- Research article
- Open Access
Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
BMC Microbiology volume 14, Article number: 9 (2014)
The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection.
We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice.
Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.
Tuberculosis (TB) is most prevalent in resource-poor countries and factors such as genetic susceptibility, malnutrition and circulating strain differences have been implicated as determinants of TB disease development in these regions [1, 2]. Compelling evidence demonstrates that many of these factors increase disease risk partly though the induction of host immune dysregulation and ultimately affect host control of Mycobacterium tuberculosis (M. tb) proliferation . The high prevalence of parasitic helminth infections in TB affected communities, has highlighted co-infection as another risk factor compromising host immunity and thus a potential determinant for development of TB [4, 5]. In support of this theory, several reports indicated that TB patients are commonly found to be co-infected with helminth species such as Trichuris trichiura and Ascaris lumbricoides and present with increased total and helminth-specific serum immunoglobulin E (IgE) .
Host control of mycobacterial or helminth infections largely rely on the induction of appropriately polarized immune responses. Protective immune responses to M. tb infection are associated with enhanced T helper 1 (TH1) type cellular immunity and the production of characteristic TH1 cytokines such as tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-12 (IL-12) . Conversely, protection against most helminths requires a T helper 2 (TH2) type cellular immune response with production of distinct TH2 cytokines such as IL-4, IL-5, IL-13 and IL-9 [9, 10]. Since TH1 and TH2 immune responses have the ability to concurrently down-regulate each other, a state of co-infection could result in inappropriate protective host immune responses to either infections . Furthermore, both pathogens have the potential to induce regulatory T cell (Treg) responses associated with production of immune suppressive cytokines such as IL-10 and transforming growth factor beta (TGF-β) [10–13].
In line with the TH1/TH2 dichotomy, hypotheses concerning helminth-mycobacterial co-infection postulate that a helminth-induced TH2 immune bias could inhibit development of protective cellular immune responses to M. tb, increase mycobacterial proliferation or lead to the failure of vaccine strategies against TB [14, 15]. This theory is supported by numerous studies which have reported a reduction in TH1 responses to be associated with poor outcomes in TB patients  and latently infected individuals  with concurrent helminth infection. Helminth-induced regulatory (Treg) responses such as TGF-β and IL-10 production have also been implicated in S. mansoni-induced progression to active TB of HIV-1 infected Ugandans . It was also established that deworming of helminth-infected individuals restores cellular immune responses to mycobacterial purified protein derivatives (PPD) [19–21]. Similarly, deworming of helminth-infected Ethiopians immigrants in Israel resulted in increased cellular immune responses against HIV- and M. tb-specific antigens compared to untreated individuals , suggesting deteriorating immune responses and poor clinical outcomes in helminth-infected individuals might not be a result of inadequate nutrition or sanitation. Several reports have also indicated helminth-mediated modulation of vaccine responses. Children with prenatal sensitization to filariae and schistosomes were reported to display a down-regulation in TH1 responsiveness to BCG vaccination  and animal co-infection models have further demonstrated that a pre-existing infection with a lung-migrating helminth, can inhibit development of protective innate anti-TB responses by inducing the IL-4 receptor pathway and accumulation of alternatively activated macrophages . In summary, most reports indicate that helminth infection significantly affects TB susceptibility. In contrast, very little data addressing the effect of mycobacterial infection on host immunity to helminth infections are available.
In the current study, we assessed the influence of co-infection on immune responses against the individual pathogens. We established a BALB/c co-infection model using Mycobacterium bovis (M. bovis) BCG and the gastrointestinal tract-restricted rodent helminth, Trichuris muris (T. muris) as TH1 and TH2 pathogenic assaults, respectively. The M. bovis BCG murine infection model is routinely used for studying anti-mycobacterial responses during latency as the associated immune response is similar to that induced during human M. tb infection , whereas T. muris infection serves as a well described model for gastrointestinal tract restricted human soil-transmitted helminth (STH) infection . We explored the possibility that concurrent infection with two pathogens, normally cleared by mice during single pathogen infection, might lead to mutually inhibitory immune dynamics and subsequent uncontrolled infection.
Specified pathogen free (SPF) female BALB/c mice (WT and IL-4 knock-out strains) between 6–8 weeks of age, were kept at the Faculty of Medicine and Health Sciences Animal Unit, Stellenbosch University (SU; South Africa) under conditions compatible with the SU guidelines for the care of animals. All procedures were approved by the SU Animal Ethics Board [Project license: 2003/186/p].
Parasite enumeration and antigen preparation
T. muris eggs were donated by Allison Bancroft (University of Manchester, UK). Egg propagation in BALB/c IL-4 knock-out mice (gift from Frank Brombacher, University of Cape Town, South Africa), helminth collection, and excretory/secretory (E/S) antigen preparations, were performed as described previously [27, 28]. Helminth burdens were determined by quantification of intestinal adult worms by examining faecal matter under a dissection microscope. Mycobacterium bovis BCG Pasteur (donated by Robin Warren, SU, South Africa) was propagated to logarithmic growth phase in Middlebrook 7H9 (Difco) liquid culture, supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Merck) at 37°C. Bacterial proliferation was assessed by manual counting of colony forming units (CFU) from serial dilutions of homogenized lungs and spleens, plated on Middelbrook 7H11 (Difco) agar plates supplemented with 0.2% glycerol and 10% oleic acid-albumin-dextrose-catalase (OADC, BD Biosciences).
