Accepted Manuscript Title: Doxycycline exerts multiple anti-allergy effects to attenuate murine allergic conjunctivitis and systemic anaphylaxis Author: Wenru Su Qian Wan Longhui Han Jingwen Huang Xiaoqing Chen Guihua Chen Song Guo Zheng Dan Liang PII: DOI: Reference:

S0006-2952(14)00456-0 http://dx.doi.org/doi:10.1016/j.bcp.2014.08.001 BCP 12051

To appear in:

BCP

Received date: Revised date: Accepted date:

5-6-2014 1-8-2014 4-8-2014

Please cite this article as: Su W, Wan Q, Han L, Huang J, Chen X, Chen G, Zheng SG, Liang D, Doxycycline exerts multiple anti-allergy effects to attenuate murine allergic conjunctivitis and systemic anaphylaxis, Biochemical Pharmacology (2014), http://dx.doi.org/10.1016/j.bcp.2014.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Doxycycline exerts multiple anti-allergy effects to attenuate murine allergic conjunctivitis and systemic anaphylaxis 1 Wenru Su MD, PHD1,2*; Qian Wan MD1*; Longhui Han MD1; Jingwen Huang MD1; Xiaoqing Chen

ip t

MD1, 3; Guihua Chen, MD, PHD2, Song Guo Zheng MD, PHD2,3#; Dan Liang MD, PHD1#

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University,

us

1

cr

Running title: Anti-allergy properties of doxycycline

Guangzhou, China; 2 Clinic Immunology Section, Sun Yat-sen University Third Affiliated Hospital,

an

Guangzhou, China; 3Division of Rheumatology, Department of Medicine, Penn State University

*

M

Hershey College of Medicine, USA

The first two authors contributed equally to this work.

ed

Author contributions: W-R. S.: conception and design, data collection and assembly, manuscript

ce pt

writing, final manuscript approval, and financial support; Q. W.: conception and design, data collection and assembly, manuscript writing, and final manuscript approval; J-W. H., L-H. H., X-Q. C. and S-J. Q: data collection and assembly; S-G. Z.: conception and design, manuscript writing, final manuscript approval; D.L.: conception and design, manuscript writing, final manuscript

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

approval, and financial support. Correspondence to: Dan Liang, MD, PhD, Phone: +86-20-87330341; Fax: +86-20-87330341, Email: [email protected] or Song Guo Zheng MD, PHD, Phone 717 531 0003, Fax: 717 531 8274, Email: [email protected] or Disclosure of potential conflicts of interest: The authors declare no competing financial conflict of interest.

Page 1 of 40

ABSTRACT Allergic diseases, which affect up to 20-30% of the world population, are still therapeutic challenge for allergists. Tetracyclines, which belong to an antibiotic drug family that possesses a striking

ip t

variety of non-antibiotic properties, have been successfully applied to a wide range of diseases. However, their roles in allergic conjunctivitis and anaphylaxis and their underlying anti-allergy

cr

mechanisms remain elusive. Here, we reported that treatment with doxycycline significantly reduced

us

IgE release from mouse B cells and the dergranulation and inflammatory cytokines production of mouse mast cells (MCs) activated by IgE-dependent way. Furthermore, doxycycline treatment

an

significantly inhibited histamine-induced vascular hyperpermeability in vitro. Mechanistically, the

M

doxycycline-mediated inhibition of B cells, MCs and histamine may occur via modulation of the PI3K/Akt pathway. In vivo, our results demonstrated that treatment with doxycycline significantly

ed

attenuated clinical symptoms of mouse models of experimental allergic conjunctivitis (EAC) with a significant decrease in inflammatory cell frequency, IgE production, histamine release, and a

ce pt

decrease in TNF-α and IL-4 production. Using mouse models of MCs-dependent passive systemic anaphylaxis (PSA), we further confirmed anti-allergy effects of doxycycline and doxycycline-mediated inhibitory effects on MCs. Furthermore, our results showed that doxycycline

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

significantly attenuate histamine-induced systemic anaphylaxis-like reaction (HISA) with a significantly downregulation of PI3K/Akt/eNOS/VE-cadherin pathway. The doxycycline-mediated anti-allergy effects during EAC, PSA and HISA were abrogated when an Akt activator, SC79, was administered. These findings suggest that doxycycline inhibits B cell, MC and histamine function and attenuates experimental allergic conjunctivitis and systemic anaphylaxis by possible modulating the PI3K/Akt pathway.

Page 2 of 40

ce pt

ed

M

an

us

cr

ip t

Key Words: Allergy, Doxycycline, B cells, Mast cells, Histamine

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Page 3 of 40

1.INTRODUCTION Allergic diseases (ADs), which affect up to 20-30% of the world population, have become a major global health problem (1, 2). Allergic patients suffer from a broad variety of symptoms, including

ip t

allergic conjunctivitis (AC), asthma, dermatitis, gastrointestinal symptoms, and systemic anaphylaxis (SA). These symptoms significantly affect patient health and can even threaten life. A number of

cr

treatment modalities have been undertaken to prevent or inhibit the development of ADs. However,

us

current treatments for patients with ADs are variable, have limited clinical success and can lead to

an

serious side effects caused by steroids (1, 2). Therefore, new safer and effective therapies are desirable.

M

Tetracyclines, an antibiotic drug family that includes tetracycline, doxycycline, minocycline and

ed

other derivative pharmaceuticals, also have a striking variety of non-antibiotic properties. Due to their multifunctional properties, there are currently over 200 ongoing clinical trials on tetracyclines

ce pt

for a wide range of diseases (3). Recently, the anti-allergy properties of tetracyclines have received increasing attention (2). Several studies have reported that the administration of doxycycline or minocycline significantly attenuates airway inflammation and hyperresponsiveness in a murine asthma model, and in humans with allergic asthma, these treatments improve asthma symptoms and

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

reduce oral steroid requirements (4-6). Therefore, tetracyclines may be a potential therapeutic option for ADs. However, the mechanisms by which tetracyclines mediate anti-allergy responses remain elusive. Moreover, the roles of tetracyclines in other ADs remain unclear. Thus, in this study, we investigated the anti-allergy mechanisms of doxycycline and the therapeutic effect of doxycycline on experimental allergic conjunctivitis (EAC) and passive systemic anaphylaxis (PSA).

