Restored Macrophage Function Ameliorates Disease Pathophysiology in a Mouse Model for IL10 Receptor-deficient Very Early Onset Inflammatory Bowel Disease

Abstract

Background and aims: Mutations in IL10 or the IL10 receptor lead to very early onset [VEO] inflammatory bowel disease [IBD], a life-threatening disease which is often unresponsive to conventional medication. Recent studies have demonstrated that defective IL-10 receptor signalling in innate immune cells is a key driver of severe intestinal inflammation in VEO-IBD. Specifically, IL10 unresponsiveness of macrophages, which govern the tight balance between pro- and anti-inflammatory responses in the intestinal system, plays a central role in the events leading to excessive inflammatory responses and the development of IBD.

Methods and results: We here evaluated haematopoietic stem cell gene therapy in a VEO-IBD mouse model and demonstrated that the therapeutic response closely correlates with gene correction of the IL10 signalling pathway in intestinal macrophages. This finding prompted us to evaluate the therapeutic efficacy of macrophage transplantation in the Il10rb-/- VEO-IBD mouse model. A 6-week regimen employing a combination of depletion of endogenous hyperinflammatory macrophages followed by intraperitoneal administration of wild-type [WT] macrophages significantly reduced colitis symptoms.

Conclusions: In summary, we show that the correction of the IL10 receptor defect in macrophages, either by genetic therapy or transfer of WT macrophages to the peritoneum, can ameliorate disease-related symptoms and potentially represent novel treatment approaches for VEO-IBD patients.

Keywords: Cell and gene therapy; IL10 receptor; macrophages.

Figure 1.

Therapeutic efficacy of haematopoietic stem cell [HSC] gene therapy in Il10rb^-/- IBD mice correlates with gene marking in lamina propria macrophages. [A] A third-generation self-inactivating [SIN] lentiviral vector was used for gene transfer of a codon optimised [co] murine Il10rb cDNA coupled to the mVenus fluorescent reporter driven by the phosphoglycerate kinase [PGK] promoter. [B] Phosphorylation of STAT3 after stimulation with IL10 analysed by flow cytometry in macrophages derived from wild type [WT] or Il10rb^-/- lin^- bone marrow cells. Il10rb^-/- lin^- cells were transduced with a PGK-eGFP [control] or PGK-IL10rb therapeutic vector. [C] Control transduced Il10rb^-/- lin^- bone marrow cells, corrected Il10rb^-/- lin^- or WT lin^- cells were transplanted into irradiated Il10rb^-/- IBD mice and body weights were analysed for 35 days. Values are given as % of initial body weight (n = 4–5 per group, mean ± range [range depicted as shaded area]). [D] Quantification after histological scoring [n = 4–5 per group, individual values with mean ± SD]. [E] Representative light microscopy of H/E stained colon paraffin sections. [F] Correlation of the frequency of gene-marked lymphocytes, granulocytes, and macrophages in the lamina propria with the histological score [mean ± 95% CI]. LTR, long terminal repeat; wPRE, woodchuck hepatitis virus post-transcriptional regulatory element, significances were calculated by two-way ANOVA with Bonferroni’s multiple comparison test, *p <0.05, **p <0.01, ***p <0.001, ****<0.0001, ns not significant. IBD, inflammatory bowel disease; SD, standard deviation; H/E, haematoxylin and eosin; CI, confidence interval; ANOVA, analysis of variance.

Figure 2.

