Helicobacter hepaticus infection in BALB/c mice abolishes subunit-vaccine-induced protection against M. tuberculosis

Highlights

• •Neonatal Hh infection of mice upregulates colonic IL10 message.
• •Neonatal Hh infection reduces lung immune responses after immunisation with Ad85A.
• •Protection against Mtb challenge induced by Ad85A is abolished in Hh infected mice.
• •IL10R blockade reverses the effects of Hh infection on Ad85A induced protection.
• •Addition of Hh to the microbiota abolishes protection induced by a subunit vaccine.

Abstract

BCG, the only licensed vaccine against tuberculosis (TB), provides geographically variable protection, an effect ascribed to exposure to environmental mycobacteria (EM). Here we show that altering the intestinal microbiota of mice by early-life infection with the commensal bacterium Helicobacter hepaticus (Hh) increases their susceptibility to challenge with Mycobacterium tuberculosis (Mtb). Furthermore Hh-infected mice immunised parenterally with the recombinant subunit vaccine, human adenovirus type 5 expressing the immunodominant antigen 85A of Mtb (Ad85A), display a reduced lung immune response and protection against Mtb challenge is also reduced. Expression of interleukin 10 (IL10) messenger RNA is increased in the colon of Hh infected mice. Treatment of Hh-infected Ad85A-immunised mice with anti-IL10 receptor antibody, following challenge with Mtb, restores the protective effect of the vaccine. These data show for the first time that alteration of the intestinal microbiota by addition of a single commensal organism can profoundly influence protection induced by a TB subunit vaccine via an IL10-dependent mechanism, a result with implications for the deployment of such vaccines in the field.

1. Introduction

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide with nearly 9 million new infections annually and 1.4 million deaths. The difficulty of reducing this disease burden is compounded by the spread of drug resistant organisms and the greatly increased susceptibility of HIV patients to TB [1].

In the face of these problems, vaccination is an attractive strategy but the protective efficacy of the only licensed vaccine, BCG, varies geographically and wanes with time. Furthermore, boosting with BCG is generally ineffective [2]. This has led to attempts either to replace BCG with a more effective vaccine or to improve its efficacy with booster vaccines.

A common hypothesis to account for the variation in BCG effectiveness in different locations is that this is due to environmental effects, including exposure to environmental mycobacteria (EM), which might may prevent growth of BCG, mask BCG-induced protection or induce regulatory T cells, as demonstrated experimentally in mice [3]. Clearly if subunit booster vaccines are to be used to improve the efficacy of BCG, it will be important to know whether their efficacy is similarly subject to environmental influences. In fact, when EM are administered orally and a subunit vaccine parenterally, protection against Mtb challenge is reduced [4]. However, in these experiments large quantities of EM were continuously administered in the drinking water, so that the question remained whether less artificial changes in the microbiota of the intestine might also influence protective immune responses to a subunit TB vaccine.

Multiple studies have highlighted the immunomodulatory effect of enteric microorganism exposure on lung immune responses. Experimental helminth infection in mice has been shown to impair innate pulmonary host defence against Mtb through the IL4 receptor signalling pathway [5] and helminth infection has been associated with increased TB incidence and reduced BCG vaccine efficacy in affected populations [6]. In contrast, epidemiological evidence suggests that infection with the gastric bacterium Helicobacter pylori is associated with protection against TB [7], while neonatal infection of mice with H. pylori prevents the induction of allergic lung disease later in life [8].

The related gram-negative bacterium, Helicobacter hepaticus (Hh), is a non-invasive organism commonly found in the murine lower intestinal tract in mouse colonies [9]. Hh does not cause histological lesions in immunocompetent mice but IL10 insufficient mice develop typhlocolitis [10]. Inflammation develops via induction of Th17 cells in the intestinal mucosa that rapidly extinguish IL17A production and become IFNγ producing Th1 cells [10–12].

In these experiments, we investigate the effect of Hh on immune response and protection induced by the recombinant subunit vaccine, human adenovirus type 5 expressing the immunodominant Antigen 85A of Mtb (Ad85A) [13].

