Granulomas microenvironment-guided sono-immunotherapy to treat and prevent recurrence of tuberculosis

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: minor_revision

Abstract
Curing tuberculosis (TB) remains challenging due to treatment complexity and high recurrence rates. Through bioinformatic analyses of the immune microenvironment within clinical non-tuberculous granulomas (NTBG)/tuberculous granulomas (TBG) samples, and peripheral blood from drug-resistant/sensitive, recurrent/non-recurrent TB patients, we identify suppression of innate immune responses, especially downregulated Toll-like receptor/NF-kappa B pathways in macrophages/dendritic cells and elevated B/T cell negative regulation, contributing to treatment failure and recurrence. Building on these insights, we develop mannose-modified organic semiconducting nano-immunostimulants (manSNI) for precise TB sono-immunotherapy. Under optimized ultrasound condition, manSNI generates massive reactive oxygen species (ROS) to eradicate Mycobacterium tuberculosis (M.tb) within lung granulomas/macrophages, while simultaneously promoting in situ release of M.tb-derived antigens. The released antigens and TLR7 agonist R837 synergistically induce potent innate/adaptive anti-TB immunity and long-lasting immune memory. This granulomas microenvironment-guided sono-immunotherapy strategy demonstrates efficient TB control and relapse/reinfection prevention, which provides a promising direction for customized therapy against high-pathogenic infections.

Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), constitutes a persistent global health crisis with an estimated 1.7 billion latent infections worldwide1. This facultative intracellular pathogen demonstrates unique survival strategies through macrophage parasitism and granulomas formation—dynamic immune cell aggregates that paradoxically contain bacterial dissemination while establishing sanctuaries for antibiotic evasion and dormancy maintenance2–4. Current first-line multidrug regimens (6–24 months) frequently fail to completely eradicate M.tb infection5, with alarming 2-year recurrence rates (6.7–18%)6–8. Consequently, challenging treatment regimens and a high incidence of TB recurrence present substantial obstacles in achieving an effective cure for this disease.

Currently, extensive research has been conducted on novel anti-TB small molecular drugs9, adjuvant immunotherapy10, and therapeutic vaccines11, as well as emerging modalities such as photothermal therapy (PTT)12 and photodynamic therapy (PDT)13,14, which hold significant promise. However, these approaches still face limitations in completely eradicating M.tb and effectively addressing the issue of TB recurrence. Furthermore, the current focus of TB mechanism primarily revolves around the heterogeneity of granulomas and the correlation between specific cell types and bacterial load15–17. Nevertheless, there remains a lack of understanding regarding the characteristics of immune responses and the intricate interplay among different immune cell populations within granulomas, particularly in patients with drug-resistant and recurrent TB. These features may serve as critical factors contributing to treatment challenges and high recurrence rates18. Therefore, it is imperative to thoroughly investigate the immune response characteristics within tuberculous granulomas in order to guide the development of novel strategies for effectively controlling TB. Moreover, it is crucial to develop new treatment approaches based on these immune characteristics that can both eliminate M.tb and prevent TB recurrence.

In this work, to gain deeper insights into the mechanisms underlying drug resistance and TB recurrence, as well as to facilitate targeted treatment strategies, we conduct bioinformatic analyses of the immune microenvironment within clinical non-tuberculous granulomas (NTBG) samples, tuberculous granulomas (TBG) samples, and peripheral blood from drug-resistant vs. drug-sensitive and recurrent vs. non-recurrent TB patients at the single-cell RNA-sequencing (scRNA-seq) and transcriptomic analysis level. Our findings identify profound immunosuppression within TBG marked by downregulated Toll-like receptor (TLR) and NF-kappa B (NF-κB) signaling pathways in antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), alongside elevated negative immune regulation of B cell and T cell activities. The impaired intercellular crosstalk between these immune cells leads to severe immunosuppression within TBG, thus contributing to chemotherapy resistance and recurrence of TB. Based on the immunological microenvironment of TBG, we developed an organic semiconducting nano-immunostimulant (manSNI) system equipped with a mannose warhead for highly precise TB sono-immunotherapy and recurrence prevention. Upon ultrasound irradiation, manSNI generates a large amount of ROS to precisely eliminate M.tb within lung granulomas and macrophages, while simultaneously promoting the in situ release of M.tb-derived antigens. The generated ROS can activate macrophages toward a pro-inflammatory state in combination with R837. Meanwhile, the release of M.tb-derived antigens and R837 from macrophages could synergistically promote DC maturation. These effects collectively trigger potent innate and adaptive anti-TB immune responses, including long-lasting immune memory. By remodeling the immunosuppressive microenvironment and augmenting the immune response, this sono-immunotherapy presents a promising avenue towards achieving a complete cure for TB while addressing the persistent issue of recurrence.

Results
Immune microenvironment characteristic analyses of TBG
Firstly, the heterogeneity of immune cells and cell-cell interactions was analyzed by collecting scRNA-seq datasets of 5 TBG samples, 12 NTBG samples, and 5 healthy lung samples from the Gene Expression Omnibus (GEO) public database16. Clinical information of these samples is summarized in Supplementary Table 1. A total of 11 immune cells and parenchymal cells were classified through cell-type-specific gene marker identification (Fig. 1a, and Supplementary Fig. 1a). Notably, TBG specimens exhibited marked alterations in cellular composition compared to both normal and NTBG subjects, particularly in immune cell populations such as macrophages, DCs, and T cells (Supplementary Fig. 1b). Among them, macrophages are the primary host cells of M.tb and key effector cells in anti-TB immunity, which can initiate the immune response through pathogen phagocytosis and cytokine secretion; DCs, as critical mediators bridging innate and adaptive immunity, facilitate antigen presentation to T cells to activate the adaptive immune response. However, TBG-associated macrophages16 and DCs19 demonstrated concurrent upregulation of immunosuppressive genes (ZFP36L2, TSC22D3, and DUSP1) and downregulation of immune activation-related genes (IL1B and CXCL8) compared to their counterparts in normal and NTBG subjects (Fig. 1b and c). Of particular significance, our analysis uncovered substantial impairment of canonical M.tb recognition and processing pathways, including NF-κB-, Nod-like receptor (NLR)-, and TLR signaling, as well as antigen processing mechanisms in TBG macrophages (Fig. 1d, e, Supplementary Fig. 2) and DCs (Fig. 1f, g, Supplementary Fig. 3). These findings collectively suggest compromised innate immune functionality characterized by defective antigen presentation in TBG, potentially contributing to delayed adaptive immunity activation. Consistent with this immunosuppressive characteristic, transcriptomic profiling of lymphocyte subsets revealed dual regulatory perturbations: upregulation of anti-inflammatory genes (such as IL10RB and DUSP1) and downregulation of pro-inflammatory genes (such as CCL20 and JUND) in CD4+ T cells within the TBG (Supplementary Fig. 4). Furthermore, CD4+ T cells and regulatory T cells (Tregs) exhibited enhanced activity of immune negative regulatory pathways (Fig. 1h and Supplementary Fig. 5). Similar functional inhibition was observed in natural killer (NK) cells in the TBG, with significant upregulation of “Negative regulation of lymphocyte activation” pathway and downregulation of “Natural killer cell mediated cytotoxicity and immunity” pathway (Fig. 1i, Supplementary Fig. 6). Notably, B cell functionality was also significantly attenuated in TBG specimens, as evidenced by suppressed activation markers and functional pathways (Fig. 1j, Supplementary Fig. 7). Cell Chat ligand-receptor interaction analysis further demonstrated disrupted intercellular communication between macrophages and other immune populations in TBG compared with the normal and NTBG groups (Fig. 1k and Supplementary Fig. 8), highlighting systemic immunosuppression through impaired immune cascades. Collectively, these findings confirm that the impairment of key M.tb recognition and antigen extraction pathways in macrophages and DCs within TBG leads to the suppression of the innate immune responses, and further weakens the overall immune function, providing a hotbed for the long-term survival and persistent infection of M.tb.

To investigate the clinical significance of immunosuppression in TBG, we further analyzed peripheral blood RNA-seq data from drug-resistant vs. drug-sensitive and recurrent vs. non-recurrent TB patients. The immune status of granulomas and peripheral blood is synchronized and correlated during TB infection20. Drug-resistant cases exhibited pronounced enrichment of immunosuppressive pathways compared with drug-sensitive cases (Fig. 1l), supported by a diagnostic model based on immune-negative regulatory differentially expressed genes (DEGs) with robust predictive accuracy across multiple machine learning algorithms (AUC 0.839; Fig. 1m; Supplementary Fig. 9). These findings strongly indicate that immunosuppression within TBG plays a critical role in influencing the drug resistance. The high recurrence rate is one of the major challenges in global TB prevention and control, and the low immunity of patients and the occurrence of drug resistance are important causes of incomplete anti-TB treatment and poor prognosis. Given the established correlation between granulomas, systemic immunity, and drug resistance, we hypothesize that TBG-mediated immunosuppression contributes to therapeutic failure and recurrence. Supporting this hypothesis, recurrent TB patients demonstrated relative upregulation of immunosuppressive genes (such as IDO1 and KIF14)18 and relative down-regulation of immune activating genes (such as CD18021, whose down-regulation can inhibit complement receptor type 3-mediated bacterial phagocytosis) compared with non-recurrent patients (Fig. 1n). Gene ontology (GO) enrichment analysis showed that many pathways associated with immune negative regulation were upregulated in recurrent patients (Fig. 1o). Meanwhile, the “Negative regulation of immune system process pathway” exhibited significant enrichment in DEGs analyses (Supplementary Fig. 10), indicating more severe immunosuppression among those with recurrent TB. Distinct separation was observed between the two sample groups based on 18 highly significant UP DEGs in the NRI pathway, and similar results were obtained using immune-active DOWN DEGs (e.g., NRCAM, RNASE1)22 (Fig. 1p and Supplementary Fig. 11). Diagnostic models incorporating these DEGs achieved near-perfect prediction of relapse outcomes (AUC 0.806-1.000; Fig. 1q; Supplementary Table 2). These multi-omics findings collectively establish TBG immunosuppression as a critical determinant of drug resistance development and post-treatment recurrence.

Collectively, our multi-omics analyses demonstrate that relative to NTBG, the immune microenvironment of TBG exhibits three hallmark suppressive features: (1) concomitant upregulation of immune checkpoint genes and functional impairment of pathogen recognition receptors (TLR/NLR/NF-κB pathways) in APCs including macrophages and DCs; (2) compromised cross-talk between innate and adaptive immunity, manifested by insufficient activation signals for cytotoxic lymphocytes (T/NK/B cells); and (3) progressive immunosuppression escalation in drug-resistant/recurrent TBG variants compared to ordinary TBG. This innate immunity impairment creates a self-perpetuating cycle-defective APC-mediated bacterial clearance synergizes with dysfunctional lymphocyte activation, collectively establishing delayed and suboptimal adaptive immune responses that permit M.tb persistence. Notably, the gradient of immunosuppressive severity (NTBG < ordinary TBG < drug-resistant/recurrent TBG) correlates directly with clinical refractoriness, suggesting microenvironmental immune paralysis as the linchpin of TB treatment failure (Fig. 2a). Taken together, our findings suggest the dual-action immune restoration-simultaneous potentiation of innate pathogen sensing and reinvigoration of cytotoxic lymphocyte functions—as a rational therapeutic strategy to overcome TBG-mediated immunosuppression, thereby breaking the vicious cycle of chronic infection and recurrence.

