Impacts of anti‐inflammatory phosphodiesterase inhibitors on a murine model of chronic pulmonary inflammation

Abstract Chronic obstructive pulmonary disease (COPD) often tends to respond poorly to glucocorticoid (GC) therapy. Reduced Histone deacetylase‐2 (HDAC‐2) activity is an important mechanism behind this GC insensitivity. In this study, we investigated the effects of three phosphodiesterase inhibitors (PDEIs), with an anti‐inflammatory propensity, on cigarette smoke (CS)‐induced pulmonary inflammation and HDAC‐2 activity. Male C57BL/6 mice were exposed to cigarette smoke (CS) over the course of 30 weeks. Administration of the PDEIs commenced from the 29th week and followed a schedule of once daily treatments, 5 days a week, for 2 weeks. Roflumilast (ROF) was administered intragastrically (5 mg·kg−1), while pentoxifylline (PTX) (10 mg·kg−1) and theophylline (THEO) (10 mg·kg−1) were administered intraperitoneally, either alone or in combination with a GC (triamcinolone acetonide or TRI, 5 mg·kg−1, i.m., single injection). Lung morphometry, as well as the activity of HDAC‐2, pro‐inflammatory cytokines and reactive oxygen species (ROS) were assessed at the end of the 30‐week course. CS exposure was associated with a reduction in HDAC‐2 activity and the up‐regulation of ROS expression. PTX, ROF, and THEO administration led to the partial restoration of HDAC‐2 activity, which was favorably associated with the reduction of ROS expression. However, combining TRI to any of these PDEIs did not synergistically augment HDAC‐2 activity. Inactivation of HDAC‐2 due to long‐term CS exposure is closely related to exaggerated oxidative stress, and this reduced HDAC‐2 activity could partially be restored through the use of PDEIs. This finding provides a potential novel approach for further clinical research.


| INTRODUC TI ON
Chronic obstructive pulmonary disease (COPD) affects approximately 13% of people over the age of 40 1 and was the fifth leading cause of death in China in 2016. 2 The 2021 Global Initiative for Chronic Obstructive Lung Disease report, 3 suggests that current pharmacological therapy for COPD is far from optimal and there is no conclusive evidence that any existing medication modifies disease progression in COPD patients, leading to a long-term decline in lung function. Unlike bronchial asthma, in which patients could possibly control using glucocorticoid steroids (GCs), the response to GCs treatment in patients with COPD is not as effective as in asthma. This insensitivity to GCs in COPD patients is thought to be due to a reduction in histone deacetylase-2 (HDAC2) activity, which correlates with COPD clinical severity. [4][5][6] Excessive inflammation, together with enhanced oxidative stress, is an important mechanism for this decrease in HDAC-2 activity in patients with COPD. 5,7 Restoration of GC sensitivity is postulated as a novel approach in COPD management. 8,9 Theophylline (THEO), pentoxifylline (PTX), and roflumilast (ROF) are three commonly prescribed phosphodiesterase (PDE) inhibitors as anti-inflammatory treatments. THEO is prescribed as a bronchodilator, while PTX is indicated in occlusive peripheral artery disease (intermittent claudication). 10 Of particular interest is that PTX has specific inhibitory effects on tumor necrotic factor (TNF)α, as the latter plays an important role in the pathogenesis of pulmonary inflammation induced by cigarette smoke (CS). Having been used for many years, PTX is also well known by clinicians for its safety and well toleration, even in long-term clinical use. ROF is a PDE4 inhibitor approved by the FDA for the treatment of adults with severe COPD. 11,12 Certain laboratory studies have claimed that GC sensitivity could be re-established by using THEO to up-regulate HDAC-2 expression, 13,14 however adding THEO to inhaled GCs did not result in improved clinical outcomes. 15 We consider whether other antiinflammatory PDE inhibitors (PDEIs), such as PTX and ROF, had a similar or even greater effect on restoring GC-sensitivity by restoring HDAC-2 activity, thereby providing a novel avenue for clinical investigation.

| Study design
To compare the effects of THEO, PTX, and ROF when used alone and in combination with a GC, the mice were randomly divided into nine groups (n = 10 for each group): (1) Sham CS exposure; (2) CS exposure; (3) CS exposure with ROF administration; (4) CS exposure with PTX administration; (5) CS exposure with THEO administration; (6) CS exposure with triamcinolone (TRI) administration; (7) CS exposure with the co-administration of ROF and TRI; (8) CS exposure with the co-administration of PTX and TRI; (9) CS exposure with the co-administration of THEO and TRI. The course set for CS exposure was 30 weeks, and the medication intervention commenced from the 29th week of the course and continued for 2 weeks.

