Background
Contrary to a number of chronic diseases, asthma incidence is on the rise [
1]. In the United States alone, more than 20 million individuals suffer from the disease. A sizable portion of these asthmatics do not respond to the existing drugs [
2]. Accordingly, the need for new drugs as mono or adjuvant therapies is immediate.
The pathogenesis of asthma involves several cellular and non-cellular factors including Th2 and Th17 CD4
+ T cells as well as B cells in addition to circulating factors such as IL-4, IL-5, IL-13 and many others [
3]. Targeting the function of these cells and the ensuing production of Th2 cytokines and IgE has been a critical objective both in the clinic and in the laboratory. Our laboratory pioneered the studies demonstrating the involvement of poly(ADP-ribose)polymerase (PARP)-1 in asthma [
4‐
8]. Our studies as well as those of others [
9‐
13] suggest that the protein may constitute a viable target for the treatment of the disease. PARP-1, a member of a large family of proteins, is a DNA repair-associated enzyme that participates in the recruitment and trafficking processes of DNA repair proteins and histones to the DNA lesions primarily through base excision repair [
14]. However, our laboratory and many others have suggested a role for the enzyme in a number of inflammatory conditions and regulation of transcription. We have shown that it controls NF-κB nuclear trafficking and thus transcription of NF-κB-dependent genes including those critical for asthma manifestation [
15‐
17]. We have also shown that PARP-1 controls the fate of STAT-6 upon IL-4 or allergen exposure both in vitro and in an animal model of the disease through a calpain-dependent mechanism [
8].
An ultimate goal of our studies is to explore the possibility that PARP can be targeted for therapy to treat asthma in human subjects. A great deal of effort has been made to generate potent inhibitors of the enzyme targeting cancer and inflammatory diseases [
18]. Recently, olaparib (AZD2281), a small molecule inhibitor of PARP-1 and PARP-2 showed great potential for the treatment of BRCA-negative breast and ovarian cancer [
19]. These neoplastic conditions were specifically targeted because the cancer cells accumulate fatal dsDNA breaks when exposed to DNA damaging agents in the absence of PARP activity leading a synthetic lethality phenotype [
20]. Because this process occurs only in BRCA-mutant cancer cells, PARP inhibition is not expected to affect normal cells. In several clinical trials, the drug showed a remarkable therapeutic efficacy with an acceptable safety index in cancer patients [
21]. It is noteworthy that other PARP inhibitors have also been developed and are currently tested in more than 20 clinical trials.
In the current study, we aimed to test the efficacy of olaparib in experimental asthma. We specifically examined whether olaparib administration at doses that can be translated to human therapy blocks some or all asthma-like traits. We also examined whether the drug blocks already established disease to mimic what actually occurs in human asthmatics.
Methods
Animals
C57Bl/6J wild type (WT) and OT-II mice (6–8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). C57BL/6 PARP-1−/− mice were generated through a backcrossing with C57BL/6 WT mice for eleven generations. The last generation was interbred to generate the C57BL/6 PARP-1−/− mice. WT mice generated through the PARP-1+/− mice breeding were also included in the experiment. Mice were bred in a specific-pathogen free facility at LSUHSC, New Orleans, LA, and allowed unlimited access to sterilized chow and water. Maintenance, experimental protocols, and procedures were approved by the LSUHSC Animal Care & Use Committee.
Ovalbumin (OVA) sensitization and challenge, Airway hyper responsiveness (AHR), organ recovery, staining, Th2 cytokine and IgE assessments, and FACS analysis
Mice were sensitized to chicken OVA (Sigma-Aldrich, St. Louis, MO, USA) as described [
6]. The mice were then challenged with aerosolized OVA for 30 min once (single challenge) or once a day for 3 days (multiple challenge). Control groups were not sensitized or challenged. Additional groups of mice received
i.p. 1, 5, or 10 mg/kg olaparib (Selleckchem, Pittsburgh, PA, USA) in saline 30 min after OVA challenge. AHR, organ recovery, histopathology, bronchoalveolar lavage (BAL), cytokine and OVA-specific IgE assessment, and FACS analysis were performed as described [
6,
22,
23]. To determine CD4
+ T cell populations, spleens were processed to generate single cell suspensions after which splenocytes were stained with antibodies to mouse CD3e (145-2c11-APC) and CD4-FITC (clone RM4-5) (both from e-Bioscience, San Diego, CA, USA). To determine T-regulatory (T-reg) cell populations, splenocytes were stained with CD4 (GK1.5-FITC) and CD25-APC (clone PC61) (from Biolegend, San Diego, CA, USA), and intracellularly with anti-mouse Foxp3 (FJK-16s)-PE (e-Bioscience) followed by FACS analysis. The multiplex assay and FACS were conducted at the LSUHSC Comprehensive Alcohol Research Center Core.
