ABSTRACT
Antibody responses to Mycoplasma pneumoniae correlate with pulmonary M. pneumoniae clearance. However, M. pneumoniae-specific IgG antibodies can cross-react with the myelin glycolipid galactocerebroside (GalC) and cause neurological disorders. We assessed whether antiglycolipid antibody formation is part of the physiological immune response to M. pneumoniae. We show that antibodies against M. pneumoniae proteins and glycolipids arise in serum of M. pneumoniae-infected children and mice. Although antibodies to M. pneumoniae glycolipids were mainly IgG, anti-GalC antibodies were only IgM. B-1a cells, shown to aid in protection against pathogen-derived glycolipids, are lacking in Bruton tyrosine kinase (Btk)-deficient mice. M. pneumoniae-infected Btk-deficient mice developed M. pneumoniae-specific IgG responses to M. pneumoniae proteins but not to M. pneumoniae glycolipids, including GalC. The equal recovery from M. pneumoniae infection in Btk-deficient and wild-type mice suggests that pulmonary M. pneumoniae clearance is predominantly mediated by IgG reactive with M. pneumoniae proteins and that M. pneumoniae glycolipid-specific IgG or IgM is not essential. These data will guide the development of M. pneumoniae-targeting vaccines that avoid the induction of neurotoxic antibodies.
INTRODUCTION
Mycoplasma pneumoniae is a major cause of community-acquired pneumonia (CAP) and can trigger immune-mediated neurological complications such as Guillain-Barré syndrome (GBS) and encephalitis (1). M. pneumoniae belongs to the smallest self-replicating microorganisms, in terms of both cellular dimensions and genome size (1). Unlike other bacteria, M. pneumoniae lacks a peptidoglycan layer and is therefore naturally resistant to cell wall synthesis inhibitors such as β-lactams. Macrolide antibiotics are recommended to treat M. pneumoniae infections in children (2). Extensive macrolide use led to an alarming worldwide increase of macrolide-resistant M. pneumoniae (MRMP) strains, with rates of over 90% in some regions (3, 4). This emergence of MRMP highlights the importance of implementing control strategies to prevent infection, such as vaccines. Vaccination primarily induces antibody responses capable of neutralizing infection (5), but attempts to develop such vaccines against M. pneumoniae using inactivated bacteria in humans (reviewed in reference 6) and live attenuated strains in an animal model (7) have been complicated by limited efficacy against respiratory disease. No serious adverse effects and only mild local reactions were reported in humans (6). However, it has been observed that reinfection or challenge after vaccination with inactivated or live attenuated strains led to exacerbation of disease in some anecdotal reports (8, 9) and animal experiments (10–14). Thus, to develop optimal approaches to vaccination against M. pneumoniae, it is critical to understand the immune mechanisms that contribute to resistance and immunopathology of M. pneumoniae disease (15).
Immune responses against M. pneumoniae have been intensively investigated in various animal models (e.g., see references 14 and 16–23). B cells are known to be involved in pulmonary M. pneumoniae clearance (22, 24–27), and we recently showed that in B cell-deficient μMT mice, M. pneumoniae infection led to chronic pulmonary disease, characterized by higher histopathology scores (28). The observed compensatory immune responses by both innate (granulocytes and monocytes) and adaptive (CD4+ and CD8+ T cells) immune cells were not able to clear M. pneumoniae infection in the absence of antibodies. In contrast, μMT mice cleared M. pneumoniae infections in the lungs when passively immunized with M. pneumoniae-specific immunoglobulin G (IgG)-containing serum from infected wild-type (WT) mice 2 weeks after infection. These findings indicate that B cells and M. pneumoniae-specific antibodies are crucial for M. pneumoniae clearance in the lungs. Furthermore, these data suggest that they may not contribute to immunopathology following primary infection given the less severe pulmonary inflammation and better outcome in WT mice than in B cell-deficient μMT mice (28).
