Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages

Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-β, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.


Introduction
Tuberculosis (TB) is still a public health problem and is one of the top 10 causes of death in the world. The COVID-19 pandemic has reversed years of progress in providing essential TB services and reducing TB disease burden. There was a large global drop in the number of people newly diagnosed with TB and reported in 2020 as compared with 2019; this fell from 7.1 million in 2019 to 5.8 million in 2020. Consequently, infected and non-diagnosed patients did not receive treatment, resulting in an increase in the number of people who died from TB in 2020. There were an estimated 1.3 million deaths among HIV-negative people and an additional 214,000 deaths among HIV-positive people [1]. The BCG vaccine does not sufficiently prevent pulmonary tuberculosis in adults [2]; thus, new prevention or treatment strategies are required to control the infection spread, and these are part of the main goals of the World Health Organization's End TB Strategy [1]. differentiation for 6 days at 37 • C/5% CO 2 , macrophages were harvested using Trypsin (Sigma-Aldrich ® , Merck KGaA, St. Louis, MO, USA) and scrapping and analyzed by flow cytometry for the expression of cell surface markers CD14, CD11b, and CD163.
One day before infection, M1 and M2 macrophages were plated in either 48= or 96-well plates at a density of 1 × 10 5 cells/well. In addition, mycobacterial cultures were diluted to a density corresponding to early log-phase growth OD600 of 0.4. On the day of infection, M1 and M2 macrophage cultures were inoculated with 100 µL of rBCG-LTAK63, BCG, or Mtb H37Rv (multiplicity of infection (MOI) = 10) and incubated at 37 • C with 5% CO 2 for 1, 24, or 48 h, as indicated.

RNA Extraction, dcRT-MLPA Assay, and Data Analysis
Macrophages were infected with mycobacterial strains for 24 or 48 h. At each time point, infected cells were collected in 350 µL of Trizol (Life Technologies-Invitrogen), and the RNA was isolated according to the manufacturer's protocol. Briefly, cells were incubated for 5 min at room temperature (RT). Chloroform (70 µL) was added, and the mixture was centrifuged for 15 min at 12,000× g at 4 • C. The aqueous phase was transferred to a 1.5 mL microcentrifuge tube. Isopropanol (175 µL) was added and incubated for 20 min at RT. The samples were spun for 10 min at 12,000× g at 4 • C, and the RNA pellets were washed twice with 1 mL of ethanol 75% before being dissolved in RNase-free H 2 O (12 µL). The concentration of RNA was measured using a Nanodrop, and the RNA was stored at −80 • C until downstream processing.
Dual-color reverse transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) was performed as described previously [19,20]. Briefly, for each targetspecific sequence, a specific RT primer was designed to hybridize immediately downstream of the left-and right-hand half-probe target sequence. Following reverse transcription of 100 ng RNA (M1 macrophages) or 25 ng RNA (M2 macrophages) using MMLV reverse transcriptase (Promega, Leiden, the Netherlands), left-and right-hand half-probes were hybridized to the cDNA at 60 • C overnight. Annealed half-probes were ligated and subsequently amplified by PCR (33 cycles of 30 s at 95 • C, 30 s at 58 • C, and 60 s at 72 • C, followed by 1 cycle of 20 min at 72 • C). Primers and probes used were from Sigma-Aldrich and MLPA reagents from MRC Holland (Amsterdam, the Netherlands). PCR amplification products were diluted 1:10 in HiDi formamide (Thermo Fisher Scientific, Waltham, MA, USA) containing GeneScan 400HD ROX (Applied Biosystems, Waltham, MA, USA) size standard and were analyzed on an Applied Biosystems 3730 capillary sequencer in GeneScan mode (Baseclear, Leiden, the Netherlands). Trace data were analyzed using GeneMapper software package 5.0 (Applied Biosystems/Life Technologies). The areas of each assigned peak (in arbitrary units) were exported for further analysis in Microsoft Excel spreadsheet software. Signals below the threshold value for noise cutoff in GeneMapper (log2-transformed peak area ≤7.64) were assigned to the threshold value for noise cutoff. Following normalization of the data to the average signal of housekeeping gene GAPDH, signals below the threshold value for noise cutoff in GeneMapper were again assigned the threshold value for noise cutoff. Primers and probes of 43 preselected immune-associated genes were designed by Leiden University Medical Center (LUMC). The fold change was calculated by comparing infected and uninfected macrophages for all genes, and statistical analysis was performed as indicated.

