The counter regulatory response induced by CpG oligonucleotides prevents bleomycin induced pneumopathy

Abstract

Bleomycin (BLM) induces life-threatening pneumonitis and pulmonary fibrosis in 20% of patients, limiting its use as a chemotherapeutic agent. Oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) stimulate cells that express Toll-like receptor 9 to initiate an inflammatory response. This short-lived inflammation is physiologically suppressed by a counter-regulatory process that peaks five days later. Using a murine model of BLM-induced lung injury, the effect of CpG ODN treatment on pulmonary inflammation, fibrosis and mortality was examined. Administering CpG ODN 5 days before BLM (so that the peak of the counter-regulatory process induced by CpG ODN coincided with BLM delivery) resulted in a dose-dependent reduction in pulmonary toxicity (p < 0.005). Delaying the initiation of therapy until the day of or after BLM administration worsened the inflammatory process, consistent with the counter-regulatory process rather than initial pro-inflammatory response being critical to CpG induced protection. The protection afforded by CpG ODN correlated with reduced leukocyte accumulation and inflammatory cytokine/chemokine production in the lungs. These changes were associated with the increased production of IL-10, a critical element of the counter-regulatory process triggered by CpG ODN, and the concomitant down-regulation of BLM-induced IL-17A and TGF-β1 (which promote pulmonary toxicity). This work represents the first example of the physiologic counter-regulation of TLR induced immune activation being harnessed to block an unrelated inflammatory response.

Figure 1

CpG ODN protect mice from lethal BLM-induced lung injury. 0.05 U of BLM was delivered into the lungs of C57BL/6 mice via the intra-tracheal route on day 0. A) Five days earlier, mice were injected i.p. with 200 μg of control or CpG ODN. B) Mice were treated with 0 - 200 μg of CpG ODN 5 days before BLM. C) Mice were treated with 200 μg of CpG ODN 5 days before, at the same time, or 5 days after BLM instillation. Data represent combined results from 2-3 independent experiments involving a total of 11-20 mice/group. Survival was analyzed with Kaplan-Meier statistics using the log rank test. *; p < 0.05, **; p < 0.005.

Figure 2

Effect of CpG ODN on BLM-induced cytokine production. Mice were injected i.p. with 200 μg of control or CpG ODN 5 days before 0.05 U of BLM was delivery intra-tracheally. The concentration of KC, MIP-2, IL-6 and IL-1β in BAL fluid collected 1 (KC and MIP-2) or 3 (IL-6 and IL-1β) days after BLM instillation was determined by ELISA. Results represent the average fold change + SE in cytokine levels compared to BLM alone in 2-3 independent experiments involving a total of 10-19 mice/group. **; p < 0.005.

Figure 3

Effect of CpG ODN on BLM-induced leukocyte accumulation in the lungs. Mice were injected i.p. with 200 μg of control or CpG ODN 5 days before 0.05 U of BLM was delivery intra-tracheally. A) The number of leukocytes present in BAL collected 1 - 7 days after BLM instillation was determined. Statistic significance was analyzed by longitudinal regression analysis. B) Cell differentials (based on 300 leukocytes/sample) in BAL collected 5 days after BLM instillation were performed on cytocentrifuge preparations after Diff-Quick staining. Data show the mean ± SE of the combined results from 2 independent experiments involving 10 mice/group/time point. **; p < 0.005.

Figure 4

Pretreatment with CpG ODN inhibits lung fibrosis. Mice were injected i.p. with 200 μg of control or CpG ODN 5 days before 0.03 U of BLM or PBS (no BLM) was delivery intra-tracheally. Lungs were removed 14 days later. A) The amount of collagen was determined analytically using the Sircol collagen assay. Data show the percent change in the amount of collagen in experimental groups vs mice that did not receive BLM. This was calculated by combining results from 3 independent experiments involving 10-15 mice/group. B) Representative histologic sections from these lungs. C) Lung tissue was fixed and stained with Masson’s Trichrome. The severity of lung fibrosis were graded as (0) none, (1) minimal, (2) mild, (3) moderate and (4) severe. Data shows the mean + SE of the combined results from 2 independent experiments involving 6-7 mice/group. *; p < 0.05, **; p < 0.005.

Figure 5

CpG ODN treatment decreases IL-17A and TGF-β1 production. Mice were treated as described in Figure 2. A) Cells were isolated from the BAL and draining thoracic lymph nodes 3 days after BLM instillation. These cells were stimulated in vitro with 50 ng/ml PMA plus 1 μg/ml ionomycin for 4 hours. The concentration of IL-17A in culture supernatants was determined by ELISA. B) The concentration of TGF-β1 in BAL fluid collected 3 days after BLM instillation was determined by ELISA. Data show the change in concentration of IL-17A and TGF-β1 (avg + SE) as a percentage of the BLM treated group alone from 2-3 independent experiments involving 9-14 mice/group. **; p < 0.005.

Figure 6

CpG ODN treatment induces the expression of IL-10. Mice were injected i.p. with 200 μg of control or CpG ODN 5 days before 0.05 U of BLM was delivery intra-tracheally. Mice were sacrificed immediately before BLM (day 0 time point) or 3 and 7 days after BLM. IL-10 mRNA expression in the lung was measured by quantitative RT-PCR. Data represent the mean + SE of combined results from 2 independent experiments involving 8-10 mice/group/each time point. **; p < 0.005.

Figure 7

Effect of anti-IL-10R Abs on CpG induced protection. Mice were injected i.p. with 200 μg of CpG ODN 5 days before 0.05 U of BLM or PBS (no BLM) was delivery intra-tracheally. Some groups were also treated with 200 μg of anti-IL-10R Abs or isotype matched Abs i.p. on the day of and 5 days after BLM instillation. Results from 5 mice/group are shown.