Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Cellular Immunology 260 (2010) 128–136

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Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis -induced macrophage death Dulfary Sánchez a,b, Mauricio Rojas a,b, Israel Hernández a,1, Danuta Radzioch c, Luis F. García a,b, Luis F. Barrera a,b,* a b c

Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Universidad de Antioquia, Medellín, Colombia Centro Colombiano de Investigación en Tuberculosis (CCITB), Medellín, Colombia Centre for the Study of Host Resistance, Departments of Experimental Medicine and Human Genetics, McGill University Health Centre Research Institute, Montreal, Canada

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Article history: Received 3 September 2009 Accepted 12 October 2009 Available online 19 October 2009 Keywords: Mycobacterium tuberculosis Macrophages Cell death TLR2 TLR4 Signal transduction

a b s t r a c t Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/ MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-jB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Mycobacterium tuberculosis (Mtb), a slow growing pathogenic intracellular microorganism, is the etiological agent of the human tuberculosis (TB), one of the main causes of morbidity and mortality worldwide. The ability of the innate immune system to respond to Mtb is essential to achieve efficient defense of the host against this infection. In the pulmonary alveoli, macrophages play an important role during the early immune response to the infection by Mtb. Macrophages express Pattern Recognition Receptors (PRRs) recognizing conserved molecular moieties that distinguish a broad specificity of microorganisms, the so-called Pathogen-Associated Molecular Patterns (PAMPs). Upon PAMP ligation by PRRs, signal transduction results in activation of transcription factors for many genes controlling innate immunity, which may lead to control Mtb intracellular replication, and eventually to its elimination [1]. Among PRRs, Toll-Like Receptors (TLRs) are recognized as members of the IL-1R superfamily and share a common activation pathway mediated by their Toll/IL-1R signaling domain, resulting in

* Corresponding author. Address: Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Universidad de Antioquia, Cra 53 No. 6130, Lab. 510, Medellín, Colombia. Fax: +57 4 210 6450. E-mail address: [email protected] (L.F. Barrera). 1 Present address: Neuroscience Research Institute (NRI), University of California, Santa Barbara, CA 93106, USA. 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.10.007

activation of MAPKs and NF-jB; however, selective pathways can be triggered by some TLRs, in particular TLR2, TLR4, and TLR9 can activate the PI-3K pathway [1]. Regarding mycobacterial infections, numerous studies have shown a role for TLR1 [2], TLR2 [1,3,4], TLR4 [3–5], TLR6 [6] and TLR9 [7] in Mtb recognition or mycobacterial cell wall-associated components. One of the most remarkable consequences of macrophage infection by Mtb is the induction of apoptosis and necrosis of both human [8,9] and murine macrophages [10,11]. While apoptosis has been interpreted as protective for the host, necrosis may lead to bacterial growth and dissemination [12] and maybe partly responsible of the local inflammation by the release of substances damaging adjacent tissue [13]. Thus, apoptosis and necrosis may have different implications on the immunopathology of TB. It has been shown that mycobacterial ligands or live Mtb induce apoptosis mainly through TLR2 [4,14]; however, it is not known if TLR2- and/or TLR4-dependent signaling pathways participate in necrosis induction in response to Mtb infection. The mechanisms associated with apoptosis have been widely studied and our group and others have shown that TNFa is implicated in apoptosis induction [10,15–17]. This cytokine seems to have a critical role modulating both apoptosis and necrosis processes of Mtb-infected macrophages or monocytes treated with purified protein derivative (PPD). Blockade of TNFa with a specific monoclonal antibody clearly inhibited apoptosis while favored necrosis in human monocytes and U937 macrophages [17,18]. TNFa production induced by

