October 2015
Volume 56, Issue 11
Free
Immunology and Microbiology  |   October 2015
Doxycycline Attenuates Endotoxin-Induced Uveitis by Prostaglandin E2-EP4 Signaling
Author Affiliations & Notes
  • Jingwen Huang
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
    Department of Ophthalmology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
  • Wenru Su
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Xiaoqing Chen
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Xiaokang Cheng
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Ye Dai
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Longhui Han
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Dan Liang
    State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Correspondence: Dan Liang, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie South Road, Guangzhou 510060, China; liangd631004@163.com
  • Wenru Su, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xianlie South Road, Guangzhou 510060, China; swrth@163.com
  • Footnotes
     JH and WS contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science October 2015, Vol.56, 6686-6693. doi:10.1167/iovs.15-17045
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      Jingwen Huang, Wenru Su, Xiaoqing Chen, Xiaokang Cheng, Ye Dai, Longhui Han, Dan Liang; Doxycycline Attenuates Endotoxin-Induced Uveitis by Prostaglandin E2-EP4 Signaling. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6686-6693. doi: 10.1167/iovs.15-17045.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We explored the anti-inflammatory effects of doxycycline in experimental uveitis and the underlying mechanisms.

Methods: Rats with endotoxin-induced uveitis (EIU) received doxycycline (1.5 mg/kg) or the control vehicle via intraperitoneal injection. Clinical scores were graded under a slit lamp. Rat peritoneal macrophages were used in vitro to further explore the anti-inflammatory mechanisms of doxycycline. The levels of nitric oxide (NO), TNF-α, IL-1β, prostaglandin E2 (PGE2), cyclooxygenase (COX)-2, I kappa B-α (IκB-α), inducible nitric oxide synthase (iNOS), Akt, caspase-3, and nuclear factor-kappa B (NF-κB) were analyzed.

Results: Treatment with doxycycline dramatically reduced the clinical scores of EIU (P < 0.001), with significant decreases in inflammatory cell infiltration, protein concentrations, and the production of NO, TNF-α, and IL-1β in the aqueous humor (AqH). In vitro, doxycycline significantly inhibited the production of NO, IL-1β, and TNF-α in peritoneal macrophages by modulating the PI3K/Akt/IκB-α/NF-κB pathway. Importantly, we found that doxycycline significantly enhanced COX2 expression and PGE2 production both in vivo and in vitro. More importantly, blockade of the EP4 receptor of PGE2 significantly reversed the doxycycline-mediated inhibition of macrophages and the PI3K/Akt pathway in vitro. Furthermore, simultaneous injection of an EP4 antagonist and doxycycline significantly blocked the doxycycline-mediated attenuation of EIU.

Conclusions: Doxycycline can ameliorate EIU, and PGE2-EP4 signaling is essential for the anti-inflammatory effects of doxycycline in vitro and in vivo.

