HIV/Mtb co-infection increases LC3B puncta formation and association of LC3B to Mtb phagosomes

Autophagy in Mtb or HIV/Mtb co-infected hMDMs was evaluated by analysing LC3B puncta formation, a characteristic of autophagosomes. Cells infected with HIV for seven days were infected for 2 h with Mtb H37Ra-GFP before they were analysed for formation of puncta as well as the puncta co-localization to Mtb phagosomes (Fig. 1a–c). Co-infection significantly increased the frequency of LC3B+ hMDMs, compared to that of single infected cells (p = 0.037) (Fig. 1a,b). Evaluating LC3B+ vs. LC3B- Mtb-phagosomes further showed an increased association of LC3B puncta to phagosomes in co-infected hMDMs (p = 0.029 for single vs. co-infected) (Fig. 1a,c). This is in line with previous findings, where HIV triggered autophagy by increased expression of LC3 II after several days of infection in hMDMs28. The increased puncta formation and co-localization to Mtb phagosomes could not be explained by a higher infection burden in co-infected cells, since there were no differences in the number of bacteria in single and co-infected cells (Fig. 1d).

Figure 1 HIV/Mtb co-infection increases LC3B puncta formation and association of LC3B to Mtb phagosomes. (a) Representative micrographs of LC3B puncta formation (arrowheads) and co-localization to Mtb phagosomes (arrows) in hMDMs infected with HIV (7 days) and/or H37Ra (MOI = 1, 2 h). Blue: DAPI, green: Mtb, red: LC3B (AlexaFluor594). (b) Percentage of LC3B positive hMDMs (3 puncta or more). (c) Percentage of LC3B positive Mtb phagosomes. (d) Percentage of hMDMs infected with the indicated number of bacteria/cell, showing no difference in Mtb infection with or without HIV. Data are mean ± SEM from 10 independent experiments in which 50–100 phagosomes were counted for each condition. *p < 0.05 using paired Student t-test. Full size image

Rapamycin accelerates H37Ra/H37Rv replication in both single and HIV co-infected hMDMs

Next, we investigated the effect of rapamycin-induced autophagy in HIV/Mtb co-infected cells, using a low MOI of Mtb. Induction of autophagy has been shown to decrease the survival of Mtb through phagolysosomal maturation9. However, the intracellular Mtb replication significantly increased upon stimulation with rapamycin, evident at day 3 and further increased at day 7, for both the avirulent (H37Ra) and the virulent (H37Rv) Mtb strain (Fig. 2a). At day 7, H37Rv increased from 6.1-fold to 30.4-fold (p = 0.012) in single infected and from 5.4-fold to 35.8-fold (p = 0.031) in HIV/H37Rv co-infected cells, without or with rapamycin treatment, respectively. Inhibition of autophagy by 3-MA on the other hand had no effect on Mtb replication in either single or co-infected hMDMs. Assessing total amount of bacteria, i.e. amounts found in cell lysate and supernatant (Fig. 2a, second panel), did not further increase the bacterial yield compared to cell lysate alone (Fig. 2a, top panel), indicating that rapamycin affected Mtb growth inside of hMDMs. In order to exclude that the differences seen were due to cell death the viability of hMDMs was measured, showing no differences over the course of infection compared to uninfected cells (Fig. 2b). Rapamycin (and 3-MA) did not have any effect on Mtb growth in absence of hMDMs (Supplementary Fig. S1), further illustrating that rapamycin affects Mtb intracellular growth by manipulating the hMDMs and not by targeting the bacteria directly.

Figure 2 mTOR inhibition using rapamycin accelerates H37Ra/H37Rv replication in both single and HIV co-infected hMDMs. (a) hMDMs were pre-infected with/without HIV for three days before infection with H37Ra or H37Rv (MOI = 1) for 2 hours. Rapamycin (rapa; 1 μM) or 3-MA (1 mM) was added for 1, 3 and 7 days and the signal from luciferase expressing H37Ra or H37Rv in cell lysates and supernatant were measured. “Total bacteria” is the combined supernatant + intracellular pool of bacteria. (b) At the indicated time-points hMDMs (MQ) viability compared to uninfected hMDMs was measured using calcein AM uptake. Data are mean ± SEM from 6 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using paired Student t-test. Full size image

Torin1 inhibits phosphorylation of mTORC1 downstream targets and induces autophagy more efficiently than rapamycin

