Allergic airway inflammation disrupts interleukin-17 mediated host defense against streptococcus pneumoniae infection

International Immunopharmacology 31 (2016) 32–38

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Allergic airway inflammation disrupts interleukin-17 mediated host defense against streptococcus pneumoniae infection Sheng Guo a,c, Liang-Xia Wu a, Can-Xin Jones b, Ling Chen a, Chun-Li Hao a, Li He c,⁎, Jian-Hua Zhang a,⁎ a b c

Department of Pediatrics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China Department of Molecular Biosciences & Bioengineering, University of Hawaii-Manoa, Honolulu, HI 96822, USA Department of Sciences and Education, Shanghai Jiao Tong University Affiliated Children's Hospital, Shanghai 200062, China

a r t i c l e

i n f o

Article history: Received 17 September 2015 Received in revised form 16 November 2015 Accepted 7 December 2015 Available online xxxx Keywords: Interleukin-17 Allergy Streptococcus pneumoniae Airway inflammation

a b s t r a c t Despite decreasing rates of invasive pneumococcal disease caused by vaccine serotypes, the prevalence of invasive pneumococcal pneumonia in asthmatic patients remains high. However, little is known about the mechanisms underlying the susceptibility of the asthmatic airway to bacterial infections. In this study, we used a combined model of allergic airway inflammation and Streptococcus pneumoniae lung infection to investigate the association between persistent allergic inflammation in the airway and antibacterial host defenses against S. pneumoniae. When challenged with S. pneumoniae, allergic mice exhibited higher airway bacterial burdens, greater eosinophil infiltration, lower neutrophil infiltration, and more severe structural damage than nonallergic mice. In sensitized mice, S. pneumoniae infection elicited higher IL-4 but lower IFN-γ, IL-17 and defensin-β2 expression than in control mice. These results indicate that persistent allergic inflammation impaired airway host defense against S. pneumoniae is associated with the insufficient IL-17 responses. To elicit IL-17 induced-anti-bacterial immune responses, mice were intranasally immunized with rIL-17. Immunized mice exhibited fewer bacterial colonies in the respiratory tract and less severe lung pathology than unimmunized mice. rIL-17 contributed to airway host defense enhancement and innate immune response promotion, which was associated with increased IL-23, MIP-2 and defensin-β2 expression. Administration of exogenous IL-17 (2 μg/mouse) suppressed eosinophil-related immune responses. The results demonstrate IL-17 plays a key role in host defenses against bacterial infection in allergic airways and suggest that exogenous IL-17 administration promotes the anti-becterial immune responses and attenuates the existed allergic inflammation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children globally [1]. Despite decreasing rates of invasive pneumococcal disease (IPD) caused by vaccine serotypes, the prevalence of IPD in asthmatic patients remains high [2,3]. Many epidemiological studies have reported that individuals with asthma have a significantly increased risk of IPD, compared to those without asthma [4–6]. Emerging evidences indicate that the ability to respond to bacteria in asthmatic patients is reduced by prior allergic airways inflammation. High levels of IgE, eosinophilia and increased inflammatory cytokine production by allergen-specific T cells are thought to contribute to a high susceptibility to bacterial infections [5,7–9]. Although many previous studies have concluded that allergic airway inflammation inhibits pulmonary antibacterial host defenses [3], the results from animal experiments are not always consistent with the clinical finding. A recent study indicates that acute allergic airway inflammation ⁎ Corresponding authors. E-mail addresses: [email protected] (L. He), [email protected] (J.-H. Zhang).

http://dx.doi.org/10.1016/j.intimp.2015.12.010 1567-5769/© 2015 Elsevier B.V. All rights reserved.

reduces the susceptibility of the airway to pneumococcal pneumonia [10], whereas another research result shows that allergic lung inflammation does not alters the susceptibility of the allergic airway to S. pneumoniae infection in mice [11]. The discrepancy results from animal researches indicate that the mechanisms underlying the susceptibility of the asthmatic airway to microbial infections are much more complex than previously revealed, and require further investigation. IL-17A, a cytokine that is primarily produced by Th17 cells, is a proinflammatory cytokine which induces differentiation and migration of neutrophils. IL-17A has been implicated to be critical for the production of cytokines, neutrophil-related chemokines and antimicrobial peptides [12]. Because of its important role in cross talk between the innate and adaptive immune systems, IL-17 is considered as an important inflammatory mediator that is critical in the development of asthma and the protection from pneumococcal colonization in airways [13,14]. In vitro and in vivo studies have shown that exposure to Th2-related cytokines suppresses antimicrobial activity, and the antimicrobial peptide human β-defensin expression in cultured airway epithelial cells downregulates the expression of IL-17A by Th17 cells [9]. The impaired IL-17 immune responses by Th2 cytokines have also been demonstrated in patients

