Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12099
Vaccine-Mediated Protection against Helicobacter pylori is not Associated with Increased Salivary Cytokine or Mucin Expression Garrett Z. Ng,*,† Yok-Teng Chionh*,† and Philip Sutton*,† *Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Vic., 3010, Australia, †Mucosal Immunology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Parkville, Vic., 3052, Australia
Keywords Vaccine, Helicobacter pylori, mucin, immune response. Reprint requests to: Philip Sutton, Mucosal Immunology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Parkville, Vic. 3052, Australia. E-mail: [email protected]
Abstract Background: The development of an effective vaccine against Helicobacter pylori is impeded by the inability to reliably produce sterilizing immunity and our lack of knowledge regarding mechanisms of protective immunity against this pathogen. It has previously been described that salivary glands are essential for vaccine-mediated protection against H. pylori, but the mechanism responsible for this effect has not been identified. In this study we tested the hypothesis that vaccines reduce H. pylori colonization by inducing an immune-mediated change in salivary gland mucin secretion. Materials and methods: Sublingual and submandibular salivary glands were removed from untreated mice, from mice infected with H. pylori and from mice vaccinated against H. pylori then challenged with live bacteria. Cytokine levels in these salivary glands were quantified by ELISA, and salivary mucins were quantified by real-time PCR. Salivary antibody responses were determined by Western blot. Results: Vaccine-mediated protection against H. pylori did not produce any evidence of a positive increase in either salivary cytokine or mucin levels. In fact, many cytokines were significantly reduced in the vaccinated/challenged mice, including IL-17A, IL-10, IL-1ß, as well as the mucin Muc10. These decreases were associated with an increase in total protein content within the salivary glands of vaccinated mice which appeared to be the result of increased IgA production. While this study showed that vaccination increased salivary IgA levels, previous studies have demonstrated that antibodies do not play a critical role in protection against H. pylori that is induced by current vaccine formulations and regimes. Conclusions: The effector mechanism of protective immunity induced by vaccination of mice did not involve immune changes within the salivary glands, nor increased production of salivary mucins.
Helicobacter pylori is an important pathogen that typically infects the human stomach during childhood, producing a chronic gastritis that is sustained for decades and is the key driver of associated pathologies such as peptic ulceration and gastric adenocarcinoma [1]. Using mouse models, it has been demonstrated that a range of vaccination strategies can produce a significant reduction in H. pylori colonization in animals subsequently challenged with live bacteria, although sterilizing immunity is only rarely achieved [2–4]. The induction of this vaccine-mediated protection requires CD4+ T cells and may be associated with IL-17, neutrophils and/or mast cells [5–8], although the potential
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role of IL-17 is uncertain [9]. However, virtually nothing is known about the direct effector mechanism by which these vaccinations actually impact upon H. pylori colonization which is a major barrier to the successful production of an effective H. pylori vaccine [10]. Identification of this effector mechanism may allow strategies to improve the effectiveness of vaccinations against this pathogen to be developed. A study published by Shirai et al. in 2000 suggested that vaccine-mediated immune protection against H. pylori challenge requires the presence of salivary glands. In that study, surgical removal of the sublingual and submandibular salivary glands (sialoadenectomy)
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from mice significantly reduced the effectiveness of vaccination against H. pylori [11]. While removal of the salivary glands from these animals also resulted in a large decrease in total IgA levels in their gastric mucosa and feces [11], numerous studies using antibody-deficient mice have shown that immunoglobulins are not required for H. pylori vaccine efficacy [5,12,13]. Thus any failure of vaccinations in sialoadenectomised mice would not be due to a loss of antibody secretion into the gastrointestinal tract. However, the actual explanation for the observation made by Shirai et al. remains unknown. Another important product of salivary glands are the secretory mucins. Previously, we have hypothesized that the effector stage of vaccine-induced protection against H. pylori may be mediated by the production of mucins [14]. Mucins comprise a family of heavily glycosylated glycoproteins that are either cell surface expressed or secreted, where they can constitute a major component of mucus. Such mucins form an intrinsic part of the barrier system lining the gastrointestinal tract that protects against bacterial infection [15]. We have previously demonstrated that the cell surface gastric mucin Muc1/MUC1 (mouse/human) plays a critical role in regulating the inflammatory response to H. pylori infection, and also restricts the ability of these bacteria to attach to the epithelial cell surface by acting as a releasable decoy [16,17]. Importantly, mucin secretion can be regulated by the acquired immune response including CD4+ T helper cells [18,19], and therefore may potentially be influenced by memory responses to previous infections, or even vaccinations. We therefore theorized that if salivary glands play a role in vaccine-mediated protection against H. pylori this could be implemented by the migration of adaptor T helper cells into the salivary glands, and modifications in the production of salivary mucins. To examine this possibility, we examined the effect of vaccination and H. pylori infection on the expression of cytokines and mucins in murine salivary glands.
