This is a study of 282 human research participants for both in vivo BCG vaccine clinical trial studies (n = 52) and in vitro mechanistic studies (n = 230). Of these total research subjects 211 had type 1 diabetes and 71 were non-diabetic control subjects. The details of all subjects and corresponding clinical subject data are depicted in Supplementary Table S1.

Long-term and stable blood sugar reduction with BCG vaccinations

BCG-vaccinated T1Ds were compared to a placebo group as well as a reference group of T1Ds receiving standard of care at the Massachusetts General Hospital in part reported after 20 weeks in the Phase I double blinded clinical trial4 (Supplementary Table S1b). We extended this trial with 8 year long monitoring (Supplementary Table 1b—8 year long subjects) and additional in vivo BCG treated subjects (Supplementary Table S1a—up to 5 year long subjects).

The 8 year long followed and BCG-treated T1Ds showed a reduction in HbA1c levels of greater than 10% after year 03, 18% at year 04, and the HbA1c remained low for the next 5 years (p = 0.0002 at year 8) (Fig. 1a, b). Subject numbers and traits are given in Supplementary Table S1a, S2. In contrast, the placebo group (n = 3) and the reference T1D groups (n = 40) had consistently higher HbA1c over the entire monitoring period of 8 years and 5 years, respectively (Fig. 1a, b, Supplementary Table S2). The efficacy of BCG was also apparent with raw HbA1c values, which show the BCG-treated TIDs by year 8 declined to a HbA1c value of 6.65% (Supplementary Table S2, Fig. 1b). BCG treatment subjects with the improved and tight blood sugar control demonstrated blood sugar stability and lowered blood sugar persistently for 5 continuous years after the initial drop in values. Semi-annual surveys confirmed that during year 03 to year 08 after BCG vaccinations there were no reports of severe hypoglycemia by any patient, even with lowered HbA1Cs near the normal range, and no change in their care as it related to new insulin pumps or continuous glucose monitoring devices. The placebo group of subjects continued to show hypoglycemia events during the same time periods of monitoring.

Fig. 1 Long-term improvement of glycemic control in T1Ds after BCG treatment. a, b Glucose control was tracked through measurements of HbA1c. HbA1c levels in the control T1D groups (saline-treated placebo group n = 3 or the untreated reference group n = 40) remained unchanged over the 8-year observation period (placebo) or 5-year observation period (untreated reference group) as measured by a % change (a) or as raw HbA1c values (b) (p = 0.73). The percentage change was calculated from pre-trial values to post-trial values measured every 6 months or yearly. In contrast, a decrease in HbA1c for the 8 year long followed BCG-treated patients uniformly occurred after year 03 and thereafter showed sustained lowering, with an 18% decrease from baseline at year 04. After the drop in HbA1c values in the BCG-treated group, HbA1c values remained lower for the next 5 years of monitoring and was statistically different from placebo (p = 0.0002 at year 8) and from reference subjects (p = 0.02 at year 5). In short, relative (percent) change rate of HbA1c was compared using the linear mixed effects model with subject-level random effects. The change rates in the control, placebo and BCG groups were compared based on the statistical significance of the interaction term between time and group indicator in the linear mixed effects model. The subject traits and sample sizes are given in Supplementary Figure S1a, S1b. c An in vivo glucagon stimulation test was performed to induce pancreatic insulin secretion as measured with C-peptide assays at trial enrollment (baseline), at 12 weeks and at 208 weeks after two BCG vaccinations in 6 T1D subjects (Supplementary Table S1b—8 year long treated subjects). The BCG-treated patients showed a clinically negligible, but statistically significant, return of stimulated serum C-peptide levels upon glucagon administration only at 208 weeks (upper panel), whereas the C-peptide response to glucagon in the reference-T1D and placebo-T1D groups remained unchanged (lower panel). For the glucagon stimulation test statistics, we used the Wilcoxon Signed Rank test. On all 8 year followed subjects with the data presented at year 04 i.e., 208 weeks. Figure inserts at 208 weeks after treatment highlight the minor changes and the standard error bars Full size image