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

with atopic eczema. IL-17-induced HBD-2 upregulation in atopic eczema keratinocytes was partially inhibited by the Th2 cytokines IL-4 and IL-13 in vitro, which partially explain why atopic eczema patients are associated with recurrent and persistent infection of skin and mucosal membranes [15].We previously reported that rIL-17F intranasal inoculation strengthen host defense against pneumococci infection in murine model [16]. Others also reported that exogenous IL-17 reduces pulmonary eosinophil recruitment and bronchial hyperreactivity, and attenuates the allergic response by inhibiting DCs and chemokine synthesis in sensitized mice [17]. A recent research demonstrated that the levels of IL-17 modulate IL-13-induced airway inflammation in a dose-dependent manner where lower doses promote inflammation and higher doses prevent/protect against disease. The research also revealed IL-17-producing γδT cells, but not CD4 T cells may play a protective role in allergic airway inflammation [18]. Some of the confusion regarding the precise role of IL-17 in allergic airway inflammation and anti-bacterial immune responses may partly due to the quantity and/or the cellular source of IL-17. Therefore, we hypothesize that IL17-dependent antibacterial cytokine and chemokine production and neutrophil response to the pathogenic bacteria are defective in an allergic airway. As a consequence of insufficient Th-17 immune responses, the innate and adaptive immune systems in allergic airway fail providing an effective protection to against pneumococcal pneumonia. To this end, two conventional murine models of allergic airway inflammation and pneumonia were combined in this study. Using this murine model system, the intricate relationship between asthma and bacterial infection was explored; furthermore, the mechanism and therapeutic potential of rIL-17 intranasal inoculation against bacterial infection and allergic airway inflammation was investigated.

33

anesthetized mice were intranasally inoculated with 2 μg of recombinant murine IL-17A (expressed and purified in our preliminary work [20]) in 20 μl of phosphate-buffered saline (PBS) containing 1% normal mouse serum as the vehicle from days 22 to 24. The control mice received i.p. injections of PBS containing the aluminum hydroxide gel and the vehicle (PBS containing 1% normal mouse serum) inoculation, but were not subjected to intranasal challenge. All immunization, sensitization, and challenge procedures were performed according to the protocol diagrammed in Fig. 1. 2.4. Mouse sacrificing and sample processing The mice were sacrificed via i.p. administration of an overdose of ketamine/xylazine and were bled out via heart puncture on the indicated day as diagrammed in Fig. 1. Using a tracheal cannula, the lungs were flushed 4 times with 0.4 ml of ice-cold PBS. The bronchoalveolar lavage fluid (BALF) and blood were analyzed for cell composition and cytokine concentrations. Half of the lung tissue was stored in liquid nitrogen for mRNA analysis; the other half was fixed overnight in 4% paraformaldehyde for histological examination. These experiments were replicated at least twice using groups of 6–8 mice. 2.5. Airway bacterial burden The bacterial burden in the airways was measured by sacrificing infected mice at the indicated time points and seeding serial 10-fold dilutions of BALF samples on blood agar plates (0.2-ml aliquots on each plate). The plates were incubated at 37 °C in 5% CO2, and the number of CFU was measured 24 h later. These results were expressed as CFU of bacteria/ml of BALF.

2. Materials and methods 2.6. Lung histology 2.1. Bacterial strains and growth conditions S. pneumoniae American Type Culture Collection (ATCC) 6303 (serotype 3) was obtained from the ATCC and stored at − 80 °C in Todd–Hewitt broth (Sigma) supplemented with 0.5% yeast extract (THY) containing 10% glycerol. Before infection, the bacteria were recovered from stock cultures and THY medium at 37 °C with 5% CO2 as described previously [16]. 2.2. Animals Female BALB/c mice (6–8 wk. old) were purchased from Sino-British SIPPR/BK Lab Animal, Ltd. (Shanghai, China), were housed under specific pathogen-free (SPF) conditions, and were provided with access to food and water ad libitum. S. pneumoniae infection was performed in a biosafety level 2 (BSL-2) biocontainment animal facility. All procedures were performed in accordance with institutional guidelines approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. 2.3. Sensitization, challenge and treatment The OVA sensitization and challenge procedures were performed as indicated previously with slight modifications [17]. Briefly, the experimental mice were sensitized via intraperitoneal (i.p.) injection of 10 μg of OVA adsorbed onto 1.6 mg aluminum hydroxide gel on days 0 and 14 and were challenged for 20 min/day from days 21 to 28 via aerosol nebulization with OVA (1% in PBS) using an Aerosol Delivery System (PARI, Midlothian, VA). The procedures for bacterial infection with S. pneumoniae (ATCC 6303 serotype 3) were modified from the methods described by Sun. et al. [19]. Briefly, the mice were slightly anesthetized using ketamine HCl (25 mg/kg) and xylazine (7.5 mg/kg) in PBS and were intranasally inoculated with S. pneumoniae (106 CFU) in 40 μl of PBS. For the rIL-17A inoculation experiment, the light

