Abstract
Introduction:
Tuberculosis (TB) continues to be one of the leading infectious causes of mortality world-wide, while the Bacillus Calmette–Guérin (BCG) vaccine provides variable protection. To address this limitation, our group developed a recombinant BCG strain expressing a detoxified Escherichia coli heat-labile toxin subunit (rBCG-LTAK63), which confers superior protection against Mycobacterium tuberculosis (Mtb). However, the mechanisms underlying this enhanced efficacy remain to be better characterized. Here, we investigated the capacity of rBCG-LTAK63 to enhance inflammasome-associated innate immune responses and its impact on T cell activation and protection against Mtb.
Methods:
Bone marrow-derived macrophages (BMDMs) from wild-type (C57Bl/6), Casp-1-/-, Nlrp3-/-, Aim-2-/- and phenotypically selected AIRmax and AIRminTT mice were used to evaluate inflammasome-associated responses using IL-1β as a primary readout. A co-culture system of inflammasome-activated BMDMs and splenocytes was employed to assess CD4+ T cell activation and polarization. Additionally, immunization of AIRmax and AIRminTT mice with BCG or rBCG-LTAK63 was performed to evaluate protection against Mtb.
Results:
rBCG-LTAK63 induced significantly higher IL-1β production than parental BCG. IL-1β production was largely ASC-associated and predominantly dependent on the NLRP3/caspase-1 axis; however, residual IL-1β production was still detected in Casp-/- and Nlrp3-/- BMDMs, indicating the contribution of additional processing pathways. Co-culture of inflammasome-activated BMDMs and splenocytes showed that rBCG-LTAK63-primed macrophages strongly promoted CD4+ T cell activation and polarization toward Th1/Th17 responses. In vivo, BCG only induced protection in AIRmax mice, while rBCG-LTAK63 induced protection in both AIRmax and AIRminTT genotypes.
Conclusion:
This demonstrates that protection is achieved in a diminished innate inflammatory response scenario, but its full engagement can further reduce pulmonary bacterial loads. Together, these findings demonstrate that rBCG-LTAK63 enhances protection against TB through a broad innate activation including inflammasome-associated mechanisms.
1 Introduction
Tuberculosis (TB) remains the leading cause of death from a single infectious agent worldwide. In 2023, according to the World Health Organization (WHO), TB was responsible for approximately 1.5 million deaths. The Bacillus Calmette–Guérin (BCG) has been the only WHO-licensed vaccine against TB for over a century (1). However, several studies have shown that BCG confers variable protection in adults. Consequently, there is an urgent need to develop more effective vaccines (2).
Successful vaccines engage innate immune pathways through the use of adjuvants to promote the development of an effective adaptive immunity. Inflammasomes are cytosolic multiprotein complexes that play a critical role in innate immunity by detecting danger signals and initiating inflammatory responses (3). Their activation is initiated by pattern recognition receptors (PRRs) that detect pathogen- (PAMPs) or damage-associated molecular patterns (DAMPs). Once activated, inflammasomes trigger the cleavage of pro-caspase-1 into its active form, leading to the maturation and release of IL-1β and IL-18, which orchestrate the early immune response against invading pathogens (4).
IL-1β is a central proinflammatory cytokine. It enhances dendritic cell activation and co-stimulation, boosts antigen presentation and cross-priming, recruits and activates neutrophils, and, together with IL-6/IL-23/TGF-β, drives RORγt in naïve T cells guiding them to Th17 differentiation. All together innate-derived cytokines, costimulatory signals, and the dose/duration of antigen presentation guide the quality of the response (e.g., Th1/Th2/Th17 polarization) (5).
Recent studies have highlighted the relevance of inflammasome activation in host defense against mycobacterial infections and in the protective efficacy of TB vaccines (6–11). One promising approach in TB vaccine development is the use of recombinant BCG strains expressing adjuvant molecules that can enhance the innate immune response and improve adaptive immune response (12, 13). Our group developed a recombinant BCG strain, rBCG-LTAK63 (rBCGΔlysA::lysA-ltak63), that uses auxotrophic complementation to stably express the genetically detoxified A subunit of Escherichia coli heat-labile toxin (14). Previous studies demonstrated that immunization with rBCG-LTAK63 provided enhanced protection against Mycobacterium tuberculosis (Mtb) H37Rv challenge compared to the parental BCG strain (15), likely due to the induction of a stronger inflammatory response (16). More recently, we showed that rBCG-LTAK63 elicits a Th1/Th17 adaptive immune response, increased memory T cell populations and long-term protection (up to 180 days after immunization) (17). Moreover, innate immune responses induced by rBCG-LTAK63 are also distinct from those generated in response to wild-type BCG. Increased numbers of macrophages and neutrophils are seen after the intraperitoneal immunization of mice with rBCG-LTAK63 (18). Stimulation of human macrophages with rBCG-LTAK63 resulted in an increased inflammatory response (19). However, the mechanisms through which LTAK63 expression in BCG augments the Th1/Th17 response are not completely understood.
Recombinant forms of the E. coli heat-labile enterotoxin are reported to activate antigen-presenting cells (APCs) and upregulate surface markers such as MHC-II, CD40, CD86, and promote the production of IL-1β (20). Based on these observations, we hypothesized that rBCG-LTAK63 enhances innate immune signaling, including inflammasome-associated pathways, thereby contributing to improved vaccine efficacy. In the present study, we investigated the capacity of rBCG-LTAK63 to induce inflammasome-associated responses and evaluated how these innate mechanisms relate to T cell activation and protection against M. tuberculosis.
