Pulmonary Colonization Resistance to Pathogens via Noncanonical Wnt and Interleukin-17A by Intranasal pep27 Mutant Immunization

Pulmonary Colonization Resistance to Pathogens via Noncanonical Wnt and Interleukin-17A by... Abstract Background Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Methods Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. Results In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Conclusions Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways. Δpep27 immunization, IL-17, Streptococcus pneumoniae, Wnt signaling Streptococcus pneumoniae infection is responsible for high morbidity and mortality worldwide [1], and it causes a variety of pneumococcal diseases, such as pneumonia, meningitis, otitis media, and sepsis [2]. There are 2 ways to protect against pneumococcal diseases: one is vaccination, which is the most effective way to prevent disease; the other is the inhibition of colonization at the first step of pneumococcal disease in the respiratory tract [3]. In previous studies, we demonstrated that immunization with an attenuated S. pneumoniae vaccine, Δpep27, confers protection against various pneumococcal serotypes by reducing colonization via immunoglobulin (Ig)A and interleukin (IL)-17A induction [4]. The Th17 response to pneumococcal colonization can prevent adherence of pneumococci on the nasopharynx [5], and together with IL-17A it inhibits mucosal colonization [6, 7]. Moreover, CD4 Th17-secreted IL-17 leads to recruitment of neutrophils [8] that directly remove infecting bacteria as part of the innate immunity in the lung [9]. Wnt signals have been studied as a part of the transduction pathways that determine cell fate or regulate cell movement [10]. Wnt signals can be divided into the canonical pathway, which is regulated mainly by β-catenin, and the noncanonical pathway, which is mediated by calcium β-catenin independently in early development [11]. In particular, nuclear factor of activated T cell (NFAT), which is a downstream molecule of the noncanonical Wnt signaling pathway, plays a critical role in T-cell development and the immune response [12]. Moreover, NFAT regulates the accumulation of neutrophils in the lung and during systemic inflammation [13], indicating that NFAT might be a beneficial target to defend against respiratory infection and sepsis. When T-cell receptors are stimulated, various transcription factors and cytokines are induced via the entry of calcium into the cytoplasm, including the calcineurin/NFAT pathways [14]; that is, NFAT binds to the IL17a promoter, followed by induction of IL-17A [12, 15]. In this study, we demonstrated that Δpep27 immunization induced NFAT and, subsequently, IL-17A secretion. Inhibition of NFAT consistently reversed the colonization inhibition by Δpep27 immunization, with reduced IL-17A secretion in the lung. Thus, NFAT-induced IL-17A contributes to the inhibition of colonization effected by Δpep27 immunization. MATERIALS AND METHODS Bacterial Strains Streptococcus pneumoniae serotype 2 (D39; NCTC7466) and its isogenic Δpep27 [4] were cultured on THY medium (Todd-Hewitt Broth with 0.5% Yeast Extract) with 5% sheep blood agar plates at 37°C overnight and then grown in THY broth with 0.5% yeast extract (BD) at 37°C. Bacterial cultures with an OD550 of 0.3 (1 × 108 colony-forming units [CFUs]/mL) were incubated and diluted with phosphate-buffered saline for infection or immunization studies. Streptococcus aureus (American Type Culture Collection [ATCC] 25923) and Klebsiella pneumoniae (ATCC 9997) were purchased from the Korean Culture Center of Microorganisms (Seoul, Korea), and cultured in THY agar supplemented with defibrinated sheep blood at 37°C overnight, then subsequently inoculated to THY broth and incubated at 37°C until the OD550 reached 0.5. Ethics Statement Four-week-old male BALB/c (Orient, Korea) were used in the infection experiments. All animal procedures were approved by the Sungkyunkwan University Animal Ethical Committee and were carried out in accordance with the guidelines of the Korean Animal Protection Law. Vaccination and Sample Collection For vaccination, mice were immunized with Δpep27 (1 × 107 to 1 × 108 CFUs, 10 μL) 3 times every 2 weeks via intranasal route. Seven days after the last immunization, serum, bronchoalveolar lavage fluid (BALF), lung, and spleen were collected [4]. Cytokine Determination The concentration of IL-17 in BALF, serum, and splenocytes were determined using an IL-17A enzyme-linked immunosorbent assay kit (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. Antibody Treatment For antibody-mediated neutralization, antibodies (Bio X Cell) were administrated via the intraperitoneal (i.p.) route at day +3 and +6 after immunization, with 200 μg of anti-IL-17A or mouse IgG1 antibody as a control [16]. Seven days postimmunization, the mice were treated with antibodies again and subsequently challenged with 2 × 108 CFUs of the D39 strain intranasally. Inhibition of Wnt Signaling The noncanonical Wnt inhibitor, 11R-VIVIT, was purchased from Tocris Bioscience, and an inactive control, 11R-VEET (RRRRRRRRRRRGGGMAGPPHIVEETGPHVI), was synthesized by Cosmogenetech (Korea). Mice received 10 mg/kg VEET or VIVIT i.p. once daily for 2 days [17]. Samples were collected 6 hours postinjection [17]. Ribonucleic Acid Isolation Total ribonucleic acid (RNA) was extracted from lungs using the TRIzol reagent (Invitrogen). The quality of the RNA was determined using an Agilent 2100 bioanalyzer with the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands), and RNA was quantified using an ND-2000 Spectrophotometer (Thermo Inc.). High-Throughput Sequencing and Transcriptomic Analyses To construct the sequencing libraries, 500 ng of total RNA was used for complementary deoxyribonucleic acid (cDNA) synthesis. The RNA libraries were then constructed using a SENSE 3′ messenger RNA (mRNA)-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria), according to the manufacturer’s protocol. Gene expression was determined by high-throughput sequencing using the Illumina NextSeq 500 system. Differentially expressed genes were selected as those that showed a 2-fold difference (upregulated) or 0.5-fold difference (downregulated) compared with the control. Gene clustering (hierarchical clustering) and heat maps were constructed based on MeV 4.9.0. These experimental and system biology analyses (network, canonical pathway, and regulatory effect) were analyzed using Ingenuity Pathway Analysis, performed by e-Biogen (Seoul, Korea). The gene expression analyses data were deposited in the National Center for Biotechnology Information database (GEO accession number GSE93718) (http://www.ncbi.nlm.nih.gov/geo/). Flow Cytometry Lungs were harvested 1 week after the last immunization, and isolated lung cells were coincubated with a cell stimulation cocktail and protein transport inhibitor cocktail (Invitrogen, Carlsbad, CA). Subsequently, the cells were resuspended in flow cytometry staining buffer (eBioscience) and then stained with a cell surface-specific marker conjugated with a fluorochrome. Fluorescein isothiocyanate-labeled anti-mouse CD4 and phycoerythrin-labeled anti-mouse CD3e were purchased from eBioscience. For intracellular staining, cells stained with the surface marker were fixed and permeabilized with an intracellular fixation and permeabilization buffer set (eBioscience) followed by allophycocyanin (APC)-labeled anti-mouse IL-17A and APC-labeled Rat IgG2a K isotype control (eBioscience). The gating of lymphocytes based on forward versus side scatter was aimed at selecting CD4+CD3e+ cells. The selected CD4+CD3e+ population was further gated for IL-17A+-positive response (Supplementary Figure 1). Flow cytometry analysis was performed using the BD FACSCanto II (BD Biosciences) using FlowJo version 10.1 software (Flowjo, LLC). Quantitative Real-Time Polymearse Chain Reaction Total RNA was isolated from the lung using an RNeasy plus mini kit (QIAGEN, Hilden, Germany), and cDNA was synthesized using EcoDry Premix kit (Takara, Japan). Quantitative real-time polymearse chain reaction (PCR) was performed according to the manufacturer’s instructions (Applied Biosystems). The reverse-transcription PCR conditions were as follows: 95°C for 15 seconds, 40 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; followed by melting curve analysis comprising 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The gene-specific primers are Tgfb1 (forward: ATG CTA AAG AGG TCA CCC GC; reverse: TGC TTC CCG AAT GTC TGA CG), IL-6 (forward: CCT CTG GTC TTC TGG AGT AC; reverse: GGA AAT TGG GGT AGG AAG G), and IL-17A (forward: CTC AAG CTC AGC GTG TCC A; reverse: ATC AGG GTC TTC ATT GCG GT), and glyceraldehyde 3-phosphate dehydrogenase (forward: TCC ACG ATG CCA AAG TTG TC; reverse: TGC ATC CTG CAC CAC CAA) served as control. Hematoxylin and Eosin Staining Isolated lungs were fixed in 10% neutral-buffered formalin. Sagittally sliced lung samples were embedded with paraffin and cut into 4-μm sections (Histoire, Ansan, Korea). The images of lungs stained with hematoxylin and eosin (H&E) were captured and analyzed by KNOTUS (Guri, Korea). Statistical Analysis Comparisons between 2 groups were performed using a Mann-Whitney U test (nonparametric). Comparisons among groups were made using one-way analysis of variance (Duncan’s method, nonparametric). Differences in median survival times between groups were analyzed using the log-rank test. Statistically significant differences were defined as *, P ≤ .05, **, P ≤ .01, and ***, P ≤ .001. RESULTS Δpep27 Immunization Induces Interleukin-17A to Protect Against Streptococcus pneumoniae Interleukin-17 produced by Th17 cells protects the host from extracellular bacterial infection [6, 18]. To investigate whether Δpep27 immunization could induce the IL-17A response in the lung, high-throughput sequencing of samples from Δpep27-immunized mouse lungs was performed. Hierarchical cluster analysis revealed that Δpep27 immunization upregulated genes related to the Th17 immune response (Figure 1A). Reverse-transcription PCR analysis confirmed significant induction of transforming growth factor beta 1 (Tgfβ1) and IL-6, which contributed to the differentiation of T cells into Th17 cells and subsequent IL-17A production [19]. In addition, ingenuity pathway analysis showed that the IL-6 in the lung centered for Th17 differentiation in the immune response after Δpep27 immunization (Supplementary Figure 2). Consistent with the RNA-sequencing data, the IL-17A protein level also increased significantly in serum, splenocytes, and BALF after Δpep27 immunization (Figure 1B). Moreover, the population of CD4+IL-17A+ cells in the lung was highly increased in the Δpep27-immunized group compared with that in nonimmunized mice (Figure 1C). These results indicated that Δpep27 immunization activated the expression of a variety of genes and proteins related to IL-17A production. Figure 1. View largeDownload slide Δpep27 immunization induces interleukin (IL)-17A. Mice (n = 3) were immunized intranasally with Δpep27 at 2-week intervals 3 times. Seven days after the last immunization, lung messenger ribonucleic acid (mRNA) was used for high-throughput sequencing. (A) Cluster analysis of gene expression identified upregulated (>2 vs control) and downregulated (<0.5 vs control) genes in the lung by Δpep27 immunization (A, left). Hierarchical clustering of Th17-related gene expression in Δpep27 immunized lungs (A, right). (B) The expression of Tgfβ1, IL-6, and IL-17A mRNA or induction of IL-17A in serum, splenocytes, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay, respectively. The experiment was performed 3 times independently. (C) The population of CD4 + IL-17A + cells was detected by flow cytometry. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed by the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 1. View largeDownload slide Δpep27 immunization induces interleukin (IL)-17A. Mice (n = 3) were immunized intranasally with Δpep27 at 2-week intervals 3 times. Seven days after the last immunization, lung messenger ribonucleic acid (mRNA) was used for high-throughput sequencing. (A) Cluster analysis of gene expression identified upregulated (>2 vs control) and downregulated (<0.5 vs control) genes in the lung by Δpep27 immunization (A, left). Hierarchical clustering of Th17-related gene expression in Δpep27 immunized lungs (A, right). (B) The expression of Tgfβ1, IL-6, and IL-17A mRNA or induction of IL-17A in serum, splenocytes, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay, respectively. The experiment was performed 3 times independently. (C) The population of CD4 + IL-17A + cells was detected by flow cytometry. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed by the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Next, to determine whether Δpep27 immunization-induced IL-17A plays an important role in protection from pneumococcal infection, we performed a colonization experiment after IL-17A neutralization using an anti-IL-17A antibody. Interleukin-17A neutralization markedly decreased the IL-17A level in BALF compared with an isotype control in the Δpep27-immunized group of mice (Figure 2A). To confirm whether IL-17A neutralization could reverse the colonization inhibition by Δpep27 immunization, IL-17A was neutralized and then the mice were challenged with D39, followed by colony counting. The results showed that IL-17A neutralization increased the bacterial CFU by approximately 100- and 10-fold in the lung and spleen, respectively, compared with the isotype control group 24 hours postinfection. This indicated that IL-17A neutralization reversed the colonization inhibition by Δpep27 immunization. Moreover, 48 hours postinfection, IL-17A neutralization increased the bacterial CFU by approximately 104-fold in the lung than in the no bacterial CFU by Δpep27 immunization (isotype control) (Figure 2B). In addition, the histology of the lung tissue showed increased inflammation predominantly in the bronchioli and alveoli 48 hours postinfection in the nonimmunized group compared with that in the immunized group (Figure 2C). Interleukin-17A neutralization showed attenuated neutrophil infiltration and bacterial clearance (Figure 2B and C). These results demonstrated that Δpep27 immunization could protect the host from pneumococcal infection using IL-17A to remove bacteria. Figure 2. View largeDownload slide Interleukin (IL)-17A provides protection after Δpep27 immunization. Mice (n = 4) were immunized intranasally with Δpep27 3 times at 2-week intervals. Interleukin-17A was neutralized by injecting an anti-IL-17A antibody on day 3 and 6 postimmunization, or immunoglobulin (Ig)G1 was injected as isotype control antibody. (A) Seven days after the last immunization, the IL-17A level in bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay. The experiment was performed 3 times independently. (B) Seven days after the last immunization, mice (n = 4) were given injections with anti-IL-17A or IgG1 followed by challenge with D39 (2 × 108 colony-forming units [CFU]). Bacterial CFU in blood, lung, and spleen were determined 24 and 48 hours postinfection. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. (C) Histopathology of lung tissue before infection or after infection were shown at 10× magnification. Scale bar, 200 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 2. View largeDownload slide Interleukin (IL)-17A provides protection after Δpep27 immunization. Mice (n = 4) were immunized intranasally with Δpep27 3 times at 2-week intervals. Interleukin-17A was neutralized by injecting an anti-IL-17A antibody on day 3 and 6 postimmunization, or immunoglobulin (Ig)G1 was injected as isotype control antibody. (A) Seven days after the last immunization, the IL-17A level in bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay. The experiment was performed 3 times independently. (B) Seven days after the last immunization, mice (n = 4) were given injections with anti-IL-17A or IgG1 followed by challenge with D39 (2 × 108 colony-forming units [CFU]). Bacterial CFU in blood, lung, and spleen were determined 24 and 48 hours postinfection. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. (C) Histopathology of lung tissue before infection or after infection were shown at 10× magnification. Scale bar, 200 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Δpep27 Immunization Induces Noncanonical Wnt Signaling Nuclear factor of activated T cell binds to Rorc (encoding RORγt) and IL17a (encoding IL-17A) promoters, leading to the activation of Th17 responses [12]. Ribonucleic acid-sequencing data in the lung revealed upregulation of noncanonical Wnt genes (Figure 3). Nfatc2/Nfat1 was markedly activated until the second Δpep27 immunization and Calm, encoding calmodulin, as a Ca2+ effector protein that ultimately leads to activation of NFAT followed by calcineurin-NFAT interaction [20] which showed significantly increased expression (Figure 3A). In addition, high-throughput sequencing data showed that 3 Δpep27 immunizations induced noncanonical Wnt pathway ligands such as Wnt4, 5b, 7a, and 7b [21] (Figure 3A). Thus, Δpep27 immunization induced noncanonical Wnt signaling. Figure 3. View largeDownload slide Δpep27 immunization induces noncanonical Wnt signaling. Mice (n = 3) were immunized intranasally with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, messenger ribonucleic acid from lung was isolated. (A) High-throughput sequencing identified up- and downregulated genes in the lung, especially genes cluster related to Wnt signal. (B) The expression of Calcineurin A and nuclear factor of activated T cells (NFAT), which are involved in noncanonical Wnt signals in lung, were detected by Western blotting. The experiment was performed 3 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed using the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05. Figure 3. View largeDownload slide Δpep27 immunization induces noncanonical Wnt signaling. Mice (n = 3) were immunized intranasally with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, messenger ribonucleic acid from lung was isolated. (A) High-throughput sequencing identified up- and downregulated genes in the lung, especially genes cluster related to Wnt signal. (B) The expression of Calcineurin A and nuclear factor of activated T cells (NFAT), which are involved in noncanonical Wnt signals in lung, were detected by Western blotting. The experiment was performed 3 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed using the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05. Next, the regulation of NFAT1 and NFAT2 expression by calcium/calcineurin [22] was analyzed to identify whether Δpep27 immunization-induced Wnt signals could activate NFAT expression. Our results showed that the Calcineurin A and NFAT1 induction-dependent Th1 [23] and Th17 cell response [24] were induced, whereas the NFAT2 induction-dependent Th2 cell response [25] was decreased in the lung (Figure 3B). Nuclear Factor of Activated T-Cell Inhibition Reversed Colonization Inhibition To verify NFAT-dependent IL-17A secretion, we inhibited NFAT and then checked for impaired IL-17A secretion followed by increased colonization. Mice were treated with an NFAT inhibitor (VIVIT) and the IL-17A level in BALF, and NFAT1 expression and the bacterial CFU in the lung were determined before D39 challenge. The NFAT inhibition diminished (1) IL-17A in BALF and (2) NFAT1 expression and the population of CD4+IL-17A+ cells in the Δpep27 immunization group (Figure 4A). Six hours postinfection with D39, NFAT inhibition significantly decreased the IL-17A level in BALF and increased the bacterial load in lung significantly in the Δpep27 immunization group (Figure 4B). Consistently, NFAT inhibition 6 hours postinfection with D39 in the Δpep27 immunization group caused an increase in inflammation compared with the inert NFAT control (VEET), as demonstrated by H&E staining of lung tissue (Figure 4C). Moreover, even 1 month after the last immunization, the Δpep27 immunization group successfully maintained high IL-17A and NFAT1 levels, and it showed suppressed colonization after D39 challenge. Conversely, NFAT inhibition reversed these results (Figure 5). Thus, activation of noncanonical Wnt signals by Δpep27 immunization could lead to protection against pneumococcal infection by inducing IL-17A at the early stage of infection. Figure 4. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in lung. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) Before D39 challenge, interleukin (IL)-17A in the bronchoalveolar lavage fluid (BALF) (A; left), NFATc2/NFAT1 expression in lung (A, middle), and CD4 + IL-17A cell population (A, right) were determined by an enzyme-linked immunosorbent assay, Western blot (WB), and flow cytometry, respectively. (B) The IL-17A level in BALF and the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. (C) Hematoxylin-eosin staining of lung tissue before or after D39 infection was shown at 10× magnification. Scale bar, 100 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance (ANOVA); ***, P < .001, **, P < .01, and *, P < .05. Figure 4. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in lung. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) Before D39 challenge, interleukin (IL)-17A in the bronchoalveolar lavage fluid (BALF) (A; left), NFATc2/NFAT1 expression in lung (A, middle), and CD4 + IL-17A cell population (A, right) were determined by an enzyme-linked immunosorbent assay, Western blot (WB), and flow cytometry, respectively. (B) The IL-17A level in BALF and the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. (C) Hematoxylin-eosin staining of lung tissue before or after D39 infection was shown at 10× magnification. Scale bar, 100 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance (ANOVA); ***, P < .001, **, P < .01, and *, P < .05. Figure 5. View largeDownload slide Interluekin (IL)-17A inhibits Streptococcus pneumoniae colonization at 1 month postimmunization via noncanonical Wnt signaling. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. One month after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) The IL-17A level in bronchoalveolar lavage fluid (BALF) and (B) the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05. Figure 5. View largeDownload slide Interluekin (IL)-17A inhibits Streptococcus pneumoniae colonization at 1 month postimmunization via noncanonical Wnt signaling. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. One month after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) The IL-17A level in bronchoalveolar lavage fluid (BALF) and (B) the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05. Protective Role of Interluekin-17A Against Other Bacteria via Noncanonical Wnt Signaling Interleukin-17A plays a critical role in protection against Gram-positive S. aureus and Gram-negative K. pneumoniae [26, 27]. Therefore, if IL-17A provides protection from these bacteria, NFAT inhibition could reverse the colonization inhibition. To test this hypothesis, Δpep27-immunized mice were treated with an NFAT inhibitor and subsequently challenged with either S. aureus or K. pneumoniae, followed by determining the bacterial load. The results revealed that in the Δpep27-immunized group, the NFAT inhibitor (VIVIT) reversed the colonization inhibition back to the level of the nonimmunized group, whereas treatment with the inert NFAT control (VEET) did not increase the bacterial load significantly (Figure 6). In the Δpep27 immunization group, VIVIT treatment increased the bacterial CFUs by approximately 2-fold and 4-fold compared with those in the VEET-treated control group 6 hours after K. pneumoniae and S. aureus infection, respectively (Figure 6). Consistently, NFAT inhibition decreased the IL-17A level significantly in BALF after challenge with either K. pneumoniae or S. aureus (Figure 6). These results demonstrated that IL-17A provides protection from these bacterial colonization. Thus, IL-17A downregulation by NFAT abolishment reverses the colonization inhibition induced by Δpep27 immunization, resulting in higher susceptibility to bacterial infection, such as by S. pneumoniae, K. pneumoniae, and S. aureus. However, 1 month after the last Δpep27 immunization, lung cells were not protected against K. pneumoniae and S. aureus infection and showed marginal increase in IL-17A level after bacterial challenge (Supplementary Figure 3). Figure 6. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in Gram (−) and (+) bacterial infection. Mice (n = 6) were immunized with Δpep27 intranasally (i.n.) 3 times at 2-week intervals. Seven days after the last immunization, the NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally. Six hours postinjection of the inhibitor, mice were challenged i.n. with (A) 6 × 106 colony-forming units [CFU] of Klebsiella pneumoniae or (B) 1 × 109 CFU of Staphylococcus aureus. The level of IL-17A in the bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay, and bacterial CFU in lung was determined 6 hours postinfection. The data are presented as mean ± standard error of the mean and performed 2 times independently. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 6. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in Gram (−) and (+) bacterial infection. Mice (n = 6) were immunized with Δpep27 intranasally (i.n.) 3 times at 2-week intervals. Seven days after the last immunization, the NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally. Six hours postinjection of the inhibitor, mice were challenged i.n. with (A) 6 × 106 colony-forming units [CFU] of Klebsiella pneumoniae or (B) 1 × 109 CFU of Staphylococcus aureus. The level of IL-17A in the bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay, and bacterial CFU in lung was determined 6 hours postinfection. The data are presented as mean ± standard error of the mean and performed 2 times independently. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. DISCUSSION Antibiotic treatment results in the clearance of susceptible intestinal microbiota and increases new infections, especially with antibiotic-resistant strains. Thus, re-establishment of a normal microbiota and subsequent colonization resistance against antibiotic-resistant infections is deemed to be a highly feasible way to eliminate potential pathogens from the intestine [28–30]. Therefore, intervention to prevent bacterial colonization or induce colonization resistance could protect against colonization by potential pathogens. Bacterial colonization in respiratory organs, such as the nasal cavity and lung, precedes diseases and occasionally results in sepsis and meningitis [31]. The first stage of respiratory infection is colonization of the nasal and pulmonary cavity; therefore, treatments should aim to reduce bacterial colonization. In a recent study, nasal colonization resistance to S. aureus was demonstrated using antibiotic-producing Staphylococcus lugdunensis strains [32]. However, antibiotic-producing strains might increase antibiotic resistance and would not be feasible for clinical implementation. An attenuated strain of Bordetella pertussis vaccine could prevent respiratory syncytial virus infection in an IL-17-dependent manner [33]. Thus, from a practical viewpoint, there is incomplete information on intranasal or respiratory resistance to respiratory bacterial colonization because of nonavailability of specific tools and methods for respiratory intervention. In the present study, we demonstrated that Δpep27 immunization could provide resistance to respiratory colonization via induction of IL-17A. Th17-deficient cells are highly vulnerable to mucosal infections by pathogens such as S aureus, Haemophilus influenzae, and S. pneumoniae [34], signifying the important role of the Th17 immune response in natural immunity. Moreover, the Th17-dependent mucosal response provides protection in the lung against Mycobacterium tuberculosis infection [35]. Interleukin-17, produced by Th17, recruits the inflammatory cells and induces the proinflammatory mediators that are required to defend against bacterial infection in the lung [18]. Immunization with the fibrinogen-binding domain of ClfA induced IL-17A and protected against S. aureus infection by enhancing neutrophil infiltration [36]. Moreover, in human lung epithelial cells, IL-17A was required for the induction of mucins and defensin-2 [37, 38]. As an antimicrobial peptide, β-defensin provides innate immunity by blocking bacteria at epithelial surfaces [39]. It is interesting to note that the RNA-sequencing data revealed a marked increase in β-defensin 1 (DefB1) mRNA expression after Δpep27 immunization (Figure 1A). Reduced IL-17A production was accompanied by a higher pneumococcal nasopharyngeal carriage compared with a high concentration of IL-17A [40]. Moreover, in infection-prone subjects, Th17-promoting cytokines rescued the reduced Th17 response to S. pneumoniae [41]. Indeed, Δpep27 immunization with neutralization of IL-17 reversed the colonization inhibition in the lung (Figure 2), suggesting that Δpep27-induced IL-17A plays a critical role in protection against pneumococcal infection. Wnt signaling is responsible for proliferation, differentiation, and migration. Moreover, activation of noncanonical Wnt signaling in the lung is critically important to maintain lung homeostasis [42]. Wnt stimulation by microbial infection has been linked to enhancement in bacterial phagocytosis; however, it promotes intracellular survival of bacteria [43] and downregulates the inflammatory response against Salmonella or Mycobacterium [44]. In contrast, Toxoplasma infection upregulates proinflammatory cytokines via Wnt signaling [45]. Moreover, activation of NFAT upregulated COX-2 and IL-6 levels, which contribute to the host defense and innate immune response at the early stage of pneumococcal infection [46]. Nuclear factor of activated T cell has been studied as a transcription factor that binds to the Rorc (encoding RORγt) and IL17a promoters [12]; therefore, we postulated that an increase in IL-17A production could be ascribed to NFAT activation. Indeed, noncanonical Wnt ligands were activated, and subsequently the expression of NFAT1 significantly increased the production of IL-17A [24, 47], which was significantly increased in the lung by Δpep27 immunization (Figure 3B). However, the expression of transcription factor 7 (TCF7), which is mainly induced by the canonical Wnt signaling pathway, showed no significant changes compared with the nonimmunized group (Supplementary Figure 4). In addition, RNA-sequencing data (Figure 3A) revealed that Δpep27 immunization highly induced CamKII (also designated as CamKIIb, Sequence ID: NC_000007.14), a negative regulator of β-catenin [48], suggesting that Δpep27 immunization does not induce canonical Wnt signaling. Recent data demonstrated that noncanonical WNT signaling inhibits canonical WNT signaling [49]. Thus, as with the noncanonical Wnt pathway, whether the canonical Wnt signal could also contribute to the protection against pneumococcal infection needs to be verified in the future. When NFAT expression was blocked by an NFAT inhibitor, IL-17A induction was significantly decreased (Figure 4A). Moreover, NFAT inhibition in the Δpep27-immunized group increased the colonization of S. pneumoniae to a level comparable to that of the nonimmunized group at 1 week and 1 month postimmunization (Figures 4B and 5A and B). These results suggested that Δpep27 immunization activates NFAT and subsequently induces IL-17A, thereby impairing colonization. Moreover, other studies demonstrated that inhibition of NFAT decreased neutrophil activity and, subsequently, increased renal susceptibility and bacterial CFUs in the kidney after uropathogenic Escherichia coli infection [50]. Thus, NFAT and IL-17A activation by Δpep27 immunization likely enhances phagocytosis of neutrophils, which has been confirmed in our previous study [4] and could result in impaired colonization. Furthermore, IL-17A also plays an important role in protection against K. pneumoniae and S. aureus [26, 27]. We were surprised to find that both K. pneumoniae and S. aureus CFUs in the lungs of the immunized mice were significantly decreased compared with those in the lungs of the nonimmunized mice. Moreover, NFAT inhibition reversed the colonization inhibition of both K. pneumoniae and S. aureus in the lungs at 1 week postimmunization (Figure 6). A reduced IL-17A level resulted in greater colonization, suggesting that IL-17A induction is a nonspecific critical protective mediator against bacterial colonization in the lung and that NFAT could augment IL-17A production. CONCLUSIONS In the present study, we demonstrated that Δpep27 immunization induced IL-17A production, which contributes to protect the lung from infectious agents by reducing colonization significantly. In addition, Δpep27 immunization induced noncanonical Wnt signaling, which can lead to induction of IL-17A. These results suggested that IL-17A responses via the noncanonical Wnt signaling pathway after Δpep27 conferred efficient protection on mice by reducing the bacterial CFU. The protective effect of Δpep27 immunization against respiratory infectious agents could attenuate or impair bacterial infection in hospitalized or immunocompromised people and provide highly feasible nonspecific preventive measures. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Disclaimer. The funding body played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments. We thank Yoe-Sik Bae for allowing us to use the fluorescence-activated cell sorting instrument. Financial support. This work was funded by the National Research Foundation (NRF-2015R1 A2 A1 A10052511; to D.-K. R.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. References 1. Klugman KP , Feldman C . Streptococcus pneumoniae respiratory tract infections . Curr Opin Infect Dis 2001 ; 14 : 173 – 9 . Google Scholar CrossRef Search ADS PubMed 2. Kadioglu A , Weiser JN , Paton JC , Andrew PW . The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease . Nat Rev Microbiol 2008 ; 6 : 288 – 301 . Google Scholar CrossRef Search ADS PubMed 3. Bogaert D , De Groot R , Hermans PW . Streptococcus pneumoniae colonisation: the key to pneumococcal disease . Lancet Infect Dis 2004 ; 4 : 144 – 54 . Google Scholar CrossRef Search ADS PubMed 4. Kim GL , Choi SY , Seon SH , et al. Pneumococcal pep27 mutant immunization stimulates cytokine secretion and confers long-term immunity with a wide range of protection, including against non-typeable strains . Vaccine 2016 ; 34 : 6481 – 92 . Google Scholar CrossRef Search ADS PubMed 5. Moffitt KL , Gierahn TM , Lu YJ , et al. T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization . Cell Host Microbe 2011 ; 9 : 158 – 65 . Google Scholar CrossRef Search ADS PubMed 6. Curtis MM , Way SS . Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens . Immunology 2009 ; 126 : 177 – 85 . Google Scholar CrossRef Search ADS PubMed 7. Zhang Z , Clarke TB , Weiser JN . Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice . J Clin Invest 2009 ; 119 : 1899 – 909 . Google Scholar PubMed 8. Kolls JK , Lindén A . Interleukin-17 family members and inflammation . Immunity 2004 ; 21 : 467 – 76 . Google Scholar CrossRef Search ADS PubMed 9. Craig A , Mai J , Cai S , Jeyaseelan S . Neutrophil recruitment to the lungs during bacterial pneumonia . Infect Immun 2009 ; 77 : 568 – 75 . Google Scholar CrossRef Search ADS PubMed 10. Katoh M , Katoh M . WNT signaling pathway and stem cell signaling network . Clin Cancer Res 2007 ; 13 : 4042 – 5 . Google Scholar CrossRef Search ADS PubMed 11. Kohn AD , Moon RT . Wnt and calcium signaling: beta-catenin-independent pathways . Cell Calcium 2005 ; 38 : 439 – 46 . Google Scholar CrossRef Search ADS PubMed 12. Hermann-Kleiter N , Baier G . NFAT pulls the strings during CD4+ T helper cell effector functions . Blood 2010 ; 115 : 2989 – 97 . Google Scholar CrossRef Search ADS PubMed 13. Zhang S , Luo L , Wang Y , Gomez MF , Thorlacius H . Nuclear factor of activated T cells regulates neutrophil recruitment, systemic inflammation, and T-cell dysfunction in abdominal sepsis . Infect Immun 2014 ; 82 : 3275 – 88 . Google Scholar CrossRef Search ADS PubMed 14. Rao A , Luo C , Hogan PG . Transcription factors of the NFAT family: regulation and function . Annu Rev Immunol 1997 ; 15 : 707 – 47 . Google Scholar CrossRef Search ADS PubMed 15. Muranski P , Restifo NP . Essentials of Th17 cell commitment and plasticity . Blood 2013 ; 121 : 2402 – 14 . Google Scholar CrossRef Search ADS PubMed 16. Deligne C , Metidji A , Fridman WH , Teillaud JL . Anti-CD20 therapy induces a memory Th1 response through the IFN-γ/IL-12 axis and prevents protumor regulatory T-cell expansion in mice . Leukemia 2015 ; 29 : 947 – 57 . Google Scholar CrossRef Search ADS PubMed 17. Noguchi H , Matsushita M , Okitsu T , et al. A new cell-permeable peptide allows successful allogeneic islet transplantation in mice . Nat Med 2004 ; 10 : 305 – 9 . Google Scholar CrossRef Search ADS PubMed 18. Ouyang W , Kolls JK , Zheng Y . The biological functions of T helper 17 cell effector cytokines in inflammation . Immunity 2008 ; 28 : 454 – 67 . Google Scholar CrossRef Search ADS PubMed 19. Ghilardi N , Ouyang W . Targeting the development and effector functions of TH17 cells . Semin Immunol 2007 ; 19 : 383 – 93 . Google Scholar CrossRef Search ADS PubMed 20. Liu JO . Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation . Immunol Rev 2009 ; 228 : 184 – 98 . Google Scholar CrossRef Search ADS PubMed 21. Siar CH , Nagatsuka H , Han PP , et al. Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma . J Oral Pathol Med 2012 ; 41 : 332 – 9 . Google Scholar CrossRef Search ADS PubMed 22. Macian F . NFAT proteins: key regulators of T-cell development and function . Nat Rev Immunol 2005 ; 5 : 472 – 84 . Google Scholar CrossRef Search ADS PubMed 23. Kiani A , García-Cózar FJ , Habermann I , et al. Regulation of interferon-gamma gene expression by nuclear factor of activated T cells . Blood 2001 ; 98 : 1480 – 8 . Google Scholar CrossRef Search ADS PubMed 24. Weigmann B , Lehr HA , Yancopoulos G , et al. The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis . J Exp Med 2008 ; 205 : 2099 – 110 . Google Scholar CrossRef Search ADS PubMed 25. Yoshida H , Nishina H , Takimoto H , et al. The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production . Immunity 1998 ; 8 : 115 – 24 . Google Scholar CrossRef Search ADS PubMed 26. Ishigame H , Kakuta S , Nagai T , et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses . Immunity 2009 ; 30 : 108 – 19 . Google Scholar CrossRef Search ADS PubMed 27. Happel KI , Dubin PJ , Zheng M , et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae . J Exp Med 2005 ; 202 : 761 – 9 . Google Scholar CrossRef Search ADS PubMed 28. Pamer EG . Resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens . Science 2016 ; 352 : 535 – 8 . Google Scholar CrossRef Search ADS PubMed 29. Buffie CG , Bucci V , Stein RR , et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile . Nature 2015 ; 517 : 205 – 8 . Google Scholar CrossRef Search ADS PubMed 30. Kim YG , Sakamoto K , Seo SU , et al. Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens . Science 2017 ; 356 : 315 – 9 . Google Scholar CrossRef Search ADS PubMed 31. Siegel SJ , Weiser JN . Mechanisms of bacterial colonization of the respiratory tract . Annu Rev Microbiol 2015 ; 69 : 425 – 44 . Google Scholar CrossRef Search ADS PubMed 32. Zipperer A , Konnerth MC , Laux C , et al. Human commensals producing a novel antibiotic impair pathogen colonization . Nature 2016 ; 535 : 511 – 6 . Google Scholar CrossRef Search ADS PubMed 33. Schnoeller C , Roux X , Sawant D , et al. Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism . Am J Respir Crit Care Med 2014 ; 189 : 194 – 202 . Google Scholar PubMed 34. Milner JD , Brenchley JM , Laurence A , et al. Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome . Nature 2008 ; 452 : 773 – 6 . Google Scholar CrossRef Search ADS PubMed 35. Gopal R , Rangel-Moreno J , Slight S , et al. Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis . Mucosal Immunol 2013 ; 6 : 972 – 84 . Google Scholar CrossRef Search ADS PubMed 36. Narita K , Hu DL , Mori F , Wakabayashi K , Iwakura Y , Nakane A . Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A . Infect Immun 2010 ; 78 : 4234 – 42 . Google Scholar CrossRef Search ADS PubMed 37. Chen Y , Thai P , Zhao YH , Ho YS , DeSouza MM , Wu R . Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop . J Biol Chem 2003 ; 278 : 17036 – 43 . Google Scholar CrossRef Search ADS PubMed 38. Kao CY , Chen Y , Thai P , et al. IL-17 markedly up-regulates beta-defensin-2 expression in human airway epithelium via JAK and NF-kappaB signaling pathways . J Immunol 2004 ; 173 : 3482 – 91 . Google Scholar CrossRef Search ADS PubMed 39. Moser C , Weiner DJ , Lysenko E , Bals R , Weiser JN , Wilson JM . beta-Defensin 1 contributes to pulmonary innate immunity in mice . Infect Immun 2002 ; 70 : 3068 – 72 . Google Scholar CrossRef Search ADS PubMed 40. Hoe E , Boelsen LK , Toh ZQ , et al. Reduced IL-17A secretion is associated with high levels of pneumococcal nasopharyngeal carriage in Fijian children . PLoS One 2015 ; 10 : e0129199 . Google Scholar CrossRef Search ADS PubMed 41. Basha S , Kaur R , Mosmann TR , Pichichero ME . Reduced T-helper 17 responses to Streptococcus pneumoniae in infection-prone children can be rescued by addition of innate cytokines . J Infect Dis 2017 ; 215 : 1321 – 30 . Google Scholar CrossRef Search ADS PubMed 42. Li C , Bellusci S , Borok Z , Minoo P . Non-canonical WNT signalling in the lung . J Biochem 2015 ; 158 : 355 – 65 . Google Scholar CrossRef Search ADS PubMed 43. Luo T , Dunphy PS , Lina TT , McBride JW . Ehrlichia chaffeensis exploits canonical and noncanonical host wnt signaling pathways to stimulate phagocytosis and promote intracellular survival . Infect Immun 2015 ; 84 : 686 – 700 . Google Scholar CrossRef Search ADS PubMed 44. Silva-García O , Valdez-Alarcón JJ , Baizabal-Aguirre VM . The Wnt/β-catenin signaling pathway controls the inflammatory response in infections caused by pathogenic bacteria . Mediators Inflamm 2014 ; 2014 : 310183 . Google Scholar CrossRef Search ADS PubMed 45. Cohen SB , Smith NL , McDougal C , et al. Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells . J Immunol 2015 ; 194 : 210 – 22 . Google Scholar CrossRef Search ADS PubMed 46. Koga T , Lim JH , Jono H , et al. Tumor suppressor cylindromatosis acts as a negative regulator for Streptococcus pneumoniae-induced NFAT signaling . J Biol Chem 2008 ; 283 : 12546 – 54 . Google Scholar CrossRef Search ADS PubMed 47. Ghosh S , Koralov SB , Stevanovic I , et al. Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis . Proc Natl Acad Sci U S A 2010 ; 107 : 15169 – 74 . Google Scholar CrossRef Search ADS PubMed 48. Flentke GR , Garic A , Hernandez M , Smith SM . CaMKII represses transcriptionally active β-catenin to mediate acute ethanol neurodegeneration and can phosphorylate β-catenin . J Neurochem 2014 ; 128 : 523 – 35 . Google Scholar CrossRef Search ADS PubMed 49. Nemeth MJ , Topol L , Anderson SM , Yang Y , Bodine DM . Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation . Proc Natl Acad Sci U S A 2007 ; 104 : 15436 – 41 . Google Scholar CrossRef Search ADS PubMed 50. Tourneur E , Ben Mkaddem S , Chassin C , et al. Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients . PLoS Pathog 2013 ; 9 : e1003152 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Pulmonary Colonization Resistance to Pathogens via Noncanonical Wnt and Interleukin-17A by Intranasal pep27 Mutant Immunization

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy158
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Abstract

