We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-α expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-α levels. Blockage of the CR4 iC3b site yielded increases in TNF-α for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-α only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-α expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways.
Mycobacterium avium, a prevalent opportunistic pathogen of immunocompromised patients, resides and replicates inside the macrophages of the infected host. Upon contact of M. avium with the host macrophage, reactions such as production of reactive oxygen and nitrogen intermediates, release of pro-inflammatory (TNF-α, IL-6), and anti-inflammatory cytokines (IL-10) are triggered [[–. It is widely accepted that the level of macrophage TNF-α expression has important consequences for host immunity [[,. In vivo, TNF-α induction by M. avium correlates with increased granuloma formation, resulting in rapid containment and clearance of the infectious focus [[,, whereas inhibition of TNF-α expression by virulent strains of Mycobacterium tuberculosis or M. avium is associated with increased bacterial survival [[,[,.
Mycobacteria can bind to a number of macrophage receptors such as the complement receptor types 1, 3 and 4 (CR1, CR3, CR4), CD14, mannose receptor (MR), transferrin receptor, and Fc receptors to gain entry into the macrophage [[,. CR3 (CD11b/CD18) in particular, maintains intimate connections with the actin cytoskeleton and is crucial to the uptake of intracellular pathogens, including mycobacteria [[1–. Opsonin-mediated uptake is partially controlled by the bacterium since M. avium has been shown to recruit complement fragment C2a to form a C3 convertase and generate active C3 breakdown products [. Receptor preference at the time of bacterial entry is considered to determine subsequent intracellular processing by the infected host cell. In fact, utilization of CRs by the intracellular pathogens Leishmania donovani, Histoplasma capsulatum, Mycobacterium leprae, and Legionella pneumophila to enter the macrophage, allows these organisms to avoid phagocytic pathways that induce the respiratory burst [[5,.
A link between receptor usage and early signal transduction events has been recently demonstrated for M. avium strain 2151 SmO. This strain utilizes CD14 to manipulate TNF-α synthesis via the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway [. While it has been demonstrated by Aderem and Underhill [ and Forsberg et al. [ that TNF-α regulates CR3-mediated MAPK activation, respiratory burst, and phagocytosis, the converse i.e., a role for CR3 in regulating TNF-α has never been studied although work done by Bohlson et al. [ suggests that complement-mediated interactions may not impact TNF-α activation.
Non-virulent strains of M. avium stimulate more TNF-α compared to virulent strains [, however, the bacterial surface structures modulating TNF-α activation have not been identified. The glycopeptidolipids (GPL) represent the most abundant cell wall component of M. avium and are situated on the outermost surfaces of the cell [, thus making them likely ligands to mediate bacterial–host interactions. Studies have suggested a role for serovar-specific GPL (ssGPL) in the pathogenesis of M. avium infection as highly antigenic molecules affecting host immune function. These data have relied on comparison of strains representing different serovars or have used purified and/or chemically modified GPL and GPL components [.
In a previous study, we disrupted the M. avium rhamnosyltransferase (rtfA) gene via homologous recombination to construct isogenic mutants devoid of serovar-8 specific GPL using an allelic exchange vector incorporating a temperature-sensitive plasmid origin of replication and sacB [. In this study, we investigate whether serum C3, and macrophage receptors such as CR3, and CR4 are involved in M. avium–macrophage interaction and whether they modulate TNF-α induction among M. avium strains differing in ssGPL expression.
Materials and methods
Bacterial strains and reagents
M. avium 920A6 is a serovar-8 bloodstream isolate cultured from a patient with AIDS that exists as smooth opaque (SmO) and smooth transparent (SmT) morphotypes [[5,. 213R.4 is a ssGPL-null (serovar-null) strain of 920A6 SmO generated by allelic exchange mutagenesis for the rtfA gene [. Strain 233R.1 created by transformation of 213R.4 with wild-type rtfA gene incorporated into an integrative vector, contains only a single-copy of rtfA, and demonstrates a pattern of ssGPL and nsGPL similar to the parent wild-type strain [.