Cytokine Production
Macrophages were infected with mycobacterial strains, and the supernatant was col-

M1 Macrophages Infected with rBCG-LTAK63 Display Marked Upregulation of Genes Associated with the IFN Signaling Pathway
The gene expression profiles of 43 immune-associated genes were evaluated by dcRT-MLPA in macrophages infected with BCG, rBCG-LTAK63, or Mtb H37Rv (Supplementary Figure S2, Figure 1B). At 24 h post-infection, 22 DEGs were identified in M1 macrophages infected with rBCG-LTAK63 (compared to uninfected controls), while 19 DEGs were identified in BCG-infected M1 cells, and 17 of these DEGs were found to be in common ( Figure 1A). Interestingly, a large set of interferon (IFN)-signaling genes was more profoundly induced in rBCG-LTAK63-infected M1 as compared to either BCG-and Mtb-infected M1 ( Figure 1B, Supplementary Table S1). The IFIT3 and OAS3 genes were significantly upregulated when rBCG-LTAK63 was compared to BCG-infected M1. CXCL9, an antimicrobial gene, was also significantly upregulated in rBCG-LTAK63-infected M1 as compared to BCG-infected M1 macrophages ( Figure 1C).  Figure 1A). Interestingly, a large set of interferon (IFN)-signaling genes was more profoundly induced in rBCG-LTAK63-infected M1 as compared to either BCG-and Mtbinfected M1 ( Figure 1B, Supplementary Table S1). The IFIT3 and OAS3 genes were significantly upregulated when rBCG-LTAK63 was compared to BCG-infected M1. CXCL9, an antimicrobial gene, was also significantly upregulated in rBCG-LTAK63-infected M1 as compared to BCG-infected M1 macrophages ( Figure 1C).

BCG Induces Downregulation of an Inflammatory Gene Profile in M2 Macrophages, While rBCG-LTAK63 Maintains or Induces Upregulation for 48 h
In contrast to M1 macrophages, only very few DEGs could be identified in mycobacteriainfected M2 macrophages at 24 h post-infection compared to uninfected controls (Supplementary Table S2, Figure 2B). Nevertheless, similar to M1, IFN-signaling genes were more profoundly induced in rBCG-LTAK63-infected M2 compared to either BCG-or Mtb-infected M2. Moreover, the IFN-signaling TAP1 and GBP1 genes were found to be significantly upregulated in rBCG-LTAK63-infected M2 when compared to BCG-infected M2 macrophages ( Figure 2B,C). After 48 h, 9 DEGs could be identified in M2 macrophages infected with rBCG-LTAK63 (compared to uninfected controls), while 2 DEGs could be identified in M2 macrophages infected with BCG, which were DEGs in common with rBCG-LTAK63-infected M2 (Figure 2A). Of note, at 48 h post-infection, BCG-infected M2 macrophages had predominantly downregulated inflammatory-correlated genes, while rBCG-LTAK63 infection maintained or further upregulated inflammatory-associated transcripts. Expression of the genes, TNIP1, SLAMF7, and IL6 was upregulated and CD14 and HCK genes downregulated when rBCG-LTAK63 was compared to BCG-infected M2 macrophages ( Figure 2C). uninfected M1 macrophages). (B) Heat map showing fold changes in expression profile of the 43 immune-related genes in M1 macrophages in response to infection with rBCG-LTAK63, BCG, or Mtb H37Rv. (C) Differentially expressed genes (p ˂ 0.05) in rBCG-LTAK63-infected M1 macrophages when compared to BCG-infected M1 cells. Statistical significance was determined by paired Student's t-test. Significant differences were observed as indicated * p ˂ 0.05.