Two infection protocols were used during this study. Each experiment consisted of 3 groups of 5–10 animals per group. Groups included M. bovis BCG-T. muris co-infected, BCG-only infected and T. muris- only infected mice. The first protocol (Figure 1A) was intended to establish a chronic, low grade M. bovis BCG infection that was subsequently followed by a TH2-inducing T. muris infection. Mice were infected intranasally (i.n.) with 1–5 × 105 CFU BCG bacilli per mouse or an equal volume of PBS. Briefly, mice were lightly anesthetized by intraperitoneal (i.p.) injection of a 200 μl mixture consisting of Ketamine (12 mg/ml Anaket-V, Centaur Labs) and Xylazine (1.6 mg/ml, Rompun, Bayer). Mice were gently lifted by the loose skin at the throat, and kept upright with its head tilted back and the nose pointed up. Using a pipette with a sterile tip, 40 μl of the declumped mycobacterial suspension was applied to the nostrils. Animals were maintained upright for another 30 seconds to ensure complete delivery to the respiratory system. Six weeks (day 42) later, mice were infected under light anaesthesia intragastrically (i.g.) with 200–250 (low dose) or 500–600 (high dose) embryonated T. muris eggs or an equal volume of PBS. At week 9 (day 63), mice were culled and the relevant organs removed for investigation. The second protocol (Figure 1B) was designed to first establish a TH2-inducing T. muris infection prior to challenge with M. bovis BCG infection. Animals were infected i.g. with 200–250 embryonated T. muris eggs or an equal amount of PBS on day 1 and every 10 days thereafter until experimental completion. On day 10, animals were infected i.n. with 1–5 × 105 CFU BCG bacilli or an equal volume of PBS. After 6 weeks (day 52), all mice were humanely euthanized and the relevant organs removed for investigation. Experiments were completed in triplicate at three separate times.
Immune phenotyping and intracellular cytokine analysis
Immune phenotyping was performed using single cell suspensions from spleens and mesenteric lymph nodes (MLNs). Intracellular cytokine expression was determined following stimulation with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma), 1 μg/ml Ionomycin (Sigma) and 10 μg/ml Brefeldin A (BFA) (Sigma) for 4 hours at 37°C and 5% CO2. Cells were resuspended in PBS containing 1% BSA and 0.1% Sodium Azide (wash buffer) and stained for 30 minutes with fluorochrome conjugated anti-mouse antibodies against CD3, CD4, CD8, CD25, B220, Foxp3, IFN-γ and IL-4 (BD Biosciences, Caltag or Biolegend). Cells were fixed with 1% formaldehyde, washed and resuspended in wash buffer. Lymphocyte populations were determined based on their Forward/Side scatter profile and gates set with the help of appropriate FMOs and Isotype controls. Acquisitions were performed on a FACSCalibur (BD Biosciences) using appropriate instrument settings, color compensation and isotype controls for all antibodies. At least 5 × 104 lymphocyte events were acquired and data analysis performed using CellQuest software (BD Bioscience).
In vitro pathogen-specific cytokine analysis
Spleen (1 × 107 cells/ml) single cell suspensions were stimulated for 24 hours with live BCG cultures (MOI 5:1), 50 μg/ml E/S antigen or culture media as control at 37°C, 5% CO2. Culture supernatants were used for cytokine concentration analyses using the luminex bead-array technology (LINCO Research) to test for the soluble cytokines IFN-γ, TNF-α, IL-4, IL-10, IL-13 and IL-17 using a Bio-Plex platform (Bio-Rad Laboratories). Background readings were controlled by subtraction of unstimulated control sample measurements. Values were checked against internal quality controls to monitor analysis accuracy within specified concentration ranges.
Nucleic acid extraction and relative quantitative real time PCR
Total RNA was extracted from the upper right lobe of mouse lungs and spleen tips using Trizol (Gibco BRL) and subsequently treated with a DNA-free kit (Ambion) to remove contaminating DNA. First strand cDNA was transcribed using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s protocols. Relative quantification of IFN-γ, IL-4, IL-10, TGF-β and Foxp3 were performed using SYBR Green PCR Master Mix kit (Roche), cDNA (500 μg) and primers (0.5 μM) on the LightCycler system v3.5 (Roche). All primers were designed to span intron-exon boundaries (Table 1). The delta-delta Ct method was used to calculate relative gene expression levels between two samples. Gene expression was assayed quantitatively and normalized to that of a housekeeping gene (GAPDH, HPRT, 18S-RNA) to obtain a RNA ratio in order to establish the relevant change in RNA expression .