Methods Helicobacter pylori Culture Helicobacter pylori strain SS1 [20] was grown on horse blood agar plates [Blood Agar Base No. 2, 2.5 lg/mL Amphotericin B (Sigma, St Louis, MO, USA) and Skirrow’s Selective Supplements (Oxoid, Basingstoke, UK) and 5% horse blood (Biolab, Melbourne, Vic, Australia)] in an anaerobic jar with a microaerophilic gas generating kit (Oxoid) for 2 days at 37 °C. For infection of mice, bacteria were subcultured into brain heart infusion broth (BHI; Oxoid) containing 0.02% Amphostat
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and 5% horse serum (Sigma) and grown in microaerophilic conditions for 24 hours at 37 °C.
Vaccination against Helicobacter pylori Animal experimentation was performed under institutional guidelines and with approval from the University of Melbourne Animal Ethics Committee. Groups of agematched, female C57BL/6 mice were dosed orogastrically with 100 lL of either 1, PBS (unvaccinated); or 2, 100 lg H. pylori SS1 lysate plus 10 lg cholera toxin (Sigma) (vaccinated). Mice received two vaccinations spaced by 3 weeks. Four weeks after the second vaccination, both groups of mice were challenged with a single oro-gastric dose of 107 H. pylori SS1 in 100 lL of BHI broth. A third group of matched mice were left unvaccinated and uninfected, as negative controls for examination of salivary mucins and cytokines. Four weeks post-challenge, stomach and salivary glands were removed for analysis. Helicobacter pylori infection levels within mouse gastric tissues were quantified by a colony-forming assay as described previously [16]. The number of colonies were counted and colony forming units calculated per stomach [21]. The submandibular and sublingual salivary glands were snap-frozen and stored at 80 °C until use.
Evaluation of Salivary Cytokine and Protein Levels One salivary gland was homogenized using a T10 homogenizer (IKA-Werke) in 1 mL of Tri Reagent (a guanidine thiocyanate and phenol solution; Ambion, Austin, TX, USA) for subsequent purification of RNA and protein. After phase separation, protein was extracted out from the organic phase as per manufacturer’s instructions and redissolved in PBS + 1% SDS. Protein concentrations were quantitated using a BCA protein assay kit (Pierce, Rockford, IL, USA) to adjust for the efficacy of extraction, and diluted with PBS down to 0.1% SDS before use. Cytokine concentrations were determined by coating 96-well Maxisorp plates (Nunc, Roskilde, Denmark) with purified anti-mouse IL-1b (0.2 lg/well; R&D Systems, Minneapolis, MN, USA), TNFa (0.1 lg/well; BioLegend, San Diego, CA, USA), IL-13 (0.05 lg/well; eBioscience, San Diego, CA, USA), IL-10 (0.1 lg/well; BD Biosciences, San Jose, CA, USA), IFNc (0.1 lg/well; BD Biosciences), IL-6 (0.05 lg/well; eBioscience), or IL-17A (0.05 lg/well; eBioscience) overnight in bicarbonate coating buffer, pH 9.6. Plates were blocked with 1% BSA (Sigma) in PBS (blocker) for one hour prior to addition of samples
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in duplicate for three hours at room temperature or 4 °C overnight. Captured cytokines were then labeled with biotinylated anti-mouse IL-1b (0.03 lg/well; R&D Systems), TNFa (0.025 lg/well; BioLegend), IL-13 (0.025 lg/well; eBioscience), IL-10 (0.05 lg/well; BD Biosciences), IFNc (0.05 lg/well; BD Biosciences), IL-6 (0.025 lg/well; eBioscience) or IL-17A (0.025 lg/well; eBioscience) in blocker for one hour prior to the addition of 50 lL horseradish peroxidase conjugated streptavidin (Pierce) 1/5000 in blocker for 30 minute. Color was developed with 100 lL of TMB solution prepared as 0.1% of 10 mg/mL TMB (Sigma) in DMSO and 0.006% hydrogen peroxide in phosphate-citrate buffer, pH 5.0, and the reaction stopped with an equal volume of 2 mol/L sulfuric acid prior to reading absorbance at 450 nm. Sample concentration was determined against a standard curve of recombinant IL-1b (R&D Systems), TNFa (BioLegend), IL-13 (eBioscience), IL-10, IFNc (BD Biosciences), IL-6 or IL-17A (eBioscience).
Evaluation of Salivary Gland Antibody by Western Blotting Salivary glands were homogenized in 3 mL of 6 mol/L guanidine HCl and dialysed into 8 mol/L urea. Twenty lg of protein (by BCA) was separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% skim milk and labelled with ImmunoPure anti-mouse IgG HRP (Pierce), anti-mouse IgA HRP (SouthernBiotech, Birmingham, AL, USA) or antib-actin then anti-rabbit HRP (both from Cell Signaling Technology, Beverly, MA, USA). Membranes were visualized using ECL chemiluminescence reagent (GE Healthcare) and an ImageQuant LAS 4000 (GE Healthcare), with densitometry performed using ImageJ software.