Page 4 of 40

2. Methods 2.1. Animals BALB/c mice were supplied by the Guangzhou Animal Testing Center and were used at 4-6 weeks

ip t

of age. All animal care and experiments were performed under institutional protocols approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen

cr

University. All procedures involving animal eye studies were conducted in accordance with the

us

guidelines provided in the Association for Research in Vision and Ophthalmology statement for the

an

use of animals in ophthalmic and vision research.

M

2.2.Cell culture

BALB/c splenic B cells were purified (purity >95% as determined by flow cytometry analysis of

ed

B220 cell surface expression) using a B cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). B

ce pt

cells (1×106/ml) were stimulated with IL-4 (50 ng/ml; Peprotech, Rocky Hill, NJ, USA) and LPS (10 μg/ml; Sigma, St Louis, MO, USA) for 5 days in RPMI 1640 containing 10% FBS (Hyclone, Logan, UT, USA), L-glutamine (Invitrogen, Carlsbad, CA, USA) and 50 μM β-mercaptoethanol (Sigma, St Louis, MO, USA). Day-6 supernatants were collected for IgE and IgG1 analysis. Mouse bone

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

marrow-derived mast cells (MCs) were obtained as previously reported (7,8). In brief, bone marrow cells were obtained by flushing bone marrow cells from the femurs of BALB/c mice. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Hyclone, Logan, UT, USA) , 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA), 25 mM HEPES, 1.0 mM sodium pyruvate, non-essential amino acids (BioSource International, Camarillo, CA, USA), 0.0035% β-mercaptoethanol and 30 ng/ml recombinant mouse IL-3 (PeproTech, Rocky Hill, NJ,

Page 5 of 40

USA). After 4 weeks, mast cell purity was evaluated by toluidine blue staining and CD117 (ebioscience, San Diego, CA, USA) surface staining by flow cytometry. The purity of the mast cells used in this study was greater than 95% (data not shown). The MCs were used following 4-6 weeks

ip t

of culture at 37°C and 5% CO2. The HUVEC line was purchased from American Type Culture Collection (Manassas, VA, USA). A specific Akt activator (9) (4 μg/ml, SC79; Sigma, St Louis, MO,

cr

USA) was used in several experiments.

us

2.3.IgE-mediated activation of MCs

an

IgE-mediated activation of MCs was performed as described in our previous study (8). In brief, MCs

M

(1×106/ml) were sensitized with 1 μg/ml of DNP-specific IgE (Sigma, St Louis, MO, USA) in medium for 4 h and challenged with DNP-BSA (100 ng/ml, Sigma, St Louis, MO, USA) for 30 min

ed

in Tyrode’s buffer [10 mM HEPES buffer (pH 7.4), 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA]. BMMCs cultured alone with and without IgE

respectively.

ce pt

sensitization and subsequent DNP challenge were used as positive and negative controls,

A β-hexosaminidase substrate assay was used to quantitate MC degranulation. For the

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

β-hexosaminidase substrate assay, IgE-presensitized MCs were challenged in Tyrode’s buffer with DNP-BSA for 30 min. Then, the β-hexosaminidase substrate assay was performed. In brief, the β-hexosaminidase substrate, p-nitrophenyl-N-acetyl-β-D-glucosaminide (50 ml/well, 1.3 mg/ml in 0.08 M sodium citrate pH 4.5; Sigma, St Louis, MO, USA), was added to 10 ml of supernatant or lysate from mast cells and incubated at 37°C overnight. Sodium hydroxide (50 ml/well, 1 N; Sigma, St Louis, MO, USA) was added to stop the reaction, and the release of the product 4-p-nitrophenol

Page 6 of 40

was detected by the absorbance at 405 nm. The percent of maximum release was calculated compared to the total amount of β-hexosaminidase from the control cell lysate after treatment with 0.2% Triton X-100 using the following formula: % maximal β-hex release = [(sample –

ip t

background)/(total cell lysate – background)] × 100.

cr

For the histamine analysis, IgE-presensitized MCs were challenged in Tyrode’s buffer with

DNP-BSA for 30 min. Then, the supernatants were collected for histamine analysis. For cytokine

us

analysis, IgE-sensitized BMMCs were cultured for 16 hr in the presence of DNP-BSA. Then, the

an

supernatants were collected for cytokine analysis.

M

2.4.Vascular permeability assay

ed

Vascular permeability assays were performed as described previously (10). In brief, HUVECs (2×105) were grown in a transwell plate (BD Biosciences, San Jose, CA, USA ) in 500 μl of medium until a

ce pt

monolayer was formed. HUVEC monolayer permeability was tested 8 h later by the addition of 7.5 μl streptavidin-HRP (1.5 μg/ml; R&D, Minneapolis, MN, USA) to the upper chamber. The monolayers were stimulated with histamine (100 μM) for 30 min prior to the addition of streptavidin-HRP. The media (50 μl) in the lower chamber was collected 5 min after the addition of

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

streptavidin-HRP and was assayed for HRP activity by the addition of 100 μl TMB substrate. Color development was detected using a microplate reader at 450 nm.

2.5.Treatment of EAC by doxycycline

EAC was developed using the following protocol: a mixture of 10 μg OVA (Sigma, St Louis, MO, USA) and 0.5 ml aluminum hydroxide gel (Pierce, Rockford, IL, USA) was administered

Page 7 of 40

intraperitoneally on the first day. On day 5, the sensitization procedure was repeated to enhance the allergic reaction. From days 10 to 14, 10ul of 2 mg OVA solution was applied to each eye daily to induce EAC. The mice in the blank group were only subjected to PBS. Mice with EAC were treated

ip t

with topical doxycycline (0.1% by instillation four times/day) on days 5-14 or were treated with the

cr

vehicle control.

Slit lamp checking was performed throughout the course of the study. After 30 min of challenge, the

us

clinical reactions were recorded and evaluated by two observers who were blinded to the treatment

an

groups. The allergic symptoms were graded using a previously published system (11). Conjunctival edema, lid swelling, tearing and conjunctival redness were graded from 0 to 4 based on the criteria.

