Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules - PubMed (original) (raw)

. 2013 Dec 20;8(12):2724-2733.

doi: 10.1021/cb400352d. Epub 2013 Oct 29.

Khoa Tran # 1, Adam B Castoreno # 2, Joanna M Peloquin 3, Kara G Lassen 2, Bernard Khor 3, Leslie N Aldrich 2 4, Pauline H Tan 1, Daniel B Graham 4, Petric Kuballa 3, Gautam Goel 3, Mark J Daly 2 5, Alykhan F Shamji 2, Stuart L Schreiber 2 4 6, Ramnik J Xavier 3 2

Affiliations

Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules

Stanley Y Shaw et al. ACS Chem Biol. 2013.

Abstract

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β and to test novel therapeutic hypotheses.

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Figures

Figure 1

Figure 1

Overview of autophagy screen and inflammatory and immune assays. a. Summary of initial small molecule screen, Compound Set Enrichment Analysis, and associated inflammatory and immune assays. b. Representative images from small molecule screen. DMSO wells show occasional cells with punctae but also homogeneous cytoplasmic LC3 signal, whereas other compounds show increases in GFP+ punctae, mCherry+ punctae, or both. Cells that exemplify autophagosome or autolysosome enrichment patterns typical of the small molecule class (see Figure 2) are marked by asterisks. (Note images do not correspond directly to CSEA analysis, which analyzed GFP+ and mCherry+ GFP- punctae.)

Figure 1

Figure 1

Overview of autophagy screen and inflammatory and immune assays. a. Summary of initial small molecule screen, Compound Set Enrichment Analysis, and associated inflammatory and immune assays. b. Representative images from small molecule screen. DMSO wells show occasional cells with punctae but also homogeneous cytoplasmic LC3 signal, whereas other compounds show increases in GFP+ punctae, mCherry+ punctae, or both. Cells that exemplify autophagosome or autolysosome enrichment patterns typical of the small molecule class (see Figure 2) are marked by asterisks. (Note images do not correspond directly to CSEA analysis, which analyzed GFP+ and mCherry+ GFP- punctae.)

Figure 2

Figure 2

CSEA-identified compound sets enriched for enhancers of basal autophagy. Each red-blue bar depicts the list of screened compounds, ranked according to their score for GFP+ punctae (top) or mCherry+ GFP- autolysosomes (bottom). Horizontal lines show the position within the ranked list of a member of a compound set. Orange segments denote the set members that contributed to enrichment by the CSEA algorithm. The permutation p-value for enrichment is shown below each bar.

Figure 3

Figure 3

Effects of small-molecule enhancers of autophagy on IL-1β production from BMDMs. Compounds were all tested at 5 μM, and IL-1β production was normalized to stimulated DMSO control (treated with IFNγ, LPS and MDP in the presence of DMSO). P-values are shown for the comparison of each compound with stimulated DMSO control.

Figure 4

Figure 4

Effects of small-molecule enhancers of autophagy on S. typhimurium autophagy and killing. a. Fractional co-localization of LC3 punctae with DsRed S. Typhimurium. All compounds were evaluated at 5 μM. b. Representative images showing increased (thioridazine, digoxin) or unchanged (fludrocortisone, cetirizine) co-localization of LC3 punctae with DsRed S. Typhimurium. c. S. Typhimurium survival, as assessed by a bacterial bioluminescence assay. P-values in panels a and c are for the comparison with DMSO control. * denotes p < 1.0 ×10−4. (Blue p-values represent comparisons where compound wells showed a modest increase in Salmonella compared to DMSO.)

Figure 5

Figure 5

Effects of small-molecule enhancers of autophagy on T cell subset differentiation. Data are presented as normalized fraction of T cell subset prevalence under maximal stimulation. Blue, orange and black outlined panels indicate predominantly Treg , TH17, or mixed effects, respectively. G+ 10+ data points (green) refer to the TR1 anti-inflammatory cell population (expressing both IFN-γ and IL-10).

Figure 6

Figure 6

Inhibition of IL-1β production in BMDMs deficient in Atg16L1. a. IL-1β production in BMDMs bearing one functional Atg16L1 allele (KO/wt). P-values are for the comparison between compound treatment and stimulated DMSO control (“stim” = stimulated control cells treated with IFNγ, LPS and MDP in the presence of DMSO; “no stim” = treated with IFNγ but neither LPS nor MDP; see Methods). b. IL-1β production in BMDMs either heterozygous (KO/wt) or null (KO/flox) for Atg16L1, expressed as a normalized ratio of the maximal stimulated value. P-values are for the comparison between the compound's normalized effect in _Atg16L1_-heterozygous vs.-null cells. c. Compound effects in Atg16L1 null cells (KO/flox). P-values are for the comparison between compound and stimulated DMSO control (“stim”). P-values in blue denote IL-1β values that are mildly increased vs. control.* denotes p < 1.0 ×10−4. Absolute levels of IL-1β production for KO/wt or KO/flox cells are shown in panels a and c, respectively, whereas normalized IL-1β levels are shown in panel b to facilitate comparison between KO/wt and KO/flox cells.

Figure 6

Figure 6

Inhibition of IL-1β production in BMDMs deficient in Atg16L1. a. IL-1β production in BMDMs bearing one functional Atg16L1 allele (KO/wt). P-values are for the comparison between compound treatment and stimulated DMSO control (“stim” = stimulated control cells treated with IFNγ, LPS and MDP in the presence of DMSO; “no stim” = treated with IFNγ but neither LPS nor MDP; see Methods). b. IL-1β production in BMDMs either heterozygous (KO/wt) or null (KO/flox) for Atg16L1, expressed as a normalized ratio of the maximal stimulated value. P-values are for the comparison between the compound's normalized effect in _Atg16L1_-heterozygous vs.-null cells. c. Compound effects in Atg16L1 null cells (KO/flox). P-values are for the comparison between compound and stimulated DMSO control (“stim”). P-values in blue denote IL-1β values that are mildly increased vs. control.* denotes p < 1.0 ×10−4. Absolute levels of IL-1β production for KO/wt or KO/flox cells are shown in panels a and c, respectively, whereas normalized IL-1β levels are shown in panel b to facilitate comparison between KO/wt and KO/flox cells.

Figure 7

Figure 7

Activity profiles of small molecule autophagy enhancers. a. Heatmap for IL-1β inhibition, enhanced _Salmonella:_LC3 co-localization, and increased Salmonella killing, depicting the negative log of the P-value for the comparison of the compound with DMSO control. Because small molecule effects could result in either direction of effect for T cell subsets, effects were noted as +1 (increased), 0 (unchanged), or −1 (decreased) if a compound altered T cell subset development at any concentration. b. Diagram of activity profiles of small molecules (colored circles) across autophagy and associated assays (grey circles). The thickness of the edges reflects the number of compounds showing these relationships; note digoxin is the only cardiac glycoside depicted for clarity.

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