Human interleukin-4-treated regulatory macrophages promote epithelial wound healing and reduce colitis in a mouse model

Abstract

Murine alternatively activated macrophages can exert anti-inflammatory effects. We sought to determine if IL-4-treated human macrophages [i.e., hM(IL4)] would promote epithelial wound repair and can serve as a cell transfer treatment for inflammatory bowel disease (IBD). Blood monocytes from healthy volunteers and patients with active and inactive IBD were converted to hM(IL4)s. IL-4 treatment of blood-derived macrophages from healthy volunteers and patients with inactive IBD resulted in a characteristic CD206⁺CCL18⁺CD14^low/- phenotype (RNA-seq revealed IL-4 affected expression of 996 genes). Conditioned media from freshly generated or cryopreserved hM(IL4)s promoted epithelial wound healing in part by TGF, and reduced cytokine-driven loss of epithelial barrier function in vitro. Systemic delivery of hM(IL4) to dinitrobenzene sulphonic acid (DNBS)-treated Rag1^-/- mice significantly reduced disease. These findings from in vitro and in vivo analyses provide proof-of-concept support for the development of autologous M(IL4) transfer as a cellular immunotherapy for IBD.

Fig. 1. IL-4 evokes substantial gene regulation in human macrophages.

Blood monocytes from three healthy donors were converted to macrophages (2 × 10⁵) with macrophage colony-stimulating factor (M-CSF) and then left untreated [M(0)] or treated with IL-4 [M(IL4)] or IFN-γ [M(IFN-γ)] for 48 hours (both 10 ng/ml). (A) RNA sequence (RNAseq) analysis revealed IL-4–evoked significant changes in 996 genes as shown in the volcano plot and confirmed some of the previously reported gene changes (Venn diagram). (B) Heat maps showing IL-4 regulation of selected genes related to M(IL4) polarization and immune function (GF, growth factors). (C) IL-4–evoked increased expression of CD206 and CCL18, and decreased CD14 mRNA was confirmed by qPCR (D) and at the protein level by ELISA or flow cytometry (MFI, mean fluorescence intensity; each dot represents macrophages from an individual donor; mean ± SEM; *, **, and ***P < 0.05, 0.01, and 0.001 compared to M0, respectively). (E) Reactome network analysis shows clusters of gene changes increased in M(IL4). (F) Comparison of hM(IL4) RNA sequence data with murine mRNA immune array shows good alignment [►, gene change is opposite direction; ●, no response in mouse M(IL4)].

Fig. 2. Human blood–derived M(IL4)s promote epithelial wound repair in vitro.

(A) Representative images of epithelia showing the original margin of wounds (X), leading edge of wounds (dashed line), and leading edge (le) of the control monolayer. (B) Treatment with TGFβ (10 ng/ml) or CM from IL-4–treated macrophages [M(IL4) CM] increased epithelial repair. The M(IL4) CM was also (C) boiled or (D) treated with trypsin, which blocked repair. (E) The hM(IL4)s spontaneously produced more TGFβ than nonactivated macrophages (M0) from the same donor. (F) Addition of TGFβ-neutralizing antibodies (1D11) to the M(IL4) CM significantly impaired epithelial repair [mean ± SEM; n = 6 monolayers from three experiments; *, #, and ɸP < 0.05 compared to control (culture medium only), ɸɸ, P < 0.01 compared to M(IL4) CM; M0 CM, and M(IL4) CM, respectively].

Fig. 3. CM from hM(IL4)s reduced IFN-γ–evoked increases in epithelial permeability.

Confluent, filter-grown T84 epithelial cell monolayers were treated with IFN-γ (10 ng/ml) ± 50% CM from hM(IL4)s (1 × 10⁶ cultured for 24 hours), and TER was measured 24 hours later (A) (n = 17 monolayers from six experiments; ****P < 0.001 compared to control; ##P < 0.01 compared to IFN-γ). (B) Following 24 hours of exposure to IFN-γ ± M(IL4) CM, 70-kDa FITC-dextran was added to the lumen aspect of the monolayers, and samples from the basolateral compartment of the culture well were collected 4 hours later and assessed (n = 4 monolayers, one experiment; inset shows TER of the monolayers under the corresponding conditions; *P < 0.001 compared to control; #P < 0.01 compared to IFN-γ).

Fig. 4. Macrophages from patients with inactive disease convert to M(IL4)s that can enhance epithelial wound repair.

