Antimycobacterial Effects of Everolimus in a Human Granuloma Model

Abstract

Mycobacterium tuberculosis (M. tb) has been historically and is currently a threat to global public health. First-line antibiotics have been effective but proven to be burdensome as they have many potential adverse side effects. There has been a recent increase in the number of active tuberculosis (TB) cases due to a prevalence of multidrug and extensively drug-resistant strains of M. tb, and an increasing number of highly susceptible people such as those with Type 2 Diabetes (T2DM) and human immunodeficiency virus (HIV) infection. Multidrug-resistant M. tb infection (MDR-TB) is challenging to treat with existing therapeutics, so novel therapeutics and treatment strategies must be developed. Host-Directed Therapy (HDT) has been a potential target mechanism for effective clearance of infection. Host cell autophagy plays an essential role in antibacterial defense. The mammalian target of rapamycin (mTOR) has been negatively correlated with autophagy induction. Everolimus is an mTOR inhibitor that induces autophagy, but with higher water solubility. Therefore, targeting the mTOR pathway has the potential to develop novel and more effective combination drug therapy for TB. This study tested the effect of everolimus, alone and in combination with current first-line antibiotics (isoniazid and pyrazinamide), on the inhibition of M. tb inside in vitro human granulomas. We found that M. tb-infected in vitro granulomas treated with everolimus alone resulted in significantly decreased M. tb burden compared to similar granulomas in the control group. Cells treated with everolimus doses of either 1 nM or 2 nM in conjunction with pyrazinamide (PZA) produced a significant reduction in intracellular M. tb burden. Treatment groups that received everolimus alone in either 1 nM or 2 nM doses experienced a significant reduction in oxidative stress. Additionally, samples treated with 2 nM everolimus alone were observed to have significantly higher levels of autophagy and mTOR inhibition as well. Results from this study indicate that everolimus is efficacious in controlling M. tb infection in the granulomas and has additive effects when combined with the anti-TB drugs, isoniazid and pyrazinamide. This study has shown that everolimus is a promising host-directed therapeutic in the context of in vitro granuloma M. tb infection. Further study is warranted to better characterize these effects.

Keywords: Autophagy; Everolimus; Host-Directed therapy; Mycobacterium tuberculosis; mTOR.

Figure 1

Viability of M. tb Erdman grown in 7H9 media in the presence or absence of everolimus added at 1 and 2 nM concentrations. The viability of M. tb was tested at eight days post-treatment by plating the bacterial cells on 7H11 agar containing albumin-dextrose-catalase (ADC). There was a significant loss in the viability of M. tb when the mycobacterial cultures were grown in the presence of everolimus (1 and 2 nM). These results indicate that everolimus is directly toxic to M. tb. ** p < 0.005 when comparing Colony-forming units (CFUs) at 8-day time point from 1 nM everolimus treated M. tb to untreated control category. ^## p < 0.005 when comparing CFUs at 8-day time point from 2 nM everolimus treated M. tb Erdman strain to untreated control category.

Figure 2

Survival of M. tb in untreated, everolimus, Isoniazid (INH), pyrazinamide (PZA), INH plus everolimus or PZA plus everolimus-treated in vitro granulomas. Peripheral Blood Mononuclear Cells (PBMCs) isolated from healthy subjects were infected with Erdman strain of M. tb and were treated as follows: sham-treatment (Figure 2A), 1 nM and 2 nM everolimus (Figure 2A), INH (0.125 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 2B), PZA (50 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 2C). Granulomas were terminated at 8-days post-infection, and cell-free supernatants were collected and stored. Granulomas were lysed with ice-cold phosphate buffer saline (PBS). Supernatants and granuloma lysates were plated on 7H11 agar plates containing ADC to determine the survival of M. tb. Analysis of figures utilized a one-way ANOVA with Tukey test. A p-value of above 0.05 is not significant; less than 0.05 is marked with one asterisk (*), less than 0.005 is marked with two asterisks (**). An asterisk indicates categories compared to a previous category directly before it. A hash mark (#) indicates categories compared to a previous category one column before it.

