Objective: To investigate the alteration of immune function and possible immunopathogenesis in the children with 2009 influenza A (H1N1) infection.
Method: Sixty patients with 2009 influenza A (H1N1) infection hospitalized in Shenzhen Children's Hospital between November 1, 2009 and January 10, 2010 and 20 age-matched healthy children were enrolled in this study. The patients were divided into two groups according to the severity of influenza A infection: 35 mild cases (mild pneumonia) and 25 severe cases (severe pneumonia, acute encephalopathy associated with influenza A, and 3 died from acute necrotizing encephalopathy with influenza A infection). Real-time PCR was used to evaluate the expression levels of pattern recognition receptor (PRRs), retinoic acid induced gene I/melanoma differentiation associated gene 5 (RIG/MDA5), Toll-like receptors (TLRs) and TLRs signaling molecules, and negative-regulator. Three color fluorescent and flow cytometry were used to investigate the apoptosis of CD3(+), CD4(+), CD8(+) and CD19(+) cells. Plasma cytokines (IL-1β, IL-6, TNF-α, IFN-γ, IFN-α, IL-10) concentrations were measured by enzyme-linked immunosorbent assay (ELISA).
Result: (1) The expression levels of RIG/MDA5, TLR2, 4 were much higher in the patients with influenza A infection, especially severe cases [TLR2 (9.69 ± 3.15) × 10(-2) vs. (3.96 ± 0.83) × 10(-2), t = 10.16, P < 0.05; TLR4 (10.23 ± 2.85) × 10(-2) vs. (7.46 ± 2.18) × 10(-2), t = 3.76, P < 0.05]. The expression levels of TLRs signal transduction molecules like MyD88 and TRAM also increased. (2) The cell counts of CD3(+), CD4(+), CD8(+) T cells and NK cells were markedly lower in the patients with influenza A infection compared to the NC group [CD3(+)(1.22 ± 0.38) × 10(9)/L vs.(3.59 ± 1.10) × 10(9)/L, t = 9.21, P < 0.05]. (3) Plasma concentrations and the mRNA expression of TNF-α, IL-6, and IL-1β were elevated in mild cases, while declined in severe cases [TNF-α (6.42 ± 1.76) × 10(-2) vs. (9.05 ± 2.51) × 10(-2), t = 4.55, P < 0.05]. Plasma concentrations of IFN-α/IFN-β were up-regulated gradually with the aggravation of the disease, especially in severe cases. Compared with healthy controls, the expression of IFN-I inducible gene IP-10, RANTES, or iNOS was significantly higher in children with mild [IP-10 (20.52 ± 6.09) × 10(-2) vs.(1.18 ± 0.34) × 10(-2), t = 18.74, P < 0.05], and relatively lower in severe cases. (4) The apoptosis of CD3(+), CD4(+), CD8(+) and NK cells significantly increased in the patients with influenza A infection than those in NC group [CD3(+)(32.90 ± 7.66)% vs. (20.21 ± 6.58)%, t = 6.21, P < 0.05]. Compared with healthy controls, the expression levels of apoptosis-related gene like TRAIL and CASPASE-3 significantly increased in the patients with influenza A infection. (5) The expression levels of negative regulator of SOCS1, SOCS3, IRAK-M, TRAF4 and FLN29 were significantly increased in the patients with influenza A, especially in severe cases than those in NC group (P < 0.05).
Conclusion: Immune function changed with the severity of the disease. The mild cases presented systemic immune activation status, while critically ill cases presented mixed immune activation and immunosuppression status.