Metabolic effects of therapy with anti-IL-6 or anti-IL-17A alone and in combination with anti-TCR in a double or triple fashion

Anti-IL-6 (0.01 mg/kg b.wt., for 5 days) or anti-IL-17A (0.1 mg/kg b.wt., for 5 days) were administered to IDDM rats within 1 day after diabetes manifestation (blood glucose > 7.5 mmol/l), either alone or in combination with anti-TCR (0.5 mg/kg b.wt., for 5 days) in a double or triple fashion. Anti-TCR therapy in combination with anti-IL-6 (Fig. 1a) or anti-IL-17A (Fig. 1b) resulted after both therapies in a return to normoglycaemia (defined as blood glucose < 7.0 mmol/l in comparison to the diabetic and the normoglycaemic healthy control animals) (Fig. 1d) after a 60-day therapy-free interval following the end of the 5-day antibody therapy course of the rats for anti-IL-6 (5 out of 7 rats) (Fig. 1a) and for anti-IL-17A (7 out of 10 rats) (Fig. 1b). The improvement was particularly pronounced after the triple combination treatment (6 out of 10 rats for anti-IL-6 and anti-IL-17A) reaching a mean blood glucose value of 5.8 mmol/l at day 65 (Fig. 1c) being not significantly different from the mean blood glucose value of 5.4 mmol/l at the same time point in the healthy control rats (Fig. 1d). In contrast, diabetic rats without therapy remained severely hyperglycaemic (Fig. 1d). No adverse events were observed during and after the treatment.

Fig. 1 Effects of anti-TCR combination therapies on the metabolic profile of IDDM rats after diabetes manifestation. a–d Blood glucose concentration (mmol/l) changes are shown for the responding rats in response to the different anti-TCR combination therapies a with anti-IL-6 (5/7), b anti-IL-17A (7/10) or c in the triple combination (6/10) compared to d the normoglycaemic healthy (n = 6) and to the acutely diabetic untreated IDDM rats (n = 6). The first dashed line at day 0 indicates the start of therapy (first biopsy) and the second dashed line at day 5 indicates the end of therapy (second biopsy). e Serum C-peptide concentration changes (pmol/l) are shown for rats responding and non-responding to the different combination therapies of anti-TCR with anti-IL-6 or anti-IL-17A alone or in combination. Data are mean values ± SEM. Comparison of the different experimental groups by one-way ANOVA followed by Bonferroni test ***p < 0.001 to the healthy control, $p < 0.05 to anti-TCR combination with anti-IL-6, §§§p < 0.001 to anti-TCR combination with anti-IL-17A, and ###p < 0.001 to triple combination for each observation time point Full size image

In the successfully treated animals with the different anti-TCR combination therapies with anti-IL-6 or anti-IL-17A alone or in a triple fashion serum C-peptide concentrations increased (Fig. 1e). Sixty days after the end of therapy, C-peptide values were more than doubled (p < 0.01) compared to the values of the diabetic animals (ranging between 200 and 300 pmol/l) before the start of therapy.

Improvement of C-peptide values was better in the combination with anti-IL-17A (757 ± 57 pmol/l) than with anti-IL-6 (615 ± 68 pmol/l). The greatest improvement of C-peptide values was achieved with the triple combination reaching values (903 ± 30 pmol/l) that were close to the C-peptide concentrations in the healthy control rats (981 ± 22 pmol/l) (Fig. 1e).

The different combination therapies were initiated within 1 day after diabetes onset at blood glucose concentrations > 7.5 mmol/l. A detailed analysis revealed interesting results regarding therapy success as documented by an increased β cell mass approaching the control values of the non-diabetic animals (around 6 mg) in relation to the initial blood glucose concentrations (Fig. 2). The exception was observed after the anti-TCR combination therapy with anti-IL-6 where maximal β cell mass values of no more than 4 mg were achieved even at moderately increased starting blood glucose concentrations in the diabetic rats and even this modest improvement was not achieved in all treated animals (Fig. 2a).