Uveitis is an intraocular inflammatory disorder that is highly prevalent worldwide and can cause severe visual loss because of its recurrence and secondary complications, such as cataracts, glaucoma, and cystoid macular edema.1,2 In clinical practice, the conventional treatment for uveitis includes the use of corticosteroids and immunosuppressive agents to control the inflammatory process.1,2 However, long-term exposure to these drugs may result in potentially significant adverse effects.2,3 Furthermore, many patients are resistant to or cannot tolerate these agents.3 Therefore, novel safe and effective therapies are desirable. 
Tetracyclines, a broad-spectrum antibiotic drug family that includes tetracycline, doxycycline, minocycline, and other derivative pharmaceuticals, exhibit an attractive variety of nonantibiotic properties. There are currently more than 200 ongoing clinical trials on the use of tetracyclines in various types of diseases.4 In particular, doxycycline is a long-acting, low-cost, semisynthetic tetracycline that has been used safely for decades in clinical settings. Recently, the anti-inflammatory and immunosuppressive properties of doxycycline have received increasing attention. Doxycycline has been reported to be useful for treating a number of inflammatory and/or immune diseases, including rheumatoid arthritis, rosacea, periodontitis, experimental autoimmune neuritis, and myocardial infarction.59 Therefore, doxycycline may represent a therapeutic option for the treatment of uveitis. However, the function of doxycycline in uveitis has not yet been explored. Moreover, the mechanisms by which doxycycline mediates anti-inflammatory responses remain elusive. Thus, in this study, we investigated the anti-inflammatory mechanisms of doxycycline and the therapeutic effect of doxycycline on endotoxin-induced uveitis (EIU). 
Materials and Methods
Animals
Inbred male Wistar rats, 6 to 8 weeks old and weighing 160 to 180 g, were obtained from the Guangzhou Animal Testing Center and maintained in an air-conditioned room with a 12-hour light–dark cycle. The animals were provided access to food and water ad libitum until they were used for experiments. All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, and all procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Antibodies and Reagents
Doxycycline, prostaglandin E2 (PGE2), EP1 to EP4 antagonists, and lipopolysaccharide (LPS; Escherichia coli 055:B5) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The following antibodies were used: anti-cyclooxygenase (COX)-2, anti-Akt, anti-phosphorylated Akt (p-Akt), anti-nuclear factor-kappa B (NF-κB) p65, anti-phosphorylated NF-κBp65 (p-NF-κBp65), anti-I kappa B-α (IκB-α), anti-inducible nitric oxide synthase (iNOS), anti-β-actin, and a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). 
Induction of EIU and Drug Treatment Protocols
Endotoxin-induced uveitis was induced as previously described.10,11 Briefly, LPS was diluted in sterile PBS (pH 7.4) at a concentration of 2 mg/mL. Eight rats were injected with a single dose of 200 μg LPS solution in the right footpad. A separate group (eight rats) received the same volume of sterilized PBS in one footpad for comparison with the LPS group. Doxycycline (1.5 mg/kg, based on a previous study)12 or vehicle was administered at the indicated time points after LPS challenge via intraperitoneal injection. Another eight rats without any treatment were used as a blank control group. 
Slit lamp evaluation was performed at 24 hours after LPS injection. The clinical reactions were recorded and evaluated by two observers who were blinded to the treatment groups. The clinical scores were evaluated according to a previously published system.13 
Quantification of Infiltrating Cells and Protein Concentrations in the Aqueous Humor
Aqueous humor (AqH, 20∼25 μL/rat) was obtained from both eyes via anterior chamber puncture with a 30-gauge needle under a surgical microscope while the animal was under deep anesthesia induced through an intraperitoneal injection of a 10% chloral hydrate solution (0.4 mL/100 g) 24 hours after LPS injection. For cell counting, the AqH samples were suspended in Trypan blue solution (1:5), and the cells were counted under light microscopy using a hemocytometer. The concentration of total protein in AqH was determined with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). 
Histopathologic Examination
For histopathologic examination, the rats were killed 24 hours after LPS injection, and their intact eyes were immediately enucleated, stored in a 10% neutral buffered formalin solution for 24 to 48 hours at room temperature, dehydrated in a graded ethanol series, and embedded in paraffin. Then, 5-μm sagittal sections were cut near the optic nerve head and stained with hematoxylin and eosin (H&E). 
