Pharmacological Stimulator of Interferon Genes Activation Enhances Autophagy-Mediated Clearance of Mycobacterium tuberculosis in Human Macrophages

Abstract

Introduction

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a critical sensor in the innate immune response to intracellular pathogens, yet its therapeutic potential for augmenting macrophage-mediated control of Mycobacterium tuberculosis (Mtb) remains incompletely understood. This study investigated whether pharmacological activation of the STING pathway could enhance autophagy to promote Mtb clearance in human macrophages.

Methods

Human THP-1 monocytes were differentiated into macrophages and infected with Mtb. The effects of the STING agonist MIW815 (ADU-S100) on Mtb phagocytosis, intracellular bacterial survival, and autophagic flux were assessed using a combination of molecular and cellular techniques, including quantitative real-time polymerase chain reaction, Western blotting, colony-forming unit (CFU) assays, and confocal immunofluorescence microscopy. The dependency on the cGAS-STING pathway was confirmed using small interfering RNA-mediated gene silencing.

Results

Pharmacological activation of STING with ADU-S100 significantly enhanced Mtb phagocytosis and subsequent intracellular clearance. This enhanced bactericidal activity was mechanistically linked to an increase in autophagic flux, as evidenced by elevated LC3-II protein levels and significantly increased colocalization of Mtb with lysosomal compartments. Importantly, treatment with the autophagy inhibitor hydroxychloroquine or silencing of cGAS significantly reversed these phenotypes, confirming the pivotal role of the STING-autophagy axis.

Conclusion

Activating the STING pathway with ADU-S100 is a potent host-directed strategy to bolster macrophage autophagy and enhance the elimination of intracellular Mtb. This provides a strong rationale for exploring STING agonists as a novel therapeutic intervention for tuberculosis, addressing a significant and clinically relevant challenge in infectious disease.

Introduction

Tuberculosis (TB) remains one of the world’s deadliest infectious diseases, exacerbated by the rise of multidrug-resistant strains of its causative agent, Mycobacterium tuberculosis (Mtb) [1]. This escalating public health crisis necessitates the development of novel therapeutic strategies, particularly host-directed therapies (HDTs) that enhance the host’s innate ability to eliminate the pathogen [2]. Autophagy, a fundamental cellular catabolic process, plays a critical role in host defense by engulfing and degrading intracellular pathogens like Mtb within autolysosomes [3, 4]. As the primary cellular niche for Mtb, macrophages are central to both containing and clearing the infection. While macrophages can initiate autophagy to combat Mtb, the bacterium has evolved sophisticated mechanisms to subvert this process, primarily by blocking phagosome-lysosome fusion, thereby ensuring its intracellular survival and replication [5–7].

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a crucial component of the innate immune system responsible for detecting cytosolic double-stranded DNA (dsDNA), a hallmark of many microbial infections [8, 9]. Upon infection, Mtb dsDNA can access the host cytosol, where it is sensed by cGAS. This triggers the synthesis of the second messenger cGAMP, which binds to and activates the endoplasmic reticulum-resident protein STING [10, 11]. Activated STING then translocates and recruits TANK-binding kinase 1 (TBK1), leading to the phosphorylation and activation of transcription factors like IRF3, culminating in the production of type I interferons (IFNs) and other inflammatory cytokines [12, 13]. The versatile functions of cGAS-STING extend beyond antiviral immunity, engaging in crosstalk with cellular processes like autophagy and apoptosis, though these interactions can be highly context-dependent and are often manipulated by pathogens like herpes simplex virus 1 [14, 15].

Recent studies have revealed a sophisticated interplay between the cGAS-STING pathway and autophagy. STING activation can directly promote autophagy, often through TBK1-mediated phosphorylation of autophagy receptors such as p62/SQSTM1, facilitating the targeting of cargo to autophagosomes [16]. Landmark work by Watson and colleagues [17] first demonstrated that cGAS detects Mtb dsDNA to induce not only type I IFNs but also autophagy, which is essential for bacterial restriction. However, Mtb can counteract this response, and the role of type I IFNs in TB is complex, with some evidence suggesting excessive production can be detrimental [18–20]. This complexity highlights the need to understand how the cGAS-STING-autophagy axis can be pharmacologically manipulated to favor bacterial clearance over pathogen persistence. Moreover, various cellular factors, such as NOD-like receptors and helicases, have been shown to modulate STING signaling, indicating a complex regulatory network that fine-tunes the immune response [21, 22].

