Changes And Clinical Significences Of Toll-like Receptors In Neonates With Infections

Objectives Neonates are vulnerable to infections because of their immature immune responses. Toll-like receptors could induce innate and acquired immune responses. The aim of the present study is to investigate the changes of TLR2/3/4 mRNA MD-2 mRNA and MyD88 mRNA on human peripheral blood mononucleate cells(PBMCs), the protein expression of TLR2/TLR4 , the percentage of TLR2/TLR4 positive cells on monocytes and neutrophils in neonatal infections, and to determine their roles in anti-infectious immune response. This study also evaluated the potential use of neutrophil CD64, the C-reactive protein (CRP), white cells counts and blood cultures in diagnosis of neonatal infections. Correlation analysis was made between TLRs and CD64.Methods Two hundrends of infants were divided into six groups: sepsis group (n=20), bacterial pneumonia group(n=71), bacterial meningitis group(n=17), urinary tract infection group(n=38), congenital syphilis group(n=11) and non-infected control group(n=48). TLR 2/3/4 MD-2 and MyD88 mRNA were determined by RT-PCR. The protein expression of TLR and the percentage of TLR positive cells were evaluated by analysis with flow cytometry. The level of CD64 was also measured by flow cytometric analysis. CRP was analyzed by immunoturbidimetry assay (ITA). Statistical analysis of the data was performed with SPSS 12.0, the significance level is P<0.05.Results 1. There were 157 infected neonates, including 94 preterms and 63 terms. In this group of infants, the symptoms of neonatal infections were atypical, with low Spo₂ fever hypothermia apnea thrombocytopenia hyperbilirubinemia feeding intolerance and bradycardia and so on.There were 21 septic pateints all with positive blood cutures, mainly coagulase-negative staphylococci. Bacterial peumonia was diagnosed in 70, pateints with positive cultures in 36. Klebsiella pneumoniae was the most common bacteria. Urinary tract infection was present in 38, patients with positive urinary cultures in 16. Klebsiella pneumoniae was also the most frequent bacteria.Bacterial meningitis was diagnosed in 17 patients, most had Escherichia Coli.2. Among the non-infected group, there was no significant difference in the mRNA expression and the protein of TLR2/3/4 among all these groups in correlation analysis with gestational age, P>0.05.3. There was no significant difference in the mRNA expression and the protein of TLR3 among all these infection groups.4. The TLR2 mRNA expression increased significantly in all the sepsis group (6.14±0.80), but was more significant in the Gram-positive sepsis group (6.43±0.74).TLR2 mRNA expression was also significantly higher in the bacterial pneumonia group (5.49±0.62) , the bacterial meningitis group (5.61±0.60) and the congenital syphilis group (5.89±0.38) . TLR2 protein expression of monocyte was highest in the sepsis group( 1.94 ±0.52) and significantly increased in the bacterial pneumonia , bacterial meningitis and the congenital syphilis groups when compared to the TLR2 protein expression of the non-infectious group(1.27±0.75). The TLR2 protein expression in the Gram-positive bacterial sepsis group was 2.54±0.68, and in the Gram-negative bacterial sepsis was 1.25±0.51, (P<0.05). The percentage of TLR2 positive cells in the neonatal infection group was not significently different when compared to the non-infected group. TLR2 protein expression of neutrophils was highest in the sepsis group(0.92±0.36). The TLR2 protein expression in the Gram-positive bacterial sepsis group was 0.97 ± 0.48, and in the Gram-negative bacterial sepsis was 0.80 ± 0.31, (P<0.05). The percentage of TLR2 positive cells between the neonatal infection group and non-infected group was significantly different. TLR2 protein expression of neutrophils was highest in the sepsis group(0.92±0.36). The TLR2 protein expression in the Gram positive bacterial sepsis group was 0.97±0.48, and in the Gram-negative bacterial sepsis was 0.80±0.31, (P<0.05). The percentage of TLR2 positive cells between the neonatal infection and in the non-infected groups was not significently different.5. The mRNA expression of TLR4 was highest in the sepsis