Haemophilus influenzae porin induces Toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages

Abstract

The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Hib porin-induced TNF-alpha and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-alpha and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

FIG. 1.

SDS-PAGE analysis of the outer membrane protein preparation and porin extract from Hib ATCC 9795. The gel was stained with Coomassie blue. Lane A, molecular mass standards (phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anydrase, 30 kDa; trypsin inhibitor, 20 kDa; α-lactalbumin, 14 kDa); lane B, Hib porin (10 μg); lane C, Hib outer membrane protein preparation (10 μg).

FIG. 2.

TNF-α and IL-6 production in peritoneal murine macrophages stimulated with Hib porin. Thioglycolate-elicited peritoneal macrophages from TLR2-deficient (TLR2^−/−), LPS-hyporesponsive (C3H/HeJ), and MyD88-defective (MyD88^−/−) mice were stimulated with different doses of Hib porin for 22 h. C57BL/6 mice were used as controls for TLR2- and MyD88-deficient mice. C3H/HeN mice were employed as wild-type (WT) controls for C3H/HeJ mice. The columns and bars represent the means ± the standard deviations of the results of three independent observations. The values for IL-6 concentrations in culture supernatants from unstimulated cells were subtracted from data obtained after porin stimulation. These basal expression values never exceeded 60 pg/ml. TNF-α was always undetectable in the supernatants of unstimulated cells. An asterisk (*) indicates results that are significantly different (P < 0.05) from those of the controls, as determined by one-way analysis of variance and the Student-Newman-Keuls test.

FIG. 3.

IL-8 release in stimulated transfected HEK 293 cells. Cells (5 × 10⁵/ml) were stimulated for 5 h with Hib porin (5 μg/ml) or heat-killed L. monocytogenes (1 μg/ml). The columns and bars represent the means ± the standard deviations of the results for three independent observations. IL-8 was undetectable in the supernatants of unstimulated cells. An asterisk (*) indicates a result significantly different (P < 0.05) from that of the controls, as determined by one-way analysis of variance and the Student-Newman-Keuls test.

FIG. 4.

IL-6 and TNF-α release in THP-1 cells treated with anti-TLR2/4 or anti-CD14 MAbs and stimulated with Hib porin. Cells (4 × 10⁶/ml) were preincubated for 30 min with anti-human TLR2 mAb (10 μg/ml), anti-human TLR4 mAb (10 μg/ml), anti-human CD14 (10 μg/ml), IgG1, IgG2a, or IgG2b as an isotype control before the Hib porin (0.05 to 5 μg/ml) was added. The columns and bars represent the means ± the standard deviations of the results of three independent observations (P < 0.05). An asterisk (*) indicates a result that is significantly different (P < 0.05) from the values observed with porins alone by one-way analysis of variance and the Student-Newman-Keuls test.