Fig. 2 Relation between initial blood glucose concentration and β cell mass after end of therapy. a After anti-TCR combination therapy with anti-IL-6 or b with anti-IL-17A or c with both cytokine antibodies together. The β cell mass showed the highest values after triple combination, followed by the double combination with anti-IL-17A and the lowest values after double combination with anti-IL-6. Remarkably, the starting blood glucose concentrations granting therapy success differed between the three analysed groups without a blood glucose concentration window for anti-TCR combination with anti-IL-6 and with starting blood glucose concentrations below 13 mmol/l for the anti-TCR combination with anti-IL-17A and 17 mmol/l for the triple combination compared to those above these glucose values Full size image

In the double combination therapy group of anti-TCR with anti-IL-17A, β cell mass values were raised without exception to values in the range between 5 and 6 mg when treatment was initiated at blood glucose values below 13 mmol/l (Fig. 2b). At higher starting blood glucose values therapy was unsuccessful (Fig. 2b).

In the triple combination therapy of anti-TCR with anti-IL-6 plus anti-IL-17A, therapy was successful at all starting blood glucose concentrations below 17 mmol/l, reaching again β cell mass values in the range between 5 and 6 mg, which were comparable to those in healthy control rats (Fig. 2c).

Animals responding with a partial increase of the beta cell mass after treatment with anti-TCR plus anti-IL-6 (Fig. 2a) were nevertheless unable to achieve sustained normoglycaemia. Though therapy was initiated at a blood glucose concentration of 9.2 ± 1.3 mmol/l (n = 2) and ended after 5 days of therapy at a blood glucose concentration of 8.2 ± 0.4 mmol/l (n = 2), the blood glucose concentration 60 days after therapy was in the hyperglycaemic range with a value of 19.6 ± 1.7 mmol/l (n = 2) due to an insufficient maintenance of the insulin secretory capacity of the beta cells as documented by the fact that the serum C-peptide levels did not increase above levels in diabetic animals (Fig. 1e). Thus, these animals are depicted as non-responders with respect to C-peptide levels (Fig. 1e) and depicted as partial responders with respect to a limited beta cell mass increase (Fig. 2a).

This is in contrast to the partial responders in the triple combination treatment (anti-TCR plus anti-IL-6 and anti-IL-17), where the glucose concentrations showed a partial reduction of hyperglycaemia from an initial high blood glucose concentration of 18.1 ± 0.3 mmol/l (n = 2) to a value to 9.1 ± 0.2 mmol/l (n = 2) and a value of 12.3 ± 3.3 mmol/l (n = 2) 60 days after therapy along with a partial increase of the beta cell mass (Fig. 2c) and a partial increase of the C-peptide levels (Fig. 1e).

In rats (n = 4 in each group) with monotherapies with anti-TCR, anti-IL-6, or anti-IL-17 as well as the combination of anti-IL-6 plus anti-IL-17 (Additional file 2: Figures S1 and Additional file 3: Figure S2) in a fashion analogous to the double and triple combinations with anti-TCR (Figs. 1 and 2), no therapy success could be achieved after diabetes manifestation with blood glucose concentrations remaining in a permanent hyperglycaemic state (> 15.0 mmol/l) during and up to the end of the observation period of 10 days after start of the treatment (Additional file 2: Figure S1a-d). All treatments were also completely ineffective in raising C-peptide concentrations above levels of diabetic animals (Additional file 2: Figure S1e). The beta cell mass of all animals in these groups did not respond to treatment remaining in a very low range in all groups (< 0.5 mg) typical for diabetic animals (Additional file 3: Figure S2a-d).

Morphometric quantification of therapeutic effects on β cells and pancreatic islet infiltration after combination therapies

Changes of proliferation and apoptosis rates in β cells

At the day of diabetes manifestation, immediately before the start of therapy at the time point of the first biopsy, the rats in the therapy groups with anti-TCR in combination with anti-IL-6 and with anti-IL-17 as well as with both anti-cytokine antibodies showed significant 6–9-fold increases of the proliferation rates analysed by Ki67 staining compared to normoglycaemic controls (Fig. 3a). The apoptosis rates analysed by TUNEL increased 24–28-fold compared to normoglycaemic controls (Fig. 3b).