Rapamycin enhanced Mtb growth in our system, which is in accordance with findings by Zullo et al.35. We therefore further investigated how effective rapamycin was at inducing autophagy. To this end we compared rapamycin to another autophagy inducer, Torin1, which is a more selective and potent ATP-competitive mTORC1 inhibitor than rapamycin21. Autophagy was evaluated by assessing the protein expression levels of the autophagy markers LC3 and SQSTM1 (also known as p62) and indirectly by assessing the inhibition of the mTORC1 pathway, as detected by decreased phosphorylation of its downstream targets S6 and 4EBP1. Torin1 was more efficient in inducing autophagy and autophagic flux compared to rapamycin (Fig. 3a). This was demonstrated by more concentration dependent de-phosphorylation of S6 and 4EBP1, as well as conversion of LC3 I to LC3 II, together with degradation of the autophagy substrate SQSTM1 (Fig. 3a).

Figure 3 The efficient mTORC1 inhibitor Torin1 causes a dose-dependent increase in Mtb growth in co-infected hMDMs at low MOI. (a) Representative immunoblots from two independent experiments showing the dose-response of Torin1 and rapamycin on the autophagy markers LC3B and SQSTM1 (p62) and phosphorylation of the mTORC1 downstream targets S6 and 4EBP1, with their respective β-actin loading controls, after 6 h treatment. Full length of the cropped blots are shown in Supplementary Fig. S3. (b) hMDMs were infected at the indicated MOI for 2 h and then treated with/without 250 nM Torin1 for 3 days. The signal from luciferase expressing H37Rv was quantified and the graph shows the ratio in total bacteria (lysate + supernatants) for Torin1 treated vs. untreated. Data are mean ± SEM from 6 independent experiments. *p < 0.05 using paired Student t-test. (c) hMDMs were pre-infected with/without HIV for seven days before infected with H37Ra (MOI = 0.2) for 2 hours. hMDMs were then incubated with/without rapamycin or Torin1 at increasing concentrations for 3 days. Graphs show the level of intracellular bacteria in cell lysates compared to day 0. Data are mean ± SEM from triplicate, representative of two independent experiments. Full size image

In a controlled infection, mTOR inhibition causes a dose-dependent increase in Mtb growth, especially in HIV co-infected hMDMs

After confirming that Torin1 was a more potent mTORC1 inhibitor and autophagy inducer in hMDMs than rapamycin, we investigated the impact of Torin1 on macrophages infected at various doses of Mtb. With decreasing dose of Mtb, autophagy induction had a greater impact on bacterial growth (Fig. 3b), indicating a higher sensitivity towards autophagy induction when infection is controlled. To examine if there were differences in the sensitivity of Mtb infected versus HIV/Mtb co-infected cells to autophagy induction, macrophages infected with a low dose of Mtb were treated with increasing concentrations of the autophagy inducers. There was a pronounced control of Mtb infection at day 3, which was lost in HIV/Mtb co-infected hMDMs with rapamycin and to higher extent when stimulated with Torin1 (Fig. 3c).

Torin1 increases Mtb replication independently of the time of stimulation

To see if changing the timing of autophagy induction also affected Mtb replication in hMDMs, we stimulated autophagy using Torin1 either directly after phagocytosis or 3 days post infection. Torin1 caused a significant increase in Mtb replication both in Mtb single (p = 0.023) and HIV/Mtb co-infected (p = 0.029) hMDMs when mTORC1 inhibition was started 2 h after infection and was maintained for the 3 first days of infection (Fig. 4). More importantly, we found that even with an established infection that was controlled at day 3, Torin1 still caused an extensive increase in Mtb replication to 6 days post infection (Fig. 4). The difference in bacterial fold between Torin1 treated and untreated at day 6 was more pronounced in HIV/Mtb co-infected hMDMs (1.67-fold increase in single infected and 3.41-fold increase in co-infected). The amount of bacteria was measured both in the lysate and in the supernatant showing that the majority of the bacteria resided inside the cells. Luciferase data of lysate was also confirmed by CFU plating of the same lysates, showing the same results (Supplementary Fig. S2).