M. pneumoniae is covered only with a cell membrane containing antigenic protein and glycolipid structures (29). The membrane-anchored proteins at the cell pole form an attachment structure important for initiating respiratory infection (30). Proteins constitute over two-thirds of the M. pneumoniae membrane mass, with the rest being membrane lipids, i.e., cholesterol, phospholipids, and glycolipids (29). M. pneumoniae glycolipid subfractions have been shown to be highly immunogenic in mice and humans (31). Their strong immunogenicity has been leveraged in diagnosis of M. pneumoniae infection, whereby antigens derived from crude culture extracts that contain large amounts of glycolipids were used in serological assays (32, 33). However, because of cross-reactions with other mycoplasmas or Gram-negative bacteria, current diagnostic assays focus on specific adhesion proteins (e.g., protein P1) rather than glycolipids (32). Importantly, M. pneumoniae glycolipids also exhibit homology with mammalian tissue compounds, which trigger cross-reactive antibodies that may target cells of multiple host organ systems (34). GBS and encephalitis constitute the most common and severe neurological diseases of M. pneumoniae extrapulmonary manifestations in which an underlying postinfectious antibody-mediated process has been proposed (33). In fact, it has been shown that galactocerebroside (GalC)-specific antibodies bind to a lipid structure present in M. pneumoniae, indicative of molecular mimicry between the major myelin glycolipid GalC and M. pneumoniae (35).
We recently showed that both IgM and IgG anti-GalC antibodies are present in the serum of GBS patients and that the presence of anti-GalC IgG correlates with GBS (36). Anti-GalC IgM was also found in 18% of anti-M. pneumoniae-seropositive control patients without neurological diseases (36). Interestingly, all anti-GalC IgM-positive individuals within this control cohort were children. This raises the question of whether the formation of antibodies to M. pneumoniae glycolipids is part of the physiological immune response and necessary to clear M. pneumoniae in children.
Antibody responses against glycolipids are thought to be driven by B-1a cells, splenic marginal zone B cells, and nodal marginal zone B cells (i.e., thymus independent [TI]) or by the help of natural killer T (NKT) cells (i.e., thymus dependent [TD]) (37–40). An important role for B-1a cells in producing antibodies to pathogen-derived glycolipid structures has been shown for Mycobacterium tuberculosis and Francisella tularensis (41–43). Interestingly, priming of Bruton tyrosine kinase-deficient (Btk−) mice with F. tularensis-derived glycolipids did not result in protection against a lethal challenge with an F. tularensis live vaccine strain (44). The lack of a protective antibody response in the Btk-deficient mice was attributed to the absence of B-1a cells (44, 45). Whether TI B cell responses, and in particular B-1a cells, are also important for protection against M. pneumoniae infection is unknown.
We set out to investigate in children which antigenic structures of M. pneumoniae are recognized by antibodies, using a well-defined cohort of children with CAP diagnosed with M. pneumoniae infection. Furthermore, employing WT and Btk− mice, we unraveled the role of TI B cell responses in the resolution of pulmonary M. pneumoniae infection.
RESULTS
IgM but not IgG to GalC is induced during M. pneumoniae infection in children.In light of our previous findings (36), we assessed whether anti-GalC IgM develops in all children with M. pneumoniae CAP. To this end, we examined the serum of children with M. pneumoniae CAP for the presence of anti-GalC antibodies by an enzyme-linked immunosorbent assay (ELISA), and M. pneumoniae-negative asymptomatic healthy control (HC) children were tested as controls. We detected anti-GalC IgM at significantly higher levels in sera of children with M. pneumoniae CAP than in sera of HC children (Fig. 1A). Moreover, anti-GalC IgG was detectable only at very low levels in both children with M. pneumoniae CAP and HC children (Fig. 1B). These findings confirm and extend previous observations that during childhood, M. pneumoniae CAP IgM against GalC develops, whereas anti-GalC IgG does not.