D. Sánchez et al. / Cellular Immunology 260 (2010) 128–136

Mtb has been previously shown to depend TLRs signaling via MyD88 [4]; however, the particular contribution of TLR2 and TLR4 signaling to TNFa production has not been clearly defined. This study provides evidence that MyD88-dependent TLR2- and TLR4-signals are critical for the induction of apoptosis by live Mtb H37Rv in murine macrophage cell lines. Results show that during in vitro Mtb infection of macrophages, the absence of TLR4 signaling favors necrosis underlining importance of the TLR4-mediated regulation of balance between apoptosis and necrotic signals induced by the macrophage infection with Mtb. In addition, apoptosis was found to be dependent on the ERK1/2 and p38 MAPKs, as well as on PI-3K and NF-jB activity, whereas necrosis process seems to be also modulated by ERK1/2 and p38 MAPKs but not by PI-3K and NF-jB activity. In addition, intracellular levels of TNFa were not correlated with the type of cell death suggesting additional modulators may be involved in cell death induction. Together this data indicate that apoptosis and necrosis are dependent on both TLR2 and TLR4 signaling, and ERK1/2, p38 MAPKs, PI-3K and NF-jB are critical for apoptosis of Mtb-infected macrophages. 2. Materials and methods 2.1. Reagents and antibodies Peptidoglycan (PGN) from Staphylococcus aureus, anhydrous dimethyl sulfoxide (DMSO), fluorescein diacetate (FDA) and trypan blue were obtained from SIGMA–Aldrich (Saint Louis, MO, USA); lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 was purchased from Alexis Biochemicals (San Diego, CA, USA); Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), Tris and Fetal Bovine Serum (FBS) from Gibco-BRL (Grand Island, NY, USA); PI-3K inhibitor wortmannin, p38 MAPK inhibitor SB203580 and NF-jB inhibitor pyrrollidine dithiocarbamate (PDTC) from Calbiochem (La Jolla, CA, USA); MEK1/2 inhibitor U0126, and Tween 20 from Promega (Madison, WI, USA); ethidium bromide (EB), and brefeldin-A from ICN Biomedical Inc. (Aurora, Ohio, USA); Middlebrook 7H9 broth and Middlebrook 7H10 agar from Becton–Dickinson (Franklin Lakes, NJ USA); Glycine electrophoresis grade and Bovine Albumin Fraction V protease free from MP Biomedicals (Solon, OH, USA); p-formaldehyde (PFA) from Fisher Scientific (Pittsburgh, PA); glycerol from Mallinckrodt Baker (Phillipsburg, NJ); Oleic acid–albumin–dextrose–catalase (OADC) from BBL & DIFCO (Becton–Dickinson, San Diego, CA, USA); ELISA Kit for murine TNFa from R&D Systems (Minneapolis, MN, USA); Limulus amoebocyte lysate (LAL) QCL-1000 (Biowhittaker, Walkersville, MD); fluorescein isothiocyanate (FITC)-conjugated annexin V and Annexin-V/PI and DIOC6 (3,30 dihexyloxacarbocyanine-iodide) from Invitrogen (Carlsbad, CA, USA); propidium iodide from (ICN Biomedicals (Cleveland, OH). R-PE-rat anti-mouse TNF (Clone MP6-XT22) or rat IgG1-PE as isotype control from BD Pharmingen (San Diego, CA, USA); antimouse TLR4/MD2 (Clone MTS510) and anti-TLR2 (Clone T2.5) from HyCult biotechnology (Canton, MA, USA); and mouse IgG1 and Rat IgG2a were purchased from eBioscience. 2.2. Mtb culture Mycobacterium tuberculosis strain H37Rv was obtained from the Instituto Nacional de Salud, Bogotá, Colombia. Mtb was grown in Middlebrook 7H9 broth supplemented with 10% and Tween 80 (0.05%) for 10 to 15 days to reach exponential growth phase [19]. Bacterial cultures were pelleted at 3000  g for 30 min and suspended in PBS 1X. Mycobacterial clumps were disrupted by sonication for 4 min at 4 °C each cycle for 7 cycles, 40 W output (CV33 Sonics Vibra Cell, Newtown, CT). The sonicate was centrifuged for

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5 min at 400  g, and the upper bacterial suspension was diluted in DMEM supplemented with 30% glycerol (vol/vol), adjusted to final absorbance of 0.1 (OD620) and frozen at 70 °C until used. The number of CFU was determined by plating 5 lL of serial dilutions onto petri dishes (Nunclon Delta, Intermed, Copenhagen, Denmark), containing Middlebrook 7H10 agar supplemented with and 1% OADC pH 7.2, and the CFU counted after 3 weeks of culture at 37 °C. 2.3. Percentage of infected macrophages In order to determine the ability of different cells lines to phagocytoze Mtb, the percentage of infected cells was evaluated by fluorescein diacetate (FDA)-labeled Mtb associated or phagocytosed by macrophages infected for 24 h and determined by flow cytometry. Mtb H37Rv was stained with 100 ng/mL FDA–acetone in PBS during 30 min at 37 °C, washed twice with PBS 1X and then frozen at 70 °C until used. FDA staining allows the evaluation of the mycobacterial viability and the estimation of the percentage of infected macrophages [20]. Also the number of macrophages associated with bacilli was counted by Ziehl–Nielssen staining [21] by light microscopy at 6 h post-infection. 2.4. Murine macrophage cell lines and Mtb infection and stimulation with TLR agonists The procedure to derive the macrophage cell lines used in this study has been described previously [22] and have been used in different study models [10,23–26]. The wild-type (WT) macrophage line (B10R) expresses surface markers and functional activities typical of tissue macrophages [23]. The TLR2- and MyD88deficient macrophage lines were derived from knockout mice as described in [22]. The TLR4 macrophage line was derived from the C57BL/10ScNcr that had a spontaneous deletion of TLR4 gene [27]. TLR2 KO, TLR4 KO and MyD88 KO mice were backcrossed to C57BL/6 background for at least several generations prior to generation of the macrophage cell lines. TLR2, TLR4 and MyD88 mRNA expression by macrophages was determined using semiquantitative PCR (data not shown) and the obtained results confirmed the absence of TLR2, TLR4 or MyD88 mRNA expression by the respective macrophage lines as previously described [26]. Cells were growth in DMEM containing 2 mM glutamine and 5% heat-inactivated FBS (complete media) without antibiotics at 37 °C, 5% CO2, 95% relative humidity. Macrophages (3  105) were cultured in 24-well polypropylene tissue culture plates (Becton– Dickinson, Franklin Lakes, NJ, USA) overnight to allow cell adherence before infection. Then, cells were washed to remove nonadherent cells and cultured with fresh complete medium. Macrophages were infected for the indicated time-points with Mtb at a Multiplicity of Infection (MOI) of 5:1, or stimulated with LPS (1 lg/mL) or PGN (10 lg/mL). All medium components contained <0.1 EU/mL final concentration of endotoxin as measured by Limulus amoebocyte lysate (LAL) 1000. 2.5. Blockade of TLRs stimulation WT macrophages were cultured for 30 min in presence of 30 lg/mL anti-TLR2 (clone T2.5), anti-TLR4/MD (clone MTS510) or mouse IgG1 and rat IgG2a as isotype control antibodies. After this time, cells were infected with Mtb and cultured for additional 24 h. 2.6. Determination of cell death Changes in the mitochondrial membrane permeability transition (MPT; [28]) and cell surface exposure of phosphatidylserine