MIW815 (also known as ADU-S100) is a potent synthetic cyclic dinucleotide agonist of STING currently being investigated in oncology for its ability to stimulate robust anti-tumor immunity [23]. Given its defined mechanism of action, we hypothesized that ADU-S100 could be repurposed as an HDT for TB by directly activating STING to augment macrophage autophagy and enhance Mtb clearance. While the link between cGAS-STING and Mtb-induced autophagy is established, the therapeutic potential of using a STING agonist to overcome Mtb’s autophagy evasion strategies remains largely unexplored. In this study, we systematically investigated the impact of ADU-S100 on Mtb-infected macrophages. We demonstrate for the first time that pharmacological activation of STING with ADU-S100 not only enhances autophagic flux but also translates into superior Mtb clearance, providing a strong rationale for its further development as a novel anti-TB agent.

Materials and Methods

Chemicals and Reagents

LC3B (D11) XP Rabbit mAb (65299S) was obtained from CST (USA). Lipofectamine 2000 (12566014) was obtained from Thermo Scientific (USA). Autophagy inhibitor 3-methyladenine (3-MA) (189490) and Tween 80 (P8074) were obtained from Sigma (USA). The cGAS-STING signaling pathway agonists ADU-S100 (HY-12885A) and autophagy inhibitor hydroxychloroquine (HCQ) (HY-B1370) were obtained from MCE (China). LysoTracker Red DND-99 (40739ES50) was obtained from Yeasen (China). 7H9 (271310), 7H10 (262710), and oleic acid-albumin-dextrose-catalase (OADC) (212351) were obtained from BD Biosciences (USA). RIPA lysis buffer was obtained from Gene-Optimal (China). Glycerol was obtained from Sangon Biotech (China).

Bacterial Strains and Cultures

The Mtb H37Rv strain (ATCC 27294; American Type Culture Collection) and its EGFP-expressing derivative (H37Rv-EGFP) were maintained in Middlebrook 7H9 liquid medium supplemented with 0.5% glycerol, 0.05% Tween 80, and 10% OADC enrichment. Cultures were grown to a mid-logarithmic phase (optical density at 600 nm of 0.6–0.8) before use. Bacterial clumps were disaggregated by passing them through a 27-gauge needle 10 times. All experiments involving live Mtb were conducted under strict biosafety level III (BSL-3) conditions.

Cell Culture and Treatment

The human acute monocytic leukemia cell line THP-1 (ATCC^® TIB-202™) was cultured in RPMI-1640 medium (Gibco™) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone™) and 1% penicillin-streptomycin at 37°C in a humidified 5% CO₂ atmosphere. For differentiation into macrophage-like cells, THP-1 monocytes were seeded at a density of 5 × 10⁵ cells/mL and treated with 20 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich^®) for 24 h. After differentiation, the cells were washed with phosphate-buffered saline (PBS) and incubated in fresh phorbol 12-myristate 13-acetate-free complete medium for another 24 h to rest before subsequent treatments and infections.

siRNA Transfection

Small interfering RNAs (siRNAs) targeting human cGAS (RefSeq: NM_138441.3) and a non-targeting negative control siRNA (si-NC) were designed and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). The sequences are listed in Table 1. Control groups in all relevant experiments were transfected with si-NC to ensure comparability. For transfection, differentiated THP-1 macrophages were seeded in 6-well plates. siRNA duplexes (50 nm final concentration) were transfected into the cells using Lipofectamine™ 2000 reagent (Invitrogen™) according to the manufacturer’s protocol. Cells were incubated with the siRNA-lipid complexes for 24 h before further experiments. Knockdown efficiency was validated by quantitative real-time polymerase chain reaction (qRT-PCR).

Table 1.

List of siRNA sequences

Target description	Primer sequence (5′→3′)
	forward	reverse
si-NC	UUC​UCC​GAA​CGU​GUC​ACG​UTT	ACG​UGA​CAC​GUU​CGG​AGA​ATT
si-cGAS-1	CAG​AAU​UCA​ACU​AGA​AGA​ATT	UUC​UUC​UAG​UUG​AAU​UCU​GTT
si-cGAS-2	GCU​GUA​ACA​CUU​CUU​AUU​ATT	UAA​UAA​GAA​GUG​UUA​CAG​CTT
si-cGAS-3	CUA​UUC​UCU​AGC​AAC​UUA​ATT	UUA​AGU​UGC​UAG​AGA​AUA​GTT

cGAS, cyclic GMP-AMP synthase; NC, negative control.