2. Materials and methods

2.1. Mice

All experiments were performed with BALB/c mice bred in house. Sentinel mice were screened every 3 months by Harlan Orlac (Blackthorn, UK) to exclude the presence of Helicobacter species and breeders were replaced after every experimental Hh infection. The experiments were approved by the animal use ethical committee of Oxford University and complied with UK Home Office guidelines.

2.2. Hh infection and quantitation

Hh NCI-Frederick isolate 1A (strain 51449) was grown as described previously [14]. Seven day old BALB/c mice from Helicobacter-free breeders were fed on two consecutive days with Hh 1A (∼2.5 × 10⁷ CFU) by oral gavage. Hh colonisation was analyzed in caecal contents collected upon sacrifice. DNA was isolated using the DNA Stool kit (QIAGEN) and SYBR Green real-time PCR with Hh-specific primers against the cdtB gene (Fwd: CCG CAA ATT GCA GCA ATA CTT; Rev: TCG TCC AAA ATG CAC AGG TG) was performed in triplicate using the CFX96 detection system (Bio-Rad Laboratories). Results represent arbitrary units normalised to uninfected control samples.

2.3. Ad85A immunisation

Four to five weeks after Hh administration, infected and matched control mice were immunised with 2 × 10⁹ virus particles of Ad85A equally divided between the two quadriceps muscles.

2.4. Infection with Mtb and determination of mycobacterial load

Five to ten mice were anesthetised with isoflurane and infected i.n. with Mtb (Erdman strain) in 40 μl PBS. The number of organisms deposited was determined 24 h after challenge (∼200 CFU). Mice were sacrificed 5 weeks post-challenge. Lungs were homogenised and 10-fold serial dilutions of tissue homogenates were plated on Middlebrook 7H11 agar plates (E&O Laboratories Ltd, Bonnybridge, UK) to determine mycobacterial load. Colony-forming units (CFU) were enumerated after 3–4 weeks of incubation at 37 °C in 5% CO₂.

2.5. Treatment with antibody to the IL-10 receptor

Naive or Hh infected mice were immunised with Ad85A i.m. or left unimmunised. Following challenge with Mtb, mice were injected intraperitoneally on day 0, 7 and 17 with 1 mg of anti-IL-10R antibody (clone 1B1.2) or PBS as control.

2.6. Histopathological assessment

Five weeks after Mtb challenge, lungs from infected mice were removed and fixed in buffered 4% formalin. 4–5 μm paraffin-embedded sections were stained with haematoxylin and eosin, and histopathology of the lungs was assessed semi-quantitatively in a blinded fashion by a trained pathologist.

2.7. Isolation of lymphocytes from lungs, spleen, gut and MLN

Lungs were perfused with PBS, cut into pieces and digested with 0.7 mg/ml collagenase type I (Sigma) and 30 μg/ml DNase I (Sigma) for 45 min at 37 °C. Digested fragments were crushed through a cell strainer using a syringe plunger, washed with PBS, layered over Lympholyte (Cederlane, Ontario, Canada) and centrifuged at 1000 × g for 25 min. Interface cells were collected and washed. Spleens and MLNs cell suspensions were prepared by mashing the tissue through a cell strainer using a syringe plunger. For spleens, red blood cells were removed with lysis buffer (Qiagen, Crawley, UK) and the cells were washed with PBS. For colonic lamina propria cell isolation, colons were opened longitudinally, washed in PBS 0.1% BSA and cut into pieces. Pieces were washed twice in HBSS supplemented with 4% FBS, 100 U/ml penicillin/streptomycin and 5 mM EDTA at 37 °C with shaking to remove epithelial cells. Tissue was then digested at 37 °C in a shaking incubator with 1 mg/ml type VIII collagenase (Sigma-Aldrich) and 0.5 mg/ml DNase I in RPMI-1640 medium supplemented with 4% FBS and 100 U/ml penicillin/streptomycin. The isolated cells were layered on a 40/80% Percoll gradient and the interface was collected and washed.