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(PS; [29]) were used as an indicative of apoptosis using the fluorochrome DIOC6 or FITC-conjugated annexin V. Cell membrane damage during necrosis was measured by the uptake of ethidium bromide (EB) or propidium iodide (PI). In brief, macrophages infected or not with Mtb were suspended in 1 mL of PBS 1X containing 10 lL of DIOC6 (7 lM) plus 25 lL of EB (1.3 mg/mL) or with 5 lL of annexin-V plus 10 lL of propidium iodide (1 lg/mL) in 100 lL of PBS 1X for 30 min at RT in the dark. Then, cells were harvested by scraping (Sarstedt, Nümbrecht, Germany), washed with PBS 1X and then suspended in PBS 1X. The apoptotic and necrotic cell proportion was assessed on an EPICS XL flow cytometer (Coulter, Hialeah, FL, USA) as described previously [24].

2.7. Inhibition of MEK/ERK1/2, p38 MAPK, PI-3K and NF-jB activity Stock solutions of the specific PI-3K inhibitor wortmannin and MEK1/2 inhibitor U0126 were prepared in anhydrous dimethyl sulfoxide (DMSO); NF-jB inhibitor PDTC and p38 MAPK inhibitor SB203580 were prepared in sterile water. Macrophages were cultured in presence or absence of U0126 (10 lM; [30]), SB203580 (5 lM; [30]), wortmannin [30] and PDTC [31] at 100 lM final concentration or DMSO (0.1% [vol/vol]) for 30 min before infection with Mtb. The effect of inhibitors on Mtb-infected macrophages was evaluated after 24 h of culture. The effect of DMSO on the percentage of death cells was negligible. Concentrations of inhibitors were chosen according to the 50% inhibitory concentration (IC50) values and specificities reported by others [32–34]. The effect of inhibitors on Mtb viability was not significant, as evaluated by counting CFU of bacilli cultured in DMEM in presence of inhibitors for 6 h (data not shown).

2.8. Staining of Mtb and determination of intracellular TNFa Fourteen hours after Mtb-FDA infection (MOI 5), macrophages were treated with 1 lg/mL brefeldin-A for 4 h at 37 °C and 5% CO2, washed twice with PBS 1X, fixed with 2% PFA in 0.1 M de NaH2PO4 during 30 min at RT and harvested. Then, cells were washed once with permeabilization buffer (PBS 1X containing 0.1% BFS and 0.1% saponin, pH 7.4), and incubated with 100 lL of permeabilization buffer containing 0.2 lg mAb anti-TNFa-PE or Rat IgG1-PE as isotype control for 30 min at RT. Cells were washed two times with PBS 1X and 2  104 cells were analyzed by flow cytometry. As control for the intracellular signal, non-permeabilized cells were stained with specific antibodies. Determination of TNFa+ macrophages was done in both, Mtb (bystander cells) and Mtb+ macrophages (infected cells).

2.9. ELISA Supernatants from macrophage cultures infected or not with Mtb were collected and centrifuged for 10 min at 12000  g and stored at 20 °C until analysis. TNFa in the culture supernatants was measured by Mouse TNFa Immunoassay Kit following the manufacturer’s instructions.

2.10. Statistics All experiments were done independently and repeated at least three times. Data were analyzed by one- or two-way ANOVA depending of type of analyses. Statistical significance was tested at p < 0.05 as critical value. Data is presented as the mean ± SEM. All analyses were done using Prism 5 software (Graphpad, San Diego, CA).