Western Blotting

Cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Roche). Total protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific™). Equal amounts of protein (20–30 µg) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore™). The membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were: anti-LC3B (1:1,000) and anti-β-actin (1:5,000, as a loading control). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1:5,000; Abcam^®) for 1 h. Protein bands were visualized using an enhanced chemiluminescence substrate (Thermo Scientific™) and imaged with a ChemiDoc™ Imaging System (Bio-Rad). Densitometric analysis was performed using ImageJ software.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from cells using the Total RNA Extraction Kit (LS1040, Promega) according to the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop spectrophotometer. One microgram of total RNA was reverse-transcribed into cDNA using the Maxima SYBR Green/ROX qPCR Master Mix kit (Thermo Fisher Scientific), which includes reverse transcriptase. qRT-PCR reactions were performed on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems). The thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The relative expression of target genes was calculated using the 2^−ΔΔCt method, with GAPDH used as the endogenous control for normalization. Primer sequences are listed in Table 2.

Table 2.

List of primers used for qRT-PCR

Primer	Primer sequence (5′→3′)		GenBank accession number
	forward	reverse
cGAS	TCA​CGT​ATG​TAC​CCA​GAA​CCC	CGC​AGT​TAT​CAA​AGC​AGA​GGC	NM_138441.3
GAPDH	ACT​CCT​CCA​CCT​TTG​ACG​CT	GGT​CTC​TCT​CTT​CCT​CTT​GTG​C	NM_002046.6
Beclin-1	CCC​CTG​AAA​CTG​GAC​ACG​AG	TCT​CCA​AAC​AGC​GTC​TGG​CT	NM_001314000.2
LC3B	AGA​AGG​CGC​TTA​CAG​CTC​AA	AGA​TTG​GTG​TGG​AGA​CGC​TG	NM_022818.5
ATG5	AGC​AAC​TCT​GGA​TGG​GAT​TGC	GGT​CTT​TCA​GTC​GTT​GTC​TGA​T	NM_004849.4
LL37	CCA​GGT​CCT​CAG​CTA​CAA​GG	CCA​GGA​GGC​GGT​AGA​GGT​TA	NM_004345.5
IFN-β	CTT​GGA​TTC​CTA​CAA​AGA​AGC​A	ATA​GAT​GGT​CAA​TGC​GGC​GT	NM_002176.3

cGAS, cyclic GMP-AMP synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-β, interferon-beta.

Flow Cytometry

Cells were harvested and washed with PBS, fixed, and permeabilized. Subsequently, cells were incubated with an anti-LC3B antibody (D11, CST) followed by a fluorescently labeled secondary antibody. Flow cytometric analysis was performed on a FACSCanto Fortessa flow cytometer (BD, NJ, USA). The gating strategy involved first identifying single-cell populations based on forward scatter (FSC-A vs. FSC-H) and side scatter (SSC-A vs. SSC-H), followed by gating on the live cell population. LC3B-positive cells were then quantified based on mean fluorescence intensity in the appropriate channel (e.g., FITC-A). Data were analyzed using FlowJo software.

Immunofluorescence Microscopy

Differentiated THP-1 cells were seeded on glass coverslips in 24-well plates. Cells were infected with H37Rv-EGFP (green fluorescence) at an MOI of 10 for 4 h. After washing to remove extracellular bacteria, cells were treated as indicated for 24 h. To visualize lysosomes, cells were incubated with 50 nm LysoTracker Red DND-99 (Thermo Fisher Scientific) for 30 min at 37°C. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and mounted on glass slides with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) to stain nuclei. Images were captured using a NIKON A1R confocal laser-scanning microscope. A scale bar (10 µm) was added to all representative images. Colocalization analysis was performed using ImageJ software with the JaCoP plugin to calculate the Pearson’s correlation coefficient from at least 10 random fields per condition.