Left upper lung lobes were fixed in 10% buffered formalin, embedded in paraffin blocks and sections (3-5 μm) stained with Haematoxylin and Eosin (H&E) for light microscopy. Pulmonary histopathological scoring was performed in a blinded fashion and calculated separately for each lung section as previously described . In brief, a scale of 0 to 4 was used to individually score the level of peribronchiolitis, perivasculitis, interstitial pneumonitis and alveolitis of each section in order to obtain an average score for each lung. A score of 0 was based upon observation of normal, uninfected mouse lung samples and a score of 4 on previous studies of greatest inflammatory change and pathology brought about by i.n M. bovis BCG infection in BALB/c mice. Scoring of gastrointestinal histopathology was achieved by measuring mucus production, presence of mast cells and mitotic body enumeration in fixed caecum tips imbedded in paraffin blocks. Sections (3-5 μm) were used for Periodic Acid Schiff (PAS) staining to score goblet cell-mucus production within caecal crypts as the percentage PAS positive stain in the crypt epithelium and lamina propria. Acidified toluidine blue staining was used for the quantification of mast cells in caecum tip samples and enumeration of mitotic bodies within caecum crypts. Scoring was conducted from two sets (cross sectional and longitudinal) of 20 caecal crypt units per animal. All slides were evaluated using the ZS300 Imaging system v.3.0 (Carl Zeiss Vision).
Data was analyzed using STATISTCA v.7 (StatSoft) software. Nonparametric analysis and Mann–Whitney U tests were performed for comparison between groups and the data presented as median values. Multiple group analysis included the multiple comparison correction (Bonferroni). Statistically significant differences were judged as p ≤ 0.05.
M. bovis BCG clearance and lung pathology is not influenced by an established or successive T. muris infection
The influence of T. muris infection on host ability to control a chronic, low grade M. bovis BCG infection in BALB/c mice was investigated for both experimental protocols (Figure 1A and B). Results demonstrated that an ongoing helminth-induced TH2 immune background, pre-established by T. muris trickle infection, failed to alter mycobacterial proliferation and dissemination when compared to M. bovis BCG-only infected mice in the lungs (Figure 2A) and spleen (data not shown). Similarly, initiation of a TH2 immune environment subsequent to BCG infection, resulted in equivalent pulmonary bacterial burdens between co-infected and BCG-only infected groups (Figure 2B). These end point CFU findings were confirmed by growth curve data demonstrating no significant difference in pulmonary mycobacterial burden between co-infected and M. bovis BCG-only infected mice at several time points post M. bovis BCG infection (Figure 2C). Histological scoring of both infection protocols indicated that T. muris-only infected mice displayed normal lung pathology with only minimal cell infiltration compared to naive mice, whereas the degree of pulmonary pathology and the cellular composition and organization in the lungs following M. bovis BCG co-infection were significantly increased (Figure 2D and E). No significant differences in pulmonary inflammatory scores could be detected between BCG-only and co-infected mice for either infection protocols (Figure 2D, 2E and Additional file 1: Figure S1).
Previously established BCG infection delays T. muris expulsion in co-infected animals
The influence of M. bovis BCG co-infection on eradication of T. muris in BALB/c mice was evaluated as worm expulsion for both experimental protocols (Figure 1A and B). In each case, susceptible IL-4KO mice with disrupted protective TH2 responses, were included as controls of delayed worm clearance . Following the infection strategy in Figure 1A, the helminth burden at experimental completion demonstrated that almost half (44%; 4/9) of mice with an established chronic BCG infection, that were subsequently co-infected with a low dose of helminth eggs, still presented with T. muris, whereas significantly more animals (88%; 7/8) from the T. muris-only infected group had cleared all helminths (Figure 3A). Both groups displayed significantly lower worm burdens compared to IL-4KO mice infected with T. muris only (Figure 3A). Similar results were observed in experimental repeats using a high dose of helminth eggs, showing helminth clearance in (100%; 0/9) T. muris-only infected BALB/c mice, whereas T. muris expulsion failed in (40%; 4/10) M. bovis BCG co-infected BALB/c mice (Figure 3B). However, when the infection sequence was reversed, where an initial T. muris infection was followed by a subsequent BCG infection (Figure 1B), repeat experiments consistently indicated helminth clearance in >90% of both co-infected and T. muris-only infected mice (data not shown).
Co-infection exacerbates cell proliferation in caecum tips
A striking observation was the massive amount of mucus present in the caeca and colons of mice co-infected according to either experimental protocol (Figure 1A and B) in comparison to T. muris-only infected mice. Although PAS stained samples failed to demonstrate significant differences in goblet cell formation or caecal crypt-mucus production between co-infected and T. muris-only infected mice (Figure 4A), acidified toluidine blue staining showed significantly increased numbers of mitotic figures in caecum crypts of co-infected animals as identified by their dense chromatic structure (Figure 4B). Very few mast cells were observed within the epithelium or lamina propria of the crypt units in co-infected mice and no significant statistical differences in mast cell recruitment were observed between infection groups (Figure 4C).