Preparation and in vitro anti-M.tb activity of manSNI
While conventional anti-TB regimens remain the cornerstone of clinical management, they face critical shortcomings in preventing disease recurrence. The protracted course of classic clinical standard chemotherapy (6–24 months) frequently results in compromised patient compliance, leading to treatment interruption, drug resistance, and relapse. Moreover, these nonspecific antibiotics induce substantial off-target toxicity, further exacerbating therapeutic challenges23. Current immunotherapeutic adjuncts (e.g., vaccine therapy, cytokine therapies, checkpoint inhibitors) attempt to potentiate antimicrobial immunity through host-directed strategies, which may reduce chemotherapy duration or mitigate adverse effects when combined with antimicrobials. However, their inability to replace antibiotics, heterogeneous patient responses, and risk of immune hyperactivation significantly limit clinical translation24. Emerging non-invasive modalities such as PTT and PDT offer mechanistically distinct advantages through an antibiotic-independent bactericidal mechanism and high spatial precision. Nevertheless, their limited tissue penetration depth (particularly problematic for pulmonary lesions) and phototoxic side effects restrict practical implementation (Fig. 2b)25. This therapeutic impasse underscores the urgent need for next-generation precision interventions capable of simultaneously achieving: (1) effective eradication of primary infection; (2) systemic immune potentiation; and (3) durable protection against recurrence. Sonodynamic therapy (SDT) is an emerging modality that employs ultrasound (US) to activate sonosensitizers for localized ROS generation26. Unlike optical modalities, US offers superior tissue penetration (>10 cm vs. <3 cm for NIR light), enabling noninvasive treatment of deep-seated lesions27. Moreover, SDT ensures precise spatiotemporal control of treatment by adjusting the location, duration, and intensity of US irradiation without causing significant tissue damage. It has also been reported that SDT can induce immunogenic cell death, enhance antigen presentation, and promote immune activation28,29. As an extension of SDT, combining SDT-mediated pathogen killing with immune stimulation to elicit systemic anti-disease immunity is referred to as sono-immunotherapy. Hence, capitalizing on these mechanisms and based on our findings, we engineered a organic semiconducting nano-immunostimulants (manSNI) system for TB sono-immunotherapy featuring (Fig. 2c): (1) mannose-mediated active targeting of infected macrophages; (2) organic semiconducting polymers with highly elongated π-π conjugated backbones represent a class of sonodynamic inducer materials30,31, exhibiting enhanced stability and efficiency in generating ROS compared to conventional counterparts32,33; and (3) TLR7 agonist Imiquimod (R837) payloads-an FDA-approved immunoadjuvant that synergistically activates type I interferon pathways APCs, thereby activating the innate and adaptive immunity34. Under US irradiation, manSNI are expected to orchestrate a tripartite therapeutic cascade: (i) focal ROS generation to clear localized M.tb and promote pathogen antigens release; (ii) release of antigens and R837 for synergistic driven APC activation to reverse immunosuppression of TBG; and (iii) pathogen-specific memory lymphocyte priming-collectively establishing a protective immune barrier against both endogenous relapse and exogenous reinfection.

Initially, an amphiphilic organic semiconducting polymer (SP) was synthesized as depicted in Fig. 3a. SPBr was obtained through palladium-catalyzed Stille polymerization by reacting monomer 1 (M1) and monomer 2 (M2) at a 1:1 ratio, followed by replacement of the bromine group with sodium azide to yield SPN3. Subsequently, SPN3 underwent click reaction with alkynyl-PEG-COOH to afford SP35,36. The amphiphilic SP can undergo self-assembly to form semiconducting nanoparticles (SN) with an averaged diameter of approximately 169 nm. Given that M.tb primarily colonizes granulomatous macrophages, D-mannosamine hydrochloride was modified on the PEG to obtain mannose-modified SP (manSP)37,38. The structures of SP and manSP were characterized and confirmed using proton nuclear magnetic resonance spectroscopy (1H NMR) (Supplementary Figs. 12–15). Subsequently, manSP could self-assemble into macrophage-targeted semiconducting nanoparticles (manSN). Moreover, hydrophobic R837 was loaded into manSN during the self-assembly process to produce macrophage-targeted organic semiconducting nano-immunostimulants (manSNI) (Fig. 3a). The resulting nanoparticles exhibited a spherical morphology and homogeneous size as observed in transmission electron microscopy (TEM) images (Fig. 3b and Supplementary Fig. 16). manSNI exhibited an averaged diameter of 178 nm and a negative surface zeta potential of −15.9 mV (Fig. 3c, d). After optimization, the final loading percentage of R837 in manSNI was determined to be around 2.3% using high-performance liquid chromatography (HPLC) (Supplementary Fig. 17).

The optical and physical properties of these semiconducting nanoparticles were then investigated. SN, manSN, and manSNI exhibited a similar maximum absorption peak at 630 nm (Fig. 3e) and characteristic emission peak at 827 nm (Fig. 3f), indicating that the modified side chain groups and loaded R837 had no impact on the optical properties of manSNI. Subsequently, the sonochemical activities of manSNI were evaluated by employing singlet oxygen sensor green (SOSG) to detect 1O2 generation. Under ultrasound irradiation (1.5 W/cm2, duty cycle of 50%, frequency of 1 MHz) for 5 min, manSNI demonstrated a significant enhancement in SOSG fluorescence intensity (F/F0) at 520 nm with an increase of ~2.1-fold compared to PBS control (Fig. 3g). Furthermore, through electron paramagnetic resonance (EPR) spectra analysis using 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) as a radical scavenger, it was observed that SN, manSN, and manSNI generated large amounts of hydroxyl radicals (•OH) with an EPR spectrum intensity ratio of approximately 1:2:2:1 (Fig. 3h, i and Supplementary Fig. 18), confirming their exceptional sonodynamic capabilities.

Encouraged by the superior ROS generation ability of manSNI under US irradiation, the anti-M.tb activity of manSNI was then investigated. Both the Bacillus Calmette-Guérin (BCG, which shares a high degree of genomic homology with M.tb and is an internationally recognized preliminary screening model for anti-TB drugs/therapies39) and the attenuated strain of M.tb H37Ra (ATCC25177) were used as the model bacteria. Firstly, the colony-forming units (CFU) assay revealed that these semiconducting nanoparticles (SN, manSN, and manSNI) were not toxic to BCG and H37Ra (Supplementary Fig. 19) in the absence of US irradiation. While even when isolated by a 5 cm thick pork tissue pad (Fig. 3j), the combination of these nanoparticles and US irradiation (1.5 W/cm2, 1 MHz, 5 min) resulted in a bacterial death rate exceeding 99% (Fig. 3k, l). In contrast, due to the poor tissue penetration of light (1.5 W/cm2, 808 nm, 5 min), the near-infrared photosensitizer represented by ICG cannot kill BCG when separated by a 5 cm-thick layer of pork tissue (Supplementary Fig. 20). Similarly, the live/dead bacterial viability assay demonstrated that manSNI exhibited excellent efficiency in killing BCG and H37Ra (Fig. 3m and Supplementary Fig. 21) under US irradiation. Then, the structural changes in BCG and H37Ra after different treatments were examined. As shown in scanning electron microscopy (SEM) images, the combination of these nanoparticles and US irradiation caused extensive damage to the body structure of BCG and H37Ra (Fig. 3n), resulting in an overall shriveled and concave shape of bacteria. The disruption of bacterial structure resulted in the leakage of cellular contents, as evidenced by protein leakage high to 0.22 μg/mL in solution from bacterial (107 CFU/mL) (Supplementary Fig. 22), which are expected to activate the body’s immune response as pathogen antigens. These results demonstrate that manSNI can kill M.tb by generating a large amount of ROS through sonodynamic effect.

Sono-immunotherapy efficacy of manSNI against intracellular M.tb
M.tb primarily colonizes within the lysosomes of macrophages in TBG, highlighting the critical importance of targeted nanomedicine accumulation in infected macrophages and intracellular bacteria for effective eradication of intracellular M.tb40. To investigate the specific targeting ability of manSNI to macrophages, mouse monocyte-derived macrophages (Raw264.7 cells) were labeled with Lyso-tracker-green and co-cultured with SN or manSNI for 12 h, and observed by confocal laser scanning microscopy (CLSM). The red fluorescence emitted by manSNI was predominantly co-localized within the lysosomes following endocytosis, exhibiting significantly stronger signal intensity compared to SN (Fig. 4a). Approximately 2.5-fold higher uptake of manSNI by macrophages compared to SN was further confirmed by flow cytometry analysis (Fig. 4b). Subsequently, an intracellular bacterial model was established by treating Raw264.7 cells infected with green fluorescent protein-labeled BCG (GFP-BCG) with either SN or manSNI for 12 h (Fig. 4c). GFP-BCG was colocalized well with manSNI, as revealed by CLSM imaging, indicating the targeting capability of manSNI towards intracellular BCG (Fig. 4d).

The intracellular anti-M.tb efficacy was subsequently investigated. At a US time of 15 min and US intensity of 1.5 W/cm2, manSNI (SN concentration: 100 μg/mL) killed over 99% of intracellular BCG (Supplementary Fig. 23) and H37Ra (Supplementary Fig. 24) with no significant decrease in the viability of Raw264.7 cells (Supplementary Fig. 27). In contrast, SN and manSN exhibited only ~49.3% and ~66.9% killing efficiency of intracellular BCG and ~49.3% and ~66.9% killing efficiency of intracellular H37Ra under the same irradiation conditions, respectively (Fig. 4e, f). Therefore, manSNI can efficiently and selectively kill intracellular M.tb but not macrophages under the optimized US conditions (Supplementary Fig. 25). To further verify sonodynamic-induced generation of intracellular ROS for M.tb killing, the ROS indicator dichlorodihydrofluorescein diacetate (DCFDA) was employed. After US irradiation, manSNI induced a significantly 1.48-fold and 3.69-fold higher level of intracellular ROS compared to manSN and SN, respectively (Fig. 4g, h). These results highlight the superior intracellular anti-M.tb efficacy achieved by manSNI compared to SN and manSN counterparts. Furthermore, it is worth noting that even at drug concentrations (based on SN) up to 320 μg/mL, both Raw264.7 cells and normal mouse fibroblast L929 cells exhibited minimal toxicity when exposed to manSNI treatment without US (Supplementary Fig. 26), confirming its excellent biocompatibility.

Appropriate levels of ROS and R837 have been reported to enhance macrophage polarization towards a pro-inflammatory (M1) phenotype and increase the release of inflammatory cytokines by activating NF-κB signaling41–43. Flow cytometry showed that manSNI induced 2.1–2.3-fold higher population of M1 macrophages (CD86+) compared to PBS, SN, and manSN in the absence of US irradiation (Fig. 4i). Moreover, in the presence of US irradiation, manSNI further increased the polarization of M1 macrophages by 5.2-fold compared to PBS group, and also induced 4.2-fold and 11.6-fold higher levels of pro-inflammatory cytokines IL-6 and TNF-α than that of PBS, respectively (Fig. 4j, k). These results indicate that manSNI + US irradiation treatment can largely activate macrophages to trigger the innate immune response. The above extracellular bacterial experiments showed the destruction of bacterial structure induced by manSNI + US irradiation, which resulted in the leakage of proteins (Supplementary Fig. 22). Here, the manSNI-mediated sonodynamic inducement of intracellular bacterial antigen presentation was further validated. The cell supernatant collected from BCG-infected macrophages after treatment was subjected to mass spectrometry analysis for identification. Compared to PBS, manSNI + US irradiation significantly enhanced the abundance of proteins and peptides in the cell supernatant, with a predominant association observed with cellular membrane and metabolic pathways (Supplementary Figs. 27–29). Importantly, abundant BCG-derived antigens, including Ag85 and PPE, were detected, which belong to a broad class of pro-inflammatory molecules known to enhance anti-TB immune response (Fig. 4l)44,45. Furthermore, GO enrichment analysis revealed that the Cellular Component (CC) category encompassed various BCG cellular structures, particularly important pathways such as the cell periphery, cell wall, and membrane (Fig. 4m). These results demonstrate that manSNI + US irradiation can kill BCG and produce a large number of BCG-derived antigens, which are released outside of macrophages through exocytosis46. These released BCG-derived antigens, along with adjuvant R837, can be transported to APCs, such as DCs, thereby promoting anti-TB adaptive immunity. Therefore, immature BMDCs and infected macrophages were co-cultured in a transwell plate (Supplementary Fig. 30). In the absence of US irradiation, manSNI treatment resulted in significantly upregulated levels of CD80 (2.81-fold) and CD86 (2.15-fold) compared to manSN treatment, indicating that loaded R837 enhanced DC maturation. In the presence of US irradiation, manSNI treatment further increased the expression levels of CD80 (1.77-fold) and CD86 (1.81-fold) compared to the non-US-irradiated manSNI group, suggesting a synergistic effect between R837 and BCG-derived antigens released on promoting the proportion of mature DCs (mDCs) (Fig. 4n). In summary, manSNI generates massive ROS under US irradiation to eradicate intracellular M.tb and to promote the in situ release of M.tb-derived antigens. The ROS and R837 enhance macrophage polarization towards a pro-inflammatory phenotype, while the antigens and R837 synergistically drive DC activation, thus inducing enhanced innate and adaptive anti-TB immune response.