3. Results 3.1. TLR2, TLR4 and MyD88 are critical for Mtb-induced macrophage death Our laboratory and others have reported that infection of human and mouse macrophages with Mtb results in the induction of both apoptosis [9,15,17,24] and necrosis [11,17]. To determine the contribution of TLR2, TLR4 and MyD88 on cell death, we used bone marrow-derived macrophage (BMDM) cell lines from either wild-type (WT), TLR2-, TLR4-, or MyD88-defficient mouse strains. Macrophages were infected at 5:1 MOI of Mtb for 24 h, and the cell percentage with mitochondrial and cell membrane damage as indicative of apoptosis and necrosis, respectively, was determined by flow cytometry using DIOC6/EB staining. As shown in Fig. 1, there was a higher percentage of WT macrophages with mitochondrial damage (DIOC6low, 43% ± 6.2) during Mtb infection comparing to TLR2- (0.9% ± 0.4; p < 0.001), MyD88- (1.8% ± 1.6; p < 0.001) and TLR4-deficient (23.8% ± 6.5; p < 0.01) macrophages, although TLR4deficient cells had significantly more DIOC6low cells than TLR2- and MyD88-deficient macrophages. In addition, TLR4-deficient macrophages showed a significant percent of cells with membrane damage (EB+, 17% ± 0.8; p < 0.05) that was almost absent in WT (2.5% ± 0.8), TLR2- (3.5% ± 1.3) and in MyD88-deficient (2.8% ± 1.8) macrophages. That these cells were indeed undergoing apoptosis and necrosis, was confirmed by Annexin-V plus PI staining and the results were essentially the same than those obtained with DIOC6/EB (data not shown). These results suggest that Mtb-induced apoptosis of macrophages requires TLR2, the adaptor molecule MyD88, and, at a lesser extent, TLR4. Furthermore, in the absence of a signal provided by TLR4, significant increase in percentage of cells undergoing necrosis is observed among Mtb infected cells. To further confirm the participation of TLR2 and TLR4 on Mtb-induced macrophage death, anti-TLR2, anti-TLR4 or both blocking monoclonal antibodies were added to WT macrophages. As shown in Fig. 2, while non-treated WT macrophages showed 52.6% ± 3.9 of DIOC6low cells, the treatment with anti-TLR2 reduced this percentage to 0.9% ± 0.5 (p < 0.001) and following the treatment with antiTLR4 the percentage of DIOC6low cells was reduced to 4.1% ± 3.6 (p < 0.001). In addition, anti-TLR4 increased percentage of EB+ cells to 17.7% ± 5 (p < 0.001) compared with untreated WT-infected cells (0%). Treatment with anti-TLR2 plus anti-TLR4 significantly reduced the percentage of DIOC6low cells (52.6% ± 3.9 to 0.3% ± 0.3; p < 0.001), and EB+ cells were not observed (1.2% ± 0.6; p < 0.001). Although the effect of isotype antibodies was minimal (data not shown), it was subtracted from the values obtained using specific antibodies. These results demonstrate the critical role of TLR2 and TLR4 in the induction of apoptosis in Mtb infected cells, and show that TLR2-induced signaling in absence of TLR4 favors necrosis. By using flow cytometry and counting infected macrophages by Ziehl–Nielssen staining, the ability of these cells to phagocytose or bind FDA-labeled Mtb was evaluated. As shown in Fig. 3A, no significant differences were observed in the percentage of infected cells among the macrophage cell lines as estimated by Mtb-FDA+ cells (WT = 56.1% ± 2; myd88/ = 44.6% ± 3.9; tlr2/ = 52.3% ± 3.8; tlr4/ = 55.2% ± 3.1) or by Ziehl-Nielssen staining (Fig. 3B; WT = 51% ± 1.7; myd88/ = 64% ± 6.2; tlr2/ = 62% ± 7.1; tlr4/  = 67% ± 4.4).

3.2. Activation of TLR2-, TLR4- and MyD88-dependent signaling pathways by Mtb induces TNFa production TNFa is a pro-apoptotic factor in Mtb-infected macrophages [10,16]. To evaluate the contribution of TLR2, TLR4 and MyD88

D. Sánchez et al. / Cellular Immunology 260 (2010) 128–136

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EB

A WT cells

myd88-/- cells

tlr2-/- cells

tlr4-/- cells

Mtb-infected WT cells

Mtb-infected myd88-/- cells

Mtb-infected tlr2-/- cells

Mtb-infected tlr4-/- cells

DIOC6

B EB+

% Cells ± SEM

100

***

***

80

*

DIOC6low

**

DIOC6highEB-

60 40 20 0

WT

myd88-/-

tlr2-/-

tlr4-/-

% Cells ± SEM

Fig. 1. TLR2, TLR4 and MyD88 are critical to Mtb-induced apoptosis. Wild-type (WT), TLR2-, TLR4- and MyD88-deficient macrophage cell lines (3  105 cells/well) were infected with Mtb H37Rv (MOI 5:1). Twenty-four hours post-infection, mitochondrial and cytoplasm membrane damage was determined by DIOC6/EB staining and cells were analyzed by flow cytometry. (A) Representative dotplot of DIOC6/EB staining. (B) Bars represent mean ± standard error (SEM) of seven independent experiments and comparisons were performed between macrophages cell lines, *p < 0.05, **p < 0.01, ***p < 0.001 with two-way ANOVA analyses.

100

***

***

80

EB+

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DIOC6

***

low

DIOC6highEB-

60 40 20 0

Mtb Anti-TLR2 Anti-TLR4

+ -

+ + -

+ +

+ + +

Fig. 2. Blockade of TLR2 inhibits apoptosis and blockade of TLR4 favors necrosis during Mtb infection. Wild-type (WT) macrophages (3  105 cells/well) were incubated in presence or absence of monoclonal anti-TLR2 and/or anti-TLR4 antibodies at a final concentration of 30 lg/ml, and 30 min later, the macrophages were infected with Mtb (MOI 5:1) for additional 24 h. Thereafter, mitochondrial damage and cytoplasm membrane damage were determined by DIOC6/EB staining and cells were analyzed by flow cytometry. Two-way ANOVA was performed comparing the effect of blocking antibodies treatment with Mtb infection; media ± SEM; ***p < 0.001; n = 3.

signals on TNFa production during Mtb infection, WT, TLR2-, TLR4and MyD88-deficient macrophages were cultured in presence or absence of FDA-labeled Mtb and 18 h later the percentage of macrophages expressing intracellular TNFa was determined by flow cytometry. It was possible to distinguish three TNFa+ macrophage populations (i) TNFa+MtbFDA, (ii) TNFa+MtbFDA+ and (iii) TNFaMtbFDA (data not shown). Although the infection with Mtb induced TNFa in both macrophage populations TNFa+MtbFDA- and TNFa+MtbFDA+, there were a higher percentage of TNFa+MtbFDA+ macrophages than TNFa+Mtb+FDA macrophages,

