Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation

Abstract

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5(fl/fl) LysM-Cre(+) mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5(fl/fl) LysM-Cre(+) mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.

Fig. 1.

Autophagy protects from excessive inflammation in a mouse model of tuberculosis infection. (A) Bacterial burden (cfu) in organs of Atg5^fl/fl LysM-Cre⁺ and Atg5^fl/fl LysM-Cre⁻ mice infected aerogenously with low-dose M. tuberculosis H37Rv [3 × 10e² cfu (±30%) of initial bacterial deposition per lung following exposure to the infectious inoculum]. The data shown are representative of more than three independent low-dose experiments. (B) Weight loss in Atg5^fl/fl LysM-Cre⁺ and Atg5^fl/fl LysM-Cre⁻ mice infected with low-dose M. tuberculosis H37Rv. (C) Gross lung pathology (low dose). (D) Lung histological sections (low dose, day 36). (i–iv) H&E staining. Arrows indicate necrotic lesions. (v and vi) Acid-fast staining. Arrows indicate bacilli. (Insets) Enlarged views of boxed areas. AFB, acid-fast bacilli. (E) Survival of Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice infected with high-dose M. tuberculosis H37Rv (Kaplan–Meier survival analysis, log-rank method). (F) Weight loss in Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice infected with high-dose M. tuberculosis H37Rv (10e⁴ cfu per lung). When not otherwise specified, data are shown as mean ± SE; *P < 0.05, **P < 0.01, ^†P > 0.05 (ANOVA; n ≥ 3).

Fig. 2.

Multiplex cytokine detection by Luminex in the lungs of Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice infected with low-dose M. tuberculosis H37Rv shows inflammatory cytokines are increased in Atg5^fl/fl LysM-Cre⁺ mice. See SI Appendix, Fig. S2 for additional cytokines. BDL, below detection limit. Data are shown as mean ± SE; *P < 0.05, **P < 0.01, ^†P > 0.05 (t test; n ≥ 3). Data in D represent the mean (± range) from a single cohort of infected mice. (See SI Appendix, Fig. S2A for pooled IL-17 data.)

Fig. 3.

Activated phenotype of CD4 T cells from uninfected Atg5^fl/fl LysM-Cre⁺ mice and their propensity to undergo polarization into IL-17–producing cells. (A) CD44 expression on lung T cells. Graph displays the percent of CD44^high CD4 and CD8 T cells in the lung of uninfected Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice. The uninfected mice were 10–12 wk of age. (B–D) Intracellular levels of IL-17A (B and C) and IFN-γ (D and E) in CD44 T cells isolated from lungs of uninfected Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice and stimulated with phorbol 12-myristate 13-acetate and ionomycin ex vivo in the presence of brefeldin A and monensin. Data are shown as mean ± SE; *P < 0.05, **P < 0.01 (t test; n ≥ 3).

Fig. 4.

In vivo and ex vivo immune response to defined M. tuberculosis antigens of Atg5^fl/fl LysM-Cre⁺ mice and IL-17 production by their splenocytes upon ex vivo restimulation. (A) DTH reaction (footpad induration) at day 21 postinfection in Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice infected i.p. with bacillus Calmette–Guérin. Mice were injected in the footpad with synthetic PPD. Data are shown as percent change in footpad thickness upon challenge with the synthetic PPD relative to the contralateral, PBS-challenged footpad. (B–E) Cytokine production by splenocytes from Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ mice (day 23 after peritoneal injection of bacillus Calmette–Guérin) restimulated for 3 d ex vivo with synthetic PPD. All mice were 10–12 wk of age at the onset of the experiment. Data are shown as mean ± SE; **P < 0.01, ^†P > 0.05 (t test; n ≥ 3).

Fig. 5.