TBG targeting and anti-TB efficacy of manSNI in vivo
The anti-TB efficacy in vivo was investigated in the mouse model of orthotopic pulmonary tuberculosis (Fig. 5a). Firstly, the targeting effect of manSNI towards lung granulomas was validated. Following the development of characteristic TBG in the lungs on day 21 post-Mcherry-BCG infection, mice were intravenously administered with SN or manSNI. As depicted in Fig. 5b, manSNI rapidly accumulated in the lung within 6 h and reached its peak after 24 h. At this time point, the intensity of manSNI in the lung was found to be 2.19-fold higher than that of SN (Fig. 5c). Immunofluorescence assay of the lung showed manSNI co-localized well with Mcherry-BCG (Supplementary Fig. 31). To further investigate the targeting ability of manSNI towards TBG and BCG, tissue-clearing technology was employed. In contrast to traditional imaging modalities (e.g., magnetic resonance imaging, computed tomography, and fluorescence imaging), tissue-clearing technology leverages high-resolution 3D organ imaging to unveil drug distribution variations in minute tissue lesions47. Post chemical clarification, lung tissues underwent comprehensive 3D fluorescence imaging via light-sheet microscope (Fig. 5d). Experimental groups were administered either SN or manSNI formulations intravenously 72 h prior to euthanasia. Advanced computational reconstruction of image stacks enabled 3D visualization of Mcherry-BCG-associated granulomas, permitting simultaneous spatial mapping of granuloma lesions, SN/manSNI distribution, and vascular architecture (Fig. 5e), and the results showed distinct biodistribution patterns: manSNI preferentially accumulated within granulomas, whereas SN exhibited significant pulmonary clearance (Fig. 5e, Supplementary Fig. 32 and Supplementary Movies 1–8). Notably, a strong spatial correlation between manSNI localization and Mcherry-BCG-associated granulomas distribution (R² = 0.9032) was observed (Fig. 5f). This optimized tissue clearing-based 3D imaging provides conclusive visual evidence that manSNI achieves precise targeting towards pulmonary TBG and BCG.

The anti-TB efficacy of manSNI was then evaluated against living mice (Fig. 5a). After the formation of characteristic TBG in the lung 21 days after Mcherry-BCG infection, mice were intravenously injected with saline, SN, manSN, or manSNI. Localized US-irradiation of the lung was performed at 1 day and 3 days post-injection. On day 21 after different treatments, mice were sacrificed, and their lung tissues were harvested for ex vivo fluorescence imaging (Fig. 5g and Supplementary Fig. 33) and colony formation assay (Fig. 5h, i). These results showed that in the absence of US irradiation, these semiconducting nanoparticles hardly induced any inhibitory effects against TB. Upon US irradiation, the anti-TB efficacy of manSNI was significantly higher than that of other treatments, nearly completely eliminating colonization of BCG within the lungs.

The anti-TB efficacy was further assessed using histological hematoxylin and eosin (H&E) staining. As depicted in Fig. 5j, the lung slices from the manSNI + US irradiation group exhibited significantly reduced inflammatory cellular infiltration, improved alveolar wall thickening and hemorrhage, decreased fibroblast migration and proliferation compared to the saline and manSN + US irradiation groups. However, persistent infiltration of BCG in the lungs was observed in the SN + US and manSN + US irradiation groups, as demonstrated by gold-standard acid-fast bacilli (AFB) staining for mycobacteria. In contrast, the manSNI + US irradiation group exhibited almost complete absence of BCG infiltration (Fig. 5k, l), indicating successful eradication of BCG in lungs. Additionally, throughout the treatment duration, body weight changes (Supplementary Fig. 34a) and hepatorenal function (Supplementary Fig. 34b, c) remained similar to those of normal mice. The manSNI + US irradiation group exhibited no significant histological damage in major organs, including the heart, liver, spleen, and kidneys (Supplementary Fig. 34d), thereby demonstrating its favorable biosafety profile for TB sono-immunotherapy.

Sono-immunotherapy elicits innate and adaptive immune responses in vivo
On day 7 post-treatment, lung tissues were collected from each group of mice to investigate the potential remodeling of the local immune microenvironment by manSNI-mediated sono-immunotherapy. RNA-seq analysis was employed to elucidate the genomic microenvironment of locoregional immune status. A total of over 2700 DEGs associated with manSNI + US irradiation treatment were identified compared with the saline treatment group, primarily linked to pro-inflammatory responses (Supplementary Fig. 35). KEGG pathway enrichment analysis revealed upregulation in antigen presentation and processing-related signaling pathways (shown in blue), such as TLR and NLR signaling pathways (Fig. 6a and Supplementary Fig. 36a, b). GSEA indicated significant upregulation in TLR and NF-κB-signaling pathways upon manSNI + US irradiation treatment compared to the saline group (Fig. 6b). Notably, TLR and NLR recognize intracellular pathogens, trigger macrophage activation, and subsequently initiate a series of downstream cascading inflammatory responses during M.tb infection48. Meanwhile, pathways associated with inflammation and immune responses (shown in green), such as the chemokine signaling pathway, Jak-stat signaling pathway, and TNF signaling pathway, were also found to be upregulated (Fig. 6a, Supplementary Fig. 36c, d). Importantly, the anti-TB pathway (shown in red) exhibited significant upregulation. The key genes, including Tnf, Tlr2, and IL1β49, in this pathway were found to be significantly upregulated and exhibited increased expression in the heatmap and protein interaction diagram (Fig. 6c), indicating the enhanced antimicrobial activity of innate immunity. The signaling pathways associated with bacterial recognition and presentation, including TLR, MHC, and CXCR receptors (shown in blue), also demonstrated upregulation in terms of molecular function (MF) (Fig. 6d). With respect to biological processes (BP), the pathways implicated in bacterial response, immune response, and lipopolysaccharides (LPS) response were all found to be upregulated. Macrophages serving as the first line of defense against M.tb infection displayed substantial stimulation (Fig. 6e and Supplementary Fig. 37), while also successfully activating the DCs, initiating an adaptive immune response (Fig. 6f, Supplementary Fig. 38). Therefore, B cell signaling pathways (Supplementary Fig. 39) and T cell signaling pathways, including T cell-mediated immunity and CD4-positive α-β T cell pathway (Supplementary Fig. 40), were also activated, suggesting a reversal of the immunosuppressive microenvironment of TBG. Most attractively, the manSNI + US irradiation treatment was able to stimulate memory T cells (Supplementary Fig. 41), which may contribute to preventing TB recurrence.

To comprehensively investigate the mechanism underlying manSNI-mediated TB sono-immunotherapy and elucidate alterations in transcriptomic profiles, immunofluorescence staining, detailed flow cytometry analysis, and cytokine level detection were performed. Firstly, macrophages within TBG underwent obvious M1 polarization following treatment with manSNI + US irradiation (Fig. 6g). Subsequently, axillary lymph nodes were collected for assessment using flow cytometry. The results demonstrated that manSNI + US irradiation treatment significantly increased the proportion of mDCs (~55.68% ± 2.31%), which was substantially higher than that observed in the saline group (~23.55% ± 2.36%) and manSN + US irradiation group (~42.35% ± 1.07%) (Fig. 6h). The high proportion of mDCs can be attributed to adjuvant stimulation and the in situ release of BCG-derived antigens from TBG during SDT. These mDCs are capable of presenting antigen information to T cells and activating them. As a result, manSNI + US irradiation treatment significantly increased the percentages of CD4+ and CD8+ T cells at the infectious site, with levels 2.44-fold and 4.54-fold higher than those observed following saline treatment, and 1.27-fold and 1.31-fold higher than those seen after manSN + US irradiation treatment, respectively (Fig. 6i and Supplementary Fig. 42). Concurrently, the manSNI + US irradiation group exhibited a significant elevation in IL-6 and TNF-α levels in the enzyme linked immunosorbent assay (ELISA) results of lung suspensions compared to the saline group (approximately an eightfold increase for IL-6 and fivefold increase for TNF-α) (Fig. 6j). Meanwhile, there was a corresponding decrease in IL-4 and IL-10 levels in the manSNI + US irradiation group relative to the saline group (only about one-quarter as much for IL-4; less than half as much for IL-10), indicating activation of specific T cell immune response (Fig. 6k). ELISA results for serum also demonstrated a significant increase in serum immunoglobulin G (lgG) and immunoglobulin M (lgM) levels in the manSNI + US irradiation group compared to that seen with saline treatment (about twofold increase for lgG and nearly threefold increase for lgM) (Fig. 6l), indicating effective activation of adaptive humoral immunity. Overall analyses suggest that manSNI-mediated sono-immunotherapy would activate robust innate and adaptive immune responses locally as well as systemically, which could serve as a prerequisite step towards BCG clearance by the host.

Sono-immunotherapy prevents endogenous TB relapse
The high recurrence rate, which includes endogenous relapse and exogenous reinfection, has consistently posed a significant challenge in the treatment of TB50–52. In the aforementioned treatment model, we have demonstrated that manSNI + US irradiation treatment exhibits remarkable therapeutic efficacy in eliminating BCG and can effectively activate adaptive immune responses, particularly immune memory. Therefore, the effectiveness of manSNI-mediated sono-immunotherapy in preventing endogenous relapse was further evaluated. The treatment protocol remained consistent with the aforementioned model. As a control group, continuous administration of the antibiotic drug rifampicin (Rif) was employed to simulate the clinical treatment strategy (i.v. Rif at 10 mg/kg every 2 days for 5 doses)53. Histological analysis using H&E and AFB staining confirmed that Rif treatment can almost completely eradicate BCG colonization in the lungs by day 21 (Supplementary Fig. 43). Subsequently, the evaluation period was extended to 42 days to investigate the anti-relapse efficacy of these treatments (Fig. 7a). At the conclusion of the experiment, evident TB recurrence was observed in the Rif group based on CFU analysis; BCG colonies and inflammation were still present in their lungs (Fig. 7b and Supplementary Fig. 44), as supported by H&E staining (Fig. 7c) and AFB staining results (Fig. 7d and Supplementary Fig. 45). Conversely, in mice subjected to manSNI + US irradiation treatment, TBG or inflammation was scarcely observed within lung tissues on day 42. Overall, the incidence of TB relapse was observed in 5 out of 6 mice in the Rif-treated group, whereas only 1 out of 6 mice exhibited relapse in the manSNI + US irradiation group (Fig. 7e). The above findings indicate that manSNI-mediated sono-immunotherapy can effectively prevent the relapse of the BCG-established TB model over a long-term period following treatment.

To further validate the underlying mechanisms of immune cells providing long-term protection, the contribution of memory T cell subsets in mice was evaluated. Firstly, the proportion of tissue-resident memory T cells (TRM, CD8+CD69+CD103+) in the lungs of mice on day 42 was analyzed using flow cytometry. TRM level in the manSNI + US irradiation group (22.23 ± 3.15%) was about 1.68-fold higher than the Rif group (13.26 ± 1.78%) (Fig. 7f, g and Supplementary Fig. 46). This high level of TRM in lung tissue is beneficial for maintaining a strong antimicrobial immune response and providing local protection against endogenous relapse54. Meanwhile, when exposed to bacterial antigens, the central memory T cells (TCM) demonstrate the capacity for proliferation and prompt differentiation into effector cells, thereby bestowing long-term protection against residual pathogens upon the host. Subsequently, the level of TCM cells in spleen tissues was analyzed. The proportion of TCM cells (CD3+CD4+ CD44+CD62L+) in the manSNI + US irradiation group (73.83 ± 3.45%) was significantly higher compared to that in the Rif group (51.58 ± 1.01%) (Fig. 7h, i and Supplementary Fig. 47). Moreover, the levels of lgG and lgM (Fig. 7j, k) in the serum of mice subjected to manSNI + US irradiation (lgG: 495.7 ± 8.8 ng/mL, lgM: 668.1 ± 34.8 ng/mL) exhibited a significant increase compared to those in the Rif group (lgG: 347.3 ± 18.7 ng/mL, lgM: 558.7 ± 5.2 ng/mL), indicating that manSNI + US irradiation treatment can also elicit a long-lasting humoral immune memory response. Consequently, manSNI-mediated sono-immunotherapy could confer enduring immune memory protection, effectively preventing endogenous relapse following treatment.

Sono-immunotherapy prevents exogenous reinfection
The recurrence of TB caused by exogenous reinfection is a prevalent clinical scenario, accounting for ~6.7–18% of recurrent cases in the clinic55,56. Inducing long-term immune memory and facilitating rapid response to invading M.tb are crucial for protection against reinfection. Compared to Rif, considering the superior efficacy of manSNI + US irradiation in clearing BCG and activating the potent innate and adaptive anti-TB immune responses, its ability to protect against exogenous reinfection was further investigated by establishing a mouse model of secondary BCG reinfection. Following treatment with manSNI + US irradiation, mice were re-challenged with a second batch of BCG on day 21 (Fig. 8a). Memory B cells are the primary mediators of humoral immunity57, effectively preventing reinfection by bacteria and viruses58. Upon subsequent exposure to the same pathogen, memory B cells exhibit a rapid and robust immune response compared to the initial encounter. Therefore, the proportion of peripheral blood memory B cells was assessed by flow cytometry analysis on day 21 prior to BCG re-challenge. Mice that were untreated after the initial infection served as the control group. The percentage of memory B cells (CD45+CD38−CD19+) in the manSNI + US irradiation group (19.23 ± 0.82%) was significantly higher than that in the control group (5.06 ± 0.75%) (Fig. 8b, c and Supplementary Fig. 48), indicating manSNI + US irradiation can induce stronger immunogenicity and protective effect. Furthermore, following re-challenge with a second batch of BCG for one week (day 28), mice in the manSNI + US irradiation group exhibited substantially elevated levels of lgG and lgM in their serum (lgG: 937.5 ± 33.3 ng/mL, lgM: 630.2 ± 23.3 ng/mL) compared to those in the control group (lgG: 450.4 ± 17.5 ng/mL, lgM: 267.7 ± 31.9 ng/mL) (Fig. 8d, e), indicating manSNI + US irradiation induced a robust humoral immune response in mice following BCG re-challenge, leading to the production of higher levels of antibodies.

