Failure of the Anti-Inflammatory Parasitic Worm Product ES-62 to Provide Protection in Mouse Models of Type I Diabetes, Multiple Sclerosis, and Inflammatory Bowel Disease

Abstract

Parasitic helminths and their isolated secreted products show promise as novel treatments for allergic and autoimmune conditions in humans. Foremost amongst the secreted products is ES-62, a glycoprotein derived from Acanthocheilonema viteae, a filarial nematode parasite of gerbils, which is anti-inflammatory by virtue of covalently-attached phosphorylcholine (PC) moieties. ES-62 has been found to protect against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus, and airway hyper-responsiveness. Furthermore, novel PC-based synthetic small molecule analogues (SMAs) of ES-62 have recently been demonstrated to show similar anti-inflammatory properties to the parent molecule. In spite of these successes, we now show that ES-62 and its SMAs are unable to provide protection in mouse models of certain autoimmune conditions where other helminth species or their secreted products can prevent disease development, namely type I diabetes, multiple sclerosis and inflammatory bowel disease. We speculate on the reasons underlying ES-62's failures in these conditions and how the negative data generated may help us to further understand ES-62's mechanism of action.

Keywords: ES-62; helminth; inflammatory bowel disease; multiple sclerosis; nematode; type 1 diabetes.

Figure 1

ES-62 and SMAs 11a and 12b do not protect against the development of type 1 diabetes. (A) Four-week-old female non-obese diabetic (NOD) mice were injected subcutaneously with 2 μg of ES-62 (n = 10) in 200 μL of sterile PBS once a week between four and 12 weeks of age. Control mice (n = 13) were injected with 200 μL of sterile PBS according to the same protocol. (B) 9–10 week-old female NOD mice were injected six times subcutaneously with sterile PBS (control) or 10 μg of ES-62 in sterile PBS over a two-week period (n = 9). Five-week-old female NOD mice received intraperitoneal injections of PBS (control) or 1 μg SMA 11a (C) or 12b (D) twice a week for 10 weeks (n = 10). The incidence of diabetes was monitored by regularly checking for the presence of glycosuria.

Figure 2

Effects of ES-62 on in vitro cytokine secretion by dendritic cells (DCs). DCs were prepared from bone marrow as described in the Materials and Methods. On day 10, they were harvested and incubated with medium alone, 500 ng/mL of LPS, 2 μg of ES-62 or with both 500 ng/mL of LPS and 2 μg of ES-62. At 72 h, supernatants were harvested and assayed for the presence of (A) IL-12p40, (B) TNF-α, (C) IL-10, and (D) IL-6. Data points correspond to the mean plus SEM of each set of samples.

Figure 3

Effects of sub-cutaneous or intra-peritoneal treatment with ES-62 on composition of BDC2.5NOD splenocyte populations. Four-week-old BDC2.5NOD mice were given two injections of 2 μg ES-62 over the course of a week. After one week, splenocytes were harvested, and the percentage of B cells, CD4⁺ cells, and CD4⁺CD25⁺ cells was assessed by staining with (A) anti-CD19, (B) anti-CD4, and (C) anti-CD25, and subsequent FACS analysis. Each dot on the scatter plot corresponds to one individual mouse.

Figure 4

Effects of sub-cutaneous or intra-peritoneal treatment with ES-62 on the ex vivo response of BDC2.5NOD splenocytes to BDC2.5 peptide. Four-week-old BDC2.5NOD mice were given two injections of 2 μg ES-62 over the course of a week. After one week, splenocytes were harvested. Equal numbers of splenocytes from each group were re-stimulated with 1 mg/mL BDC2.5 peptide. Culture supernatants were harvested at 48 h and assessed for the presence of (A) IFN-γ, (B) IL-4, and (C) IL-10 by ELISA. Each dot on the scatter plot corresponds to the average cytokine secretion of splenocytes from one individual mouse.

Figure 5

ES-62 and SMAs 11a and 12b do not prevent experimental autoimmune encephalomyelitis. (A) Age 7–9 week-old mixed sex C57BL/6 mice were immunised with MOG_35–55 peptide and treated with PBS (control), ES-62, or SMAs 11a or 12b and clinical disease scores were monitored twice daily (n = 5–7). There were no statistically significant differences amongst groups, and hence, SD values have been omitted to ensure the clarity of the figure. Spinal cords were collected from treatment groups and immunohistochemical analysis of CD4 (B) or F4/80 (C) was performed and representative images are shown. Magnification = 20×.

Figure 6

ES-62, and SMAs 11a and 12b do not prevent the induction of chronic colonic inflammation in the DSS-colitis model. (A) Body weight changes (as percentage of the initial weight) from the initiation of DSS-colitis was measured during four cycles of DSS with thrice weekly treatments of ES-62 or SMAs (11a or 12b). (B) Colon length (cm) was measured at cull. (C) Total cell counts and the proportion of CD4⁺ T cells (D) present in the lamina propria mononuclear cells (LPMC) were measured. (E) Representative Heamatoxylin and Eosin (H & E) staining of colons at time of sacrifice. Magnification = 100×. Data in (A–D) represent the means ± SD (n = 5) and statistical significance as determined by student’s t test (PBS versus ES-62) is denoted by * p < 0.05.

Figure 7

ES-62 SMAs do not attenuate T cell transfer colitis in mice. Naïve CD4⁺ T cells from wild type (WT) mice were injected into Rag1^−/− mice. PBS or SMAs (11a, 12b) were injected twice per week. (A) Body weight changes as a percentage of the initial weight at day 1. (B) Colon length. (C) Total cell counts in lamina propria mononuclear cells. (D) Representative H & E staining of colons at the time of sacrifice. Magnification = 100×. For (A–C) data represent means ± SD. N = 5/group. * p < 0.05 as determined by Student’s t-test for comparisons between PBS- and 12b-treated mice.