Co-infection increases CD4+ splenocyte frequencies and modifies the TH1/TH2 immune balance
Flow cytometric analysis demonstrated that co-infection according to either infection protocol (Figure 1A and B) did not impact lymphocyte composition in the spleen or MLN, since no significant differences between infection groups were observed for populations of CD3+ T cells or B220+ B cells (data not shown). However, analysis of ex-vivo lymphocyte subpopulations in BALB/c mice infected according to Figure 1A, revealed an increase in CD4+ T helper cell population in the spleens of mice co-infected according to the protocol in Figure 1A, when compared to BCG-only infected mice (Figure 5A). Although no differences in the percentages of natural regulatory T cell (CD4+CD25+Foxp3+) populations were observed between infection groups in either the spleen or MLN (data not shown), co-infection significantly increased the percentage of IL-4-producing CD4+ and CD8+ splenocytes in comparison to M. bovis BCG-only infected controls (Figure 5B). IL-4-producing CD4+ and CD8+ MLN cells from co-infected mice were however significantly reduced in comparison to T. muris-only infected mice (Figure 5C). A marked decrease in CD8+IFNγ+ MLN cells was also observed in co-infected mice in comparison to mice infected only with T. muris, whereas frequencies of CD4+ IFNγ+ MLN cells were measured at similar levels between co-infected and T.muris-only infected mice (Figure 5D).
When the infection order was reversed during trickle infection to address the effect of introduction of co-infection with M. bovis BCG into an established helminth-induced TH2 environment (Figure 1B), a significant increase in activated effector T cell (CD4+CD25+Foxp3-) percentages in MLNs of co-infected animals was observed in comparison to T. muris-only infected controls (Figure 5E). A trend towards decreased frequencies of inducible regulatory T cells (iTreg) (CD4+CD25-Foxp3+) was also observed in the MLNs of co-infected compared to T. muris-only infected mice (Figure 5F). No significant differences in ex vivo cytokine production between infection groups were observed for CD4+ and CD8+ lymphocytes in the spleen or MLNs (data not shown).
Co-infection reduces pathogen-specific TH1 and TH2 immune responses
Pathogen-specific TH1/TH2/TH17/Treg cytokine immune responses in the spleen were analyzed only in BALB/c mice infected according to the protocol in Figure 1A, since no significant differences in ex vivo T cell cytokine production between infection groups were observed in the spleens or lungs of mice infected according to the protocol in Figure 1B.
E/S stimulated splenocytes from both co-infected and BCG-only infected mice displayed a prominent reduction in TH2/Treg (IL-4, IL-13 and IL-10) cytokine production when compared to T. muris-only infected animals, although IL-4 levels were significantly increased in co-infected compared to BCG-only infected mice (Figure 6A). Similarly, E/S-specific TH1 cytokines (TNF-α and IFN-γ) were reduced in both the co-infected and BCG-only infected groups with respect to T. muris-only infected animals (Figure 6A). No notable differences between the infection groups were observed for helminth-specific IL-17 production (data not shown).
BCG-stimulated splenocytes displayed notably low concentrations of TH2 (IL-4 and IL-13) cytokines in all infection groups. Although no significant differences in concentrations of the cytokines, IFN-γ and IL-17 (Figure 6B) were measured between infection groups, co-infection significantly decreased production of the cytokines TNF-α, IL-10 and IL-4 in comparison to T. muris-only and/or BCG-only infected mice (Figure 6B).
Co-infection reduces the pulmonary cytokine gene expression profile relative to BCG-only infected animals
To assess whether the immunological changes observed in mice infected according to the infection protocol indicated in Figure 1A, also extends to alterations in pulmonary and splenic gene expression levels, the relative gene expression of co-infected mice and BCG-only infected mice was determined. At week 9, the relative gene expression ratios from co-infected mice demonstrated significantly decreased RNA levels in the lungs for TGF-β (p = 0.034), Foxp3 (p = 0.042) and IFN-γ (p = 0.012) relative to BCG-only infected mice (Figure 7). The levels of IL-10 (p = 0.072) also showed a trend towards decreased expression across these two groups (Figure 7). Analysis of RNA profiles in the spleen failed to show significant variations in expression levels for any of the genes measured, between co-infected and BCG-only infected groups (data not shown).