thus, next experiments were focused on the TNFa+MtbFDA+ macrophages. As shown in Table 1, infection with Mtb resulted in a significant increase in the percentage of TNFa+ cells, as well as in the mean fluorescence intensity (MFI) compared to non-infected macrophages in all macrophage lines. However, there was a higher production of TNFa per cell by WT cells (MFI; 310.7 ± 34.7) than by TLR2- (71.4 ± 3.8; p < 0.001), TLR4- (62.9 ± 11.5; p < 0.001), and MyD88-deficient macrophages (81.1 ± 18; p < 0.001). No significant differences in the percentage of TNFa+ cells were observed among all infected deficient cell lines. Determination of accumulated TNFa in the supernatants of non-infected and Mtb-infected macrophages by ELISA confirmed these observations (data not shown). These results demonstrate that TNFa is produced in response to Mtb through TLR2 and TLR4 in a MyD88-dependent pathway. 3.3. A single signal through TLR2 or TLR4 is not enough to induce cell death Since TLR2 and TLR4 seem to be necessary for Mtb-induced macrophage apoptosis, we wondered whether other TLR2 and TLR4 agonist could activate death signals. Treatment of WT macrophages with peptidoglycan (PGN) or lipopolysaccharide (LPS) did not induce mitochondrial or cell membrane damage (0.03% ± 0.03; Fig. 4); however, treatment with PGN plus LPS resulted in a significant percentage of cells with mitochondrial damage (38.4% ± 1.2; p < 0.001), suggesting that apoptosis depends of the activation of both TLR2 and TLR4 pathways. In order to answer whether treatment with PGN or LPS affected pro-apoptotic events induced by Mtb, we tested the effect of PGN or LPS treatment

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B 80

80

60

60

% of infection ( Mtb -ASF+ cells)

% of infection ( Mtb -FDA+ cells)

A

40

20

0

40

20

0

myd88-/-

WT

tlr2 -/-

tlr4 -/-

WT

myd88-/-

tlr2 -/-

tlr4 -/-

Fig. 3. Macrophage cell lines do not differ in phagocytic capacity to Mtb. Macrophages (3  105) were infected with live Mtb stained with FDA (MOI 5:1) and 24 h later, the percentage of Mtb-FDA associated macrophages was determined by (A) flow cytometry and by (B) counting of cells Mtb-Ziehl-Neelsen acid-fast staining (AFS) positive by light microscopy. Results are shown as the media ± SEM from nine independent experiments and the level of significance was calculated by one-way ANOVA.

WT

% MFI % MFI % MFI % MFI

myd88/ tlr2/ tlr4/

Non- infected

Mtb (infected)

p-Valuea

4.3 ± 1.7 6.6 ± 0.7 3.9 ± 1 9.6 ± 1 1.1 ± 0.7 5.6 ± 0.8 1.1 ± 0.3 7.1 ± 1.7

36.4 ± 6.3 310.7 ± 34.7 37.8 ± 5.9 81.1 ± 18 49.7 ± 2.6 71.4 ± 3.8 40.8 ± 5.9 62.9 ± 11.5

p < 0.001 p < 0.001 p < 0.001 p < 0.01 p < 0.001 p < 0.01 p < 0.001 p < 0.05

p-Valueb

p < 0.001 p < 0.001 p < 0.001

Data shows media ± SEM of nine independent experiments. a p-Value obtained comparing TNFa production by infected cells with noninfected cells (two-way ANOVA). b p-Value obtained comparing TNFa production by KO- with WT-infected cells (two-way ANOVA).

***

*** ***

***

EB+

% Cell ± SEM

100

DIOC6low

80

DIOC6highEB-

60 40 20 0

LPS PGN Mtb

+ -

+ -

+ + -

+

+ +

+ +

Fig. 4. Both TLR2 and TLR4 receptors ligation is required to induce apoptosis. WT macrophages (3  105 cells/well) were incubated in presence or absence of LPS (1 lg/mL) and/or PGN (10 lg/mL), or pre-incubated or not for 30 min with LPS (1 lg/mL) or PGN (10 lg/mL), previous to Mtb infection (MOI 5:1). Twenty-four hours after agonist treatment or infection, mitochondrial and membrane damage were determined by DIOC6/EB staining and cells were analyzed by flow cytometry. Two-way ANOVA was performed comparing the effect of different treatments and/ or Mtb infection; media ± SEM; ***p < 0.001; n = 3.

(Figs. 1 and 2); however, it is possible that it could be a late consequence of mitochondrial damage, the so called secondary necrosis [35]. In order to evaluate whether membrane and mitochondrial damage detected in TLR4-deficient macrophages occur at different time periods, the percent of EB+ cells and mitochondrial damage was determined at 6, 12, 18, and 24 h post-infection. As shown in Fig. 5, at 12 h post-Mtb infection of TLR4-deficient macrophages there was significant increase of percentage of macrophages EB+ compared with non-infected cells (0% to 15.4% ± 2, p 6 0.05). At this time, no significant changes in the percentage of cells with mitochondrial damage (0% to 6% ± 1.6) were observed. Cells with mitochondrial damage were detected only after 18 h of infection. 3.4. MAPKs, PI-3K and NF-jB have a critical role in cell death induction and TNFa production Foregoing results suggest that signals through TLR2 and TLR4 lead to apoptosis, and TLR4 protects from Mtb-mediated necrosis. Infection of human or murine macrophages with Mtb or treatment with mycobacterial derivates such as heat shock proteins (HSPs) 65 and 70, and glycopeptidolipids (GPL), results in activation of p38 and ERK1/2 MAPKs, PI-3K, and NF-jB and this activation correlated with engagement of TLR2 and TLR4 [1]. To identify critical points associated with the type of cell death, WT and TLR4-deficient macrophages were treated 30 min with MEK/ERK1/2 inhibitor U0126 [30], p38 MAPK inhibitor SB203680 [30], PI-3K inhibitor wortmannin [30] and NF-jB inhibitor PDTC [31], and then the inhibitor treated or untreated macrophages were infected with Mtb. As

DIOC6low

100

% Cells ± SEM

Table 1 Mtb-induced TNFa production is dependent of TLR2, TLR4 and MyD88.