Figure 4 Same effect of early and late autophagy induction in Mtb infected hMDMs. hMDMs were pre-infected with/without HIV for seven days before infected with H37Rv (MOI = 1) for 2 hours. hMDMs were then incubated with/without Torin1 (250 nM) for 3 days, added either directly or 3 days post infection. The graph shows the level of bacteria in cell lysates and supernatants. Data are mean ± SEM from 5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were * represent the significance for lysate while (*) represents the significance for total bacteria (lysate + supernatant) using paired Student t-test. (See also Supplementary Fig. S2 for CFU confirmation of these lysates). Full size image

HIV/Mtb co-infection inhibits phagosomal fusion with lysosomes, which is further decreased upon mTORC1 inhibition

Since mycobacterial killing is subsequent to phagosomal maturation36 and increased killing has been correlated with increased acidification9, we hypothesized that the decreased killing of Mtb during autophagy induction was due to a decreased phagosomal acidification. In order to study maturation of Mtb phagosomes and how HIV affects this process, LysoTracker Deep Red co-localization was assessed. This probe stains organelles with low pH such as lysosomes37. HIV pre-infected hMDMs were infected with Mtb for 6 h or 24 h before co-localization of phagosomes with LysoTracker was analysed. The positive phagolysosome control, yeast, showed a near to complete co-localization of LysoTracker (97%) after 6 h whereas co-localization was greatly reduced for Mtb with approximately 50% for both single and HIV/H37Ra co-infected cells (Fig. 5b) and a further reduction in H37Rv infected/co-infected cells at both 6 h and 24 h (Fig. 5a,c).

Figure 5 HIV/Mtb co-infection inhibits phagosomal fusion with lysosomes, which is further decreased upon autophagy induction. (a) Representative micrographs of LysoTracker Deep Red (LTDR) co-localization to phagosomes in hMDMs infected with yeast (MOI = 5) or co-infected with HIV/H37Rv for 6 h (7 days pre-infection with HIV), unstimulated and stimulated with Torin1 (250 nM) the last 4 h. The arrows in the HIV/H37Rv co-infected micrographs indicate co-localization to some of the Mtb phagosomes. All yeast particles in the lower micrographs exhibited co-localization. Green: Mtb or yeast, red: LTDR, yellow: co-localization. (b) Percentage of LTDR co-localization with yeast or H37Ra (MOI = 1) phagosomes 6 h post infection. (c) Percentage of LTDR co-localization with H37Rv (MOI = 1) phagosomes 6 and 24 h post infection. (d) hMDMs were pretreated with bafilomycin (baf; 100 nM) for 1h before infection with luciferase-expressing H37Rv (MOI = 1) for 2 h. Extracellular bacteria were washed away and baf was re-added every 12 h. The combined luminescence signal from supernatant and hMDM-lysate (=total bacteria) are shown. Data are mean ± SEM with *p < 0.05 and **p < 0.01 using paired Student t-test, of 3 independent experiment for (d) and six independent experiments for LysoTracker data at 6 h and eight independent experiments for 24 h (n = 6 for yeast) in which 100–200 phagosomes were counted for each condition. Full size image

Although Torin1-treatment had no effect on yeast phagosome maturation (Supplementary Fig. S4a), autophagy induction caused an additional 10% decrease in phagolysosome fusion in H37Ra infected hMDMs (exhibiting 40% co-localization with LysoTracker; p = 0.002) and a 20% decrease in co-infected cells (exhibiting 30% co-localization with LysoTracker; p = 0.004), similar to the levels of the negative control bafilomycin (Fig. 5b). CD63 co-localization was analysed in addition to LysoTracker co-localization and revealed a Mtb/CD63 co-localization that was similar to that of the bafilomycin control (~15%) (Supplementary Fig. S4b). mTORC1 inhibition of Mtb-single or HIV co-infected hMDMs did not further improve this co-localization (Supplementary Fig. S4b). Torin1-reduced phagolysosome fusion, e.g. LysoTracker co-localization, in H37Rv infected hMDMs was similar to that in H37Ra. At 6 h H37Rv infected hMDMs exhibited a 1.7-fold increase to bafilomycin (p = 0.006), while co-infected cells showed no increase (1.02-fold increase; p = 0.95 compared to bafilomycin), indicating that HIV co-infection by itself inhibits phagosomal maturation. To monitor differences in LysoTracker co-localization with Torin1 stimulation also during HIV co-infection with H37Rv, phagosomal maturation were additionally analysed at 24 h. At this time point Torin1 significantly inhibited LysoTracker co-localization in both single (p = 0.025) and co-infected (p = 0.035) hMDMs (Fig. 5c). In both the uninfected and H37Rv infected cells, mTOR inhibition did not affect the total protein levels of proteins involved in phagosome/autophagosome maturation, i.e. levels of LAMP-1 and CD63 were unchanged (Supplementary Fig. S4c,d). These results reveal that autophagy induction further impair the already deficient phagosomal maturation in HIV co-infected hMDMs. Such a decrease in phagosomal maturation/acidification can reduce the control of Mtb33 and we found that bafilomycin treatment had this effect as the hMDMs ability to control replication of H37Rv decreased and gave a more than 2.6-fold higher bacterial load compared to untreated cells day 3 post infection (p = 0.02) (Fig. 5d). These data would explain the increased sensitivity towards mTOR inhibition that results in accelerated Mtb growth particularly in HIV co-infected cells (as seen in Fig. 3c and with day 3 to 6 treatment in Fig. 4).