We presumed at this point that the return of near normoglycemia in the BCG treated human T1D (8 year long followed subjects) was by the same mechanism as was observed in the NOD mouse treated with BCG, i.e., restored insulin from pancreas regeneration. In the genetically prone non-obese diabetic (NOD) mouse model of type 1 diabetes, the mechanism of stable blood sugar restoration after BCG is driven in large part by the regeneration of insulin-secreting islets in the pancreas.17,18 As previously published, the elevations in tumor necrosis factor (TNF) from the BCG vaccine stimulate cytotoxic T cell death and beneficial Treg expansion.4,29,30 We sought proof of a similar mechanism in humans of pancreatic islet regeneration as the cause for restored blood sugar control by measuring endogenous insulin secretion through the co-secreted C-peptide levels. C-peptide is co-secreted with insulin from the pancreas and can be used to selectively detect the secretion of endogenous insulin. Insulin levels cannot be used to look for pancreas regeneration since all subjects take exogenous insulin.

Stimulated C-peptide was measured with a glucagon challenge in the BCG and placebo type 1 diabetic subject groups at three time points (pre-BCG, post-BCG 12 weeks, and 208 weeks) to look for pancreas recovery or regeneration (Fig. 1c, Supplementary Table S1b). C-peptide is co-secreted with insulin from the pancreas and can be used to selectively detect the secretion of endogenous insulin. Insulin levels cannot be used to look for pancreas regeneration since all subjects take exogenous insulin.

In this study we observe the long term and stable lowering of blood sugars in humans after BCG vaccinations. In the human, this stable blood sugar control was not driven primarily in these human subjects by pancreas recovery or regeneration. The human pancreas after BCG even at four years after repeat vaccinations did not secrete significant insulin as clinically measured by C-peptide. The mechanism for lowered HbA1c values was not equivalent to the NOD diabetic mouse pancreas regeneration after BCG treatment, despite equally restored and long term improved blood sugar control. The BCG-treated type 1 diabetic subjects at year 4 after glucagon challenge had a negligible to no return of clinically significant C-peptide. The C-peptide values after glucagon were in the range of 2–3 pmol/L of C-peptide (Fig. 1c), but with no known clinical significance. Therefore we concluded that BCG vaccinations did not induce a clinically meaningful return of C-peptide levels in the pancreas by regeneration, as observed in the NOD mouse model of diabetes17,18 Thus pancreas rescue or regeneration could not fully account for the persistent and long term HbA1c lowering in humans receiving BCG.

After BCG vaccinations, regulatory T cell signature genes are de-methylated in vivo resulting in enhanced mRNA expression

The beneficial effect of BCG in humans, as previously documented in mouse experiments, could be due to an induction of the beneficial Treg cells. The co-evolution of Mycobacterium and humans has resulted in Mycobacterium-modulated host cell machinery, including de-novo host gene expression by de-methylation of important immune response genes.31,32,33,34 Chronic infections by Mycobacterium evade host recognition on a cellular level by measurable increases in Treg cell numbers and cellular functions.35 Treg cells are believed deficient in numbers or function in diverse autoimmune diseases and induction through BCG therapy would be a first step in restoring the immune balance that has been quantified by only flow cytometric methods after BCG.4,36 Transcriptional start site (TSS) clusters are located within the Treg-specific demethylation region (TSDR) that is critical for Treg function and that are modulated by de-methylation as was monitored in this study (Supplementary Table S3).

BCG’s impact on methylation was studied at various methylation sites of the following Treg signature genes: Foxp3, TNFRSF18, IL2RA, IKZF2, IKZF4 and CTLA4. T1D subjects were studied before and after (at week 8) in vivo BCG dosing (Supplementary Table S1a). CD4 cells were isolated and genomic DNA was prepared, bisulphite-converted, and then analyzed on Illumina Infinium Human Methylation450 BeadChips (Fig. 2a, Supplementary Fig. S1). Results are expressed in Fig. 2a as change in methylation (after BCG treatment minus before BCG treatment) of the multiple methylation target sites for each gene represented on the BeadChip (Mean ± SEM) or as total change in methylation of all targets per gene (Supplementary Fig. S1). After BCG treatment, the majority of the targets of all six signature genes showed demethylation of most of the methylation control sites. For Foxp3, the false discovery rate (FDR) adjusted p-value was 0.004; for TNFRSF18, the FDR adjusted p-value was 0.0008; for CTLA4, the FDR adjusted p-value was >0.51; for IL2RA, the FDR adjusted p-value was 0.003; for IKZF2, the FDR adjusted p-value was 0.10; for IKZF4, the FDR adjusted p-value was 0.04.