In this study, we demonstrate the capability of the gastrointestinal tract restricted helminth, T. muris, to induce local and systemic TH2 immune responses that affect immunity to M. bovis BCG. Of particular interest was the significant reduction in BCG-specific TNF-α and IL-10 cytokine concentrations and significant increase in IL-4-producing CD4+ and CD8+ T cells in the spleens of co-infected mice, in comparison to BCG-only infected mice. In addition, we show that co-infection significantly reduced pulmonary IFN-γ, TGF-β and Foxp3 gene expression, relative to BCG-only infected mice. Collectively, our data show a down-regulation in pulmonary TH1 and Treg-associated responses and the induction of systemic TH2 responsiveness following co-infection. Nevertheless, lung and systemic bacterial burdens remained unaffected in co-infected mice and did not translate into alterations in pulmonary histopathology with respect to BCG-only infected mice, suggesting that protective host immune responses could be sufficiently compartmentalized to appropriately respond to the mycobacterial infection. Previous reports have demonstrated the host’s ability to fully compartmentalize immunity during co-infection with TH1 and TH2-inducing pathogens at different sites of the mammalian body . While helminth co-infection has been shown to negatively influence host control of other intracellular pathogens, several reports suggest that this outcome is specific to the helminth species investigated [35–38]. Even so, T. muris infection marginally increased pulmonary cellular infiltration with respect to naive mice, likely due to systemic inflammation caused by the helminth infection or the presence of helminth antigens. Although not discussed here, work done by us shows that neither adoptive transfer of splenocytes or MLN leukocytes from helminth-only infected animals, or abrogation of IL-4 in IL-4 deficient mice, resulted in altered mycobacterial burden (unpublished data). These transfer experiments could however not exclude a role for suppressive MLN or spleen cell subsets since purified populations were not used in these experiments. Also, the timing of transfer and the absence of continual pathogen-derived antigen stimulation in the recipient host could play a role in the effector responses and activation status of these cells.
Interestingly, our results show that prior pulmonary M. bovis BCG infection also significantly affected local and systemic protective host immune responses to a subsequent T. muris infection. Although the lack of ex vivo phenotyping data from BCG-only infected mice is a weakness in this infection protocol, co-infected mice displayed a significant reduction in E/S-specific TH1 and TH2 cytokine responses in the spleen, and significantly reduced IL-4 producing CD4+ and CD8+ T cells and IFN-γ-producing CD8+ T cells in the mesenteric lymph nodes when compared to T. muris-only infected mice. In support of a functional role for this reduction in T. muris-specific immunity, we demonstrated an associated delay in helminth clearance and increased helminth-related intestinal pathology in co-infected mice, when compared to T. muris-only infected mice. These intestinal pathological changes were characterized by increased cell turnover, suggesting increased apoptosis or cell damage, necessitating cell replacement . Intestinal crypt cell apoptosis was previously reported to occur following T. muris infection and subsequently shown to be reduced following neutralization of IFN-γ and TNF-α . In parallel with this we observed an increase in intestinal mucus production, which likely operates as a compensatory mechanism to aide expulsion of persisting parasites. Our results verify reports illustrating that M. bovis co-infection increase helminth parasite burden and correlates with decreased IL-4 and IL-13 cytokine production . Our findings also agree with early reports demonstrating a reduction in protective immune responses and a delay in T. muris expulsion during other co-infections with Nematospiroides dubius, Plasmodium berghei or Trypanosoma brucei[42–44]. It is well established that resolution of T. muris infection is characterized by the production of TH2 cytokines, resulting in intestinal goblet cell hyperplasia and increased intestinal epithelial cell turnover [45, 46]. On the other hand, mast cells, γδ T cells and eosinophils are suggested as dispensable for T. muris expulsion [45, 47] and the contribution of B cells and antibody responses remains controversial [48–50]. Previous reports convincingly show that T. muris infection is delayed following depletion of CD4 T cells , inhibition/down-regulation of TH2 cytokines [33, 45] and increased TH1 polarization . It is therefore likely that our observation of reduced helminth-specific TH2 responses in this co-infection model could, at least in part, explain the delay in T. muris expulsion, although induction of TH1 immune responses to M. bovis BCG following T. muris infection would also influence parasite expulsion. Interestingly, altering the infection sequence to elucidate the effect of a subsequent mycobacterial infection on an established helminth-induced TH2 immune response did not have any negative influence on mycobacterial or helminth clearance by the host. This is most likely to be due to the rapid clearance of the helminth infection and development of resistance to re-infection, or due to the presence of an established TH1 immune response for altering helminth clearance .
These modified pathogen-specific and non-specific immune responses following co-infection provide clear evidence that both pathogens have the ability to reciprocally modulate immune responses towards each other at their individual infection foci. More importantly, the down-regulation of overall immune responsiveness in the context of both infections suggests co-infection-induced immune suppression as a possible mechanism. Several reports confirm that chronic immune activation during helminth infections could initiate immune suppression or anergy . Here, we show significant increases in the frequency of systemic CD4+ T cells and effector T cells in MLN of co-infected animals, suggesting increased immune activation following co-infection. Although the presence of immune suppressive regulatory cell populations was investigated, no differences in the frequencies of Treg populations could be detected between infection groups in either of the BALB/c co-infection models. As Treg cells exert their suppressive function in a cytokine dependent manner and also interact with other T cells and APC directly, the implications of co-infection on regulatory immune mechanisms are not clear. Changes in IL-10, Foxp3 and TGF-β gene expression reveal that the role of Tregs cannot be excluded. Our results could point towards a role for other immune regulatory cell populations, and current research efforts are focused towards the involvement of innate nuocytes and myeloid derived suppressor cells (MDSCs) [54, 55].