M

The clinical score was the sum of the four parameters. The scratching times of each animal fifteen

ed

minutes after the 30 min challenge were also counted by observers who were blinded to the treatment protocol. The scratching response was defined as rapid movements of the hind paws that were

ce pt

precisely directed toward the eye.

Ophthalmic lavage fluid (OLF) was collected after the last OVA exposure. PBS (10 μl) was applied to the eye using a micropipette without touching the eye. After 2 or 3 forced blinks, the OLF was

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

collected. The lavage was repeated 5 times in each eye. OLF was centrifuged at 400 g for 10 min and the supernatant was separated for further analysis. 1 h after the last challenge, the eyeballs was removed after euthanasia for further analysis. In some experiments, SC79 or the vehicle was injected intraperitoneally (40 mg/kg) 5 min prior to every challenge (9).

2.6.Treatment of PSA and histamine-induced systemic anaphylaxis-like reaction (HISA) by doxycycline

Page 8 of 40

For PSA, the mice were sensitized for 24 h by intravenous injection of 5 μg anti-DNP IgE (SPE-7) and were subsequently challenged by an intravenous injection of 100 μg DNP-BSA. For HISA, histamine (10 μm in 200 μl PBS) was intravenously injected into the mice. After challenge with

ip t

DNP-BSA or histamine stimulation, blinded researchers measured and recorded the body temperature and respiratory frequency at the indicated time points for 1 h using a physiological

cr

signal measurement system (RM-6280C, Chengdu instrument plant, Chengdu, China). After 1 h,

us

blood and lung tissue were harvested for further analysis. Doxycycline (1.5 mg/kg) or vehicle was injected intraperitoneally 30 min prior to DNP-BSA or histamine stimulation (12, 13). In some

an

experiments, SC79 or the vehicle was injected intraperitoneally (40 mg/kg) 5 min prior to DNP-BSA

M

or histamine stimulation (9).

ed

2.7.Immunohistochemistry

H&E staining was performed on paraffin-embedded sections for histological examination. For

ce pt

semi-quantification, positive signals in at least 5 random high-power fields were visualized and calculated with Image Pro-Plus 5.1 (Media Cybernetics cell numbers, Silver Spring, MD, USA ).

2.8.Western blot analysis, ELISA and NO assay

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Cell lysates or mouse ear lysates (50-100 μg of total protein) were separated on polyacrylamide-SDS gels and electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with TBS/5% nonfat dry milk, the membrane was incubated with an antibody against Akt, VE-cadherin， eNOS，PLCγor Lyn (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with a HRP-conjugated secondary antibody. The signals were visualized by enhanced

Page 9 of 40

chemiluminescence (PIERCE, Rockford, IL, USA). The blots were then reprobed with a specific antibody against β-actin (Cell Signaling Technology).

The concentrations of IgE, IgG1, TNF-α and IL-4 in the OLF or supernatants of cultured cells were

ip t

detected by ELISA (eBioscience). The histamine concentration in the supernatants, OLF or plasma

cr

was determined using an EIA kit (Cayman Chemicals, Ann Arbor, MI, USA). The activity of PI3K was determined using a PI3K activity ELISA: Pico kit (Echelon Biosciences Inc. Salt Lake, UT,

us

USA). The supernatants or plasma NO levels were measured using the Griess reaction (14). In brief,

M

sulfanilamide at room temperature for 10 minutes.

an

50 μl of the samples were mixed with 0.1% N-1-napthylethylenediamine dihydrochloride and 1%

2.9. Real-time PCR. Total RNA from the tissue lysates was extracted with the RNeasy Mini Kit

ed

(Qiagen, Valencia, CA, USA), and cDNA was generated using an Omniscript RT kit (Qiagen, Valencia, CA, USA). TNF-α and IL-4 mRNA expression was quantified with ABsolute SYBR Green

ce pt

ROX mix (Thermo Scientific,Waltham, MA, USA). The samples were run in triplicate, and the relative expression of TNF-α and IL-4 was determined by normalizing the expression of each target to β-actin using the 2-ΔΔCt method.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2.10.Statistical analysis

Student’s t test was used to analyze statistical difference (SPSS 16.0). p < 0.05 was considered significant.

Page 10 of 40

3.RESULTS

3.1.The PI3K/Akt pathway is involved in the doxycycline-mediated inhibition of IgE release by B cells

ip t

B cells play important roles in the pathophysiology of ADs, primarily through their ability to produce

cr

IgE (1). Thus, we first explored whether doxycycline had any inhibitory effects on IgE release by B

us

cells. For this purpose, purified mouse splenic B cells were cultured with IL-4 (50 ng/ml) and LPS (10 μg/ml) in the presence or absence of doxycycline at increasing concentrations for 5 days. IgE and

an

IgG1 levels in the culture supernatants were measured by ELISA. Our results showed that doxycycline treatment led to a dose-dependent inhibition of IgE and IgG1 release by activated B

M

cells (Figure 1 A and B). Compared to 40 μM doxycycline, 80 μM doxycycline inhibited IgE

ed

releasing more effectively, but B cell viability was affected (Figure 1C). Therefore, we chose 40 μM doxycycline in the following experiments unless specifically indicated.

ce pt

We next determined the mechanisms involved in the doxycycline-mediated inhibition of B cells. The PI3K/Akt pathway plays important roles in B cell differentiation and function (15, 16). Previous studies have shown that doxycycline can effectively inhibit PI3K/Akt activation (4, 17). Thus, we

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

asked whether doxycycline could modulate the PI3K/Akt pathway in B cells. To this end, B cells were cultured with LPS/IL-4 in the presence or absence of doxycycline (40 μM) for 5 days. PI3K activity and the expression of phosphorylated Akt (p-Akt) were analyzed by ELISA and Western blot, respectively. Although the stimulation of B cells with LPS/IL-4 resulted in increased PI3K activity and p-Akt protein expression (Figure 1D and 1E), treatment with doxycycline significantly inhibited PI3K activity and p-Akt expression in B cells. To further confirm the role of the PI3K/Akt pathway