80

EB+

**

**

*

*

60

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DIOC6highEB-

40 20

*

0

30 min before and during the infection. As shown in Fig. 4, LPS (1.2% ± 1.2) and PGN (3.6% ± 1.8) prevented Mtb-induced mitochondrial damage (58.8% ± 5.4). These results reinforce our previous conclusion that pro-apoptotic signaling is induced by concomitant TLR2 and TLR4 signaling activation upon ligands recognition. Membrane damage was observed in TLR4-deficient macrophages and WT cells treated with anti-TLR4 upon Mtb infection

0 6 12 18 24 Post-infection Time (hours) Fig. 5. Membrane damage in tlr4/ macrophages infected with Mtb is not a secondary effect of mitochondrial damage. tlr4/ macrophages were infected with Mtb at MOI 5:1 at the indicated time-points. Determination of macrophages with mitochondrial and membrane damage was determined by DIOC6/EB staining and cells were analyzed by flow cytometry. Two-way ANOVA was performed comparing between 0 h and different time-points of Mtb infection; media ± SEM; *p < 0.05, ** p < 0.01, ***p < 0.001; n = 3.

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MyD88-deficient macrophages induced by Mtb prevented any detection of the inhibitors activity on cytokine production. In addition, these data show that reduction or increasing of TNFa by inhibitors does not correlate with the blockade of mitochondrial damage associated with apoptosis in WT-infected cells or membrane damage associated with necrosis in TLR4-deficient macrophages.

shown in Fig. 6A, while Mtb infection of WT macrophages induced 43% ± 6.2 of DIOC6low and 2.5% ± 0.8 of EB+ cells, the treatment with SB203580, U0126, wortmannin and PDTC significantly reduced the DIOC6low percentage to 7.3% ± 4.6, 5.5% ± 4.7, 5.3% ± 2.5 and 3.7% ± 0.7, respectively. On the contrary, none of the inhibitors affected significantly the percentage of EB+ cells. Similar to WT macrophages, the percentage of cells with DIOC6low induced by Mtb in TLR4-deficient macrophages (Fig. 6B; 23.8% ± 6.5) was reduced when macrophages were treated with each inhibitor (3 ± 1.5, 11.2 ± 3, 6.6 ± 2, 8.8 ± 4.1 for SB203580, U0126, wortmannin and PDTC, respectively). In addition, although all tested inhibitors did not affect significantly the percentage of EB+ cells, p38 MAPK inhibitor SB203580 reduced the percentage of EB+ cells approximately 2.7-fold (6.4% ± 1.6) whereas the MEK/ ERK1/2 inhibitor U0126 increased this percentage about 1.9-fold (32.8% ± 4.6) as compared to untreated and infected TLR4-deficient macrophages (17 ± 0.8). Comparing the effect of each inhibitor on cell death between WT and TLR4-deficient macrophages, U0126 induced significantly higher percentage of EB+ in TLR4-deficient cells as compared to WT cells (p < 0.001). Overall, these results indicate that mitochondrial damage induced by Mtb involves ERK1/2, p38, PI-3K and NF-jB, and these results suggest that p38 and ERK1/2 MAPKs may play an opposite role in regulation of cell necrosis, since p38 inhibition reduces necrosis in TLR4-deficient macrophages while inhibition of ERK1/2 increased it. To establish if p38 and ERK1/2 MAPKs, PI-3K and NF-jB are also involved in TLR2- and TLR4-mediated TNFa production in response to Mtb, macrophages were pre-treated for 30 min with U0126, SB203580, wortmannin, and PDTC, and then infected with Mtb for 24 h. As shown in Table 2, the percentage of TNFa+ cells was not affected by treatment with any of the inhibitors; however, the mean fluorescence intensity (MFI) was significantly decreased when Mtb-infected WT macrophages were treated with U0126, wortmannin, and PDTC, compared to Mtb-infected WT cells cultured in the absence of the inhibitors. On the contrary, the treatment with SB203580 of Mtb-infected WT cells resulted in a significant increase of TNFa MFI. These results suggest that ERK1/2, p38, PI-3K and NF-jB are essential for TNFa production; however, while ERK1/2, PI-3K, and NF-jB are positive regulators, p38 MAPK is a negative regulator of its production during Mtb infection. The low production of TNFa by TLR2-, TLR4- and