Upon re-exposure to foreign antigens, T cells can undergo rapid proliferation and differentiation into effector memory T cells (TEM, CD3+CD4+CD62L−CD44+) that produce crucial cytokines such as TNF-α and IL-6, thereby inducing a robust immune memory protection effect59. Therefore, the percentage of TEM cells in the spleen was analyzed after re-challenge with the second batch of BCG for one week (day 28). Notably, the manSNI + US irradiation group exhibited a 5.02-fold higher percentage of TEM cells (43.96 ± 4.72%) than that of the control group (11.65 ± 2.43%) (Fig. 8f, g). ELISA analysis revealed significantly elevated levels of IL-6 and TNF-α in the serum of mice treated with manSNI + US irradiation compared to those of the control (Fig. 8h, i). These findings suggest that manSNI + US irradiation confers long-term immune memory protection by enabling a rapid immune response to reinfection. Therefore, following re-challenge with the second batch of BCG for three weeks (day 42), four out of six mice within the manSNI + US irradiation-treated group exhibited no M.tb presence and inflammation as evidenced by CFU assay (Fig. 8j and Supplementary Fig. 49), H&E staining (Fig. 8k), and AFB staining of lung tissues (Fig. 8l and Supplementary Fig. 50), and only two out of six mice exhibited slight TBG or inflammation in lung tissue on day 42 in the manSNI + US irradiation group (Fig. 8m). Thus, manSNI-mediated sono-immunotherapy also elicited robust immune memory to effectively prevent reinfection by BCG.

Methods
M.tb strains and culture
Bacillus Calmette-Guérin strains, with or without fluorescent reporter gene constructs, namely BCG, GFP-BCG, and Mcherry-BCG, were utilized in this study. The cultivation of BCG was conducted in 7H9 medium supplemented with 10% oleic albumin dextrose catalase (OADC) at a temperature of 37°C. GFP-BCG was cultured in 7H9 broth medium supplemented with kanamycin at a concentration of 30 μg/mL. Similarly, Mcherry-BCG was grown in 7H9 broth medium containing kanamycin at the same concentration. The attenuated strain of M.tb H37Ra (ATCC25177) was grown in Middlebrook 7H10 solid medium supplemented with 10% oleic acid–albumin–dextrose–catalase enrichment (BD Difco Laboratories). The in vitro and in vivo experiments associated with Mycobacterium were conducted in a biosafety level II laboratory located at Southern Medical University. All the strains were purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

manSNI targets macrophages and intracellular M.tb
To target macrophages, Raw264.7 cells were seeded at a density of 1×106 cells/well in confocal dishes overnight. Subsequently, the SN and manSNI (at a concentration of 100 μg/mL based on SN) were added to the confocal dishes. After 12 h, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min. Lysosomal probes were used to label the lysosomes of Raw264.7 cells following the manufacturer’s instructions. Cell nuclei were stained using an anti-fluorescence quencher containing DAPI. Finally, images were acquired using a confocal laser scanning microscope (FV3000, OLYMPUS).

The intracellular infection model was modified based on previous methods60. To establish an intracellular bacterial model for targeting BCG within macrophages, Raw264.7 cells were seeded at a density of 1×106 cells/well in confocal dishes and incubated overnight. Subsequently, the cells were infected with GFP-BCG for 3 h, followed by removal of the medium and three washes with PBS. Fresh medium was then added to the cells, which were subsequently treated with SN and manSNI (at a concentration of 100 μg/mL based on SN). After 24 h of treatment, the medium was removed, and the cells were washed three times with PBS before fixation using 4% paraformaldehyde for 15 min. Finally, DAPI staining was performed, and images were captured using a confocal laser scanning microscope (FV3000, OLYMPUS).

Effectiveness of SN, manSN, and manSNI in killing intracellular M.tb
Raw264.7 cells were seeded at a density of 1×106 cells/well in confocal dishes overnight. Subsequently, the cells were infected with BCG (MOI=10). After 3 h, the uninfected cells were washed with PBS to remove bacteria. Following this, the cells were treated with SN, manSN, and manSNI (at a concentration of 100 μg/mL based on SN). After 12 h, the drug-containing medium was replaced with fresh medium. The US-irradiation groups (PBS, SN, manSN and manSNI) underwent US irradiation using a small-animal sonicator (Intelect Portable Ultrasonic Therapeutic Instrument) for 10 min at an intensity of 1.5 W/cm2; whereas the non-US-irradiation groups (PBS, SN, manSN, and manSNI) remained untreated. The cells were then incubated at 37°C with 5% CO2 for an additional 24 h. Finally, cell lysis was performed using 0.1% Triton X-100 for 15 min followed by dilution with PBS to obtain specific concentrations of cell lysates, which were subsequently inoculated on Middlebrook’s solid medium (7H10). The plates were incubated at 37°C for a period of time ranging from 21 to 30 days before counting and recording colony numbers.

Phenotyping cultures of macrophages and BMDCs in vitro
To evaluate the activation of macrophages by manSNI, an intracellular infection model of BCG was established following the aforementioned protocol. Subsequently, cells were treated with PBS, SN, manSN, and manSNI (at a concentration of 100 μg/mL based on SN). After 12 h, the medium was replaced with fresh medium. The US-irradiation groups underwent sonication using a small animal sonicator for 10 min (at a power density of 1.5 W/cm2). The non-US-irradiation groups remained untreated. Following this treatment regimen, the samples were incubated at 37°C in a CO2 incubator for an additional 24 h. The culture medium was collected to measure the expression levels of inflammatory cytokines (IL-6 and TNF-α) using enzyme-linked immunosorbent assay (ELISA), while flow cytometry analysis was performed on the collected cells to assess their differentiation.

The macrophages were processed using the aforementioned method. Subsequently, BMDCs were isolated following established protocols61. BMDCs were then seeded in transwell plates at a density of 50,000 cells per well and cultured overnight. Afterwards, the cells were collected and subjected to flow cytometry analysis in accordance with the manufacturer’s recommendations to evaluate the maturation status of BMDCs.

Detailed ultrasound parameters used in animal experiments
The ultrasound device used in this study was the Intelect Portable Ultrasonic Therapeutic Instrument (Chattanooga Group, USA) equipped with a 2776 transducer, operating at a fixed frequency of 1 MHz with an adjustable power density range of 0.5–3.0 W/cm2.

Parameter calibration and verification
Before the experiments, ultrasound intensity was calibrated using a hydrophone (HNR-0500, Onda Corporation, USA) in a simulated body fluid environment at 37°C. The calibration was performed at a vertical distance of ~1 cm between the ultrasound probe and the target tissue (mouse lung), consistent with the irradiation distance during the treatment. This ensured that the nominal intensity (1.5 W/cm2) matched the actual intensity acting on the lesions.

During lung irradiation, the thickness of the acoustic couplant layer (2–5 mm) between the probe and the skin was kept consistent across animals to minimize variability in intensity attenuation. The US-irradiation groups received ultrasound irradiation using a small-animal sonicator for 10 min at 24 and 72 h post-injection, with a duty cycle of 50% and continuous-wave irradiation mode, at a power density of 1.5 W/cm2.

Anti-TB efficacy of manSNI + US irradiation treatment in vivo
The animal experimental protocol was reviewed by the Experimental Animal Ethics Committee of Guangdong Medical Laboratory Animal Center and complied with the relevant ethical norms (approval number C202401-2). Mice were raised in a specific pathogen-free (SPE) grade animal laboratory. Mice were housed at light/dark cycle: 12 h/12 h; temperature: 22–24°C; humidity: 50 ± 5%. A mouse tuberculosis model was established by nebulized inhalation of Mcherry-BCG (107 CFU/mL, 100 μL). Intravenous injections of saline, SN, manSN, and manSNI solutions (based on an SN concentration of 10 mg/kg) were administered 21 days after infection. The US-irradiation groups underwent ultrasound irradiation using a small animal sonicator for 10 min at 24 and 72 h post-injection with a power density of 1.5 W/cm2. The non-US-irradiation groups remained untreated. After another 21 days had elapsed from the injection time point, the mice were euthanized to collect their lungs for subsequent analysis. A portion of the lung tissue was imaged using a small animal in vivo imaging system to quantify the fluorescence intensity emitted by Mcherry-BCG as an indicator of bacterial load in the lungs. Simultaneously, another portion was subjected to colony-forming unit determination assay. Additionally, lung tissue sections were prepared for H&E staining along with immunohistochemical and AFB staining.

In vivo activation of immune responses by manSNI treatment
A mouse tuberculosis model was established as described above. After one week of treatment, the lungs, lymph nodes, and spleens were collected from the mice. Subsequently, these organs were cut into small pieces and enzymatically degraded for 1 h at 37°C using 1 mg/mL Collagenase IV. The resulting suspensions were then passed through 70 μm cell filters to obtain individual cell suspensions after removing erythrocytes with erythrocyte lysate (Gibco). To analyze the composition of immune cells, the obtained cell suspensions were incubated with CD16/32 for 10 min followed by staining with fluorescent antibodies (FITC-CD103, PE-CD69, APC-CD8, PE-Cy5.5-CD62L, APC-CD44, FITC-CD4, PE-CD3, PE-CD45, APC-CD19, and FITC-CD38) on ice for 30 minutes. The same staining protocol used in vitro was applied for CD206 staining as well. Cytokine levels (IL-6, TNF-α, IL-4, and IL-10) and serum antibody levels (IgM and IgG) in the lungs were determined using ELISA kits according to the manufacturer’s instructions. For RNA-seq analysis, total RNA was extracted using Trizol reagent and subsequently subjected to RNA-seq analysis conducted by BGI Genomics.

Prevention of TB relapse
A mouse TB model was established using the aforementioned method, and mice were treated with Rif and manSNI. Rifampicin (Rif) was dissolved in dimethyl sulfoxide (DMSO) (100 mg Rif/mL) and diluted with saline to a final DMSO concentration of 1% before use. Mice in the Rif control group received intravenous injection of Rif at 10 mg/kg (0.1 mL per mouse) every 2 days for a total of 5 doses. manSNI solutions ([SN]=10 mg/kg) were administered 21 days after infection. US irradiation was applied using a small animal sonicator for 10 min at 24 and 72 h post-injection of manSNI with a power density of 1.5 W/cm2. After a period of 42 days, lung, spleen, and blood samples were collected and processed following the protocol described in “In vivo activation of immune effects by manSNI. Flow analysis was conducted to assess the induction of long-lasting immunity in mice. Simultaneously, lung tissues from mice were collected for CFU assay as well as H&E staining and AFB staining to evaluate tuberculosis recurrence.

Prevention of BCG reinfection
A mouse TB model was established as previously described, and mice were treated with manSNI + US. After 21 days, a portion of the spleen and lymph nodes was collected and processed according to the method “In vivo activation of immune effects by manSNI. Flow cytometry analysis was performed to determine the composition of immune cells prior to secondary BCG reinfection. Simultaneously, another group of mice was used for reinfection experiments where BCG (107 CFU/mL, 100 μL) was nebulized and inhaled. After 7 days, a portion of spleens and lymph nodes was collected and processed using the method “In vivo activation of immune effects by manSNI for flow cytometry analysis to determine the composition of immune cells after reinfection. Cytokine levels (IL-6 and TNF-α) as well as serum antibody levels (IgM and IgG) were measured using ELISA kits following the manufacturer’s instructions. Finally, at indicated time points (21 days after reinfection), mice were euthanized, and lung tissues were collected for evaluation of bacterial load through histopathological sections and acid-fast staining.

