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

Kinjo Takeshi, Tomaru Koji, Haines, Klinman

Description

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.

Sujets

• Bleomycin
• Pneumonitis
• Fibrosis

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Kinjo et al. Respiratory Research 2012, 13:47
http://respiratory-research.com/content/13/1/47
RESEARCH Open Access
The counter regulatory response induced by CpG
oligonucleotides prevents bleomycin induced
pneumopathy
1 1 2 1*Takeshi Kinjo , Koji Tomaru , Diana C Haines and Dennis M Klinman
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.
Keywords: Bleomycin, Pneumonitis, Fibrosis, CpG oligonucleotide
Introduction dose-limiting side effect of this therapy is interstitial
Bleomycin is effective in the chemotherapy of various pneumonitis progressing to fibrosis [7-9]. Approximately
cancers including squamous cell carcinomas of the head 20% of patients treated with BLM develop pneumonitis,
and neck, cervix, and esophagus, germ cell tumors and with a mortality that approaches 20% [7,10-12].
both Hodgkins and non-Hodgkins lymphoma [1-3]. The lung cells damaged by BLM release uric acid and
BLM exerts its anti-tumor activity by generating oxygen other factors that trigger alveolar macrophages to secrete
and free radicals via the reduction of Fe (II) to Fe (III). pro-inflammatory cytokines and chemokines (including
These free radicals cause DNA strand breakage and cell IL-1β, IL-6, KC and MIP-2). These recruit additional in-
death [4-6]. BLM is metabolized/inactivated under flammatory cells to the lung that contribute to the devel-
physiologic conditions by bleomycin hydrolase. As there opment of bleomycin-induced pneumonitis (BIP) and
is a paucity of this enzyme in the lungs, they are un- pulmonary fibrosis [13-15]. Recent studies indicate that
usually susceptible to BLM-induced toxicity. While IL-17A, TGF-β, and IL-10 are critical regulators of
several pulmonary syndromes associated with BLM BLM-induced pneumopathy. IL-17A and TGF-β
administration have been described, the most common synergistically promote the development of BIP whereas
IL-10 down-regulates IL-17A and thus abrogates disease
[16]. The importance of these factors was confirmed in
* Correspondence: [e-mail]
1 animal models showing that BLM-induced lung injuryCancer and Inflammation Program, National Cancer Institute, Bldg 567, Rm
205, NCI at Frederick, Frederick, MD 21702, USA was reduced in mice in which IL-17A or its receptor
Full list of author information is available at the end of the article
© 2012 Kinjo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Kinjo et al. Respiratory Research 2012, 13:47 Page 2 of 8
http://respiratory-research.com/content/13/1/47
were deleted/blocked, while IL-10 KO mice developed In vitro stimulation of LN and BAL cells
more severe BLM-induced lung inflammation and A single cell suspension prepared from the draining
5
fibrosis [16-19]. thoracic LN and BAL was plated at 0.5 - 4 x 10 cells
The unmethylated CpG motifs present in bacterial per well in 96-well microtiter plates and stimulated with
DNA stimulate cells that express Toll-like receptor 9 50 ng/ml PMA and 1 μg/ml Ionomycin (Sigma-Aldrich)
(TLR9). This interaction triggers a short-lived innate for 4 hr in RPMI 1640 medium supplemented with 5%
immune response characterized by the production of FCS, 50 U/ml penicillin and 50 μg/ml streptomycin.
pro-inflammatory cytokines and chemokines [20-22].
The immune stimulation induced by bacterial DNA is Elisa
duplicated by synthetic oligonucleotides expressing CpG KC, MIP-2, IL-1β, IL-6 and IL-17A levels in BAL and
motifs (CpG ODN). culture supernatants were measured by ELISA, as previ-
Microarray identification of the genes triggered by CpG ously described [28]. TGF-β1 was measured by ELISA
ODN showed that the inflammatory response peaked on (R&D Systems).
day 1, but that>96% of these genes were suppressed by a