Rat Peritoneal Macrophage Isolation and Culture
Male Wistar rats were injected intraperitoneally with 2 mL sterile 3% thioglycollate medium (Sigma-Aldrich Corp.), and peritoneal macrophages were harvested 3 days later. The cells were suspended in antibiotic-free RPMI 1640 medium containing 10% fetal bovine serum and cultured in 24-well plates in a humidified incubator at 37°C in 5% CO2. Two hours later, nonadherent cells were removed, and the medium was replaced. The resultant cell preparation consisted of more than 95% macrophages (data not shown), as verified by F4/80 (eBioscience, San Diego, CA, USA) surface staining and flow cytometry. For LPS activation, the cells were preincubated with or without doxycycline (40 μM) for 1 hour, followed by incubation with LPS (1 μg/mL) for 16 hours. The supernatants and cells were collected for further analysis. An EP1 receptor antagonist (SC-51322, 10 μM), EP2 receptor antagonist (PF-04418948, 10 μM), EP3 receptor antagonist (L-798106, 10 μM), and EP4 receptor antagonist (L-161,982, 10 μM) were used in several experiments. 
Enzyme-Linked Immunosorbent Assay and NO Assay
The concentrations of TNF-α and IL-1β in AqH or the supernatants of cultured cells were measured via ELISA (eBioscience, San Diego, CA, USA). The activity of PI3K was determined using a PI3K activity ELISA: Pico kit (Echelon Biosciences, Inc., Salt Lake City, UT, USA). The concentrations of PGE2 in the supernatants or AqH were determined using ELISA kits (Cayman Chemical, Ann Arbor, MI, USA). The AqH from the eyes of two to three rats or supernatants were pooled, and 50 μL was used for a single assay. All measurements were performed in duplicate. The levels of NO in the supernatants or AqH were measured using the Griess reaction.14 Briefly, 50-μL aliquots of the samples were mixed with 0.1% N-1-napthylethylenediamine dihydrochloride and 1% sulfanilamide at room temperature for 10 minutes. 
Western Blotting
Cell lysates or rat iris ciliary body (ICB) lysates (50–100 μg total protein) were separated on polyacrylamide-SDS gels and electroblotted/transferred onto a nitrocellulose filter membrane (Bio-Rad, Hercules, CA, USA). After blocking with TBS/5% defatted milk powder, the nitrocellulose membrane was incubated with antibodies against COX2, Akt, p-Akt, NF-κBp65, p-NF-κBp65, IκB-α, iNOS, and β-actin, followed by incubation with an HRP-conjugated secondary antibody for 1 hour. The signals were visualized using enhanced chemiluminescence detection (Pierce). 
Real-Time PCR
Total RNA from the tissue lysates was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and cDNA was generated using the Omniscript RT kit (Qiagen). Tumor necrosis factor α and IL-1β mRNA expression was quantified with Absolute SYBR Green ROX mix (Thermo Scientific, Waltham, MA, USA). The samples were run in triplicate, and the relative expression of TNF-α and IL-1β was determined by normalizing the expression of each target to β-actin using the 2−ΔΔCt method. 
Statistical Analysis
Student's t-test was used to analyze significant differences (SPSS 16.0; IBM SPSS Statistics, IBM Corporation, Chicago, IL, USA), with P < 0.05 considered statistically significant. 
Results
Doxycycline Treatment Attenuates EIU
To examine the therapeutic effects of doxycycline on EIU, doxycycline was injected intraperitoneally in rats with EIU following LPS injection. Lipopolysaccharide administration generated typical signs mimicking human uveitis, including ciliary congestion, iris blood vessel dilatation, pupil occlusion and fibrinous membrane formation. Clinical scores were evaluated at 24 hours after LPS injection. As shown in Figure 1A, doxycycline administered at 6 hours and 12 hours after LPS injection reduced the inflammatory scores of the EIU rats compared with the untreated group. However, the suppressive effects were not as significant as those observed in animals treated with doxycycline administered immediately after LPS injection. Therefore, we chose the immediate administration of doxycycline after LPS injection in the following studies. Representative images of ocular symptoms demonstrate that doxycycline treatment significantly attenuated iris blood vessel dilatation, pupil occlusion and fibrinous membrane formation (Fig. 1B). Lipopolysaccharide application induced a significant increase in the number of cells and the levels of protein in the AqH compared with control rats, and doxycycline administration significantly decreased the number of cells and protein levels in the AqH in treated rats (Figs. 1C, 1D). 
Figure 1
 