The lungs were fixed overnight in 4% buffered paraformaldehyde and embedded in paraffin. Lung sections (4 μm) were stained with hematoxylin and eosin (HE) and examined under a Leica microscope (× 40 magnification). Immunohistochemistry (IHC) was performed using the Biomodule IHC staining kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. To detect the target protein, goat anti-mouse defensin-β2 (Santa Cruz Biotech, TX) was used as the primary antibody. Peribronchial inflammatory cell infiltrate and goblet cell hyperplasia were assessed by two independent observers. 2.7. ELISA for cytokines, chemokines, and ovalbumin (OVA)-specific antibodies The cytokine and chemokine concentrations in BALF or sera were determined via ELISA using commercial kits (IL-17A, IL-4, and INF-γ ELISA kits from BioLegend, San Diego, CA; MIP-2β ELISA kits from PeproTech, Rocky Hill, NJ). OVA-specific IgE was detected as described previously, with a slight modification [21,22]. Briefly, 96-well microtiter plates (COSTAR, NY) were coated with 20 μg/ml OVA (Sigma), the serum sample was diluted 1:20, and antibody detection was performed using biotinylated rat anti-mouse IgE (1:5000, eBioscience) at 37 °C for 1 h. Total IgE was measured according to standard ELISA procedures using rat anti-mouse IgE (1:5000, eBioscience) as the primary antibody. 2.8. RNA isolation and RT-PCR analysis Total RNA was extracted from liquid nitrogen-frozen lung tissue (right lower lobe) using TRIzol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer's protocol. Total RNA (100 ng) was converted to cDNA using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany). Mouse defensin-β2 (mBD-2), eotaxin-1, and macrophage inflammatory protein 2 (MIP-2) mRNA expressions were determined via real-time PCR

34

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

Fig. 1. Sensitization, challenge, and immunization protocols. To investigate the influence of persistent allergic inflammation on the progression of S. pneumoniae infection in mice, BALB/c mice were sensitized via i.p. injection of two doses of OVA on days 0 and 14, followed by challenge for 20 min per day from days 21 to 28 via aerosol nebulization of OVA. At 3 days before the end of OVA challenge, the mice were intranasally (i.n.) infected with 106 CFU of S. pneumoniae. To determine the effect of rIL-17 inoculation on airway inflammation in S. pneumoniaeinfected OVA-allergic mice, 2 μg of rIL-17A (n = 8 mice) were inoculated i.n. per day from days 22 to 24. The control group received PBS with 1% normal mouse serum in the same manner.

analyses using SYBR Green (TOYOBO, Osaka, Japan). The specific primer pairs used were as follows: defensin-β2: 5′-CTGATATGCTGCCTCCTTTT CT-3′ (sense) and 5′-CTTGCAACAGGGGTTCTTCTC-3′ (antisense); eotaxin-1: 5′-CTTCTATTCCTGCTGCTCACG-3′ (sense) and 5′-TTGTAGCT CTTCAGTAGTGTGTTGG-3′ (antisense); MIP-2: 5′-CACCAACCACCAGG CTACAGGG-3′ (sense) and 5′-GGGCTTCAGGGTCAAGGCAAAC-3′ (antisense); and Gapdh: 5′-TGCAGTGGCAAAGTGGAGATTGTTG-3′ (sense) and 5′-GGTCTCGCTCCTGGAAGATGGTGAT-3′ (antisense). The samples were amplified for 40 cycles according to the following program: 94 °C for 5 min, followed by 40 cycles of 94 °C for 15 s, 57.6 °C for 15 s, and 72 °C for 45 s. Signal detection was performed using an Applied Biosystems 7700 Sequence Detector. Gapdh was used as an internal control, and mRNA expression was normalized to Gapdh.

2.9. Statistical analysis The data are expressed as the means ± SEM. Differences were analyzed via Student's t test or one-way ANOVA followed by Dunn's post-test. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA), and differences were considered to be significant at p b 0.05.

3. Results 3.1. Persistent allergic airway inflammation disrupts IL-17 expression in response to S. pneumonia infection To determine whether allergic airway inflammation modulates cytokine expression in response to S. pneumoniae infection, we developed a model of persistent allergic airway inflammation followed by S. pneumoniae airway infection based on OVA sensitization and challenge. At 72 h post-infection, the highly expressed Th2-related cytokine IL-4 was decreased in the BALF after S. pneumoniae infection, but its expression remained higher than that in S. pneumoniae-infected nonsensitized mice. IFN-γ expression in the BALF in response to S. pneumoniae infection was reduced due to pre-existing allergic airway inflammation(Fig. 2A). To assess the impact of airway allergic inflammation on IL-17 expression, we compared IL-17 levels between OVAallergic mice and control mice after S. pneumoniae infection. Subjected to S. pneumoniae infection, the non-sensitized mice expressed a considerably higher concentration of IL-17 in the BALF (≈2.5-fold increase) and serum (≈ 2.8-fold increase) than the allergic mice (≈ 1.1-fold increase in BALF and ≈1.6-fold increase in serum), (Fig. 2B). To examine whether insufficient IL-17 secretion upon S. pneumoniae infection

Fig. 2. OVA-induced allergic airway inflammation disrupts IL-17 modulated host defense against S. pneumoniae. The levels of cytokines in S. pneumoniae-infected mice with or without OVA-induced airway inflammation were measured via ELISA. The mice with or without airway inflammation were used as controls. (A) IL-4 and IFN-γ secretion in the BALF. (B) IL17A secretion in the BALF or serum. (C) IL-6 secretion in the BALF. (D) IL-23 secretion in the BALF. (*P b 0.05, **P b 0.01, and ***P b 0.001, n = 8).