Data analysis
The quantitative data were presented as the mean ± s.d. All experiments were performed with a minimum of three replicates. Statistical analysis between the two groups was conducted using two-sided unpaired t-tests. One-way ANOVA followed by post-hoc correction was employed for multiple comparisons. A probability value <0.05 (P < 0.05) was considered statistically significant62.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_3": "Summary: The goal of this work is to demonstrate a sonodynamic therapy approach to treat tuberculosis granulomas obtained from TB patients. The authors report a nano-immunostimulant agent equipped with mannose for targeted sonodynamic tuberculosis (TB) immunotherapy aimed at preventing recurrence. Ultrasonic stimulation caused bactericide along with release of antigens that enhance innate and adaptive anti-TB immune responses. These studies aim to serve as a step towards translating sonodynamic bactericide for TB applications.\nGeneral comments: This manuscript is focused on a clinically significant problem. The work is fairly detailed, with in vitro and in vivo results. The paper is well written and comprehensive. However, as presented, the paper has some major deficiencies. Most notably, it appears that the authors did not perform any characterization/hydrophone calibration of the ultrasound transducer. It appears that only the nominal intensity provided by the manufacturer is used. Even here, the details are missing. There is no mention of the instrumentation used for sonication including the transducer. The intensity can vary with the distance and it is unclear at which location the nominal intensity was assumed/measured. This is a major flaw which will affect the reproducibility and future translation of the study. I have provided more details below about the interpretation of results.\nDetailed comments:\n1. This therapeutic ultrasound exposure information is not enough information to interpret and replicate the work reported in this paper and represents a major flaw. Please see the work published by ter Haar et al. \"Guidance on reporting ultrasound exposure conditions for bio-effects studies\" Ultrasound Med. Biol. 2011 for guidelines on why these parameters are critical. Also, ultrasound is a key part of sonodynamic therapy and it should be included in detail in the main text and not in the supplementary text.\n2. In the Methods, kanamycin is listed at 30 mg/mL for selective media (line 894). Did the authors mean 30 μg/mL? 30 mg/mL appears to be too high.\n3. All experiments were performed with M. bovis BCG, but the claims appear to be made for M. tuberculosis. The manuscript should be rewritten to clearly state that these findings need to be extended for M.tuberculosis.\n4. In the organ histology images, the scale bar is “100 nm.” This appears to be a typo as it is too small. Check figure S32.\n5. Details of rifampicin dosage appear missing. Details should be added in the methods section.\n6. The text mentions reactive oxygen species (ROS) a few times mentioning them as immunogenic. ROS are immunomodulators that cause stress and damage, but they are not antigens and cannot directly affect adaptive immunity. The Main Text (mechanistic explanation of immune activation) should be revised to state that ROS may enhance immune responses, rather than suggesting ROS themselves are immunogens.\n7. The number of independent experiments is low for several arms. This point should be clearly mentioned in the limitations, and the figure legends should clearly report N values.\n8. The manuscript uses both terms “sonodynamic therapy” (SDT) and “sonoimmunotherapy”. It can be confusing to the readers. Please clarify the difference and be consistent.\n9. The limitations of the manuscript, and pathway to clinical translation should be clearly detailed.", "reviewer_4": "This interesting manuscript describes a novel therapeutic approach to mycobacterial infection using a construct design to target mannose receptors on macrophages and to deliver a payload that on ultrasound treatment generates ROS and releases a TLR7 agonist. The authors argue that this strategy can overcome the ability of MTB to remain dormant and evade chemotherapy and host defenses. My main concern is the reliance on BCG as the organism for this infection model. BCG generally does not become dormant or cause disease except under the most unusual circumstances (profound immunosuppression or overwhelming infection after therapeutic use). I believe it is also true that BCG is substantially more susceptible to ROS than MTB (which is triggered by ROS to enter dormancy). I therefore am not at all sure that an acute treatment model using BCG followed by a very short course of anti-TB drugs is a good model in which to test this concept.\nMinor concerns\nLine 176-7. It is surprising that the immune profiles of drug-resistant cases differ from drug sensitive. Are the authors sure this was not the result of infection with different MTB families (Beijing vs non-Beijing), or the effect of drug treatment (BDQ+pretomanid vs rifampin)?\nIn multiple lines, the authors refer to \"eliminating M.tb\" or \"TB relapse\" or \"anti-TB activity of SNI\". I am concerned demonstrating activity against BCG does not ensure activity against MTB."}, "author_response": "Response letter to reviewers\nReviewer #3 (Remarks to the Author):\nSummary: The goal of this work is to demonstrate a sonodynamic therapy approach to treat\ntuberculosis granulomas obtained from TB patients. The authors report a nano-immunostimulant\nagent equipped with mannose for targeted sonodynamic tuberculosis (TB) immunotherapy aimed\nat preventing recurrence. Ultrasonic stimulation caused bactericide along with release of antigens\nthat enhance innate and adaptive anti-TB immune responses. These studies aim to serve as a step\ntowards translating sonodynamic bactericide for TB applications.\nGeneral comments: This manuscript is focused on a clinically significant problem. The work is\nfairly detailed, with in vitro and in vivo results. The paper is well written and comprehensive.\nHowever, as presented, the paper has some major deficiencies. Most notably, it appears that the\nauthors did not perform any characterization/hydrophone calibration of the ultrasound transducer.\nIt appears that only the nominal intensity provided by the manufacturer is used. Even here, the\ndetails are missing. There is no mention of the instrumentation used for sonication including the\ntransducer. The intensity can vary with the distance and it is unclear at which location the nominal\nintensity was assumed/measured. This is a major flaw which will affect the reproducibility and\nfuture translation of the study. I have provided more details below about the interpretation of\nresults.\nResponse：Thank you for accurately identifying this critical issue! We fully agree that the detailed\ncharacterization of ultrasound exposure parameters, transducer calibration, and standardized\noperations are core prerequisites for research reproducibility and clinical translation. We sincerely\napologize for failing to fully elaborate on these key details previously. In response to your\nconcerns, we have completed systematic supplementary verification and information\nsupplementation, and the specific revisions are elaborated in detail in the following response.\nDetailed comments:\nQuestion 1：This therapeutic ultrasound exposure information is not enough information to\ninterpret and replicate the work reported in this paper and represents a major flaw. Please see the\nwork published by ter Haar et al. \"Guidance on reporting ultrasound exposure conditions for bio-effects studies\" Ultrasound Med. Biol. 2011 for guidelines on why these parameters are\ncritical. Also, ultrasound is a key part of sonodynamic therapy and it should be included in detail\nin the main text and not in the supplementary text.\nResponse：We appreciate the reviewers for pointing out this important issue. We fully agree that\ndetailed reporting of ultrasound exposure parameters is crucial for reproducibility and for\n1\nfacilitating future clinical translation. We have added a comprehensive description of all\nultrasound parameters to the \"Methods\" section of the revised manuscript. The revised content is\nsummarized below:\nDetailed ultrasound parameters used in animal experiments:\nThe ultrasound device used in this study was the Intelect Portable Ultrasonic Therapeutic\nInstrument (Chattanooga Group, USA) equipped with a 2776 transducer, operating at a fixed\nfrequency of 1 MHz with an adjustable power density range of 0.5–3.0 W/cm².\nParameter calibration and verification:\nBefore the experiments, ultrasound intensity was calibrated using a hydrophone (HNR-0500,\nOnda Corporation, USA) in a simulated body fluid environment at 37℃. The calibration was\nperformed at a vertical distance of approximately 1 cm between the ultrasound probe and the\ntarget tissue (mouse lung), consistent with the irradiation distance during the treatment. This\nensured that the nominal intensity (1.5 W/cm²) matched the actual intensity acting on the lesions.\nDuring lung irradiation, the thickness of the acoustic couplant layer (2-5 mm) between the\nprobe and the skin was kept consistent across animal to minimize variability in intensity\nattenuation. The US-irradiation groups received ultrasound irradiation using a small-animal\nsonicator for 10 minutes at 24 h and 72 h post-injection, with a duty cycle of 50% and\ncontinuous-wave irradiation mode, at a power density of 1.5 W/cm2.\nMeanwhile, we have added a discussion of the limitations and future perspectives of ultrasound\ntherapy in the Conclusion section of the revised manuscript as follows:\nFinally, the US devices used in small-animal studies differ substantially from clinical systems in\nparameter ranges and tissue compatibility. To further enhance the translational relevance of this\nstrategy, future studies will involve collaborations with accredited laboratories to evaluate the\nefficacy of manSNI-mediated sono-immunotherapy against a panel of clinically relevant M.tb\nstrains. Additionally, we plan to extend the preclinical assessment to large animal models,\nincluding guinea pigs and non-human primates, which more accurately recapitulate the\npathological characteristics of human TB. These studies will employ appropriately expanded\ngroup sizes (n ≥ 10 per group) to ensure robust statistical power and will systematically optimize\nultrasound parameters—including intensity, duration, and frequency—to align with the\noperational specifications of clinical ultrasound systems.\nQuestion 2: In the Methods, kanamycin is listed at 30 mg/mL for selective media (line 894). Did\nthe authors mean 30 μg/mL? 30 mg/mL appears to be too high.\nResponse：We appreciate the reviewers for pointing out this oversight. The original \"30 mg/mL\"\nwas indeed a unit typographical error. We have corrected it to 30 μg/mL in the Methods section of\n2\nthe revised manuscript. We also reviewed the corresponding experimental records and confirmed\nthat all GFP-BCG and Mcherry-BCG cultures were screened using 30 μg/mL kanamycin. This\ncorrection does not affect any experimental results or conclusions of the study.\nQuestion 3: All experiments were performed with M. bovis BCG, but the claims appear to be\nmade for M. tuberculosis. The manuscript should be rewritten to clearly state that these findings\nneed to be extended for M.tuberculosis.\nResponse: We thank the reviewers for raising this important point and fully agree that our\nfindings should not be generalized to Mycobacterium tuberculosis (M.tb) without proper\nclarification. We also recognize the biological differences between BCG and M.tb. To address this\nconcern, we have made the following revisions and additions to the revised manuscript:\n(1) Clarified the rationale for using BCG:\nIn the revised introduction, we now emphasize that BCG is an internationally recognized\npreliminary screening model for anti-tuberculosis drugs and therapies[1-2] because it shares more\nthan 90% genomic homology with M.tb[3].\n(2) Added validation using an attenuated M.tb strain:\nTo strengthen the relevance of our findings, we have supplemented the study with in vitro\nand intracellular bactericidal activities against the attenuated strain Mycobacterium tuberculosis\nH37Ra. These added data showed below confirm that manSNI+US irradiation also retains potent\nbactericidal activity against H37Ra.\nRevisions added to the revised manuscript：\nEncouraged by the superior ROS generation ability of manSNI under US irradiation, the\nanti-M.tb activity of manSNI was then investigated. Both the Bacillus Calmette-Guérin (BCG,\nwhich shares a high degree of genomic homology with M.tb and is an internationally recognized\npreliminary screening model for anti-TB drugs/therapies39) and the attenuated strain of M.tb\nH37Ra (ATCC25177) were used as the model bacteria. BCG, Firstly, the colony-forming units\n(CFU) assay revealed that these semiconducting nanoparticles (SN, manSN, and manSNI) were not\ntoxic to BCG and H37Ra (Supplementary Fig. 19) in the absence of US irradiation. While, even\nwhen isolated by a 5 cm thick pork tissue pad (Fig. 3j), the combination of these nanoparticles and\nUS irradiation (1.5 W/cm2, 1 MHz, 5 min) resulted in a bacterial death rate exceeding 99% (Fig.