CD4+ T cell purification, Th1/Th2 skewing, TCR stimulation, Adoptive transfer, and RT-PCR
OT-II or WT mice were sacrificed and splenic CD4
+ T cells were isolated by negative selection (Stem Cell Technologies, Vancouver, Canada). Purified CD4
+ T cells were stimulated on coated plates with antibodies to CD3 (1 μg/ml) and CD28 (0.5 μg/ml) (e-bioscience, San Diego, CA, USA) then skewed toward a Th1 or Th2 phenotype as described [
23]. WT CD4
+ T cells were skewed in the absence or presence of 5 μM olaparib. RNA was extracted using Qiagen RNA extraction kit according to the manufacturer instructions. The extracted total RNA was used for the generation of cDNA using reverse transcriptase III (Invitrogen) and quantitative PCR was conducted using primer sets (IDT, San Jose, CA, USA) specific for mouse
gata-
3,
il-
4,
t-
bet,
ifn-
γ, or
β-
actin as described [
23,
24]. Quantitative determination of gene expression levels using a 2-step cycling protocol was conducted on a MyIQ Cycler (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the 2[−Delta Delta C(T)] method [
25]. Quantities of all targets were normalized to the mouse β-actin gene.
Th2-like cells from OT-II mice were administered i.v. into the tail vein of recipient mice (1 × 106 cells/mouse). All mice were subjected to OVA challenge daily for 4 days. Mice were sacrificed 48 h after the last challenge.
Data analysis
All data are expressed as means ± SEM of values from at least five mice per group unless stated otherwise. PRISM software (GraphPad, San Diego, CA, USA) was used to analyze the differences between experimental groups by one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Discussion
In this study, we show that olaparib administration is highly efficient in blocking established AAI and AHR, which constitute two major components of asthma. We also provide evidence for an important role for PARP-1 in CD4+ T cell function without a prominent effect on B cell function. Moreover, our results support the possibility that PARP inhibition may also influence T-reg cell accumulation as an additional mechanism in dampening allergic response in our experimental models. Lastly, the effect of olaparib on CD4+ T cell function may be strongly linked to the ability of PARP-1 to control expression of the transcription factor GATA-3.
Olaparib treatment was very effective in blocking repeated challenges to OVA in mice. Remarkably, a dose as low as 1 mg/kg of the PARP inhibitor was sufficient to confer protection against the manifestation of several asthma-like traits including AHR. As very recently shown by one of us [
26], olaparib is also effective in reducing lung inflammation induced by LPS and inhibits expression of several inflammatory factors including VCAM-1 and TNF-α. Our results show that a major role of PARP may be in the function of CD4
+ T cells. This is supported by the finding that an adoptive transfer of OT-II CD4
+ T cells was sufficient to reverse lung cellularity and production of Th2 cytokines and IgE to levels comparable to those detected in similarly treated WT mice. The Th1 cytokines were also elevated. An increase in IL-2 is expected as it is critical for CD4
+ T activation [
27]. However, the production in IFN-γ was surprising. Although speculative, it is possible that the increase in IFN-γ was mediated by PARP-1
−/− CD4
+ T cells in the presence of IL-2 produced by the adoptively transferred WT CD4
+ T cells. It is also possible that the increase in IFN-γ may be mediated by PARP-2. This is based on the observation that PARP-1 gene knockout only slightly increase the expression of the Th1 cytokine while treatment with olaparib, which inhibits both PARP-1 and PARP-2, substantially increased it (Figure
3c). It is important to acknowledge that the study, as conducted, does not cover all the aspects of asthma manifestation and it remains to be determined whether the transfer of WT CD4
+ T cells is sufficient to reverse AHR and mucus production in PARP-1
−/− mice. Although more specific experimentation is required, it is tempting to conclude from the adoptive transfer study that PARP-1 may not play a direct role in B cell function. The adoptive transfer of OT-II CD4
+ T cells was sufficient to induce substantial levels of OVA-specific IgE. Such immunoglobulin production could have only been produced by PARP-1
−/− B cells clearly suggesting that the function of these cells is comparable to that of WT B cells in response to OVA challenge. We speculated in our previous studies that the primary reason for the reduced production of IgE upon PARP inhibition is the effect on IL-4 production [
5,
6]. We cannot, however, exclude the possibility that PARP plays a role in B cell trafficking especially when considering the effect of PARP inhibition, pharmacologically or by gene knockout, on the overall recruitment of lymphocytes to the lung as shown in Figures
1b and
2b. The role of PARP-1 in GATA-3 expression may be the driving cause for the ability of PARP inhibition to reduce IL-4, IL-5, and IL-13 production. It is noteworthy that GATA-3 is the master regulator for the development of Th2 cells [
28] through its ability to control the activation of the
Il4/Il5/Il13 cytokine locus.