The antibody response induced by pulmonary M. pneumoniae infection in children is directed against GalC and M. pneumoniae protein and glycolipid fractions. (A and B) The presence of antigalactocerebroside (anti-GalC) IgM (A) and anti-GalC IgG (B) in serum of children with M. pneumoniae (Mp) community-acquired pneumonia (CAP) and healthy controls (HC) was determined by an ELISA. Sera of children with M. pneumoniae CAP were obtained at a median of 34 days (interquartile range, 30 to 42 days) after the onset of symptoms (n = 5). Dots represent data for individual children, and the horizontal line in each graph represents the median. OD, optical density. (C) Total M. pneumoniae and purified M. pneumoniae protein fractions were analyzed by SDS-PAGE followed by silver staining. Lane a, marker; lane b, total untreated M. pneumoniae M129 (125 ng); lane c, M. pneumoniae protein fractions (125 ng). (D) Thin-layer chromatography (TLC) was used to analyze purified M. pneumoniae lipids. Lane a, cerium(IV) sulfate stain for total M. pneumoniae lipids; lane b, orcinol stain for M. pneumoniae glycolipids. C, cholesterol; CE, cholesterol esters. (E to G) IgG and IgM levels in sera of children with M. pneumoniae CAP (n = 5) at the indicated time points after the onset of symptoms and reactivity against total M. pneumoniae (E) or the isolated M. pneumoniae proteins (F) and M. pneumoniae glycolipids (G). Not all children had sera available at each time point beyond 30 days. Data are expressed as fold increases over controls (levels in sera of HC children; n = 5). Control levels are indicated by a dashed line. The means ± standard deviations (SD) are shown. (H) IgG subclasses against M. pneumoniae glycolipids in children with M. pneumoniae CAP and HC children (serum samples as the ones used for panel A). Dots represent data for individual children. **, P < 0.01 (A, B, and H) (by a Mann-Whitney U test).
IgM and IgG recognize M. pneumoniae protein and glycolipid structures during M. pneumoniae infection in humans.In addition to the antiglycolipid response to GalC, we next investigated the antibody response against the complete M. pneumoniae glycolipid as well as M. pneumoniae protein fractions. First, M. pneumoniae proteins and glycolipids were separated from M. pneumoniae cultures using chloroform-methanol (MeOH) extraction (2:1, vol/vol). The separation of proteins and glycolipids from the M. pneumoniae lysate was analyzed by SDS-PAGE followed by silver staining (Fig. 1C) and by thin-layer chromatography (TLC) followed by orcinol staining (Fig. 1D), respectively. The reactivity and kinetics of M. pneumoniae-specific IgM and IgG antibodies toward these structures were subsequently determined with an ELISA. To this end, we incubated sera of children with M. pneumoniae CAP with either M. pneumoniae glycolipid or M. pneumoniae protein fractions and an M. pneumoniae lysate as a control (referred to as total M. pneumoniae). As for total M. pneumoniae, a specific antibody response of both IgM and IgG isotypes against M. pneumoniae proteins and glycolipids could be detected in all M. pneumoniae CAP patients within 9 days after the onset of the first CAP symptoms and peaked at around 1 month (20 to 35 days) (Fig. 1E to G). The specific IgM and IgG response returned to baseline values around 3 months after the onset of CAP symptoms. Analysis of IgG subclasses revealed that both IgG1 and IgG2 subclasses were present in the anti-M. pneumoniae glycolipid IgG pool (Fig. 1H). These findings show that the human IgM and IgG response to M. pneumoniae is directed against both M. pneumoniae glycolipids and M. pneumoniae proteins.
IgG against glycolipids also predominates during M. pneumoniae infection in mice.We previously showed that passive immunization of B cell-deficient μMT mice with serum of WT mice containing M. pneumoniae-specific IgG enabled μMT mice to clear pulmonary M. pneumoniae infection (28). In fact, the detection of M. pneumoniae-specific IgG in bronchoalveolar lavage fluids (BALFs) correlated with bacterial clearance in the lungs of μMT recipient mice after WT serum transfer. Here, we demonstrate that upon M. pneumoniae infection, antibodies are generated against both M. pneumoniae protein and glycolipid structures. However, it is unclear whether both the anti-M. pneumoniae protein and glycolipid antibodies are important for clearance of M. pneumoniae from the lungs. To evaluate this, we examined pulmonary M. pneumoniae infection in WT and Btk− mice, as it has been shown that Btk− mice cannot mediate protective antibody responses to pathogen-derived glycolipid structures (43).