2 Materials and methods
2.1 Ethical statement and mice
All animal procedures were approved by the Ethics Committee on Animal Use of the Butantan Institute (CEUA/IB protocol 2012250722) and conducted in compliance with the Brazilian National Council for Animal Experimentation Control (CONCEA) guidelines. Female C57Bl/6 mice (4–8 weeks old) were obtained from the Butantan Institute’s Central Animal Facility. Knockout strains, Casp-1-/-, Nlpr3-/-, and Aim-2-/- (C57Bl/6 background) were kindly provided by Dr. Sérgio Costa Oliveira (University of São Paulo). AIRmax and AIRminTT mice are bred under standardized conditions at the Laboratório de Imunogenética (21). Mice were housed in the Laboratório de Desenvolvimento de Vacinas facility with ad libitum access to food and water. Environmental conditions were maintained at 20–24 °C, 40–70% relative humidity, and a 12-h light/dark cycle.
2.2 Bacterial strains and culture conditions
The recombinant rBCG-LTAK63 strain (ΔlysA::lysA-ltak63), generated from BCG Danish as previously described (14) and control strain (BCG Danish ATCC 35733, American Type Culture Collection, Rockville, MD, USA) were cultured in Middlebrook 7H9 (BD Difco™, Detroit, MI, USA) medium supplemented with 10% OADC (oleic acid–albumin–dextrose–catalase; BD BBL, Cockeysville, MD, USA), 0.05% Tween 80 (Sigma-Aldrich®, Merck KGaA, St. Louis, MO, USA), and 0.5% glycerol (Sigma-Aldrich) at 37 °C with 5% CO2 for 7 days. Cultures were expanded to 50 mL and grown to mid-log phase (OD600 = 0.8). Bacterial cells were harvested by centrifugation at 3,180 × g, washed twice with ice-cold 10% glycerol, and resuspended in 1 mL of the same solution. Aliquots (100 µL) were cryopreserved at -80 °C for long-term storage. Post-preservation viability was determined by colony-forming unit (CFU) counts. Serial dilutions were plated on Middlebrook 7H10 (BD Difco™) agar supplemented with 10% OADC (BD BBL™) and incubated for 21 days at 37 °C with 5% CO2.
2.3 RNA-seq
The dataset is deposited in GEO under accession GSE278523, and the corresponding raw sequencing data are available in NCBI SRA under BioProject PRJNA1159865. BALB/c mice were immunized with rBCG-LTAK63 or BCG (106 CFU, s.c.) and lymph node samples collected at 7 days post-immunization. Gene expression was analyzed using EdgeR (22). Raw gene counts were normalized using the trimmed mean of M-values (TMM) method to correct for library size variations. Differentially expressed genes (DEGs) were identified using a false discovery rate (FDR) threshold of < 0.05, with multiple testing correction applied via the Benjamini-Hochberg method. To investigate functional relationships among DEGs, protein-protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database v11.5, applying a medium confidence interaction score (≥ 0.4). Subsequently, KEGG pathway enrichment analysis was conducted to identify significantly overrepresented biological pathways within the DEG dataset, with statistical significance defined as FDR < 0.05.
2.4 Isolation and differentiation of murine bone marrow macrophages
C57Bl/6, Casp-1-/-, Nlrp3-/-, Aim-2-/-, AIRmax, and AIRminTT mice were euthanized with an overdose of anesthetic (300 mg/kg ketamine + 30 mg/kg xylazine) by intraperitoneal route, and tibias and femurs were aseptically collected. Bone marrow cells were flushed from the bones using RPMI-1640 medium (Gibco, Life Technologies, Paisley, UK) and a 10 mL disposable syringe (BD Plastipak™) with a 25G needle (BD PrecisionGlide™). The resulting cell suspension was centrifuged at 200 × g for 10 min at 4 °C. The pellet was resuspended in complete R10 medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich®)), an antibiotic solution (penicillin 100 U/mL, streptomycin 100 µg/mL), and 30% L929-conditioned medium as a source of macrophage colony-stimulating factor (M-CSF). L929 cells were originally obtained from ATCC. Cells were seeded into 6-well plates (Corning®, NY, USA) and incubated at 37 °C with 5% CO2 for 6 days, with half of the medium replaced on day 4. On day 6, non-adherent cells were removed by washing twice with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.2), and adherent cells were detached using a cell scraper (KASVI, Pinhais, PR, Brazil) in ice-cold RPMI-1640. Cells were centrifuged at 200 × g for 10 min and resuspended in complete R10 medium.
Cell viability was assessed by Trypan Blue (Sigma-Aldrich®) exclusion, and viable cells were counted using a Neubauer chamber. Macrophages were adjusted to 1 × 106 cells/mL and seeded into 96-well plates (100 µL/well) for subsequent experiments. An aliquot was stained to confirm differentiation to BMDM by flow cytometry using anti-mouse CD11b conjugated to PE (BD Biosciences, San Diego, CA, USA) and anti-mouse F4/80 conjugated to Bv421 (BioLegend, San Diego, CA, USA).
2.5 Inflammasome activation assay
BMDMs (1 × 105 cells/well) were plated in 96-well plates (Corning®) and either primed with LPS (500 ng/mL, Sigma-Aldrich®) for 4 h or left unstimulated, followed by infection with BCG or rBCG-LTAK63 strains at a multiplicity of infection (MOI) 10:1 for 6 h. Appropriate positive controls were included for each experiment: Nigericin (20 μM, 40 min) or ATP (5 mM, 30 min; both from Sigma-Aldrich®) for NLRP3 inflammasome activation. Methodologically, replacing Nigericin with ATP enhanced experimental consistency. Culture supernatants were collected and stored at -80 °C until use.