Fig. 2 BCG treatment reduces DNA methylation and upregulates expression of Treg signature genes. a CD4 T cells were isolated from T1D patients before and after BCG treatment (n = 3 subjects; Supplementary Table S1a). DNA was isolated, bisulfite converted and analyzed on the Illumina Infinium HumanMethylation 450 BeadChip array. The data shows that after BCG treatment all six Treg signature genes are demethylated at multiple CpG methylation sites. For a table of the CpG sites used please see Supplementary Table S3. This data compares the methylation state in BCG-treated diabetics 8 weeks after administration of the two BCG vaccines against their pre-treatment baseline. For the Foxp3 gene, all nine methylation sites on the BeadChip were significantly demethylated after BCG treatment (FDR adjusted p = 0.004). For the TNFRSF18 gene (also known as GITR receptor), 16 out of 17 methylation sites on the Beadchip were demethylated after BCG and one site was unchanged (FDR adjusted p = 0.0008). For the IL2RA gene all 9 methylation sites on the chip showed decreases in methylation after BCG treatment (FDR adjusted p = 0.003). For the IKZF2 gene, also known as IKAROS family zinc finger 2 (Helios), there are 17 sites on the chip. After BCG treatment, 13 of those sites were de-methylated, 1 site showed augmented methylation, and 3 sites were unchanged. Overall, demethylation of the IKZF2 sites after BCG treatment was not statistically significant with FDR adjusted p = 0.106. For the IKZF4 gene, also known as IKAROS family zinc finger 4 (Eos), there are 11 methylation sites represented on the chip. After BCG treatment, 8 sites were de-methylated and 3 sites showed augmented methylation. Overall the IKZF4 sites were significantly demethylated after BCG treatment (FDR adjusted p = 0.038). For the CTLA4 gene, there were 7 sites represented on the chip. After BCG treatment, 5 sites were demethylated and 2 sites showed increases in methylation. Overall there was no significant difference in CTLA4 sites before and after BCG treatment (FDR adjusted p = 0.509). This data is from 3 subjects receiving BCG therapy (Supplementary Table S1a). b RNA was isolated from PBLs of three T1D before and after in vitro culture with BCG for 48 h and analyzed using RNAseq or transcription profiling. BCG treatment caused a sharp increase in the amount of mRNA as expressed by the number of RNAseq reads for each of the six Treg signature genes that promote Treg function and correlated with the de-methylation patterns Full size image

To confirm the observed in vivo epigenetic de-methlyation in the many Treg signature genes after BCG administration, we next profiled the ability of BCG to reciprocally turn on mRNA levels of the same genes. Using transcription profiling we confirmed that the BCG-induced demethylation at the gene level did cause increased mRNA expression of the Treg signature genes; the methylation trends inversely correlated with the mRNA expression levels (Fig. 2b). T1D peripheral blood lymphocytes (PBLs) were cultured for 48 h. with and without BCG. For comparison, averaged data across all methylation sites per Treg signature gene also inversely correlated with increased mRNA for the gene products (Supplementary Fig S1).

A novel mechanism for treatment of hyperglycemia: induction of the early steps of aerobic glycolysis