2.8. Flow cytometry

For surface staining, cells were washed and incubated with CD16/CD32 monoclonal antibody to block Fc binding. Cells were stained with a fixable viability dye and a combination of the following antibodies: CD45 (30-F11), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14) and CD25 (PC61.5) surface markers. For Tregs staining, cells were fixed and permeabilised with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions and stained with Foxp3 (FJK-16s). For intracellular cytokine staining in response to 85A stimulation, cells from Ad85A immunised animals were stimulated for 6 h with a pool of 66 15-mer peptides overlapping by 10 amino acids, covering the 85A protein sequence (Peptide Protein Research Ltd, Fareham, UK), in Hepes buffered RPMI with 10% heat-inactivated FCS, l-glutamine, penicillin and streptomycin. Each peptide was 2 at μg/ml. After 2 h at 37 °C, Golgi Plug (BD Biosciences, Oxford, UK) was added according to the manufacturer's instructions. Cells were stained for surface markers then for intracellular IFNγ (XMG1.2) and TNFα (MP6-XT22) (eBioscience, Hatfield, UK) using the BD Cytofix/Cytoperm kit according to the manufacturer's instructions. All cells were run on a LSRII (BD Biosciences) and analysed using FlowJo software (Tree Star Inc, Ashland, Oregon, USA).

2.9. Quantitation of gene expression using TaqMan qPCR

RNA was isolated from snap-frozen lung and colonic tissue using the RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions, including an on-column DNase I digestion step. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) for the candidate genes was performed using TaqMan gene expression assays (Life Technologies). cDNA samples were analysed in duplicate using the CFX96 detection system (Bio-Rad Laboratories) and values were normalised to Hprt according to the Δ-Ct method. TaqMan Gene Expression Assays were performed for mouse Hprt (Mm01545399_m1), Il10 (Mm00439614_ml), Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), Rorc (Mm01261022_m1) and Foxp3 (Mm00475162_m1).

2.10. Statistical analysis

Data were analysed using one-way ANOVA followed by Tukey's multiple comparison tests. Immune responses and histopathological scores were assessed by Mann–Whitney U with Bonferroni's post-test.

3. Results

3.1. Hh infection increases Mtb bacterial load and abolishes the protective effect of Ad85A immunisation

In order to understand how early life exposure to a bacterial trigger could influence the response to Mtb infection and vaccine efficacy, we assessed the impact of neonatal infection with Hh on Mtb challenge in mice. One week old BALB/C mice were infected with Hh orally and challenged intranasally at ten weeks of age with Mtb along with uninfected littermates. Mice were assessed for mycobacterial load in the lungs 5 weeks post-challenge (Fig. 1A). We observed that Hh infected animals had an average 0.6 log increase of Mtb CFU in the lungs compared to their uninfected littermates (Fig. 1B).

Fig. 1.

Protection against Mtb challenge. BALB/c mice were infected with Hh and 5 weeks later immunised with 2 × 10⁹ v.p. Ad85A i.m. Controls were uninfected naïve or uninfected Ad85A immunised animals. Mice were challenged with Mtb 5 weeks after immunisation and lung Mtb CFU were determined 5 weeks after challenge. (A) Schematic depiction of the treatment regimen. (B) Lung Mtb CFU. Symbols show individual mice and horizontal lines indicate the mean. *** p < 0.001, ** p < 0.01 between the indicated groups as determined by one-way ANOVA with Tukey's post-test. Data are representative of two independent experiments with similar results.

Similarly, groups of Hh infected or uninfected mice were immunised with Ad85A at weeks 6 of age and challenged 5 weeks later with Mtb (Fig. 1A). As previously reported, Ad85A immunisation efficiently reduced the Mtb colonisation level in the lungs of uninfected mice by ∼0.5 log₁₀ [4]. However, early-life infection with Hh strikingly abolished the protective effect of Ad85A (Fig. 1B).