Quantification of Salivary Gland Mucin Gene Expression RNA from one submandibular and one sublingual salivary gland was extracted using Tri Reagent (Ambion), then converted to cDNA using the Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany) which was diluted out to 150 lL in Tris-EDTA buffer. For qPCR, duplicate reactions of 25 lL containing 12.5 lL Quanti-
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Figure 1 Protective immunity induced by vaccination of C57BL/6 mice. C57BL/6 mice (n = 5) were orally vaccinated with H. pylori SS1 lysate plus CT. Unvaccinated, infected controls (n = 8) were sham treated with PBS. Four weeks post-vaccination, mice were challenged with live H. pylori. Efficacy of vaccination was determined 4 weeks after bacterial challenge by colony-forming assay. Vaccinated mice had significant lower H. pylori colonization when compared with unvaccinated controls (*ANOVA). Box-plots present the median (horizontal bar), interquartile range (box) and 10th and 90th percentiles (bars).
Tect SYBR Green PCR Master Mix (Qiagen), 0.2 lmol/L primers and 3 lL of cDNA were performed in an Mx3000P cycler (Stratagene, La Jolla, CA, USA). Primer efficiencies within each run were determined with LinRegPCR [22] and gene expression calculated relative to Actb.
Statistics For statistical analyses, data were log-transformed then compared by analysis of variance (ANOVA), with Dunnett’s post hoc analysis using SPSS software, version 20.0 (IBM, Armonk, NY, USA).
Results To examine whether changes in salivary cytokine or mucin expression correlated with vaccine-mediated protection, mice were immunized orally with H. pylori lysate and CT adjuvant. Vaccination was confirmed to induce a significant reduction in H. pylori colonization upon subsequent challenge with live bacteria, when compared with unimmunized controls (Fig. 1).
Figure 2 Vaccination does not increase salivary gland cytokine levels. Salivary glands were removed from 1, uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori challenged mice (as in Fig. 1). Cytokine data shown are total cytokine levels per salivary gland, quantitated by ELISA. Salivary gland protein levels were quantified by BCA. Vaccinated/challenged mice had significantly reduced total levels of many cytokines in their salivary glands, relative to both uninfected and infected mice (*ANOVA). This inversely correlated with a significant increase in total protein content of the salivary glands from vaccinated/challenged mice (*ANOVA). There was a trend towards increased protein levels in salivary glands from infected mice when compared with uninfected controls, but this did not reach significance (p = 0.072; ANOVA).
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To determine whether this protective response correlated with an increase in immune activity in the salivary glands, cytokine levels were compared in these glands from infected and immunized/challenged mice, as well as from negative controls (uninfected/unimmunized). Not only was there no evidence of an increase, but surprisingly the total levels of many cytokines (IL-1ß, TNFa, IL-10, IL-6 and IL-17A) were significantly reduced in the salivary glands of immunized, infected mice (Fig. 2). Further analysis revealed that salivary glands from the immunized/challenged mice in this experiment contained significantly more total protein than non-immunized mice (Fig. 2). Salivary gland weights were not recorded, so it was not possible to determine whether this was due to an increase in salivary gland size (although no obvious increase was noted at extraction), or increased protein concentrations within the glands. Given salivary glands are a major source of mucosal secretory antibody, in particular IgA, we theorized the increase in protein concentration in immunized infected mice was most likely to be due to increased levels of IgA production, and this was confirmed by Western blot (Fig. 3). The key aim of this study was to evaluate the effect of vaccination on salivary mucin production. The salivary glands of mice produce three mucins, Muc5b,
Figure 3 Vaccination-induced increase in salivary IgA. Levels of IgG and IgA in salivary glands from 1, uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori SS1 challenged mice were assessed by Western blot relative to ß-actin housekeeping control. Graph plots show densitometry analysis of the Western blots. No difference was noted in total IgG levels between the groups but vaccinated mice had a significant increase in total IgA (*ANOVA).
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Muc10, and Muc19. Similar to cytokines above, quantitation revealed that vaccinations did not increase the levels of salivary mucin expression, with, if anything, a decrease in mucin expression relative to infected, nonvaccinated mice (Fig. 4).