Blood-derived macrophages from healthy volunteers and patients with Crohn’s disease (CD) or ulcerative colitic (UC) with active (CD-A) or inactive (CD-I) disease was exposed to IL-4 (10 ng/ml, 48 hours) and (A) CD206, (B) CCL18, and (C) CD14 mRNA assessed by qPCR and normalized to nontreated macrophages (M0) from the same individual. (D) Confluent monolayers of the Caco2 epithelial cell line were mechanically wounded and exposed to culture media or a 50% CM from the various macrophage groups, and the area recovered was measured 24 hours later. (E) Setting a 1.5-fold increase in CD206 mRNA in response to IL-4 predicted an M(IL4) CM that would significantly increase epithelial wound repair [mean ± SEM; *P < 0.05 compared to M0 (A to C) or culture medium only (D) by analysis of variance (ANOVA) followed by Tukey’s test; each dot represents macrophages from a different donor]. (F) Correlation of patient mRNA expression of TGFβ and CD206 with their wound healing capacity (% wound healing compared to control).

Fig. 5. hM(IL4)s reduce DNBS-induced colitis in mice.

Male C57BL/6 Rag1^−/− mice were treated with freshly generated hM(IL4)s or murine (m) M(IL4)s or unstimulated macrophages (M0) [10⁶ cells intraperitoneally (i.p.)] 48 hours before intrarectal delivery of 5 mg of DNBS. Seventy-two hours later, mice were necropsied and (A) a macroscopic disease score calculated, (B) colon length recorded, and (C) histopathology assessed on H&E sections. In separate studies, hM(IL4)’s cryopreserved for 1 month were thawed and delivered by i.p. or intravenous (i.v.) injection 48 hours before DNBS and subsequently, (D) disease activity score, (E) colon length, and (F) histopathology were assessed [mean ± SEM; each dot represents a mouse; macrophages from 10 healthy donors in five experiments (A to C) and 5 to 6 donors in two experiments for cryopreserved hM(IL4) (D to F); * and #P < 0.05 compared to control (con) and DNBS, respectively, by ANOVA followed by Tukey’s test (colon length) or the Kruskal-Wallis test (disease and histopathology scores)].

Fig. 6. hM(IL4)s reduce apoptosis and promote anti-inflammatory conditions in mice with DNBS-induced colitis.

Male C57/bl6 Rag1^−/− were treated with cryopreserved hM(IL4)s (10⁶ cells i.p. or i.v.) and 48 hours later received 5 mg of DNBS intrarectally. Seventy-two hours later, mice were necropsied and (A) cryosections of mid-colon assessed by TUNEL staining for apoptotic cells [red, 4′,6-diamidino-2-phenylindole; green, TUNEL⁺; original objective, 20×)], (B) murine TNFα and IL-10 mRNA assessed by qPCR, (C) the ratio of TNFα:IL-10, and protein levels of (D) murine TNFα assessed by ELISA [mean ± SEM; each dot represents a mouse; macrophages from four to six healthy donors in two experiments; * and #P < 0.05 compared to control (con) and DNBS, respectively, by ANOVA followed by Tukey’s test; m, muscle; l, lumen; arrow, TUNEL⁺ cell].

Fig. 7. Administration of hM(IL4) increases colonic TGFβ.

Fresh or cryopreserved hM(IL4) (10⁶ cells i.p.) were administered to male C57BL/6 Rag1^−/− mice and 48 hours later received intrarectal delivery of 5 mg of DNBS. Three days later, mice were necropsied and (A) colonic protein levels of TGFβ assessed by ELISA (mean ± SEM; each dot represents a mouse; macrophages from four healthy donors in two experiments; * and #P < 0.05 compared to control and DNBS, respectively, by ANOVA followed by Tukey’s test). (B) CM (50%) from unstimulated M(0)s and M(IL4)s, and mouse and human TGFβ (10 ng/ml) were applied to serum-starved (1 hour) confluent mouse IEC.4 cells, total protein was isolated at 30 min, and SMAD2 phosphorylation was detected by immunoblotting. β-actin was used as a control.

Fig. 8. Mice receiving hM(IL4) 1 month before DNBS have reduced disease severity.

Male C57BL/6 Rag1^−/− mice were administered cryopreserved hM(IL4)s (10⁶ cells i.p.) and 30 days later received intrarectal delivery of 5 mg of DNBS. Seventy-two hours later, mice were necropsied and (A) a macroscopic disease score was calculated, (B) colon length was recorded, and (C) histopathology assessed on H&E sections was assessed [data are mean ± SEM; each dot represents a mouse; macrophages from three healthy donors in two experiments; * and #P < 0.05 compared to control (con) and DNBS, respectively, by ANOVA followed by Tukey’s test (colon length) or the Kruskal-Wallis test (disease and histopathology scores)]. (D) presents representative histological images from the treatment groups (M, outer layers of muscle; L, gut lumen; *, ulceration; triangle, inflammatory infiltrate; original objective, 20×). (E) shows that male BALB/c mice treated with murine M(IL4)s 2 to 21 days before DNBS are protected from the pro-colitic stimulus [mean ± SEM; * and #P < 0.05 compared to control (con) and DNBS, respectively, by ANOVA followed by Kruskal-Wallis test].