BCG Induces Downregulation of an Inflammatory Gene Profile in M2 Macrophages, While rBCG-LTAK63 Maintains or Induces Upregulation for 48 h
In contrast to M1 macrophages, only very few DEGs could be identified in mycobacteria-infected M2 macrophages at 24 h post-infection compared to uninfected controls (Supplementary Table S2, Figure 2B). Nevertheless, similar to M1, IFN-signaling genes were more profoundly induced in rBCG-LTAK63-infected M2 compared to either BCGor Mtb-infected M2. Moreover, the IFN-signaling TAP1 and GBP1 genes were found to be significantly upregulated in rBCG-LTAK63-infected M2 when compared to BCG-infected M2 macrophages ( Figure 2B,C). After 48 h, 9 DEGs could be identified in M2 macrophages infected with rBCG-LTAK63 (compared to uninfected controls), while 2 DEGs could be identified in M2 macrophages infected with BCG, which were DEGs in common with rBCG-LTAK63-infected M2 (Figure 2A). Of note, at 48 h post-infection, BCG-infected M2 macrophages had predominantly downregulated inflammatory-correlated genes, while rBCG-LTAK63 infection maintained or further upregulated inflammatory-associated transcripts. Expression of the genes, TNIP1, SLAMF7, and IL6 was upregulated and CD14 and HCK genes downregulated when rBCG-LTAK63 was compared to BCG-infected M2 macrophages ( Figure 2C). Cytokines are mediators and effector molecules of immune responses against pathogens. We evaluated the production of cytokines associated with inflammatory responses, cell growth, and repair in the culture supernatants of M1 and M2 macrophages infected with rBCG-LTAK63 or BCG at 48 h post-infection, or uninfected macrophages as a control.

M1 and M2 Macrophages Infected with rBCG-LTAK63 Display Increased Production of Inflammatory Cytokines
Cytokines are mediators and effector molecules of immune responses against pathogens. We evaluated the production of cytokines associated with inflammatory responses, cell growth, and repair in the culture supernatants of M1 and M2 macrophages infected with rBCG-LTAK63 or BCG at 48 h post-infection, or uninfected macrophages as a control.
Overall, the levels of secreted cytokines in rBCG-LTAK63-infected M1 macrophages clearly surpassed the levels observed in BCG-infected M1. Furthermore, the production of IL-17A, IFNA2, TNF-α, Fractalkine, IL-10, VEGF, and FLT3L was strongly induced in both rBCG-LTAK63-and BCG-infected M1 when compared with uninfected M1 macrophages but, more interestingly, the production of IL-15 and proinflammatory cytokines IL-12(p70) and TNF-β was significantly higher in rBCG-LTAK63-compared to BCG-infected M1 macrophages ( Figure 3).  . M1 macrophages infected with rBCG-LTAK63 display higher secretion levels of inflammatory cytokines. M1 macrophages derived from 4 different donors were infected with rBCG-LTAK63 or BCG for 48 h and the supernatants were analyzed for cytokine production by Luminex and compared to uninfected control cells. Statistical significance was determined by RM-ANOVA (p < 0.05). Significant differences were observed as indicated * p < 0.05, ** p < 0.01. Similar to M1-infected cells, overall, the levels of secreted cytokines in rBCG-LTAK63infected M2 macrophages exceeded the levels observed in BCG-infected M2 (Figure 4). The production of MCP-3 and EGF was higher in rBCG-LTAK63 when compared with BCG- infected M2 macrophages; the production of IFN-γ was higher only when rBCG-LTAK63 was compared with uninfected M2 macrophages; and the production of IL-6, IL-9, IL-7, Fractalkine, TGF-α, and IL-12 (p70) was higher in rBCG-LTAK63 or BCG when compared with uninfected M2 macrophages. Similar to M1-infected cells, overall, the levels of secreted cytokines in rBCG-LTAK63-infected M2 macrophages exceeded the levels observed in BCG-infected M2 (Figure 4). The production of MCP-3 and EGF was higher in rBCG-LTAK63 when compared with BCG-infected M2 macrophages; the production of IFN-γ was higher only when rBCG-LTAK63 was compared with uninfected M2 macrophages; and the production of IL-6, IL-9, IL-7, Fractalkine, TGF-α, and IL-12 (p70) was higher in rBCG-LTAK63 or BCG when compared with uninfected M2 macrophages. Figure 4. M2 macrophages infected with rBCG-LTAK63 display higher secretion levels of inflammatory cytokines. M2 macrophages derived from 4 different donors were infected with rBCG-LTAK63 or BCG for 48 h and the supernatants were analyzed for cytokine production by Luminex and compared to uninfected controls. Statistical significance was determined by RM-ANOVA (p ˂ 0.05). Significant differences were observed as indicated * p ˂ 0.05, ** p ˂ 0.01.