Page 11 of 40

in the doxycycline-mediated inhibition of B cells, an Akt activator (SC79) was used (9). Our results showed that SC79 significantly reversed the doxycycline-mediated inhibition of IgE release from B cells stimulated with LPS/IL-4 (Figure 1F). As expected, SC79 significantly induced Akt

ip t

phosphorylation and reversed doxycycline-mediated inhibition of Akt phosphorylation (Figure 1G). Taken together, these results suggest that doxycycline inhibits IgE and IgG1 release from B cells by

cr

modulating the PI3K/Akt pathway.

us

3.2.Doxycycline suppresses IgE-mediated activation of MCs by modulating the PI3K/Akt

an

pathway

MCs have been recognized as key players in IgE-dependent type I or immediate allergic responses,

M

primarily through their ability to release granule-stored preformed mediators and to produce

ed

inflammatory mediators (1, 18). Therefore, our next experiments explored the impact of doxycycline on the IgE-mediated activation of MCs. For this purpose, MCs were pretreated with doxycycline (40

ce pt

μM) for 24 h, followed by anti-DNP IgE-sensitization and DNP-BSA challenge in the presence of doxycycline. Degranulation was measured by the release of the MC granule-associated enzyme β-hexosaminidase and histamine. As shown in Figure 2A, doxycycline treatment significantly inhibited MC degranulation from 34.6% ± 5.8% to 19.3% ± 4.6%. Similarly, results from the EIA

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

assay showed that doxycycline significantly reduced histamine release by IgE-antigen-stimulated MCs (Figure 2B). The ELISA results showed that doxycycline also significantly decreased the production of TNF-α and IL-4 by IgE-antigen-stimulated MCs (Figure 2C and 2D). Doxycycline (40 μM) did not affect MCs viability (Figure 2E). These findings suggest that doxycycline can inhibit the IgE-mediated activation of MCs.

Page 12 of 40

The PI3K/Akt pathway also plays a central role in mast cell biology (19). Thus, to further explore the mechanism of doxycycline-mediated MC inhibition, PI3K activity and p-Akt expression were analyzed by ELISA and Western blot, respectively. As shown in Figure 2F and G, treatment with

ip t

doxycycline significantly reduced PI3K activity and p-Akt expression in MCs. The Akt activator SC79 significantly reversed the doxycycline-mediated inhibitory effects on degranulation and

cr

inflammatory cytokines production (Figure 2H, 2I, 2J and 2K). To further elucidate mechanism, the

us

IgE-driven upstream of PI3K/Akt pathway were examed. Western blot results demonstrated that doxycycline treatment reduced the phosphorylation of PLCγand Lyn, but not as notable as the

an

decrease of p-Akt (Figre 2L and 2M). Taken together, these results indicate that the PI3K/Akt

ed

MCs activated in an IgE-dependent manner.

M

pathway and its upstream signaling plays an important role in the doxycycline-mediated inhibition of

3.3.Doxycycline exerts anti-histamine effects by modulating the PI3K/Akt/eNOS/VE-cadherin

ce pt

pathway

As a key mediator secreted by activated mast cells, histamine plays an important role in the development of ADs (20-22). Histamine causes the signs of allergic reactions predominantly by acting on vascular smooth muscle cells and endothelial cells, leading to vasodilatation and vascular hyperpermeability

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(20-22). Therefore, we asked whether doxycycline has inhibitory effects on histamine-induced vascular hyperpermeability. For this purpose, HUVECs were cultured with or without doxycycline using a transwell system for 24 h. Cells from each culture condition were stimulated with histamine for 30 min and were subsequently tested in the permeability assay. Our results showed that doxycycline led to a significant decrease in vascular hyperpermeability to streptavidin-HRP than HUVECs cultured alone

Page 13 of 40

(Figure 3A), and did not affect HUVECs viability (Figure 3B).

Next, we sought to determine the possible mechanisms involved in the doxycycline-mediated inhibition of vascular hyperpermeability. VE-cadherin-VE-cadherin homophilic interactions are

ip t

crucial for maintaining normal vascular integrity and permeability. Previous studies have shown that

cr

histamine can induce tyrosine phosphorylation of VE-cadherin and disrupt VE-cadherin-VE-cadherin homophilic interactions to cause vascular hyperpermeability (23-25). Thus, we analyzed the expression

us

of total VE-cadherin and phosphorylated VE-cadherin (p-VE-cadherin) of HUVECs under different

an

conditions. Our results revealed that culturing with doxycycline significantly increased total

M

VE-cadherin but reduced p-VE-cadherin in HUVECs (Figure 3C and 3D).

The PI3K/Akt/eNOS pathway is critical for VE-cadherin regulation, endothelium-derived NO release

ed

and histamine-mediated vascular hyperpermeability and acute inflammation (26-28). Thus, we examined whether the PI3K/Akt/eNOS pathway is involved in doxycycline-mediated anti-histamine

ce pt

effects. Following stimulation with histamine, the supernatants or cells from each culture condition were harvested and subsequently subjected to a Griess assay, Western blot and ELISA. The results showed that the addition of doxycycline significantly reduced PI3K activity and the expression of

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

p-Akt and phosphorylated eNOS (p-eNOS), and NO release was lower in doxycycline treated HUVECs than in HUVECs treated with a control (Figure 3E, 3F, 3G and 3H). In addition, the Akt activator SC79 significantly reversed the doxycycline-mediated inhibitory effects on histamine-induced vascular hyperpermeability (Figure 3I). These findings suggest that doxycycline is capable of blocking histamine-induced vascular hyperpermeability by modulating the PI3K/Akt/eNOS/VE-cadherin pathway.