2.6 In vivo immunization and ex vivo co-culture assay
Female C57Bl/6 mice (5–7 weeks old) were randomly divided into three groups (n=5/group) receiving either: Saline (100 µL, subcutaneous (s.c.)), BCG Danish (100 µL, 106 CFU, s.c.), or rBCG-LTAK63 (100 µL, 106 CFU, s.c.). Thirty days later, spleens were aseptically collected for co-culture assays. Splenocytes were isolated by mechanical dissociation (Corning, Pyrex® Ten Broeck Homogenizer), erythrocytes lysed by incubation with sterile ultrapure water for 10 s, and resuspended in complete R10 medium after centrifugation (200 × g, 10 min, 4 °C). Cell viability was assessed by trypan blue exclusion (Neubauer chamber), adjusting to 1 × 106 cells/mL.
For co-culture, after differentiation BMDMs were seeded in 48-well culture plates and primed with LPS (500 ng/mL, 4 h) or left unprimed, then infected with BCG or rBCG-LTAK63 (MOI 10:1, 6 h). Controls included: unstimulated BMDMs (negative) and nigericin (48 µM, 40 min). After several PBS washes, BMDMs (2 × 105/well) were co-cultured with CD28-stimulated splenocytes (2 × 106/well; anti-CD28 1 µg/mL, 4 h) in 48-well plates (Corning®). Monensin (3 µM) was added after 48 h, and the cultures were maintained for an additional 12 h prior to analysis.
2.7 Immunization and challenge in AIRmax and AIRminTT
Female AIRmax and AIRminTT mice (5–7 weeks old) were randomly divided into three groups receiving either: Saline (100 µL, subcutaneous (s.c.)), BCG Danish (100 µL, 106 CFU, s.c.), or rBCG-LTAK63 (100 µL, 106 CFU, s.c.). Thirty days later, aliquots of Mtb (H37Rv) maintained at –80 °C were thawed, and the concentration adjusted to 1.25 x 104 CFU/mL using saline. Mice under mild anesthesia were instilled with 40 µL (500 CFU) into the nostrils with aid of a micropipette. Thirty days after challenge, mice were euthanized and lungs collected. For CFU counts, the cranial and median lobes of the lungs were homogenized, diluted in PBS and plated on Middlebrook 7H10 agar (BD Difco™), supplemented with 0.5% glycerol (Sigma-Aldrich®), 10% OADC (BD BBL™), 5 µg/ml of TCH (2-thiophenecarboxylic acid hydrazide, Sigma-Aldrich®, a BCG growth inhibitor) and incubated at 37 °C and 5% CO2 for up to 3 weeks to evaluate the bacterial load.
2.8 Quantification cytokine production
Samples were thawed and assayed for IL-1β by ELISA using the DuoSet® Mouse IL-1β/IL-1F2 (R&D Systems, Inc., Minneapolis, MN, USA). For inflammation-related cytokines (IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70) a Cytometric Beads Array (CBA mouse inflammatory kit, BD Biosciences) was used according to the manufacturer’s instructions. The assay lower limits of detection were between 2.5 and 17.5 pg/mL, depending on the cytokine, and the higher limit was 5,000 pg/mL. Data were acquired using FACS Canto II equipment and analyzed using FCAP Array Software (BD Biosciences).
2.9 Flow cytometry
For immunophenotyping of co-culture cells, the following extracellular markers were used: BV510-CD3 (clone: 145-2C11, BD Horizon™), FITC-CD4 (clone: GK1.5, BD Pharmingen™), PerCP-CD69 (clone: H1.2F3, BD Pharmingen™), and APC-Cy7-CD44 (clone: IM7, BioLegend). Cells were incubated with antibodies for 30 min, washed with PBS, and fixed/permeabilized using Fix/Perm Buffer (BD Biosciences™). For intracellular staining, the following markers were used: PE-IFN-γ (clone: XMG1.2, BD Pharmingen™), and BV421-IL-17 (clone: TC11-18H10, BD Horizon™). Cells were incubated with intracellular markers for 30 min, followed by washes with Perm Wash buffer (BD Biosciences™). All analyses were performed by acquiring 100,000 events on a FACSCanto II flow cytometer. Data were analyzed using FlowJo v10. Lymphocyte selection was based on size (FSC) and granularity (SSC) characteristics. CD4+ were gated to evaluate T cell activation markers (CD44+ and CD69+) and Th1/Th17-related intracellular cytokines (IFN-γ and IL-17). Gating strategy is depicted in Supplementary Figure S1.
2.10 Statistical analysis
Data was analyzed using GraphPad Prism 8.0 (GraphPad Software) and expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Multiple group comparisons were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. p values < 0.05 were considered statistically significant. The experimental design figures were created with http://biorender.com.
3 Results
3.1 rBCG-LTAK63 upregulates the transcriptional signature of an early inflammatory response in mouse lymph nodes
We analyzed the transcriptomic profile of differentially expressed genes (DEGs) from the draining lymph nodes of mice at seven days post-immunization, comparing rBCG-LTAK63 to BCG using STRING analysis. A cluster of upregulated genes was revealed, indicating the transcriptional signature linked to early immune activation (Fosb, Fos, Atf3, Ptgs2), cellular stress (Hspa1a, Hspa1b, Dnajb1) and leukocyte recruitment (Cxcl1, Cxcl2) (Figure 1A). The protein-protein interaction map (Figure 1B) shows two main hubs. A chaperone-centered hub was revealed, which indicates an active and highly stressed environment, involving core (Hspa1a, Hspa1b, Hspa8), assistant co-chaperones (Dnaja4, Dnajb13, Dnajb4, Dnajb1) and other important members (Hsp90a1, Hsph1). The other hub reinforces this active immune environment linking transcription factors (Fos, Jun, Junb, Klf4, Klf6), chemokines (Cxcl1, Cxcl2), stress (Atf4, Hspa1a, Hspa1b) and inflammation resolution (Dusp1, Atf3) (Figure 1B).