Histological analysis shows that naive mice have multifocal coalescing granulomatous lesions composed of macrophages and lymphocytes with small aggregates of neutrophils and scattered multinucleate cells, making up 50–70% of the sections in most animals (Fig. 2A). In contrast, Ad85A immunised mice show significantly smaller areas of interstitial inflammation with a low number of lymphohistiocytic aggregates (Fig. 2B). Lungs from Hh-infected animals show multifocal lesions with mild interstitial infiltrates and foci of peribronchial lymphocytes, often with focal extravasated red blood cells (haemorrhage), mild oedema and congestion. Interestingly, the pathological appearance in Hh-infected animals immunised with Ad85A is similar to their non-immunised counterparts, but in several animals the inflammation is severe, with intra-alveolar inflammatory infiltrates in addition to areas of haemorrhage and oedema. Thus, the histopathological appearances reflect the abolition of the protective effect of Ad85A by Hh infection.

Fig. 2.

Histopathology. (A) Representative histopathology of the lungs of mice 5 weeks after Mtb challenge. Naïve mice are compared to the lungs of mice immunised with Ad85A or mice infected with Hh or infected with Hh and immunisd with Ad85A. Haematoxylin/eosin-stained paraffin-embedded lung sections. Bar is 100 μm. (B) Histograms show mean and standard error of semi-quantitative scoring of the extent of pathology in groups of 10 mice as 10–15%, 15–25%, 25–50%, 50–70% and >70%. ** p < 0.01, * p < 0.05 between the indicated groups by Mann Whitney U test.

3.2. Effect of Hh infection on antigen specific responses and lung cell composition

To characterise in more detail the effect of Hh infection on Ad85A immunisation, we measured 85A-specific responses in the lungs and spleen 4–5 weeks after immunisation, at the peak of the response to Ad85A. Intramuscular administration of Ad85A has previously been reported to induce a strong CD8 but weaker CD4T cell response [13,15] and both Hh-infected and uninfected control mice show a similar increase in the frequency of CD8⁺ effector memory T cells in the spleen following immunisation (Fig. 3A). In the lungs, the total number of leukocytes is similar in control and Hh-infected mice immunised with Ad85A and the frequencies of CD4 and CD8 cells are unchanged (Fig. 3B). However, while in uninfected mice immunised with Ad85A, 4–5% of CD8 and ∼0.2% of CD4 lung T cells produce IFNγ and TNF upon antigenic restimulation with 85A peptides, 85A-specific CD8T cell IFNγ responses are significantly reduced in immunised mice that have been previously infected with Hh (Fig. 3C).

Fig. 3.

Antigen 85A specific T cells responses. BALB/c mice were infected with Hh and 5 weeks later immunised with Ad85A. Controls were uninfected naïve or uninfected Ad85A-immunised animals. Lung and splenic lymphocytes were isolated 4 weeks post immunisation. (A) Frequencies of CD44^hi CD62L^lo effector memory T cells among splenic CD8⁺ and CD4⁺ T cells and representative flow-cytometry staining gated on live CD45⁺ CD3⁺ CD8⁺ T cells. (B) Absolute numbers of lung leukocytes and frequencies of lung CD8⁺ and CD4⁺ T cells among total CD45⁺ cells. (C) Frequency of lung of lung CD8⁺ and CD4⁺ T cells expressing IFNγ and TNFα determined by flow-cytometry following 6 h in vitro stimulation with 85A peptides. Data points represent individual mice and horizontal bars represent medians. * p < 0.05 as determined by one-way ANOVA with Tukey's post-test.

Taken together, these results indicate that Hh infection of neonatal BALB/c mice increases the lung mycobacterial load after Mtb challenge. Furthermore, Hh infection reduces antigen specific lung immune responses after Ad85A i.m. immunisation and abolishes protection against Mtb challenge.