Two clinical methods are typically used for improved blood sugar control: increased insulin delivery or decreased peripheral insulin resistance. Because long-term monitoring after BCG treatment failed to show a substantial increase in pancreatic insulin secretion (Fig. 1c), we sought evidence for BCG-induced change in peripheral insulin resistance equivalent to the commercially available Quantose™ IR test (Metabolon, Morrisville, NC). BCG-treated diabetic samples were studied before BCG and up until 4 years after BCG treatment looking for a decrease in insulin resistance. (Supplementary Table S1b; 8 year long followed subjects). In the commercially available Quantose IR test, four metabolites were quantified that are indicative of changes in insulin resistance: alpha-hydroxybutyrate (α-HB), also named 2-hydroxybutyrate, 1 and 2-linoleoyl-glycerophospocholine (L-GPC), and oleic acid.37 We therefore analyzed our metabolomics data for changes in these 4 insulin resistance markers. Lowered α-HB is associated with decreased insulin resistance. In our subjects average pre-BCG values were not significantly different from post-BCG values at any time during weeks 4 through year 04 after BCG vaccinations (1.14 ± 0.08 vs. 1.09 ± 0.10; p = 0.34). L-GPC is negatively correlated with insulin resistance and impaired glucose tolerance. Pre-treatment diabetic values for 1-LGPC and 2-LGPC were 1.03 ± 0.05 and 1.07 ± 0.05, respectively, whereas post-BCG treatment values were 1.120 ± 0.07 and 1.14 ± 0.07 (p = 0.16 and 0.20). Oleic acid is positively correlated with increased lipolysis and insulin resistance. The average pre-BCG value was 1.18 ± 0.10 and post-BCG was 1.38 ± 0.16 (p = 0.15). Since the differences for all 4 metabolites were thus not significant, it was unlikely that decreased insulin resistance defined at least from this screening test could explain the reduction in HbA1c due to BCG vaccinations.

We then investigated a full panel of serum metabolites in BCG-treated and Placebo T1Ds (n = 6; sampled bi-weekly from 7 to 20 weeks and then yearly through year 05 compared to untreated T1D diabetic and non-diabetic control subjects (Supplementary Table S1b,c; 8 year long followed subjects). We found two categories of metabolites significantly altered by BCG treatment as compared to the placebo group: glucose processing metabolites and pathway intermediates for de novo purine synthesis (Fig. 3a, b). 1,5-Anhydroglucitol, a sugar metabolite known to increase with lower blood sugar, was lower in untreated T1Ds compared to non-diabetic controls (p < 0.001, q = 0.001). After BCG treatment it showed a significant increase compared to untreated T1Ds (Fig. 3a, b, p = 0.007, q < 0.001). Sugar metabolite alpha-ketobutyrate is known to be higher in T1D as compared to non-diabetic controls; we observed this as well (p = .026, q < 0.001). After BCG treatment alpha-ketobutyrate levels were even slightly lower than control suggesting a meaningful lowering of blood sugars in those cohorts (p = 0.008, q < 0.001). To demonstrate that the changes after BCG in glucose metabolism and purine synthesis were likely due to the BCG vaccine, the same comparisons of metabolites from the placebo T1D compared to untreated T1D yielded no statistically significant trends toward corrections thus correlating the specificity of the metabolic shifts to BCG vaccinations (Fig. 3b). The T1D to T1D post-placebo comparison for alpha-ketobutyrate yielded a significant p and q value but this was due to the placebo group having a very low and uncorrected level of this metabolite. While the age of the untreated T1D was slightly shorter (34+/−2 years) compared to the untreated nondiabetic controls (39+/−2 years), all other clinical parameters of age of onset, duration of diabetes and age were closely matched for the BCG-treated T1D subjects compared to the T1D placebo subjects (Supplementary Table S1c).

Fig. 3 BCG treatment switches cellular metabolism from oxidative phosphorylation to early aerobic glycolysis. a, b Metabolomic comparisons of the relative levels of intermediates of glucose metabolism and purine synthesis for non-diabetic controls, untreated T1D subjects and T1D patients after treatment with BCG or placebo (sampled biweekly from week 7 to 20 and then yearly through year 5). The results indicate that glucose metabolism is shifted towards aerobic glycolysis in the BCG treated T1D. Asterisks indicate statistically significant differences, which are listed in Fig. 3b. For all metabolomics data, we used an unpaired one-tail Student’s t-test that was then corrected for the multiple comparisons with p and q values. p values are given in Fig. 3b. Note that q values maintained significance for the T1D results for the BCG treated cohorts. c The systemic lowering of blood sugars in T1Ds after BCG vaccines combined with the increased glucose uptake and purine synthesis is consistent with BCG switching cellular metabolism to early aerobic glycolysis. This hypothesis holds that BCG causes downregulation of the Krebs cycle, accelerated aerobic glycolysis, increased glucose uptake, and shunting of glucose to the Pentose Phosphate Shunt for augmented purine biosynthesis Full size image