Fig. 3 Morphometric analyses of β cells and immune cells in IDDM rats after anti-TCR combination therapies. Changes in the rate of a proliferation, b apoptosis, c islet infiltration score and d pancreatic β cell mass after successful anti-TCR combination therapy with anti-IL-6 or anti-IL-17A alone or combined together after diabetes manifestation. Measurements were performed immediately before therapy (first biopsy), at the end of therapy (second biopsy) and 60 days after the end of therapy Data are mean values ± SEM. Comparison of the different experimental groups by one-way ANOVA followed by Bonferroni test ***p < 0.001, **p < 0.01 and *p < 0.05 to the healthy control, $$$p < 0.001 to anti-TCR combination with anti-IL-6, §§§p < 0.001 and §§p < 0.01 to anti-TCR combination with anti-IL-17A, ###p < 0.001 and ##p < 0.01 to triple combination for each observation time point. Numbers of pancreases analysed as given in Fig. 1 Full size image

Immediately after the end of the different combination therapies at the time point of the second biopsy, only the anti-TCR combination therapy together with anti-IL-6 or with anti-IL-6 plus anti-IL-17A showed a further increase of the proliferation rate (Fig. 3a). In all anti-TCR combination therapies with anti-IL-6 or anti-IL-17A alone or with both antibodies the apoptosis rate was reduced by around 50% already at the end of the 5-day therapy (Fig. 3b).

At 60 days after the end of therapy, rats successfully treated with the combination of anti-TCR with anti-IL-6 alone or in the triple therapy showed still a slight doubled proliferation rate whereas in the anti-TCR combination with anti-IL-17A the proliferation rate remained significantly increased by a factor of 4 as compared to healthy controls (Fig. 3a). In the triple combination therapy or in the anti-TCR combination with anti-IL-6 the apoptosis rate was no more significantly increased when compared to healthy controls whereas the anti-TCR combination with anti-IL-17A showed still a significantly increased apoptosis rate compared also to the triple combination (Fig. 3b).

Changes in proliferation/apoptosis ratios in β cells

Calculation of the proliferation/apoptosis ratios revealed a decrease to half of the values of the healthy controls in both double combination therapies; only in rats responding to the triple combination therapy the ratio was identical to that in the healthy controls (Additional file 1: Table S4).

Infiltration score

On the day of diabetes manifestation, before the start of therapy, the infiltration score of the islets was high with values above 2.5 for all combination therapies (Fig. 3c). The insulitis score was not significantly reduced after the end of all combination therapies (Fig. 3c). At 60 days after the end of therapy, the infiltration score in the regenerated endocrine pancreases was reduced to values < 1.0 for the anti-TCR combination with anti-IL-6 as well as the triple combination and < 2.0 for the anti-TCR combination with anti-IL-17A (Fig. 3c).

β cell mass

On the day of diabetes manifestation, before the start of therapy, the β cell mass of the pancreases was reduced in all diabetic rats to around 1/3 of the value in the controls (Fig. 3d). Immediately after the end of the anti-TCR combination therapies with anti-IL-6 and anti-IL-17A alone or in the triple fashion, the pancreatic β cell mass was moderately increased to half of the values in healthy control rats (Fig. 3d). Sixty days after the end of the different combination therapies with anti-IL-17A, the β cell mass had attained values in the normal range (Fig. 3d) whereas the β cell mass in the anti-TCR combination with anti-IL-6 reached only around 2/3 of the control values (Fig. 3d).

In summary, thus when compared to the pre-treatment situation, the β cell mass of the pancreases (Fig. 3d) was increased and the rates of proliferation, apoptosis and islet infiltration were reduced (Fig. 3a–c) 60 days after the end of therapy to levels comparable to the healthy control situation. On the other hand, in the monotherapy treatment groups, positive effects were observed at the end of the observation period neither on the mass of the beta cells nor on proliferation, apoptosis and insulitis score (Additional file 4: Figure S3a-d). This is in clear contrast to the positive effects of the combination therapies presented in Fig. 3.

Effects of therapies on the immune cell infiltration pattern in pancreatic islets

In the diabetic control rats, the islet infiltrate (Fig. 4a) was composed of around 37% CD8 T cells and 45% CD68 macrophages, with a smaller amount of 4% CD4 and 4% of γ,δ T cells each as well as 10% of other immune cell types in the acutely diabetic rats (Table 1).