Mtb inhibits autophagic flux and Torin1 treatment leads to a further build-up of the autophagic substrate SQSTM1

Autophagic flux was then analysed, focusing on the degradation or accumulation of the autophagy substrate SQSTM1, which delivers polyubiquitinated protein aggregates and bacteria to the autophagosome13,14,23,24. SQSTM1 co-localization was evaluated by confocal microscopy in infected or co-infected macrophages. Phagosomes in hMDMs containing yeast for 6 h had a low level of SQSTM1 co-localization (~10%; Fig. 6a,b), unless cells were pre-treated with bafilomycin which increased the co-localization more than 2-fold (p = 0.004; Fig. 6b). Bafilomycin was used to prevent degradation of SQSTM1 in case of lysosomal fusion leading to autophagic flux and reveals an active flux in macrophages infected with yeast. These differences with and without bafilomycin treatment was not seen in macrophages infected with Mtb, indicating that Mtb indeed inhibits the autophagic flux (Fig. 6b). Similar degree of SQSTM1 co-localization between yeast+ bafilomycin-treated hMDMs and Mtb infected hMDMs indicates that autophagy is present in both samples, although Mtb inhibits the autophagic flux while yeast does not. Furthermore, Torin1 increased SQSTM1 positive phagosomes with 1.3-fold in the single Mtb infected hMDMs (p = 0.006) and with 1.6-fold in the HIV co-infected hMDMs (p = 0.003). This is in agreement with LysoTracker data, indicating that Torin1 further inhibits acidification and maturation of Mtb-phagosomes causing reduced autophagic flux with build-up of SQSTM1, which is more pronounced in HIV co-infected hMDMs. Furthermore, LC3-LysoTracker-Mtb co-localization studies indicated that the level of maturing autophagosomes, e.g. LC3+LysoTracker+Mtb+ autophagolysosomes, were lower in HIV co-infected than in Mtb-single infected hMDMs. In addition, mTORC1 inhibition increased the number of autophagosomes (LC3+Mtb+), without increasing the number of autophagolysosomes (Supplementary Fig. S5). Although Torin1 induced autophagy and flux on a cellular level (whole cell lysates of uninfected hMDMs; Fig. 3a), we found that when the cells are infected with Mtb, there is still a decreased flux on the subcellular level, localized specifically to the Mtb phagosomes.

Figure 6 Mtb inhibits autophagic flux and autophagy induction cause further build-up of SQSTM1 in Mtb phagosomes. (a) Representative micrographs of SQSTM1 recruitment/accumulation to phagosomes in hMDMs infected with yeast (MOI = 5) or co-infected with HIV and Mtb (MOI = 1) for 6 h (7 days pre-infection with HIV), unstimulated and stimulated with Torin1 (250 nM) the last 4 h. Some cells were treated with bafilomycin (baf; 100 nM) 1 h prior to Mtb/yeast infection. The arrows indicate SQSTM1 co-localization to Mtb or yeast phagosomes. Green: Mtb or yeast, red: SQSTM1. (b) Percentage of SQSTM1 co-localization with yeast or H37Ra phagosomes. Data are mean ± SEM with **p < 0.01 using paired Student t-test, of six independent experiments (n = 3 for yeast+baf) in which 100–300 phagosomes were counted for each condition. Full size image

Torin1-induced autophagy and flux is cellular and not localized to Mtb phagosomes