| Animals
Six-to seven-week-old male wild-type C57BL/6 mice (18-22 g body weight) were purchased from Vital River Laboratory and Animals Co., Ltd.. The animals were housed in a specific pathogenfree environment, at a temperature of 22-26℃ with a humidity of 60%-70%, where a 12-h day/night cycle was maintained. The mice had free access to standard laboratory food and fresh water. The laboratory animal management rules of Tongji Medical College of Huazhong University of Science and Technology were strictly adhered to.

| Cigarette smoke exposure
The animals in different groups were caged separately in a series of transparent chambers (each sized 28 × 21 × 17 cm) that were ar- The mice allocated to the sham CS exposure were caged in the same environment but exposed only to fresh air for 60 min. These 60-min CS and sham -CS exposure episodes were conducted twice a day, 5 days a week, for 30 weeks.
Determination of the TRI dosage was done by converting the equivalent dose for human beings, i.e., 24 mg TRI for a 60 kg adult, based on body surface area and adapting it accordingly for the mice. 19 Medication was commenced from the 29th week. ROF was suspended in sterilized water and given intragastrically once a day, 5 days a week for 2 weeks while PTX and THEO were dissolved in sterilized water and given intraperitoneally once a day, 5 days a week for 2 weeks, TRI (5 mg·kg −1 ) was administered intramuscularly through a single injection at the 29th week due to its long-lasting effect. All the mice were humanely euthanized with an overdose of sodium pentobarbitone (50 mg·kg −1 ) and exsanguinated via retinal blood vessels immediately after death at the end of 30 weeks.

| Morphometry and biomarker assessment
The animals underwent endotracheal intubation soon after euthanasia. For further morphologic and morphometric study, one side of the lungs was fixed through intratracheal instillation of 4% paraformaldehyde (PFA) solution with a constant hydrostatic pressure of 20 cm for at least 20 min and then immersed in PFA solution for at least 4 h before further histological processing. Slices, 4 µm thick, were excised in the sagittal plane from the mid-portion of the paraffin-embedded lung sample and stained with hematoxylin and eosin. Morphometric studies were conducted in accordance with previously reported methods. 20 In brief, 20 non-overlapping fields in each slice were systemically selected using an Olympus DP72 camera system (×40 magnification power). The degree of airspace (the entire acinar air space complex, that is, alveoli and alveolar ducts combined) enlargement or emphysema was assessed by mean linear intercept (Lm, μm) with the assistance of software used for digital image evaluation (SETPanizer). The alveolar surface area (I A ) was measured first; then, intercepts of alveolar septa (P A ) and alveolar ducts (P duct ) across a test line with a given length of D in a microscopic field were counted. Lm was calculated using the formula:

| Body weight
The body weight gained in animals with long-term CS exposure was slower than the animals in the sham CS exposure group. The intergroup difference in body weight attained statistical significance from the 24th week of the course (p < .05, Figure 1).

F I G U R E 1
Mice with long-term CS exposure (presented as black dots) had gained body weight slower than the animals in the sham CS exposure group (presented as circles), a significant difference in body weight was noted from the 24th week (p < .05). Data presented as mean body weight ± SD (n = 10 in each group)

| Pulmonary morphology and morphometry
As expected, emphysema-like structural destruction was observed in mice with long-term CS-exposure (Figure 2, panel A), and was associated with a greater Lm compared to the mice in sham CS exposure group (52.70 ± 0.31 μm vs. 32.39 ± 3.55 μm, p < .05). Two weeks' treatment with any of these PDEIs or combined TRI with any of these PDEIs did not mitigate this structural damage (Figure 2, panel A and B, p > .05).

| Pro-inflammatory cytokines in BALF
Long-term CS exposure induced the up-regulation of TNFα

| ROS expression in situ
Expression of ROS in the lung tissue was also augmented by longterm CS exposure (Figure 4, panel A, Table 1). The expression of ROS was decreased by the administration of PTX, THEO, or TRI but not by ROF. The addition of TRI to PTX or ROF enhanced this inhibition ( Figure 4, Table 1). Combining TRI with PTX tended to be more effective than with the other PDEIs when considering ROS inhibition ( 1.88 ± 0.12 AU, as compared to TRI-ROF or TRI-THEO combination).

| HDAC-2 activity in lung tissue
As shown in Figure 5 and Table 1, the lungs of mice with long-term CS exposure had a greater reduction in HDAC-2 activity as compared to that of the sham CS exposure group. ROF, PTX, or THEO partially restored the HDAC-2 activity. The addition of TRI to any of these PDEIs, however, did not synergistically increase the HDAC-2 activity further (p > .05).