Fig. 4 Immune cell infiltration in pancreatic islets of IDDM rats after successful anti-TCR combination therapies. a–d β Cells (green) and immune cells (red) were examined in islets from animals successfully treated with anti-TCR and anti-IL-6 (b) or with anti-TCR and anti-IL-17A (c) or with anti-TCR and both cytokine antibodies (d) after diabetes manifestation and compared to the untreated diabetic situation (a). Islets were immunostained for insulin (green) and CD68 macrophages (red), CD8 T cells (red), or γ,δ T cells (red) and counterstained with DAPI (blue). Erythrocytes were identified by yellow to orange colour through auto-fluorescence in the red and green channel. In each group, 40 to 80 islets in the pancreases were analysed Full size image

Table 1 Presence of different immune cell types in the pancreatic islets Full size table

Sixty days after the end of the different combination therapies, islet immune cell infiltration in the responding rats was very markedly reduced (Fig. 4b–d and Table 1). After anti-TCR combination therapy with anti-IL-6, only a very few γ,δ T cells and CD68 macrophages were still residing in the periphery of the islets (Fig. 4b and Table 1). After anti-TCR combination with anti-IL-17A, very few CD4 T cells and some CD8 T cells and CD68 macrophages were still visible inside and around the islets along with a complete absence of γ,δ T cells (Fig. 4c and Table 1). In view of this islet immune cell infiltration, the insulitis score (Fig. 3c) remained distinctly higher in the combination with anti-IL-17A than with anti-IL-6. The triple combination resulted in contrast to the double combination therapies in a complete disappearance of all T cell subtypes in islets (Fig. 4d and Table 1). Only very occasionally single infiltrating CD68 macrophages were still detectable in the islets after triple combination therapy in a quantity identical to the small number of in the healthy control pancreas (Table 1). These CD68 macrophages are not activated (i.e. not a site of pro-inflammatory cytokine production) and act as scavengers for cell debris.

Changes in the gene expression pattern of cytokines in pancreatic islets after combination therapies

Immune cells infiltrating the islets were activated in diabetic rats with blood glucose concentrations > 15 mmol/l without antibody therapies. This was documented by high gene expression levels of the pro-inflammatory cytokines, Tnf, Il1b, Ifng, Il2, Il6 and Il17 as well as the anti-inflammatory cytokines Il4 and Il10 in the infiltrating immune cells (Table 2). Sixty days after the end of anti-TCR combination therapy with anti-IL-6 or anti-IL-17A or in the triple fashion with both antibodies, the pro-inflammatory cytokine gene expression was strongly reduced. The combination of anti-TCR with anti-IL-6 was clearly more effective than the combination with anti-IL-17A. However, only the triple combination abolished the diabetes induced pro-inflammatory cytokine increases completely, identical with the situation in islets from healthy control animals (Table 2). Interestingly, expression of the anti-inflammatory cytokines Il4 and Il10 was present still at a low level after combination of anti-TCR with anti-IL-17A (Table 2). The same was true for a significant expression of the anti-inflammatory cytokine Il10 after combination of anti-TCR with anti-IL-6 and anti-IL-17A (Table 2).

Table 2 Pro-and anti-inflammatory cytokine gene expression by in situ RT-PCR in the islet immune cell infiltrate Full size table

Changes of pro- and anti-inflammatory cytokines in the circulation after combination therapies

In the animals responding to double and triple combination therapies, the serum protein concentrations of the pro-inflammatory cytokines, TNF-α and IL-1β, were 60 days after the end of the therapies as low as in healthy control animals with a significant decrease compared to the diabetic controls (Fig. 5a, b). The pro-inflammatory cytokine interferon gamma (IFN-γ) showed the same values in all groups including the control groups (Fig. 5c). The increased levels of the immune cell activating cytokine IL-2 in diabetic control rats decreased in all animals after the different combination therapies but with a significant reduction only in the triple combination compared to the diabetic controls (Fig. 5d). Only after triple combination therapy of anti-TCR with anti-IL-6 and anti-IL-17A the protein concentration of the anti-inflammatory cytokine IL-4 was somewhat increased (Fig. 5e). The anti-inflammatory cytokine IL-10 increased even to values comparable to those in healthy controls after triple combination therapy (Fig. 5f). The serum concentrations of IL-6 and IL-17A decreased after double and triple combination therapies to a range not higher than in healthy controls (Fig. 5g, h).