First, we investigated whether M. pneumoniae infection also results in a specific antibody response against M. pneumoniae proteins and glycolipids in WT mice. We therefore tested the reactivity and kinetics of murine serum antibodies of M. pneumoniae-infected C57BL/6 WT mice against M. pneumoniae proteins or glycolipids as for children with M. pneumoniae CAP. Consistent with our data from children, a specific antibody response of both IgM and IgG isotypes against M. pneumoniae proteins and glycolipids could be detected in all mice within 7 days postinfection (p.i.) and peaked at around day 28 p.i. (Fig. 2A to C). These findings in mice parallel the data from children by demonstrating a specific antibody response targeting both M. pneumoniae proteins and glycolipids.
Antibody response against GalC and M. pneumoniae protein and glycolipid fractions during pulmonary M. pneumoniae infection in mice. (A to C) IgG and IgM levels in sera of M. pneumoniae-infected C57BL/6 WT mice at the indicated time points postinfection (p.i.) and reactivity against total M. pneumoniae (A) or M. pneumoniae proteins (B) and M. pneumoniae glycolipids (C). Data are expressed as fold increases over controls (i.e., levels in sera of mock-infected control mice). Data for control levels are indicated by a dashed line. The means ± SD are shown (n = 6 to 12 mice/time point). (D and E) Presence of anti-GalC IgM (D) and absence of anti-GalC IgG (E) in sera of M. pneumoniae-infected and mock-infected mice obtained at 28 days p.i. (n = 6). Dots represent data for individual mice, and the horizontal line in each graph represents the median.
Furthermore, we determined whether anti-GalC IgM is also detectable during the course of M. pneumoniae infection in mice. Sera of M. pneumoniae-infected WT mice, isolated at 28 days p.i., were incubated with GalC. Indeed, compared to mock-infected control WT mice, levels of IgM antibodies to GalC were high in M. pneumoniae-infected WT mice, although significance was not reached (P = 0.19) (Fig. 2D). In agreement with the data from children, anti-GalC IgG was detectable at equally very low levels after pulmonary M. pneumoniae infection as in uninfected control WT mice (Fig. 2E).
IgG against M. pneumoniae proteins but not M. pneumoniae glycolipids is crucial to resolve M. pneumoniae infection in mice.We next evaluated whether a potential reduction in antiglycolipid antibodies in Btk− mice affects M. pneumoniae clearance from the lungs. We thus compared Btk− mice with WT mice in terms of the outcomes of M. pneumoniae infection and antiglycolipid antibody responses. Surprisingly, CFU counts of M. pneumoniae in BALF of Btk− mice were not different from those in infected WT mice (Fig. 3A). Btk− mice showed even better control of pulmonary infection than WT mice at day 3 p.i. Nevertheless, both WT and Btk− mice were able to clear M. pneumoniae within 42 days. Analysis of serum antibodies revealed that M. pneumoniae-specific antibody levels increased over time in both WT and Btk− mice, which were much more pronounced for IgG than for IgM, but the levels in Btk− mice were significantly lower than those in WT mice (Fig. 3B and C). The same pattern was observed for total IgG and IgM antibody levels in Btk− mice (data not shown). Although Btk− mice contained lower IgG levels after M. pneumoniae infection, the avidity index of IgG antibodies against M. pneumoniae-derived proteins was not different from that in WT mice (Fig. 3D).