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

via the IL-23 or IL-6 pathway, IL-6 and IL-23 expressions in BALF were assessed via ELISA. The results revealed that, compared with control mice, infection stimulated IL-6 in allergic mice was sufficient (1.59-fold vs. 1.22-fold, Fig. 2C), whereas IL-23 expression was significantly suppressed by allergic inflammation (1.47-fold vs. 7.65-fold, Fig. 2D). 3.2. Exogenous IL-17 intranasal administration protects allergic mice from S. pneumoniae infection To address whether allergic airway inflammation disrupted the IL-17 expression will leading to more-severe S. pneumoniae infection in allergic mice, the airway bacterial burden and lung histopathology were evaluated. At 72 h post-infection, we detected a significant increase in the bacterial burden in the BALF from S. pneumoniae-infected OVA-sensitized mice compared with that from control S. pneumoniae-infected mice (Fig. 3A). Histological assessment of lung sections via H&E staining revealed that S. pneumoniae infection in mice subjected to allergic inflammation resulted in severe immune cell infiltration into the airspace and the airway lumen (Fig. 3B-c); alternatively, in control mice, after S. pneumoniae infection, neutrophil infiltration was primarily localized to the airspace (Fig. 3B-a). To determine the role of IL-17 in host defense against S. pneumonia in allergic airway, a rescue experiment using rIL-17 intranasal administration prior to S. pneumonia inoculation was performed. At 72 h after S. pneumoniae infection, the bacterial burden in the BALF from mice immunized with rIL-17 was significantly reduced compared with that from untreated mice (Fig. 3A). Compared with untreated mice, lung sections of rIL-17 inoculated mice displayed significantly decreased airway lumen mucus secretion and cell infiltration around blood vessels and bronchus (Fig. 3B). Our data strongly suggested that intranasal administration with rIL-17 promoted airway bacteria clearance in allergen sensitive mice. 3.3. Facilitating neutrophil recruitment and β-defensins expression plays a crucial role in IL-17-mediated immune response against pneumococcal infection in allergic mice Recruitment of neutrophils in the lung is one of the most important components of the innate immune responses against bacterial infection [23]. Here, we examined whether intranasal rIL-17 immunization will mediate the transmigration of neutrophils into airway during allergic inflammation combined with bacterial infection. BALF analysis revealed

35

a significant increase in neutrophils but decrease in eosinophils in the BALF from rIL-17 treated mice compared with that control mice (Fig. 4A). The chemotaxis gradient formation is the initial critical step of airway neutrophils infiltration, so we investigated the MIP-2 expression in allergic mice in response to S. pneumoniae infection. MIP-2 is belonging to the CXC chemokine family. In murine, MIP-2 is a potent neutrophil attractant and activator. MIP-2 concentration and mRNA expression was increased when allergic mice were infected with S. pneumoniae compared to that from mice without infection. MIP-2 concentration in BALF and mRNA expression in lung tissues was further enhanced by rIL-17 in nasal inoculation (Fig. 4B). The potential effect of rIL-17 treatment on the bacterial infection-mediated induction of mBD-2 expression in lungs pulmonary was investigated via IHC and real-time RT-PCR analysis. Compared with untreated mice, the immunoreactivity for mouse mBD-2 in S. pneumoniae-infected lung tissue sections was significantly increased in rIL-17 treated allergic mice with pneumonia, predominantly in the surface epithelium of the bronchi (Fig. 4D). Likewise, RT-PCR analysis revealed that the bacterial infection-mediated increase in the mRNA expression of mBD-2 in lung tissues from the rIL-17 treated mice were higher than that from the untreated allergic mice (Fig. 4C). These results indicate that IL-17 increase in the airway promotes MIP-2 and mBD-2 expression, which in turn enhances neutrophil recruitment and anti-bacterial immune response. 3.4. Exogenous IL-17 does not exacerbate pulmonary eosinophil chemokine and IgE expressions Although interleukin-17 exerts a host-defensive role in protecting the host from infection, and promotes inflammatory pathology in the neutrophil-predominant asthma, the contributions of IL-17 to the outcome of allergic airway inflammation remain controversial [24–26]. To determine whether intranasal rIL-17 exerts beneficial or harmful effects on the airway allergic inflammation, the levels of total serum IgE and OVA-specific IgE were determined via ELISA and the expressions of eotaxin-1 mRNA were assayed via real-time RT-PCR. The elevated levels of both total and OVA-specific IgE antibodies in the allergic mice were markedly reduced in S. pneumoniae-infected sensitized mice (Fig. 5A). Consistent with the decreased IgE levels, eotaxin-1, also called CCL11, a major chemokine for eosinophils, its mRNA expression in lungs was significantly decreased in S. pneumoniae-infected mice compared with allergic mice without respiratory infection(Fig. 5B). No significant

