Modulation of CD39 and Exogenous APT102 Correct Immune Dysfunction in Experimental Colitis and Crohn's Disease

Abstract

Background and aims: CD39/ENTPD1 scavenges pro-inflammatory nucleotides, to ultimately generate immunosuppressive adenosine, which has a central role in immune homeostasis. Global deletion of Cd39 increases susceptibility to experimental colitis while single nucleotide polymorphisms within the human CD39 promoter, and aberrant patterns of expression during experimental hypoxia, predispose to Crohn's disease. We aimed to define the impact of transgenic human CD39 [hTG] overexpression in experimental colitis and to model therapeutic effects using the recombinant apyrase APT102 in vivo. We also determined the in vitro effects of APT102 on phenotypic and functional properties of regulatory T-lymphocytes derived from patients with Crohn's disease.

Methods: Colitis was induced by administration of dextran sulfate sodium in wild-type [WT] or hTG mice, and, in another model, by adoptive transfer of CD45RBhigh cells with or without WT or hTG regulatory T cells [Treg]. In additional experiments, mice were treated with APT102. The effects of APT102 on phenotype and function of Treg and type-1 regulatory T [Tr1] cells were also evaluated, after purification from peripheral blood and lamina propria of Crohn's disease patients [n = 38].

Results: Overexpression of human CD39 attenuated experimental colitis and protected from the deleterious effects of systemic hypoxia, pharmacologically induced by deferoxamine. Administration of APT102 in vivo enhanced the beneficial effects of endogenous Cd39 boosted by the administration of the aryl hydrocarbon receptor [AhR] ligand unconjugated bilirubin [UCB]. Importantly, supplemental APT102 restored responsiveness to AhR stimulation by UCB in Treg and Tr1 cells, obtained from Crohn's disease patients.

Conclusions: hCD39 overexpression ameliorated experimental colitis and prevented hypoxia-related damage in vivo. Exogenous administration of APT102 boosted AhR-mediated regulatory effects in vivo while enhancing Treg functions in Crohn's disease in vitro.

Keywords: ATPase/ADPase; Crohn’s disease; ectonucleotidase; regulatory cells.

Figure 1.

Treg and Tr1 cells from hTG mice display higher frequencies of mCD39⁺ cells and show increased proliferative capacity. [A] Histogram plots of human CD39 [hCD39] MFI of Treg and Tr1 cells obtained from a representative WT, Entpd1^−/− and hTG mouse. Numerical values of hCD39 MFI are indicated within the histogram plots. [B] Mean ± SEM frequency of hCD39⁺ cells within Treg and Tr1 cells of WT [n = 3–5], Entpd1^−/− [n = 3–5] and hTG mice [n = 6–9]. [C] Mean ± SEM frequency of mCD39⁺ cells within Treg and Tr1 cells of WT [n = 5], Entpd1^−/− [n = 5] and hTG mice [n = 8]. [D] Representative hCD39 and mCD39 staining of frozen sections from the spleen and colon of WT and hTG mice. Staining from one of three independent experiments is shown [original magnification, ×20]. [E] Proliferation of Treg and Tr1 cells obtained from WT [n = 9] and hTG [n = 18] mice was measured by ³H-thymidine incorporation. Mean ± SEM counts (cpm) are shown. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 2.

Treg and Tr1 cells from hTG mice display heightened ectoenzymatic activity. [A] Mean ± SEM frequency of CD73⁺ cells within Treg and Tr1 cells from WT [n = 5] and hTG [n = 8] mice. [B] Cell ADPase ectoenzymatic activity was determined upon cell incubation with¹⁴C-labelled ADP by TLC. One representative of four independent experiments is shown. [C] Mean + SEM ADP/AMP and AMP/adenosine [ADO] ratio of Treg and Tr1 cells [WT n = 4; hTG n = 4]. Mean ± SEM percentage suppression of Treg and Tr1 cells over IFNγ [D] and IL17 [E] production by CD4⁺CD25⁻ cells [WT n = 5–8; hTG n = 12–13]. Mean ± SEM fold change in Foxp3 mRNA expression in untreated and UCB-treated hTG Treg [n = 8] [F], or in untreated, UCB-, APT102- or UCB plus APT102-treated WT Treg [n = 13]. *p ≤ 0.05;**p ≤ 0.01.