Doxycycline treatment suppresses EIU. (A) The clinical scores of EIU were determined at 24 hours after LPS challenge with a slit lamp microscope in different groups (n = 8). (B) Representative images of ocular symptoms in the indicated experimental group at 24 hours after LPS challenge (white arrow: pupil occlusion and fibrinous membrane formation). (CD) Effects of doxycycline on protein concentrations and cellular infiltration in AqH collected 24 hours after LPS challenge (n = 6). The data are presented as the mean ± SD. **P < 0.01.
Figure 1
 
Doxycycline treatment suppresses EIU. (A) The clinical scores of EIU were determined at 24 hours after LPS challenge with a slit lamp microscope in different groups (n = 8). (B) Representative images of ocular symptoms in the indicated experimental group at 24 hours after LPS challenge (white arrow: pupil occlusion and fibrinous membrane formation). (CD) Effects of doxycycline on protein concentrations and cellular infiltration in AqH collected 24 hours after LPS challenge (n = 6). The data are presented as the mean ± SD. **P < 0.01.
Doxycycline Suppresses Inflammation in EIU
Next, we investigated the effects of doxycycline treatment on the inflammatory profiles in EIU. Histologic analyses revealed significantly decreased inflammatory cell numbers in the ICB in doxycycline-treated rats compared with control animals (Fig. 2A). Next, we determined the effects of doxycycline on the levels of local inflammatory mediators in the AqH using ELISA. These results demonstrated a significant increase in TNF-α, IL-1β, and NO production in the AqH of rats with EIU, whereas doxycycline suppressed the production of TNF-α, IL-1β, and NO (Figs. 2B–D). Western blot analysis of the ICB showed that doxycycline treatment reduced the phosphorylation of NF-κBp65 and expression of iNOS (Figs. 2E, 2F) but increased COX2 expression in the ICB of rats with EIU (Fig. 2G). In addition, doxycycline treatment significantly increased the level of PGE2 in the AqH of EIU rats compared with control animals (Fig. 2H). 
Figure 2
 
Doxycycline attenuates inflammation in EIU. (A) Representative images of H&E staining of ICB samples from rats in the indicated experimental group at 24 hours after LPS challenge (scale bars: 200 μm). (BD) The levels of TNF-α, IL-1β, and NO in AqH were measured by ELISA and the Griess reaction (n = 6). (EG) The levels of iNOS, (phosphorylated) NF-κBp65, and COX2 in the ICB were determined via Western blotting (n = 6). (H) The levels of PGE2 in AqH were measured by ELISA. The data are presented as the mean ± SD. **P < 0.01.
Figure 2
 
Doxycycline attenuates inflammation in EIU. (A) Representative images of H&E staining of ICB samples from rats in the indicated experimental group at 24 hours after LPS challenge (scale bars: 200 μm). (BD) The levels of TNF-α, IL-1β, and NO in AqH were measured by ELISA and the Griess reaction (n = 6). (EG) The levels of iNOS, (phosphorylated) NF-κBp65, and COX2 in the ICB were determined via Western blotting (n = 6). (H) The levels of PGE2 in AqH were measured by ELISA. The data are presented as the mean ± SD. **P < 0.01.
Doxycycline Inhibits the Release of Proinflammatory Mediators by Macrophages In Vitro
To explore the anti-inflammatory mechanism of doxycycline in EIU, rat peritoneal macrophages were isolated and used as an in vitro model. For this purpose, purified rat peritoneal macrophages were plated in six-well plates for 24 hours and subsequently stimulated with LPS (1 μg/mL) for another 16 hours in the presence or absence of doxycycline at increasing concentrations. The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. The results showed that doxycycline treatment led to a dose-dependent inhibition of TNF-α and IL-1β release by peritoneal macrophages activated by LPS (Figs. 3A, 3B). Compared with 40 μM doxycycline, 80 μM doxycycline inhibited IgE release more effectively, but B-cell viability was affected (Fig. 3C). Therefore, we chose 40 mM doxycycline in the following experiments unless specifically indicated. Additionally, the mRNA expression of TNF-α and IL-1β in peritoneal macrophages was significantly reduced after doxycycline treatment compared with untreated peritoneal macrophages (Figs. 3E, 3F). Nitric oxide production and iNOS expression were determined via the Greiss reaction and Western blotting, respectively. As shown in Figures 3E and 3F, doxycycline treatment significantly suppressed the levels of LPS-induced NO release and iNOS expression. 
Figure 3
 
Doxycycline inhibits proinflammatory mediator release by peritoneal macrophages in vitro. (A, B) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (C, D) The expression of TNF-α and IL-1β mRNA in peritoneal macrophages was determined using real-time PCR. (E, F) The levels of NO and iNOS in the culture supernatants were determined through the Griess reaction and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 3
 