IgG against M. pneumoniae proteins but not M. pneumoniae glycolipids is crucial to resolve M. pneumoniae infection in mice. WT C57BL/6 mice (n = 6 to 12 mice/time point) and Btk− mice (n = 6 to 12 mice/time point) were infected intranasally with M. pneumoniae. Control WT mice received SP4 medium alone. Dots represent data for individual mice, and the horizontal lines represent the medians. (A to C) At the indicated time points, bacterial loads in BALF (A) and serum levels of M. pneumoniae-specific IgM (B) and IgG (C) were determined. Bacterial loads are expressed as median CFU per milliliter with interquartile ranges. AU, arbitrary units. (D) Avidity index of IgG against M. pneumoniae proteins at day 42 p.i. The bars represent the means ± SD. (E and F) Serum levels of M. pneumoniae-specific IgM (E) and IgG (F) against total M. pneumoniae (gray and black) or the separated M. pneumoniae proteins (light red and red) and M. pneumoniae glycolipids (light blue and blue) in sera of M. pneumoniae-infected C57BL/6 WT mice (circles) and Btk− mice (triangles) and additionally IgG in sera of CD19-hBtk mice (diamonds) at day 14 p.i. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (determined by a Kruskal-Wallis test with Dunn’s post hoc multiple-comparison test [A to C, E, and F] or Welch’s t test [D]).
The increases in levels of IgM against M. pneumoniae proteins or glycolipids compared to controls were similar between WT and Btk− mice (Fig. 3E). Also, comparable IgG responses to M. pneumoniae proteins were measured in sera from WT and Btk− mice (Fig. 3F, left). In contrast, the IgG response to M. pneumoniae glycolipids in Btk− mice was strikingly different from that in WT mice: no IgG antibodies against M. pneumoniae glycolipids were detected (Fig. 3F, middle). The generation of IgG antibodies against M. pneumoniae glycolipids was partially restored in CD19-hBtk mice (Fig. 3F, right), in which Btk is selectively rescued in B cells and is lacking only in myeloid cells (46). These findings could be corroborated by measuring IgM and IgG to M. pneumoniae proteins or glycolipids of WT and Btk− mice in BALF. As previously observed (28), we found very low levels of IgM, which were comparable between mouse strains for M. pneumoniae proteins and glycolipids (Fig. 4A). As in serum, the IgG antibody levels to M. pneumoniae glycolipids in Btk− mice were significantly lower than those in BALF of WT mice and close to control levels (Fig. 4B). Serum anti-GalC IgM and IgG antibodies were not produced by M. pneumoniae-infected Btk− mice (Fig. 5).
Local antibody response against M. pneumoniae protein and glycolipid fractions during pulmonary M. pneumoniae infection in mice. Shown are BALF levels of M. pneumoniae-specific IgM (A) and IgG (B) against total M. pneumoniae (gray and black) or the separated M. pneumoniae proteins (light red and red) and M. pneumoniae glycolipids (light blue and blue) of M. pneumoniae-infected C57BL/6 WT mice (circles) (n = 6) and Btk− mice (triangles) (n = 6) at day 14 p.i. Dots represent data for individual mice, and the horizontal lines represent the medians. *, P < 0.05; ***, P < 0.001 (by a Kruskal-Wallis test with Dunn’s post hoc multiple-comparison test).
Comparison of the antibody responses against GalC between WT and Btk− mice during pulmonary M. pneumoniae infection. Shown are serum anti-GalC IgM (A) and anti-GalC IgG (B) responses of M. pneumoniae-infected WT mice (n = 5 mice) and Btk− mice (n = 4 mice) at 42 days p.i. ***, P < 0.001 (by a Mann-Whitney U test).
DISCUSSION
Here, we extend previous findings on the essential role of M. pneumoniae-specific IgG antibodies in pulmonary clearance (27, 28) by demonstrating that IgG antibodies reactive with M. pneumoniae proteins alone seem to be sufficient to clear M. pneumoniae in the lungs. We show that Btk− mice clear M. pneumoniae infections comparably to WT mice but do not generate a detectable humoral response to M. pneumoniae glycolipids. These data also indicate that even low levels of M. pneumoniae protein-specific IgG antibodies, albeit of sufficient avidity, are able to mediate protection in the lungs of Btk− mice. Notably, Btk− mice showed better control of pulmonary M. pneumoniae infection than did WT mice at day 3 p.i. At this time point, significantly higher numbers of alveolar macrophages and NK cells were observed in lungs of Btk− mice than in WT mice (data not shown). B-1a cells have been shown to inhibit macrophage-NK cell cross talk (47). The observed improved control of M. pneumoniae replication in Btk− mice may thus result from the absence of B-1a cells in these mice (48, 49). We speculate that in WT mice, M. pneumoniae triggers the activation of B-1a cells, which dampen M. pneumoniae clearance by inhibiting the activation of macrophages and/or NK cells. However, the inhibitory effect of B-1a cells is not as sufficient and/or long lasting, as M. pneumoniae is cleared from the lungs of WT mice within 4 to 6 weeks.