Page 14 of 40

3.4.Doxycycline treatment attenuates EAC Allergic conjunctivitis (AC) involves ocular inflammation associated with type I hypersensitivity reactions accompanied by the characteristic symptoms (29, 30). To examine the therapeutic effect of

ip t

doxycycline on AC, doxycycline was administered to mice with OVA-induced EAC by topical applications. The OVA challenges generated typical signs that mimic human AC, including lid

cr

swelling, conjunctival edema, redness, tearing, and frequent eye scratching. The effect of

us

doxycycline on these ocular symptoms in mice was evaluated after exposure to OVA. The symptoms in the EAC mice were significantly suppressed with doxycycline compared to the EAC controls

an

(Figure 4A and 4C). Treatment with doxycycline significantly inhibited the scratch response of mice

M

for 15 min compared to the EAC controls (Figure 4B). Further analysis of conjunctival specimens harvested 24 h after the last challenge showed a clear reduction of inflammatory infiltrates in the

ed

doxycycline-treated mice compared to the EAC controls (Figure 4D and 4E). We next investigated the in vivo effects of doxycycline on the production of IgE, histamine and local inflammatory

ce pt

cytokines in mice with EAC. As shown in Figures 4F and 4G, the application of OVA significantly increased the levels of IgE and histamine in the EAC mice compared to the control mice, whereas doxycycline treatment significantly reduced the levels of IgE and histamine. Real-time PCR results

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

showed that the administration of doxycycline significantly reduced TNF-α and IL-4 expression in EAC mice (Figure 4H and 4I). As these results showed that modulation of the PI3K/Akt pathway is essential for the doxycycline-mediated inhibition of B cells, MCs and histamine, we next asked whether the PI3K/Akt pathway is implicated in the doxycycline-mediated attenuation of EAC. Our results showed that doxycycline treatment significantly decreased conjunctival p-Akt expression in mice with EAC (Figure 4J), whereas the Akt activator SC79 partially reversed the

Page 15 of 40

doxycycline-mediated attenuation of EAC (Figure 4K, 4L, 4M and 4N). Taken together, these findings indicate that doxycycline attenuates EAC at least partially by modulating the PI3K/Akt pathway.

ip t

3.5.Doxycycline treatment prevents PSA

cr

Anaphylaxis, a life-threatening systemic allergic reaction, is responsible for more than 1500 deaths

us

per year in the US (31). To explore the role of doxycycline in SA and the direct effects of doxycycline on IgE-mediated activation of MCs in vivo, IgE-dependent PSA (a well-established

an

animal model for SA) was used. Doxycycline was injected intraperitoneally 30 min prior to a

M

DNP-BSA challenge. After challenge with DNP-BSA, changes in body temperature and respiratory frequency were monitored. Compared with the PSA controls, body temperature declined

ed

significantly less in the doxycycline-treated mice (Figure 5A). Similarly, a significantly smaller decrease in respiratory frequency was observed in the doxycycline-treated mice compared to the PSA

ce pt

controls (Figure 5B). Treatment with doxycycline showed significant inhibitory effects on histamine production in the plasma compared to the PSA controls (Figure 5C). Histological analysis of the lung showed that pulmonary perivascular edema, a characteristic feature of airway anaphylactic reactions,

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

was significantly attenuated in the doxycycline-treated mice compared to the PSA controls (Figure 5D). We next asked whether the PI3K/Akt pathway is implicated in the doxycycline-mediated attenuation of PSA. Our results showed that doxycycline treatment significantly decreased Akt phosphorylation in the lungs of mice with PSA (Figure 5E), whereas the Akt activator SC79 reversed the doxycycline-mediated attenuation of PSA (Figure 5F, 5G, 5H and 5I). These results indicate that doxycycline is capable of attenuating PSA and MC activation in vivo, possibly by modulating the

Page 16 of 40

PI3K/Akt pathway.

3.6.Inhibitory effects of doxycycline on HISA Next, we wanted to further explore the direct effects of doxycycline on histamine function in vivo.

ip t

Histamine injections typically decrease body temperature and respiratory frequency (32). When

cr

doxycycline was injected intraperitoneally 30 min prior to histamine stimulation, we observed that

us

body temperature and respiratory frequency declined significantly less in the doxycycline-treated HISA mice than in the control mice (Figure 6A and 6B). Treatment with doxycycline also

an

significantly suppressed NO production in the plasma compared to the HISA controls (Figure 6C). Histological analysis of the lung showed that pulmonary perivascular edema was significantly

M

attenuated in the doxycycline-treated mice compared to the HISA controls (Figure 6D). Treatment

ed

with doxycycline significantly inhibited p-Akt, p-eNOS and p-VE-cadherin in the lungs of mice with HISA (Figure 6E, 6F and 6G). Intraperitoneal injections of SC79 significantly reversed the inhibitory

ce pt

effects of doxycycline on HISA (Figure 6H, 6I, 6J and 6K). Taken together, these results indicate that doxycycline can directly inhibit the histamine-mediated pathological effects in vivo, at least partially by modulating the PI3K/Akt/eNOS/ VE-cadherin pathway.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Page 17 of 40

ip t cr

4.DISCUSSION

us

Following the discovery of a striking variety of non-antibiotic properties of tetracyclines, therapeutic applications of these drugs have been successfully extended (3). In past decades, tetracyclines have

an

been successfully used to treat various diseases, including rheumatoid arthritis, periodontal disease,

M

aortic aneurysm, stroke, shock, nasal polyps and tumor metastases (3). In this study, we investigated the anti-allergy properties of doxycycline in EAC and PSA. Using an OVA-induced EAC model, we

ed

demonstrated that doxycycline treatment attenuated the allergic symptoms of EAC, coupled with a significant decrease in IgE, histamine and inflammatory cytokine production. In PSA, doxycycline

ce pt

also significantly ameliorated clinical symptoms and lung edema with a significant decrease in histamine production. Taken together, these compelling findings suggest that doxycycline is capable of inhibiting ADs.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

In ADs, after antigen stimulation, allergen-specific CD4+ Th2 cells produce Th2 cytokines to initiate B cell production of allergen-specific IgE, which binds to the high-affinity IgE receptor (FcɛRI) on MCs. Allergen crosslinking of allergen-specific IgE leads to the release of preformed and granule-stored allergic mediators, such as histamine, which then initiates the early acute inflammatory responses and de novo synthesis of mediators, such as inflammatory cytokines chemokines and cysteinyl leukotrienes. Eventually, this leads to a sustained, late phase inflammatory