Fig. 3. Exogenous IL-17 intranasal administration protects allergic mice from S. pneumoniae infection. (A) rIL-17 vaccination reduced the bacterial burden (CFU) in OVA-sensitive and S. pneumoniae-infected mice. The bacterial burdens were calculated using serial dilutions seeded on blood agar plates as described in section 2. (B) 3 days after S. pneumoniae infection, paraformaldehyde-fixed lung tissue sections were stained with HE to visualize cell recruitment and inflammatory lesions. (a) The mice without OVA-induced airway inflammation. (b) The mice without OVA-induced airway inflammation received rIL-17 treatment. (c) The mice with OVA-induced airway inflammation. (b) The mice with OVA-induced airway inflammation received rIL-17 treatment. (Scale bars = 100 μm, 400×) (*P b 0.05, **P b 0.01, and ***P b 0.001, n = 8).

36

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

Fig. 4. IL-17 enhances neutrophil recruitment and anti-bacterial peptide expression in OVA-sensitive mice with S. pneumoniae-infection. (A) Cell counts and differentiation in the BALF. rIL17 inoculation enhances neutrophil recruitment but reduces eosinophil recruitment in the BALF. The cells were pelleted onto glass slides, stained with Wright Giemsa stain, and observed under a microscope. The numbers of total cells and of different cell types are presented as the means ± SEM. (B) The expression levels of mouse MIP-2 in the BALF and lungs were measured via ELASA or quantitative RT-PCR. (C) The expression levels of mBD-2 in the lungs were measured via quantitative RT-PCR. Relative gene expression (ratio) was determined using Gapdh as a reference gene and showed as fold increase relative to control. (D). IHC for mBD-2 in bronchial sections of OVA-sensitized (a), S. pneumoniae-infected-OVA sensitized (b) and S. pneumoniae-infected-OVA sensitized mice received rIL-17 treatment (c) were obtained from paraformaldehyde-fixed, paraffin-embedded lung tissue that were prepared and stained using an anti- mBD-2 antibody (Scale bars = 100 μm, 400× n = 8); (*P b 0.05, **P b 0.01 and ***P b 0.001).

influences of rIL-17 immunization on eotaxin-1 and IgE expressions were detected in lung tissues and sera. 4. Discussion S. pneumoniae, a Gram-positive coccus, is the predominant cause of acute bacterial pneumonia. In healthy adults and children, S. pneumoniae

is a transient commensal bacterium that colonizes the throat and upper respiratory tract. In contrast, in patients with asthma and other atopic conditions, S. pneumoniae often causes invasive pneumococcal disease. The risk of severe pneumococcal pneumonia among individuals with asthma is at least double that among controls [3,27,28]. Consistent with this epidemiological finding, in the present study, mice with allergic airway inflammation displayed a significantly higher bacterial burden

Fig. 5. Influence of exogenous rIL-17 immunization on eotaxin-1 and IgE expressions. (A) The total IgE (left panel) and OVA-specific IgE (right panel) expression levels in sera were measured via ELISA. These measurements were performed at A405 or A450 and are expressed as the means ± SEM (n = 8). (B) mRNA expression of eotaxin-1 in lung tissues. mRNA expression was analyzed via real-time RT-PCR. The relative expression levels of the target genes were normalized to those of Gapdh. The results are expressed as the fold-change in mRNA expression relative to the corresponding expression level in normal control mice. The results are presented as the means ± SEM. (n = 8). Significant differences are denoted as *P b 0.05, **P b 0.01, or ***P b 0.001.