Figure 3.

hCD39 overexpression in mice ameliorates DSS colitis in vivo. WT [n = 6] and hTG [n = 6] mice were treated with 3% DSS for 6 days. DSS treatment was then replaced with normal water for an additional 4 days. [A] Mean+SEM disease activity index in WT and hTG mice. [B] Mean ± SEM colon length [cm] at the time of harvesting. [C] Haematoxylin and eosin staining of colon sections [original magnification, ×10]; arrows indicate the area magnified in the insets [×20]; mean ± SEM histology score at the time of harvesting is also shown. Mean ± SEM frequency of [D] CD4⁺FOXP3⁺, [E] CD4⁺mCD39⁺, [F] CD4⁺IL17⁺, [G] CD4⁺IFNγ ⁺ and [H] CD4⁺IL10⁺ lymphocytes among spleen, MLN, IEL and LP mononuclear cells. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 4.

hCD39 overexpressing Treg ameliorate experimental colitis, induced by adoptive transfer. CD45RB^high lymphocytes, obtained from the spleen of WT mice, were injected into Rag-2^−/− recipients, alone or in association with WT or hTG Treg. Mice were kept for up to 10 weeks and disease activity index was monitored throughout. [A] Mean+SEM disease activity index in Rag-2^−/− mice injected with CD45RB^high [n = 5], CD45RB^high plus WT Treg [n = 10] or CD45RB^high plus hTG Treg [n = 10]. [B] Mean ± SEM colon length [cm] at the time of harvesting. [C] Haematoxylin and eosin staining of colon sections [original magnification, ×10]; arrows indicate the area magnified in the insets [×20]; mean ± SEM histology score at the time of harvesting is also shown. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 5.

hCD39 overexpression protects against deleterious systemic effects of deferoxamine-induced hypoxia. WT [n = 24] and hTG [n = 24] mice were exposed to 3% DSS for 6 days and then to normal water for four additional days. For the whole duration of the experiment, mice were administered vehicle [n = 6], UCB [n = 6], deferoxamine [DFO, n = 6], or UCB plus DFO [n = 6]. [A] Mean+SEM disease activity index in vehicle, UCB-, DFO- and UCB plus DFO-treated WT and hTG mice. [B] Mean ± SEM colon length [cm] at the time of harvest. [C] Haematoxylin and eosin staining of colon sections [original magnification, ×10]; arrows indicate the area magnified in the insets [×20]; mean ± SEM histology score at the time of harvesting is also shown. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 6.

APT102 boosts UCB immunoregulatory properties in DSS colitis in vivo. WT mice were treated with 2% DSS for 6 days. DSS treatment was then replaced with normal water for an additional 24 days. For the whole duration of the experiment, mice were exposed to vehicle [n = 4], UCB [n = 6], APT102 [n = 4] or UCB plus APT102 [n = 6]. [A] Mean+SEM disease activity index. Asterisks indicate statistical significance as determined by ANOVA. [B] Mean ± SEM colon length [cm]. [C] Haematoxylin and eosin staining of colon sections [original magnification, ×10]; mean ± SEM histology score at the time of harvesting is also shown. [D] Mean ± SEM frequency of CD4⁺FOXP3⁺, CD4⁺mCD39⁺, CD4⁺CD49b and CD4⁺LAG-3⁺ lymphocytes within spleen, MLN, IEL and LP mononuclear cells. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 7.

APT102 promotes additional AhR activation in Treg and Tr1 cells from Crohn’s disease patients. Treg and Tr1 cells were obtained from peripheral blood-derived CD4⁺ lymphocytes of healthy subjects and Crohn’s disease patients. Mean ± SEM [A] FOXP3, LAG-3 and CD39 MFI in untreated, UCB- or UCB plus APT102-treated Treg; [B] CD49b, LAG-3 and CD39 MFI in untreated, UCB- or UCB plus APT102-treated Tr1 cells [healthy subjects, n = 11 for Treg, n = 12 for Tr1 cells; Crohn’s patients, n = 11]. [C] Mean ± SEM CD73 MFI of untreated, UCB- and UCB plus APT102-treated CD39⁺ Treg and CD39⁺ Tr1 cells [healthy subjects, n = 6 for Treg and n = 5 for Tr1 cells; Crohn’s patients, n = 6]. Mean ± SEM frequency of [D] IL17 and [E] IFNγ-producing cells within CD4⁺CD25⁻ cells in the absence or presence of untreated, UCB- or UCB plus APT102-treated Treg and Tr1 cells [healthy subjects n = 5; Crohn’s patients n = 5]. *p ≤ 0.05;**p ≤ 0.01;***p ≤ 0.001.

Figure 8.

Exposure to APT102 and UCB boosts the regulatory phenotype of lamina propria-derived Tr1 cells in Crohn’s disease. Treg and Tr1 cells were derived from lamina propria CD4 lymphocytes of Crohn’s disease patients [n = 6] and exposed to APT102 for 12 h in combination with UCB, added for the last 6 h of culture. Treg and Tr1 cells were obtained from both inflamed and non-inflamed biopsied areas. Mean ± SEM [A] FOXP3, LAG-3 and CD39 MFI in Treg; and [B] CD49b, LAG-3 and CD39 MFI in Tr1 cells. Addition of APT102 to UCB boosts CD49b, LAG-3 and CD39 MFI in Tr1 cells obtained from non-inflamed biopsied areas. *p ≤ 0.05;**p ≤ 0.01.