Abstract Background Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Methods Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. Results In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Conclusions Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways. Δpep27 immunization, IL-17, Streptococcus pneumoniae, Wnt signaling Streptococcus pneumoniae infection is responsible for high morbidity and mortality worldwide [1], and it causes a variety of pneumococcal diseases, such as pneumonia, meningitis, otitis media, and sepsis [2]. There are 2 ways to protect against pneumococcal diseases: one is vaccination, which is the most effective way to prevent disease; the other is the inhibition of colonization at the first step of pneumococcal disease in the respiratory tract [3]. In previous studies, we demonstrated that immunization with an attenuated S. pneumoniae vaccine, Δpep27, confers protection against various pneumococcal serotypes by reducing colonization via immunoglobulin (Ig)A and interleukin (IL)-17A induction [4]. The Th17 response to pneumococcal colonization can prevent adherence of pneumococci on the nasopharynx [5], and together with IL-17A it inhibits mucosal colonization [6, 7]. Moreover, CD4 Th17-secreted IL-17 leads to recruitment of neutrophils [8] that directly remove infecting bacteria as part of the innate immunity in the lung [9]. Wnt signals have been studied as a part of the transduction pathways that determine cell fate or regulate cell movement [10]. Wnt signals can be divided into the canonical pathway, which is regulated mainly by β-catenin, and the noncanonical pathway, which is mediated by calcium β-catenin independently in early development [11]. In particular, nuclear factor of activated T cell (NFAT), which is a downstream molecule of the noncanonical Wnt signaling pathway, plays a critical role in T-cell development and the immune response [12]. Moreover, NFAT regulates the accumulation of neutrophils in the lung and during systemic inflammation [13], indicating that NFAT might be a beneficial target to defend against respiratory infection and sepsis. When T-cell receptors are stimulated, various transcription factors and cytokines are induced via the entry of calcium into the cytoplasm, including the calcineurin/NFAT pathways [14]; that is, NFAT binds to the IL17a promoter, followed by induction of IL-17A [12, 15]. In this study, we demonstrated that Δpep27 immunization induced NFAT and, subsequently, IL-17A secretion. Inhibition of NFAT consistently reversed the colonization inhibition by Δpep27 immunization, with reduced IL-17A secretion in the lung. Thus, NFAT-induced IL-17A contributes to the inhibition of colonization effected by Δpep27 immunization. MATERIALS AND METHODS Bacterial Strains Streptococcus pneumoniae serotype 2 (D39; NCTC7466) and its isogenic Δpep27 [4] were cultured on THY medium (Todd-Hewitt Broth with 0.5% Yeast Extract) with 5% sheep blood agar plates at 37°C overnight and then grown in THY broth with 0.5% yeast extract (BD) at 37°C. Bacterial cultures with an OD550 of 0.3 (1 × 108 colony-forming units [CFUs]/mL) were incubated and diluted with phosphate-buffered saline for infection or immunization studies. Streptococcus aureus (American Type Culture Collection [ATCC] 25923) and Klebsiella pneumoniae (ATCC 9997) were purchased from the Korean Culture Center of Microorganisms (Seoul, Korea), and cultured in THY agar supplemented with defibrinated sheep blood at 37°C overnight, then subsequently inoculated to THY broth and incubated at 37°C until the OD550 reached 0.5. Ethics Statement Four-week-old male BALB/c (Orient, Korea) were used in the infection experiments. All animal procedures were approved by the Sungkyunkwan University Animal Ethical Committee and were carried out in accordance with the guidelines of the Korean Animal Protection Law. Vaccination and Sample Collection For vaccination, mice were immunized with Δpep27 (1 × 107 to 1 × 108 CFUs, 10 μL) 3 times every 2 weeks via intranasal route. Seven days after the last immunization, serum, bronchoalveolar lavage fluid (BALF), lung, and spleen were collected [4]. Cytokine Determination The concentration of IL-17 in BALF, serum, and splenocytes were determined using an IL-17A enzyme-linked immunosorbent assay kit (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. Antibody Treatment For antibody-mediated neutralization, antibodies (Bio X Cell) were administrated via the intraperitoneal (i.p.) route at day +3 and +6 after immunization, with 200 μg of anti-IL-17A or mouse IgG1 antibody as a control [16]. Seven days postimmunization, the mice were treated with antibodies again and subsequently challenged with 2 × 108 CFUs of the D39 strain intranasally. Inhibition of Wnt Signaling The noncanonical Wnt inhibitor, 11R-VIVIT, was purchased from Tocris Bioscience, and an inactive control, 11R-VEET (RRRRRRRRRRRGGGMAGPPHIVEETGPHVI), was synthesized by Cosmogenetech (Korea). Mice received 10 mg/kg VEET or VIVIT i.p. once daily for 2 days [17]. Samples were collected 6 hours postinjection [17]. Ribonucleic Acid Isolation Total ribonucleic acid (RNA) was extracted from lungs using the TRIzol reagent (Invitrogen). The quality of the RNA was determined using an Agilent 2100 bioanalyzer with the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands), and RNA was quantified using an ND-2000 Spectrophotometer (Thermo Inc.). High-Throughput Sequencing and Transcriptomic Analyses To construct the sequencing libraries, 500 ng of total RNA was used for complementary deoxyribonucleic acid (cDNA) synthesis. The RNA libraries were then constructed using a SENSE 3′ messenger RNA (mRNA)-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria), according to the manufacturer’s protocol. Gene expression was determined by high-throughput sequencing using the Illumina NextSeq 500 system. Differentially expressed genes were selected as those that showed a 2-fold difference (upregulated) or 0.5-fold difference (downregulated) compared with the control. Gene clustering (hierarchical clustering) and heat maps were constructed based on MeV 4.9.0. These experimental and system biology analyses (network, canonical pathway, and regulatory effect) were analyzed using Ingenuity Pathway Analysis, performed by e-Biogen (Seoul, Korea). The gene expression analyses data were deposited in the National Center for Biotechnology Information database (GEO accession number GSE93718) (http://www.ncbi.nlm.nih.gov/geo/). Flow Cytometry Lungs were harvested 1 week after the last immunization, and isolated lung cells were coincubated with a cell stimulation cocktail and protein transport inhibitor cocktail (Invitrogen, Carlsbad, CA). Subsequently, the cells were resuspended in flow cytometry staining buffer (eBioscience) and then stained with a cell surface-specific marker conjugated with a fluorochrome. Fluorescein isothiocyanate-labeled anti-mouse CD4 and phycoerythrin-labeled anti-mouse CD3e were purchased from eBioscience. For intracellular staining, cells stained with the surface marker were fixed and permeabilized with an intracellular fixation and permeabilization buffer set (eBioscience) followed by allophycocyanin (APC)-labeled anti-mouse IL-17A and APC-labeled Rat IgG2a K isotype control (eBioscience). The gating of lymphocytes based on forward versus side scatter was aimed at selecting CD4+CD3e+ cells. The selected CD4+CD3e+ population was further gated for IL-17A+-positive response (Supplementary Figure 1). Flow cytometry analysis was performed using the BD FACSCanto II (BD Biosciences) using FlowJo version 10.1 software (Flowjo, LLC). Quantitative Real-Time Polymearse Chain Reaction Total RNA was isolated from the lung using an RNeasy plus mini kit (QIAGEN, Hilden, Germany), and cDNA was synthesized using EcoDry Premix kit (Takara, Japan). Quantitative real-time polymearse chain reaction (PCR) was performed according to the manufacturer’s instructions (Applied Biosystems). The reverse-transcription PCR conditions were as follows: 95°C for 15 seconds, 40 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; followed by melting curve analysis comprising 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The gene-specific primers are Tgfb1 (forward: ATG CTA AAG AGG TCA CCC GC; reverse: TGC TTC CCG AAT GTC TGA CG), IL-6 (forward: CCT CTG GTC TTC TGG AGT AC; reverse: GGA AAT TGG GGT AGG AAG G), and IL-17A (forward: CTC AAG CTC AGC GTG TCC A; reverse: ATC AGG GTC TTC ATT GCG GT), and glyceraldehyde 3-phosphate dehydrogenase (forward: TCC ACG ATG CCA AAG TTG TC; reverse: TGC ATC CTG CAC CAC CAA) served as control. Hematoxylin and Eosin Staining Isolated lungs were fixed in 10% neutral-buffered formalin. Sagittally sliced lung samples were embedded with paraffin and cut into 4-μm sections (Histoire, Ansan, Korea). The images of lungs stained with hematoxylin and eosin (H&E) were captured and analyzed by KNOTUS (Guri, Korea). Statistical Analysis Comparisons between 2 groups were performed using a Mann-Whitney U test (nonparametric). Comparisons among groups were made using one-way analysis of variance (Duncan’s method, nonparametric). Differences in median survival times between groups were analyzed using the log-rank test. Statistically significant differences were defined as *, P ≤ .05, **, P ≤ .01, and ***, P ≤ .001. RESULTS Δpep27 Immunization Induces Interleukin-17A to Protect Against Streptococcus pneumoniae Interleukin-17 produced by Th17 cells protects the host from extracellular bacterial infection [6, 18]. To investigate whether Δpep27 immunization could induce the IL-17A response in the lung, high-throughput sequencing of samples from Δpep27-immunized mouse lungs was performed. Hierarchical cluster analysis revealed that Δpep27 immunization upregulated genes related to the Th17 immune response (Figure 1A). Reverse-transcription PCR analysis confirmed significant induction of transforming growth factor beta 1 (Tgfβ1) and IL-6, which contributed to the differentiation of T cells into Th17 cells and subsequent IL-17A production [19]. In addition, ingenuity pathway analysis showed that the IL-6 in the lung centered for Th17 differentiation in the immune response after Δpep27 immunization (Supplementary Figure 2). Consistent with the RNA-sequencing data, the IL-17A protein level also increased significantly in serum, splenocytes, and BALF after Δpep27 immunization (Figure 1B). Moreover, the population of CD4+IL-17A+ cells in the lung was highly increased in the Δpep27-immunized group compared with that in nonimmunized mice (Figure 1C). These results indicated that Δpep27 immunization activated the expression of a variety of genes and proteins related to IL-17A production. Figure 1. View largeDownload slide Δpep27 immunization induces interleukin (IL)-17A. Mice (n = 3) were immunized intranasally with Δpep27 at 2-week intervals 3 times. Seven days after the last immunization, lung messenger ribonucleic acid (mRNA) was used for high-throughput sequencing. (A) Cluster analysis of gene expression identified upregulated (>2 vs control) and downregulated (<0.5 vs control) genes in the lung by Δpep27 immunization (A, left). Hierarchical clustering of Th17-related gene expression in Δpep27 immunized lungs (A, right). (B) The expression of Tgfβ1, IL-6, and IL-17A mRNA or induction of IL-17A in serum, splenocytes, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay, respectively. The experiment was performed 3 times independently. (C) The population of CD4 + IL-17A + cells was detected by flow cytometry. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed by the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 1. View largeDownload slide Δpep27 immunization induces interleukin (IL)-17A. Mice (n = 3) were immunized intranasally with Δpep27 at 2-week intervals 3 times. Seven days after the last immunization, lung messenger ribonucleic acid (mRNA) was used for high-throughput sequencing. (A) Cluster analysis of gene expression identified upregulated (>2 vs control) and downregulated (<0.5 vs control) genes in the lung by Δpep27 immunization (A, left). Hierarchical clustering of Th17-related gene expression in Δpep27 immunized lungs (A, right). (B) The expression of Tgfβ1, IL-6, and IL-17A mRNA or induction of IL-17A in serum, splenocytes, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay, respectively. The experiment was performed 3 times independently. (C) The population of CD4 + IL-17A + cells was detected by flow cytometry. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed by the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Next, to determine whether Δpep27 immunization-induced IL-17A plays an important role in protection from pneumococcal infection, we performed a colonization experiment after IL-17A neutralization using an anti-IL-17A antibody. Interleukin-17A neutralization markedly decreased the IL-17A level in BALF compared with an isotype control in the Δpep27-immunized group of mice (Figure 2A). To confirm whether IL-17A neutralization could reverse the colonization inhibition by Δpep27 immunization, IL-17A was neutralized and then the mice were challenged with D39, followed by colony counting. The results showed that IL-17A neutralization increased the bacterial CFU by approximately 100- and 10-fold in the lung and spleen, respectively, compared with the isotype control group 24 hours postinfection. This indicated that IL-17A neutralization reversed the colonization inhibition by Δpep27 immunization. Moreover, 48 hours postinfection, IL-17A neutralization increased the bacterial CFU by approximately 104-fold in the lung than in the no bacterial CFU by Δpep27 immunization (isotype control) (Figure 2B). In addition, the histology of the lung tissue showed increased inflammation predominantly in the bronchioli and alveoli 48 hours postinfection in the nonimmunized group compared with that in the immunized group (Figure 2C). Interleukin-17A neutralization showed attenuated neutrophil infiltration and bacterial clearance (Figure 2B and C). These results demonstrated that Δpep27 immunization could protect the host from pneumococcal infection using IL-17A to remove bacteria. Figure 2. View largeDownload slide Interleukin (IL)-17A provides protection after Δpep27 immunization. Mice (n = 4) were immunized intranasally with Δpep27 3 times at 2-week intervals. Interleukin-17A was neutralized by injecting an anti-IL-17A antibody on day 3 and 6 postimmunization, or immunoglobulin (Ig)G1 was injected as isotype control antibody. (A) Seven days after the last immunization, the IL-17A level in bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay. The experiment was performed 3 times independently. (B) Seven days after the last immunization, mice (n = 4) were given injections with anti-IL-17A or IgG1 followed by challenge with D39 (2 × 108 colony-forming units [CFU]). Bacterial CFU in blood, lung, and spleen were determined 24 and 48 hours postinfection. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. (C) Histopathology of lung tissue before infection or after infection were shown at 10× magnification. Scale bar, 200 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 2. View largeDownload slide Interleukin (IL)-17A provides protection after Δpep27 immunization. Mice (n = 4) were immunized intranasally with Δpep27 3 times at 2-week intervals. Interleukin-17A was neutralized by injecting an anti-IL-17A antibody on day 3 and 6 postimmunization, or immunoglobulin (Ig)G1 was injected as isotype control antibody. (A) Seven days after the last immunization, the IL-17A level in bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay. The experiment was performed 3 times independently. (B) Seven days after the last immunization, mice (n = 4) were given injections with anti-IL-17A or IgG1 followed by challenge with D39 (2 × 108 colony-forming units [CFU]). Bacterial CFU in blood, lung, and spleen were determined 24 and 48 hours postinfection. The experiment was performed 2 times independently. The data are presented as the mean ± standard error of the mean. (C) Histopathology of lung tissue before infection or after infection were shown at 10× magnification. Scale bar, 200 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Δpep27 Immunization Induces Noncanonical Wnt Signaling Nuclear factor of activated T cell binds to Rorc (encoding RORγt) and IL17a (encoding IL-17A) promoters, leading to the activation of Th17 responses [12]. Ribonucleic acid-sequencing data in the lung revealed upregulation of noncanonical Wnt genes (Figure 3). Nfatc2/Nfat1 was markedly activated until the second Δpep27 immunization and Calm, encoding calmodulin, as a Ca2+ effector protein that ultimately leads to activation of NFAT followed by calcineurin-NFAT interaction [20] which showed significantly increased expression (Figure 3A). In addition, high-throughput sequencing data showed that 3 Δpep27 immunizations induced noncanonical Wnt pathway ligands such as Wnt4, 5b, 7a, and 7b [21] (Figure 3A). Thus, Δpep27 immunization induced noncanonical Wnt signaling. Figure 3. View largeDownload slide Δpep27 immunization induces noncanonical Wnt signaling. Mice (n = 3) were immunized intranasally with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, messenger ribonucleic acid from lung was isolated. (A) High-throughput sequencing identified up- and downregulated genes in the lung, especially genes cluster related to Wnt signal. (B) The expression of Calcineurin A and nuclear factor of activated T cells (NFAT), which are involved in noncanonical Wnt signals in lung, were detected by Western blotting. The experiment was performed 3 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed using the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05. Figure 3. View largeDownload slide Δpep27 immunization induces noncanonical Wnt signaling. Mice (n = 3) were immunized intranasally with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, messenger ribonucleic acid from lung was isolated. (A) High-throughput sequencing identified up- and downregulated genes in the lung, especially genes cluster related to Wnt signal. (B) The expression of Calcineurin A and nuclear factor of activated T cells (NFAT), which are involved in noncanonical Wnt signals in lung, were detected by Western blotting. The experiment was performed 3 times independently. The data are presented as the mean ± standard error of the mean. The significance of the differences was analyzed using the Mann-Whitney U test; ***, P < .001, **, P < .01, and *, P < .05. Next, the regulation of NFAT1 and NFAT2 expression by calcium/calcineurin [22] was analyzed to identify whether Δpep27 immunization-induced Wnt signals could activate NFAT expression. Our results showed that the Calcineurin A and NFAT1 induction-dependent Th1 [23] and Th17 cell response [24] were induced, whereas the NFAT2 induction-dependent Th2 cell response [25] was decreased in the lung (Figure 3B). Nuclear Factor of Activated T-Cell Inhibition Reversed Colonization Inhibition To verify NFAT-dependent IL-17A secretion, we inhibited NFAT and then checked for impaired IL-17A secretion followed by increased colonization. Mice were treated with an NFAT inhibitor (VIVIT) and the IL-17A level in BALF, and NFAT1 expression and the bacterial CFU in the lung were determined before D39 challenge. The NFAT inhibition diminished (1) IL-17A in BALF and (2) NFAT1 expression and the population of CD4+IL-17A+ cells in the Δpep27 immunization group (Figure 4A). Six hours postinfection with D39, NFAT inhibition significantly decreased the IL-17A level in BALF and increased the bacterial load in lung significantly in the Δpep27 immunization group (Figure 4B). Consistently, NFAT inhibition 6 hours postinfection with D39 in the Δpep27 immunization group caused an increase in inflammation compared with the inert NFAT control (VEET), as demonstrated by H&E staining of lung tissue (Figure 4C). Moreover, even 1 month after the last immunization, the Δpep27 immunization group successfully maintained high IL-17A and NFAT1 levels, and it showed suppressed colonization after D39 challenge. Conversely, NFAT inhibition reversed these results (Figure 5). Thus, activation of noncanonical Wnt signals by Δpep27 immunization could lead to protection against pneumococcal infection by inducing IL-17A at the early stage of infection. Figure 4. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in lung. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) Before D39 challenge, interleukin (IL)-17A in the bronchoalveolar lavage fluid (BALF) (A; left), NFATc2/NFAT1 expression in lung (A, middle), and CD4 + IL-17A cell population (A, right) were determined by an enzyme-linked immunosorbent assay, Western blot (WB), and flow cytometry, respectively. (B) The IL-17A level in BALF and the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. (C) Hematoxylin-eosin staining of lung tissue before or after D39 infection was shown at 10× magnification. Scale bar, 100 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance (ANOVA); ***, P < .001, **, P < .01, and *, P < .05. Figure 4. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in lung. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. Seven days after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) Before D39 challenge, interleukin (IL)-17A in the bronchoalveolar lavage fluid (BALF) (A; left), NFATc2/NFAT1 expression in lung (A, middle), and CD4 + IL-17A cell population (A, right) were determined by an enzyme-linked immunosorbent assay, Western blot (WB), and flow cytometry, respectively. (B) The IL-17A level in BALF and the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. (C) Hematoxylin-eosin staining of lung tissue before or after D39 infection was shown at 10× magnification. Scale bar, 100 μm. The arrow denotes neutrophils in aveoli or bronchioles. The number at the left-bottom corner represents the inflammation score. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance (ANOVA); ***, P < .001, **, P < .01, and *, P < .05. Figure 5. View largeDownload slide Interluekin (IL)-17A inhibits Streptococcus pneumoniae colonization at 1 month postimmunization via noncanonical Wnt signaling. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. One month after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) The IL-17A level in bronchoalveolar lavage fluid (BALF) and (B) the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05. Figure 5. View largeDownload slide Interluekin (IL)-17A inhibits Streptococcus pneumoniae colonization at 1 month postimmunization via noncanonical Wnt signaling. Mice (n = 6) were immunized intranasally (i.n.) with Δpep27 3 times at 2-week intervals. One month after the last immunization, NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally, and after 6 hours the mice were challenged i.n. with D39 (2 × 108 colony-forming units [CFU]). (A) The IL-17A level in bronchoalveolar lavage fluid (BALF) and (B) the bacterial CFU in the lung were determined 6 hours postinfection. The experiments were performed 3 times independently. The data are presented as mean ± standard error of the mean. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05. Protective Role of Interluekin-17A Against Other Bacteria via Noncanonical Wnt Signaling Interleukin-17A plays a critical role in protection against Gram-positive S. aureus and Gram-negative K. pneumoniae [26, 27]. Therefore, if IL-17A provides protection from these bacteria, NFAT inhibition could reverse the colonization inhibition. To test this hypothesis, Δpep27-immunized mice were treated with an NFAT inhibitor and subsequently challenged with either S. aureus or K. pneumoniae, followed by determining the bacterial load. The results revealed that in the Δpep27-immunized group, the NFAT inhibitor (VIVIT) reversed the colonization inhibition back to the level of the nonimmunized group, whereas treatment with the inert NFAT control (VEET) did not increase the bacterial load significantly (Figure 6). In the Δpep27 immunization group, VIVIT treatment increased the bacterial CFUs by approximately 2-fold and 4-fold compared with those in the VEET-treated control group 6 hours after K. pneumoniae and S. aureus infection, respectively (Figure 6). Consistently, NFAT inhibition decreased the IL-17A level significantly in BALF after challenge with either K. pneumoniae or S. aureus (Figure 6). These results demonstrated that IL-17A provides protection from these bacterial colonization. Thus, IL-17A downregulation by NFAT abolishment reverses the colonization inhibition induced by Δpep27 immunization, resulting in higher susceptibility to bacterial infection, such as by S. pneumoniae, K. pneumoniae, and S. aureus. However, 1 month after the last Δpep27 immunization, lung cells were not protected against K. pneumoniae and S. aureus infection and showed marginal increase in IL-17A level after bacterial challenge (Supplementary Figure 3). Figure 6. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in Gram (−) and (+) bacterial infection. Mice (n = 6) were immunized with Δpep27 intranasally (i.n.) 3 times at 2-week intervals. Seven days after the last immunization, the NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally. Six hours postinjection of the inhibitor, mice were challenged i.n. with (A) 6 × 106 colony-forming units [CFU] of Klebsiella pneumoniae or (B) 1 × 109 CFU of Staphylococcus aureus. The level of IL-17A in the bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay, and bacterial CFU in lung was determined 6 hours postinfection. The data are presented as mean ± standard error of the mean and performed 2 times independently. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. Figure 6. View largeDownload slide Nuclear factor of activated T-cell (NFAT) inhibitor reversed colonization inhibition in Gram (−) and (+) bacterial infection. Mice (n = 6) were immunized with Δpep27 intranasally (i.n.) 3 times at 2-week intervals. Seven days after the last immunization, the NFAT inhibitor (VIVIT) or control peptide (VEET) was injected intraperitoneally. Six hours postinjection of the inhibitor, mice were challenged i.n. with (A) 6 × 106 colony-forming units [CFU] of Klebsiella pneumoniae or (B) 1 × 109 CFU of Staphylococcus aureus. The level of IL-17A in the bronchoalveolar lavage fluid (BALF) was detected by an enzyme-linked immunosorbent assay, and bacterial CFU in lung was determined 6 hours postinfection. The data are presented as mean ± standard error of the mean and performed 2 times independently. The significance of the differences was analyzed by analysis of variance; ***, P < .001, **, P < .01, and *, P < .05 compared with the nonimmunized group. DISCUSSION Antibiotic treatment results in the clearance of susceptible intestinal microbiota and increases new infections, especially with antibiotic-resistant strains. Thus, re-establishment of a normal microbiota and subsequent colonization resistance against antibiotic-resistant infections is deemed to be a highly feasible way to eliminate potential pathogens from the intestine [28–30]. Therefore, intervention to prevent bacterial colonization or induce colonization resistance could protect against colonization by potential pathogens. Bacterial colonization in respiratory organs, such as the nasal cavity and lung, precedes diseases and occasionally results in sepsis and meningitis [31]. The first stage of respiratory infection is colonization of the nasal and pulmonary cavity; therefore, treatments should aim to reduce bacterial colonization. In a recent study, nasal colonization resistance to S. aureus was demonstrated using antibiotic-producing Staphylococcus lugdunensis strains [32]. However, antibiotic-producing strains might increase antibiotic resistance and would not be feasible for clinical implementation. An attenuated strain of Bordetella pertussis vaccine could prevent respiratory syncytial virus infection in an IL-17-dependent manner [33]. Thus, from a practical viewpoint, there is incomplete information on intranasal or respiratory resistance to respiratory bacterial colonization because of nonavailability of specific tools and methods for respiratory intervention. In the present study, we demonstrated that Δpep27 immunization could provide resistance to respiratory colonization via induction of IL-17A. Th17-deficient cells are highly vulnerable to mucosal infections by pathogens such as S aureus, Haemophilus influenzae, and S. pneumoniae [34], signifying the important role of the Th17 immune response in natural immunity. Moreover, the Th17-dependent mucosal response provides protection in the lung against Mycobacterium tuberculosis infection [35]. Interleukin-17, produced by Th17, recruits the inflammatory cells and induces the proinflammatory mediators that are required to defend against bacterial infection in the lung [18]. Immunization with the fibrinogen-binding domain of ClfA induced IL-17A and protected against S. aureus infection by enhancing neutrophil infiltration [36]. Moreover, in human lung epithelial cells, IL-17A was required for the induction of mucins and defensin-2 [37, 38]. As an antimicrobial peptide, β-defensin provides innate immunity by blocking bacteria at epithelial surfaces [39]. It is interesting to note that the RNA-sequencing data revealed a marked increase in β-defensin 1 (DefB1) mRNA expression after Δpep27 immunization (Figure 1A). Reduced IL-17A production was accompanied by a higher pneumococcal nasopharyngeal carriage compared with a high concentration of IL-17A [40]. Moreover, in infection-prone subjects, Th17-promoting cytokines rescued the reduced Th17 response to S. pneumoniae [41]. Indeed, Δpep27 immunization with neutralization of IL-17 reversed the colonization inhibition in the lung (Figure 2), suggesting that Δpep27-induced IL-17A plays a critical role in protection against pneumococcal infection. Wnt signaling is responsible for proliferation, differentiation, and migration. Moreover, activation of noncanonical Wnt signaling in the lung is critically important to maintain lung homeostasis [42]. Wnt stimulation by microbial infection has been linked to enhancement in bacterial phagocytosis; however, it promotes intracellular survival of bacteria [43] and downregulates the inflammatory response against Salmonella or Mycobacterium [44]. In contrast, Toxoplasma infection upregulates proinflammatory cytokines via Wnt signaling [45]. Moreover, activation of NFAT upregulated COX-2 and IL-6 levels, which contribute to the host defense and innate immune response at the early stage of pneumococcal infection [46]. Nuclear factor of activated T cell has been studied as a transcription factor that binds to the Rorc (encoding RORγt) and IL17a promoters [12]; therefore, we postulated that an increase in IL-17A production could be ascribed to NFAT activation. Indeed, noncanonical Wnt ligands were activated, and subsequently the expression of NFAT1 significantly increased the production of IL-17A [24, 47], which was significantly increased in the lung by Δpep27 immunization (Figure 3B). However, the expression of transcription factor 7 (TCF7), which is mainly induced by the canonical Wnt signaling pathway, showed no significant changes compared with the nonimmunized group (Supplementary Figure 4). In addition, RNA-sequencing data (Figure 3A) revealed that Δpep27 immunization highly induced CamKII (also designated as CamKIIb, Sequence ID: NC_000007.14), a negative regulator of β-catenin [48], suggesting that Δpep27 immunization does not induce canonical Wnt signaling. Recent data demonstrated that noncanonical WNT signaling inhibits canonical WNT signaling [49]. Thus, as with the noncanonical Wnt pathway, whether the canonical Wnt signal could also contribute to the protection against pneumococcal infection needs to be verified in the future. When NFAT expression was blocked by an NFAT inhibitor, IL-17A induction was significantly decreased (Figure 4A). Moreover, NFAT inhibition in the Δpep27-immunized group increased the colonization of S. pneumoniae to a level comparable to that of the nonimmunized group at 1 week and 1 month postimmunization (Figures 4B and 5A and B). These results suggested that Δpep27 immunization activates NFAT and subsequently induces IL-17A, thereby impairing colonization. Moreover, other studies demonstrated that inhibition of NFAT decreased neutrophil activity and, subsequently, increased renal susceptibility and bacterial CFUs in the kidney after uropathogenic Escherichia coli infection [50]. Thus, NFAT and IL-17A activation by Δpep27 immunization likely enhances phagocytosis of neutrophils, which has been confirmed in our previous study [4] and could result in impaired colonization. Furthermore, IL-17A also plays an important role in protection against K. pneumoniae and S. aureus [26, 27]. We were surprised to find that both K. pneumoniae and S. aureus CFUs in the lungs of the immunized mice were significantly decreased compared with those in the lungs of the nonimmunized mice. Moreover, NFAT inhibition reversed the colonization inhibition of both K. pneumoniae and S. aureus in the lungs at 1 week postimmunization (Figure 6). A reduced IL-17A level resulted in greater colonization, suggesting that IL-17A induction is a nonspecific critical protective mediator against bacterial colonization in the lung and that NFAT could augment IL-17A production. CONCLUSIONS In the present study, we demonstrated that Δpep27 immunization induced IL-17A production, which contributes to protect the lung from infectious agents by reducing colonization significantly. In addition, Δpep27 immunization induced noncanonical Wnt signaling, which can lead to induction of IL-17A. These results suggested that IL-17A responses via the noncanonical Wnt signaling pathway after Δpep27 conferred efficient protection on mice by reducing the bacterial CFU. The protective effect of Δpep27 immunization against respiratory infectious agents could attenuate or impair bacterial infection in hospitalized or immunocompromised people and provide highly feasible nonspecific preventive measures. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Disclaimer. The funding body played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments. We thank Yoe-Sik Bae for allowing us to use the fluorescence-activated cell sorting instrument. Financial support. This work was funded by the National Research Foundation (NRF-2015R1 A2 A1 A10052511; to D.-K. R.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. References 1. Klugman KP , Feldman C . Streptococcus pneumoniae respiratory tract infections . Curr Opin Infect Dis 2001 ; 14 : 173 – 9 . Google Scholar CrossRef Search ADS PubMed 2. Kadioglu A , Weiser JN , Paton JC , Andrew PW . The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease . Nat Rev Microbiol 2008 ; 6 : 288 – 301 . Google Scholar CrossRef Search ADS PubMed 3. Bogaert D , De Groot R , Hermans PW . Streptococcus pneumoniae colonisation: the key to pneumococcal disease . Lancet Infect Dis 2004 ; 4 : 144 – 54 . Google Scholar CrossRef Search ADS PubMed 4. Kim GL , Choi SY , Seon SH , et al. Pneumococcal pep27 mutant immunization stimulates cytokine secretion and confers long-term immunity with a wide range of protection, including against non-typeable strains . Vaccine 2016 ; 34 : 6481 – 92 . Google Scholar CrossRef Search ADS PubMed 5. Moffitt KL , Gierahn TM , Lu YJ , et al. T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization . Cell Host Microbe 2011 ; 9 : 158 – 65 . Google Scholar CrossRef Search ADS PubMed 6. Curtis MM , Way SS . Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens . Immunology 2009 ; 126 : 177 – 85 . Google Scholar CrossRef Search ADS PubMed 7. Zhang Z , Clarke TB , Weiser JN . Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice . J Clin Invest 2009 ; 119 : 1899 – 909 . Google Scholar PubMed 8. Kolls JK , Lindén A . Interleukin-17 family members and inflammation . Immunity 2004 ; 21 : 467 – 76 . Google Scholar CrossRef Search ADS PubMed 9. Craig A , Mai J , Cai S , Jeyaseelan S . Neutrophil recruitment to the lungs during bacterial pneumonia . Infect Immun 2009 ; 77 : 568 – 75 . Google Scholar CrossRef Search ADS PubMed 10. Katoh M , Katoh M . WNT signaling pathway and stem cell signaling network . Clin Cancer Res 2007 ; 13 : 4042 – 5 . Google Scholar CrossRef Search ADS PubMed 11. Kohn AD , Moon RT . Wnt and calcium signaling: beta-catenin-independent pathways . Cell Calcium 2005 ; 38 : 439 – 46 . Google Scholar CrossRef Search ADS PubMed 12. Hermann-Kleiter N , Baier G . NFAT pulls the strings during CD4+ T helper cell effector functions . Blood 2010 ; 115 : 2989 – 97 . Google Scholar CrossRef Search ADS PubMed 13. Zhang S , Luo L , Wang Y , Gomez MF , Thorlacius H . Nuclear factor of activated T cells regulates neutrophil recruitment, systemic inflammation, and T-cell dysfunction in abdominal sepsis . Infect Immun 2014 ; 82 : 3275 – 88 . Google Scholar CrossRef Search ADS PubMed 14. Rao A , Luo C , Hogan PG . Transcription factors of the NFAT family: regulation and function . Annu Rev Immunol 1997 ; 15 : 707 – 47 . Google Scholar CrossRef Search ADS PubMed 15. Muranski P , Restifo NP . Essentials of Th17 cell commitment and plasticity . Blood 2013 ; 121 : 2402 – 14 . Google Scholar CrossRef Search ADS PubMed 16. Deligne C , Metidji A , Fridman WH , Teillaud JL . Anti-CD20 therapy induces a memory Th1 response through the IFN-γ/IL-12 axis and prevents protumor regulatory T-cell expansion in mice . Leukemia 2015 ; 29 : 947 – 57 . Google Scholar CrossRef Search ADS PubMed 17. Noguchi H , Matsushita M , Okitsu T , et al. A new cell-permeable peptide allows successful allogeneic islet transplantation in mice . Nat Med 2004 ; 10 : 305 – 9 . Google Scholar CrossRef Search ADS PubMed 18. Ouyang W , Kolls JK , Zheng Y . The biological functions of T helper 17 cell effector cytokines in inflammation . Immunity 2008 ; 28 : 454 – 67 . Google Scholar CrossRef Search ADS PubMed 19. Ghilardi N , Ouyang W . Targeting the development and effector functions of TH17 cells . Semin Immunol 2007 ; 19 : 383 – 93 . Google Scholar CrossRef Search ADS PubMed 20. Liu JO . Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation . Immunol Rev 2009 ; 228 : 184 – 98 . Google Scholar CrossRef Search ADS PubMed 21. Siar CH , Nagatsuka H , Han PP , et al. Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma . J Oral Pathol Med 2012 ; 41 : 332 – 9 . Google Scholar CrossRef Search ADS PubMed 22. Macian F . NFAT proteins: key regulators of T-cell development and function . Nat Rev Immunol 2005 ; 5 : 472 – 84 . Google Scholar CrossRef Search ADS PubMed 23. Kiani A , García-Cózar FJ , Habermann I , et al. Regulation of interferon-gamma gene expression by nuclear factor of activated T cells . Blood 2001 ; 98 : 1480 – 8 . Google Scholar CrossRef Search ADS PubMed 24. Weigmann B , Lehr HA , Yancopoulos G , et al. The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis . J Exp Med 2008 ; 205 : 2099 – 110 . Google Scholar CrossRef Search ADS PubMed 25. Yoshida H , Nishina H , Takimoto H , et al. The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production . Immunity 1998 ; 8 : 115 – 24 . Google Scholar CrossRef Search ADS PubMed 26. Ishigame H , Kakuta S , Nagai T , et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses . Immunity 2009 ; 30 : 108 – 19 . Google Scholar CrossRef Search ADS PubMed 27. Happel KI , Dubin PJ , Zheng M , et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae . J Exp Med 2005 ; 202 : 761 – 9 . Google Scholar CrossRef Search ADS PubMed 28. Pamer EG . Resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens . Science 2016 ; 352 : 535 – 8 . Google Scholar CrossRef Search ADS PubMed 29. Buffie CG , Bucci V , Stein RR , et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile . Nature 2015 ; 517 : 205 – 8 . Google Scholar CrossRef Search ADS PubMed 30. Kim YG , Sakamoto K , Seo SU , et al. Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens . Science 2017 ; 356 : 315 – 9 . Google Scholar CrossRef Search ADS PubMed 31. Siegel SJ , Weiser JN . Mechanisms of bacterial colonization of the respiratory tract . Annu Rev Microbiol 2015 ; 69 : 425 – 44 . Google Scholar CrossRef Search ADS PubMed 32. Zipperer A , Konnerth MC , Laux C , et al. Human commensals producing a novel antibiotic impair pathogen colonization . Nature 2016 ; 535 : 511 – 6 . Google Scholar CrossRef Search ADS PubMed 33. Schnoeller C , Roux X , Sawant D , et al. Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism . Am J Respir Crit Care Med 2014 ; 189 : 194 – 202 . Google Scholar PubMed 34. Milner JD , Brenchley JM , Laurence A , et al. Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome . Nature 2008 ; 452 : 773 – 6 . Google Scholar CrossRef Search ADS PubMed 35. Gopal R , Rangel-Moreno J , Slight S , et al. Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis . Mucosal Immunol 2013 ; 6 : 972 – 84 . Google Scholar CrossRef Search ADS PubMed 36. Narita K , Hu DL , Mori F , Wakabayashi K , Iwakura Y , Nakane A . Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A . Infect Immun 2010 ; 78 : 4234 – 42 . Google Scholar CrossRef Search ADS PubMed 37. Chen Y , Thai P , Zhao YH , Ho YS , DeSouza MM , Wu R . Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop . J Biol Chem 2003 ; 278 : 17036 – 43 . Google Scholar CrossRef Search ADS PubMed 38. Kao CY , Chen Y , Thai P , et al. IL-17 markedly up-regulates beta-defensin-2 expression in human airway epithelium via JAK and NF-kappaB signaling pathways . J Immunol 2004 ; 173 : 3482 – 91 . Google Scholar CrossRef Search ADS PubMed 39. Moser C , Weiner DJ , Lysenko E , Bals R , Weiser JN , Wilson JM . beta-Defensin 1 contributes to pulmonary innate immunity in mice . Infect Immun 2002 ; 70 : 3068 – 72 . Google Scholar CrossRef Search ADS PubMed 40. Hoe E , Boelsen LK , Toh ZQ , et al. Reduced IL-17A secretion is associated with high levels of pneumococcal nasopharyngeal carriage in Fijian children . PLoS One 2015 ; 10 : e0129199 . Google Scholar CrossRef Search ADS PubMed 41. Basha S , Kaur R , Mosmann TR , Pichichero ME . Reduced T-helper 17 responses to Streptococcus pneumoniae in infection-prone children can be rescued by addition of innate cytokines . J Infect Dis 2017 ; 215 : 1321 – 30 . Google Scholar CrossRef Search ADS PubMed 42. Li C , Bellusci S , Borok Z , Minoo P . Non-canonical WNT signalling in the lung . J Biochem 2015 ; 158 : 355 – 65 . Google Scholar CrossRef Search ADS PubMed 43. Luo T , Dunphy PS , Lina TT , McBride JW . Ehrlichia chaffeensis exploits canonical and noncanonical host wnt signaling pathways to stimulate phagocytosis and promote intracellular survival . Infect Immun 2015 ; 84 : 686 – 700 . Google Scholar CrossRef Search ADS PubMed 44. Silva-García O , Valdez-Alarcón JJ , Baizabal-Aguirre VM . The Wnt/β-catenin signaling pathway controls the inflammatory response in infections caused by pathogenic bacteria . Mediators Inflamm 2014 ; 2014 : 310183 . Google Scholar CrossRef Search ADS PubMed 45. Cohen SB , Smith NL , McDougal C , et al. Beta-catenin signaling drives differentiation and proinflammatory function of IRF8-dependent dendritic cells . J Immunol 2015 ; 194 : 210 – 22 . Google Scholar CrossRef Search ADS PubMed 46. Koga T , Lim JH , Jono H , et al. Tumor suppressor cylindromatosis acts as a negative regulator for Streptococcus pneumoniae-induced NFAT signaling . J Biol Chem 2008 ; 283 : 12546 – 54 . Google Scholar CrossRef Search ADS PubMed 47. Ghosh S , Koralov SB , Stevanovic I , et al. Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis . Proc Natl Acad Sci U S A 2010 ; 107 : 15169 – 74 . Google Scholar CrossRef Search ADS PubMed 48. Flentke GR , Garic A , Hernandez M , Smith SM . CaMKII represses transcriptionally active β-catenin to mediate acute ethanol neurodegeneration and can phosphorylate β-catenin . J Neurochem 2014 ; 128 : 523 – 35 . Google Scholar CrossRef Search ADS PubMed 49. Nemeth MJ , Topol L , Anderson SM , Yang Y , Bodine DM . Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation . Proc Natl Acad Sci U S A 2007 ; 104 : 15436 – 41 . Google Scholar CrossRef Search ADS PubMed 50. Tourneur E , Ben Mkaddem S , Chassin C , et al. Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients . PLoS Pathog 2013 ; 9 : e1003152 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of Infectious DiseasesOxford University Press

Published: Mar 22, 2018

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