Human monoclonal antibodies against Clostridium difficile toxins A and B inhibit inflammatory and histologic responses to the toxins in human colon and peripheral blood monocytes

Abstract

Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage.

Fig 1

MK3415 attenuated toxin A-mediated TNF-α expression in PBMCs. (A and B) PBMCs were incubated with no antibody (blank), control IgG, or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 0 to 1 μg/ml for 4 h. (C and D) PBMCs were incubated with control IgG or MK3415 (0.1 to 50 mg/ml) for 30 min, followed by incubation with toxin A at 0.1 μg/ml for 4 h. Conditioned media were collected for TNF-α ELISA. TNF-α mRNA expression was detected by real-time RT-PCR. All experiments are representative of 3 independent experiments.

Fig 2

MK6072 attenuated toxin B-mediated TNF-α expression in PBMCs. (A and B) PBMCs were incubated with no antibody (blank), control IgG, or MK6072 (30 μg/ml) for 30 min, followed by incubation with toxin B at 0 to 10 μg/ml for 4 h. (C and D) PBMCs were incubated with control IgG or MK6072 (0.1 to 50 μg/ml) for 30 min, followed by incubation with toxin B at 1 μg/ml for 4 h. Conditioned media were collected for TNF-α ELISA. TNF-α mRNA expression was detected by real-time RT-PCR. All experiments are representative of 3 independent experiments.

Fig 3

MK3415 attenuated toxin A-mediated IL-1β expression in PBMCs. (A and B) PBMCs were incubated with no antibody (blank), control IgG, or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 0 to 1 μg/ml for 4 h. (C and D) PBMCs were incubated with control IgG or MK3415 (0.1 to 50 μg/ml) for 30 min, followed by incubation with toxin A at 0.1 μg/ml for 4 h. Conditioned media were collected for IL-1β ELISA. IL-1β mRNA expression was detected by real-time RT-PCR. All experiments are representative of 3 independent experiments.

Fig 4

MK6072 attenuated toxin B-mediated IL-1β expression in PBMCs. (A and B) PBMCs were incubated with no antibody (blank), control IgG, or MK6072 (30 μg/ml) for 30 min, followed by incubation with toxin B at 0 to 10 μg/ml for 4 h. (C and D) PBMCs were incubated with control IgG or MK6072 (0.1 to 50 μg/ml) for 30 min, followed by incubation with toxin B at 1 μg/ml for 4 h. Conditioned media were collected for IL-1β ELISA. IL-1β mRNA expression was detected by real-time RT-PCR. All experiments are representative of 3 independent experiments.

Fig 5

MK3415 and MK6072 inhibited toxin A- and toxin B-mediated NF-κB p65 phosphorylation in PBMCs. (A) PBMCs were incubated with control IgG or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 0 to 1 μg/ml for 1 h. (B) PBMCs were incubated with control IgG or MK3415 (0.1 to 50 mg/ml) for 30 min, followed by incubation with toxin A at 0.1 μg/ml for 1 h. (C) PBMCs were incubated with control IgG or MK6072 (30 μg/ml) for 30 min, followed by incubation with toxin B at 0 to 10 μg/ml for 1 h. (D) PBMCs were incubated with control IgG or MK6072 (0.1 to 50 mg/ml) for 30 min, followed by incubation with toxin B at 1 μg/ml for 1 h. Equal amounts of cellular protein were used for detection of phospho-p65 (p-p65) expression. All experiments are representative of 3 independent experiments.

Fig 6

MK3415 and MK6072 attenuated toxin A- and toxin B-mediated TNF-α and IL-1β mRNA expression in human colonic explants. (A) Fresh human colonic explants were incubated with control IgG or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 0 to 1 μg/ml for 16 h. (B) Fresh human colonic explants were incubated with control IgG or MK6072 (30 μg/ml) for 30 min, followed by addition of toxin B at 0 to 10 μg/ml for 16 h. TNF-α mRNA expression was detected by real-time RT-PCR. (C) Fresh human colonic explants were incubated with control IgG or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 0 to 1 μg/ml for 16 h. (D) Explants were incubated with control IgG or MK6072 (30 μg/ml) for 30 min, followed by addition of toxin B at 0 to 10 μg/ml for 16 h. IL-1β mRNA expression was detected by real-time RT-PCR. All experiments are representative of 5 patient samples.

Fig 7

MK3415 and MK6072 attenuated toxin A- and toxin B-mediated epithelial damage in human colonic explants. (A) Fresh human colonic explants were incubated with control IgG or MK3415 (30 μg/ml) for 30 min, followed by incubation with toxin A at 1 μg/ml for 16 h. (C) Explants were incubated with control IgG or MK6072 (30 μg/ml) for 30 min, followed by addition of toxin B at 10 μg/ml for 16 h. (B and D) Epithelial tissue damage was evaluated semiquantitatively as described in Materials and Methods. All experiments are representative of 5 patient samples.