The bleomycin model is one of the best-characterized models of fibrosis since this chemotherapeutic invokes a highly reproducible inflammatory response that ultimately leads to fibroblast proliferation and collagen deposition 
. IL-13 is one of the dominant pro-fibrotic cytokines produced during the development of fibrosis in the bleomycin model since it directly activates fibroblasts 
. Puri and colleagues 
have previously shown that tumor cells expressing IL-13Rα2 are susceptible to the cytotoxic effects of IL13-PE making this chimeric protein a highly effective anti-tumor agent, and we have extended these findings to show that targeting IL-13 responsive cells such as fibroblasts is also highly effective in treating established fibrotic responses in the lung 
. Prior to further development of IL13-PE as a therapeutic in clinical disease we undertook the present study in order to evaluate whether prior exposure to P. aeruginosa
pathogen or IL13-PE altered the therapeutic potential of IL-13 immunotoxin. Our study highlighted two important findings: 1) immune responses to Pseudomonas
and/or its exotoxin actually protect mice from bleomycin-induced pulmonary fibrosis; 2) neutralizing antibodies against Pseudomonas
and/or its exotoxin do not hamper the therapeutic effect of IL13-PE delivered into the fibrotic lung.
is a pathogen of significant clinical importance because it can elicit a severe pneumonia in affected individuals. No prior association between this pathogen and fibrosis was apparent in our literature searches so we undertook experiments to better understand how this micro-organism might affect the lung subsequently exposed to bleomycin sulfate. While antibody responses to live Pseudomonas
infections in the lung have been described previously 
, little was known about the specificity of these antibody responses against Pseudomonas
exotoxin. From the present study it was apparent that relatively small intrapulmonary inoculations of live Pseudomonas
bacilli evoked strong IgA and IgG2a anti-exotoxin antibody systemic (in serum) and local (in lung) responses. When we next examined the impact of a prior P. aeruginosa
infection on the susceptibility of the lung to fibrosis, we were surprised to observe that prior infection protected mice from severe pulmonary fibrosis. Protection from fibrosis did not correlate with the retention of bacilli in the lungs of these mice as no bacteria were recovered from these mice prior to bleomycin injection day (data not show). These findings were surprising in light of several studies that have shown that repeated exposure or chronic infections with pathogens such as Paracoccidioides brasiliensis 
, Saccharopolyspora rectivirgula 
and many others 
leads to the development of pulmonary fibrosis. The divergence observed between the present and previous studies might be explained by the rapid clearance of P. aeruginosa
in the present study prior to the induction of pulmonary fibrosis with bleomycin. It is possible that acute bacterial infection drives a protective immune response that tempers bleomycin-induced fibrosis. We are presently addressing the hypothesis that this protective effect observed in acutely infected mice involves pathogen-associated molecular pattern (PAMP) activation of toll like receptors (TLRs). Specifically, we have observed that PAMPS such as bacterial hypomethylated DNA or CpG motifs inhibit the development and progression of bleomcyin-induced pulmonary fibrosis (CMH, unpublished findings). When these CpG motifs bind TLR9 in lung immune cells, this leads to the generation of type 1 interferons, which have potent anti-fibrotic effects. Further studies will address the possibility that CpG and TLR9 activation in immune cells accounts, at least in part, for the protective effects of a prior P. aeruginosa
Systemic but not local IL13-PE dosing led to the development of neutralizing antibodies against this chimeric protein, which effectively blocked the cytotoxic action of IL13-PE in vitro
toward tumor cells. Using a well-characterized tumor toxicity assay 
, we analyzed the ability of various serum samples from mice systemically sensitized to IL13-PE to block the cytotoxicity of IL13-PE against IL-13Rα2-expressing tumor cells. We observed that serum from IL13-PE-sensitized mice markedly dampened the IL13-PE-induced toxicity against target tumor cells suggesting the presence of neutralizing antibodies directed against IL13-PE in serum. Accordingly, the development of neutralizing antibodies to IL13-PE was a concern given that these neutralizing antibodies might interfere with IL13-PE therapy in pulmonary fibrosis. However, despite the presence of the neutralizing antibodies in sensitized mice, intranasal IL13-PE therapy worked effectively as a therapeutic in the bleomycin-induced pulmonary fibrosis model. The manner in which IL13-PE continues to work in the lung despite the presence of these neutralizing antibodies is not presently clear but we have observed that the numbers of IL-13Rα2-positive cells during pulmonary fibrosis are markedly increased (
and unpublished findings). Under these conditions it is possible that the neutralizing titers of antibody are not sufficient to prevent IL13-PE from binding to this high affinity IL-13 receptor. Further studies will address this possibility in the lung.
Thus, using a well-established bleomycin pulmonary fibrosis model, we have observed that prior Pseudmonas infection or systemic sensitization to IL13-PE does not impair the therapeutic anti-fibrotic properties of IL13-PE. Surprisingly, both of these events provide protective effects either by reducing the fibrotic response (as in the case of infection) or sparing mice from the lethal effects of pulmonary fibrosis (as in the case of systemic IL13-PE sensitization). Together, these findings further bolster the prospects of IL13-PE as a clinically useful therapeutic in the treatment of pulmonary fibrosis.