Three purine intermediates measured by mass spectrometry also showed changes after BCG treatment (Fig. 3a, b, Supplementary Table S1c). Adenine was lower in untreated T1Ds compared to controls (p = 0.002, q = 0.002). After BCG treatment, adenine levels were closer to normal and were significantly higher than untreated T1D levels (p = 0.029, q = 0.001). The same trends were observed for two additional purine synthesis intermediates: N6-carbamoylthreonyladenosine (p = 0.003, q = 0.0005) and methylguanine (p < 0.001, q = 0.0006) that were both lower in untreated T1D compared to controls. After BCG treatment, N6-carbamoylthreonyladenosine levels were closer to normal and were significantly higher than untreated T1D levels (p = 0.013, q = 0.001). After BCG treatment, methylguanine levels were closer to normal and were significantly higher than untreated T1D levels (p = 0.014, q = 0.001). The same comparisons were done at the same monitoring time points for the simultaneously studied placebo subjects and no significant changes in purines were observed (Fig. 3b; Supplementary Table S4).

The finding that BCG treatment improved glucose metabolism and augmented purine biosynthesis suggests a novel mechanism to explain the BCG-induced reduction of HbA1c: a cellular switch from primarily oxidative phosphorylation, a low glucose utilization state, to augmented early aerobic glycolysis, a high glucose utilization state associated with high purine metabolism (Fig. 3c). It is known that aerobic glycolysis is active at the site of many local infections with low oxygen, including tuberculosis, but not on a systemic level. Indeed the local environment of infections often have augmented glucose utilization and enhanced purine synthesis.38 Aerobic glycolysis fuels a dramatic uptake of glucose through regulated cell surface glucose transport and secondary utilization of the pentose phosphate shunt for increased purine synthesis. Our data enabled us to test the hypothesis that the apparently long-term lowering of blood sugars after BCG, even in advanced diabetes, was due to BCG’s systemic induction of aerobic glycolysis and switch from an overactive oxidative phosphorylation metabolism (Fig. 3c).

To help demonstrate that the BCG vaccine was inducing a state of augmented aerobic glycolysis resulting in higher systemic glucose utilization, we examined PBLs and monocyte cultures at baseline and after brief BCG exposures (48 h) for induction of Hypoxia-Inducible Factor 1 Alpha subunit (HIF1A). HIF1A is a master transcription factor that regulates the conversion to high aerobic glycolysis states.39 We found that, after brief BCG exposures in vitro, HIF1A mRNA was rapidly upregulated in both PBLs and monocytes of both T1Ds and controls compared to baseline (Supplementary Fig. S2). While this was not a functional outcome, the findings supported our hypothesis of a BCG-mediated switch to augmented aerobic glycolysis on a systemic level similar to what has been observed at local infection sites in the lungs for tuberculosis.40

Further corroboration of the concept that BCG treatment switches cellular metabolism towards aerobic glycolysis came from transcription profiling studies (mRNAseq) performed on freshly isolated lymphocytes from T1Ds after BCG treatment. Figure 4b shows the sequential steps of the pathway of glucose metabolism from transport across the cell membrane, early glycolysis, late glycolysis and finally entry into the Krebs cycle both as it relates to enzymes and to metabolites. We examined mRNA for alterations in key enzymes of glucose metabolism in vitro and also for corresponding changes for in vivo metabolites after BCG vaccinations (Fig. 4).