Figure 3

CellROX fluorescent staining and oxidative stress measurement of in vitro granulomas. CellROX staining was performed on the in vitro granulomas derived from PBMCs of healthy subjects and treated with: 1 nM and 2 nM everolimus (Figure 3A), INH (0.125 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 3E), PZA (50 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 3J). Sham-treated granulomas served as the control group. The CellROX mean fluorescent intensity values were corrected with the values from 4′,6-diamidino-2-phenylindole (DAPI) mean fluorescent intensity. Images represent CellROX fluorescent staining in untreated control granulomas (Figure 3B,F,K), granulomas treated with: 1 nM (Figure 3C) and 2 nM everolimus (Figure 3D), granulomas treated with INH alone (Figure 3G), INH along with 1 nM everolimus (Figure 3H), INH along with 2 nM everolimus (Figure 3I), granulomas treated with PZA alone (Figure 3L), PZA along with 1 nM everolimus (Figure 3M) and PZA along with 2 nM everolimus (Figure 3N). Analysis of figures utilized a one-way ANOVA with Tukey test. A p-value of above 0.05 is not significant, under 0.05 is marked with one asterisk (*), less than 0.005 is marked with two asterisks (**), under 0.0005 is marked with three asterisks (***), under 0.0001 is marked with four asterisks (****). An asterisk indicates categories compared to a previous category directly before it. A hash mark (#) indicates categories compared to a previous category one column before it. A dollar sign ($) indicates categories compared to a previous category two columns before it.

Figure 4

Mean LC3B fluorescent staining and measurement of autophagy. LC3B staining was performed in the in vitro granulomas derived from PBMCs of healthy subjects and treated with: 1 nM and 2 nM everolimus (Figure 4A), INH (0.125 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 4E), PZA (50 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 4J). Sham-treated granulomas served as a control group. The LC3B mean fluorescent intensity values were corrected with the values from DAPI mean fluorescent intensity. Images represent immunofluorescent staining of LC3B in untreated control granulomas (Figure 4B,F,K), granulomas treated with: 1 nM (Figure 4C) and 2 nM everolimus (Figure 4D), granulomas treated with INH alone (Figure 4G), INH along with 1 nM everolimus (Figure 4H), INH along with 2 nM everolimus (Figure 4I), granulomas treated with PZA alone (Figure 4L), PZA along with 1 nM everolimus (Figure 4M) and PZA along with 2 nM everolimus (Figure 4N). Analysis of figures utilized a one-way ANOVA with Tukey test. A p-value of above 0.05 is not significant, under 0.05 is marked with one asterisk (*), less than 0.005 is marked with two asterisks (**), under 0.0005 is marked with three asterisks (***), under 0.0001 is marked with four asterisks (****). An asterisk indicates categories compared to a previous category directly before it. A hash mark (#) indicates categories compared to a previous category one column before it. A dollar sign ($) indicates categories compared to a previous category two columns before it.

Figure 5

Mean mTOR fluorescent staining. mTOR staining was performed on the in vitro granulomas derived from PBMCs of healthy subjects and treated with: 1 nM and 2 nM everolimus (Figure 5A), INH (0.125 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 5E), PZA (50 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 5J). Sham-treated granulomas served as a control group. The mTOR mean fluorescent intensity values were corrected with the values from DAPI mean fluorescent intensity. Images represent immunofluorescent staining of mTOR in untreated control granulomas (Figure 5B,F,K), granulomas treated with: 1 nM (Figure 5C) and 2 nM everolimus (Figure 5D), granulomas treated with INH alone (Figure 5G), INH along with 1 nM everolimus (Figure 5H), INH along with 2 nM everolimus (Figure 5I), granulomas treated with PZA alone (Figure 5L), PZA along with 1 nM everolimus (Figure 5M) and PZA along with 2 nM everolimus (Figure 5N). Analysis of figures utilized a one-way ANOVA with Tukey test. A p-value of above 0.05 is not significant, under 0.05 is marked with one asterisk (*), less than 0.005 is marked with two asterisks (**), under 0.0005 is marked with three asterisks (***), under 0.0001 is marked with four asterisks (****). An asterisk indicates categories compared to a previous category directly before it. A hash mark (#) indicates categories compared to a previous category one column before it. A dollar sign ($) indicates categories compared to a previous category two columns before it.

Figure 6

Levels of TNF-α in the supernatants of untreated, everolimus, INH, PZA, INH plus everolimus and PZA plus everolimus-treated granulomas. PBMCs isolated from healthy subjects were infected with Erdman strain of M. tb and were treated as follows: sham-treatment (Figure 6A), 1 nM and 2 nM everolimus (Figure 6A), INH (0.125 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 6B), PZA (50 μg/mL) in the presence or absence of everolimus added at 1 nM and 2 nM concentrations (Figure 6C). Data were analyzed by one-way ANOVA with the Tukey test. A p-value of above 0.05 is not significant, under 0.05 is marked with one asterisk (*), less than 0.005 is marked with two asterisks (**), under 0.0005 is marked with three asterisks (***), under 0.0001 is marked with four asterisks (****). An asterisk indicates categories compared to a previous category directly before it. A hash mark (#) indicates categories compared to a previous category one column before it. A dollar sign ($) indicates categories compared to a previous category two columns before it.