In summary, the work presented here supports the hypothesis that co-infection by two unrelated and anatomically separated pathogens can reciprocally alter the host’s immune response to either infection. Co-infection altered host pathology and the host’s ability to expel invading helminth parasites; however the magnitude of the impact was dependent on the sequence of co-infection. These phenotypic changes were associated with alterations in organ-restricted TH1/TH2/Treg immune balance, immune suppression and pathogen-specific and non-specific cytokine responses. It is likely that multiple mechanisms may operate concurrently and further research is needed to identify the critical factors involved, although our results strongly support a mechanism whereby chronic immune activation leads to hyporesponsiveness resulting in reduced pathogenic control during co-infection. These findings demonstrate the complexity of immune response regulation and systemic interaction between innate and adaptive immunity and thereby hightlights the need for greater understanding of the role of infection history on the evolution of host immunity.
Hendrik J Nel and Nelita du Plessis co-first author.
Bellamy R: Genetic susceptibility to tuberculosis. Clin Chest Med. 2005, 26: 233-246. 10.1016/j.ccm.2005.02.006. vi
Hanekom M, van Pittius NC G, McEvoy C, Victor TC, Van Helden PD, Warren RM: Mycobacterium tuberculosis Beijing genotype: a template for success. Tuberculosis. 2011, 91: 510-523. 10.1016/j.tube.2011.07.005.
Schluger NW, Rom WN: The host immune response to tuberculosis. Am J Respir Crit Care Med. 1998, 157: 679-691. 10.1164/ajrccm.157.3.9708002.
WHO | The world health report 1999 - making a difference.http://www.who.int/whr/1999/en/index.html,
Elias D, Mengistu G, Akuffo H, Britton S: Are intestinal helminths risk factors for developing active tuberculosis? . Trop Med Int Health. 2006, 11: 551-558. 10.1111/j.1365-3156.2006.01578.x.
Hotez PJ, Molyneux DH, Fenwick A, Ottesen E, Ehrlich Sachs S, Sachs JD: Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med. 2006, 3: e102-10.1371/journal.pmed.0030102.
Adams JF, Schölvinck EH, Gie RP, Potter PC, Beyers N, Beyers AD: Decline in total serum IgE after treatment for tuberculosis. Lancet. 1999, 353: 2030-2033. 10.1016/S0140-6736(98)08510-9.
Flynn JL, Chan J: Immunology of tuberculosis. Annu Rev Immunol. 2001, 19: 93-129. 10.1146/annurev.immunol.19.1.93.
Dixon H, Blanchard C, Deschoolmeester ML, Yuill NC, Christie JW, Rothenberg ME, Else KJ: The role of Th2 cytokines, chemokines and parasite products in eosinophil recruitment to the gastrointestinal mucosa during helminth infection. Eur J Immunol. 2006, 36: 1753-1763. 10.1002/eji.200535492.
Yazdanbakhsh M, van den Biggelaar A, Maizels RM: Th2 responses without atopy: immunoregulation in chronic helminth infections and reduced allergic disease. Trends Immunol. 2001, 22: 372-377. 10.1016/S1471-4906(01)01958-5.
Maizels RM, Balic A, Gomez-Escobar N, Nair M, Taylor MD, Allen JE: Helminth parasites–masters of regulation. Immunol Rev. 2004, 201: 89-116. 10.1111/j.0105-2896.2004.00191.x.
McKee AS, Pearce EJ: CD25 + CD4+ Cells contribute to Th2 polarization during helminth infection by suppressing Th1 response development. J Immunol. 2004, 173: 1224-1231.
Hesse M, Piccirillo CA, Belkaid Y, Prufer J, Mentink-Kane M, Leusink M, Cheever AW, Shevach EM, Wynn TA: The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells. J Immunol. 2004, 172: 3157-3166.
Borkow G, Weisman Z, Leng Q, Stein M, Kalinkovich A, Wolday D, Bentwich Z: Helminths, human immunodeficiency virus and tuberculosis. Scand J Infect Dis. 2001, 33: 568-571. 10.1080/00365540110026656.
Bentwich Z, Kalinkovich A, Weisman Z, Borkow G, Beyers N, Beyers AD: Can eradication of helminthic infections change the face of AIDS and tuberculosis? . Immunol Today. 1999, 20: 485-487. 10.1016/S0167-5699(99)01499-1.
Resende Co T, Hirsch CS, Toossi Z, Dietze R, Ribeiro-Rodrigues R: Intestinal helminth co-infection has a negative impact on both anti-mycobacterium tuberculosis immunity and clinical response to tuberculosis therapy. Clin Exp Immunol. 2007, 147: 45-52.
Babu S, Bhat SQ, Kumar NP, Jayantasri S, Rukmani S, Kumaran P, Gopi PG, Kolappan C, Kumaraswami V, Nutman TB: Human type 1 and 17 responses in latent tuberculosis are modulated by coincident filarial infection through cytotoxic T lymphocyte antigen–4 and programmed death–1. J Infect Dis. 2009, 200: 288-298. 10.1086/599797.
Brown M, Mawa PA, Joseph S, Bukusuba J, Watera C, Whitworth JAG, Dunne DW, Elliott AM: Treatment of schistosoma mansoni infection increases helminth-specific type 2 cytokine responses and HIV-1 loads in coinfected Ugandan adults. J Infect Dis. 2005, 191: 1648-1657. 10.1086/429668.