Fig. 4 Analysis of mRNA expression and of Metabolites corroborates with the switch from the Krebs Cycle to augmented early aerobic glycolysis after BCG. a mRNA expression analysis of type 1 diabetic PBLs before and after BCG treatment in vitro. BCG causes upregulation of mRNA for early glycolysis (HK2, PFKB3), downregulation for late glycolysis (ALDOAP2, PGM1), and a strong downregulation of mRNA for late Krebs cycle steps (bottom). p and q values for the genes shown are HK2 (p = 0.049, q = 0.017), PFKB3 (p = 0.016, q = 0.017), ALDOAP2 (p = 0.113, q = 0.017), PGM1 (p = 0.022, q = 0.017), DLST (p = 0.074, q = 0.017), IDH3B (p = 0.084, q = 0.017), IDH3G (p = 0.084, q = 0.017), MDH2 (p = 0.090, q = 0.017) and OGDH (p = 0.070, q = 0.017). Combined p values for the Krebs cycle genes using Fisher’s method is 0.005. b The schematic summarizes the normal pathway for glucose oxidation via the Krebs cycle and the connecting nodes to the Pentose Phosphate Shunt. Blue rectangles and blue ovals represent upregulated metabolites and mRNA, respectively, after BCG. Gray rectangles and gray ovals represent downregulated metabolites and mRNA, respectively, after BCG. Three BCG-treated T1D subjects were studied. Bars on Fig. 4a, c, d depict SD. The blue shading in Fig. 4b represents the upregulated mRNAs (ovals) and metabolites (rectangles). c Serum lactate levels in Phase 1 BCG-treated vs. placebo patients as determined by Metabolon’s GC/HPLC and MassSpec platform one year after treatment. Lactate levels were significantly higher in the BCG-treated patients than placebo-treated patients (p = 0.001 and q = 0.003) (data from subjects represented in Supplementary Table S1e). d Cultured CD4 cells from T1D subjects in the presence or absence of BCG for 48 h (n = 25 paired samples) showed both augmented lactate production and accelerated glucose uptake. A short 4-hour collection time of media from either glucose uptake or for lactate secretion after 48 h of BCG or media exposures (control) showed significantly more lactate production after BCG exposures (p = 0.025, n = 25) and accelerated glucose consumption (p = 0.02, n = 27) as compared to control lymphocytes that were cultured without BCG Full size image

T1D PBLs (n = 6 samples; 3 paired samples before vs. after in vitro BCG) were isolated and cultured in the presence of BCG in culture for 48 h. Cells were collected before and after BCG and analyzed by transcription profiling. BCG upregulates expression of early glucose transporters and enzymes involved in early glycolysis (HK2, PFKFB3) (Fig. 4a). BCG downregulates enyzmes involved in late glycolysis (ALDOAP2, PGM1) (Fig. 4a). Consistent with a switch from oxidative phosphorylation to high aerobic glycolysis, Krebs cycle enzymes were also down-regulated (DLST, IDH3B, IDH3G, MDH2, OGDH) (overall Fisher’s p-value of 0.005) (Fig. 4a). This data supports a BCG-driven process of a shift in energy metabolism. To further confirm this shift to accelerated glucose utilization through aerobic glycolysis, we studied in vivo serum lactate production and in vitro cellular lactate production and glucose uptake rates after BCG exposures to cultured lymphocytes. Serum lactate levels in Phase I BCG-treated subjects (n = 3) vs. untreated T1D (n = 50) was determined with mass spectrometry analysis up to the one year time point compared to baseline values. The post-treatment lactate levels rose more in BCG-treated subjects as compared to T1D subjects that received placebo vaccines (p = 0.001) (Fig. 4c). This metabolic switch after BCG to aerobic glycolysis could also be observed in vitro (Fig. 4d). T1D PBLs incubated with BCG for 48 h followed by a fresh 4-h collection of media again demonstrated more lactate production (p = 0.025, n = 25 paired samples) and also accelerated glucose uptake even with even a short 4 h monitoring time (p = 0.02, n = 27 paired samples). Taken together these results suggest BCG both in vitro and in vivo activated early steps in glucose transport and downregulated the Krebs cycle. This creates in BCG-treated T1Ds an increase in serum lactate. In culture with BCG, lactate was higher as well as the rapid increase in glucose uptake into the T1D lymphoid cells.