Excess cytokine secretion is a cell-autonomous property of autophagy-deficient macrophages, and IL-1α hypersecretion by Atg5^fl/fl LysM-Cre⁺ macrophages depends on reactive oxygen intermediates and calpain. (A–C) In vitro cytokine [IL-1α (A), CXCL1 (B), and IL-12p70 (C)] release (ELISA) from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ BMM. (D) CXCL1 released (ELISA) from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁺ BMM in the absence of presence of IL-1RA (0.5 µg/mL). (E) Fraction (flow cytometry) of 7-AAD⁺ BMM after LPS and IFN-γ stimulation in vitro. (F and G) IL-1α (ELISA) released from LPS- and IFN-γ−stimulated Atg5^fl/fl LysM-Cre⁻ BMM in the presence of 50 μg/mL rapamycin (Rap) or 10 mM 3-MA (F) or 100 nM Baf A1 (G) after 12 h of treatment. (H) IL-1α secretion during inflammasome activation. Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ BMM were pretreated overnight with LPS (100 ng/mL) and then were stimulated for 1 h in the absence or presence of the inflammasome agonist silica (250 μg/mL) in EBSS. (I) IL-1α secretion in the presence of caspase 1 inhibitor YVAD. Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ BMM were pretreated overnight with LPS (100 ng/mL) and then were stimulated for 1 h in the absence or presence of YVAD (50 μM) during inflammasome activation with silica as in H. (J–L) Effects of caspase 1 siRNA knockdown (immunoblot, J) on IL-1α release (graphs, K and L) from Atg5^fl/fl LysM-Cre⁺ BMM. (K) IL-1α release was measured (ELISA) from LPS-stimulated and siRNA-treated Atg5^fl/fl LysM-Cre⁺ BMM in full medium or EBSS (Starv) (K) or in full medium only (L). Casp 1, caspase 1 siRNA; Scr, scrambled siRNA (control). (M and N) Effects of NLRP3 and ASC siRNA knockdown (immunoblot, M) on IL-1α release (N). IL-1α release was measured (ELISA) from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁺ BMM knocked down with siRNA for inflammasome components ASC and NLRP3. (O and P) ROS inhibition and IL-1 secretion. IL-1α (O) and IL-1β (P) released (ELISA) from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ BMM in the absence or presence of the ROS antagonist APDC (50 μM) after 12 h of incubation. (Q) Calpain and IL-1α hypersecretion phenotype. IL-1α (ELISA) released from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁻ and -Cre⁺ BMM in the absence or presence of the calpain inhibitor ALLN (100 μM) after 12 h of stimulation. (R) IL-1α release from LPS- and IFN-γ–stimulated Atg5^fl/fl LysM-Cre⁻ BMM and Atg5^fl/fl LysM-Cre⁺ BMM knocked down with siRNA for Calpain S1. Data are shown as mean ± SE; *P < 0.05, **P < 0.01, ^†P > 0.05 (t test; n ≥ 3).

Fig. P1.

Autophagy protects against M. tuberculosis infection and excessive inflammation in vivo. (A) The M. tuberculosis-infected lung in mice defective for autophagy (Atg5⁻) shows increased pathology, necrosis, neutrophil infiltration, and excessive production of the proinflammatory cytokines IL-1α, IL-12, CXCL1, and IL-17. (B) Macrophages defective for autophagy (Atg5⁻) secrete proinflammatory cytokines IL-1α, IL-12, and CXCL1 in a cell-autonomous fashion. (C) Specialized organelles called “autophagosomes” (double-membrane organelles enveloping mitochondria) remove invading microbes from the eukaryotic cytoplasm or disused organelles such as depolarized mitochondria. Cells that cannot continuously remove depolarized (Δψ_m↓) mitochondria through autophagy are prone to IL-1α hypersecretion caused by reactive oxygen intermediates (ROS; small red dots). This state is known to induce inflammasome (composed of NLRP3, ASC, and caspase 1) activation and IL-1β secretion; however, IL-1α hypersecretion differs, because it uses a calpain-dependent pathway independently of the inflammasome.