A

4. Discussion Our study focused on the role of TLR2 and TLR4 on macrophage death upon engagement with Mtb. Our results show an important role of TLR2, TLR4 and their adaptor molecule MyD88 in the proapoptotic signaling induced by Mtb infection. While in TLR2- and MyD88-deficient macrophages do not undergo apoptosis, in TLR4-deficient cells the apoptosis is decreased but necrosis is enhanced. These findings may explain our previous published results by hypothesizing that monocytes from TB patients may have a dysfunction of TLR4-dependent signaling that could explain the necrosis observed after in vitro infection with Mtb [9]. The pro-apoptotic effect of Mtb was compared with TLR2 and TLR4 ligands. Independent signaling activation by TLR2 or TLR4 with PGN or LPS did not induce cell death but combined stimuli triggered apoptosis, suggesting that activation of the apoptotic pathway depends on simultaneous activation of TLR2 and TLR4 pathways. Several evidences support our finding that LPS does not induce apoptosis [36,37]. LPS stimulation prevents apoptosis induced by dexamethasone and anti-Fas antibody [37], live Mtb [24] or TNFa [38]. However, the simultaneous administration of LPS and protein synthesis inhibitors [39], or when NF-jB activation is blocked, as is observed during Y. enterocolitica and Y. pseudotuberculosis infection [40], apoptosis is induced, suggesting that blocking the synthesis of endogenous survival molecules induced by LPS may activate the apoptotic program. TLR2 agonists have different effects on cell death [37,41,42]. It is possible that the balance between survival and cell death signals is due to cooperation of different PRRs signals. TLR2 forms heterodimers with TLR1, TLR6, CD36, and co-receptor CD14 [43,44] facilitating the interaction with different microbial components and possibly promoting different intracellular signaling pathways. Thus differences in apoptosis induction could depend upon the

B

EB+ DIOC6

100

***

***

***

***

% cells ± SEM

100 75 50

* 75 50

25

25

0

0

Mtb SB203580 U0126 Wortmannin PDTC

tlr4-/- tlr2+/+myd88+/+

DIOC6highEB-

WT

% cells ± SEM

low

+ -

+ + -

+ + -

+ + -

+ +

Mtb SB203580 U0126 Wortmannin PDTC

+ -

+ + -

+ + -

+ + -

+ +

Fig. 6. MAPKs, PI-3K and NF-jB activity are necessary to apoptosis of Mtb-infected macrophage. WT (A) and tlr4/ (B) macrophages were treated or not with 10 lM U0126, 10 lM SB203580, 100 lM wortmannin or 100 lM PDTC 30 min before infection with Mtb (5:1), 24 h later mitochondrial damage and cytoplasm membrane damage were determined by DIOC6/EB staining and cells were analyzed by flow cytometry. Comparisons were performed between non-treated controls and treated cells and between WT versus tlr4/ cells by two-way ANOVA; media ± SEM; *p < 0.05, ***p < 0.001; n = 3–7.

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Table 2 ERK1/2, p38, PI-3K and NF-jB have opposite effect on TNFa production induced by Mtb infection.

WT myd88 tlr2/ tlr4/

/

% MFI % MFI % MFI % MFI

Mtb

SB203580 + Mtb

Mtb

U0126 + Mtb

Mtb

Wortmannin + Mtb

Mtb

PDTC + Mtb

27.3 ± 7.9 349.2 ± 61.2 34.7 ± 14.3 67.8 ± 17.5 51 ± 3 74.1 ± 7.5 48 ± 10 47.9 ± 16.2

17 ± 5.2 1112 ± 387  31.3 ± 3.4 171.9 ± 64.2 39.7 ± 6.7 473.4 ± 158 40 ± 6.5 240.8 ± 45.3

36 ± 9.1 266.9 ± 44.6 37 ± 15.7 55.6 ± 12 52.7 ± 2.3 64.1 ± 2.9 48.3 ± 9.9 41.1 ± 9.7

50 ± 13.5 8.9 ± 1.8à 55.7 ± 8.8 10.9 ± 1.6 51 ± 4.6 9.3 ± 1.3 48.7 ± 5.7 8.2 ± 1.1

43.5 ± 13.8 316.1 ± 83.9 40.7 ± 5.8 120 ± 47.5 46.5 ± 5.9 75.9 ± 8 29.7 ± 9.8 99.8 ± 13.8

52 ± 12.4 38.6 ± 19.3à 45 ± 4.7 12.1 ± 4.3 53.2 ± 7 12.6 ± 2.6 37 ± 10.2 16.1 ± 4.4

43.5 ± 13.8 316.1 ± 83.9 40.7 ± 5.8 120 ± 47.5 46.5 ± 5.9 75.9 ± 8 29.7 ± 9.8 99.8 ± 13.8

52.2 ± 11.7 37.5 ± 12à 42.5 ± 4.7 10.2 ± 2.2 52.5 ± 6.2 15.6 ± 4.9 38.7 ± 8.4 19.2 ± 6.5

The symbols   and à are showing the p-values p < 0.05 or p < 0.001, respectively, resulting after two-way ANOVA comparing TNFa production by infected and treated cells with the indicated inhibitor with the corresponding infected and non-treated cells. Data shows media ± SEM of three independent experiments.