Mtb Infection and Intracellular Survival Assay

For bacterial uptake and survival experiments, differentiated THP-1 macrophages were seeded in 24-well plates. Cells were pretreated with ADU-S100, HCQ, or transfected with si-cGAS as indicated before infection. Cells were then infected with Mtb H37Rv at an MOI of 10. For uptake (2 h): After 2 h of infection, cells were washed three times with PBS to remove extracellular bacteria and then lysed with 0.1% Triton X-100 in sterile water. For intracellular survival (24 and 48 h): After 4 h of infection, cells were washed and incubated with amikacin (200 µg/mL) for 2 h to kill any remaining extracellular bacteria. The cells were then washed again and incubated in fresh medium with the respective treatments. At 24 and 48 h post-infection, cells were lysed as described above. Cell lysates were serially diluted in 7H9 broth and plated on Middlebrook 7H10 agar plates supplemented with 10% OADC. The plates were incubated at 37°C for 3–4 weeks, and the number of colony-forming units (CFUs) was counted.

Statistical Analysis

All data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism V8.0.0 software. For comparisons between two groups, an unpaired, two-tailed Student’s t test was used. For comparisons involving more than two groups, a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons was performed. A p value <0.05 was considered statistically significant.

Results

Efficient Knockdown of cGAS by siRNA in THP-1 Cells

To investigate the role of the cGAS-STING pathway, we first established a knockdown model in THP-1 macrophages. We tested three different siRNA sequences to silence cGAS expression. qRT-PCR analysis 24 h post-transfection revealed that si-cGAS-3 achieved the most significant and consistent knockdown of cGAS mRNA levels compared to the negative control (si-NC) (Fig. 1, p < 0.001). Consequently, si-cGAS-3 was selected for all subsequent experiments to investigate the role of cGAS in this pathway.

Fig. 1.

Efficient knockdown of cGAS expression in THP-1 cells. THP-1 cells were transfected with three different siRNA sequences targeting cGAS (si-cGAS-1, -2, -3) or a negative control (si-NC) for 24 h. Relative cGAS mRNA expression was determined by qRT-PCR. si-cGAS-3 showed the highest knockdown efficiency. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. ***p < 0.001 compared to the si-NC group.

Mtb Infection Induces a Robust Autophagic Response in Macrophages

To establish a baseline for the role of autophagy during Mtb infection, we evaluated the autophagic response in THP-1-derived macrophages. Following a 24-h infection with Mtb, we observed a significant upregulation in the mRNA expression of key autophagy-related genes, including ATG5, BECN1, and LC3B, as well as the antimicrobial peptide cathelicidin (LL37), compared to uninfected controls (Fig. 2a, p < 0.01). This induction was abrogated when cells were pretreated with the autophagy inhibitor 3-MA, confirming the specificity of the response. The induction of autophagy was further validated by flow cytometry, which showed a significant increase in LC3B protein levels in Mtb-infected cells, an effect that was also reversed by 3-MA (Fig. 2b). These results collectively demonstrate that Mtb infection potently induces autophagy in macrophages.

Fig. 2.

Mtb infection induces autophagy in macrophages. THP-1 cells were treated with 3-MA or left untreated, followed by Mtb infection for 24 h. a Expression of ATG5, BECN1, LC3B, and LL37 was determined by qRT-PCR. b Quantitative analysis of the mean fluorescence intensity (MFI) from flow cytometry data, showing LC3B protein levels. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

Activation of the cGAS-STING Pathway Enhances Mtb Phagocytosis and Subsequent Clearance by Macrophages

To investigate the functional consequences of cGAS-STING pathway modulation on the macrophage’s ability to handle Mtb, we first determined the nontoxic concentrations of the STING agonist ADU-S100 and the autophagy inhibitor HCQ using a CCK-8 assay (Fig. 3a). Based on these results, nontoxic concentrations were selected for subsequent assays. We then assessed the impact of these compounds on Mtb uptake and intracellular survival. At 2 h post-infection, a time point reflecting initial phagocytosis, macrophages treated with 4 µm ADU-S100 showed a significantly higher number of intracellular CFUs compared to the control group, indicating enhanced bacterial uptake (Fig. 3b, p < 0.05). Conversely, treatment with HCQ or silencing of cGAS significantly reduced Mtb uptake (p < 0.05), confirming the role of this pathway in bacterial entry.

Fig. 3.