Discussion Vaccination strategies against H. pylori have thus far proven inadequate due to their typical inability to reliably produce complete protection. The development of a truly effective vaccine against H. pylori is severely hindered by our lack of understanding of host mechanisms involved in protective immunity against this infection. In this study we followed on from a previous observation by Shirai et al. which indicated that vaccine-mediated protection against H. pylori requires salivary glands, but for which a mechanism was lacking [11]. As the majority of H. pylori reside in mucus lining the gastric mucosa where they are exposed to secreted mucins, we theorized that mucin secretion by salivary glands could explain this observation. However, we found no evidence to suggest that vaccination produces any increase in immune response in the salivary glands. Cytokine and mucin expression levels were actually lower in vaccinated/challenged mice compared with infected or uninfected controls, which in fact demonstrates an inverse correlation with the effector stage of protection. Shirai et al. concluded that salivary glands were important in the induction of protective immunity, as sialoadenectomy prior to vaccination against H. pylori removed vaccine efficacy [11]. However, they also found that salivary glands were important for long-term maintenance of vaccine-induced protection. The current study was not designed to evaluate the role of mucins in the initial induction of protective immunity, as we envisage that secreted salivary mucins are unlikely to play a role in immune induction. Hence a theoretical role for mucins in vaccine-mediated induction of protection against H. pylori cannot be completely ruled out by this study, although the mechanism by which this may occur is unclear. Rather, by their nature, we hypothesized that secreted salivary mucins may play an important role in the effector/maintenance stage of this protection; the data presented in this study argue against this theory. Another major product of salivary glands is immunoglobulin A (IgA) which is the main antibody isotype secreted into the gastrointestinal tract. The increased protein levels found in the salivary glands of vaccinated mice in this study correlated with an increase in IgA production which is completely consistent with current
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Figure 4 Vaccination does not increase salivary gland mucin expression. Salivary glands were removed from 1, Uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori SS1 challenged mice. Mucin mRNA expression levels were quantitated by real-time PCR. Vaccination did not increase salivary gland mucin mRNA levels. Salivary gland Muc10 mRNA levels were significantly lower than those of infected mice (*ANOVA).
dogma regarding mucosal immune response to gastrointestinal vaccination and infection. This is further supported by Shirai et al., who showed that sialoadenectomized mice had less than half the levels of gastric and fecal IgA than did control animals [11]. Given numerous previous studies have clearly demonstrated that antibody levels, including IgA, are not involved in vaccinemediated protection against H. pylori [5,12,13,23], we do not believe this increase in IgA levels is responsible for the protection induced by vaccination in this study. For many infections, this would be an effective strategy, but in the case of H. pylori, clearly this response is ineffective as we have recently discussed in detail [10]. Another important related point is that we have quantified salivary protein levels in two other vaccine experiments, involving mice that were vaccinated either intranasally or subcutaneously. In both experiments, vaccination induced a level of protection similar to that presented in this study, there was no concurrent increase in salivary protein levels (data not shown). Hence, the increased salivary protein levels may be a consequence of the route of vaccination, only occurring following oral delivery, and does not seem to be associated with, or required for, protective immunity. In conclusion, we have evaluated the cytokine and mucin response of the salivary glands of mice vaccinated against H. pylori and found no evidence to suggest that immunization induced any positive change in salivary cytokines or mucins during the effector stage of the ensuing protective immune response. The explanation for the observation of Shirai et al. [11], therefore remains unknown. More research is clearly needed to identify the mechanisms by which vaccinations target H. pylori. It is essential that we overcome our ignorance regarding these protective immune
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mechanisms, if we are to realize the development of an effective human H. pylori vaccine.
Acknowledgements and Disclosures Competing interests: the authors have no competing interests.
References 1 Kandulski A, Selgrad M, Malfertheiner P. Helicobacter pylori infection: a clinical overview. Dig Liver Dis 2008;40:619–26. 2 Every AL, Stent A, Moloney MB, Ng GZ, Skene CD, Edwards SJ, Sutton P. Evaluation of superoxide dismutase from Helicobacter pylori as a protective vaccine antigen. Vaccine 2011;29:1514–18. 3 Chionh YT, Wee JL, Every AL, Ng GZ, Sutton P. M-cell targeting of whole killed bacteria induces protective immunity against gastrointestinal pathogens. Infect Immun 2009;77:2962– 70. 4 Garhart CA, Redline RW, Nedrud JG, Czinn SJ. Clearance of Helicobacter pylori infection and resolution of postimmunization gastritis in a kinetic study of prophylactically immunized mice. Infect Immun 2002;70:3529–38. 5 Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R, Lee CK, Kleanthous H, Monath TP. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses. J Exp Med 1998;188:2277–88. 6 DeLyria ES, Redline RW, Blanchard TG. Vaccination of mice against H pylori induces a strong Th-17 response and immunity that is neutrophil dependent. Gastroenterology 2009;136:247–56. 7 Velin D, Bachmann D, Bouzourene H, Michetti P. Mast cells are critical mediators of vaccine-induced Helicobacter clearance in the mouse model. Gastroenterology 2005;129:142–55. 8 Velin D, Favre L, Bernasconi E, Bachmann D, Pythoud C, Saiji E, Bouzourene H, Michetti P. Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model. Gastroenterology 2009;136:2237–46.