In addition to their important role in the clearance of M. pneumoniae in the lungs, the induced M. pneumoniae protein-specific antibodies may also prevent reinfection or reduce horizontal transmission. It was shown previously that antibodies from M. pneumoniae-immunized guinea pigs targeting recombinant P1 and P30 adhesion proteins inhibit the adherence of M. pneumoniae to human bronchial epithelial cells in vitro (23). This indicates that an M. pneumoniae-specific vaccine for high-risk individuals, i.e., schoolchildren and elderly people, should aim at inducing potent antibodies directed against M. pneumoniae proteins. The data obtained with Btk− mice suggest that such a vaccine may even be effective in children with common variable immunodeficiency or hypogammaglobulinemia with reduced B cells, who have been reported to be at an increased risk for M. pneumoniae pulmonary disease and/or extrapulmonary manifestations (50–54).
Our data reveal that the humoral response against M. pneumoniae glycolipids is redundant for the clearance of M. pneumoniae in the lungs of mice. This is rather unexpected given the fact that levels of IgG against M. pneumoniae glycolipids were higher than those against M. pneumoniae proteins and were of the IgG1 and IgG2 subclasses, which indicates potential neutralization and complement-dependent killing of M. pneumoniae. Furthermore, within the pool of M. pneumoniae glycolipid-specific IgM antibodies, some also bear cross-reactivity to self-tissue (i.e., GalC-like M. pneumoniae structure [33]). Indeed, we previously elucidated cross-reactivity between M. pneumoniae and GalC and associated the presence of anti-GalC IgG with GBS triggered by M. pneumoniae (36). However, here, we confirmed that in the absence of neuropathy, anti-GalC IgG was not formed during pulmonary M. pneumoniae infection. In contrast, the formation of anti-GalC IgM, which seems not to inflict neurotoxicity, was part of the physiological response.
Cross-reactivity of M. pneumoniae glycolipid-specific antibodies with self-tissue, causing immunopathology, may be one possible reason why BALB/c mice vaccinated with live attenuated M. pneumoniae developed more-severe pulmonary disease following infection with wild-type M. pneumoniae (10). This could also be observed after repeated infections with wild-type M. pneumoniae in BALB/c mice but not in C57BL/6 mice (11). It is known that not only rechallenge after vaccination but also primary M. pneumoniae infection of BALB/c mice led to worse pulmonary inflammation than in C57BL/6 mice (18), which simply reflects heterogeneity in susceptibility to M. pneumoniae infection in mice as in humans (1, 18).
The observation that M. pneumoniae glycolipid-specific antibodies are not generated in Btk− mice suggests that the production of lipid-specific antibodies may be, at least in part, mediated by B-1a cells, as these cells are lacking in Btk− mice (48, 49). Notably, in contrast to M. pneumoniae infection, antibodies to pathogen-derived glycolipid structures were shown previously to protect against infections with M. tuberculosis and F. tularensis (41–43). Immunization of Btk− mice with F. tularensis glycolipids did not result in protection against a lethal challenge with an F. tularensis live vaccine strain, which was attributed to the absence of B-1a cell-produced glycolipid-specific antibodies (44, 45). Apart from TI B cell responses, a potential role for nonclassical T helper cells, the NKT cells, in the production of M. pneumoniae lipid antibodies cannot be ruled out.