Elias D, Britton S, Kassu A, Akuffo H: Chronic helminth infections may negatively influence immunity against tuberculosis and other diseases of public health importance. Expert Rev Anti-Infect Ther. 2007, 5: 475-484. 10.1586/14787220.127.116.115.
Stewart GR, Boussinesq M, Coulson T, Elson L, Nutman T, Bradley JE: Onchocerciasis modulates the immune response to mycobacterial antigens. Clin Exp Immunol. 1999, 117: 517-523. 10.1046/j.1365-2249.1999.01015.x.
Elias D, Wolday D, Akuffo H, Petros B, Bronner U, Britton S: Effect of deworming on human T cell responses to mycobacterial antigens in helminth‐exposed individuals before and after bacille calmette–guérin (BCG) vaccination. Clin Exp Immunol. 2001, 123: 219-225. 10.1046/j.1365-2249.2001.01446.x.
Borkow G, Bentwich Z: Chronic immune activation associated with chronic helminthic and human immunodeficiency virus infections: role of hyporesponsiveness and anergy. Clin Microbiol Rev. 2004, 17: 1012-1030. 10.1128/CMR.17.4.1012-1030.2004.
Malhotra I, Mungai P, Wamachi A, Kioko J, Ouma JH, Kazura JW, King CL: Helminth- and bacillus calmette-guérin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. J Immunol. 1999, 162: 6843-6848.
Potian JA, Rafi W, Bhatt K, McBride A, Gause WC, Salgame P: Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway. J Exp Med. 2011, 208: 1863-1874. 10.1084/jem.20091473.
Fulton SA, Martin TD, Redline RW, Henry Boom W: Pulmonary immune responses during primary mycobacterium bovis- calmette-guerin bacillus infection in C57Bl/6 mice. Am J Respir Cell Mol Biol. 2000, 22: 333-343. 10.1165/ajrcmb.22.3.3776.
Klementowicz JE, Travis MA, Grencis RK: Trichuris muris: a model of gastrointestinal parasite infection. Semin Immunopathol. 2012, 34: 815-828. 10.1007/s00281-012-0348-2.
Wakelin D: Acquired immunity to trichuris muris in the albino laboratory mouse. Parasitology. 1967, 57: 515-524. 10.1017/S0031182000072395.
Else KJ, Wakelin D, Roach TI: Host predisposition to trichuriasis: the mouse--T. muris model. Parasitology. 1989, 98 (Pt 2): 275-282.
Arocho A, Chen B, Ladanyi M, Pan Q: Validation of the 2-DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts. Diagn Mol Pathol. 2006, 15: 56-61. 10.1097/00019606-200603000-00009.
Wang X, Seed B: A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 2003, 31: e154-10.1093/nar/gng154.
Aubin E, Lemieux R, Bazin R: Absence of cytokine modulation following therapeutic infusion of intravenous immunoglobulin or anti-red blood cell antibodies in a mouse model of immune thrombocytopenic purpura. Br J Haematol. 2007, 136: 837-843. 10.1111/j.1365-2141.2007.06495.x.
Hamelin M-È, Yim K, Kuhn KH, Cragin RP, Boukhvalova M, Blanco JCG, Prince GA, Boivin G: Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol. 2005, 79: 8894-8903. 10.1128/JVI.79.14.8894-8903.2005.
Bancroft AJ, Artis D, Donaldson DD, Sypek JP, Grencis RK: Gastrointestinal nematode expulsion in IL-4 knockout mice is IL-13 dependent. Eur J Immunol. 2000, 30: 2083-2091. 10.1002/1521-4141(200007)30:7<2083::AID-IMMU2083>3.0.CO;2-3.
Lamb TJ, Graham AL, Le Goff L, Allen JE: Co-infected C57BL/6 mice mount appropriately polarized and compartmentalized cytokine responses to litomosoides sigmodontis and leishmania major but disease progression is altered. Parasite Immunol. 2005, 27: 317-324. 10.1111/j.1365-3024.2005.00779.x.
Sangaré LR, Herrin BR, Herrin BR, John-Stewart G, Walson JL: Species-specific treatment effects of helminth/HIV-1 co-infection: a systematic review and meta-analysis. Parasitology. 2011, 138: 1546-1558. 10.1017/S0031182011000357.
Erb KJ, Trujillo C, Fugate M, Moll H: Infection with the helminth nippostrongylus brasiliensis does not interfere with efficient elimination of mycobacterium bovis BCG from the lungs of mice. Clin Diagn Lab Immunol. 2002, 9: 727-730.
Frantz FG, Rosada RS, Turato WM, Peres CM, Coelho-Castelo AAM, Ramos SG, Aronoff DM, Silva CL, Faccioli LH: The immune response to toxocariasis does not modify susceptibility to mycobacterium tuberculosis infection in BALB/C mice. Am J Trop Med Hyg. 2007, 77: 691-698.