Figure 1
The cell specific enrichment analysis of these genes (using human orthologues) points to expression of these genes primarily in macrophages (Figure 1C). The lack of direct up-regulation of IL-1 or core inflammasome genes may reflect the cellular composition of the lymph nodes, mainly T and B cells or even the time-point used (7 days after immunization). Pathway enrichment analysis (Figure 1D) demonstrated strong activation of key immune and inflammatory pathways, including IL-17 (mmu04657), TNF (mmu04668) and NK-κB signaling (mmu04064), antigen processing and presentation (mmu04612) and NOD-like receptor signaling (mmu04621). This molecular signature suggests a coordinated engagement of innate and adaptive immune responses, supported by an intense cellular activity demonstrated by the enrichment of Protein processing in endoplasmic reticulum (mmu04141), MAPK signaling (mmu04010) and apoptosis (mmu04210) pathways. Interestingly, the induction of pathogen-response pathways (mmu05145: Toxoplasmosis, mmu05134: Legionellosis, mmu05162: Measles) indicates broad-spectrum immune priming against intracellular pathogens.
Collectively the transcriptional analysis indicates that rBCG-LTAK63 promotes a potent and early immune response in comparison to BCG and points to macrophages as the main responsible for this response.
3.2 Inflammasome activation is a key innate immune mechanism triggered by the rBCG-LTAK63 vaccine
To characterize the contribution of inflammasome-associated pathways to the immune response induced by rBCG-LTAK63, we combined in vitro assays using murine macrophages with in vivo infection models. These analyses were performed in wild-type mice, in knockout strains lacking key inflammasome components, and in AIRmax and AIRminTT mouse lines, selected for maximal and minimal inflammatory responsiveness including inflammasome-associated responses, respectively. Bacterial burden was assessed in these models through CFU counts (Figure 2).
Figure 2
To evaluate inflammasome activation by rBCG-LTAK63, we measured IL-1β concentration in the supernatants of BMDMs according to standard protocols (23). Our results demonstrate that only rBCG-LTAK63 was able to induce detectable levels of IL-1β in the supernatant of non-primed cells (Figure 3A). On the other hand, when the BMDMs were primed with LPS, BCG induced IL-1β expression as compared to unstimulated cells, while rBCG-LTAK63 induced even higher levels (Figure 3B).
Figure 3
3.3 rBCG-LTAK63 induces IL-1β secretion through inflammasome-associated pathways
In order to identify the inflammasome pathway activated by rBCG-LTAK63 we used inflammasome-deficient macrophages. In Casp-1-/- or Nlrp3-/- macrophages, neither BCG nor the positive control nigericin could induce IL-1β secretion. In contrast, rBCG-LTAK63 stimulation of either Casp-1-/- or Nlrp3-/- macrophages induced IL-1β (Figures 4A, B). The production of IL-1β in Aim-2-deficient macrophages was maintained by both stimulus (Figure 4C). Comparative analyses across genotypes (e.g., wild-type vs. knockout) showed that Casp-1-/- or Nlrp3-/- cells exhibited an ~10-fold reduction in IL-1β compared to wild-type cells, whereas IL-1β production in Aim-2-/- macrophages was comparable to wild-type levels (Figures 4D–F).
Figure 4
Together, these results indicate that rBCG-LTAK63 induces IL-1β secretion predominantly through the canonical NLRP3/caspase-1 inflammasome pathway, while also engaging caspase-1 and NLRP3-independent mechanisms that partially compensate for the absence of these components.
3.4 Crosstalk between inflammasome-activated macrophages and T cells
To investigate the crosstalk between innate and adaptive immunity, we established a co-culture system combining inflammasome-activated BMDMs with naïve splenocytes or splenocytes from immunized mice (Figure 2B).
When co-cultured with naïve splenocytes, macrophages stimulated with either BCG or rBCG-LTAK63 increased the percentage of memory-phenotype (CD44+, Figure 5A) and activated (CD69+, Figure 5B) CD4+ T cells, and promoted direction toward a Th1 phenotype (IFN-γ+, Figure 5C). However, no significant differences were observed between the BCG and rBCG-LTAK63 groups in these subsets, including Th17 (IL-17+) cells (Figure 5D).
Figure 5
In contrast, when the co-culture was performed using splenocytes from immunized mice (Figure 2B) a more pronounced effect was observed. rBCG-LTAK63-stimulated macrophages induced a significantly higher percentage of both activated (CD69+) and memory-phenotype (CD44+) CD4+ T cells compared to its own basal control (Supplementary Figures S2D, E). Under these conditions, rBCG-LTAK63 stimulation also led to a robust increase in IFN-γ+ (Th1) and IL-17+ (Th17) CD4+ T cells relative to its basal group (Supplementary Figures S2F, G). These findings demonstrate that pre-existing immunity unleashes rBCG-LTAK63’s unique capacity to drive a robust and polyfunctional adaptive immune response.