In fact, cross-linking of the B cell receptor (BCR) on B-1a cells by lipid-based antigens leads to an innate-like polyreactive IgM response (so-called natural antibody response) important for early protection against mucosal pathogens (37). B-1 cells can also provide long-lasting TI IgM memory (43, 55). One might speculate that the presence of anti-GalC IgM but not IgG after M. pneumoniae infection in children and mice indicates that a GalC-like structure in M. pneumoniae triggered specifically B-1a cells to produce natural IgM. Natural antibodies are characterized by low affinity (37, 56), which may explain why M. pneumoniae glycolipid-specific antibodies were redundant for clearance of pulmonary M. pneumoniae infection. Apart from affinity, the level of anti-GalC IgG may be critical for the initiation of M. pneumoniae-associated neurological disease. It is possible that low levels of GalC IgG are removed from the circulation by target-mediated clearance (57). Interestingly, B-1a cells triggered by glycolipid antigens can also undergo activation-induced deaminase-dependent class switching (43). However, this is a rare event (43), as is the development of GBS following M. pneumoniae infection (36). Thus, these findings further support the hypothesis that a GalC-like M. pneumoniae structure may trigger B-1a cells, which undergo class switching in rare cases, and produce potentially neurotoxic anti-GalC IgG. However, the exact events that lead to the induction of autoreactive B cells and anti-GalC IgG remain to be identified.
In conclusion, our data extend previous findings on the essential role of M. pneumoniae-specific antibodies in clearing M. pneumoniae from the lungs (27, 28) by suggesting that the IgG response to M. pneumoniae-derived proteins is important for pulmonary clearance of M. pneumoniae. The finding that M. pneumoniae glycolipid-specific IgM and IgG antibodies are redundant for M. pneumoniae clearance but can also target the myelin glycolipid GalC is of importance not only for the understanding of M. pneumoniae-associated immune-mediated diseases but also for the design of M. pneumoniae-targeting vaccines. Based on our results, such vaccine formulations should include M. pneumoniae protein antigens rather than M. pneumoniae lipids, thereby avoiding the induction of potential autoimmune antiglycolipid antibodies.
MATERIALS AND METHODS
Mice.C57BL/6 mice were purchased from Charles River Laboratories and used at 8 to 12 weeks of age. Btk− mice (Btk−/− or Btk−/Y, on the C57BL/6 background [48]) and CD19-hBtk mice (backcrossed on Btk− C57BL/6 mice for >10 generations [46]) were bred and housed in the animal facilities of the Erasmus MC under specific-pathogen-free conditions. All experiments were conducted according to Dutch guidelines for animal experimentation and approved by the Animal Experiments Committee of the Erasmus MC, Rotterdam, The Netherlands (protocol numbers 103-13-05 and 103-14-02).
Patients.Children with CAP and asymptomatic controls, admitted for a planned elective surgical procedure, from 3 to 18 years of age were enrolled from 1 May 2016 to 30 April 2017 during a CAP study at the University Children’s Hospital Zurich (P. M. Meyer Sauteur, unpublished data). Diagnosis was based on the detection of M. pneumoniae DNA in pharyngeal swab specimens by PCR, specific serum IgM antibodies by an ELISA, and circulating IgM antibody-secreting cells by an enzyme-linked immunospot (ELISpot) assay (Meyer Sauteur, unpublished). Asymptomatic controls tested negative for M. pneumoniae by PCR and serum IgM. Sera of these M. pneumoniae-positive CAP patients and M. pneumoniae-negative asymptomatic controls were used in this study, since no further information about respiratory disease characteristics was available from previous M. pneumoniae-seropositive controls without neurological diseases (36), and M. pneumoniae infection can present with a wide range of respiratory tract symptoms apart from CAP. The study was approved by the ethics committee of the Canton Zurich, Switzerland (BASEC number 2016-00148). Written informed consent was obtained from all parents and from children above the age of 14 years.
Bacteria.M. pneumoniae reference strain M129 (subtype 1; ATCC 29342) was cultured as previously described (28).
Infection.Mice were inoculated intranasally with 1 × 109 CFU of M. pneumoniae M129 diluted in 50 µl SP4 medium. Control mice were inoculated with 50 µl SP4 medium.
M. pneumoniae quantification.The presence of M. pneumoniae was detected by either PCR or culture of BALF on SP4 agar plates (58).