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9 Delyria ES, Nedrud JG, Ernst PB, Alam MS, Redline RW, Ding H, Czinn SJ, Xu J, Blanchard TG. Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A. Helicobacter 2011;16:169–78. 10 Sutton P, Chionh YT. Why can’t we make an effective vaccine against Helicobacter pylori? Expert Rev Vaccines 2013;12:433–41. 11 Shirai Y, Wakatsuki Y, Kusumoto T, Nakata M, Yoshida M, Usui T, Iizuka T, Kita T. Induction and maintenance of immune effector cells in the gastric tissue of mice orally immunized to Helicobacter pylori requires salivary glands. Gastroenterology 2000;118:749–59. 12 Blanchard TG, Czinn SJ, Redline RW, Sigmund N, Harriman G, Nedrud JG. Antibody-independent protective mucosal immunity to gastric Helicobacter infection in mice. Cell Immunol 1999;191:74–80. 13 Sutton P, Wilson J, Kosaka T, Wolowczuk I, Lee A. Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies. Immunol Cell Biol 2000;78:28–30. 14 Sutton P. Helicobacter pylori vaccines and mechanisms of effective immunity: is mucus the key? Immunol Cell Biol 2001;79:67–73. 15 McGuckin MA, Linden SK, Sutton P, Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Microbiol 2011;9:265–78. 16 McGuckin MA, Every AL, Skene CD, et al. Muc1 mucin limits both Helicobacter pylori colonization of the murine gastric mucosa and associated gastritis. Gastroenterology 2007;133:1210– 18.
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17 Linden SK, Sheng YH, Every AL, Miles KM, Skoog EC, Florin TH, Sutton P, McGuckin MA. MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy. PLoS Pathog 2009;5:e1000617. 18 Khan WI, Abe T, Ishikawa N, Nawa Y, Yoshimura K. Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice. Parasite Immunol 1995;17:485–91. 19 Hasnain SZ, Evans CM, Roy M, et al. Muc5ac: a critical component mediating the rejection of enteric nematodes. J Exp Med 2011;208:893–900. 20 Lee A, O’Rourke J, De Ungria MC, Robertson B, Daskalopoulos G, Dixon MF. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 1997;112:1386–97. 21 Sutton P. Considering increased mouse stomach mass, when calculating prophylactic vaccine efficacy against Helicobacter pylori. Helicobacter 2007;12:210–12. 22 Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 2003;339:62–6. 23 Garhart CA, Nedrud JG, Heinzel FP, Sigmund NE, Czinn SJ. Vaccine-induced protection against Helicobacter pylori in mice lacking both antibodies and interleukin-4. Infect Immun 2003;71:3628–33.
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Vaccine-Mediated Protection against Helicobacter pylori is not Associated with Increased Salivary Cytokine or Mucin Expression Garrett Z. Ng,*,† Yok-Teng Chionh*,† and Philip Sutton*,† *Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Vic., 3010, Australia, †Mucosal Immunology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Parkville, Vic., 3052, Australia
Keywords Vaccine, Helicobacter pylori, mucin, immune response. Reprint requests to: Philip Sutton, Mucosal Immunology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Parkville, Vic. 3052, Australia. E-mail: [email protected]
Abstract Background: The development of an effective vaccine against Helicobacter pylori is impeded by the inability to reliably produce sterilizing immunity and our lack of knowledge regarding mechanisms of protective immunity against this pathogen. It has previously been described that salivary glands are essential for vaccine-mediated protection against H. pylori, but the mechanism responsible for this effect has not been identified. In this study we tested the hypothesis that vaccines reduce H. pylori colonization by inducing an immune-mediated change in salivary gland mucin secretion. Materials and methods: Sublingual and submandibular salivary glands were removed from untreated mice, from mice infected with H. pylori and from mice vaccinated against H. pylori then challenged with live bacteria. Cytokine levels in these salivary glands were quantified by ELISA, and salivary mucins were quantified by real-time PCR. Salivary antibody responses were determined by Western blot. Results: Vaccine-mediated protection against H. pylori did not produce any evidence of a positive increase in either salivary cytokine or mucin levels. In fact, many cytokines were significantly reduced in the vaccinated/challenged mice, including IL-17A, IL-10, IL-1ß, as well as the mucin Muc10. These decreases were associated with an increase in total protein content within the salivary glands of vaccinated mice which appeared to be the result of increased IgA production. While this study showed that vaccination increased salivary IgA levels, previous studies have demonstrated that antibodies do not play a critical role in protection against H. pylori that is induced by current vaccine formulations and regimes. Conclusions: The effector mechanism of protective immunity induced by vaccination of mice did not involve immune changes within the salivary glands, nor increased production of salivary mucins.