group (6.20±1.59), with the Gram-negative bacteial sepsis group (6.78±1.79), higher than the Gram-positive bacterial group (5.39 ±0.78), (t=2.29,P=0.037). TLR4 mRNA expression increasedsignificantly in the bacterial pneumonia (5.33±1.07) , the bacterial meningitis (5.87±0.70) and the urinary tract infection groups (5.38±0.91) . There were no significant differences in TLR4 protein expression between these groups. The percentage of TLR4 positive cells in the neonatal infection groups was (0.71±0.31)%, higher than the percentage of non-infected group (0.29 ± 0.36%). TLR4 protein expression of neutrophils was highest in the sepsis group (3.25 ± 1.25) and significantly increased in the bacterial pneumonia , bacterial meningitis and urinary tract infection group.All were higher than the TLR4 protein expression of the non-infection group (1.16 ± 0.36). The TLR4 protein expression in the Gram positive bacterial sepsis group was 2.97±1.48, and in the Gram negative bacterial sepsis was 3.75 ± 1.01, (P<0.05). The percentage of TLR4 positive cells in the neonatal infection group was not significently different to the non-infection group.6. The mRNA expression of MD-2 was highest in the sepsis group (5.57±1.16). The mRNA expression of MD-2 of the Gram positive bacteial sepsis group was 5.43 ± 1.19, and was not significently different to the Gram negative bacterial group 5.76±1.17,(t=0.594, P=0.56) . MD-2 mRNA expression increased significantly in the bacterial meningitis (5.10±0.92) and urinary tract infection groups (4.98±0.42) . The mRNA expression of MyD88 was highest in the sepsis group (5.71±0.94). The mRNA expression of MyD88 of the Gram positive bacteial sepsis group was 5.92 ±1.01, and not significently different to the Gram negative bacterial group 5.79 ± 0.85, (t=0.793, P=0.86), The MyD88 mRNA increased in all neonatal infection groups.7. In the Gram-positive bacterial sepsis group, the mRNA expression of TLR2 (6.43±0.74) was higher than the mRNA expression of TLR4 (5.39 ± 0.78), (t=1.56,P=0.024). In the Gram negative bacterial sepsis group, the mRNA expression of TLR4 (6.78 ± 0.79)was significantly higher than the mRNA expression of TLR2 (5.64 ± 0.68) (t=2.63, P=0.011). In the sepsis group, the TLR2 protein expression was significantly higher than the expression of TLR4 (t=1.06, P=0.044) . The percentage of TLR4 positive cells was lower than the percentage of TLR2 positive cells among all these groups, P<0.01. In all the infection groups, the TLR4 protein expression wassignificantly higher than the expression of TLR2 , P<0.05. The percentage of TLR4 positive cells was lower than the percentage of TLR2 posivive cells among all these groups, P<0.01.8. The levels of CD64 in patients with sepsis, pneumonia and other infections were 7845.25 ± 1790.07 M/U, 6162.82±944.11 M/U, 4003.86±1790.07 M/U, higher than the ones in patients without infection (2533.00±1355.20 M/U) . Using the ROC curve for statistical analysis, the markers are considered positive if CD64 ≥3000 M/U. Among these indices of the diagnosis the neonatal sepsis, the positive rate of CD64(91 %) and the sensitivity 92% was highest. When neutrophil protein of TLR2/TLR4 was correlated with the CD64, only in sepsis group, the TLR2/TLR4 protein was significantly correlated with CD64, TLR2/TLR4 protein could distinguish the Gram-positive infection from the Gram-negative infection, while CD64 could not do this.Conclusions The mRNA and the protein expression of TLR2/TLR4 increased significantly in the neonatal infection groups, especially in the severe sepsis groups. The mRNA expression of TLR2 increased mainly in the Gram-positive bacterial infection groups, and the mRNA expression of TLR4 increased in the Gram-negative bacterial infection groups. The TLR was significantly correlated with the MD-2 and MyD88, suggesting that both the TLR2 and TLR4 signal pathway participanted in the immune mechanism of neonatal infections. The CD64 may be a new diagnosis marker of neonatal infections, if the neonatal sepsis is associated with TLR2/TLR4, which made help to indentify the type of bacteria. This study provides a new concept and experimental basis for further understanding of the immune mechanisms of neonatal infection.