Activation of the cGAS-STING pathway enhances Mtb phagocytosis and intracellular clearance. a Effects of different concentrations of ADU-S100 and HCQ on THP-1 cell viability, assessed by CCK-8 assay after 24 h of treatment. b Quantification of Mtb CFU counts at 2 h post-infection to assess bacterial uptake. Both HCQ and si-cGAS significantly reduced uptake compared to control (*p < 0.05; ***p < 0.001). c Time-course analysis of intracellular Mtb survival at 24 and 48 h post-infection. ADU-S100 treatment significantly reduced intracellular bacterial load over time, while HCQ and si-cGAS treatments favored bacterial survival. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

More importantly, we performed a time-course intracellular survival assay to determine whether this enhanced uptake translated into better bacterial clearance. After removing extracellular bacteria, we monitored the intracellular bacterial load at 24 and 48 h. While the control and HCQ/si-cGAS groups showed bacterial persistence or growth, the ADU-S100-treated group exhibited a substantial and significant reduction in intracellular CFUs over time (Fig. 3c). This critical result demonstrates that activating the cGAS-STING pathway not only boosts initial phagocytosis but, more crucially, empowers macrophages to effectively clear the intracellular Mtb.

ADU-S100 Activates the STING Pathway and Enhances Autophagic Flux in Mtb-Infected Macrophages

To provide direct evidence that ADU-S100 activates the STING pathway and modulates autophagy at the molecular level, we first examined a key signaling outcome. As ADU-S100 is a known STING agonist, we confirmed pathway engagement by measuring the transcription of interferon-beta (IFN-β), a canonical downstream target. Indeed, ADU-S100 treatment robustly induced IFNB1 expression (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000550530). We next assessed the impact on autophagic flux. Western blot analysis revealed that treatment with 4 µm ADU-S100 significantly increased the levels of the lipidated form of LC3B (LC3-II), a hallmark of autophagosome formation (Fig. 4a, b, p < 0.05). In contrast, both HCQ treatment and cGAS silencing led to a significant decrease in LC3-II levels. To visualize the functional outcome of this enhanced autophagy, we performed immunofluorescence microscopy to track the fate of Mtb within the cell. In ADU-S100-treated macrophages, we observed a significant increase in the colocalization of EGFP-Mtb (green) with LysoTracker-stained acidic compartments (lysosomes, red), indicating enhanced fusion of Mtb-containing autophagosomes with lysosomes to form autolysosomes (Fig. 4c). Quantitative analysis confirmed this observation with a significantly higher Pearson’s correlation coefficient in the ADU-S100 group (online suppl. Fig. 2). Taken together, these data provide a strong mechanistic link, showing that ADU-S100 activates the STING pathway to enhance autophagic flux and promote the delivery of Mtb to lysosomes for degradation.

Fig. 4.

Activation of the STING signaling pathway enhances autophagy in Mtb-infected macrophages. a Western blot analysis of LC3-I to LC3-II conversion in THP-1 cells treated with 4 µm ADU-S100, 20 µm HCQ, or si-cGAS for 24 h during Mtb infection. β-actin was used as a loading control. b Densitometric analysis of the LC3-II/β-actin ratio from three independent experiments (n = 3). ADU-S100 significantly increased LC3-II levels (*p < 0.05). c Representative confocal microscopy images showing the colocalization of Mtb-EGFP (green) and LysoTracker Red-stained lysosomes (red). Nuclei were stained with DAPI (blue). Scale bar = 10 µm. Quantitative analysis is provided in online suppl. Fig. 2. Data in (b) are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05; ***p < 0.001.

Discussion

The emergence of multidrug-resistant Mtb strains has underscored the urgent need for novel therapeutic strategies against TB, particularly those that bolster the host's own defense mechanisms [1, 2]. In this study, we investigated the therapeutic potential of pharmacologically activating the cGAS-STING pathway, a central hub of innate immunity, to enhance macrophage-mediated control of Mtb. Our key finding is that the STING agonist ADU-S100 significantly promotes the clearance of intracellular Mtb by augmenting macrophage autophagy. This work provides a strong proof-of-concept for repurposing STING agonists, originally developed for cancer immunotherapy, as a novel host-directed therapy for TB.