Page 18 of 40

response (1). Therefore, B cell-derived IgE and MCs play key roles in the development of ADs. In this study, we demonstrated that doxycycline significantly reduced IgE production by B cells activated in vitro with LPS/IL-4. Additionally, we observed that doxycycline significantly inhibited

ip t

degranulation and inflammatory mediator production by MCs activated through an IgE trigger in vitro. Mechanistically, we found that the PI3k/Akt pathway was involved in the

cr

doxycycline-mediated inhibition of B cells and MCs. In vivo, we found that the administration of

us

doxycycline significantly decreased IgE and histamine production during OVA-induced EAC. During IgE-induced PSA, doxycycline treatment significantly reduced histamine production. These

an

results indicate that the inhibition of B cell and MC function at least partially contributes to the

M

anti-allergy properties of doxycycline.

ed

Histamine is synthesized and released primarily by MCs, as well as basophils, lymphocytes, macrophages, platelets, dendritic cells and neutrophils, and plays critical roles in ADs (20-22). In

ce pt

allergic pathology, histamine triggers acute symptoms due to its rapid effects on the vascular endothelium and bronchial and smooth muscle cells, leading to vasodilatation, vascular hyperpermeability, bronchoconstriction, hypotension, itching, cramping, diarrhea or cutaneous weal and flare responses (20-22). In the present study, we demonstrated that doxycycline could

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

significantly inhibit histamine-induced vascular hyperpermeability. Importantly, in vivo treatment with doxycycline also significantly attenuated HISA and lung edema. Mechanistically, our results implicate the PI3k/Akt1/eNOS/VE-cadherin pathway in the doxycycline-mediated effects of anti-histamine in vitro and in vivo. These findings suggest that doxycycline possesses anti-histamine properties that may play important roles in the anti-allergy properties of doxycycline. To our knowledge, this is the first time that tetracyclines have been shown to inhibit histamine function,

Page 19 of 40

further expanding the understanding of the biological properties of tetracyclines.

Currently, MC inhibitors and anti-histamine drugs, which remain the mainstay of therapy for ADs, have demonstrated variable and limited clinical success (1, 2). This could be due to factors other than

ip t

MCs and histamine, as T cells, B cells, macrophages, platelets, dendritic cells and neutrophils also

cr

play important roles in allergic diseases. Therefore, simultaneously targeting multiple cellular

components or inflammatory mediators may be a promising modality for the treatment of ADs.

us

Unlike MC inhibitors and anti-histamine drugs, our results showed that doxycycline could

an

simultaneously act on multiple targets, including B cells, MCs and histamine. Furthermore, it has also been reported that doxycycline possesses multifunctional immunosuppressive and

M

anti-inflammatory functions through the inhibition of T cells, dendritic cells, natural killer cells,

ed

neutrophils and macrophages (3, 33-36). Steroids with potent anti-inflammatory and anti-allergy activity also remain the mainstay of therapy for ADs. However, numerous studies have shown that

ce pt

steroid treatment can lead to serious side effects and complications. Unlike steroids, tetracyclines have been used safely for decades in clinical settings with few side effects. Furthermore, the combined therapy of tetracyclines and steroids has shown promising therapeutic effects in allergic asthmatic humans (5). These unique properties render doxycycline a promising therapeutic tool for ADs.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Tetracyclines are broad-spectrum antimicrobial agents, raising the possibility of the induction of bacterial resistance to doxycycline by the clinical anti-allergic application of doxycycline. Previous studies have demonstrated that antimicrobial doses of tetracycline have a qualitative effect on the normal microflora (37). However, in the clinical setting, substantial evidence indicates that a

Page 20 of 40

sub-antimicrobial dose of doxycycline (40 mg/day) provides a significant benefit in the treatment of inflammatory or/and immunological diseases, include rheumatoid arthritis and periodontal disease. At a sub-antimicrobial dose, long-term treatment with doxycycline has no antibacterial effect and

ip t

does not cause the appearance of resistant bacteria (38, 39). Therefore, the clinical use of a sub-antimicrobial dose of doxycycline as an anti-allergic drug is unlikely to induce the appearance of

cr

resistant bacteria. Further clinical studies are warranted to evaluate the therapeutic effects of

us

sub-antimicrobial doses of doxycycline in allergic subjects.

an

In summary, our study demonstrates for the first time the anti-allergy properties of tetracyclines on EAC and PSA. Our results showed that doxycycline reduced IgE release from B cells and reduced

M

IgE-stimulated MC degranulation and inflammatory mediator production by modulating the

ed

PI3K/Akt pathway. Importantly, we provide the first evidence that doxycycline possesses the anti-histamine property of inhibiting histamine-stimulated vascular hyperpermeability and HISA.

ce pt

through modulation of the PI3K/Akt/eNOS/VE-cadherin pathway in vitro and in vivo. These findings provide compelling evidence that doxycycline can inhibit allergic reactions and support the notion that tetracyclines are an appropriate and ideal therapeutic option for allergic diseases.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Acknowledgments

This study was funded by the Natural Science Foundation of China (81271051) and the Natural Science Foundation of China (81300740). Conflict of interest None.

Page 21 of 40

Abbreviations: ADs: allergic diseases; AC: allergic conjunctivitis;

ip t

SA: systemic anaphylaxis; EAC: experimental allergic conjunctivitis;

cr

PSA: passive systemic anaphylaxis;

us

HISA histamine-induced systemic anaphylaxis like reaction;

BMMCs, bone marrow derived mast cells;

TNF-α, tumor necrosis factor-alpha;

NO, nitric oxide;

ce pt

LPS, lipopolysaccharides;

ed

IL-4, interleukin-4;

M

HUVEC: human umbilical vein endothelial cells;

an

MC: Mast cell;

DNP: dinitrophenyl.

OLF: Ophthalmic lavage fluid

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

References

1. Holgate ST, Polosa R. Treatment strategies for allergy and asthma. Nat Rev Immunol 2008; 8： 218-30. 2. Joks R, Durkin HG. Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs. Pharmacol Res 2011; 64：602-9. 3. Bahrami F, Morris DL, Pourgholami MH. Tetracyclines: drugs with huge therapeutic potential.