A consequence of elevated or restored early aerobic glycolysis should be greater utilization of the pentose phosphate shunt (Fig. 5a, Supplementary Fig. S3). The pentose phosphate shunt yields increased synthesis of purine and pyrimidine metabolites.41,42 Our transcription profiling of 48-hour BCG-treated PBLs from T1D subjects showed upregulation of the pentose phosphate shunt consistent with the in vivo metabolomics data (Fig. 5a). There are changes in gene expression for positive regulators and negative regulators of the pentose phosphate shunt, as well as downstream enzymes that divert the pentose phosphate pathway away from purines. A schematic of this pathway and connecting nodes is provided (Supplementary Fig. S3). Seven positive pentose phosphate shunt protein regulators were upregulated (ATM, HSP27, PI3K, SRC, SREBP, K-ras, and TAp73) after BCG treatment of T1D lymphocytes. Fisher’s method combined p-value for the positive regulators was 0.005. For example, TAp73 was upregulated by more than 650%. Increases in expression of positive regulators suggest increased throughput of glucose via the pentose phosphate pathway. In contrast, gene expression for negative regulators, such as PTEN of the pentose phosphate shunt, was almost two fold lower (p = 0.06), and the downstream pentose phosphate shunt also appeared to be downregulated, consistent with a switch to aerobic glycolysis and diversion towards purine pathways.

Fig. 5 Effect of BCG on induction of the Pentose Phosphate shunt pathway and resulting purine and pyrimidine synthesis. a mRNA expression analysis of T1D PBLs treated in vitro with BCG. The graph shows the percent change in gene expression before vs. after in vitro treatment with BCG for 48 h. Positive regulators are mostly upregulated, whereas negative regulators and downstream mediators are mostly downregulated. Three BCG-treated T1D subjects were studied. Bars on the Fig. 5 represent means + /− SEM. Fisher’s Method combined p-value for the positive regulators is 0.005. The p-values for PTEN in the negative regulators and for TKT in the downstream regulators are almost significant at 0.06 and 0.08, respectively. b In vivo metabolomics data of BCG-treated T1D supports the BCG-induced utilization of the Pentose Phosphate Shunt and shows that purine and pyrimidine metabolism are upregulated after BCG treatment. This is consistent with accelerated pentose shunt utilization. Data shows metabolite levels in serum from CTRL (non-diabetic controls) (n = 25), T1D subjects (n = 50) and post-BCG T1D (n = 3 subjects after treatment with two BCG vaccinations). The asterisks represents statistical differences of <0.05; actual statistical values for both p and q values are given in Supplementary Table S4 Full size image

We studied serum metabolites of BCG-treated T1Ds to confirm the mRNA expression findings of augmented pentose phosphate shunt utilization and increased purine and pyrimidine metabolites (Fig. 5b, Supplementary Table S1c). Non-diabetic control subjects have significantly higher serum levels of purine and pyrimidine intermediates than untreated TIDs (Fig. 5b, Supplementary Table S4). After two vaccinations of BCG in T1D subjects almost all purine intermediates increased. In the purine pathway, adenine, N6-carbamoylthreonyladenosine, 7-methylguanine and N2,N2-dimethylguanosine all statistically showed significant increases in BCG-treated T1Ds compared to untreated T1Ds (p = 0.029, q = 0.001; p = 0.013, q = < 0.001; p = 0.014, q = < 0.001; p = 0.002, q = <0.001, respectively). Allantoin and N1-methyladenosine also rose in BCG-treated T1Ds, but not significantly. Pseudouridine, a member of the pyrimidine metabolic pathway, was significantly lower in the untreated T1D group as compared to the non-diabetic controls, again suggesting underutilization of early aerobic glycolysis (p < 0.001, q = 0.001). After BCG, pseudouridine showed a small increase in BCG-treated as compared to untreated T1D that approached statistically significance (p = 0.057, q = 0.002). p and q values for all comparisons are listed in Supplementary Table S4. The BCG-induced switch to high glucose transport and shunting to the pentose phosphate pathway is illustrated in Supplementary Fig. S3 as it relates to the monitored proteins (ovals) and metabolites (rectangles) of this pathway.