F I G U R E 2
Long-term cigarette smoke (CS) exposure induced emphysemalike pulmonary morphometry (panel A, 10× magnification), illustrated by a greater mean liner intercept (Lm) when compared to the sham CS exposure group (panel B, p < .05). Two-week course of treatment with roflumilast, pentoxifylline, theophylline, or a combination of a glucocorticoid (Triamcinolone) with any of these phosphodiesterase inhibitors, did not mitigate pulmonary emphysema (panel B, p > .05)

| DISCUSS ION
The murine model of CS-induced pulmonary emphysema has many similarities to human COPD, including growth delay, 23 The PDEs are classified into 11 super-families. 11 The involvement of PDE isoforms such as PDE3, PDE4, PDE5 and PDE7 has been demonstrated in the pathogenesis of airway inflammation and hyper-responsiveness. [29][30][31] Similar to previous studies, 17,32 the expression of ROS, IL-8, and TNFα in our mice exposed to long-term CS was elevated, which was associated with a reduction in HDAC-2 activity and emphysema-like pulmonary destruction. As in human COPD, in which no therapeutic modalities are able to repair pulmonary emphysematous destruction, neither the PTX nor the ROF therapy in our present study was able to mitigate the CS-induced pulmonary damage (Lm remained unchanged) despite these PDEIs having demonstrated some anti-inflammatory merits, that is IL-8 expression being down-regulated, and the HDAC-2 activity being partially restored after 2 weeks of monotherapy with each of these PDEIs. This restoration was preferentially associated with the overall decreased expression of ROS, rather than the inhibition of any single cytokine.
PTX is the strongest anti-inflammatory agent among the three PDEIs, if their inhibitory effects on IL-8, TNFα, and ROS are considered. PTX has never been recommended for COPD treatment, as early clinical trials on PTX showed no significant clinical benefits when oxygenation and amelioration of pulmonary function in patients with COPD were taken into account. 33,34 Unfortunately, until now, no pharmacological agent has been found to reverse the natural course of COPD. 3  When considered together, we concluded that the reduction of HDAC-2 activity is associated with the up-regulation of ROS. THEO, as well as PTX and ROF, can restore HDAC-2 activity by alleviating oxidative stress. Particular attention should be paid to PTX for its selective inhibitory effect on TNFα.
This study does have some limitations. First, CS-induced murine pulmonary inflammation is gender and strain sensitive. 40 Female mice are more susceptible to tobacco exposure 41 though the male C57BL/6J strain we selected in the present study was also susceptible to CS exposure. 42 Second, the up-regulation of ROS only partially mirrors (as reactive nitrogen species are not evaluated) the augmented oxidative stress as a consequence of F I G U R E 3 Expression of TNFα (panel A) and IL-8 (panel B) in bronchoalveolar lavage fluid (BALF) samples was up-regulated by long term cigarette smoke (CS) exposure. Only PTX down-regulated the expression of TNFα, while the addition of TRI did not increase this inhibition. PTX, THEO, and TRI but not ROF down-regulated the expression of IL-8. The addition of TRI intensifies the inhibition, not only for PTX and THEO but also for ROF. Results presented as mean ± SD (n = 5 mice for each group). * p < .05, compared to the Sham-CS group; † p < .05, compared to the CS group exaggerated pulmonary inflammation due to long-term CS exposure. The mechanism that the PDEIs restore the activity of HDAC-2 is not explained simply by referring to the dynamic changes of IL-8, TNFα and oxidative stress. Third, the efficacy of the PDEIs was not evaluated with reference to pharmacokinetic and pharmacodynamic information in vivo.

F I G U R E 4
Expression of ROS in the lung tissues was augmented by long-term CS-exposure (panel A, p < .05). PTX, THEO and TRI but not ROF inhibited ROS expression. The addition of TRI to ROF, however, increased the inhibition (panel B). the addition of TRI to PTX resulted in the synergistic inhibition on ROS expression (panel B, p < .05). Data presented as mean ± SD (n = 5 mice per group). * p < .05, compared to the Sham-CS group; † p < .05, compared to the CS group; ¶ p < .05, compared to the CS+TRI group TA B L E 1 Measurements of biological variables in the control and study groups (data presented as mean ± SD)

D I SCLOS U R E
The authors declare no conflict of interest.

R I G O R
This declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigor of pre-clinical research as recommended by funding agencies, publishers and other organizations engaged with supporting research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.