3.5 Innate immune competence is required for the enhanced immunogenicity of rBCG-LTAK63
We investigated the specific involvement of innate inflammatory responses using macrophages (BMDMs) from AIRmax and AIRmin mouse strains. These strains were genetically selected for high and low acute inflammatory responses, respectively. The AIRminTT strain is a subline with a characterized loss-of-function mutation in ASC (24).
In AIRmax macrophages, rBCG-LTAK63 triggered significantly higher IL-1β secretion than BCG or unstimulated cells (Figure 6A). In AIRminTT macrophages, the IL-1β response was completely abolished for all stimuli including the positive control ATP (Figures 6B, C). rBCG-LTAK63 was statistically different from BCG group but not in comparison to unstimulated cells (Figure 6B).
Figure 6
We also investigated whether rBCG-LTAK63 would also stimulate a cytokine response beyond IL-1β. The results showed a significant reduction in the production of IL-6 (Figure 6D), TNF-α (Figure 6E), and MCP-1 (Figure 6F) was observed in AIRminTT in comparison to AIRmax macrophages. Collectively, these data demonstrate that rBCG-LTAK63 can induce IL-1β secretion in both macrophage lineages but with a reduced efficiency in AIRmin BMDMs.
3.6 Protective effect of rBCG-LTAK63 immunization in AIRmax and AIRminTT mice
rBCG-LTAK63 has consistently demonstrated increased protection of mice against Mtb challenge including in different BCG background strains (Moreau or Danish), expression systems (auxotrophic complementation or antibiotic-maintained), challenge models (intratracheal or intranasal) and Mtb strains (H37Rv or Beijing) (14, 15). Here, we used AIRmax and AIRmin mouse models to assess whether inflammasome-associated innate immune competence influences vaccine-induced protection in vivo. AIRmax and AIRminTT mice were immunized with either BCG or rBCG-LTAK63, or received saline, and subsequently challenged with Mtb H37Rv. The pulmonary bacterial burden was quantified 30 days post-infection (Figure 7A).
Figure 7
Non-immunized mice (Saline group) from both AIRmax and AIRminTT genotypes exhibited a similarly high bacterial load in the lungs of challenged mice (Figure 7B). In AIRminTT mice, BCG did not induce protection, but in AIRmax mice, protection was similar to that obtained in naïve mice. More importantly, rBCG-LTAK63 induced higher protection as compared to BCG in both genotypes. Comparison between genotypes demonstrates that AIRmax mice exhibit significantly lower CFU counts in comparison to AIRminTT mice in both BCG and rBCG-LTAK63 groups. Nevertheless, the ability of rBCG-LTAK63 to confer protection in AIRminTT mice indicates that this recombinant vaccine can partially overcome limitations imposed by a genetically attenuated inflammatory background.
4 Discussion
Inflammasomes play a critical role in innate immunity and the subsequent developed adaptive responses. Significant advances have been made in understanding inflammasome biology in the context of vaccine-induced immune responses. Inflammasome activation is not only important in mycobacterial infections but also contributes to the protective effect elicited by BCG, both as a vaccine and as immunotherapy in bladder cancer (25–27).
From the draining lymph nodes of immunized mice clear differences between rBCG-LTAK63 and the parental BCG were noted as early as 7 days post-immunization. The overrepresentation of a chaperone-centered hub is a response to a highly active and stressed cellular environment. In conjunction with the upregulation of the chemokines CXCL1, CXCL2 and COX-2 (Ptgs2), this signature implies an early and robust innate immune response induced by rBCG-LTAK63. The absence of a direct IL-1-related hub may be due to the predominance of T and B lymphocytes in the lymph nodes at 7 days post-immunization (28).
Our study demonstrates that rBCG-LTAK63 induces an inflammasome-associated innate immune response in a distinct manner compared to the parental BCG, characterized by the enhanced IL-1β secretion linked to the adjuvant LTAK63. IL-1β is observed even without LPS priming and suggests that rBCG-LTAK63 may simultaneously act as signal 1 (priming) and signal 2 (activation). While canonical NLRP3 signaling contributes to this response, the residual IL-1β production detected in caspase-1-/- and Nlrp3-/- macrophages indicates that additional pathways are participating. These may include caspase-8–dependent mechanisms can be recruited after the activation of certain surface receptors such as TLR-2 and Dectin-1. These mechanisms have not been associated with LT or LTA, but they have been described in the context of mycobacterial infection (29–32).
It also supports the evidence that even for BCG, a known immunostimulant, which comprises a milieu of multiple PAMPs that activate TLR, NLR, CLR and RLR downstream signaling (33), further reinforcement of innate immune pathways can be beneficial. The combination of BCG and a RIG-I/NOD2-activating molecule (Inarigivir) increased the secretion of pro-inflammatory cytokines, including IL-1β, and resulted in improved protection against Mtb (34). Similarly, VPM1002 (BCGΔureC::hly), which provides improved protection against Mtb, also triggers the AIM-2 inflammasome and increases IL-1β in comparison to parental BCG (8). In fact, combinatorial inflammasome activation using multiple ligands can trigger mechanisms not accessed by a single ligand (35). Therefore, co-activation of multiple innate pathways including inflammasomes may result in improved immune outcomes.
The interplay between inflammasome activation and adaptive immunity was also evaluated. Overall, rBCG-LTAK63-stimulated macrophages drove a significant CD4+ T cell activation and Th1/Th17 polarization in co-culture experiments. These observations extend previous reports linking IL-1β to Th17 differentiation (36). Furthermore, a robust innate response is not only important to promote proper adaptive responses but may directly provide innate-mediated protection independent of T cells (37).