3.3. IL10 is a key determinant of the Hh-mediated suppression of Ad85A vaccine-induced protection against Mtb challenge

IL10 plays a key role in preventing pathological intestinal inflammation induced by Hh [10–12] and IL10^−/− mice infected with Hh develop severe inflammation in the caecum and colon. In contrast, Hh-infected wild-type mice mount an antigen-specific regulatory T cell response to the bacterium that prevents bacteria-induced colitis in an IL10-dependent manner [16]. We therefore examined the expression of Il10 mRNA in the colon and lungs of naive and Hh-infected mice. We first confirmed that Hh-infected animals are similarly colonised by Hh eight weeks after their initial infection (Fig. 4A). Both unimmunised and Ad85A-immunised mice infected with Hh display significantly up-regulated IL10 mRNA expression levels in the colon but not the lungs (Fig. 4B), with a trend towards reduced T-bet and GATA3 mRNA expression, transcription factors associated with Th1 and Th2 cell differentiation, respectively (Fig. 4C). However, Hh infection does not affect Foxp3 tissue expression level (Fig. 4D) or the frequency of Foxp3⁺CD4⁺ regulatory T cells in the colon, lung, spleen and mesenteric lymph nodes (Fig. 4E) in Ad85A-immunised mice.

Fig. 4.

Protection against Mtb challenge depends on IL10R signalling. Control or neonatally Hh-infected BALB/c mice were immunised with Ad85A and were compared to unimmunised controls. (A) Hh colonisation levels quantified by SYBR qPCR (arbitrary units). (B–D) IL10, T-bet, GATA-3 and RORγt mRNA expression of total colonic and lung tissue quantified by TaqMan qPCR and normalised to Hprt. (E) Frequencies of CD4⁺ CD25⁺ Foxp3⁺ regulatory T-cells among total live CD45⁺ cells in the colon, lungs, mesenteric lymph nodes (MLNs) and spleen. Data are representative of or pooled from two independent experiments. (F) Naive or Hh infected mice were immunised with Ad85A or left unimmunised. Following challenge with Mtb, mice were treated with anti-IL10R mAb or PBS on day 0, 7 and 17. Lung CFU were evaluated 5 weeks post challenge. Symbols show individual mice and horizontal lines indicate the means. ** p < 0.01, * p < 0.05 between the indicated groups as determine by Mann–Whitney U test (A–E) or one-way ANOVA with Tukey's post-test (F).

Because IL10 has been shown to play an important role in regulating immunity to Mtb infection [17], we investigated whether this pathway was involved in the Hh-mediated abrogation of parenteral Ad85A-induced protection. We therefore assessed the effect of IL10R signalling blockade during Mtb challenge in control, uninfected immunised or Hh-infected immunised mice, using a treatment regime shown capable of inducing colitis in normal mice infected with Hh [16]. Anti-IL10R treatment of unimmunised naive mice does not alter lung Mtb CFU numbers, nor does it affect protection in uninfected mice immunised with Ad85A (Fig. 4F). However, the protective effect of Ad85A is restored in Hh-infected mice treated with anti-IL10R, strongly suggesting that IL10 is a crucial determinant of the Hh-mediated suppression of Ad85A vaccine protection against Mtb challenge.

4. Discussion

BCG has long been known to vary in efficacy in different geographical locations, an effect often ascribed to exposure to EM [18]. As many candidate TB vaccines are now entering clinical trials [19], it will be important to determine whether these will also show variable efficacy in different locations and whether this variability is linked to EM exposure only or reflects larger differences in the gut microbiota composition across populations. Understanding the mechanisms underlying variable vaccine efficacy may allow the development of strategies to overcome it.

Experimentally, parenteral administration of EM to mice does not affect protection induced by subunit vaccines (ESAT-6 or Ag85B-ESAT-6 proteins), also administered parenterally [3]. In contrast, concurrent oral administration of EM or BCG abrogates protection induced by the parenterally administered subunit vaccine, Ad85A [4]. That oral but not parenteral administration of EM affects protection is in agreement with recent observations indicating the importance of the intestinal microbiota in both, development of the immune system and immune regulation.