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

than control mice. It is likely that the interaction between host innate immunity and the adaptive immune response to bacterial infection is disturbed in the OVA-sensitized and -challenged mice. Beisswenger's research showed that the antimicrobial activity of the airway epithelium is inhibited by Th2-related cytokines. When airway epithelial cells were incubated in Th2-related cytokines, the mRNA expression levels of hBD-2 were significantly suppressed [9]. In addition to S. pneumoniae, allergic airway inflammation also impaired host defenses against other bacteria, such as Mycoplasma pneumoniae and Pseudomonas aeruginosa [9,29,30]. By impairing lung IL-6 and Toll-like receptor 2 expression, the Th2-related cytokines IL-4 and IL-13 increased the M. pneumoniae burden in the airways of allergic mice [30]. Decreased lung IL-6 and IL-1 expression due to allergic inflammation has been demonstrated to impair the lung clearance of P. aeruginosa in an acute infection mouse model [9]. Although allergic airway inflammation is generally considered to increase the risk for bacterial pneumonia in humans [2–4,31], experimental studies have revealed conflicting results concerning the effect of allergic airway inflammation on bacterial infection in the lungs. Clement and colleagues previously reported that allergic airway inflammation neither enhanced nor impaired lung bacterial clearance or mortality when infected with S. pneumoniae serotype 4 [11], whereas Kang et al. showed that pre-existing allergic inflammation reduced the risk for pneumococcal pneumonia and the bacterial burden in the lung at the time of pneumonia onset [10]. Additionally, Dulek's group showed that allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality [29]. In present study, we revealed that persistent allergic airway inflammation disrupts IL-17 expression in response to S. pneumonia infection. The elevation of IL-17 in allergic airway is insufficient upon bacterial infection, this result is similar with Juhn's report [32]. Corresponding to increased Th2-related cytokine expression and insufficient Th1 and Th17-related cytokine and antimicrobial peptide expression, mice with allergic inflammation experienced an increased bacterial burden in the airway after S. pneumoniae infection. These results suggested that persistent allergic airway inflammation impaired the host responses to S. pneumoniae infection. The interpretation of the discrepancies in the results from these different studies should consider these differences in the models used. There are several differences between their model and ours. In our mouse model, to maintain persistent allergic inflammation, OVA challenge was performed for 2 weeks and was continued until 72 h after bacterial infection; alternatively, in the 3 studies noted above, OVA challenge was performed only once before infection, and acute allergic inflammation was transiently induced by OVA challenge. Potentially, acute allergic airway inflammation may reduce the risk of bacterial pneumonia during the acute phase, while chronic allergic airway inflammation may be a risk factor for bacterial pneumonia. Our data revealed that when allergic inflammation persistently dominated the airway immune response, S. pneumoniae infection failed to promote host immunity against the microbe by insufficiently inducing the expression of IL-17 associated cytokines and chemokines and recruiting neutrophils. As a major proinflammatory cytokine, IL-17 plays an important role in the induction of neutrophil-mediated protective immune responses to S. pneumoniae infection. IL-17 promotes pneumococcal clearance by neutrophils, even in the absence of antibodies or complement; however, in the absence of neutrophils, IL-17 did not induce any pneumococcal death in vitro [14]. In the present study, the accumulation of eosinophils, but not neutrophils, was the distinctive characteristic of allergic inflammation in the lungs in our OVA-sensitized mouse model. The initial critical mechanism supporting the airway mucosal host defense against pneumococci is sufficient neutrophil recruitment to the infected airway. However, mice with allergic inflammation challenged with S. pneumoniae in our model failed to recruit a sufficient number of neutrophils to

37

their airways compared with control mice. Studies have shown that IL-4 inhibits Th17 cell differentiation and IL-17 expression [33]; in allergic airways, this impaired neutrophilia may be due to an insufficient IL-17 response. Using a combined model of allergic airway inflammation and K. pneumoniae lung infection, Dulek et al. demonstrated that allergic airway inflammation impairs the expression of IL-17A in the lungs and airway neutrophilia following challenge with Klebsiella pneumoniae [29]. In this regard, treatment with IL-17A might rescue the impaired response of neutrophils in the allergic airways, and our data confirmed this hypothesis. In the present study, via intranasal administration, exogenous IL17 aided allergic mice in eliciting an immune response against S. pneumoniae without exacerbating the pre-existing airway allergic inflammation, which was associated with increased IL-23, MIP-2 and defensin-β2 expression. In our model, OVA-allergic mice administrated with rIL-17 exhibited enhanced bacterial clearance, increased expression of cytokines and defensin-β2 and increased neutrophil recruitment in airway lumen and lung tissues in response to S. pneumoniae infection. In our previous study, with a S. pneumoniae infection murine model we demonstrated rIL-17F inoculation prior to infection significantly reduced S. pneumoniae colonization in lungs through a similar mechanism but less powerful. These studies indicate that IL-17A and IL-17F have overlapping yet distinct roles in host immune and defense mechanisms against bacterial infection [16]. Via the regulation of neutrophil recruitment and antimicrobial protein expression in the epithelium, IL-17 plays a vital role in lung mucosal immunity against bacterial infection. However, overexpression of IL-17 causing inflammation associated with neutrophil infiltration is involved in the pathogenesis of asthma [34]. IL-17A was shown to be expressed in bronchial biopsies, the BALF and sputum from patients with asthma, and increased numbers of IL-17A+ cells were detected in bronchial biopsies from mild asthmatics compared with healthy controls (reviewed in [28,35]). Although this increase was demonstrated in several studies, it remains unclear whether IL-17 (or IL-17 + cells) is (are) increased in an attempt to protect the individual or whether it might cause localized damage [28]. In allergic asthma, the inflammatory process is primarily mediated by allergen-specific Th2 lymphocytes, whereas in Th17 cell-dominant asthmatic inflammation, neutrophils contribute to inflammation more than eosinophils [35]. In our OVA-sensitized and -challenged mouse model, allergic airway inflammation is characterized by a dominant eosinophil response. Administration of rIL-17 efficiently suppressed the infiltration of eosinophils, and slightly suppressed the expression of eotaxin-1 and IgE (compared with placebo the decrease was not significant). The results indicate that the exogenous IL-17 is a negative regulator of allergic airway inflammation in this model. Schnyder and colleagues also demonstrated that neutralization of IL-17 augmented the allergic response in sensitized mice; conversely, exogenous IL-17 reduced pulmonary eosinophil recruitment and bronchial hyperreactivity [17]. In summary, our results support that IL-17-dependent responses to the S. pneumonia infection are defective in the chronic allergic airway. Insufficient Th-17 immune responses fail to provide an effective protection to against pneumococcal pneumonia. Exogenous IL-17 administration is helpful for rescue the impaired immune response to bacterial infection in the allergic airways. However, further investigation must be conducted to determine the precise mechanisms underlying the interaction between allergic asthma and bacterial infection and the different effects of exogenous IL-17 administration on the pathogenesis of non-atopic and atopic asthma.