Genetic models revealed that IL-1β secretion was dependent on the mouse phenotype, with AIRmax mice mounting stronger inflammatory responses than AIRmin (24). Accordingly, not only IL-1β but other inflammatory cytokines/chemokines are upregulated in AIRmax mice upon stimulation (e.g. Cxcl1, Cxcl2, Tnf-α, Il-6, Ccl2) (38). The naturally occurring Pycard missense mutation (E19K) present in AIRminTT mice impairs, but does not abolish, ASC function, resulting in an attenuated inflammasome-associated response influenced by genetic background (24). Together, these characteristics make AIRmax and AIRminTT strains valuable models for studying the relationship between innate inflammation and immune-mediated outcomes (21, 24). Interestingly, when challenged with Mtb, rBCG-LTAK63 induced protection in both genotypes, whereas BCG was only effective in AIRmax mice. Even if other mechanisms are present aside differences in inflammatory responses, these results suggests that rBCG-LTAK63 can bypass certain genetic restraints to induce protection. Engagement of inflammasome-associated innate pathways correlates with a reduced pulmonary bacterial burden in both BCG and rBCG-LTAK63 immunized mice, suggesting a contributory, rather than exclusive, role for inflammasome signaling in protection. How rBCG-LTAK63 induces protection in AIRminTT mice despite their impaired innate inflammatory response remains to be evaluated. The renewed interest in the innate responses induced by BCG or its recombinant derivatives seems to point towards common pathways such as cGAS/STING (39) or autophagy (40). Autophagy can be induced in situations of cellular stress, and the transcriptional profile of rBCG-LTAK63-immunized mice suggests an intense cellular stress. In fact, a recent work describes the induction of autophagy by rBCG-LTAK63 in RAW264.7, a murine monocyte/macrophage-like cell line (41). Additionally, other T cell subsets (memory-like or effector function) can be generated independent of inflammasomes or other innate mechanisms and may be involved in the induced protection (17).
Overall, our findings demonstrate that rBCG-LTAK63 enhances innate immune activation, including inflammasome-associated pathways, and that this heightened innate response correlates with improved protection against M. tuberculosis. Rather than establishing a direct causal dependence on NLRP3 or ASC, our data support a model in which broad and effective activation of innate immune mechanisms contributes to the superior efficacy of rBCG-LTAK63.
5 Conclusion
Our findings demonstrate that the rBCG-LTAK63 vaccine promotes robust innate immune activation, including inflammasome-associated pathways, which likely contributes to its enhanced protective efficacy against M. tuberculosis. Future studies will be required to define the precise contribution of individual inflammasome components and to investigate the involvement of other innate immune mechanisms. This work highlights the importance of innate immune activation as a central axis in tuberculosis vaccine development and supports the continued evaluation of rBCG-LTAK63 as a promising next-generation tuberculosis vaccine.
Statements
Data availability statement
The RNA-seq data are available in the NCBI Sequence Read Archive under accession number GSE278523.
Ethics statement
The animal study was approved by Ethics Committee on the Use of Animals (Comissão de Ética no Uso de Animais – CEUA, protocol 2012250722), Instituto Butantan, São Paulo, Brazil. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
AC: Writing – review & editing, Methodology, Investigation, Writing – original draft, Visualization, Validation, Formal analysis, Data curation, Conceptualization. MT: Writing – review & editing, Methodology, Visualization, Validation, Investigation. DR: Methodology, Writing – review & editing, Investigation. LM-N: Investigation, Writing – review & editing, Methodology. PS: Investigation, Writing – review & editing, Methodology. NS: Investigation, Writing – review & editing, Resources. SO: Investigation, Resources, Writing – review & editing. LL: Funding acquisition, Conceptualization, Resources, Visualization, Supervision, Project administration, Writing – review & editing, Data curation. AK: Supervision, Methodology, Investigation, Validation, Resources, Writing – review & editing, Conceptualization, Data curation, Funding acquisition, Formal analysis, Writing – original draft, Project administration, Visualization.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2022/13917-9 and 2017/24832-6, and Fundação Butantan. The author(s) thank the Coordination for the Improvement of Higher Education Personnel (CAPES), Fundação Butantan, and FAPESP (2023/02577-5). AC is the recipient of CAPES and FAPESP scholarships.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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References
1
World Health Organization. Global Tuberculosis Report 2024. 1st ed. Geneva: World Health Organization; (2024). p. 1.