functional interactions between TLR2 and other receptors. Cooperation between PRRs allowing a balance between survival and cell death signals was previously demonstrated in a study of atherosclerosis. Apoptosis induced by endoplasmic reticulum stress, as a model of macrophage death in atherosclerotic plaque of advanced lesions, is driven by cooperation between the type A scavenger receptor (SRA) and TLR4 [45]. Ligation of SRA silences the pro-survival TLR4/IRF3/IFNb pathway triggering the pro-apoptotic TLR4/MyD88/JNK pathway, suggesting that SRA redirects TLR4 signaling from pro-survival to pro-apoptotic. Our results show that both TLR2 and TLR4 signals cooperate in order to induce apoptosis during Mtb infection. Functional cooperation between TLR2 and TLR4 has been demonstrated. The ERK1/2 activation and posterior CXCL8 secretion by epithelial cells were shown to be dependent on simultaneous recognition of M. bovis BCG by TLR2 and TLR4 [46]. Recently, it was shown that during in vitro infection with Mtb H37Ra there are terminal steps required to complete apoptosis process [47]. These steps include the formation of an apoptotic envelope on infected cell surface that promotes the preservation of the integrity of the membrane during the formation of apoptotic bodies. When the arrangement of this envelope is suppressed, necrosis is induced. It is tempting to speculate that recognition of Mtb by TLR2 could trigger the early events of apoptosis while TLR4 activates signaling pathways that mediate late events, and when TLR4 signaling is absent, necrosis may occur. This suggestion is in agreement with the hypothesis that necrosis is a consequence of a failed autophagy or apoptosis [48]. On the other hand, since TLR4 may induce two types of signal cascades, one pro-apoptotic and other one pro-survival, we speculate that while TLR2 interact with Mtb and the cell death pathway is activated, the interaction of Mtb with TLR4 could induce signals that could control the TLR2-mediated apoptotic program. Our results using pharmacological inhibitors of ERK1/2 and p38 MAPKs, PI-3K, and NF-jB, indicate that cell death induced by Mtb infection may result from the disruption of signaling pathways mediated by ERK1/2, p38, PI-3K, NF-jB and that the ability of cells to survive or die by apoptosis or necrosis could be dictated by a critical balance among these molecules activity. ERK1/2, PI-3K, and NF-jB are clearly required for macrophage apoptosis in WT macrophages. In contrast the effects of inhibitors of p38 and ERK1/2 MAPK seem to induce opposite effects on the regulation of necrosis in TLR4-deficient macrophages. ERK1/2 inhibition modestly promoted cell necrosis whereas the inhibitor of p38 MAPK seem to slightly inhibit necrosis in TLR4-deficient macrophages. There is controversy regarding the role of ERK1/2, p38, NF-jB, and PI-3K as pro-survival or pro-death molecules. ERK1/2, p38, PI-3K, and NF-jB have been shown to participate in both antiapoptotic [49–51] and pro-apoptotic [49,52–54] programs in different experimental models. The precise intracellular mechanisms that determine the switch from to anti- to pro-apoptotic role of those molecules have not been yet clearly defined.

There is enough evidence showing the critical role of TNFa in apoptosis induced by Mtb [10,16,17,55]. We evaluated the effect of the TLR2, TLR4, and MyD88 signaling and the inhibition of the ERK1/2, p38, PI-3K, and NF-jB activity on intracellular TNFa protein levels induced by Mtb infection. We found that ERK1/2, PI3K, and NF-jB are necessary for TNFa intracellular production, while p38 MAPK is associated with inhibition of TNFa production during Mtb infection. Our data are consistent with the reported role of ERK1/2 in the TNFa production by murine macrophages infected with M. avium and Mtb-infected human monocytes [56,57]. However, there is evidence that human monocytes produce TNFa by a p38-dependent way in response to the mycobacterial 38 kDa glycoprotein [58]. However, our data did not show correlation between the TNFa levels and apoptosis of macrophages infected with Mtb comparing different cell lines and treatment with different inhibitors. We hypothesize that there could be a threshold of TNFa required for triggering apoptosis which is not reached when the TLR2 or TLR4 pathways are blocked. In addition, TNFa levels also did not correlate with apoptosis when MAPKs, PI3K or NF-jB were inhibited, suggesting that TNFa is necessary but not sufficient to activate the apoptotic program in macrophages infected with Mtb. Our result could be also be explained in different way whether other critical components for apoptosis, such as Bcl-2-family molecules or caspases [59], were blocked by the tested inhibitors, suggesting that if TNFa is necessary to induce apoptosis, its levels are not high enough to complete the apoptotic process. On the other way, the activation of pro-necrotic molecules could be modulated by TLRs. Previous studies from our group showed an important role of cytoplasmic phospholipase A2 (cPLA2) in favoring necrosis [18]. It would be interesting to evaluate whether TLR2 and TLR4 modulate cPLA2 activation in Mtb-induced necrosis. Although our results suggest a hierarchical role of TLR2 and TLR4 on apoptosis in response to Mtb and that other PRRs for Mtb may be dispensable in this process, PRRs such as C-type lectins [60] and the scavenger receptor MARCO [61] might also participate in cell death signaling and different experimental systems should be required to evaluate the role of these PRRs. In summary, our data indicate that simultaneous activation of TLR2 and TLR4 signaling pathways induce apoptosis of macrophages and that the induction of apoptosis and necrosis in response to Mtb is dependent of different signaling. After Mtb engagement by TLR2 and TLR4, the activation of ERK1/2, p38, PI3K and NF-jB is important to induce apoptosis, but absence of a TLR4-derived signal(s) favors necrosis, that is modulated by ERK1/2 and p38 MAPK activation. Although Mtb infection induces TNFa production and it is dependent on TLR2 and TLR4 signaling, no close correlation between the activation of signaling molecules tested and the TNFa production and macrophage apoptosis outcome was observed, implying others players in apoptosis and necrosis induction by Mtb depending of TLR2 and TLR4 signaling.

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Further work is necessary to assess how these pathways modulation alter the capacity of these macrophages in respect to their microbicidal activity.

Acknowledgments This work was funded by grants 1115-04-12950 and CT 4312004 from Colciencias, Bogotá, Colombia.

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