Doxycycline inhibits proinflammatory mediator release by peritoneal macrophages in vitro. (A, B) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (C, D) The expression of TNF-α and IL-1β mRNA in peritoneal macrophages was determined using real-time PCR. (E, F) The levels of NO and iNOS in the culture supernatants were determined through the Griess reaction and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
PI3K/Akt/IκB-α/NF-κB Signaling Is Involved in the Doxycycline-Mediated Inhibition of Macrophages
Nuclear factor κB is a key heterodimeric transcription factor that is responsible for inflammatory and immune processes including uveitis.15,16 Thus, we asked whether NF-κB signaling is involved in the doxycycline-mediated inhibition of peritoneal macrophages. We found that stimulation of macrophages with LPS increased phosphorylated NF-κBp65 protein expression, whereas doxycycline treatment significantly inhibited phosphorylated NF-κBp65 expression in macrophages (Fig. 4A). IκB-α degradation exposes a nuclear localization signal that leads to the phosphorylation of NF-κB. Therefore, we also investigated whether doxycycline modulates NF-κB activity in peritoneal macrophages by inhibiting IκB-α degradation. As expected, doxycycline significantly inhibited IκB-α degradation in peritoneal macrophages (Fig. 4B). Because the PI3K/Akt pathway plays a critical role in macrophage activation and NF-κB activation, we next evaluated whether doxycycline could modulate the PI3K/Akt pathway in rat peritoneal macrophages. These results showed that although stimulation of macrophages with LPS resulted in increased PI3K activity and p-Akt protein expression, treatment with doxycycline significantly reduced PI3K activity and p-Akt expression in peritoneal macrophages (Figs. 4C, 4D). 
Figure 4
 
PI3K/Akt/IκB-α/NF-κB signaling is involved in the doxycycline-mediated inhibition of macrophages. (A, B) The levels of phosphorylated NF-κBp65 and IκB-α in peritoneal macrophages were determined through Western blotting. (C, D) PI3K activity and p-Akt expression in peritoneal macrophages were measured via ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 4
 
PI3K/Akt/IκB-α/NF-κB signaling is involved in the doxycycline-mediated inhibition of macrophages. (A, B) The levels of phosphorylated NF-κBp65 and IκB-α in peritoneal macrophages were determined through Western blotting. (C, D) PI3K activity and p-Akt expression in peritoneal macrophages were measured via ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Prostaglandin E2-EP4 Signaling Is Responsible for the Doxycycline-Mediated Anti-Inflammatory Effects on Peritoneal Macrophages
Our in vivo results showed that doxycycline reduced TNF-α, IL-1β, and NO production but increased PGE2 production and COX2 expression. Thus, we next asked whether COX2/PGE2 signaling was involved in the doxycycline-mediated anti-inflammatory effects on peritoneal macrophages. As shown in Figures 5A and 5B, doxycycline treatment also significantly increased COX2 expression and PGE2 production in peritoneal macrophages. Typically, PGE2 exerts its biological functions via four subtypes of prostaglandin E receptors, EP1 to EP4. Thus, to further confirm and elucidate the roles of COX2/PGE2 in the doxycycline-mediated suppression of inflammatory mediator release in peritoneal macrophages, antagonists of EP1 to EP4 were used. We observed that although the addition of an antagonist of EP1, EP2, or EP3 did not affect the anti-inflammatory effects of doxycycline on peritoneal macrophages, the addition of an EP4 antagonist significantly reversed the doxycycline-mediated inhibitory effects on peritoneal macrophages following LPS stimulation (Figs. 5C, 5D). Additionally, treatment with the EP4 antagonist led to an increase in inflammatory cytokine release in macrophages stimulated with LPS, but this increase was not as significant as that observed in macrophages treated with LPS and doxycycline (Figs. 5E, 5F). As shown in Figures 5G and 5H, EP4 blockade also significantly decreased the ability of doxycycline to inhibit the PI3k/Akt pathway. Together, these results indicate that PGE2-EP4 signaling plays important roles in the doxycycline-mediated inhibition of macrophages. 
Figure 5
 
Prostaglandin E2-EP4 signaling is responsible for the doxycycline-mediated anti-inflammatory effects on peritoneal macrophages. (A, B) Doxycycline significantly increased COX2 expression and PGE2 production in peritoneal macrophages. (CF) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (G, H) PI3K activity and p-Akt expression in the culture supernatants were measured through ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 5
 
