Therapeutic efficacy of Cintredekin Besudotox (IL13-PE38QQR) in murine lung fibrosis is unaffected by immunity to Pseudomonas aeruginosa exotoxin A

Abstract

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/principal findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

Figure 1. P. aeruginosa infection induced an antibody response against PE and IL13-PE.

A: IgA levels in BAL; B: IgG2a in BAL; C: IgA in serum. C57Bl/6 mice were infected by oropharingeal route with one of a range of bacterial doses and their bronchoalveolar fluid (BAL) and blood were collected at 14 days post infection. Antibody levels were evaluated by ELISA using plates coated with PE (open bars) or IL13-PE (closed bars). Statistical comparisons were determined between infected and non-infected groups with the same coating on the ELISA plate. *p≤0.05 compared with biologic sample added to PE-coated wells. ^#p≤0.05 compared with biologic sample added to IL13-PE-coated wells.

Figure 2. IL13-PE treatment reduced the histological appearance of pulmonary fibrosis regardless of the prior P. aeruginosa infection status of the mouse.

C57Bl/6 mice were infected by oropharingeal route with two bacterial doses (1.5×10⁵ or 3×10⁵ bacilli/mouse), fourteen days after infection mice were injected with bleomycin to exacerbate fibrosis and at days 21, 23, 25 and 27 after bleomycin mice received intranasal IL13-PE treatments (1000 ng/dose). At day 28 after bleomycin, lungs were collected and processed using routine histological techniques and stained with Masson's trichrome for histopathological analysis. Magnification 100×.

Figure 3. Effect of systemic IL13-PE sensitization on the development of pulmonary fibrosis and the subsequent intranasal IL13-PE therapy.

Mice were sensitized with IL13-PE via an intraperitoneal immunization protocol. Controls received saline alone. One week after last dose of IL13-PE or saline, all mice were injected i.t. with bleomycin. Twenty-one days after the induction of pulmonary fibrosis, treatment started with saline or IL13-PE intranasal instillation. At day 28 after bleomycin, lungs were collected for histopathological or biochemical assessment of pulmonary fibrosis. Magnification 100× (A). Hydroxyproline content in lung homogenate from saline (closed bars) or IL13-PE (hatched bars) sensitized animals are shown (B).