Discussion
In order to uncover the immunological mechanisms or pathways activated by rBCG-LTAK63 in human cells and in more depth, we investigated the gene expression profiles induced in infected primary human macrophages. Since the expression of heterologous proteins in bacteria can be influenced by several factors in both in vivo and in vitro conditions [21][22][23], we first demonstrated that the recombinant strain is stable during infection of primary human macrophages.
Compared to BCG-infected M1 cells, the rBCG-LTAK63-infected M1 macrophages displayed stronger upregulation of IFIT3 and OAS3 genes, which are part of the IFN signaling pathway. The role of the IFN signaling pathway is complex, as it can both be beneficial or harmful to the host, depending on the experimental context [24]. The IFIT3 gene is part of the interferon-induced proteins with tetratricopeptide repeats (IFITs) family, strongly induced due to activation of type I IFN [25]. Initially, the role of this gene was described in antiviral defense, as well as in the activation of several components of the IFN signaling pathway [24]. However, it has also been described to be positively regulated in bacterial infections, such as mycobacteria infections [26][27][28][29]. . M2 macrophages infected with rBCG-LTAK63 display higher secretion levels of inflammatory cytokines. M2 macrophages derived from 4 different donors were infected with rBCG-LTAK63 or BCG for 48 h and the supernatants were analyzed for cytokine production by Luminex and compared to uninfected controls. Statistical significance was determined by RM-ANOVA (p < 0.05). Significant differences were observed as indicated * p < 0.05, ** p < 0.01.