Prostaglandin E2-EP4 signaling is responsible for the doxycycline-mediated anti-inflammatory effects on peritoneal macrophages. (A, B) Doxycycline significantly increased COX2 expression and PGE2 production in peritoneal macrophages. (CF) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (G, H) PI3K activity and p-Akt expression in the culture supernatants were measured through ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Doxycycline Attenuates EIU via PGE2-EP4 Signaling
Our in vitro studies showed that PGE2-EP4 signaling plays a critical role in the doxycycline-mediated inhibition of peritoneal macrophages. Therefore, we next determined whether PGE2-EP4 signaling was also involved in the doxycycline-mediated inhibition of EIU. To this end, the EP4 antagonist (L-161,982, 1 mg/kg) and doxycycline were injected simultaneously following LPS administration. These results showed that treatment with the EP4 antagonist significantly blocked the doxycycline-mediated decrease in the clinical scores of EIU, cell numbers and protein levels in the AqH (Figs. 6A–C). As shown in Figures 6D through 6F, the EP4 antagonist also significantly decreased the inhibitory ability of doxycycline with regard to inflammatory mediator production. These findings suggest that the upregulation of COX2/PGE2-EP4 signaling might contribute, at least in part, to the doxycycline-mediated attenuation of EIU. 
Figure 6
 
Doxycycline attenuates EIU via PGE2-EP4 signaling. (A) An EP4 antagonist significantly blocked the doxycycline-mediated decrease in the clinical scores of EIU (n = 8). (B, C) The EP4 antagonist significantly reversed the doxycycline-mediated decreases in protein concentrations and cellular infiltration in the AqH (n = 6). (DF) The levels of TNF-α, IL-1β, and NO in AqH were measured via ELISA and the Griess reaction (n = 6). The data are presented as the mean ± SD. **P < 0.01. Error bars: means ± SEMs.
Figure 6
 
Doxycycline attenuates EIU via PGE2-EP4 signaling. (A) An EP4 antagonist significantly blocked the doxycycline-mediated decrease in the clinical scores of EIU (n = 8). (B, C) The EP4 antagonist significantly reversed the doxycycline-mediated decreases in protein concentrations and cellular infiltration in the AqH (n = 6). (DF) The levels of TNF-α, IL-1β, and NO in AqH were measured via ELISA and the Griess reaction (n = 6). The data are presented as the mean ± SD. **P < 0.01. Error bars: means ± SEMs.
Discussion
Endotoxin-induced uveitis is an established animal model of some types of uveitis in humans, particularly acute anterior uveitis.11 Endotoxin-induced uveitis is induced via the systemic injection of a single sublethal dose of LPS, and the maximum inflammatory response is achieved at 24 hours after LPS injection.10,11 In the present study, we investigated the anti-inflammatory effects and mechanisms of doxycycline in EIU. The results of this study demonstrated that doxycycline could significantly attenuate the ocular inflammatory response in EIU rats at 24 hours after LPS injection, with significant decreases in inflammatory cell infiltration and the production of NO, TNF-α, and IL-1β in the AqH. These findings suggest that doxycycline is capable of inhibiting EIU. Importantly, compared with the drugs currently used to treat uveitis, which generate certain serious side effects and complications, doxycycline has been used relatively safely for decades in clinical settings with fewer side effects. Furthermore, combined therapy with tetracyclines and steroids has shown promising therapeutic effects in asthmatic patients. In addition, doxycycline has been used in ocular diseases, such as meibomian gland disease and rosacea,6,17 and recurrent corneal erosions.18,19 In a randomized, proof-of-concept clinical trial of participants with diabetic retinopathy who received oral doxycycline monohydrate, 50 mg, daily for 24 months was associated with improved frequency doubling the perimetry foveal sensitivity compared with placebo.20 Thus, doxycycline may be a potential therapeutic alternative for uveitis treatment. 