\n3k, l). In contrast, due to the poor tissue penetration of light (1.5 W/cm2, 808 nm, 5 minutes), the\nnear-infrared photosensitizer represented by ICG cannot kill BCG when separated by a 5 cm thick\nlayer of pork tissue (Supplementary Fig. 20). Similar, the live/dead bacterial viability assay\ndemonstrated that manSNI exhibited excellent efficiency in killing BCG and H37Ra (Fig. 3m and\nSupplementary Fig. 21) under US irradiation. Then, the structural changes in BCG and H37Ra\n3\nafter different treatments were examined. As shown in scanning electron microscopy (SEM)\nimages, the combination of these nanoparticles and US irradiation caused extensive damage to the\nbody structure of BCG and H37Ra (Fig. 3n), resulting in an overall shriveled and concave shape\nof bacteria. The disruption of bacterial structure resulted in the leakage of cellular contents as\nevidenced by protein leakage high to 0.22 μg/mL in solution from bacterial (107 CFU/mL)\n(Supplementary Fig. 22), which are expected to activate the body’s immune response as pathogen\nantigens. These results demonstrate that manSNI can kill M.tb by generating a large amount of\nROS through sonodynamic effect.\nFig.3 | Preparation and in vitro anti-M.tb activity of manSNI. j, Schematic diagram of the anti-M.tb action\nmechanism of ICG-mediated PTT and manSNI-mediated SDT. 5 cm thick pork tissue placed between the\nlight/ultrasonic instrument and the test tube containing M.tb (1.5 W cm-2, 1 MHz, 5 min). k, Representative images\nshowing the survival BCG and H37Ra in the culture plate. l, Quantitative statistics of the number of BCG colonies\nand H37Ra colonies through standard plate counting assay. Data were presented as mean ± SD (n=3). m, SYTO\n9/PI staining assay of BCG and H37Ra after treated with PBS, SN, manSN and manSNI under US irradiation. n,\nSEM images of BCG and H37Ra after treated with PBS, SN, manSN and manSNI under US irradiation.\nSupplementary Fig. 19. a, Representative images showing the survival BCG and H37Ra in the\nculture plate. b, Quantitative statistics of the number of BCG and H37Ra colonies through\nstandard plate counting assay. Data were presented as mean ± SD (n=3).\n4\nk\n6\n0\n)601×(\nUFC\nj\nPBS SN manSN manSNI\nl m n\nPBS SN manSN manSNI PBS SN manSN manSNI\nBCG H37Ra\n5\n4\n3\n2\nGCB\naR73H\nGCB\naR73H\n+US\nLight 808 nm US\nPork tissue,thickness 5 cm Membrane rupture\nROS\nROS\nPathogen antigens\nLive Mtb Dead M.tb\n+US +US\n50 μm 1 μm\n1 μm\nGCB\naR73H\n50 μm\nGCB\naR73H\nNo US\nPBS SN manSN manSNI 6\n0\n)601×(\nUFC\na b\nBCG H37Ra\n5\n4\n3\n2\nPBS SN\nnoitartnecnoc\nnietorP\n)Lm/gμ(\n0.3 -US\nP<0.0001 P<0.0001\n+US\n0.2\n0.1\n0\nmanSN manSNI PBS SN\nnoitartnecnoc\nnietorP\n)Lm/gμ(\na b\nBCG H37Ra\n0.3 -US P<0.0001 P<0.0001\n+US\n0.2\n0.1\n0\nmanSN manSNI\nSupplementary Fig. 24. a, b, Colony photographs (a) and CFU counting (b) of H37Ra at\ndifferent US times and US intensities.\n(3) Expanded discussion on limitations and clinical translation:\nWe have added a discussion on the limitations and clinical translation in the conclusion\nsection: we will clearly state the model limitations, point out that there are essential differences in\npathogenicity and dormancy characteristics between BCG and human-derived M.tb, and note that\nthe results of this study need to be further verified in human-derived M.tb models. Subsequent\nstudies will use M.tb standard strain (H37Rv) to verify the eradication efficacy of this strategy\nagainst M.tb.\nRevisions added to the Conclusion Section of revised manuscript：\nDespite the promising findings, this study also has several limitations. First, due to biosafety\nconstraints, the anti-TB efficacy was validated using attenuated strains (BCG and H37Ra) rather\nthan virulent M.tb strains such as H37Rv strain or clinical isolates. Clinical M.tb strains exhibit\ndistinct pathogenicity, immune evasion mechanisms, and cell wall components, which may affect\nthe translatability of our findings. Second, the impact of distinct M.tb strain lineages (e.g. the\nBeijing family vs. non-Beijing strains) on the immune landscape of drug-resistant and\ndrug-sensitive patients was overlooked. Due to the lack of strain typing data in the GEO datasets,\nwe were unable to dissect whether strain-specific characteristics affect the immune differences\nbetween drug-resistant and drug-sensitive patients. Third, the mouse model used in our study may\nnot fully recapitulate the complexity of human TB, including granuloma structure and\nimmune-cell diversity. Fourth, the relatively small sample sizes (n = 3-4 for in vitro assays, n = 6\nSU+\nPBS SN manSN manSNI\n)301×(\nUFC\na\nb\nPBS SN\n250\nmanSN manSNI\n200\n100\n50\n0\n-US +US\nfor in vivo experiments) may limit statistical power. Finally, the US devices used in small-animal\nstudies differ substantially from clinical systems in parameter ranges and tissue compatibility. To\nfurther enhance the translational relevance of this strategy, future studies will involve\ncollaborations with accredited laboratories to evaluate the efficacy of manSNI-mediated\nsono-immunotherapy against a panel of clinically relevant M.tb strains. Additionally, we plan to\nextend the preclinical assessment to large animal models, including guinea pigs and non-human\nprimates, which more accurately recapitulate the pathological characteristics of human TB. These\nstudies will employ appropriately expanded group sizes (n ≥ 10 per group) to ensure robust\nstatistical power and will systematically optimize ultrasound parameters—including intensity,\nduration, and frequency—to align with the operational specifications of clinical ultrasound\nsystems.\nReferences\n1. Padilla-Carlin DJ, McMurray DN, Hickey AJ. The Guinea pig as a model of infectious diseases.\nComp Med. 2008;58(4):324–40.\n2. Li, H., Li, H. Animal Models of Tuberculosis. In: Christodoulides, M. (eds) Vaccines for\nNeglected Pathogens: Strategies, Achievements and Challenges. Springer, Cham. 2023.\nhttps://doi.org/10.1007/978-3-031-24355-4_7.\n3. Behr, M. A. BCG — different strains, different vaccines? The Lancet Infect. Dis. 2, 86-92\n(2002))\nQuestion 4: In the organ histology images, the scale bar is “100 nm.” This appears to be a typo as\nit is too small. Check figure S32.\nResponse: We appreciate the reviewers for pointing out this error. The \"100 nm\" label in Figure\nS32 (now is Figure S36) was indeed a typographical mistake, and the correct scale bar should be\n100 μm. We have thoroughly checked all histology-related figures, corrected the scale bar units,\nand ensured consistency across all images in accordance with standard histological magnification\nlevels.\nRevisions in the text：\ndays. b, c, Plasma biochemistry index (b) and blood routine (c) of healthy mice after manSNI\ntreatment for 14 days. ALT, alanine transaminase. AST, aspartate aminotransferase. ALB, albumin.\nUREA, urea nitrogen. CR, creatinine. WBC, white blood cells. RBC, red blood cells, PLT,\nplatelets. LYM, lymphocytes. NE, neutrophils. d, H&E staining of the different organs obtained\nfrom the healthy mice after manSNI treatment for 14 days. Scale bar, 100 μm.\nQuestion 5: Details of rifampicin dosage appear missing. Details should be added in the methods\nsection.\nResponse：Thanks for your suggestion. We have added the complete rifampicin administration\ndetails to the Methods section of the revised manuscript. The added description is as follows:\n“Rifampicin was dissolved in dimethyl sulfoxide (DMSO) (100 mg Rif/mL) and diluted with\nsaline to a final DMSO concentration of 1% before use. Mice in the Rif control group received\nintravenous injection of Rif at 10 mg/kg (0.1 mL per mouse) every 2 days for a total of 5 doses.”\nRevisions in the revised manuscript：\nPrevent TB relapse\nA mouse TB model was established using the aforementioned method, and mice were treated with\nRif and manSNI. Rifampicin (Rif) was dissolved in dimethyl sulfoxide (DMSO) (100 mg Rif/mL)\nand diluted with saline to a final DMSO concentration of 1% before use. Mice in the Rif control\ngroup received intravenous injection of Rif at 10 mg/kg (0.1 mL per mouse) every 2 days for a\ntotal of 5 doses. manSNI solutions ( [SN] = 10 mg/kg) were administered 21 days after infection.\nUS irradiation was applied using a small animal sonicator for 10 minutes at 24 h and 72 h\npost-injection of manSNI with a power density of 1.5 W/cm2. After a period of 42 days, lung,\n8\nspleen, and blood samples were collected and processed following the protocol described in “In\nvivo activation of immune effects by manSNI. Flow analysis was conducted to assess the induction\nof\nthe induction\nof long-lasting immunity in mice. Simultaneously, lung tissues from mice were collected for CFU\nassay as well as H&E staining and antacid staining to evaluate tuberculosis recurrence.\nQuestion 6: The text mentions reactive oxygen species (ROS) a few times mentioning them as\nimmunogenic. ROS are immunomodulators that cause stress and damage, but they are not\nantigens and cannot directly affect adaptive immunity. The Main Text (mechanistic explanation of\nimmune activation) should be revised to state that ROS may enhance immune responses, rather\nthan suggesting ROS themselves are immunogens.\nResponse: We thank the reviewers for this important clarification. We agree that ROS act as\nimmunomodulators rather than antigens, and that their role should be described as enhancing\nimmune responses rather than directly eliciting adaptive immunity. We have revised the\nmechanistic description throughout the main text to accurately reflect the immunomodulatory\nfunction of ROS:\nThe original sentence: \"Upon ultrasound irradiation, manSNI precisely eliminates M.tb within\nlung granulomas and macrophages. Additionally, it facilitates the simultaneous in situ release of\nM.tb-derived antigens. This combined with sonodynamic reactive oxygen species (ROS) and\nencapsulated TLR7 agonist R837 induces robust innate and adaptive anti-TB immune responses,\nincluding long-term immune memory responses. \"\nThe revised sentence: \"Upon ultrasound irradiation, manSNI generates a large amount of ROS to\nprecisely eliminate M.tb within lung granulomas and macrophages, while simultaneously\npromoting the simultaneous in situ release of M.tb-derived antigens. The generate ROS can\nactivate macrophages toward a pro-inflammatory state in combination with R837. Meanwhile, the\nrelease M.tb-derived antigens and R837 from macrophages could synergistically promote DC\nmaturation. These effects collectively trigger potent innate and adaptive anti-TB immune\nresponses, including long-lasting immune memory.\"\nThe original sentence: \"The appropriate levels of ROS and R837 have been reported to enhance\nthe polarization of macrophages towards pro-inflammatory states (M1 macrophages) and increase\nthe release of inflammatory cytokines\"\nThe revised sentence: \"Appropriate levels of ROS and R837 have been reported to enhance\nmacrophage polarization towards a pro-inflammatory (M1) phenotype and increase the release of\ninflammatory cytokines by activating NF-κB signaling.\"\nThe original sentence: \"In summary, manSNI-mediated SDT activates macrophages into a\npro-inflammatory state and generates BCG-derived antigens by killing BCG, which further\n9\nenhances anti-TB adaptive immune response, thus achieving sonodynamic TB immunotherapy.\"\nThe revised sentence: \"In summary, manSNI generates massive ROS under US irradiation to\neradicate intracellular M.tb and to promote the in situ release of M.tb-derived antigens. The ROS\nand R837 enhance macrophage polarization towards a pro-inflammatory phenotype, while the\nantigens and R837 synergistically drive DC activation, thus inducing enhanced innate and\nadaptive anti-TB immune response.\"\nThe original sentence: \"Additionally, it induced the release of BCG-derived antigens in situ and,\nin conjunction with the sonodynamic ROS and encapsulated TLR7 agonist R837, triggered potent\ninnate and adaptive anti-TB immune responses at the infectious site, thereby successfully\nreversing the immunosuppression of TBG.\"\nThe revised sentence: \"Additionally, the generate ROS can activate macrophages toward a\npro-inflammatory state in combination with of R837. Meanwhile, the in situ release of\nM.tb-derived antigens and R837 from macrophages could synergistically promote DC maturation.\nThese effects collectively trigger potent innate and adaptive anti-TB immune responses at the\ninfection site, effectively reversing the immunosuppressive microenvironment of TBG.\"\nQuestion 7: The number of independent experiments is low for several arms. This point should be\nclearly mentioned in the limitations, and the figure legends should clearly report N values.\nResponse: Thanks for your suggestion. In the revised manuscript, we have made the following\nrevisions:\n(1) Clarify N-value in all figure legends:\nComplete N-value information has been added to every figure legend to ensure transparency.\nFor example:\nFig.3 | Preparation and in vitro anti-BCG activity of manSNI. a, A schematic illustration\nshowing the preparation of manSNI. b, TEM image of manSNI. c, DLS of SN, manSN and manSNI. d,\nSurface Zeta potential of SN, manSN and manSNI. Data were presented as mean ± SD (n=3). e,\nUV-vis absorption spectra of SN, manSN and manSNI. f, Fluorescence emission spectra of SN,\nmanSN and manSNI. g, Detection of 1O generation measured by SOSG probe. Data were presented\n2\nas mean ± SD (n=3). h, i, Electron paramagnetic resonance spectra for manSN (h) and (i) manSNI\nwith or without US irradiation. j, Schematic diagram of the anti-BCG action mechanism of\nICG-mediated PTT and manSNI-mediated SDT. 5 cm thick pork tissue placed between the\nlight/ultrasonic instrument and the test tube containing M.tb (1.5 W cm-2, 1 MHz, 5 min). k,\nRepresentative images showing the survival BCG in the culture plate. l, Quantitative statistics of\nthe number of BCG colonies through standard plate counting assay. Data were presented as\nmean ± SD (n=3). m, SYTO 9/PI staining assay of BCG by PBS, SN, manSN and manSNI with and\n10\nwithout US irradiation. n, SEM images of BCG after incubation with PBS, SN, manSN and manSNI\nunder US irradiation.