in TLR2gene and the central role of them in the progress of AD, there might be an association between the plasma protein levels and AD. Therefore, we hypothesize that there may be significant changes in TLR2expressions on peripheral blood mononuclear cells (PBMCs) from patients with LOAD when compared to healthy control subjects.Methods:A total of60sporadic LOAD (age at onset≥65years) patients (women33; mean age=80.2±8.2years) and60healthy controls subjects matched for sex and age (female34; mean age=80.6±6.3years) were recruited for this study; all participants were Northern Han Chinese in origin. A clinical diagnosis of probable AD fulfilled the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association (N1NCDS-ADRDA).5.5mL of venous blood was drawn with heparin anticoagulant tubc without trauma at once from both healthy controls and from patients. Total RNA from PBMCs was extracted and purified using Trizol reagent kit (Promega, USA). Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. Flow cytometry (FCM) was used to detect expressions of TLR2proteins and real-time quantitative RT-PCR was performed to determine TLR2mRNAs. Statistical analysis was performed using SPSS11.5software. Differences in the characteristics between cases and controls were analyzed using the Student t-test or the chi-square test. Repeated measures one-way analysis of variance (ANOVA) followed by Bonferroni was used for comparisons within groups. The correlation between mRNA, protein and MMSE were performed with Pearson’s coefficient. The criterion for significant difference is P<0.05.Results:60LOAD patients were well-matched with the60controls in terms of age (P=0.75) and gender (P=0.85). Expression of TLR2mRNA showed significantly difference between AD patients and healthy controls (0.390±0.204vs.0.281±0.167, P<0.01). FCM results showed that the expressions of TLR2proteins on PMBCs were higher in LOAD group than that of control group (97.12±1.67%vs.41.07±18.44%, P<0.01), respectively. Meanwhile, positive correlations were found between expression of mRNA and protein in both cases (TLR2:r=0.980and0.976, P<0.01). There was a significant negative correlation between TLR2levels and and MMSE score (r=-0.32; P=0.01). A negative correlation between TLR4levels and MMSE was also observed (r=-0.29; P=0.02, data not shown).Conclusion:In conclusion, this is the first time to study plasma levels of TLR2on PBMCs in AD. Our results indicated that the expressions of TLR2proteins were increased in AD patients than in healthy controls. Further and larger studies in Han Chinese as well as in other ethnic populations are necessary to confirm this. Background:Toll-like receptor play a key role to our understanding of how innate immune cells recognize a variety of exogenous (i.e., microbial) as well as endogenous (i.e., host-derived) pathogenic molecules since it was found20years ago,. Subsequently, the mechanisms by which these receptors trigger inflammatory responses that are appropriately tailored to the insult have been revealed. These studies provided a quantum leap that spurred the field of innate immunity into a renaissance after many years of neglect. As a result of this renewed research interest, it has become apparent that phylogenetically conserved innate immunity not only provides the first line of defense against invading microbes but also governs the response to tissue injury and controls the activity of adaptive immunity.Alzheimer’s disease (AD) is characterized by the formation of insolubledeposits of b-amyloid (Ab) within the parenchyma of the brain. These deposits areassociated with a robust microglia-mediated inflammatory response. Recent workhas demonstrated that Toll-like receptors (TLRs) participate in this inflammatoryresponse.Toll-like receptors (TLRs) are transmembrane pattern recognition receptors that initiate signals in response to diverse pathogen-associated molecular patterns (PAMPs) in the innate immune system [1-2]. A number of studies have reported an association between TLRs and AD, especially TLR4, that play a key role in inflammatory neurodegeneration binding the highly hydrophobic amyloid peptides or lipopolysaccharide (LPS). Moreover, TLR4are involved in the microglia-mediated inflammatory response, Aβ plaque formation and Aβ clearance in Alzheimer’s disease (AD). Our previous studies have shown that variants in the TLR4genes are associated with the risk of AD. In view of the functionality of the variants in TLR4gene and the central role of them in the progress of AD, there might be an association between the plasma protein levels and AD. Therefore, we hypothesize that there may be significant changes in TLR4expressions on peripheral blood mononuclear cells (PBMCs) from patients with LOAD when compared to healthy control subjects.Methods:A total of60sporadic LOAD (age at onset≥65years) patients (women33; mean age=80.2±8.2years) and60healthy controls subjects matched for sex and age (female34; mean age=80.6±6.3years) were recruited for this study; all participants were Northern Han Chinese in origin. A clinical diagnosis of probable AD fulfilled the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association (NINCDS-ADRDA).5.5mL of venous blood was drawn with heparin anticoagulant tube without trauma at once from both healthy controls and from patients. Total RNA from PBMCs was extracted and purified using Trizol reagent kit (Promega, USA). Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. Flow cytometry (FCM) was used to detect expressions of TLR4proteins and real-time quantitative RT-PCR was performed to determine TLR4mRNAs. TLR4/11367polymorphism was genotyped using single-tube, bidirectional, allele-specific amplification. Statistical analysis was performed using SPSS11.5software. Differences in the characteristics between cases and controls were analyzed using the Student t-test or the chi-square test. Repeated measures one-way analysis of variance (ANOVA) followed by Bonferroni was used for comparisons within groups. The correlation between mRNA, protein and MMSE were performed with Pearson’s coefficient. The criterion for significant difference is P<0.05.Results:60LOAD patients were well-matched with the60controls in terms of age (P=0.75) and gender(P=0.85). Expression of TLR4mRNA was also up-regulated in LOAD patients (0.503±0.195vs.0.322±0.183, P<0.01). FCM results showed that the expressions of TLR4proteins on PMBCs were higher in LOAD group than that of control group (66.56±23.74%vs.14.83±4.31%, P<0.01), respectively. Meanwhile, positive correlations were found between expression of mRNA and protein in both cases (TLR4:r=0.938and0.970, P<0.01). A negative correlation between TLR4levels and MMSE was also observed (r=-0.29; P=0.02, data not shown). Our previous study demonstrated an association between TLR4/11367polymorphism and LOAD, here we analyzed TLR4plasma level in individuals with different genotypes using ANOVA followed by Bonferroni analyses. The number of individuals with GG, GC, and CC genotypes in AD group was20,20, and20, respectively, while in control group it was35,20, and5. We found that TLR4level in AD patients with the GG genotype was significantly lower than that with CC genotype (P<0.001), and that with GC (P<0.001). In control group, TLR4plasma level didn’t vary with TLR4genotypes.Conclusion:In conclusion, this is the first time to study plasma levels of TLR4on PBMCs in AD. Our results indicated that the expressions of TLR4proteins were increased in AD patients than in healthy controls and CC genotype can increase the expression of TLR4in AD patients. Further and larger studies in Han Chinese as well as in other ethnic populations are necessary to confirm this.