Cryopreserved Interleukin-4-Treated Macrophages Attenuate Murine Colitis in an Integrin β7 - Dependent Manner

Abstract

The adoptive transfer of alternatively activated macrophages (AAMs) has proven to attenuate inflammation in multiple mouse models of colitis; however, the effect of cryopreservation on AAMs, the ability of previously frozen AAMs to block dinitrobenzene sulfonic acid (DNBS) (Th1) and oxazolone (Th2) colitis and their migration postinjection remains unknown. Here we have found that while cryopreservation reduced mRNA expression of canonical markers of interleukin (IL)-4-treated macrophages [M(IL-4)], this step did not translate to reduced protein or activity, and the cells retained their capacity to drive the suppression of colitis. The anticolitic effect of M(IL-4) adoptive transfer required neither T or B cell nor peritoneal macrophages in the recipient. After injection into the peritoneal cavity, M(IL-4)s migrated to the spleen, mesenteric lymph nodes and colon of DNBS-treated mice. The chemokines CCL2, CCL4 and CX3CL1 were expressed in the colon during the course of DNBS-induced colitis. The expression of integrin β7 on transferred M(IL-4)s was required for their anticolitic effect, whereas the presence of the chemokine receptors CCR2 and CX3CR1 were dispensable in this model. Collectively, the data show that M(IL-4)s can be cryopreserved M(IL-4)s and subsequently used to suppress colitis in an integrin β7-dependent manner, and we suggest that these proof-of-concept studies may lead to new cellular therapies for human inflammatory bowel disease.

Figure 1

Comparison of fresh and cryopreserved (Cryo.) mouse and human M(IL-4)s. (A) Mouse macrophages were assessed by their capacity to express the mannose receptor (MRC1) by flow cytometry (n = 3), arginase activity and ability to produce nitric oxide in response to LPS, as measured by nitrite levels in a Griess assay (n = 7, two experiments). (B) Human M(IL-4)s from healthy control blood donor monocytes were similarly assessed by their capacity to downregulate CD14 and upregulate MRC1 as measured by flow cytometry (n = 3) and spontaneously secrete CCL18 into the culture supernatant, as measured by ELISA (n = 5, two experiments). MFI, mean fluorescence intensity. p < 0.05: ^†compared with M(−); ^#compared with Cryo. M(IL-4). Data are represented as the mean ± SEM.

Figure 2

Transfer of cryopreserved (Cryo.) M(IL-4)s protect against murine colitis. One million M(IL-4)s were transferred intraperitoneally 48 h before the induction of colitis. The extent of damage in DNBS (Th1) colitis was measured by weight loss, shortened colon lengths and extent of colon macroscopic damage (A) and histopathology (B). Experiments with DNBS were repeated twice: naive control (n = 9), DNBS (n = 10) and DNBS + Cryo. M(IL-4) (n = 10). (C, D) By using the same experimental timeline, M(IL-4)-mediated protection against oxazolone (Th2)-induced colitis was assessed by the same parameters. Experiments with oxazolone were repeated four times: naive control (n = 15), Oxa (n = 14), Oxa + M(IL-4) (n = 17). p < 0.05: ^†compared with naive control; ^#compared with DNBS + Cryo. M(IL-4). Bar represents 50 μm; M, muscularis; L, lumen. Data are represented as the mean ± SEM.

Figure 3

Intraperitoneal transfer of M(IL-4)s does not require host lymphocytes to protect against DNBS colitis. The M(IL-4) transfer model was repeated in RAG1 KO mice (lacking T and B cells) to determine whether host lymphocytes were essential for the anticolitic effect. The data show that wild-type M(IL-4) were still able to attenuate (A) colon shortening, and only mice treated with DNBS alone were significantly different from naive controls in terms of macroscopic damage, MPO activity, and (B) histological damage. Experiments were repeated twice: naive control (n = 5), DNBS (n = 10), DNBS + M(IL-4) (n = 10). p < 0.05: ^†compared with naive control; ^#compared with DNBS + M(IL-4). Bar represents 50 μm; M, muscularis; L, lumen. Data are represented as the mean ± SEM.

Figure 4

Transferred M(IL-4)s home to the colon, liver, mesenteric lymph nodes and spleen during DNBS colitis. (A) Macrophages and spleen cells (as a nonspecific control cell) were labeled with fluorescent dye VivoTrack 680 NIR and localized using whole-body imaging; fluorescence on d 3 just before the necropsy is depicted overlying the X-ray images. Throughout the course of the experiment, the majority of cells remained within the peritoneal cavity in a pattern similar to d 3 (data not shown). (B) Whole organs were dissected and reexamined by using the whole-body imaging system to confirm localization to the liver, spleen, mesenteric lymph nodes and colon in M(IL-4) + DNBS–treated mice. Arrows indicate the areas of peak fluorescence. (C) Cellular localization in the liver, spleen and colon was confirmed further with spinning disk confocal microscopy on mounted organ sections. VivoTrack 680–labeled macrophages at 20× magnification were enumerated in triplicate random fields of view. Whole mounted colons from DNBS mice treated with M(IL-4)s contained significantly more macrophages compared with M(IL-4)s into naive control mice. Data are representative of two to three experiments: DNBS, n = 2; DNBS + M(IL-4), n = 3–4; M(IL-4), n = 3–4; DNBS + splenocytes, n = 2. ^†p < 0.05 (Mann-Whitney U test, one-tailed). Data are represented as the mean ± SEM.

Figure 5

Alternative activation of bone marrow–derived macrophages from WT and Itgb7^null mice is comparable. (A) Expression of AAM markers Arg1 and MRC1 are not significantly different in Itgb7^null mouse macrophages, as measured by flow cytometry, although there is a significant increase in Ly6C expression in all Itgb7^null macrophages compared with WT. (B) This result was confirmed at the functional level where arginase activity was similar between WT and KO M(IL-4)s. All groups (n = 3), one experiment. p < 0.05: ^†compared with WT. Data are represented as the mean ± SEM.

Figure 6

ITGB7 expression on transferred M(IL-4)s is required for protection against DNBS colitis. Protection mediated by pretransfer of M(IL-4)s intraperitoneally was measured on d 3 of DNBS colitis. (A) In these experiments, mice treated with WT but not Itgb7^null M(IL-4)s were significantly protected against colitis-associated cachexia, colon shortening, macroscopic damage and (B) histopathology. Experiments were repeated twice: naive control, n = 5; DNBS, n = 7; DNBS + WT M(IL-4), n = 10; DNBS + Itgb7^null M(IL-4); n = 10. p < 0.05: ^†compared with naive control; ^#compared with DNBS + WT M(IL-4). Bar represents 50 μm; M, muscularis; L, lumen. Data are represented as the mean ± SEM.