\n(2) Added a brief limitations description in the Conclusion section as your suggestion: A new\nsection has been included in the \"Conclusion\" part:\n\"Fourth, the relatively small sample sizes (n = 3-4 for in vitro assays, n = 6 for in vivo experiments)\nmay limit statistical power. Finally, the US devices used in small-animal studies differ\nsubstantially from clinical systems in parameter ranges and tissue compatibility. To further\nenhance the translational relevance of this strategy, future studies will involve collaborations with\naccredited laboratories to evaluate the efficacy of manSNI-mediated sono-immunotherapy against a\npanel of clinically relevant M.tb strains. Additionally, we plan to extend the preclinical assessment\nto large animal models, including guinea pigs and non-human primates, which more accurately\nrecapitulate the pathological characteristics of human TB. These studies will employ appropriately\nexpanded group sizes (n ≥ 10 per group) to ensure robust statistical power and will systematically\noptimize ultrasound parameters—including intensity, duration, and frequency—to align with the\noperational specifications of clinical ultrasound systems.\"\nQuestion 8: The manuscript uses both terms “sonodynamic therapy” (SDT) and\n“sonoimmunotherapy”. It can be confusing to the readers. Please clarify the difference and be\nconsistent.\nResponse: Thank you for your suggestion. In the revised manuscript, we have clarified the\ndefinitions of “sonodynamic therapy” (SDT) and “sono-immunotherapy” and have standardized\ntheir usage to avoid confusion.\nFirst, we added a clear explanation in the revised manuscript to distinguish the two terms:\n\"Sonodynamic therapy (SDT) is an emerging modality that employs ultrasound (US) to activate\nsonosensitizers for localized ROS generation.26 Unlike optical modalities, US offers superior\ntissue penetration (>10 cm vs. <3 cm for NIR light), enabling noninvasive treatment of\ndeep-seated lesions.27 Moreover, SDT ensures precise spatiotemporal control of treatment by\nadjusting the location, duration, and intensity of US irradiation without causing significant tissue\ndamage. It has also been reported that SDT can induce immunogenic cell death, enhance antigen\npresentation, and promote immune activation.28,29 As an extension of SDT, combining\nSDT-mediated pathogen killing with immune stimulation to elicit systemic anti-disease immunity\nis referred to as sono-immunotherapy.\"\nSecond, we unified all terminology in the manuscript. \"sono-immunotherapy\" is now\nconsistently used when describing the therapeutic strategy developed in this study. \"SDT\" is only\nused when referring specifically to the ultrasound-triggered ROS-generation mechanism.\nThe original article title: “Granulomas Immune Landscape-Guided Sonodynamic Tuberculosis\nImmunotherapy and Recurrence Prevention Using Organic Semiconducting\nNano-Immunostimulants”\nThe revised article title: “Granulomas Immune Landscape-Guided Tuberculosis\nSono-Immunotherapy and Recurrence Prevention Using Organic Semiconducting\nNano-Immunostimulants”\nThe original Fig. 2:\nFig.2 | Concept design of granulomas immune landscape-guided sonodynamic TB immunotherapy. a,\nSchematic diagram of the immunosuppressive landscape of ordinary TBG, drug-resistant and recurrent TBG\ncompared with NTBG. b, Evaluation of current TB treatments, including chemotherapy, immunotherapy, PTT and\nPDT, based on, anti-drug resistance, recurrence prevention, immunity activation, tissue penetration and biosafety.\nc, Schematic diagram of the structure of manSNI and action mechanism of manSNI-mediated sonodynamic TB\nimmunotherapy.\nFig.2 | Concept design of granulomas immune landscape-guided TB sono-immunotherapy. a, Schematic\ndiagram of the immunosuppressive landscape of ordinary TBG, drug-resistant and recurrent TBG compared with\nNTBG. b, Evaluation of current TB treatments, including chemotherapy, immunotherapy, PTT and PDT, based on,\nanti-drug resistance, recurrence prevention, immunity activation, tissue penetration and biosafety. c, Schematic\ndiagram of the structure of manSNI and action mechanism of manSNI-mediated TB sono-immunotherapy.\nThe original sentence: “Building on these insights, we developed an organic semiconducting\nnano-immunostimulants (manSNI) system equipped with mannose warhead for highly targeted and\nprecise sonodynamic TB immunotherapy and recurrence prevention.”\nThe revised sentence: “Based on the immunological landscape of TBG, we developed an organic\nsemiconducting nano-immunostimulants (manSNI) system equipped with mannose warhead for\nhighly precise TB sono-immunotherapy and recurrence prevention.”\nThe original sentence: “On day 7 post-treatment, lung tissues were collected from each group of\nmice to investigate the potential remodeling of the local immune microenvironment by\nmanSNI-mediated sonodynamic TB immunotherapy.”\nThe revised sentence: “On day 7 post-treatment, lung tissues were collected from each group of\nmice to investigate the potential remodeling of the local immune microenvironment by\nmanSNI-based sono-immunotherapy.”\nThe original sentence: “To comprehensively investigate the mechanism underlying\nmanSNI-mediated sonodynamic TB immunotherapy and further elucidate alterations in\ntranscriptomic profiles, immunofluorescence staining, detailed flow cytometry analysis, and\ncytokine level detection were performed.”\nThe revised sentence: “To comprehensively investigate the mechanism underlying\n13\nmanSNI-mediated TB sono-immunotherapy and elucidate alterations in transcriptomic profiles,\nimmunofluorescence staining, detailed flow cytometry analysis, and cytokine level detection were\nperformed.”\nQuestion 9: The limitations of the manuscript, and pathway to clinical translation should be\nclearly detailed.\nResponse：Thank you for your suggestions. In the revised manuscript, we have added a clear and\nbalanced discussion of the study’s limitations and the anticipated pathway toward clinical\ntranslation. The updated paragraph in the Conclusion section is as follows:\nDespite the promising findings, this study also has several limitations. First, due to biosafety\nconstraints, the anti-TB efficacy was validated using attenuated strains (BCG and H37Ra) rather\nthan virulent M.tb strains such as H37Rv strain or clinical isolates. Clinical M.tb strains exhibit\ndistinct pathogenicity, immune evasion mechanisms, and cell wall components, which may affect\nthe translatability of our findings. Second, the impact of distinct M.tb strain lineages (e.g. the\nBeijing family vs. non-Beijing strains) on the immune landscape of drug-resistant and\ndrug-sensitive patients was overlooked. Due to the lack of strain typing data in the GEO datasets,\nwe were unable to dissect whether strain-specific characteristics affect the immune differences\nbetween drug-resistant and drug-sensitive patients. Third, the mouse model used in our study may\nnot fully recapitulate the complexity of human TB, including granuloma structure and\nimmune-cell diversity. Fourth, the relatively small sample sizes (n = 3-4 for in vitro assays, n = 6\nfor in vivo experiments) may limit statistical power. Finally, the US devices used in small-animal\nstudies differ substantially from clinical systems in parameter ranges and tissue compatibility. To\nfurther enhance the translational relevance of this strategy, future studies will involve\ncollaborations with accredited laboratories to evaluate the efficacy of manSNI-mediated\nsono-immunotherapy against a panel of clinically relevant M.tb strains. Additionally, we plan to\nextend the preclinical assessment to large animal models, including guinea pigs and non-human\nprimates, which more accurately recapitulate the pathological characteristics of human TB. These\nstudies will employ appropriately expanded group sizes (n ≥ 10 per group) to ensure robust\nstatistical power and will systematically optimize ultrasound parameters—including intensity,\nduration, and frequency—to align with the operational specifications of clinical ultrasound\nsystems.\nRemarks to the Author: This interesting manuscript describes a novel therapeutic approach to\nmycobacterial infection using a construct design to target mannose receptors on macrophages and\nto deliver a payload that on ultrasound treatment generates ROS and releases a TLR7 agonist. The\nauthors argue that this strategy can overcome the ability of MTB to remain dormant and evade\nchemotherapy and host defenses. My main concern is the reliance on BCG as the organism for this\ninfection model. BCG generally does not become dormant or cause disease except under the most\nunusual circumstances (profound immunosuppression or overwhelming infection after therapeutic\nuse). I believe it is also true that BCG is substantially more susceptible to ROS than MTB (which\nis triggered by ROS to enter dormancy). I therefore am not at all sure that an acute treatment\nmodel using BCG followed by a very short course of anti-TB drugs is a good model in which to\ntest this concept.\nResponse: We thank the reviewers for raising this important point and fully agree that our\nfindings should not be generalized to Mycobacterium tuberculosis (M.tb) without proper\nclarification. We also recognize the biological differences between BCG and M.tb. To address this\nconcern, we have made the following revisions and additions to the revised manuscript:\n(1) Clarified the rationale for using BCG:\nIn the revised introduction, we now emphasize that BCG is an internationally recognized\npreliminary screening model for anti-tuberculosis drugs and therapies[3] because it shares more\nthan 90% genomic homology with M.tb[3].\n(2) Added validation using an attenuated M.tb strain:\nTo strengthen the relevance of our findings, we have supplemented the study with in vitro\nand intracellular bactericidal activities against the attenuated strain Mycobacterium tuberculosis\nH37Ra. These added data showed below confirm that SNI-mediated sonodynamic therapy also\nretains potent bactericidal activity against M.tb.\nRevisions added to the revised manuscript：\nEncouraged by the superior ROS generation ability of manSNI under US irradiation, the anti-M.tb\nactivity of manSNI was then investigated. Both the Bacillus Calmette-Guérin (BCG, which shares a\nhigh degree of genomic homology with M.tb and is an internationally recognized preliminary\nscreening model for anti-TB drugs/therapies39) and the attenuated strain of M.tb H37Ra\n(ATCC25177) were used as the model bacteria. BCG, Firstly, the colony-forming units (CFU)\nassay revealed that these semiconducting nanoparticles (SN, manSN, and manSNI) were not toxic to\nBCG and H37Ra (Supplementary Fig. 19) in the absence of US irradiation. While, even when\nisolated by a 5 cm thick pork tissue pad (Fig. 3j), the combination of these nanoparticles and US\nirradiation (1.5 W/cm2, 1 MHz, 5 min) resulted in a bacterial death rate exceeding 99% (Fig. 3k, l).\nnear-infrared photosensitizer represented by ICG cannot kill BCG when separated by a 5 cm thick\nlayer of pork tissue (Supplementary Fig. 20). Similar, the live/dead bacterial viability assay\ndemonstrated that manSNI exhibited excellent efficiency in killing BCG and H37Ra (Fig. 3m and\nSupplementary Fig. 21) under US irradiation. Then, the structural changes in BCG and H37Ra\nafter different treatments were examined. As shown in scanning electron microscopy (SEM)\nimages, the combination of these nanoparticles and US irradiation caused extensive damage to the\nbody structure of BCG and H37Ra (Fig. 3n), resulting in an overall shriveled and concave shape\nof bacteria. The disruption of bacterial structure resulted in the leakage of cellular contents as\nevidenced by protein leakage high to 0.22 μg/mL in solution from bacterial (107 CFU/mL)\n(Supplementary Fig. 22), which are expected to activate the body’s immune response as pathogen\nantigens. These results demonstrate that manSNI can kill M.tb by generating a large amount of\nROS through sonodynamic effect.\nFig.3 | Preparation and in vitro anti-M.tb activity of manSNI. j, Schematic diagram of the anti-M.tb action\nmechanism of ICG-mediated PTT and manSNI-mediated SDT. 5 cm thick pork tissue placed between the\nlight/ultrasonic instrument and the test tube containing M.tb (1.5 W cm-2, 1 MHz, 5 min). k, Representative images\nshowing the survival BCG and H37Ra in the culture plate. l, Quantitative statistics of the number of BCG colonies\nand H37Ra colonies through standard plate counting assay. Data were presented as mean ± SD (n=3). m, SYTO\n9/PI staining assay of BCG and H37Ra after treated with PBS, SN, manSN and manSNI under US irradiation. n,\nSEM images of BCG and H37Ra after treated with PBS, SN, manSN and manSNI under US irradiation.\n16\nk\n6\n0\n)601×(\nUFC\nj\nPBS SN manSN manSNI\nl m n\nPBS SN manSN manSNI PBS SN manSN manSNI\nBCG H37Ra\n5\n4\n3\n2\nGCB\naR73H\nGCB\naR73H\n+US\nLight 808 nm US\nPork tissue,thickness 5 cm Membrane rupture\nROS\nROS\nPathogen antigens\nLive Mtb Dead M.tb\n+US +US\n50 μm 1 μm\n1 μm\nGCB\naR73H\n50 μm\nGCB\naR73H\nNo US\nPBS SN manSN manSNI 6\n0\n)601×(\nUFC\na b\nBCG H37Ra\n5\n4\n3\n2\nSupplementary Fig. 19. a, Representative images showing the survival BCG and H37Ra in the\nculture plate. b, Quantitative statistics of the number of BCG and H37Ra colonies through\nstandard plate counting assay. Data were presented as mean ± SD (n=3).