After analysing autophagy and flux on a subcellular level (Figs 1,6 and Fig. S5) we further analysed these processes in single and HIV/Mtb co-infected cells at a cellular level. Analysing the total protein expression, we observed an induction of autophagy with inhibited flux in Mtb and co-infected cells (Fig. 7), seen by the increased conversion of LC3 I into LC3 II and the accumulation of the autophagy substrate SQSTM1. When adding Torin1, autophagy was induced in both single and co-infected cells, as seen by the complete conversion of LC3 I into LC3 II, which together with SQSTM1 had decreased considerably indicating autophagic flux. Similarly, autophagy induction in dendritic cells could also overcome the cellular Mtb-inhibited flux38. However, co-infected hMDMs stimulated with Torin1 retained an increased level of SQSTM1 (p = 0.042) compared to its Torin1 stimulated control (Fig. 7a,d). In order to further evaluate if HIV manipulates autophagy in co-infected cells, autophagic flux was assessed using the flux inhibitor bafilomycin. For both SQSTM1 and LC3 II, there was no cellular flux (baf/without baf = 1 or less) in Mtb and HIV/Mtb co-infected hMDMs in absence of Torin1. Torin1 stimulation on the other hand increased the cellular flux of both SQSTM1 (p = 0.029) and LC3II (p = 0.047) in Mtb infected but not in HIV/Mtb co-infected hMDMs (Supplementary Fig. S7a,b). Furthermore, HIV infected hMDMs displayed enhanced 4EBP1 phosphorylation, as well as partial resistance to de-phosphorylation by Torin1 in HIV only and HIV co-infected hMDMs (Fig. 7a,f). The decreased phosphorylation of the mTORC1 targets S6 and 4EBP1 by Torin1 reflects the initiation of early steps in the autophagy process, consistent with a previous study39. In the absence of Torin1 this was counteracted by the pathogens in both single and co-infected hMDMs (Fig. 7a,e,f), although Mtb only increased S6 phosphorylation without affecting 4EBP1, as also seen previously40.

Figure 7 Torin1-induced autophagy and flux is cellular and not localized to Mtb phagosomes . (a) Representative immunoblots from seven independent experiments showing the autophagy markers LC3B and SQSTM1 (p62) and the phosphorylation of the mTORC1 downstream targets S6 and 4EBP1, with their respective β-actin loading controls. The hMDMs were pre-infected for seven days with HIV before 6 h Mtb infection (MOI = 1), with the addition of Torin1 (250 nM) the last 4 h. Full length of the cropped blots are shown in Supplementary Fig. S6. (b–f) Densitometry measurements normalized to their respective β-actin control and presented as ratio over control without Torin1, shown as mean ± SEM with *p < 0.05 and **p < 0.01 using repeated measurement ANOVA comparing all treatments against its control (n = 7). Full size image

By making a distinction between overall cellular autophagy and subcellular autophagy, our findings indicate that Torin1-induced flux in hMDMs only affects the overall cellular autophagy, but do not overcome the flux inhibited by Mtb inside the phagosome. As shown in previous figures, this process was especially accentuated in phagosomes of HIV co-infected hMDMs leading to increased replication of Mtb.

HIV modulated expression of essential ATG genes during co-infection

Gene expression analyses were performed to evaluate differential expression of ATG genes and whether the decrease in autophagy proteins during Torin1 treatment correlates with a lowered gene expression. Similar to the protein levels, SQSTM1 showed an increased gene expression upon co-infection (Fig. 8a), but in contrary to the protein levels gene expression analysis revealed that SQSTM1 was unaffected or even increased with Torin1 treatment (Fig. 8a). In contrast to the evident differences of LC3B (ATG8) at protein level (Fig. 7a–c) there were no significant differences in LC3B gene expression levels in the absence of Torin1 (Fig. 8b). Upon Torin1 treatment, HIV pre-infected hMDMs resisted the modest increase in LC3B expression seen in the uninfected control (p = 0.0303). Beclin1/ATG6, ATG4A, 5, 12 and ATG16L2 have previously shown to be differentially expressed during infection with the intracellular pathogen Francisella tularensis41. Although no significant changes were detected for these genes (Fig. 8c–g), there was a noteworthy increase in Beclin1/ATG6 and ATG12 expression during co-infection, which was not seen in Mtb single infected hMDMs even when stimulated with Torin1 (Fig. 8c,f).

Figure 8 HIV modulated expression of essential ATG genes during co-infection. hMDMs were pre-infected with/without HIV for seven days before 6 h Mtb infection (MOI = 1), adding Torin1 (250 nM) the last 4 h. Trizol was added to the infected and stimulated hMDMs to extract RNA. RNA was extracted from the same samples as analyzed for protein expression in Fig. 7. The gene expression were analyzed for: (a) SQSTM1, (b) LC3B, (c) Beclin1, (d) ATG4A, (e) ATG5, (f) ATG12 and (g) ATG16L2. The changes in gene expression are presented as ratios over control without Torin1, shown as mean ± SEM with *p < 0.05 and **p < 0.01 using repeated measures ANOVA (n = 5–6). Full size image

Together the gene expression analyses indicates that some ATG genes (Beclin1/ATG6 and ATG12) could be differentially expressed during co-infection, but that the degradation of LC3B and SQSTM1 proteins caused by Torin1 do not correlate with decreased gene expression of these autophagy markers, but instead is due to an overall increased cellular autophagic flux.