2
DockrellHMSmithSG. What have we learnt about BCG vaccination in the last 20 years? Front Immunol. (2017) 8:1134. doi: 10.3389/fimmu.2017.01134
3
BultéDRigamontiCRomanoAMortellaroA. Inflammasomes: mechanisms of action and involvement in human diseases. Cells. (2023) 12:1766. doi: 10.3390/cells12131766
4
HuangYXuWZhouR. NLRP3 inflammasome activation and cell death. Cell Mol Immunol. (2021) 18:2114–27. doi: 10.1038/s41423-021-00740-6
5
MantovaniADinarelloCAMolgoraMGarlandaC. Interleukin-1 and related cytokines in the regulation of inflammation and immunity. Immunity. (2019) 50:778–95. doi: 10.1016/j.immuni.2019.03.012
6
GengenbacherMNieuwenhuizenNVogelzangALiuHKaiserPSchuererSet al. Deletion of nuoG from the Vaccine Candidate Mycobacterium bovis BCG ΔureC::hly Improves Protection against Tuberculosis. mBio. (2016) 7:e00679–16. doi: 10.1128/mBio.00679-16
7
GröschelMISayesFShinSJFriguiWPawlikAOrgeurMet al. Recombinant BCG expressing ESX-1 of mycobacterium marinum combines low virulence with cytosolic immune signaling and improved TB protection. Cell Rep. (2017) 18:2752–65. doi: 10.1016/j.celrep.2017.02.057
8
SaigaHNieuwenhuizenNGengenbacherMKoehlerABSchuererSMoura-AlvesPet al. The recombinant BCG ΔureC::hly vaccine targets the AIM2 inflammasome to induce autophagy and inflammation. J Infect Dis. (2015) 211:1831–41. doi: 10.1093/infdis/jiu675
9
TheobaldSJMüllerTALangeDKeckKRybnikerJ. The role of inflammasomes as central inflammatory hubs in Mycobacterium tuberculosis infection. Front Immunol. (2024) 15:1436676. doi: 10.3389/fimmu.2024.1436676
10
WawrockiSDruszczynskaM. Inflammasomes in mycobacterium tuberculosis-driven immunity. Can J Infect Dis Med Microbiol J Can Mal Infect Microbiol Médicale. (2017) 2017:2309478. doi: 10.1155/2017/2309478
11
MishraAKhanASinghVKGlydeESaikolappanSGarnicaOet al. The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice. Front Immunol. (2024) 15:1321657. doi: 10.3389/fimmu.2024.1321657
12
StoverCKde la CruzVFFuerstTRBurleinJEBensonLABennettLTet al. New use of BCG for recombinant vaccines. Nature. (1991) 351:456–60. doi: 10.1038/351456a0
13
Marques-NetoLMPiwowarskaZKannoAIMoraesLTrentiniMMRodriguezDet al. Thirty years of recombinant BCG: new trends for a centenary vaccine. Expert Rev Vaccines. (2021) 20:1001–11. doi: 10.1080/14760584.2021.1951243
14
MoraesLTrentiniMMFousterisDEtoSFChudzinski-TavassiAMLeite LC deCet al. CRISPR/cas9 approach to generate an auxotrophic BCG strain for unmarked expression of LTAK63 adjuvant: A tuberculosis vaccine candidate. Front Immunol. (2022) 13:867195. doi: 10.3389/fimmu.2022.867195
15
NascimentoIPRodriguezDSantosCCAmaralEPRofattoHKJunqueira-KipnisAPet al. Recombinant BCG Expressing LTAK63 Adjuvant induces Superior Protection against Mycobacterium tuberculosis. Sci Rep. (2017) 7:2109. doi: 10.1038/s41598-017-02003-9
16
Carvalho Dos SantosCRodriguezDKanno IssamuACezar De Cerqueira LeiteLPereira NascimentoI. Recombinant BCG expressing the LTAK63 adjuvant induces increased early and long-term immune responses against Mycobacteria. Hum Vaccines Immunother. (2019) 16:673–83. doi: 10.1080/21645515.2019.1669414
17
Marques-NetoLMTrentiniMMKannoAIRodriguezDLeite LC deC. Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice. Front Immunol. (2023) 14:1205449. doi: 10.3389/fimmu.2023.1205449
18
Carvalho Dos SantosCRodriguezDKanno IssamuACezar De Cerqueira LeiteLPereira NascimentoI. Recombinant BCG expressing the LTAK63 adjuvant induces increased early and long-term immune responses against Mycobacteria. Hum Vaccin Immunother. (2020) 16:673–83. doi: 10.1080/21645515.2019.1669414
19
Dos SantosCCWalburgKVvan VeenSWilsonLGTrufenCEMNascimentoIPet al. Recombinant BCG-LTAK63 vaccine candidate for tuberculosis induces an inflammatory profile in human macrophages. Vaccines (Basel). (2022) 10:831. doi: 10.3390/vaccines10060831
20
ValliEBaudierRLHarriettAJNortonEB. LTA1 and dmLT enterotoxin-based proteins activate antigen-presenting cells independent of PKA and despite distinct cell entry mechanisms. PloS One. (2020) 15:e0227047. doi: 10.1371/journal.pone.0227047
21
IbañezOMStarobinasNMonteleoneLFBorregoAIcimotoMYCabreraWKet al. Novel Pycard gene polymorphism impairs Nlpr3 inflammasome-induced IL-1β production in mice selected for low inflammatory response. J Immunol. (2018) 200:115.10. doi: 10.4049/jimmunol.200.Supp.115.10
22
RobinsonMDMcCarthyDJSmythGK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. (2010) 26:139–40. doi: 10.1093/bioinformatics/btp616
23
BanerjeeI. In vitro Assays to Study Inflammasome Activation in Primary Macrophages. In: Abdul-SaterAA, editor. The Inflammasome. Methods in Molecular Biology, vol. 2459 . Humana, New York, NY (2022). doi: 10.1007/978-1-0716-2144-8_2
24
BorregoAColomboFde SouzaJGJensenJRDassanoAPiazzaRet al. Pycard and BC017158 candidate genes of irm1 locus modulate inflammasome activation for IL-1β Production. Front Immunol. (2022) 21:899569. doi: 10.3389/fimmu.2022.899569