The NF-κB proteins are a ubiquitously expressed family of transcription factors that are crucial for the synthesis and release of various inflammatory mediators.21,22 Most agents, including LPS, activate NF-κB through the phosphorylation or degradation (or both) of IκB-α.2123 IκB-α degradation exposes a nuclear localization signal and leads to the activation of NF-κB.2123 In EIU, LPS binds to Toll-like receptor 4 (TLR4) on macrophages and other cells to activate the IκB-α/NF-κB pathway.2428 The subsequent increase in the release of inflammatory mediators contributes to the development of uveitis. In this study, our in vivo results showed that doxycycline inhibits the expression of phosphorylated NF-κBp65 protein. In vitro, using peritoneal macrophages, we found that doxycycline reduced the LPS-induced release of inflammatory mediators by modulating the IκB-α/NF-κB pathway. Furthermore, we demonstrated the inhibitory effects of doxycycline on the PI3K/Akt pathway, which is the upstream signaling pathway for NF-κB activation. These findings are concordant with previous studies29,30 suggesting that doxycycline may attenuate EIU by modulating the PI3K/Akt/NF-κB pathway. 
Prostaglandin E2, a short-lived potent bioactive lipid mediator, is a major downstream product of arachidonic acid metabolism via the cyclooxygenase COX-2.31 Prostaglandin E2 exerts a wide variety of biological effects, including anti-inflammatory and proinflammatory effects, by binding to the prostaglandin E receptors, EP1 to EP4.31,32 In the current study, our results showed that doxycycline increased COX2 expression and PGE2 production in the eyes of rats with EIU. It was further confirmed in rat peritoneal macrophages that COX2/PGE2 signaling was upregulated following doxycycline treatment. Importantly, we found that the addition of an EP4 antagonist significantly reversed the doxycycline-mediated inhibitory effects on peritoneal macrophages. Even more importantly, simultaneous injection of the EP4 antagonist and doxycycline abolished the therapeutic effects of doxycycline on EIU. Collectively, these compelling findings suggest that PGE2-EP4 signaling contributes, at least in part, to the doxycycline-mediated attenuation of EIU. Although the anti-inflammatory function of doxycycline was discovered several decades ago, the underlying molecular mechanisms remain elusive. Thus, it is noteworthy that PGE2-EP4 signaling was implicated in the anti-inflammatory effects of doxycycline. 
Based on the doxycycline dose of 1.5 mg/kg used in this study, this is equivalent to approximately 90 mg per day for human subjects (approximately 60 kg). This is a lower dose compared with the typical antimicrobial dose of doxycycline given to humans (200 mg per day, Food and Drug Administration). However, it is similar to a dose in previous studies that showed that the subantimicrobial dose of doxycycline (40–100 mg per day) provided significant benefit in the treatment of human patients.5,3335 
Although the EIU model is a good model of auto-inflammatory uveitis, translation into clinical application should proceed with caution. The EIU model evaluates uveitis and response to treatment in a short period of time (24 hours), whereas uveitis in human patients is a chronic disease with peaks and relapses of activity. Uveitis treatment requires long-term therapies. Although a small pilot study has shown that oral doxycycline monohydrate at 50 mg daily for 24 months is safe and improved the frequency of doubling perimetry foveal sensitivity in patients with diabetic retinopathy, further study is still needed to verify this in patients with uveitis. 
In summary, our study is the first to demonstrate the therapeutic effects of doxycycline on experimental uveitis. Our results showed that doxycycline reduced inflammatory mediator release from peritoneal macrophages, possibly by modulating the PI3K/Akt/NF-κB pathway. Importantly, we further elucidated the anti-inflammatory mechanisms of doxycycline by demonstrating that PGE2-EP4 signaling is essential for doxycycline-mediated anti-inflammatory effects in vitro and in vivo. 
Acknowledgments
Supported by the Natural Science Foundation of China (81271051 and 81300740). The authors alone are responsible for the content and the writing of the paper. 
Disclosure: J. Huang, None; W. Su, None; X. Chen, None; X. Cheng, None; Y. Dai, None; L. Han, None; D. Liang, None 
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Figure 1
 