Discussion
In order to uncover the immunological mechanisms or pathways activated by rBCG-LTAK63 in human cells and in more depth, we investigated the gene expression profiles induced in infected primary human macrophages. Since the expression of heterologous proteins in bacteria can be influenced by several factors in both in vivo and in vitro conditions [21][22][23], we first demonstrated that the recombinant strain is stable during infection of primary human macrophages.
Compared to BCG-infected M1 cells, the rBCG-LTAK63-infected M1 macrophages displayed stronger upregulation of IFIT3 and OAS3 genes, which are part of the IFN signaling pathway. The role of the IFN signaling pathway is complex, as it can both be beneficial or harmful to the host, depending on the experimental context [24]. The IFIT3 gene is part of the interferon-induced proteins with tetratricopeptide repeats (IFITs) family, strongly induced due to activation of type I IFN [25]. Initially, the role of this gene was described in antiviral defense, as well as in the activation of several components of the IFN signaling pathway [24]. However, it has also been described to be positively regulated in bacterial infections, such as mycobacteria infections [26][27][28][29].
Expression of IFIT family genes depends on pattern recognition and the JAK-STAT pathway. Pathogen-associated molecular patterns (PAMPs) are molecules associated with different groups of pathogens, including viruses, bacteria, fungi, and others. Pattern recognition receptors (PRRs) recognize different PAMPs during infection by pathogens and activate signaling molecules. As a result, activation of receptors known as Toll-like Vaccines 2022, 10, 831 9 of 14 (TLRs) and RIG-like receptors (RLRs) induce gene expression of the IFIT family [25]. The IFIT3 gene has been described as a protective molecule against virus infection [27]. It has been reported that upon viral or bacterial infection, the IFN signaling activation induces the production of the 2 -5 -oligoadenylate synthetase (OAS) family [30], which includes the OAS3 gene, that was upregulated by infection with rBCG-LTAK63. The role of the OAS family in bacterial infections is not well defined yet. However, it has been reported to induce intracellular survival of mycobacteria, IFN secretion, and autophagy [31].
Another gene significantly upregulated by infection with rBCG-LTAK63 compared to BCG is the IFN-induced monokine-encoding gene, CXCL9, that is induced in response to IFN-γ and induces inflammation together with recruitment of activated lymphocytes [32,33]. CXCL9 is a pleiotropic molecule involved in innate and adaptive immune response mechanisms [34][35][36].
The higher protein secretion levels of IL-12(p70), TNF-β, and IL-15 displayed in rBCG-LTAK63-infected M1 are in agreement with the findings at the transcriptomic level in that infection with rBCG-LTAK63 enhanced the inflammatory and activated cellular immune response profile compared to BCG-infected M1 macrophages. IL-12(p70) is a proinflammatory cytokine that has been described to have an important role in the control of mycobacterial infections [37][38][39]. The proinflammatory cytokines, TNF-β and TNF-α, are homotrimers, and they have similar structures and functions [40,41]. It has also been reported that mycobacteria-infected macrophages secrete IL-15 [42]. It is mainly produced by macrophages and dendritic cells and acts in the recruitment of T lymphocytes, stimulating CD4 T cells [43] and specific subpopulations of memory lymphocytes, such as CD8 T cells [44,45]. In support of a possible role for IL-15 during rBCG-LTAK63 vaccination, we have previously shown that rBCG-LTAK63 induces CD4+ and CD8+ T cells in vivo [16]. However, the induction of memory and activation markers for T cells still need to be evaluated.
It is interesting to note the unique feature of the rBCG-LTAK63 induced transcriptomic signature, which separates it from BCG and Mtb, and which involves the strong induction of a large set of IFN signaling genes. Although excessive inflammation may be damaging to the host [46], it is evident that neither BCG nor Mtb induces an immune response that effectively eliminates Mtb. On the other hand, our previous results have shown that immunization with rBCG-LTAK63 induced reduced immunopathology in Mtb-infected mouse lungs [16]. Therefore, our results suggest that the superior induction of IFN signaling genes/proinflammatory cytokines by rBCG-LTAK63 correlates with, and may at least partly explain, the improved protection in mice.
Since M2 macrophages usually have an immunomodulatory phenotype, a different gene signature and cytokine production profile would be expected. While genes associated with an inflammatory profile were rapidly upregulated in mycobacteria-infected M1 macrophages (within 24 h), mycobacteria-infected M2 macrophages showed only few DEGs 24 h post-infection. Substantially more DEGs were identified at 48 h post-infection, suggesting that the default immunomodulatory profile of M2 macrophages delays the switch towards an inflammatory profile.
In M2 macrophages, genes associated with an inflammatory profile were more extensively upregulated by infection with rBCG-LTAK63, compared to infection with BCG, including the TAP1 and GBP1 genes, which are both part of the IFN signaling pathway. TAP1-transporter associated with antigen processing-1-is important for MHC-I function and has a key role in adaptive immunity [47,48]. GBP1-guanylate-binding protein-1 precursor-is part of the IFN-induced GTPase family. This protein is associated with protection against bacterial infections, host defense, and intracellular pathogen killing, including mycobacteria [49][50][51][52].
The levels of expression of the TNIP1, SLAMF7, and IL6 genes were also higher in M2 infected with rBCG-LTAK63 when compared to BCG. TNIP1 is one of the major regulators of the NF-Kβ signaling pathway and is implicated in cell inflammation [53]. A wide repertoire of functions is suggested for TNIP1, including modulation of cellular activation and enhancement of CD4 T-cell levels [54]. IL-6 is a pleiotropic cytokine, secreted by a variety of cell types, playing a role in inflammation, response to infections, and repair of cellular tissues [55]. SLAMF7 is a receptor present in NK cells and is related to cell activation [56,57]. Furthermore, the cytokine MCP-3 was produced at elevated levels in M2 macrophages infected with rBGC-LTAK63. The chemokine MCP-3 is structurally and functionally similar to MCP-1, and both are potent monocyte chemoattractants for T cells and NK cells [58]. On the whole, these results could suggest that rBCG-LTAK63 activated M2 cells may have a role in signaling to NK and CD4 T cells; however, the involvement of NK cells has not been investigated. The cytokine EGF was produced at elevated levels in M2 macrophages infected with rBGC-LTAK63 as compared to those infected with BCG. Epidermal growth factor (EGF) promotes proliferation, differentiation, survival, and repair in several cell types [59].
In conclusion, rBCG-LTAK63, when compared to BCG, induced a dominant inflammatory profile in M1 macrophages, while a combined inflammatory and repair profile was induced in M2 macrophages. In this study, we used three different mycobacterial strains: Mtb H37Rv, a virulent strain; BCG, the current attenuated strain used as a vaccine for TB; and the rBCG-LTAK63, a recombinant BCG strain that has been shown to confer increased protective properties against TB in a murine TB challenge model. In general, we observed only few differences between the attenuated BCG and the virulent Mtb strains, but this is in agreement with other studies that have shown that infection, even with inactivated pathogens, is able to activate immune responses [60]. Although this underlines the complexity of understanding the immune mechanisms related to pathogenicity or protection induced by mycobacteria, we clearly found distinct transcript and protein secretion profiles in human macrophages infected with rBCG-LTAK63 compared to cells infected with either BCG or Mtb. Previously, rBCG-LTAK63 vaccination was shown to increase the recruitment of neutrophils, macrophages, and lymphocytes and induce enhanced innate immune response with higher levels of mediators of macrophage activation (nitric oxide, hydrogen peroxide, and inflammatory cytokines) in vivo in a murine TB infection model [16]. Here, our data in human in vitro infection models indicate an important immunological profile induced in human macrophages by rBCG-LTAK63 ( Figure 5) that can be correlated with a protective immune response against TB. Further studies will be needed to confirm this hypothesis.
The distinct immunological profiles induced in human macrophages indicate that the mechanism of protection may be different from BCG and the currently studied vaccines, reinforcing the potential of rBCG-LTAK63 as a vaccine candidate. Vaccines 2022, 9, x 11 of 14 The distinct immunological profiles induced in human macrophages indicate that the mechanism of protection may be different from BCG and the currently studied vaccines, reinforcing the potential of rBCG-LTAK63 as a vaccine candidate.