Lipid extraction.Lipids were extracted from the M. pneumoniae M129 culture by the addition of chloroform-methanol-water (2:1:1, vol/vol/vol). After a 1-h incubation at 4°C, the mixture was vortexed and centrifuged at 2,000 × g for 1 min. After repeating the extraction procedure, cells were sonicated for 30 min and centrifuged at 10,000 × g for 5 min. Lipid extracts contained in the chloroform layer were pooled and evaporated under nitrogen. Lipids were dissolved in ethanol, and extraction was verified by the Liebermann-Burchard reaction. The purification of M. pneumoniae glycolipids was confirmed by TLC. Here, extracted lipids (5 µg) were applied to a silica glass plate (Merck Millipore) after activation for 2 h at 110°C. The TLC plate was developed using CHCl3–MeOH–Milli-Q (60:35:8, vol/vol/vol). Total lipids were visualized by using 10% cerium(IV) sulfate in 15% H2SO4, and glycolipids were visualized by using 0.1% orcinol in 5% H2SO4, with heating up to 110°C.
Protein extraction.After delipidation of the M. pneumoniae lysate by chloroform-methanol-water (2:1:1, vol/vol/vol) extraction, cold acetone was added to the aqueous (upper) layer and the insoluble interface, followed by incubation at 4°C for 2 h. Proteins were collected by centrifugation at 10,000 × g for 5 min. Pellets were washed twice with acetone, air dried on ice, and dissolved in 4 M urea–250 mM ammonium bicarbonate. The protein concentration was determined by a bicinchoninic acid assay. Additionally, proteins (125 ng) were analyzed by SDS-PAGE, followed by silver staining (Merck).
Quantification of M. pneumoniae-specific antibodies against M. pneumoniae antigens and GalC.To assess the presence of specific antibodies against total M. pneumoniae, M. pneumoniae proteins and glycolipids, and GalC, 96-well half-area polystyrene plates (Corning Costar) were coated overnight at 4°C with an M. pneumoniae lysate (0.5 µg) (28) or M. pneumoniae proteins (normalized to 1 ng adhesion protein P1 per well present in both total M. pneumoniae and M. pneumoniae protein fractions) in a 100 mM sodium carbonate-bicarbonate buffer (pH 9.6) containing 1 M urea or with M. pneumoniae glycolipids (normalized to 0.6 µg cholesterol per well present in both total M. pneumoniae and M. pneumoniae glycolipid fractions) and GalC (900 pmol) dissolved in ethanol. Nonspecific binding was blocked using phosphate-buffered saline (PBS)–1% bovine serum albumin (BSA) (Sigma). Serum samples, diluted in PBS–0.1% BSA, and undiluted BALF samples were added and incubated overnight at 4°C. Bound antibodies were detected by the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM or rabbit anti-mouse IgG (Thermo Scientific), biotinylated goat anti-human IgM with HRP-conjugated streptavidin (Thermo Scientific), or HRP-conjugated rabbit anti-human IgG (Invitrogen) and IgG1 to -4 (Sanquin). Reactions were visualized by using tetramethylbenzidine (TMB; Sigma) as the substrate and stopped by using 1 M H2SO4. The optical density was measured at 450 nm with an ELISA microplate reader (VersaMax). The results were analyzed by microplate data collection and analysis software (VersaMax).
Statistical analysis.The R software environment (version 3.4.0) was used for statistical analysis. Welch’s t test, the Mann-Whitney U test, and the Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test of selected pairs were used to determine statistical significance. Statistical significance was defined as a P value of <0.05.
ACKNOWLEDGMENTS
A.M.C.V.R. was supported by a grant from the Sophia Scientific Research Foundation (SSWO 2014-150/WO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank the Erasmus Medical Center experimental animal facility for their care for our mice and assistance with experiments.
We have no conflict of interest.
FOOTNOTES
- Received 10 October 2018.
- Accepted 28 October 2018.
- Accepted manuscript posted online 5 November 2018.
- Copyright © 2019 American Society for Microbiology.