M. avium was grown in Middlebrook 7H9 sucrose broth or 7H11 agar (Difco Laboratories, Detroit, MI) supplemented with 10% oleic acid dextrose complex (OADC) at 37°C. Bacteria were grown to an optical density at 600 nm of 0.4, diluted with an equal volume of 10% glycerol, frozen on dry ice–ethanol, and stored as 1 ml aliquots at −80°C. One aliquot of frozen bacteria of each type was serially diluted to determine baseline colony forming units (cfu) and to confirm colony morphotype. Prior to infections, bacteria were thawed on ice and repeatedly passaged through a 27-gauge syringe to disengage any clumps.
Macrophage infections and TNF-α measurements
The murine macrophage cell line, J774A.1 (ATCC, TIB-67) was used in this study and propagated in RPMI medium containing 10% fetal bovine serum (FBS, Sigma, St. Louis, MO) without added antibiotics. To rule out variability in results concerning the involvement of serum proteins, only one source of FBS was used throughout the study to generate heat-inactivated and complement-depleted serum. Heat-inactivated serum was generated by heating FBS at 56°C for 30 min. In studies where C3-depleted serum was used, FBS was incubated with fractionated C3 antiserum (Sigma, St. Louis, MO) at a ratio of 8:1 (FBS:antiserum) for 30 min at 37°C. To investigate the role of serum proteins and serum opsonins in TNF-α synthesis, J774A.1 cells were grown to confluence in RPMI with 10% heat-inactivated FBS or 10% C3-depleted serum as indicated above.
To derive murine bone-marrow derived macrophages (BMDMs), bone marrow cells from femurs were cultured (5 × 106 cells ml−1, 37°C, 5% CO2), in complete DMEM medium containing 10% fetal calf serum, 2 mM l-glutamine, penicillin (100 units ml−1), streptomycin (100 μg ml−1), and 30% (vol/vol) L-929 cell-conditioned medium, to provide macrophage growth factor. To eliminate contaminating fibroblasts, nonadherent bone marrow cells were transferred after 24 h to nontissue culture petri dishes and grown in L929 cell-conditioned complete DMEM medium for 7 days. Adherent BMDMs were lifted by incubation in PBS (20 min, 4°C, 5% CO2) and used for this study.
Endotoxin-free anti-CR3 Mac-1 and anti-CR4 monoclonal antibodies (moAb) M1/70 and HL3 against the iC3b epitope of the I domain of murine CD11b (CR3) and CD11c (CR4), respectively, were purchased from Pharmingen (BD Biosciences, San Diego, CA) and used at a concentration of 15 μg ml−1 [.
To investigate the role of serum proteins and serum opsonins in TNF-α synthesis, J774A.1 cells previously grown in RPMI medium with 10% FBS were subcultured into culture flasks and grown to confluence (?3 days) at 37°C in the presence of 5% CO2 in RPMI medium containing either 10% FBS, 10% heat-inactivated FBS or 10% C3-depleted FBS. The cells were then subcultured into 24-well tissue culture plates (with three independent wells per test condition) and grown for ?1 day to confluence (5 × 105 cells per well) under the same culture conditions. Identical steps were taken to determine the involvement of serum proteins and M. avium GPL in TNF-α induction in murine BMDMs.
On the day of infection, for blocking studies using anti-CR3 moAb M1/70 and anti-CR4 moAb HL3, the J774A.1 cells were washed twice, and fresh medium added, including moAbs M1/70 or HL3 to a final concentration of 15 μg ml−1. The cells were incubated for 1 h at 37°C in the presence of 5% CO2 after which time the supernatant was discarded, the cells were washed twice to remove any residual moAb, and were ready for infection with isogenic M. avium strains.
M. avium strains were used to infect murine macrophage J774A.1 and BMDMs at a multiplicity of infection (MOI) of 5 bacteria per cell. After a 1 h infection period at 37°C in the presence of 5% CO2, supernatants were collected and frozen at −20°C until analysis. TNF-α expression was determined by enzyme-linked immunosorbent assay (ELISA) using the murine TNF-α kit as per the manufacturer's directions (OptEIA, Pharmingen, San Diego, CA). The limit of detection was 10 pg TNF-α ml−1. All conditions were repeated twice to ensure consistency of observations.
Data were compared across strains and culture conditions using 1-way ANOVA, with subsequent pairwise comparisons via Tukey's Studentized Range Test. An alpha value of p≤ 0.05 was used to assess statistical significance. Data are presented as means ± standard deviation (SD).
Several cytokines appear to play a critical role in host defense against intracellular pathogens, and TNF-α, a cytokine produced primarily by macrophages, may be involved in the early host response against M. avium[[–. Thus, the purpose of our present study was to explore whether serum proteins and/or macrophage CRs modulate TNF-α synthesis during the initial phases of M. avium–macrophage interaction.
TNF-α induction differs between isogenic strains of M. avium differing in GPL expression
For M. avium, numerous studies have shown that SmT morphotypes are highly virulent relative to isogenic SmO forms [. SmT strains are typically cultured from AIDS patients and convert to a SmO morphotype in vitro. We compared early (1 h) TNF-α induction for SmT and SmO morphotypes of M. avium serovar-8 strain, 920A6. Stimulation of J774A.1 macrophages by 920A6 SmO induced 3-fold more TNF-α relative to 920A6 SmT (Fig. 1(a), p < 0.05). These results are consistent and confirm the results of other investigators [. We then determined TNF-α induction for the ssGPL-null mutant of 920A6. This strain has been shown to be devoid of serovar-8 specific GPL but retains the ability to express the same array of non-specific GPL as wt serovar-8 SmO and SmT strains [. J774A.1 macrophages stimulated with the ssGPL-null mutant yielded TNF-α levels ?2-fold less than the wt parent SmO (Fig. 1(a), p < 0.05) and ?2-fold more than the virulent SmT (Fig. 1(a), p < 0.05) strain. Complementation of rtfA with a single copy integrant restored ssGPL expression for 233R.1 to a pattern similar to wild-type M. avium [. Infection of J774A.1 macrophages with the complemented M. avium strain induced TNF-α levels similar to the wild-type M. avium parent strain (Fig. 1(b), p > 0.05). Experiments performed with murine BMDMs demonstrated results identical to the murine macrophage cell line J774A.1 (Fig. 1(c)). Thus, for reproducibility and feasibility purposes, all future experiments in this study were conducted using the murine macrophage cell line, J774A.1.
Heat inactivation of serum proteins result in increased TNF-α synthesis
Relative to infections performed in the presence of serum, infections performed in heat-inactivated serum yielded a ?3-fold increase in TNF-α expression for the SmO strain (Fig. 2, p < 0.05), a ?9-fold increase for the SmT strain (Fig. 2, p < 0.05) and a ?4-fold increase for the ssGPL-null strain (Fig. 2, p < 0.05). Comparison of TNF-α activation between the three strains in conditions using heat-inactivated serum showed relatively little difference (Fig. 2, p > 0.05 for all pair wise comparisons). These results suggested that the increase in TNF-α expression observed in heat-inactivated serum related to interactions with host macrophage surface components/receptors mediated by heat-labile serum proteins. In separate experiments, we found that in contrast to cells incubated with heat-treated FBS over 3–4 days, a single overnight incubation of J774A.1 cells in medium supplemented with heat-treated FBS did not yield maximal increases in TNF-α expression, suggesting that residual bound serum proteins remained (data not shown). Experiments performed with murine BMDMs using shorter incubation periods in serum-free conditions demonstrated results identical to the J774A.1 cells (data not shown), thus validating our use of this cell line as a model of infection.
Increased TNF-α synthesis in heat-treated serum conditions relates to loss of C3
It has been previously reviewed that C3 breakdown components, C3b and iC3b bind to the mycobacterial surface to initiate macrophage uptake via CR1, CR3, and CR4 [. We next sought to determine whether complement-mediated opsonization could be responsible for differences in TNF-α expression observed between serum and heat-inactivated serum conditions. As observed in Fig. 3, use of C3-depleted serum was associated with increased levels of TNF-α for 920A6 SmO (?2-fold increase, p < 0.05), SmT (?8-fold increase, p < 0.05), and 213R.4 (4-fold increase, p < 0.05) relative to infections performed in the presence of normal serum. Between-strain comparisons in C3-depleted serum were not significant. Similar to heat-treated serum, we found that prolonged incubation of J774A.1 cells with multiple changes of medium was necessary to fully eliminate C3 (data not shown).
Opsonization of M. avium via CR3 and CR4 is necessary to downregulate TNF-α synthesis
The observation that depletion of serum C3 resulted in increased TNF-α expression for SmO, SmT, and serovar-null infected cells suggested that for all three serovar-8 strains, the loss of opsonic interaction of M. avium via iC3b to macrophage receptors CR3 and/or CR4 resulted in higher TNF-α levels. Interaction through CR1 was not considered since murine macrophages do not express this receptor [.
Although CR3 has consistently been implicated as a key macrophage receptor for M. avium uptake, its role in TNF-α activation has not been studied. We, therefore first assessed the role of CR3 in TNF-α modulation. The I-domain of CD11b contains the iC3b opsonic binding site [. To confirm that opsonic binding of M. avium to CD11b suppressed TNF-α induction, bacterial infections were performed while blocking the iC3b binding site with anti-CD11b moAb M1/70. Blockage increased TNF-α levels 4-fold for 920A–6 SmT and serovar-null strains (Fig. 4, p < 0.05). In contrast, the SmO strain demonstrated no difference in TNF-α induction when CR3 is blocked. The increase in TNF-α was not due to activation of CR3 by moAb M1/70 since control wells containing moAb alone yielded TNF-α levels no different than control wells containing medium alone (Fig. 4).
Since all strains manifested increases in TNF-α expression under opsonin-free conditions (Figs. 2 and 3) and CR3 blockade did not cause an increase in TNF-α for SmO strains, this would suggest that for the SmO strain, regulation of TNF-α expression was mediated via CR4. We therefore investigated the role of CR4 in TNF-α activation. Bacterial infections were performed while blocking the iC3b binding site of CR4 with anti-CD11c moAb HL3. Blockage of the I-domain of CD11c with moAb HL3 significantly increased TNF-α levels for all 920A6 strains (Fig. 5, p > 0.05 for all intra-strain pair wise comparisons), comparable to levels obtained during heat-inactivated and C3-depleted serum conditions. Between-strain comparisons were not statistically different (p > 0.05). To rule out non-specific Fc-mediated activation in TNF-α induction, control antibodies were used at 15 μg ml−1. The level of host TNF-α induction was not statistically significant (date not shown, p > 0.05).
Like M. tuberculosis, M. avium can survive within macrophages and evade the host immune response. Because TNF-α appears to be involved in host immunity, early bacterial down-regulation of TNF-α expression in infected macrophages may be an important mechanism for intracellular survival of virulent M. avium. One of the goals of this research was to determine if serum proteins, and receptors on the infected macrophage affect TNF-α expression and thus trigger pathways early on during M. avium infection that could ultimately decide the fate of the invading bacterium. Also, we wanted to determine if strains differing in GPL expression differentially induced TNF-α during the early phase of M. avium–macrophage interaction and if possible, construct consistent hypotheses and future experiments to understand the variability in cytokine expression among the isogenic M. avium strains.
The role of serum proteins, in particular iC3b, to prevent M. avium induced TNF-α expression is suggested by our observation that M. avium infection of murine macrophages in the presence of heat-inactivated serum resulted in significantly higher levels of TNF-α than its whole, active serum counterparts. Support for C3 opsonization in regulating TNF-α expression was demonstrated by our use of C3-depleted serum, where murine macrophage cells infected with M. avium wt and ssGPL-null strains produced higher levels of TNF-α. The complement-mediated opsonic interaction between the mycobacterium and the infected macrophage via the CR3 and/or CR4 could be advantageous to the bacterium by avoidance of potentially harmful reactions, such as TNF-α production, which could lead to increased bacterial survival. Our results are in apparent contrast with the study of Bohlson et al. [ that demonstrated no difference in TNF-α induction of BMDM macrophages derived from C3 +/+ and C3 −/− C57BL/6 mice and J774A.1 macrophages. There are two important differences between studies. First, the study by Bohlson et al. studied two serovar-2 strains of M. avium, TMC724 and 2–151, whereas our study investigated a virulent serovar-8 strain and highlights the possibility of GPL-related differences in the activation of macrophage signaling pathways to affect the host immune response. Moreover, unpublished results from our laboratory using TMC724 have also demonstrated a lack of difference in TNF-α induction in conditions varying in the presence of complement. Second, Bohlson et al. studied TNF-α induction of cells propagated in the presence of FBS. Prior to M. avium infection, cells were washed and then incubated with C3-deficient medium for 2 h. We have shown here that such short incubation times in C3-deficient medium are insufficient to demonstrate differences between conditions.
Intracellular pathogens, including mycobacteria, bind CR1, CR3, CR4 as a first step in the invasion of mammalian cells [, linking the receptors cytoplasmic domains to the actin cytoskeleton and proximal components of the cell signaling pathways [. While this process is regulated by TNF-α, we show for the first time that C3 opsonization of M. avium with subsequent binding to host macrophage receptors CR3 and CR4 is necessary to decrease TNF-α secretion by macrophages.
In addition to differences observed in TNF-α synthesis by M. avium infected J774A.1 cells during various culture conditions, we also noted differences in TNF-α induction among the three isogenic M. avium serovar-8 strains. Effects were similar for the murine macrophage cell line J774A.1 and the BMDMs demonstrating the utility of this cell line to model in vivo infections. We noted that the serovar-null mutant activated lower levels of TNF-α compared to the wt parent SmO strain under control conditions and that the serovar-null strain complemented with wild-type ssGPL induced macrophage TNF-α to levels identical to the wild type M. avium parent strain. Absence of M. avium ssGPL could be the reason for host TNF-α suppression by the serovar-null strain since prior studies have demonstrated the role of ssGPL as a potent immunogen and its ability to trigger higher levels of TNF-α [. Our results confirm the involvement of M. avium ssGPL in macrophage TNF-α expression. We also noted that the wt SmT strain activated lower levels of TNF-α compared to wt SmO strain under control (normal serum) conditions, this data is in agreement with previous research [. One possible explanation could be due to the differences in the non-specific (ns) GPL/ssGPL ratios between the wt SmO and SmT strains. However, the full difference in the expression profiles between SmO and SmT morphotypes is unknown, and thus we cannot rule out other bacterial factors modulating host cell signaling pathways. The latter may be likely since SmT morphotypes typically express greater levels of ssGPL than SmO strains [.
Regulation of TNF-α induction mediated through CR-interaction differed among strains. Wild-type SmO parent strain and the derived serovar-null mutant varied as to CR utilization responsible for regulation of TNF-α levels. While the iC3b domain of CR4 was used by wt SmO for down-regulation of TNF-α, the serovar-null mutant was able to utilize the iC3b binding domains of both CR3 and CR4 to downregulate TNF-α synthesis. Since these are isogenic strains, the differences in receptor binding and TNF-α modulation between the parent SmO and the ssGPL-null mutant are attributable to the absence of terminal serovar specific sugars in the mutant strain. We have demonstrated that the presence of M. avium ssGPL triggers higher levels of host TNF-α. Whether alterations in TNF-α expression correlate with survival is unknown and is being investigated.
Receptor preference has been shown to be crucial for bacterial survival [[5,. Little is known about the involvement of CR4 or whether it is preferred over CR3 during the M. avium infection process. In this study, we have shown that the receptor preference depends on the type of infecting M. avium strain. Preliminary results on simultaneous blockade of the iC3b domains of CR3 and CR4 suggests that on M. avium infection, modulation of TNF-α synthesis via these macrophage receptors occurs via independent MAPK p38 and p42/44 pathways (data not shown), thus raising the possibility that these M. avium strains could trigger different host MAPK signaling pathways which could ultimately result in a different intracellular fate for each infecting strain.
In summary, serum protein C3, macrophage receptors CR3, CR4, and M. avium ssGPL are involved in modulation of TNF-α induction during the early stages of M. avium–macrophage interaction. This is the first reported study that demonstrates the involvement of CR3 and CR4 in suppression of TNF-α synthesis during M. avium–macrophage interaction.
Y. Patterson and L. Buxbaum are gratefully acknowledged for the gift of J774A.1 cells and BMPMs, respectively. Paul M. Nealen is gratefully acknowledged for assistance in statistical analyses. Thomas Glaze and Andrea Rossi are acknowledged for technical assistance. Support for this study was provided through Merit Review and VISN 4 grant from the Veterans Affairs, and University Foundation Grant from the University of Pennsylvania, 5-UO1-AI32783, P30-AI-045008–06 to JNM.