Macrophage transplantation reduces colitis in Il10rb^-/- IBD mice. [A] Schematic representation of the experimental set-up. Macrophages were generated from WT BM cells and 5‐10 x 10⁶ cells were transplanted into IBD animals by intraperitoneal [i.p.] injection every 2 weeks [green arrows]. In some experimental groups, clodronate liposomes were administered i.p. [red arrows] to deplete endogenous macrophages before macrophage transplantation. The experiment comprised two to three treatment cycles. [B] Colon histology scoring was performed for untreated WT mice, untreated IBD animals or IBD animals treated two to three treatment cycles with clodronate liposomes [Clodro], bone marrow-derived macrophages [BMDMs], or a combination therapy of clodronate liposomes followed by BMDMs [Clodro/BMDMs]. Data represent five independent experiments, individual values with mean ± SD, n = 8–10 per group. [C] Representative light microscopy of H/E-stained colon paraffin sections. [D and E] Immuno-phenotyping of lamina propria [LP] macrophage populations [pre-gated on: CD45⁺/CD11b⁺/CD103^-/ Ly6G^-/CD64⁺ monocyte/macrophages, see Supplementary Figure 2 for gating strategy, available as Supplementary data at ECCO-JCC online]. [D] Representative flow cytometry plots showing the frequency of Ly6C⁺/MHCII⁺ inflammatory and Ly6C^-/MHCII⁺ anti-inflammatory LP monocyte/macrophages in different treatment groups; and [E] quantification of four independent experiments [individual values with mean ± SD, n = 5–10 per group]. Significances calculated by one-way ANOVA with Dunnett’s multiple comparison test, *p <0.05, **p <0.01, ***p <0.001, ****<0.0001, ns not significant. IBD, inflammatory bowel disease; BM, bone marrow; SD, standard deviation; H/E, haematoxylin and eosin; WT, wild type; ANOVA, analysis of variance.

Figure 3.

Detection of transplanted WT macrophages in vivo and cytokine profiling. [A and B] Polymerase chain reaction [PCR] was used to detect wild-type macrophages harbouring the Il10rb gene; glycosyltransferase-like domain containing 1 [Gtdc1] was used as a control in [A] peritoneal cavity macrophages and [B] cells isolated from the lamina propria of animals treated with: SV129 IL10rb^-/- IBD animals treated with BMDMs, or clodronate liposomes, or a combination of clodronate liposomes and BMDMs, or clodronate liposomes only. Untreated IBD animals, dilutions of WT gDNA and H₂0 are shown as controls [two individual animals/group are shown]. [C and D] Flow cytometry analysis of CD45.1⁺ expression on donor-derived macrophages 6 days after transplantation into Bl6 Il10rb^-/- IBD animals. Animals were either transplanted with BMDMs only or with a combination of clodronate liposomes and BMDMs [Clodro/BMDMs]. [C] Frequency of CD45.1⁺ donor-derived cells among F480^high/CD11b^high peritoneal cavity macrophages; and [D] Frequency of CD45.1+ donor-derived cells among LP macrophages [mean ± SD]. [E] Secretion of IL1b, IL17a, IL27, and GM-CSF by macrophages derived from the peritoneal lavage fluid of different experimental groups [WT untreated, IBD untreated, and IBD+clodro/BMDMs] after LPS stimulation [data represent two independent experiments, individual values with mean ± SD, n = 4–6 per group, cells isolated after 4–6 weeks of treatment]. Significances are calculated by one-way ANOVA with Dunnett’s multiple comparison test, *p <0.05, **p <0.01, ***p <0.001, ****<0.0001, ns not significant. IBD, inflammatory bowel disease; SD, standard deviation; WT, wild-type; LP, laminapropria; LPS, lipopolysaccharide; BBMDM, bone-marrow derived macrophages; ANOVA, analysis of variance.

Figure 4.

Macrophage-based therapy normalises peripheral blood cell composition in Il10rb^-/- mice. Flow cytometric analysis was used to calculate the frequency of [A] granulocytes [CD45⁺/CD3^-/B220^-/CD11b⁺/Gr1^high] and [B] T cells [CD45⁺/ CD11b^-/Gr1^-/CD3⁺/B220^-] in peripheral blood [data represent five independent experiments, individual values with mean ± SD, n = 8–10 per group]. Significances calculated by one-way ANOVA with Dunnett’s multiple comparison test, *p <0.05, **p <0.01, ***p <0.001, ****<0.0001, ns not significant. SD, standard deviation; ANOVA, analysis of variance.