The role of PARP-1 in T-reg cell accumulation has been reported in mice, which was associated with an increase in Foxp3 [
29]. We confirm these results in the experimental AAI setting. Although olaparib increased the T-reg (CD4
+/CD25
+/Foxp3
+) cells upon a single or repeated OVA challenge, T-reg cells were increased in PARP-1
−/− mice regardless of challenge with OVA. This suggests that PARP-1 moderately regulates T-reg cells but not upon an inflammatory response. Whether the slight increase in T-reg cells is a major driving force in the anti-inflammatory effect of PARP inhibition is not clear. Interestingly, a recent study demonstrated that T-reg cells isolated from PARP-1
−/− mice are as functional as those isolated from WT mice [
30]. Overall, the present studies provide critical information on the role of PARP-1 upon an acute or established AAI and AHR and provide support to the notion that PARP can be targeted for the treatment of some aspects of human asthma.
Almost two dozen clinical trials most of which are in phase II or III are currently examining the possibility of establishing olaparib as a mono or adjuvant therapy for some specific cancers with BRCA mutation [
19]. It is noteworthy that there are additional drugs with varying potency in inhibiting PARP under clinical trials most of which focus on the synthetic lethality induced by the drugs in BRCA-mutant cancer cells [
19]. This phenomenon, as stated above, spares normal cells while targeting specifically the mutant cancer cells leading to their demise as a result of the accumulation of a fatal level of dsDNA breaks. It is important to note that the overarching assumption of these clinical trials is that these drugs do not have any important negative effects on normal cells and tissues. According to a clinical trial conducted by Fong et al. [
21], a total of 200 mg olaparib, daily for more than 24 weeks, did not cause any side effects. This dose represents a 2.3 mg/kg for men with an average weight of 87 and 2.69 mg/kg for women with an average weight of 74.4. These doses fall between the 1 and 5 mg/kg doses used in the current study with which we observed substantial protection against experimental asthma. It is important to note that in the aforementioned clinical study and others [
31‐
33] on higher doses of olaparib for patients with breast or ovarian cancer, the most common side effects were nausea, vomiting, fatigue and anemia. Despite these effects, discontinuation of the drug due to these side effects was a rare event. Additionally, patients with advanced cancer may be more prone to adverse events than asthma patients. Nevertheless, this would need to be tested closely in any human clinical study. The likely reduced side effects associated with the use of low doses of olaparib or other PARP inhibitors is very promising for the potential use of these drugs in treatment regimens against human asthma. Furthermore, treatment regimens may be extensive and lengthy in cancer, which may not be the case in asthma predicting that the use of olaparib in asthma may be associated with lesser side effects. Perhaps the therapeutic potential of olaparib may become more relevant to difficult to treat asthma especially those that do not respond to corticosteroids. Although we remain cautious, our study suggests that olaparib and potentially other PARP inhibitors are ready for testing on human asthma.
Authors’ contributions
MAG is the graduate student who led the study, conducted most of the experiments and the statistical analyses. KP conducted some the in vitro experiments and the statistical analyses. SVI conducted the skewing experiments, RT-PCR, and the statistical analyses. AAA assisted with some of the animal studies and FACS analysis. JW assisted with the animal studies. PR conducted some of the in vitro experiments and help in the troubleshooting. HFR assisted with some of the animal studies and FACS analysis. AHE assisted with the animal studies. MRL edited the manuscript and contributed to discussion. MSM contributed to the training of the first author. KAG contributed to the training of the first author. AR provided reagents and assistance with FACS. AO helped in the discussion and provided reagents and access to instruments. ASN contributed to the training of several members of the laboratory, experimental design, and troubleshooting. AHB is the principal investigator; contributed to the design of the experiment and the training of researchers as well as provided the financial support for the conducted work. All authors read and approved the final manuscript.