Page 22 of 40

Mini Rev Med Chem 2012; 12:44-52. 4. Lee KS, Jin SM, Kim SS, Lee YC. Doxycycline reduces airway inflammation and hyperresponsiveness in a murine model of toluene diisocyanate-induced asthma. J Allergy Clin

ip t

Immunol 2004;113:902-9.

cr

5. Daoud A, Gloria CJ, Taningco G, Hammerschlag MR, Weiss S, Gelling M, Roblin PM, Joks R. Minocycline treatment results in reduced oral steroid requirements in adult asthma. Allergy

us

Asthma Proc 2008; 29:286-94.

an

6. Smith-Norowitz TA, Bluth MH, Drew H, Norowitz KB, Chice S, Shah VN, Nowakowski M,

Allergy Asthma Immunol 2002;89:172-9.

M

Josephson AS, Durkin HG, Joks R. Effect of minocycline and doxycycline on IgE responses. Ann

ed

7. Jensen BM, Swindle EJ, Iwaki S, Gilfillan AM. Generation, isolation, and maintenance of rodent

ce pt

mast cells and mast cell lines. Curr Protoc Immunol 2006; Chapter 3：Unit 3.23. 8. Su W, Fan H, Chen M, Wang J, Brand D, He X, Quesniaux V, Ryffel B, Zhu L, Liang D, Zheng SG. Induced CD4(+) forkhead box protein-positive T cells inhibit mast cell function and established contact hypersensitivity through TGF-beta1. J Allergy Clin Immunol 2012;130:444-52.e7.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

9. Jo H, Mondal S, Tan D, Nagata E, Takizawa S, Sharma AK, Hou Q, Shanmugasundaram K, Prasad A, Tung JK, Tejeda AO, Man H, Rigby AC, Luo HR. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci USA. 2012; 109:10581-6. 10. Chuang YC, Lei HY, Liu HS, Lin YS, Fu TF, Yeh TM. Macrophage migration inhibitory factor

Page 23 of 40

induced by dengue virus infection increases vascular permeability. Cytokine 2011; 54：222-31. 11. Merayo-Lloves J, Zhao TZ, Dutt JE, Foster CS. A new murine model of allergic conjunctivitis and effectiveness of nedocromil sodium. J Allergy Clin Immunol 1996;97：1129-40.

cr

mortality to lethal endotoxemia by reducing nitric oxide synthesis via an

ip t

12. D'Agostino P, La Rosa M, Barbera C, Arcoleo F, Di BG, Milano S, Cillari E. Doxycycline reduces

us

interleukin-10-independent mechanism. J Infect Dis 1998;177：489-92.

13. Milano S, Arcoleo F, D'Agostino P, Cillari E. Intraperitoneal injection of tetracyclines protects

an

mice from lethal endotoxemia downregulating inducible nitric oxide synthase in various organs

M

and cytokine and nitrate secretion in blood. Antimicrob Agents Chemother 1997;41：117-21. 14. Grisham MB, Johnson GG, Lancaster JR. Quantitation of nitrate and nitrite in extracellular fluids.

ed

Meth Enzymol 1996;268：237-46.

ce pt

15. Donahue AC, Fruman DA. PI3K signaling controls cell fate at many points in B lymphocyte development and activation. Semin Cell Dev Biol 2004;15：183-97. 16. So L, Fruman DA. PI3K signalling in B- and T-lymphocytes: new developments and therapeutic advances. Biochem J 2012;442:465-81.

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

17. Su W, Li Z, Li F, Chen X, Wan Q, Liang D. Doxycycline-mediated inhibition of corneal angiogenesis: an MMP-independent mechanism. Invest Ophthalmol Vis Sci 2013; 54：783-8. 18. Bischoff SC. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 2007;7：93-104. 19. Kim MS, Radinger M, Gilfillan AM. The multiple roles of phosphoinositide 3-kinase in mast cell

Page 24 of 40

biology. Trends Immunol 2008;29：493-501. 20. Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov 2008;7:41-53.

ip t

21. Jutel M, Akdis M, Akdis CA. Histamine, histamine receptors and their role in immune pathology.

cr

Clin Exp Allergy 2009;39：1786-800.

us

22. Jones BL, Kearns GL. Histamine: new thoughts about a familiar mediator. Clin Pharmacol Ther 2011;89：189-97.

an

23. Andriopoulou P, Navarro P, Zanetti A, Lampugnani MG, Dejana E. Histamine induces tyrosine

M

phosphorylation of endothelial cell-to-cell adherens junctions. Arterioscler Thromb Vasc Biol 11999;9：2286-97.

ed

24. Shasby DM, Ries DR, Shasby SS, Winter MC. Histamine stimulates phosphorylation of adherens

L1330-8.

ce pt

junction proteins and alters their link to vimentin. A J Physiol Lung Cell Mol Physiol 2002; 282：

25. Winter MC, Shasby SS, Ries DR, Shasby DM. Histamine selectively interrupts VE-cadherin adhesion independently of capacitive calcium entry. Am J Physiol Lung Cell Mol Physiol. 2004;

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

287：L816-23.

26. Cauwels A, Janssen B, Buys E, Sips P, Brouckaert P. Anaphylactic shock depends on PI3K and eNOS-derived NO. J Clin Invest. 2006;116：2244-51. 27. Evora PR, Simon MR. Role of nitric oxide production in anaphylaxis and its relevance for the treatment of anaphylactic hypotension with methylene blue. Ann Allergy Asthma Immunol 2007;

Page 25 of 40

99：306-13. 28. Di LA, Fernandez-Hernando C, Cirino G, Sessa WC. Akt1 is critical for acute inflammation and histamine-mediated vascular leakage. Proc Natl Acad Sci USA 2009;106：14552-7.

ip t

29. Ozaki A, Seki Y, Fukushima A, Kubo M. The control of allergic conjunctivitis by suppressor of

cr

cytokine signaling (SOCS)3 and SOCS5 in a murine model. J Immunol 2005;175：5489-97.

treatment. J Allergy Clin Immunol 2005; 115：118-22.

us

30. Ono SJ, Abelson MB. Allergic conjunctivitis: update on pathophysiology and prospects for future

an

31. Neugut AI, Ghatak AT, Miller RL. Anaphylaxis in the United States: an investigation into its

M

epidemiology. Arch Intern Med 2001;161：15-21.