Conflict of interest statement None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

38

S. Guo et al. / International Immunopharmacology 31 (2016) 32–38

Acknowledgments The authors thank Dr. Pin Li (Shanghai Jiao Tong University, China) for assistance in histology and Dr. Xiaoping Jing (Shanghai Jiao Tong University, China) for technical assistance. This research was partially supported by a grant (12ZR1425700) from the National Natural Science Foundation of China and a grant (No. 10ZR1423000) from the Technology Commission of Shanghai Municipality. References [1] K.L. O'Brien, L.J. Wolfson, J.P. Watt, E. Henkle, M. Deloria-Knoll, N. McCall, et al., Burden of disease caused by streptococcus pneumoniae in children younger than 5 years: global estimates, Lancet 374 (2009) 893–902. [2] M. Habibzay, G. Weiss, T. Hussell, Bacterial superinfection following lung inflammatory disorders, Future Microbiol 8 (2013) 247–256. [3] T.R. Talbot, T.V. Hartert, E. Mitchel, N.B. Halasa, P.G. Arbogast, K.A. Poehling, et al., Asthma as a risk factor for invasive pneumococcal disease, N. Engl. J. Med. 352 (2005) 2082–2090. [4] Y.J. Juhn, H. Kita, B.P. Yawn, T.G. Boyce, K.H. Yoo, M.E. McGree, et al., Increased risk of serious pneumococcal disease in patients with asthma, J Allergy Clin Immunol. 122 (2008) 719–723. [5] Y.J. Juhn, Risks for infection in patients with asthma (or other atopic conditions): is asthma more than a chronic airway disease? J. Allergy Clin. Immunol. 134 (2014) 247–257 258-9. [6] P. Klemets, O. Lyytikainen, P. Ruutu, J. Ollgren, T. Kaijalainen, M. Leinonen, et al., Risk of invasive pneumococcal infections among working age adults with asthma, Thorax 65 (2010) 698–702. [7] S. Mrabet-Dahbi, M. Maurer, Does allergy impair innate immunity? Leads and lessons from atopic dermatitis, Allergy 65 (2010) 1351–1356. [8] P.F. Cheung, C.K. Wong, C.W. Lam, Molecular mechanisms of cytokine and chemokine release from eosinophils activated by IL-17A, IL-17F, and IL-23: implication for Th17 lymphocytes-mediated allergic inflammation, J. Immunol. 180 (2008) 5625–5635. [9] C. Beisswenger, K. Kandler, C. Hess, H. Garn, K. Felgentreff, M. Wegmann, et al., Allergic airway inflammation inhibits pulmonary antibacterial host defense, J. Immunol. 177 (2006) 1833–1837. [10] C.I. Kang, M.S. Rouse, R. Patel, H. Kita, Y.J. Juhn, Allergic airway inflammation and susceptibility to pneumococcal pneumonia in a murine model with real-time in vivo evaluation, Clin. Exp. Immunol. 156 (2009) 552–561. [11] C. Clement, M. Tuvim, C. Evans, D. Tuvin, B. Dickey, S. Evans, Allergic lung inflammation alters neither susceptibility to streptococcus pneumoniae infection nor inducibility of innate resistance in mice, Respir. Res. 10 (2009) 1–8. [12] G. Matsuzaki, M. Umemura, Interleukin-17 as an effector molecule of innate and acquired immunity against infections, Microbiol. Immunol. 51 (2007) 1139–1147. [13] J.J. Yu, S.L. Gaffen, Interleukin-17: a novel inflammatory cytokine that bridges innate and adaptive immunity, Front. Biosci. 13 (2008) 170–177. [14] Y.J. Lu, J. Gross, D. Bogaert, A. Finn, L. Bagrade, Q. Zhang, et al., Interleukin-17A mediates acquired immunity to pneumococcal colonization, PLoS Pathog. 4 (2008) e1000159. [15] K. Eyerich, D. Pennino, C. Scarponi, S. Foerster, F. Nasorri, H. Behrendt, et al., IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response, J. Allergy Clin. Immunol. 123 (2009) 59–66. [16] L. Chen, S. Guo, L. Wu, C. Hao, W. Xu, J. Zhang, Effects of recombinant IL-17F intranasal inoculation against Streptococcus pneumoniae infection in a murine model, Biotechnol. Appl. Biochem. (2014).

[17] S. Schnyder-Candrian, D. Togbe, I. Couillin, I. Mercier, F. Brombacher, V. Quesniaux, et al., Interleukin-17 is a negative regulator of established allergic asthma, J. Exp. Med. 203 (2006) 2715–2725. [18] M.W. Kinyanjui, J. Shan, E.M. Nakada, S.T. Qureshi, E.D. Fixman, Dose-dependent effects of IL-17 on IL-13-induced airway inflammatory responses and airway hyperresponsiveness, J. Immunol. 190 (2013) 3859–3868. [19] K. Sun, S.L. Salmon, S.A. Lotz, D.W. Metzger, Interleukin-12 promotes gamma interferon-dependent neutrophil recruitment in the lung and improves protection against respiratory Streptococcus pneumoniae infection, Infect. Immun. 75 (2007) 1196–1202. [20] G. Sheng, F. Xiao-yong, H. Chun-li, M.A. Hui, C. Ling, Z. Jian-hua, Prokaryotic expression of mouse interleukin 17A and its ability of stimulating RAW264.7 cell inflammatory factors expression, Chin. J. Microbiol. Immunol. 30 (2010) 635–640. [21] A. de Vries, L. Hazlewood, P.M. Fitch, J.R. Seckl, P. Foster, S.E. Howie, High-fat feeding redirects cytokine responses and decreases allergic airway eosinophilia, Clin. Exp. Allergy 39 (2009) 731–739. [22] J. Xu, W. Dai, Z. Wang, B. Chen, Z. Li, X. Fan, Intranasal vaccination with chitosanDNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by Streptococcus pneumoniae, Clin. Vaccine Immunol. 18 (2011) 75–81. [23] A. Craig, J. Mai, S. Cai, S. Jeyaseelan, Neutrophil recruitment to the lungs during bacterial pneumonia, Infect. Immun. 77 (2009) 568–575. [24] B.N. Lambrecht, H. Hammad, The immunology of asthma, Nat. Immunol. 16 (2015) 45–56. [25] R.M. Onishi, S.L. Gaffen, Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease, Immunology 129 (2010) 311–321. [26] B.P. Tian, W. Hua, L.X. Xia, Y. Jin, F. Lan, J.J. Lee, et al., Exogenous interleukin-17A inhibits eosinophil differentiation and alleviates allergic airway inflammation, Am. J. Respir. Cell Mol. Biol. 52 (2015) 459–470. [27] J.A. Jung, H. Kita, B.P. Yawn, T.G. Boyce, K.H. Yoo, M.E. McGree, et al., Increased risk of serious pneumococcal disease in patients with atopic conditions other than asthma, J. Allergy Clin. Immunol. 125 (2010) 217–221. [28] D.M. Bullens, A. Decraene, S. Seys, L.J. Dupont, IL-17A in human respiratory diseases: innate or adaptive immunity? Clinical implications, Clin. Dev. Immunol. 2013 (2013) 840315. [29] D.E. Dulek, D.C. Newcomb, K. Goleniewska, J. Cephus, W. Zhou, S. Reiss, et al., Allergic airway inflammation decreases lung bacterial burden following acute Klebsiella pneumoniae infection in a neutrophil- and CCL8-dependent manner, Infect. Immun. 82 (2014) 3723–3739. [30] Q. Wu, R.J. Martin, S. Lafasto, B.J. Efaw, J.G. Rino, R.J. Harbeck, et al., Toll-like receptor 2 down-regulation in established mouse allergic lungs contributes to decreased mycoplasma clearance, Am. J. Respir. Crit. Care Med. 177 (2008) 720–729. [31] U. Jounio, R. Juvonen, A. Bloigu, S. Silvennoinen-Kassinen, T. Kaijalainen, H. Kauma, et al., Pneumococcal carriage is more common in asthmatic than in non-asthmatic young men, Clin. Respir. J. 4 (2010) 222–229. [32] H. Zhao, C.I. Kang, M.S. Rouse, R. Patel, H. Kita, Y.J. Juhn, The role of IL-17 in the association between pneumococcal pneumonia and allergic sensitization, Int. J. Microbiol. 2011 (2011) 709509. [33] L.A. Cooney, K. Towery, J. Endres, D.A. Fox, Sensitivity and resistance to regulation by IL-4 during Th17 maturation, J. Immunol. 187 (2011) 4440–4450. [34] Y. Iwakura, S. Nakae, S. Saijo, H. Ishigame, The roles of IL-17A in inflammatory immune responses and host defense against pathogens, Immunol. Rev. 226 (2008) 57–79. [35] L. Cosmi, F. Liotta, E. Maggi, S. Romagnani, F. Annunziato, Th17 cells: new players in asthma pathogenesis, Allergy 66 (2011) 989–998.