BCG-induced aerobic glycolysis: implications for all forms of hyperglycemia

We next tested the hypothesis that BCG should be able to lower blood sugars regardless of the cause of hyperglycemia. The data presented above suggests a novel way to systemically regulate blood sugars, independent of insulin. This novel approach for blood sugar regulation appears to hinge on a systemic switch from primarily oxidative phosphylation to early aerobic glycolysis resulting in the acceleration of glucose uptake. If this novel mechanism of systemic blood sugar control is driving the return towards normoglycemia after BCG treatment, the data would suggest that this mechanism is independent of the underlying etiology of hyperglycemia—in this case autoimmune type 1 diabetes. Autoimmunity was not essential for BCG to lower blood sugars.

To test the hypothesis that BCG in vivo can induce a switch to systemic aerobic glycolysis with clinical significance, i.e., sufficient to lower blood sugars, we turned to murine testing. We used Streptozotocin (STZ) as a chemical that selectively destroys the insulin secreting islet β-cells in the pancreas to induce hyperglycemia as a way to elevate blood sugars and test for glucose utilization. We wanted to determine the impact of BCG vaccinations in normal mice with and without hyperglycemia and in the absence of autoimmunity. Normal C57BL/6 mice (n = 24) were divided into two groups. One group (n = 12) was treated with 0.1 mg BCG whereas the other group (n = 12) remained untreated. For this series of experiments all the mice remained normoglycemic. Weight and blood sugars were determined weekly for 6 weeks. Figure 6a (upper left) shows that BCG-treated mice gained weight at the same rate as controls. Likewise, blood sugars in the BCG-treated mice were indistinguishable from controls (Fig. 6a, lower left). Thus BCG alone affected neither weight nor glycemic control in healthy mice.

Fig. 6 BCG pre-administration reduces hyperglycemia in chemically-induced (Streptozocin) mice but does not induce hypoglycemia in normal mice. a Normal BALB/c mice were first studied in a normoglycemic state with (n = 12) and without BCG (n = 12) treatment for blood sugars and weight (left panels). BALB/c mice were rendered chemically diabetic (arrows) and studied with and without BCG treatment six weeks earlier with preventative pre-injections (right panels). Most mice became severely hyperglycemic after treatment with streptozocin (STZ) which selectively kills the insulin-secreting cells in the pancreas. All mice were monitored for blood sugar levels and weighed on a weekly basis. BCG-treated mice gained weight at the same rate as untreated control mice and had normal blood sugars with no indication of hypoglycemia (left panels, blue lines). After STZ induction of hyperglycemia, the control mice rapidly started to lose weight and became severely hyperglycemic within one week (right, black lines). In contrast, mice first treated with BCG before STZ treatment were able to maintain their weight and had markedly lower levels of hyperglycemia (right, blue line). b Measurements of HbA1c values in STZ-treated BALB/c mice after 6 weeks with and without prior BCG treatment show the protection afforded by BCG and the resulting lower HbA1c values of 85 ± 6.6 mmol/mol (9.9 ± 0.6% NGSP) without BCG vs. 67 ± 5.5 mmol/mol (8.3 ± 0.5% NGSP) with BCG treatment; p = 0.02, n = 19 surviving mice). c At 8 weeks after the induction of hyperglycemia, the BCG-treated mice had statistically lowered HbA1c values Full size image

We tested the effects of hyperglycemia induction with STZ when one group of mice received BCG (n = 12) and one group of mice received saline (n = 12) six weeks earlier (Fig. 6b). Control mice given only STZ rapidly started to lose weight (Fig. 6b, upper right) and became severely hyperglycemic (lower right). In contrast, the BCG-treated mice maintained their weight at a constant level (upper right; p < .0001) and their blood sugars did not rise as high as the controls and plateaued at a much lower level (Fig. 6b, lower right; p = 0.002). At week 8 after STZ treatment, the BCG-treated mice had a significantly lower HbA1C as compared to the control mice (Fig. 6b, p = 0.02). It should be noted, that the at least 6 week dosing lag in the administration of BCG prior to a glucose challenge was obligatory to see the maximal protective effect of improved blood sugar control. BCG can thus significantly lower blood sugars without underlying autoimmunity, and BCG has no deleterious effect by lowering blood sugars lower than normal. BCG treatment does not carry the risk of hypoglycemia as is the case for intense insulin therapy.