Helicobacter pylori is an important pathogen that typically infects the human stomach during childhood, producing a chronic gastritis that is sustained for decades and is the key driver of associated pathologies such as peptic ulceration and gastric adenocarcinoma [1]. Using mouse models, it has been demonstrated that a range of vaccination strategies can produce a significant reduction in H. pylori colonization in animals subsequently challenged with live bacteria, although sterilizing immunity is only rarely achieved [2–4]. The induction of this vaccine-mediated protection requires CD4+ T cells and may be associated with IL-17, neutrophils and/or mast cells [5–8], although the potential
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role of IL-17 is uncertain [9]. However, virtually nothing is known about the direct effector mechanism by which these vaccinations actually impact upon H. pylori colonization which is a major barrier to the successful production of an effective H. pylori vaccine [10]. Identification of this effector mechanism may allow strategies to improve the effectiveness of vaccinations against this pathogen to be developed. A study published by Shirai et al. in 2000 suggested that vaccine-mediated immune protection against H. pylori challenge requires the presence of salivary glands. In that study, surgical removal of the sublingual and submandibular salivary glands (sialoadenectomy)
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from mice significantly reduced the effectiveness of vaccination against H. pylori [11]. While removal of the salivary glands from these animals also resulted in a large decrease in total IgA levels in their gastric mucosa and feces [11], numerous studies using antibody-deficient mice have shown that immunoglobulins are not required for H. pylori vaccine efficacy [5,12,13]. Thus any failure of vaccinations in sialoadenectomised mice would not be due to a loss of antibody secretion into the gastrointestinal tract. However, the actual explanation for the observation made by Shirai et al. remains unknown. Another important product of salivary glands are the secretory mucins. Previously, we have hypothesized that the effector stage of vaccine-induced protection against H. pylori may be mediated by the production of mucins [14]. Mucins comprise a family of heavily glycosylated glycoproteins that are either cell surface expressed or secreted, where they can constitute a major component of mucus. Such mucins form an intrinsic part of the barrier system lining the gastrointestinal tract that protects against bacterial infection [15]. We have previously demonstrated that the cell surface gastric mucin Muc1/MUC1 (mouse/human) plays a critical role in regulating the inflammatory response to H. pylori infection, and also restricts the ability of these bacteria to attach to the epithelial cell surface by acting as a releasable decoy [16,17]. Importantly, mucin secretion can be regulated by the acquired immune response including CD4+ T helper cells [18,19], and therefore may potentially be influenced by memory responses to previous infections, or even vaccinations. We therefore theorized that if salivary glands play a role in vaccine-mediated protection against H. pylori this could be implemented by the migration of adaptor T helper cells into the salivary glands, and modifications in the production of salivary mucins. To examine this possibility, we examined the effect of vaccination and H. pylori infection on the expression of cytokines and mucins in murine salivary glands.
Methods Helicobacter pylori Culture Helicobacter pylori strain SS1 [20] was grown on horse blood agar plates [Blood Agar Base No. 2, 2.5 lg/mL Amphotericin B (Sigma, St Louis, MO, USA) and Skirrow’s Selective Supplements (Oxoid, Basingstoke, UK) and 5% horse blood (Biolab, Melbourne, Vic, Australia)] in an anaerobic jar with a microaerophilic gas generating kit (Oxoid) for 2 days at 37 °C. For infection of mice, bacteria were subcultured into brain heart infusion broth (BHI; Oxoid) containing 0.02% Amphostat
© 2013 John Wiley & Sons Ltd, Helicobacter 19: 48–54
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and 5% horse serum (Sigma) and grown in microaerophilic conditions for 24 hours at 37 °C.
Vaccination against Helicobacter pylori Animal experimentation was performed under institutional guidelines and with approval from the University of Melbourne Animal Ethics Committee. Groups of agematched, female C57BL/6 mice were dosed orogastrically with 100 lL of either 1, PBS (unvaccinated); or 2, 100 lg H. pylori SS1 lysate plus 10 lg cholera toxin (Sigma) (vaccinated). Mice received two vaccinations spaced by 3 weeks. Four weeks after the second vaccination, both groups of mice were challenged with a single oro-gastric dose of 107 H. pylori SS1 in 100 lL of BHI broth. A third group of matched mice were left unvaccinated and uninfected, as negative controls for examination of salivary mucins and cytokines. Four weeks post-challenge, stomach and salivary glands were removed for analysis. Helicobacter pylori infection levels within mouse gastric tissues were quantified by a colony-forming assay as described previously [16]. The number of colonies were counted and colony forming units calculated per stomach [21]. The submandibular and sublingual salivary glands were snap-frozen and stored at 80 °C until use.
