The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells

Abstract

The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor alpha [TNF-alpha]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.

FIG. 1.

Cytokine induction by the LT-II toxins and CT. THP-1 cells were incubated for 16 h in the absence or presence of heat-labile enterotoxins (LT-IIa, LT-IIb, and CT; all at 2 μg/ml) or with E. coli LPS (10 ng/ml; positive control). Culture supernatants were assayed for cytokine content by ELISA. Results are presented as means ± standard deviations of triplicate determinations. Values that are statistically significantly different (P < 0.05) from those of controls treated only with medium are indicated by an asterisk.

FIG. 2.

LT-II toxins and CT regulate cytokine induction in activated cells. THP-1 cells were pretreated for 1 h with medium only or with 2-μg/ml concentrations of LT-IIa, LT-IIb, or CT. The cells were subsequently incubated for an additional 16 h with medium only, E. coli LPS (Ec-LPS), P. gingivalis LPS (Pg-LPS), or FimA. Culture supernatants were assayed for TNF-α (A) or IL-1β (B) responses by ELISA. Results are shown as means ± standard deviations of triplicate determinations. Asterisks indicate statistically significant (P < 0.05) inhibition of TNF-α (A) or enhancement of IL-1β (B) responses in LPS- or FimA-activated cells by the toxins.

FIG. 3.

AB₅ toxins inhibit, whereas their B pentamers promote, IL-8 induction. THP-1 cells were pretreated for 1 h with medium only or with 2-μg/ml concentrations of LT-IIa, LT-IIb, CT, or their respective B pentamers. The cells were subsequently incubated for an additional 16 h with 1 μg of Ec-LPS/ml (A) or were left without further treatment (B). The insert summarizes the results of an independent experiment in which THP-1 cells were incubated for 16 h with medium only or with B pentamers in the absence or presence of 10 μg of polymyxin B (PMB)/ml. Induction of IL-8 release in culture supernatants was assayed by ELISA, and data shown are means ± standard deviations of triplicate determinations. (A) Statistically significant (P < 0.05) inhibition or enhancement of LPS-induced IL-8 release is indicated by an asterisk or a black circle, respectively. (B) B-pentamer-induced IL-8 responses that are statistically significantly (P < 0.05) higher than those corresponding to their respective holotoxins are indicated by asterisks, while IL-8 responses that are statistically significantly (P < 0.05) elevated over medium-only-treated controls are indicated by black circles.

FIG. 4.

LT-IIbB is a more potent cytokine inducer than LT-IIaB or CTB. THP-1 cells were incubated for 16 h in the absence or presence of LT-IIaB, LT-IIbB, or CTB (all at 2 μg/ml) or with E. coli LPS (10 ng/ml; positive control). Induction of TNF-α, IL-1β, or IL-6 release in culture supernatants was assayed by ELISA. Results are presented as means ± standard deviations of triplicate determinations. Values that are statistically significantly different (P < 0.05) from those of medium-only-treated controls are indicated by an asterisk.

FIG. 5.

The LT-II toxins and CT synergize with proinflammatory stimuli in IL-10 induction. THP-1 cells were pretreated for 1 h with medium only or with 2-μg/ml concentrations of LT-IIa, LT-IIb, or CT. The cells were subsequently incubated for an additional 16 h with medium only, Ec-LPS, Pg-LPS, or FimA. Induction of IL-10 release in culture supernatants was assayed by ELISA. Results are presented as means ± standard deviations of triplicate determinations. Asterisks indicate statistically significant (P < 0.05) enhancement of IL-10 induction compared to treatment with proinflammatory stimuli in the absence of LT-II toxins or CT.

FIG. 6.

Cytokine induction by the LT-IIb B pentamer is regulated by LT-IIb holotoxin. THP-1 cells were incubated for 16 h with medium only, LT-IIbB alone, LT-IIbB plus LT-IIb, or LT-IIb alone (all at 2 μg/ml). Culture supernatants were assayed for cytokine content by ELISA. Results are presented as means ± standard deviations of triplicate determinations. Cytokine responses in cells treated with both LT-IIbB and LT-IIb holotoxin that are statistically significantly different (P < 0.05) from those for treatment with LT-IIbB alone are indicated by asterisks.