counter-regulatory process that peaked on day 5 [23,24]. Evaluation of lung fibrosis
IL-10 was identified as a key mediator of this down- Total soluble lung collagen was detected using the Sircol
regulatory process, based on both bioinformatic and func- collagen assay (Biocolor, Northern Ireland, UK). Lung
tional studies of CpG-induced inflammation [23,25,26]. As tissue was fixed and stained with Masson’s Trichrome.
noted above, IL-10 also reduces BLM-induced pulmonary The severity of lung fibrosis were evaluated by a path-
inflammation by down-regulating IL-17A production. This ologist blinded to the origin of each sample.
combination of findings raised the possibility that the
counter-regulatory process induced by CpG ODN might RNA isolation and quantitative RT-PCR
be harnessed to limit BLM-dependent pneumonitis. A RNA was extracted from homogenized lung using the
murine model was used to examine the effect of CpG RNeasy Mini Kit (QIAGEN,Valencia, CA) and cDNA was
ODN on BLM-induced morbidity and mortality, and generated using the Reverse Transcription Kit (QIAGEN).
analyze theimpact of this treatment on the production and The cDNA from each sample was used for a quantitative
regulation of pro-inflammatorycytokines and chemokines. RT-PCR based on TaqMan chemistry using the Step One
Plus real-time PCR System (Applied Biosystems). The
reaction mixture contained Taqman Gene Expression
Materials and methods Master Mix with Taqman probes for the IL-10 gene with
Oligodeoxynucleotides FAM dye (Mm00439616m1) and for the GAPDH gene
Phosphorothioate CpG ODN 1555 (sequence: GCTA withVIC dye(AppliedBiosystems, FosterCity, CA).
GACGTTAGCGT) and control ODN 1612 (sequence:
GCTAGAGCTTAGGCT) were synthesized at the Cen- Statistical analysis
ter for Biologics Core Facility. ODNs were administered Differences between groupswere assessedusing a one-way
by intraperitoneal injection in all experiments. All ODNs ANOVA and differences in survival rate were determined
were free of endotoxin and protein contamination. using the Kaplan-Meier log-rank test. P values≤0.05 were
considered significant for all analyses.
BLM-induced lung injury model
ResultsFemale C57BL/6 mice were studied at 7-12 wk of age.
Mice pre-treated with CpG ODN survive BLM-inducedBLM (Sigma-Aldrich, St. Louis, MO) in 50 μl of PBS was
lung injuryinstilled endotracheally into the lungs of anesthetized mice
Preliminary studies established that intra-tracheal instilla-using a 24-gauge catheter. All experiments were approved
tion of 0.05 U of BLM consistently induced severeby the NCI Animal Care and Use Committee, and mice
pulmonary inflammation. The severity of the resultantweremonitored daily.
pneumonitis required that 80% of the animals be eutha-
nized within 3 wk. As CpG ODN treatment induces a
Collection of bronchoalveolar lavage (BAL) fluid and cells short-term inflammatory response that is down-regulated
The trachea was cannulated with a 22-gauge catheter and under physiological conditions after 3 - 5 days, the effect of
BAL fluid collected as previously described [27]. BAL was CpG ODN administration on BLM induced toxicity was
centrifuged and supernatants analyzed as described below. evaluated. As seen in Figure 1A, lethal pulmonary inflam-
Cell pellets were re-suspended and analyzed histologically mation was prevented by treating mice with 200μgofCpG
after cytocentrifugation and Diff-Quick staining (Dade ODN 5 days before BLM instillation (p<0.005). This effect
Behring, Deerfield, IL). wasCpG specific, ascontrol ODN had no impact onKinjo et al. Respiratory Research 2012, 13:47 Page 3 of 8
http://respiratory-research.com/content/13/1/47
To evaluate the effect of CpG treatment on BLM-
(A) induced lung injury, BAL fluid was collected 1 - 7 days
100
after BLM instillation. Consistent with previous reports,CpG ODN80
BLM triggered a significant increase in the concentra-60 ** **
tion of KC and MIP-2 (peaking on day 1 and persist