Evaluation of Salivary Cytokine and Protein Levels One salivary gland was homogenized using a T10 homogenizer (IKA-Werke) in 1 mL of Tri Reagent (a guanidine thiocyanate and phenol solution; Ambion, Austin, TX, USA) for subsequent purification of RNA and protein. After phase separation, protein was extracted out from the organic phase as per manufacturer’s instructions and redissolved in PBS + 1% SDS. Protein concentrations were quantitated using a BCA protein assay kit (Pierce, Rockford, IL, USA) to adjust for the efficacy of extraction, and diluted with PBS down to 0.1% SDS before use. Cytokine concentrations were determined by coating 96-well Maxisorp plates (Nunc, Roskilde, Denmark) with purified anti-mouse IL-1b (0.2 lg/well; R&D Systems, Minneapolis, MN, USA), TNFa (0.1 lg/well; BioLegend, San Diego, CA, USA), IL-13 (0.05 lg/well; eBioscience, San Diego, CA, USA), IL-10 (0.1 lg/well; BD Biosciences, San Jose, CA, USA), IFNc (0.1 lg/well; BD Biosciences), IL-6 (0.05 lg/well; eBioscience), or IL-17A (0.05 lg/well; eBioscience) overnight in bicarbonate coating buffer, pH 9.6. Plates were blocked with 1% BSA (Sigma) in PBS (blocker) for one hour prior to addition of samples
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in duplicate for three hours at room temperature or 4 °C overnight. Captured cytokines were then labeled with biotinylated anti-mouse IL-1b (0.03 lg/well; R&D Systems), TNFa (0.025 lg/well; BioLegend), IL-13 (0.025 lg/well; eBioscience), IL-10 (0.05 lg/well; BD Biosciences), IFNc (0.05 lg/well; BD Biosciences), IL-6 (0.025 lg/well; eBioscience) or IL-17A (0.025 lg/well; eBioscience) in blocker for one hour prior to the addition of 50 lL horseradish peroxidase conjugated streptavidin (Pierce) 1/5000 in blocker for 30 minute. Color was developed with 100 lL of TMB solution prepared as 0.1% of 10 mg/mL TMB (Sigma) in DMSO and 0.006% hydrogen peroxide in phosphate-citrate buffer, pH 5.0, and the reaction stopped with an equal volume of 2 mol/L sulfuric acid prior to reading absorbance at 450 nm. Sample concentration was determined against a standard curve of recombinant IL-1b (R&D Systems), TNFa (BioLegend), IL-13 (eBioscience), IL-10, IFNc (BD Biosciences), IL-6 or IL-17A (eBioscience).
Evaluation of Salivary Gland Antibody by Western Blotting Salivary glands were homogenized in 3 mL of 6 mol/L guanidine HCl and dialysed into 8 mol/L urea. Twenty lg of protein (by BCA) was separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% skim milk and labelled with ImmunoPure anti-mouse IgG HRP (Pierce), anti-mouse IgA HRP (SouthernBiotech, Birmingham, AL, USA) or antib-actin then anti-rabbit HRP (both from Cell Signaling Technology, Beverly, MA, USA). Membranes were visualized using ECL chemiluminescence reagent (GE Healthcare) and an ImageQuant LAS 4000 (GE Healthcare), with densitometry performed using ImageJ software.
Quantification of Salivary Gland Mucin Gene Expression RNA from one submandibular and one sublingual salivary gland was extracted using Tri Reagent (Ambion), then converted to cDNA using the Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany) which was diluted out to 150 lL in Tris-EDTA buffer. For qPCR, duplicate reactions of 25 lL containing 12.5 lL Quanti-
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Figure 1 Protective immunity induced by vaccination of C57BL/6 mice. C57BL/6 mice (n = 5) were orally vaccinated with H. pylori SS1 lysate plus CT. Unvaccinated, infected controls (n = 8) were sham treated with PBS. Four weeks post-vaccination, mice were challenged with live H. pylori. Efficacy of vaccination was determined 4 weeks after bacterial challenge by colony-forming assay. Vaccinated mice had significant lower H. pylori colonization when compared with unvaccinated controls (*ANOVA). Box-plots present the median (horizontal bar), interquartile range (box) and 10th and 90th percentiles (bars).
Tect SYBR Green PCR Master Mix (Qiagen), 0.2 lmol/L primers and 3 lL of cDNA were performed in an Mx3000P cycler (Stratagene, La Jolla, CA, USA). Primer efficiencies within each run were determined with LinRegPCR [22] and gene expression calculated relative to Actb.
Statistics For statistical analyses, data were log-transformed then compared by analysis of variance (ANOVA), with Dunnett’s post hoc analysis using SPSS software, version 20.0 (IBM, Armonk, NY, USA).
Results To examine whether changes in salivary cytokine or mucin expression correlated with vaccine-mediated protection, mice were immunized orally with H. pylori lysate and CT adjuvant. Vaccination was confirmed to induce a significant reduction in H. pylori colonization upon subsequent challenge with live bacteria, when compared with unimmunized controls (Fig. 1).
Figure 2 Vaccination does not increase salivary gland cytokine levels. Salivary glands were removed from 1, uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori challenged mice (as in Fig. 1). Cytokine data shown are total cytokine levels per salivary gland, quantitated by ELISA. Salivary gland protein levels were quantified by BCA. Vaccinated/challenged mice had significantly reduced total levels of many cytokines in their salivary glands, relative to both uninfected and infected mice (*ANOVA). This inversely correlated with a significant increase in total protein content of the salivary glands from vaccinated/challenged mice (*ANOVA). There was a trend towards increased protein levels in salivary glands from infected mice when compared with uninfected controls, but this did not reach significance (p = 0.072; ANOVA).