Macrophages are the primary battleground for Mtb infection, and autophagy is a critical weapon in their arsenal [10, 24]. We first confirmed that Mtb infection itself is a potent inducer of autophagy, consistent with previous reports [4, 25]. However, the pathogen’s ability to evade this process necessitates strategies to amplify the host’s autophagic response. Recent research has highlighted the potential of small molecule agonists to reactivate suppressed immune pathways in chronic infections, as exemplified by the development of PPM1A-targeting compounds that activate autophagy for host-directed therapy [26]. Our study demonstrates that the STING pathway is a key regulatable node in this process. While the pathway’s activation by Mtb dsDNA is known, our work highlights that its pharmacological hyperactivation via ADU-S100 can tip the balance in favor of the host. This finding aligns with studies by Dwivedi and Baindara [27], who demonstrated that STING activation can override Mtb-mediated inhibition of autophagy. Furthermore, the robust induction of IFN-β confirms that ADU-S100 effectively engages the canonical signaling cascade, which has been shown to cooperate with TBK1 to phosphorylate autophagy receptors like p62 [28].

A crucial contribution of our study is the clear distinction between bacterial uptake and clearance. Our finding that ADU-S100 enhances initial phagocytosis is consistent with recent observations that STING signaling can remodel the actin cytoskeleton to facilitate pathogen engulfment [29]. More importantly, the subsequent clearance phase distinguishes effective immunity from mere containment. Our data showing sustained bacterial killing over 48 h contrasts with the effects of autophagy inhibition, which exacerbated bacterial burden. This supports the concept that the flux through the autophagic pathway is a critical determinant of bacterial clearance, a principle emphasized in methodologies for measuring autophagosome flux [30]. The ability of ADU-S100 to promote lysosomal fusion, as evidenced by our colocalization data, suggests it may help bypass the Mtb-induced blockade of phagolysosome maturation, a mechanism also observed with other HDT candidates like metformin [31].

Interestingly, while cGAS is the primary sensor for Mtb DNA, our use of a direct STING agonist suggests that therapeutic efficacy can be achieved by bypassing the sensor step, which is often targeted by viral and bacterial virulence factors [32]. This “bypassing” strategy might be particularly advantageous in strains of Mtb that have evolved to mask their DNA or degrade cGAS [33]. Future studies should investigate whether ADU-S100 retains efficacy against clinical isolates with varying virulence profiles [34].

However, our study has limitations. First, the experiments were conducted in the THP-1 cell line. While this is a widely accepted model for human macrophages, validation in primary human monocyte-derived macrophages and, crucially, in in vivo animal models is required to confirm the therapeutic window and safety profile [35]. Additionally, while flow cytometry showed clear shifts in LC3B intensity, the inclusion of isotype controls in future studies would further rigorise the exclusion of nonspecific binding, although our Western blot data corroborates the autophagic upregulation. Finally, we focused on autophagy; however, STING activation also triggers inflammasome activation and cell death pathways [36], which were not explored here but could contribute to bacterial clearance [35].

Conclusion

This study provides the first evidence that the pharmacological STING agonist ADU-S100 enhances macrophage-mediated clearance of Mtb. We demonstrate that this effect is driven by the activation of the STING signaling pathway, which leads to an increase in autophagic flux and promotes the delivery of Mtb to lysosomes for degradation. These findings identify the STING-autophagy axis as a promising therapeutic target and support the further development of STING agonists as a novel host-directed therapy for TB.

Statement of Ethics

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Medical Ethics Committee of Wuhan No. 1 Hospital (Approval No. WZ21M02). The human acute monocytic leukemia cell line THP-1 (ATCC^® TIB-202™) used in this study is a commercially available, established cell line. The Medical Ethics Committee of Wuhan No. 1 Hospital granted an exemption from requiring specific written informed consent for the use of this cell line in the present research.

Conflict of Interest Statement

The authors declare that they have no competing interests.

Funding Sources

This work is supported by Major Scientific Research Project of Wuhan Municipal Health Commission (No. WX21M02, WZ21C04, WZ21C18).

Author Contributions

Jing Feng and Ping Xia developed the idea for the study. Ronghao Zhong and Fei Niu conducted relevant experiments and analyzed and interpreted the experiments data. Ronghao Zhong was a major contributor in writing the manuscript. Xiyong Dai and Junwen Wang supervised and guided the experimental process. All authors discussed the results and revised the manuscript.

Funding Statement

This work is supported by Major Scientific Research Project of Wuhan Municipal Health Commission (No. WX21M02, WZ21C04, WZ21C18).

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. Further inquiries can be directed to the corresponding author.