Doxycycline treatment suppresses EIU. (A) The clinical scores of EIU were determined at 24 hours after LPS challenge with a slit lamp microscope in different groups (n = 8). (B) Representative images of ocular symptoms in the indicated experimental group at 24 hours after LPS challenge (white arrow: pupil occlusion and fibrinous membrane formation). (CD) Effects of doxycycline on protein concentrations and cellular infiltration in AqH collected 24 hours after LPS challenge (n = 6). The data are presented as the mean ± SD. **P < 0.01.
Figure 1
 
Doxycycline treatment suppresses EIU. (A) The clinical scores of EIU were determined at 24 hours after LPS challenge with a slit lamp microscope in different groups (n = 8). (B) Representative images of ocular symptoms in the indicated experimental group at 24 hours after LPS challenge (white arrow: pupil occlusion and fibrinous membrane formation). (CD) Effects of doxycycline on protein concentrations and cellular infiltration in AqH collected 24 hours after LPS challenge (n = 6). The data are presented as the mean ± SD. **P < 0.01.
Figure 2
 
Doxycycline attenuates inflammation in EIU. (A) Representative images of H&E staining of ICB samples from rats in the indicated experimental group at 24 hours after LPS challenge (scale bars: 200 μm). (BD) The levels of TNF-α, IL-1β, and NO in AqH were measured by ELISA and the Griess reaction (n = 6). (EG) The levels of iNOS, (phosphorylated) NF-κBp65, and COX2 in the ICB were determined via Western blotting (n = 6). (H) The levels of PGE2 in AqH were measured by ELISA. The data are presented as the mean ± SD. **P < 0.01.
Figure 2
 
Doxycycline attenuates inflammation in EIU. (A) Representative images of H&E staining of ICB samples from rats in the indicated experimental group at 24 hours after LPS challenge (scale bars: 200 μm). (BD) The levels of TNF-α, IL-1β, and NO in AqH were measured by ELISA and the Griess reaction (n = 6). (EG) The levels of iNOS, (phosphorylated) NF-κBp65, and COX2 in the ICB were determined via Western blotting (n = 6). (H) The levels of PGE2 in AqH were measured by ELISA. The data are presented as the mean ± SD. **P < 0.01.
Figure 3
 
Doxycycline inhibits proinflammatory mediator release by peritoneal macrophages in vitro. (A, B) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (C, D) The expression of TNF-α and IL-1β mRNA in peritoneal macrophages was determined using real-time PCR. (E, F) The levels of NO and iNOS in the culture supernatants were determined through the Griess reaction and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 3
 
Doxycycline inhibits proinflammatory mediator release by peritoneal macrophages in vitro. (A, B) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (C, D) The expression of TNF-α and IL-1β mRNA in peritoneal macrophages was determined using real-time PCR. (E, F) The levels of NO and iNOS in the culture supernatants were determined through the Griess reaction and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 4
 
PI3K/Akt/IκB-α/NF-κB signaling is involved in the doxycycline-mediated inhibition of macrophages. (A, B) The levels of phosphorylated NF-κBp65 and IκB-α in peritoneal macrophages were determined through Western blotting. (C, D) PI3K activity and p-Akt expression in peritoneal macrophages were measured via ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 4
 
PI3K/Akt/IκB-α/NF-κB signaling is involved in the doxycycline-mediated inhibition of macrophages. (A, B) The levels of phosphorylated NF-κBp65 and IκB-α in peritoneal macrophages were determined through Western blotting. (C, D) PI3K activity and p-Akt expression in peritoneal macrophages were measured via ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 5
 
Prostaglandin E2-EP4 signaling is responsible for the doxycycline-mediated anti-inflammatory effects on peritoneal macrophages. (A, B) Doxycycline significantly increased COX2 expression and PGE2 production in peritoneal macrophages. (CF) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (G, H) PI3K activity and p-Akt expression in the culture supernatants were measured through ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 5
 
Prostaglandin E2-EP4 signaling is responsible for the doxycycline-mediated anti-inflammatory effects on peritoneal macrophages. (A, B) Doxycycline significantly increased COX2 expression and PGE2 production in peritoneal macrophages. (CF) The levels of TNF-α and IL-1β in the culture supernatants were measured via ELISA. (G, H) PI3K activity and p-Akt expression in the culture supernatants were measured through ELISA and Western blotting, respectively. The data are presented as the mean ± SD. **P < 0.01.
Figure 6
 
Doxycycline attenuates EIU via PGE2-EP4 signaling. (A) An EP4 antagonist significantly blocked the doxycycline-mediated decrease in the clinical scores of EIU (n = 8). (B, C) The EP4 antagonist significantly reversed the doxycycline-mediated decreases in protein concentrations and cellular infiltration in the AqH (n = 6). (DF) The levels of TNF-α, IL-1β, and NO in AqH were measured via ELISA and the Griess reaction (n = 6). The data are presented as the mean ± SD. **P < 0.01. Error bars: means ± SEMs.
Figure 6
 
Doxycycline attenuates EIU via PGE2-EP4 signaling. (A) An EP4 antagonist significantly blocked the doxycycline-mediated decrease in the clinical scores of EIU (n = 8). (B, C) The EP4 antagonist significantly reversed the doxycycline-mediated decreases in protein concentrations and cellular infiltration in the AqH (n = 6). (DF) The levels of TNF-α, IL-1β, and NO in AqH were measured via ELISA and the Griess reaction (n = 6). The data are presented as the mean ± SD. **P < 0.01. Error bars: means ± SEMs.
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