25
FremondCMTogbeDDozERoseSVasseurVMailletIet al. IL-1 receptor-mediated signal is an essential component of myD88-dependent innate response to mycobacterium tuberculosis infection. J Immunol. (2007) 179:1178–89. doi: 10.4049/jimmunol.179.2.1178
26
SugawaraIYamadaHHuaSMizunoS. Role of interleukin (IL)-1 type 1 receptor in mycobacterial infection. Microbiol Immunol. (2001) 45:743–50. doi: 10.1111/j.1348-0421.2001.tb01310.x
27
ZhouHZhangLLuoWHongHTangDZhouDet al. AIM2 inflammasome activation benefits the therapeutic effect of BCG in bladder carcinoma. Front Pharmacol. (2022) 13:1050774. doi: 10.3389/fphar.2022.1050774
28
SenderRWeissYNavonYMiloIAzulayNKerenLet al. The total mass, number, and distribution of immune cells in the human body. Proc Natl Acad Sci U S A. (2023) 120:e2308511120. doi: 10.1073/pnas.2308511120
29
GringhuisSIKapteinTMWeversBATheelenBvan der VlistMBoekhoutTet al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1β via a noncanonical caspase-8 inflammasome. Nat Immunol. (2012) 13:246–54. doi: 10.1038/ni.2222
30
AbdallaHSrinivasanLShahSMayer-BarberKDSherASutterwalaFSet al. Mycobacterium tuberculosis Infection of Dendritic Cells Leads to Partially Caspase-1/11-Independent IL-1β and IL-18 Secretion but Not to Pyroptosis. PloS One. (2012) 7:e40722. doi: 10.1371/journal.pone.0040722
31
de AraujoACVSCde QueirozNMGPMarinhoFVOliveiraSC. Bacillus Calmette-Guérin-Trained Macrophages Elicit a Protective Inflammatory Response against the Pathogenic Bacteria Brucella abortus. J Immunol. (2023) 211:791–803. doi: 10.4049/jimmunol.2200642
32
Mayer-BarberKDBarberDLShenderovKWhiteSDWilsonMSCheeverAet al. Cutting edge: caspase-1 independent IL-1β Production is critical for host resistance to mycobacterium tuberculosis and does not require TLR signaling in vivo. J Immunol Baltim Md 1950. (2010) 184:3326–30. doi: 10.4049/jimmunol.0904189
33
MishraAAkhtarSJagannathCKhanA. Pattern recognition receptors and coordinated cellular pathways involved in tuberculosis immunopathogenesis: Emerging concepts and perspectives. Mol Immunol. (2017) 87:240–8. doi: 10.1016/j.molimm.2017.05.001
34
KhanASinghVKMishraASoudaniEBakhruPSinghCRet al. NOD2/RIG-I activating inarigivir adjuvant enhances the efficacy of BCG vaccine against tuberculosis in mice. Front Immunol. (2020) 11:592333. doi: 10.3389/fimmu.2020.592333
35
OhSLeeJOhJYuGRyuHKimDet al. Integrated NLRP3, AIM2, NLRC4, Pyrin inflammasome activation and assembly drive PANoptosis. Cell Mol Immunol. (2023) 20:1513–26. doi: 10.1038/s41423-023-01107-9
36
ArboreGWestEESpolskiRRobertsonAABKlosARheinheimerCet al. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4 + T cells. Science. (2016) 352:aad1210. doi: 10.1126/science.aad1210
37
BickettTEMcLeanJCreissenEIzzoLHaganCIzzoAJet al. Characterizing the BCG induced macrophage and neutrophil mechanisms for defense against mycobacterium tuberculosis. Front Immunol. (2020) 11:1202. doi: 10.3389/fimmu.2020.01202
38
FernandesJGCanhameroTBorregoAJensenJRCabreraWHCorreaMAet al. Distinct gene expression profiles provoked by polyacrylamide beads (Biogel) during chronic and acute inflammation in mice selected for maximal and minimal inflammatory responses. Inflammation Res. (2016) 65:313–23. doi: 10.1007/s00011-016-0918-1
39
NingHWangLZhouJLuYKangJDingTet al. Recombinant BCG With Bacterial Signaling Molecule Cyclic di-AMP as Endogenous Adjuvant Induces Elevated Immune Responses After Mycobacterium tuberculosis Infection. Front Immunol. (2019) 10:1519. doi: 10.3389/fimmu.2019.01519
40
JagannathCLindseyDRDhandayuthapaniSXuYHunterRLJrEissaNT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med. (2009) 15:267–76. doi: 10.1038/nm.1928
41
Marques-NetoLMTrentiniMMMorenoACREtoSFCarvalhoACONetoAPSet al. rBCG-LTAK63 enhances protection against tuberculosis by inducing autophagy and circadian gene regulation. Front Immunol. (2025) 14:1695560. doi: 10.3389/fimmu.2025.1695560
Summary
Keywords
BCG vaccine, inflammasome, innate immunity, rBCG-LTAK63, recombinant BCG, tuberculosis
Citation
Carvalho ACO, Trentini MM, Rodriguez D, Marques-Neto LM, Silveira PHS, Starobinas N, Oliveira SC, Leite LCC and Kanno AI (2026) Broad innate immune activation enhances the protective efficacy of rBCG-LTAK63 against Mycobacterium tuberculosis. Front. Immunol. 17:1758476. doi: 10.3389/fimmu.2026.1758476
Received
01 December 2025
Revised
30 January 2026
Accepted
03 February 2026
Published
04 March 2026
Volume
17 - 2026
Edited by
Abel A. Ramos Vega, National Polytechnic Institute (IPN), Mexico
Reviewed by
Abhishek Mishra, Houston Methodist Research Institute, United States
Jae-Hun Ahn, Seoul National University Hospital, Republic of Korea
Updates
Copyright
© 2026 Carvalho, Trentini, Rodriguez, Marques-Neto, Silveira, Starobinas, Oliveira, Leite and Kanno.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Alex Issamu Kanno, alex.kanno@butantan.gov.br
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.