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To determine whether this protective response correlated with an increase in immune activity in the salivary glands, cytokine levels were compared in these glands from infected and immunized/challenged mice, as well as from negative controls (uninfected/unimmunized). Not only was there no evidence of an increase, but surprisingly the total levels of many cytokines (IL-1ß, TNFa, IL-10, IL-6 and IL-17A) were significantly reduced in the salivary glands of immunized, infected mice (Fig. 2). Further analysis revealed that salivary glands from the immunized/challenged mice in this experiment contained significantly more total protein than non-immunized mice (Fig. 2). Salivary gland weights were not recorded, so it was not possible to determine whether this was due to an increase in salivary gland size (although no obvious increase was noted at extraction), or increased protein concentrations within the glands. Given salivary glands are a major source of mucosal secretory antibody, in particular IgA, we theorized the increase in protein concentration in immunized infected mice was most likely to be due to increased levels of IgA production, and this was confirmed by Western blot (Fig. 3). The key aim of this study was to evaluate the effect of vaccination on salivary mucin production. The salivary glands of mice produce three mucins, Muc5b,
Figure 3 Vaccination-induced increase in salivary IgA. Levels of IgG and IgA in salivary glands from 1, uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori SS1 challenged mice were assessed by Western blot relative to ß-actin housekeeping control. Graph plots show densitometry analysis of the Western blots. No difference was noted in total IgG levels between the groups but vaccinated mice had a significant increase in total IgA (*ANOVA).
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Muc10, and Muc19. Similar to cytokines above, quantitation revealed that vaccinations did not increase the levels of salivary mucin expression, with, if anything, a decrease in mucin expression relative to infected, nonvaccinated mice (Fig. 4).
Discussion Vaccination strategies against H. pylori have thus far proven inadequate due to their typical inability to reliably produce complete protection. The development of a truly effective vaccine against H. pylori is severely hindered by our lack of understanding of host mechanisms involved in protective immunity against this infection. In this study we followed on from a previous observation by Shirai et al. which indicated that vaccine-mediated protection against H. pylori requires salivary glands, but for which a mechanism was lacking [11]. As the majority of H. pylori reside in mucus lining the gastric mucosa where they are exposed to secreted mucins, we theorized that mucin secretion by salivary glands could explain this observation. However, we found no evidence to suggest that vaccination produces any increase in immune response in the salivary glands. Cytokine and mucin expression levels were actually lower in vaccinated/challenged mice compared with infected or uninfected controls, which in fact demonstrates an inverse correlation with the effector stage of protection. Shirai et al. concluded that salivary glands were important in the induction of protective immunity, as sialoadenectomy prior to vaccination against H. pylori removed vaccine efficacy [11]. However, they also found that salivary glands were important for long-term maintenance of vaccine-induced protection. The current study was not designed to evaluate the role of mucins in the initial induction of protective immunity, as we envisage that secreted salivary mucins are unlikely to play a role in immune induction. Hence a theoretical role for mucins in vaccine-mediated induction of protection against H. pylori cannot be completely ruled out by this study, although the mechanism by which this may occur is unclear. Rather, by their nature, we hypothesized that secreted salivary mucins may play an important role in the effector/maintenance stage of this protection; the data presented in this study argue against this theory. Another major product of salivary glands is immunoglobulin A (IgA) which is the main antibody isotype secreted into the gastrointestinal tract. The increased protein levels found in the salivary glands of vaccinated mice in this study correlated with an increase in IgA production which is completely consistent with current
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Figure 4 Vaccination does not increase salivary gland mucin expression. Salivary glands were removed from 1, Uninfected; 2, H. pylori infected; and 3, vaccinated then H. pylori SS1 challenged mice. Mucin mRNA expression levels were quantitated by real-time PCR. Vaccination did not increase salivary gland mucin mRNA levels. Salivary gland Muc10 mRNA levels were significantly lower than those of infected mice (*ANOVA).
dogma regarding mucosal immune response to gastrointestinal vaccination and infection. This is further supported by Shirai et al., who showed that sialoadenectomized mice had less than half the levels of gastric and fecal IgA than did control animals [11]. Given numerous previous studies have clearly demonstrated that antibody levels, including IgA, are not involved in vaccinemediated protection against H. pylori [5,12,13,23], we do not believe this increase in IgA levels is responsible for the protection induced by vaccination in this study. For many infections, this would be an effective strategy, but in the case of H. pylori, clearly this response is ineffective as we have recently discussed in detail [10]. Another important related point is that we have quantified salivary protein levels in two other vaccine experiments, involving mice that were vaccinated either intranasally or subcutaneously. In both experiments, vaccination induced a level of protection similar to that presented in this study, there was no concurrent increase in salivary protein levels (data not shown). Hence, the increased salivary protein levels may be a consequence of the route of vaccination, only occurring following oral delivery, and does not seem to be associated with, or required for, protective immunity. In conclusion, we have evaluated the cytokine and mucin response of the salivary glands of mice vaccinated against H. pylori and found no evidence to suggest that immunization induced any positive change in salivary cytokines or mucins during the effector stage of the ensuing protective immune response. The explanation for the observation of Shirai et al. [11], therefore remains unknown. More research is clearly needed to identify the mechanisms by which vaccinations target H. pylori. It is essential that we overcome our ignorance regarding these protective immune
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mechanisms, if we are to realize the development of an effective human H. pylori vaccine.
Acknowledgements and Disclosures Competing interests: the authors have no competing interests.
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