In germ free mice most populations of lymphocytes are greatly reduced in number, while infection with host specific microbiota leads to re-population of the immune system [20]. However, this is not true of all lymphocyte populations; for example, CXCR6⁺ iNKT cells are specifically increased in the colon and lung of germ free mice, due to higher expression of CXCL16 at mucosal surfaces [21]. The link between intestinal and lung immunity is supported by many other studies and manipulation of the microbiota by administration of a single bacterial species can greatly alter immune responses. For example, monocolonisation of germ-free animals with Bacteroides fragilis increases the induction of IL10-producing Tregs [22]. Furthermore, there is an inverse relationship between numbers of Foxp3⁺ Tregs and Th17 cells in the intestinal lamina propria [23] and differentiation of Th17 cells, which are important in protection against bacterial and fungal infections at mucosal surfaces, is critically dependent on the presence of segmented filamentous bacteria in the intestine [24]. These data indicate that production of two cytokines, IL10 and IL17A that play important roles in immunity to TB [17,25] is regulated by components of the intestinal microbiota.

In other experiments, neonatal H. pylori infection of mice has been shown to prevent the induction of lung allergic responses through the reprograming of dendritic cells and induction of highly suppressive Tregs in an IL10-dependant manner [8,26]. IL10 also plays a key role in preventing the pathological colonic inflammation induced by infection with Hh [10–12], an effect attributed mainly to the induction of Hh antigen-specific regulatory T cells [16]. However, additional mechanisms may be involved, since Hh infection leads to its dominant presence among the local bacterial pool and has been associated with a decreased overall diversity of the intestinal micobiota [27].

Irrespective of whether the effect is directly attributable to Hh itself, here we show that neonatal infection of BALB/c mice with Hh up-regulates IL10 mRNA expression within the colonic lamina propria and increases susceptibility to later Mtb challenge, as evidenced by higher lung Mtb CFU. Hh-infected mice immunised parenterally with the recombinant subunit vaccine Ad85A further display reduced protection against Mtb challenge compared to their non-infected counterparts, an effect that is dependent on IL10R signalling.

IL10 has been shown to modify protective immunity to several organisms including Mtb. In Mtb infection there are two main effects. Protection against infection is generally increased in IL10 insufficient mice and mycobacterial counts are lower. This is thought to be mainly due to the effects of IL-10 on myeloid cells, which become less responsive to IFNγ activation and therefore less able to kill mycobacteria, while antigen presentation to T cells and induction of Th17 responses are also inhibited. In contrast, IL10 may dampen down Th1-mediated protective responses later in infection and lessen immune mediated pathology. However these effects are both mouse and Mtb strain dependent as well as subject to environmental effects (reviewed in [17]).

In our experiments, Hh infection alone increases significantly Mtb lung CFU after challenge, a result in accord with the known effects of IL10 on Mtb infection. However, the abolition of parenteral Ad85A-induced protection is much more surprising (Fig. 1B). The exact mechanisms of this reduction of vaccine-induced protection remain to be determined, as there was no increase in lung Il10 message. In an earlier study, protection induced by parenteral but not intranasal administration of Ad85A was abolished by oral administration of EM, suggesting that priming of Ad85A specific cells in peripheral lymph nodes or migration of cells into the lung, rather than expression of local immunity, may be affected. Recent experiments on the effects of intestinal flagellin-bearing bacteria on systemic immune responses suggest that the mechanism involves changes in myeloid cell function in lymph nodes [28]. Here we show here that following parenteral immunisation, numbers of antigen specific CD8⁺ T cells (the principal population induced by Ad85A in BALB/c mice) in the lung are reduced in Hh infected mice. However it remains to be determined whether this reduction is due to impaired priming in systemic lymph nodes or if altered migration is responsible.

Irrespective of the exact mechanisms involved, this is the first direct evidence that experimental Hh infection can profoundly influence the magnitude and protective efficacy of immune responses to Ad85A, a candidate TB vaccine, which has already entered clinical trials [29]. These data indicate that it may be expected that not only BCG but subunit vaccines against TB will suffer from geographical variation in efficacy. Furthermore these results add to the arguments for further exploring the use of the pulmonary route for administration of TB vaccines [30], since at least under some circumstances this can overcome the suppressive effect of alterations in intestinal flora [4].

Conflict of interest statement

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following are the supplementary data to this article: