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Keywords:

  • Alveolar macrophage;
  • Autophagy;
  • Innate immunity;
  • NOD2;
  • Tuberculosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1β, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

The recognition of pathogen-associated molecular patterns by innate immune receptors is essential for the initiation and coordination of the immune mechanisms responsible for host protection against lung-invading pathogens [[1]]. The innate immune recognition is based on a limited repertoire of pattern recognition receptors, which sense conserved microbial components known as pathogen associated molecular patterns. The pattern recognition receptors include the members of the Toll-like receptor (TLR) family and the nucleotide-binding oligomerization domain (NOD) proteins (NOD-like receptors, NLRs), among others [[2, 3]].

The NLR proteins have been demonstrated to play an important role in the defense against respiratory agents, including Pseudomonas aeruginosa, Streptococcus pneumoniae, Legionella pneumophila, and Mycobacterium tuberculosis [[1, 4, 5]]. The NOD proteins, members of the NLR family, are cytoplasmic receptors implicated in the recognition of bacterial molecules produced during the synthesis and/or degradation of peptidoglycan. Specifically, NOD2 senses the cytosolic presence of muramyldipeptide (MDP) [[6, 7]]. Activation of NOD2 by bacterial products can stimulate two major signaling pathways to activate caspase-1 and pro-inflammatory responses, including the NF-κB pathway and the inflammasome pathway [[5]]. Upon ligand recognition, NOD2 activates the receptor-interacting protein-2 kinase (Rip2), which forms a multiprotein complex via its caspase activation recruitment domains (CARDs). These CARD-CARD interactions lead to NF-κB nuclear translocation with the consequent initiation of both innate and acquired immune responses and the enzymatic cleavage of pro-IL-1β, which releases the biologically active form of IL-1β [[8, 9]]. Murine macrophages and dendritic cells release IL-6, IL-1β, IL-8, and RANTES following MDP-induced NOD2 activation [[10, 11]]. Human blood mononuclear cells produce the NOD2-dependent inflammatory cytokines, TNF-α, IL-1β, and IL-6 [[12, 13]], and chemokines that recruit neutrophils [[14, 15]]. Moreover, the induction of antimicrobial effectors, such as cryptidins and beta-defensins, in human epithelial cells has been associated with NOD2 signaling [[16-18]].

The contribution of NOD2 to the host response against M. tuberculosis and the pathogenesis of mycobacterial infections have both been established at different levels. Polymorphisms in the NOD2 gene confer susceptibility to leprosy and tuberculosis in human populations [[19, 20]]. Monocytes from patients with Crohn's disease that are homozygous for the 3020insC NOD2 mutation lack functional NOD2 and have a severe reduction in their pro-inflammatory cytokine response to M. tuberculosis infection, despite an intact TLR response [[12, 21]]. NOD2 recognizes M. tuberculosis and not only initiates a cascade of pro-inflammatory cytokines after recognition of the infection but also contributes to M. tuberculosis clearance. Data from the NOD2-deficient mouse model suggest that macrophages might control the replication of virulent M. tuberculosis during the late stages of the infection in a NOD2-dependent manner [[22]]. Moreover, recent evidence indicates that pretreatment with the ligand of NOD2 augments pro-inflammatory cytokine production from M. tuberculosis-infected human alveolar macrophages, which supports the control of M. tuberculosis growth [[23]]. However, the mechanisms responsible for how NOD2 contributes to antimicrobial responses during a M. tuberculosis infection remain unknown.

In this study, we investigated the role of two antimicrobial components in NOD2-driven innate defense in human alveolar macrophages: the autophagy enzyme immunity-related GTPase M protein (IRGM), which has been involved in the clearance of intracellular bacteria including Mycobacterium bovis [[24, 25]], and the human cathelicidin peptide LL37, which has been shown to contribute to bactericidal activity against M. tuberculosis in macrophages [[26, 27]]. To determine the function of NOD2 in the response of human alveolar macrophages against virulent M. tuberculosis, we evaluated whether NOD2 is involved in the induction of these antimicrobial effectors, namely, IRGM and LL37, and their relative contribution to the control of intracellular growth of M. tuberculosis. Here, we provide evidence that NOD2 plays a role in human pulmonary defense.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

Human alveolar macrophages express functional NOD2

We examined the presence of NOD2 in the cytosol of alveolar macrophages and evaluated its function after stimulation with MDP. Flow cytometric analysis demonstrated that the NOD2 protein was expressed in freshly isolated alveolar macrophages (Fig. 1A). MDP induced an increase in NOD2 protein levels and a slight increase in IκBα in the cytosolic fractions of alveolar macrophages (Fig. 1B). MDP stimulation also led to increased NOD2 gene expression (Fig. 1C). We examined the localization of NOD2 by immunoelectron microscopy and observed that the NOD2 protein was homogeneously distributed in the cytosol of unstimulated alveolar macrophages, but migrated to plasma membrane after MDP stimulation of NOD2 (Fig. 1D).

image

Figure 1. NOD2 accounts for intracellular pathogen sensing receptors in human alveolar macrophages. (A) Intracellular detection of NOD2 (bold line in the histogram plot) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells (R1, dot plot). One representative experiment out of three experiments is presented. (B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported. (C) The upregulation of NOD2 gene expression was assessed by quantitative PCR. The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells (n = 11). (D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP-stimulated cells (right, ×63,000) using an anti-NOD2 antibody and detected with a secondary antibody coupled to 5-nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.

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NOD2 induces a release of pro-inflammatory cytokines

To investigate the contribution of NOD2 ligation in the early release of pro-inflammatory mediators, we measured the production of the following cytokines in the supernatant of MDP-stimulated alveolar cell cultures: IL-1β, IL-6, IL-10, IL-12p70, IL-17, IFN-α2, and TNF-α. MDP induced a significant release of IL-1β, IL-6, and TNF-α in comparison to unstimulated alveolar macrophages (p < 0.01, Fig. 2). IL-1β, IL-6, and IL-10 production was also significantly induced by LPS compared with unstimulated cells and cells stimulated with MDP. The LPS-dependent production of TNF-α was similar to that of MDP stimulation. IL-12p70, IL-17, and IFN-α2 were not detected in either MDP- or LPS-stimulated cells (data not shown).

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Figure 2. NOD2 ligation in alveolar macrophages induces pro-inflammatory cytokine release. Cells were incubated for 24 h in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS. Production of IL-1β, IL-6, TNF-α, and IL-10 was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p < 0.05 and **p <0.01 using the two-tailed Wilcoxon signed-rank test.

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LL37 and IRGM are induced after NOD2 activation with MDP

We investigated the potential of the NOD2 receptor to induce the antimicrobial peptide LL37 and the autophagy enzyme IRGM (an ortholog of mouse LRG47) in human alveolar macrophages. Our results showed that MDP treatment resulted in the upregulation of LL37 and IRGM gene expression, which ranged from a 1.43-fold to 30.71-fold increase and a 1.67-fold to 83.71-fold increase, respectively, relative to unstimulated cells (Fig. 3A and D). In addition, immunoblot analysis revealed a slight increase in the protein expression of the LL37 peptide and an increment in IRGM protein expression in the cytosolic fraction of the cells after MDP treatment (Fig. 3B, C, E, and F).

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Figure 3. LL37 and IRGM gene expression is upregulated following NOD2 activation (A-D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7–12). Bold lines indicate median values. (B, E) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis. (C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.

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NOD2 activation in M. tuberculosis-infected alveolar macrophages improved control of intracellular growth

Mycobacterium tuberculosis survives and replicates inside macrophages, therefore we aimed to determine the contribution of NOD2 activation in the control of M. tuberculosis growth after treating infected human alveolar macrophages with MDP. Figure 4A shows that M. tuberculosis intracellular growth significantly increased between day 1 and day 4 (intracellular growth increased by 12.2-fold, median 5.5, between day 1 and day 4, p < 0.05). However, when the alveolar macrophages were treated with MDP, the macrophages were able to limit the intracellular growth of M. tuberculosis (intracellular growth increased by only 0.5-fold, median 2.8, between day 1 and day 4).

image

Figure 4. NOD2 activation after M. tuberculosis infection induces an antibacterial innate activation profile. Alveolar macrophages were infected with M. tuberculosisH37Rv at an infection ratio of 1–2 bacteria/20 macrophages for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were then treated with 10 μg/mL of MDP or left with medium alone. (A) Intracellular bacterial burden was measured by quantifying CFU, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments. The production of (B) TNF-α, (C) IL-6 and (D) IL-1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments. (E) LL37 and (F) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p <0.05 and **p <0.01 using the two-tailed Wilcoxon signed-rank test. (G) LL37 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented. (H) The amount of protein normalized to tubulin relative to infected-only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.

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MDP treatment of M. tuberculosis-infected alveolar macrophages increased the production of TNF-α and IL-6 and upregulated LL37 and IRGM gene expression

Because alveolar macrophages could limit M. tuberculosis growth following NOD2 activation, we examined NOD2-induced cytokine expression to understand the mechanism behind controlled M. tuberculosis growth. We measured the release of various cytokines into the supernatant of infected cell cultures following treatment with MDP, including IL-1β, IL-6, IL-10, IL-12p70, IL-17, IFN-α2, and TNF-α. The addition of MDP to infected cells induced a significant increase in the production of TNF-α and IL-6, although the production of IL-1β was not significantly altered (Fig. 4B–D). Stimulation with MDP did not induce the production of IL-10, IL-12p70, IL-17, or IFN-α2 (data not shown).

In previous experiments, we had shown that LL37 and IRGM were upregulated in alveolar macrophages in response to NOD2 stimulation by MDP (Fig. 3). Infection of alveolar macrophages alone with M. tuberculosis induced a slight change in LL37 and IRGM gene expression. However, infected alveolar macrophages significantly increased the gene expression of these antimicrobial molecules after stimulation with MDP (p < 0.05, Fig. 4E and F). MDP treatment also induced an increase in LL37 and IRGM amounts (Fig. 4G and H). Our results suggest that MDP stimulation of infected macrophages via NOD2 can activate the pathways that lead to both antimicrobial peptide production and autophagy.

Activation of NOD2 recruits autophagy proteins to M. tuberculosis-containing vesicles

To investigate the presence of IRGM in M. tuberculosis-containing vesicles, we used immunoelectron microscopy. Our results showed that the alveolar macrophages in the infected-only cells did not recruit IRGM into M. tuberculosis-containing vesicles (Fig. 5A). Rather, IRGM was confined in separate vacuoles that were not in contact with the bacilli. In contrast, when alveolar macrophages were treated with MDP, IRGM was co-localized with the M. tuberculosis-containing vesicles (arrowheads, Fig. 5B). The quantitative morphometric analysis of IRGM recruitment, measured as gold particles/bacterium, proved to be significant (Fig. 5C, p < 0.05). These results suggest a role for NOD2 in the induction of autophagy in human alveolar macrophages.

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Figure 5. NOD2 activation of M. tuberculosis-infected macrophages induces the recruitment of autophagy proteins IRGM, LC3, and ATG16L1 to the bacteria-containing vesicles. Alveolar macrophages were infected with M. tuberculosisH37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h or left with medium alone. (A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP-treated cells using (A, B) anti-IRGM, (D, E) anti-LC3, and (G, H) anti-ATG16L1 antibodies and detected with a secondary antibody coupled to 5-nm gold particles indicated with arrowheads; bar represents 150 nm. (C, F, I) Gold particles co-localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p <0.05 using a two-tailed paired t-test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.

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In addition, the autophagy molecule LC3 was minimally recruited to M. tuberculosis-containing vesicles in infected alveolar macrophages, although treatment of these cells with MDP post-infection induced a massive recruitment of LC3 to these vesicles (Fig. 5D–F, p < 0.05). The same pattern was observed for the autophagy protein ATG16L1 (Fig. 5G–I, p < 0.05). Further, supplementing infected cultures with the Rip2/p38 inhibitor SB203580 prior to treatment with MDP limited the recruitment of IRGM, LC3, and ATG16L1 to mycobacteria-containing vesicles, thereby demonstrating that their co-localization was NOD2-dependent (Supporting Information Fig. 1). The localization of IRGM, LC3, and ATG16L1 to M. tuberculosis-containing vesicles indicated that M. tuberculosis was either contained in an autophagosome or in a phagosome that had acquired autophagy enzymes.

To characterize the M. tuberculosis-containing vesicles, we used conventional electron microscopy. We found large vesicles containing organelles and degraded cell structures in the alveolar macrophage cytosol (Fig. 6A) and in lamellar multivesicular bodies, which are characteristic of autophagy (Fig. 6B). M. tuberculosis was identified in large vesicles that also contained these structures. (Fig. 6C and D). We confirmed the autophagy completion by determining the cleavage of soluble LC3-I to form LC3-II (Supporting Information Fig. 2) [[28]]. Thus, we concluded that M. tuberculosis was contained in autophagosomes.

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Figure 6. Macrophages treated with MDP after M. tuberculosis infection develop autophagy vacuoles. Alveolar macrophages were infected with M. tuberculosisH37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h. Autophagy indicators (arrowheads) such as (A) vesicles containing organelles (TEM magnification ×20,000) and (B) onion skin-like lamellar multivesicular bodies (TEM magnification ×16,000) were identified by ultrastructural analysis. (C, D) Autophagosomes containing mycobacteria (Mtb) are indicated with arrows (C, TEM magnification ×40,000; D, TEM magnification ×12,000). Micrographs were obtained from one experiment. Bar represents 300 nm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

There has been an increasing interest in the role of NLRs in the innate defense against intracellular pathogens. The NOD2 receptor has been found to be important for the detection of intracellular pathogens and the initiation of bactericidal responses against them, including M. tuberculosis [[22, 29]]. It was recently reported that NOD2 is expressed by human alveolar macrophages [[23]], although the role that NOD2 plays in the control of intracellular pathogens in human alveolar macrophages is poorly characterized. In this study, we investigated whether NOD2 is involved in the production of pro-inflammatory cytokines, the induction of antimicrobial responses or cellular autophagy of infected human alveolar macrophages.

First, we demonstrated that NOD2 was functional in primary alveolar macrophages. NOD2 was present in unstimulated alveolar macrophages, both at the protein and mRNA level, and NOD2 gene expression was upregulated following MDP stimulation. In addition, MDP can induce NOD2 protein redistribution toward the plasma membrane. It has been described that NOD2 recruitment to the plasma membrane initiates downstream signaling [[30]].

Our results indicate that NOD2 is included among the repertoire of lung macrophage receptors used in innate immune responses, and these data also show that significant levels of IL-1β, IL-6, and TNF-α, but not anti-inflammatory cytokines such as IL-10, are produced following MDP binding to NOD2. The TNF-α response was similar to that observed for LPS stimulation, indicating that MDP elicits a strong inflammatory response from human alveolar macrophages and contrasts with the almost nonexistent TNF-α response previously reported for MDP-treated murine macrophages [[22]]. The canonical IL-1β released after stimulation of alveolar macrophages with MDP was consistent with the release of IL-1β from human monocytes in a NOD2-dependent manner [[12, 13]].

We demonstrate for the first time that the bactericidal effector molecule LL37 is upregulated in human alveolar macrophages after NOD2 ligand recognition. We have previously reported that cathelicidin LL37 is induced in human alveolar macrophages by TLR2, TLR4, and TLR9 signaling [[31]]; however, the NOD2-dependent induction is smaller than that observed after stimulation of TLR4. Thus, we now include NOD2 to broaden the spectrum of innate signals capable of cathelicidin induction by the human alveolar immune response. We also report for the first time that IRGM is overexpressed in alveolar macrophages after NOD2 activation. IRGM has been thought to play an important role in autophagy, suggesting that in addition to its antibacterial effects, NOD2 may play a complex role in linking the innate response to autophagy.

NOD2 stimulation induced a pro-inflammatory and antimicrobial profile in alveolar macrophages. Therefore, we sought to investigate whether this NOD2-dependent antibacterial response could be induced in human alveolar macrophages following infection with virulent M. tuberculosis. A previous study performed in human macrophages had reported that knocking down NOD2 leads to an increase in intracellular M. tuberculosis [[23]]. Here, we observed that alveolar macrophages infected with a virulent strain of M. tuberculosis permit the intracellular growth of the pathogen, but that these infected macrophages could be activated to prevent intracellular mycobacteria replication by the addition of MDP. The infection alone was insufficient to elicit a protective innate response. However, the treatment with MDP for 24 h post infection induced the release of significant amounts of IL-6 and TNF-α as well as the overexpression of LL37 and IRGM. This antimicrobial response was expected to result in effective control of intracellular growth during a M. tuberculosis infection because LL37 had previously exhibited direct bactericidal effects against M. tuberculosis [[26]]. Moreover, the role of IRGM in the autophagy process might contribute to the elimination of intracellular M. tuberculosis as demonstrated by others [[32, 33]]. Although the MDP of M. tuberculosis cell wall activates NOD2, the availability of this ligand in the cytosol is not immediate. Thereby, we consider that infection alone, especially at a low multiplicity of infection (MOI), was insufficient to induce antimicrobial effectors expression, and the addition of MDP after infection enhanced the macrophages antimicrobial response. We cannot exclude that this effect is a result of activation of bystander macrophages.

Autophagy is emerging as an important mechanism in the defense of mammals against several bacterial infections, especially those that are dependent on the activation of innate immune responses. Autophagy can be triggered by the activation of pattern recognition receptors, mainly TLRs, and by immunity-related GTPases downstream of IFN-γ activation, such as IRGM [[34]]. The NOD2 receptor may be critical for the autophagy response to invading pathogens by promoting the recruitment of enzymes such as ATG16L1 to the site of pathogen entry in murine macrophages and human epithelial cells [[35]]. Here, we also demonstrate that NOD2 induces autophagy in human alveolar macrophages. We show that NOD2 activation of M. tuberculosis-infected human alveolar macrophages leads to the conversion of LC3-I into LC3-II as indicative of autophagosome formation and also to the recruitment of the autophagy markers IRGM, LC3, and ATG16L1 to M. tuberculosis-containing vesicles in a Rip2/p38-dependent manner. Finally, we found that bacilli were actually enclosed in autophagosomes.

Taken together, our findings suggest that autophagy is a key NOD2-dependent mechanism by which human alveolar macrophages eliminate M. tuberculosis. In human alveolar macrophages, the activation of NOD2 may be involved in the early innate control of M. tuberculosis primary infections, thereby preventing the development of active disease. In comparison, the study of the role of NOD2 during M. tuberculosis infection investigated in NOD2-deficient mice revealed that NOD2-dependent immune responses only partially limited M. tuberculosis growth in the acute phase of infection. In contrast, the major contribution of NOD2 was perceived during the chronic phase of the disease [[22]].

In conclusion, NOD2 is a receptor that participates in the early control of intracellular pathogens in human alveolar macrophages and is associated with the activation of an antibacterial response. We hypothesize that the influence of NOD2 in early immune responses to M. tuberculosis prevents the onset of active disease, which may be important for the development of vaccine adjuvants and immunological therapies.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

Study group

We recruited 20 healthy, HIV-1 seronegative residents of Mexico City, who had normal chest radiographs and no history of prior exposure to TB patients, at the National Institute for Respiratory Diseases (INER) in Mexico City. Fiberoptic bronchoscopy was performed on all study subjects with bronchoalveolar lavage. Approval to perform these studies was granted by the Institutional Review Board of INER. Written informed consent was obtained from all study subjects prior to any procedure, according to the guidelines of the U.S. Department of Health and Human Services and the declaration of Helsinki.

Human cell preparations

Bronchoalveolar cells were obtained by bronchoalveolar lavage, as described previously [[36]]. Briefly, bronchoalveolar lavage fluid was centrifuged and bronchoalveolar cells were resuspended in culture medium. More than 93% of the bronchoalveolar cells were alveolar macrophages in every case according to flow cytometric analysis using a gate on size and granularity. Therefore, the cells are hereafter referred to as alveolar macrophages. All cell cultures were carried out in RPMI 1640 (Lonza, Walkersville, MD, USA) supplemented with 50 μg/mL gentamycin sulfate, 200 mM L-glutamine, and 10% heat-inactivated pooled human AB serum (Gemini Bioproducts, Sacramento, CA, USA) at 37°C in 5% CO2.

Stimulation of alveolar macrophages with NOD2 ligand

To assess ligand-induced responses in alveolar macrophages, 106 cells were cultured in duplicate wells in ultralow attachment polystyrene 24-well plates (Corning Inc., NY, USA). Cells were stimulated with 10 μg/mL of synthetic MDP (InvivoGen, San Diego, CA, USA) for 24 h. Following stimulation, one set of alveolar macrophage cultures was harvested and prepared for flow cytometry, and one set was harvested and prepared for mRNA extraction. Supernatants were collected and kept frozen until NOD2 ligand-induced cytokine release was assessed. Culture medium alone was used as a negative control. In selected experiments, 10 μM SB203580 Rip2/p38 inhibitor (Promega, Madison, WI, USA) was added to the cells 30 min prior to MDP stimulation to block NOD2 signaling.

Intracellular NOD2 expression by flow cytometry

To assess NOD2 expression, 106 alveolar macrophages were incubated in phosphate-buffered saline (Lonza) with 50% rabbit serum for 10 min at room temperature with agitation (30 rpm) to block nonspecific Fc receptor binding. Cells were then treated with permeabilizing buffer (Becton Dickinson, San José, CA, USA), and saturating amounts of either goat antihuman NOD2 or corresponding goat isotype control (Imgenex, San Diego, CA, USA) were added in the presence of 50% rabbit serum. The cells were then incubated for 20 min at 4°C. After one washing step, secondary fluorescein isothiocyanate (FITC)-coupled donkey anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added, and the cells were incubated for 20 min at 4°C in the dark. Following another wash step, the cells were fixed with 1% paraformaldehyde. Acquisition of 20,000 cells was achieved using a FACSCalibur flow cytometer (Becton Dickinson). Flow cytometric analysis was performed using a gate set on large granular cells (high FSC and SSC). The FITC voltage detector was set to the minimum level that discriminates between autofluorescence and specific antibody staining. Isotype controls were used to define settings in the histogram plot analyses.

Real-time PCR for NOD2, LL37, and IRGM gene expression

Quantitative real-time PCR (qRT-PCR, TaqMan) was performed to determine the NOD2, LL37, and IRGM mRNA expression levels using the comparative threshold cycle (ΔΔCt) method of relative quantitation, as described previously [[36]]. Briefly, real-time PCR reactions were performed in duplicate wells according to the manufacturer's protocol for the following Taqman predesigned gene assays: NOD2 (Hs00223394_m1), LL37 (Hs00189038_m1), and IRGM (Hs01013699_s1) (Applied Biosystems, Carlsbad, CA, USA). PCR reactions were performed on an ABI Prism 7500 Sequence Detection System (Applied Biosystems). The Ct values for each gene were normalized to 18S rRNA (4319413E) as an endogenous control. To analyze ligand-induced expression changes of NOD2, LL37, and IRGM mRNA, the expression of unstimulated alveolar macrophages was set as 1, and the mRNA expression of the ligand-stimulated cells was reported relative to that of unstimulated cells.

Immunoblot

Cells were washed once in PBS and then immediately frozen in liquid nitrogen and stored at −70°C until use. For extraction of cytoplasmic proteins, the frozen cell pellet was lysed by vigorous pipeting with lysis buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 5 μg/mL of leupeptin, 5 μg/mL of aprotinin, and 0.5 mM PMSF (all from Sigma-Aldrich, St. Louis, MO, USA). The lysates were centrifuged at 3500 rpm for 10 min at 4°C, and the supernatants containing cytosolic proteins were recovered. The total amount of protein was determined using the Bradford method, with BSA used as a standard.

For western blot analyses, cytosolic and nuclear extracts were resuspended in Laemmli buffer and boiled for 5 min. Proteins were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 1% BSA in blocking buffer (TBS, 50 mM Tris.Cl, pH 7.5; 150 mM NaCl; 0.1% Tween 20), the membranes were incubated with anti-NOD2 (Millipore, Billerica, MA, USA), anti-IRGM (Abcam, Cambridge, MA, USA), anti-IkB (Santa Cruz Biotechnology), anti-LC3 (Novus Biologicals, Littleton, CO, USA) or anti-alpha tubulin (Sigma-Aldrich) antibodies for 2 h followed by incubation with peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody for 1 h at room temperature. Specific bands were detected by chemiluminiscence using the SuperSignal system (Thermo, Rockford, IL, USA) and revealed on autoradiographic films. Densitometry was performed with ImageJ 1.44o (National Institutes of Health, USA).

Cytokine detection

The cytokines IL-1β, IL-6, IL-10, IL-12p70, IL-17, IFN-α2, and TNF-α were measured in the culture supernatants using a custom-designed Milliplex human cytokine kit (Millipore), according to the manufacturer's instructions.

Infection with M. tuberculosis

The M. tuberculosis strain H37Rv (ATCC 25618) was grown for 28 days in Middlebrook 7H9 broth, counted, and stored at −70°C until use. For the in vitro infection, 105 bronchoalveolar cells were allowed to adhere for 1 h in round bottom 96-well polystyrene plates. After removal of nonadherent cells, alveolar macrophages were infected with M. tuberculosis in RPMI with 30% nonheat-inactivated, pooled human AB serum at an infection ratio of 1–2 bacteria/20 macrophages, which yielded an average of 8.4% cells infected with four bacilli. Cells were then incubated for 1 h followed by three washes to remove nonphagocytosed bacteria. The cells were then cultured for another hour in RPMI supplemented with 10% heat-inactivated pooled human serum with or without 10 μg/mL of MDP. To evaluate macrophage control of mycobacterial intracellular growth, infected macrophages were incubated for 1 h (Day 0), 24 h (Day 1), and 96 h (Day 4). The cells were lysed with 1% SDS followed by 20% BSA, serially diluted in Middlebrook 7H9 medium and plated in triplicate over 7H10 medium to quantify colony forming units (CFUs). To take into account the phagocytosis of individual cells, the fold change of intracellular growth was calculated as the CFU at Day 1 or 4/CFU at Day 0.

For the modulation of gene expression and cytokine production following M. tuberculosis infection, alveolar macrophages were infected at the same infection ratio using 5 × 105 cells plated in 24-well plates under the same conditions described above. The cells were treated with MDP for 24 h post infection, and supernatants were collected to assess cytokine production. Cells were lysed, and mRNA was extracted. For transmission electron microscopy (TEM) examination, alveolar macrophages were infected at MOI 5 using 4 × 106 cells in polypropylene tubes under the same conditions described above, which yielded an average of 42.6% of cells infected with four bacilli. The cells were treated with MDP for 24 h post infection and fixed in preparation for electron micro-scopy detection of subcellular localization of proteins and ultrastructure.

Electron microscopy

Cells were prepared for TEM by centrifugation for 1 min/6000 rpm followed by fixation of the various cell preparations for either immunodetection of protein subcellular localization or conventional ultrastructure observation. An analysis of the subcellular localization of NOD2, IRGM, LC3, and ATG16L1 proteins was performed as previously described [[37]]. Briefly, cells were fixed in 4% paraformaldehyde in 0.2 M Sörensen buffer; the samples were dehydrated with different concentrations of ethylic alcohol and infiltrated with LR-White hydrosoluble resin (London Resin Co., Hampshire, UK). Sections 60 to 80 nm thick were placed on nickel grids. The grids were incubated overnight at 4°C with specific polyclonal rabbit anti-NOD2 (Millipore), anti-IRGM (Abcam), anti-LC3, or anti-ATG16L1 (Novus Biologicals) antibodies. After rinsing with PBS, the grids were incubated for 2 h at room temperature with goat anti-rabbit IgG (Sigma-Aldrich) conjugated to 5-nm gold particles (Sigma-Aldrich) and diluted 1:20 in PBS. The grids were contrasted with uranyl acetate (Electron Microscopy Sciences, Fort Washington, PA, USA) and examined with an M-10 Zeiss electron microscope (Karl Zeiss, Jena, Germany). To quantitatively assess the recruitment of autophagy proteins to the mycobacteria-containing vesicle, we performed the morphometric analysis by counting the gold particles co-localizing with the bacteria contained in 10 randomly selected cells from each condition (10–17 bacteria/condition) using ImageJ software.

For conventional ultrastructure observations, the cells were fixed in 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7), postfixed in 2% osmium tetroxide in 100 mM cacodylate buffer, dehydrated with increasing concentrations of ethanol and gradually infiltrated with Epon resin (Pelco International, Redding, CA, USA). Thin sections were contrasted with uranyl acetate and lead citrate before TEM examination.

Statistical analysis

Data were analyzed by the non-parametric two-tailed Wilcoxon signed-rank test. Boxplots displaying median and quartiles (min to max) or means ± standard error of the mean (SEM) are presented were indicated. TEM data were analyzed using a two-tailed paired t-test. Values were statistically different when p < 0.05. Analysis was performed using SPSS 15.0 for Windows (SPSS, Chicago, IL, USA) and GraphPad prism 5.0 for Mac (GraphPad Software Inc., La Jolla, CA, USA).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

This work was supported by the National Council of Science and Technology (CONACYT) grants SEP2004-CO1–47745 and CB-2008–01-101948. We acknowledge the Facultad de Ciencias, Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México and the CONACYT for the scholarship awarded to Esmeralda Juárez. We are thankful for the excellent technical assistance of Ricardo Poblano and Edmundo Olivares.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information
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Abbreviations
CARD

caspase activation recruitment domain

IRGM

immunity-related GTPase M

MDP

muramyldipeptide

NLR

nod-like receptor

NOD

nucleotide-binding oligomerization domain 2 receptor

NOD2

nucleotide-binding oligomerization domain 2 receptor

Rip2

receptor interacting protein 2

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2213-sup-0001.ppt2643K

Supporting information. Figure 1. The recruitment of autophagy proteins to the site of bacteria localization was NOD2- dependent. Human primary alveolar macrophages were infected with M. tuberculosis H37Rv (Mtb) at an MOI of 5 for 1 h. Non-phagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour in presence of 10 mM SB203580, a Rip2/p38 inhibitor, to block NOD2 signaling prior to MDP stimulation. Cells were then treated with 10 μg/ml of MDP for 24 h. The subcellular localization of autophagy proteins was ascertained using anti-IRGM, anti-LC3 and anti-ATG16L1 antibodies and detected with a secondary antibody coupled to 5 nm gold particles (indicated with arrowheads; bar represents 150 nm, TEM magnification x 80,000). Data were generated from one experiment.

Supporting information. Figure 2. MDP administered after infection induces LC3-II conversion. Human primary alveolar macrophages were infected with M. tuberculosis H37Rv at an infection ratio of 1-2 bacteria/20 macrophages for 1 h. Non-phagocytosed bacteria were washed away, and the macrophages were then treated with 10 μg/ml of MDP or left with medium alone. (A) LC3 I/II protein expression was assessed in cytoplasmic extracts by western blot; tubulin was used as loading control. (B) The LC3 II/I ratio and (C) the amount of LC3 II normalized to tubulin relative to that of infected-only cells was calculated by densitometry using the ImageJ software. Data are depicted as the means ± SEM of n=3 subjects.

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