32. Makabe-Kobayashi Y, Hori Y, Adachi T, Ishigaki-Suzuki S, Kikuchi Y, Kagaya Y, Shirato K, Nagy

ed

A, Ujike A, Takai T, Watanabe T, Ohtsu H. 2002. The control effect of histamine on body

ce pt

temperature and respiratory function in IgE-dependent systemic anaphylaxis. J Allergy Clin Immunol 110：298-303.

33. Takeshita S, Ono Y, Kozuma K, Suzuki M, Kawamura Y, Yokoyama N, Furukawa T, Isshiki T. Modulation of oxidative burst of neutrophils by doxycycline in patients with acute myocardial

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

infarction. J Antimicrob Chemother 2002;49：411-3. 34. Krakauer T, Buckley M. Doxycycline is anti-inflammatory and inhibits staphylococcal exotoxin-induced cytokines and chemokines. Antimicrob Agents Chemother 2003;47：3630-3. 35. Lindeman JH, Abdul-Hussien H, Van-Bockel JH, Wolterbeek R, Kleemann R. Clinical trial of doxycycline for matrix metalloproteinase-9 inhibition in patients with an abdominal aneurysm

Page 26 of 40

doxycycline selectively depletes aortic wall neutrophils and cytotoxic T cells. Circulation 2009;119：2209-16. 36. Goh DH, Ferrante A. In vitro inhibition of natural killer cell activity by doxycycline. Int J

ip t

Immunopharmacol 1984;651-54

cr

37. Edlund C, Nord CE. Ecological impact of antimicrobial agents on human intestinal microflora.

us

Alpe Adria Microbiol J 1993; 3: 137–64

38. Walker C, Preshaw PM, Novak J, Hefti AF, Bradshaw M, Powala C. Long-term treatment with

an

sub-antimicrobial dose doxycycline has no antibacterial effect on intestinal flora. J Clin

M

Periodontol. 2005;32: 1163-9.

39. Rashid MU, Panagiotidis G, Backstrom T, Weintraub A, Nord CE. Ecological impact of

ce pt

Agents. 2013; 41: 352-7.

ed

doxycycline at low dose on normal oropharyngeal and intestinal microflora. Int J Antimicrob

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Page 27 of 40

Figure legends

Figure 1. The PI3K/Akt pathway is involved in the doxycycline-mediated inhibition of IgE release from B cells. B cells were cultured with doxycycline for 24 h (A-D) in the absence or presence of a

ip t

specific Akt activator, SC79 (F). Following stimulation with LPS/IL-4 for 5 days, IgE and IgG1 levels in the supernatants was determined by ELISA (A, B, F); cell viability was assessed before and

cr

after treatment using Trypan blue exclusion and examined using phase contrast microscopy. (C);

us

PI3K activity and phosphorylated Akt expression in B cells were determined by ELISA and Western

an

blot, respectively (D, E，G). **: p < 0.01. Error bars: mean ±SEMs. Experiments were repeated twice with similar results.

M

Figure 2. Doxycycline suppresses IgE-mediated activation of MCs by modulating the PI3K/Akt

ed

pathway. MCs were cultured with doxycycline for 24 h (A-G) in the absence or presence of a specific Akt activator, SC79 (H-K). Following IgE sensitization for 4 h and IgE-antigen stimulation

ce pt

for 30 min, degranulation was measured by release of the MC granule-associated enzyme β-hexosaminidase (A, H) and histamine (B, I). Following IgE sensitization for 4 h and IgE-antigen stimulation for 16 h, TNF-α (C, J) and IL-4 (D, K) levels in the supernatants were determined by ELISA. PI3K activity and phosphorylated Akt expression in MCs were determined by ELISA and

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Western blot, respectively (F, G). Following IgE sensitization for 4 h and IgE-antigen stimulation for 24 h, cell viability was assessed before and after treatment using Trypan blue exclusion and examined using phase contrast microscopy (E). Phosphorylated PLCγ and Lyn expression in MCs were determined by Western blot (L, M) **: p < 0.01. Error bars: mean ±SEMs. Experiments were repeated twice with similar results.

Page 28 of 40

Figure 3. Doxycycline exerts anti-histamine effects by modulating the PI3K/Akt/eNOS/VE-cadherin

ip t

pathway. HUVECs were cultured with doxycycline for 24 h (A-G) in the presence or absence of a

cr

specific Akt activator, SC79 (I). Following stimulation with histamine for 30 min, vascular

permeability assays were performed (A, I); cell viability was assessed before and after treatment

us

using Trypan blue exclusion and examined using phase contrast microscopy (B); the expression of

an

total VE-cadherin (C), phosphorylated VE-cadherin (D), phosphorylated Akt (F) and phosphorylated eNOS (G) was determined by Western blot; PI3K activity was determined by ELISA (E); the nitrite

M

content in the supernatants was measured using the Griess reaction (H). **: p < 0.01. Error bars:

ed

mean ±SEMs. Experiments were repeated twice with similar results. Figure 4. Doxycycline treatment attenuates EAC. Ocular clinical symptoms of EAC (A, K, L) and

ce pt

scratch times (B, M, N) (n=6/group) were evaluated and recorded at the indicated time points after OVA challenge in the indicated experimental groups. (C): Representative images of ocular symptoms in the indicated experimental group 30 min after the last challenge (red arrow: lids swelling; blue arrow: conjunctival edema). (D): Representative images of H&E staining of conjunctival samples

Ac

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

from mice in the indicated experimental group. (E): Quantification of cellular components in the conjunctivas of mice in the indicated experimental groups. The levels of IgE (F) and histamine (G) in the ophthalmic lavage fluid were determined by ELISA and EIA, respectively. TNF-α mRNA (H) and IL-4 mRNA (I) expression in conjunctival tissue was determined by real-time PCR. (J): The expression of phosphorylated Akt in the conjunctival tissue were determined by Western blot. **: p