\nPBS SN manSN\nSupplementary Fig. 21. a, SYTO 9/PI staining assay of BCG and H37Ra after different\ntreatments. b, c, fluorescence quantification of PI in BCG (b) and H37Ra (c) after different\ntreatments.\nSupplementary Fig. 22. a,b, Protein leakage from BCG (a) and H37Ra (b) after different\ntreatments.\nThe intracellular anti-M.tb efficacy was subsequently investigated. At a US time of 15 min and\nUS intensity of 1.5 W/cm2, manSNI (SN concentration: 100 μg/mL) killed over 99% of intracellular\nBCG (Supplementary Fig. 23) and H37Ra (Supplementary Fig. 24) with no significant decrease in\nthe viability of Raw264.7 cells (Supplementary Fig. 27). In contrast, SN and manSN exhibited only\n~49.3% and ~66.9% killing efficiency of intracellular BCG and ~49.3% and ~66.9% killing\nefficiency of intracellular H37Ra under the same irradiation conditions, respectively (Fig. 4e-f).\nTherefore, manSNI can efficiently and selectively kill intracellular M.tb but not macrophages under\nthe optimized US conditions (Supplementary Fig. 25).\n17\n).u.a(\nLF\nb\n200 -US\n+US P<0.0001 P<0.0001\n150\n100\n50\n50 μm 0\nmanSNI\nGCB\naR73H\na No US\nPBS SN manSN manSNI\n50 μm\n).u.a(\nLF\nc\nBCG H37Ra\n100 -US\nP<0.0001 P<0.0001 80 +US\n60\n40\n20\nPBS SN\nnoitartnecnoc\nnietorP\n)Lm/gμ(\n0.3 -US\nP<0.0001 P<0.0001\n+US\n0.2\n0.1\n0\nmanSN manSNI PBS SN\nnoitartnecnoc\nnietorP\n)Lm/gμ(\na b\nBCG H37Ra\n0.3 -US P<0.0001 P<0.0001\n+US\n0.2\n0.1\n0\nmanSN manSNI\nSupplementary Fig. 24. a, b, Colony photographs (a) and CFU counting (b) of H37Ra at\ndifferent US times and US intensities.\n(3) Expanded discussion on limitations and clinical translation:\nWe have added a discussion on the limitations and clinical translation in the conclusion\nsection: we will clearly state the model limitations, point out that there are essential differences in\npathogenicity and dormancy characteristics between BCG and human-derived M.tb, and note that\nthe results of this study need to be further verified in human-derived M.tb models. Subsequent\nstudies will use M.tb standard strain (H37Rv) to verify the eradication efficacy of this strategy\nagainst M.tb.\nRevisions added to the revised manuscript：\nConclusion\nDespite the promising findings, this study also has several limitations. First, due to biosafety\nconstraints, the anti-TB efficacy was validated using attenuated strains (BCG and H37Ra) rather\nthan virulent M.tb strains such as H37Rv strain or clinical isolates. Clinical M.tb strains exhibit\ndistinct pathogenicity, immune evasion mechanisms, and cell wall components, which may affect\nthe translatability of our findings. Second, the impact of distinct M.tb strain lineages (e.g. the\nBeijing family vs. non-Beijing strains) on the immune landscape of drug-resistant and\ndrug-sensitive patients was overlooked. Due to the lack of strain typing data in the GEO datasets,\nwe were unable to dissect whether strain-specific characteristics affect the immune differences\nSU+\nPBS SN manSN manSNI\n)301×(\nUFC\na\nb\nPBS SN\n250\nmanSN manSNI\n200\n100\n50\n0\n-US +US\nbetween drug-resistant and drug-sensitive patients. Third, the mouse model used in our study may\nnot fully recapitulate the complexity of human TB, including granuloma structure and\nimmune-cell diversity. Fourth, the relatively small sample sizes (n = 3-4 for in vitro assays, n = 6\nfor in vivo experiments) may limit statistical power. Finally, the US devices used in small-animal\nstudies differ substantially from clinical systems in parameter ranges and tissue compatibility. To\nfurther enhance the translational relevance of this strategy, future studies will involve\ncollaborations with accredited laboratories to evaluate the efficacy of manSNI-mediated\nsono-immunotherapy against a panel of clinically relevant M.tb strains. Additionally, we plan to\nextend the preclinical assessment to large animal models, including guinea pigs and non-human\nprimates, which more accurately recapitulate the pathological characteristics of human TB. These\nstudies will employ appropriately expanded group sizes (n ≥ 10 per group) to ensure robust\nstatistical power and will systematically optimize ultrasound parameters—including intensity,\nduration, and frequency—to align with the operational specifications of clinical ultrasound\nsystems.\nMinor concerns：\nQuestion 1: Line 176-7. It is surprising that the immune profiles of drug-resistant cases differ\nfrom drug sensitive. Are the authors sure this was not the result of infection with different MTB\nfamilies (Beijing vs non-Beijing), or the effect of drug treatment (BDQ+pretomanid vs rifampin)?\nResponse: Thanks for your valuable comment. Regarding the immune profiles of tuberculosis\n(TB), we referred to the study by Heyckendorf et al. (doi.org/10.1183/13993003.03492-2020;\nRNA data publicly available in the Gene Expression Omnibus database under accession number\nGSE147689) and obtained the RNA sequencing data from this work. Although the study did not\nspecify the classification of TB strains (e.g., Beijing family vs. non-Beijing family), it clearly\nstated that the samples were collected from patients receiving different treatment regimens.\nHeyckendorf et al. included both drug-susceptible TB (DS-TB) and multidrug-resistant TB\n(MDR-TB) patients in their cohort: “Anti-TB therapy regimens were based on comprehensive\ndrug-susceptibility testing and consistent with current therapy recommendations [5, 17, 18].\nTreatment outcomes were assessed following the TB net definitions, where relapse-free cure is\ndefined by having a negative M. tuberculosis culture status at 6 months after treatment initiation\nwithout positive cultures thereafter and no disease recurrence during the follow-up period of 1\nyear after therapy end [14].” Below are the clinical characteristics of TB patients in the\ndoi.org/10.1183/13993003.03492-2020 (Figure R1).\ndoi.org/10.1183/13993003.03492-2020.\nIn references [18] (https://doi.org/10.1055/s-0043-105954) in Heyckendorf´s work, the anti-TB\ntreatment regimen is based on comprehensive drug susceptibility testing and is administered\naccording to WHO, German Central Committee against Tuberculosis (DZK), German Respiratory\nSociety (DGP) for TB treatment guidelines and recommendations. Below are the relevant table\ndata (Figure R2-R4) in the references 18:\nFigure R2. Treatment recommendations for adult tuberculosis patients.\nhttps://doi.org/10.1055/s-0043-105954.\nFigure R3. Doses of drugs in standard treatment. https://doi.org/10.1055/s-0043-105954.\nFigure R4. Multidrug resistance treatment recommendations according to WHO.\nhttps://doi.org/10.1055/s-0043-105954.\nMDR-TB patients during model development, incorporating \"drug resistance status\" as a key\ncovariate into the algorithm. Be examining their results in detail, we found that compared with\ndrug-susceptible patients, those with drug-resistant TB exhibited stronger immune suppression,\nparticularly the downregulation of Toll-like receptor (TLR) and NF-κB signaling pathways in\nantigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs). From an\nimmunological perspective, this impaired immune response may reduce the ability to clear\nMycobacterium tuberculosis (M.tb), leading to persistently high bacterial loads. This increases the\nlikelihood of resistance mutations emerging under antibiotic selection pressure. Both the study by\nHeyckendorf et al. and our additional analyses demonstrate that this distinct pattern of immune\nsuppression may be a critical host factor driving the development of clinical drug-resistant\nphenotypes, rather than simply a consequence of different strains or treatment regimens.\nRegarding the impact of drug therapy, from the perspective of direct drug action, anti-TB drugs\nprimarily work by directly killing or inhibiting bacteria rather than regulating host immunity. For\nexample, rifampicin blocks transcription by inhibiting bacterial RNA polymerase, isoniazid\ndisrupts the cell wall by suppressing mycolic acid synthesis, and bedaquiline depletes bacterial\nenergy by targeting mycobacterial ATP synthase. These drugs act on bacteria-specific biochemical\npathways and are designed to maximize M.tb killing while minimizing direct effects on host cells.\nUnlike glucocorticoids or immunosuppressants, which directly modulate immune responses by\nacting on host immune signaling pathways, traditional anti-TB chemotherapeutic agents do not\ndirectly target host immune systems. Therefore, drug driven immunomodulation is unlikely to be a\nkey factor explaining differences between drug resistant and drug susceptible TB patients. Instead,\ninherent or early infection-established immune defects in patients may by limiting the initial\nclearance of M.tb, weakening antibiotic efficacy, and promoting the development of TB drug\nresistance. Our study specifically analyzes the critical role of host immune factors in the treatment\nof DS-TB and MDR-TB patients.\nFurthermore, we fully agree that different M.tb strains (e.g., the Beijing family) may possess\ndistinct immunomodulatory properties. However, the study and database by Heyckendorf et al. did\nnot systematically investigate the impact of M.tb strain lineages on host transcriptomic profiles,\nand strain-specific information is not available for the data we used. Thus, the lack of such\nanalysis represents a limitation of our current study. Nevertheless, the \"immune suppression\nsignature\" we identified reflects a comprehensive host response state. Even if different strains\nexhibit varying virulence, inducing or exploiting this host immune suppression state is likely one\nof the common pathways ultimately leading to clinical drug-resistant phenotypes. In future studies,\nwe will attempt to incorporate strain typing to further analyze the characteristics of host-pathogen\n21\ninteractions. We will discuss this limitation related to strain lineage analysis in the conclusion\nsection.\nIn summary, our analysis of peripheral blood from DS-TB and MDR-TB patients in the\ndatabase reveals more pronounced immune suppression signatures, which may reflect an inherent\nimmune susceptibility that predisposes individuals to the development of drug resistance. This\nfinding provides a new perspective for understanding the host mechanisms underlying TB drug\nresistance. Additionally, we have added discussion of different drug treatments and strain lineages\nas potential confounding factors in the manuscript, highlighting their importance for baseline\nimmune characteristics.\nQuestion 2: In multiple lines, the authors refer to \"eliminating M.tb\" or \"TB relapse\" or \"anti-TB\nactivity of SNI\". I am concerned demonstrating activity against BCG does not ensure activity\nagainst MTB.\nResponse: Thank you for your suggestions. We supplemented the investigations on the in vitro\nand intracellular bactericidal activities against the attenuated strain Mycobacterium tuberculosis\nH37Ra to more accurately evaluate the bactericidal effect of SNI-mediated sono-immunotherapy\non Mycobacterium tuberculosis (As shown in Response 1). Meanwhile, BCG is an internationally\nrecognized preliminary screening model for anti-tuberculosis drugs/therapies since it shares > 90%\ngenomic homology with M.tb. Many researches utilize animals infected with BCG to establish\nBCG-based tuberculous granulomas for drug screening[1-2]. Therefore, the BCG animal model is a\nhighly suitable tuberculosis (TB) model, which can to a certain extent reflect the anti-TB efficacy\nof our treatment strategy. Meanwhile, in accordance with your suggestions, to ensure precise\nexpression, the relevant experimental descriptions involving BCG that were previously stated as\n\"eliminating M.tb\" have been revised to \"eliminating BCG\". For example:\nThe original sentence: \"In the aforementioned treatment model, we have demonstrated that\nmanSNI+US irradiation treatment exhibits remarkable therapeutic efficacy in eliminating M.tb and\ncan effectively activate adaptive immune responses, particularly immune memory.\"\nThe revised sentence: \"In the aforementioned treatment model, we have demonstrated that\nmanSNI+US irradiation treatment exhibits remarkable therapeutic efficacy in eliminating BCG and\ncan effectively activate adaptive immune responses, particularly immune memory.\"\nThe original sentence: \"The high recurrence rate, which includes endogenous relapse and\nexogenous reinfection, has consistently posed a significant challenge in the treatment of TB.50-52\nIn the aforementioned treatment model, we have demonstrated that manSNI+US irradiation\ntreatment exhibits remarkable therapeutic efficacy in eliminating M.tb and can effectively activate\nadaptive immune responses, particularly immune memory.\"\nexogenous reinfection, has consistently posed a significant challenge in the treatment of TB.50-52\nIn the aforementioned treatment model, we have demonstrated that manSNI+US irradiation\ntreatment exhibits remarkable therapeutic efficacy in eliminating BCG and can effectively activate\nadaptive immune responses, particularly immune memory.\"\nReferences\n1. Padilla-Carlin DJ, McMurray DN, Hickey AJ. The Guinea pig as a model of infectious diseases.\nComp Med. 2008;58(4):324–40.\n2. Li, H., Li, H. Animal Models of Tuberculosis. In: Christodoulides, M. (eds) Vaccines for\nNeglected Pathogens: Strategies, Achievements and Challenges. Springer, Cham. 2023.\nhttps://doi.org/10.1007/978-3-031-24355-4_7.\n23"}, {"round": 2, "reviewer_comments": {"reviewer_3": "I am satisfied with the authors response, particularly with respect to ultrasound probe calibration. I have no further comments.", "reviewer_4": "I believe this revised manuscript addresses the concerns raised in the first review."}, "author_response": ""}]

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