Patents
I.P.N. and L.C.C.L. have a patent application involving rBCG-LTKA63 use as Mtb vaccine.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Kinetics of the intracellular bacilli survival and rBCG-LTAK63 stability in M1 and M2 macrophages infection; Figure S2: Genes analyzed by dcRT-MLPA and their predominant functions; Table S1: Statistical analysis of expression of all genes evaluated by dcRT-MLPA in M1 macrophages in response to infection with rBCG-LTAK63, BCG, or Mtb H37Rv compared to uninfected macrophages; Table S2: Statistical analysis of expression of all genes evaluated by dcRT-MLPA in M2 macrophages in response to infection with rBCG-LTAK63, BCG, or Mtb H37Rv compared to uninfected macrophages.  Informed Consent Statement: Monocytes were isolated from buffy coats obtained from Sanquin blood products (Amsterdam, the Netherlands), as approved by the medical ethical review board of

Patents
I.P.N. and L.C.C.L. have a patent application involving rBCG-LTKA63 use as Mtb vaccine.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/vaccines10060831/s1, Figure S1: Kinetics of the intracellular bacilli survival and rBCG-LTAK63 stability in M1 and M2 macrophages infection; Figure S2: Genes analyzed by dcRT-MLPA and their predominant functions; Table S1: Statistical analysis of expression of all genes evaluated by dcRT-MLPA in M1 macrophages in response to infection with rBCG-LTAK63, BCG, or Mtb H37Rv compared to uninfected macrophages; Table S2: Statistical analysis of expression of all genes evaluated by dcRT-MLPA in M2 macrophages in response to infection with rBCG-LTAK63, BCG, or Mtb H37Rv compared to uninfected macrophages.  Informed Consent Statement: Monocytes were isolated from buffy coats obtained from Sanquin blood products (Amsterdam, the Netherlands), as approved by the medical ethical review board of the LUMC. Healthy donors provided written informed consent for scientific use of their blood products.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.