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

Abstract

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.

Keywords: Influenza; Toll-like receptors.

Figure 1

Expressions of Toll‐like receptors (TLRs 2, 3, 4, 7, 8, 9) in monocytes and dendritic cells (DCs) in influenza A patients and age‐gender matched controls, measured by quantitative flow cytometry. The mean fluorescence intensity (MFI) values are represented in logarithmic scale (median values indicated by horizontal bars in red). Each point represents the MFI in an individual study subject. ‘Monocytes’ [patients versus controls, MFI median (IQR)]: TLR8, 587·0 (202·0, 3374·0) versus 328·0 (1·0, 469·5), P = 0·006; TLR9, 2056·0 (819·0, 9996·0) versus 293·0 (1·0, 954·0), P < 0·001; TLR2, 23·0 (1·0, 296·5) versus 840·0 (119·0, 2159·5), P = 0·031; TLR4, 1·0 (1·0, 155·0) versus 388·0 (1·0, 842·5), P = 0·061. ‘Total Dendritic Cells, DCs’: TLR3, 711·0 (415·5, 1304·5) versus 420·0 (48·5, 859·0), P = 0·079; TLR8, 1389·0 (401·8, 4412·3) versus 118·0 (1·0, 852·0), P = 0·024; TLR9, 1906·0 (1411·5, 3817·0) versus 676·0 (1·0, 1046·0), P = 0·001; TLR7, 70·0 (1·0, 694·8) versus 1·0 (1·0, 1·0), P = 0·147. Detection of TLR7 (positive), 50·0% versus 18·2%, P = 0·078. Representative flow‐cytometry histograms (including isotypic control) and subgroup analyses on mDC and pDC are provided in Data S2. Available convalescentphase samples from 6 influenza patients showed normalizing TLR8, 9 and TLR2, 4 levels (also see Table 3).

Figure 2

Level of gene expression of RIG‐1 and MDA‐5 in peripheral blood mononuclear cells in influenza A patients and age‐gender matched controls, measured by quantitative real‐time PCR. Each point represents the Relative Quantitation (RQ) in an individual study subject. *P = 0·038, ***P < 0·001, Mann‐Whitney U‐test ‘RQ’ (‘Relative Quantitation’) = mRNA (RIG‐1 or MDA‐5)/GAPDH.

Figure 3

Negative correlations between expressions of TLRs and influenza ‘viral load’ in the respiratory tract. Both mean fluorescence intensity (MFI) values and viral RNA concentrations (measured by real‐time, quantitative reverse‐transcription PCR) are shown in logarithmic scale. mDC , myeloid dendritic cells; pDC, plasmacytoid dendritic cells. Trends of negative correlations between viral RNA concentration and TLR7 expression were also observed: ‘Total DC’, r = −0·34, P = 0·12; ‘pDC’, r = −0·31, P = 0·21; ‘mDC’, r = −0·34, P = 0·18. There was no significant correlation found between viral RNA concentration and TLR2 or TLR4 expression. r = Spearman's rank coefficient (rho).