Role of human Toll-like receptors in naturally occurring influenza A infections.

BACKGROUND: We investigated the roles of Toll-like receptors (TLRs) in naturally occurring influenza. METHODS: A prospective, case - control study was conducted. Adults hospitalized with virologically confirmed influenza A infections (onset <48 hours, before treatment) were compared with age-/gender-matched controls. TLRs (2, 3, 4, 7, 8, 9) expression in monocytes and dendritic cells (DCs - total, myeloid, plasmacytoid) was quantitated using flow cytometry. Gene expression of RLRs (RIG-1, MDA-5) was evaluated using real-time PCR. Concomitant signaling molecules expression, plasma cytokine/chemokine concentrations, and respiratory tract viral loads were measured. PBMCs were cultured and stimulated ex vivo with TLR-specific ligands for cytokine responses. RESULTS: Forty two patients with influenza (24 A/H3N2, 18 A/H1N1pdm09) and 20 controls were studied. Patients' mean age was 68 ± 16 years; 81% had respiratory/cardiovascular complications. There were increased cellular expressions of TLR9, TLR8, TLR3, and TLR7 during influenza; TLR2 and TLR4 were suppressed. Results were similar for both virus strains. Higher TLR expression levels at presentation significantly correlated with lower viral loads (Spearman's rho: -0.46 to -0.69 for TLR9, TLR8, and TLR3; P-values <0.05). Multivariate regression models (adjusted for age, comorbidity, disease severity, time from onset) confirmed their independent associations. Increased signaling molecules (phospho-MAPKs, IκB) and inflammatory cytokines (IL-6, sTNFR-1, CCL2/MCP-1; CXCL10/IP-10, IFN-γ) correlated with increased TLR expression. RLRs were upregulated simultaneously. PBMCs of patients with influenza showed significant, dynamic changes in their cytokine responses upon TLR stimulation, compared with controls. CONCLUSIONS: Our results suggest that TLRs play an important role in early, innate viral inhibition in naturally occurring influenza. Inflammatory cytokine responses are concomitantly induced. These findings support investigation of TLR targeting as a novel intervention approach for prophylaxis against influenza.

Cytokine response patterns in severe pandemic 2009 H1N1 and seasonal influenza among hospitalized adults.

BACKGROUND: Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. METHODS: Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. RESULTS: 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2-15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2-27 times lower than seasonal influenza; P-values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3-4, 55% vs 85%; day 6-7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6-61.5, per log(10)unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman's rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their 'responsiveness' to stimuli was shown to change dynamically during the illness course. CONCLUSIONS: A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.