Elias D, Akuffo H, Thors C, Pawlowski A, Britton S: Low dose chronic Schistosoma mansoni infection increases susceptibility to mycobacterium bovis BCG infection in mice. Clin Exp Immunol. 2005, 139: 398-404. 10.1111/j.1365-2249.2004.02719.x.
Artis D, Potten CS, Else KJ, Finkelman FD, Grencis RK: Trichuris muris: host intestinal epithelial cell hyperproliferation during chronic infection is regulated by interferon-γ. Exp Parasitol. 1999, 92: 144-153. 10.1006/expr.1999.4407.
Cliffe LJ, Potten CS, Booth CE, Grencis RK: An increase in epithelial cell apoptosis is associated with chronic intestinal nematode infection. Infect Immun. 2007, 75: 1556-1564. 10.1128/IAI.01375-06.
Carmo AM, Vicentini MA, Dias AT, Alves LL, Alves CCS, Brandi JS, De Paula ML, Fernandes A, Barsante MM, Souza MA, Teixeira HC, Negrão-Corrêa D, Ferreira AP: Increased susceptibility to strongyloides venezuelensis in mice due to mycobacterium bovis co-infection which modulates production of Th2 cytokines. Parasitology. 2009, 136: 1357-1365. 10.1017/S0031182009990655.
Jenkins SN, Behnke JM: Impairment of primary expulsion of Trichuris muris in mice concurrently infected with nematospiroides dubius. Parasitology. 1977, 75: 71-78. 10.1017/S0031182000048332.
Legesse M, Erko B, Balcha F: Increased parasitaemia and delayed parasite clearance in Schistosoma mansoni and plasmodium berghei co-infected mice. Acta Trop. 2004, 91: 161-166. 10.1016/j.actatropica.2004.04.002.
Phillips RS, Selby GR, Wakelin D: The effect of plasmodum berghei and trypanosoma brucei infections on the immune expulsion of the nematode Trichuris muris from mice. Int J Parasitol. 1974, 4: 409-415. 10.1016/0020-7519(74)90050-2.
Cliffe LJ, Humphreys NE, Lane TE, Potten CS, Booth C, Grencis RK: Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion. Science. 2005, 308: 1463-1465. 10.1126/science.1108661.
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: 485-491. 10.1111/j.1365-3024.1995.tb00919.x.
Else KJ, Hültner L, Grencis RK: Cellular immune responses to the murine nematode parasite Trichuris muris: II differential induction of TH-cell subsets in resistant versus susceptible mice. Immunology. 1992, 75: 232-237.
Else KJ, Grencis RK: Antibody-independent effector mechanisms in resistance to the intestinal nematode parasite Trichuris muris. Infect Immun. 1996, 64: 2950-2954.
Wakelin D, Selby GR: Immune expulsion of Trichuris muris from resistant mice: suppression by irradiation and restoration by transfer of lymphoid cells. Parasitology. 1976, 72: 41-50. 10.1017/S0031182000043171.
Lee TD, Wakelin D, Grencis RK: Cellular mechanisms of immunity to the nematode Trichuris muris. Int J Parasitol. 1983, 13: 349-353. 10.1016/S0020-7519(83)80039-3.
Koyama K, Tamauchi H, Ito Y: The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris. Parasite Immunol. 1995, 17: 161-165. 10.1111/j.1365-3024.1995.tb01018.x.
Else KJ, Entwistle GM, Grencis RK: Correlations between worm burden and markers of Th1 and Th2 cell subset induction in an inbred strain of mouse infected with Trichuris muris. Parasite Immunol. 1993, 15: 595-600.
Bancroft AJ, Else KJ, Humphreys NE, Grencis RK: The effect of challenge and trickle Trichuris muris infections on the polarisation of the immune response. Int J Parasitol. 2001, 31: 1627-1637. 10.1016/S0020-7519(01)00281-8.
Nagaraj S, Collazo M, Corzo CA, Youn J-I, Ortiz M, Quiceno D, Gabrilovich DI: Regulatory myeloid suppressor cells in health and disease. Cancer Res. 2009, 69: 7503-7506. 10.1158/0008-5472.CAN-09-2152.
Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TKA, Bucks C, Kane CM, Fallon PG, Pannell R, Jolin HE, McKenzie ANJ: Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature. 2010, 464: 1367-1370. 10.1038/nature08900.
This work was supported by the South African National Research Foundation and the South African Medical Research Council (MRC) through financial contributions to this project. We thank N. Brown for her technical assistance.
The authors declare that they have no competing interests.
Study concept & design – GW, HJN. Acquisition of data – HJN, LK. Statistical analysis – HJN, NDP. Analysis and interpretation of data – GW, HJN, NDP. Drafting of the manuscript – HJN, NDP. Critical revisions to the manuscript – GW, AGL, NDP, PVH. Obtained Funding – GW, HJN. Study Supervision – GW. All authors read and approved the final manuscript.
Electronic supplementary material
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Nel, H.J., du Plessis, N., Kleynhans, L. et al. Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens. BMC Microbiol 14, 9 (2014). https://doi.org/10.1186/1471-2180-14-9