Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

Abstract

Background: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, diarrhoea, weight loss and bloody stools, where sustained remission is not currently achievable. Dextran Sulphate Sodium (DSS)-induced colitis is an animal model that closely mimics human UC. Ultrasound (US) has been shown to prevent experimental acute kidney injury through vagus nerve (VN) stimulation and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients may present dysfunctional VN activity, our aim was to determine the effects of therapeutic ultrasound (TUS) in DSS-induced colitis.

Methods: Acute colitis was induced by 2% DSS in drinking water for 7 days and TUS was administered to the abdominal area for 7 min/day from days 4-10. Clinical symptoms were analysed, and biological samples were collected for proteomics, macroscopic and microscopic analysis, flow cytometry and immunohistochemistry.

Findings: TUS attenuated colitis by reducing clinical scores, colon shortening and histological damage, inducing proteomic tolerogenic response in the gut during the injury phase and early recovery of experimental colitis. TUS did not improve clinical and pathological outcomes in splenectomised mice, while α7nAChR (α7 nicotinic acetylcholine receptor - indicator of CAIP involvement) knockout animals presented with disease worsening. Increased levels of colonic F4/80+α7nAChR+ macrophages in wild type mice suggest CAIP activation.

Interpretation: These results indicate TUS improved DSS-induced colitis through stimulation of the splenic nerve along with possible contribution by VN with CAIP activation. FUND: Intramural Research Programs of the Clinical Centre, the National Institute of Biomedical Imaging and Bioengineering at the NIH and CAPES/Brazil.

Keywords: Acute ulcerative colitis; Cholinergic anti-inflammatory pathway; Dextran Sulphate sodium; Inflammatory bowel disease; Therapeutic ultrasound; α7 nicotinic acetylcholine receptor.

Published by Elsevier B.V.

Figures

Graphical abstract

Fig. 1

Schematics of the methods section. Mice were shaved over the abdomen and a water-based ultrasound gel was applied. The 5 cm2 transducer was placed on top of the gel and held motionless by a clamp holder while the animal was under isoflurane anaesthesia. The treatment lasted for 7 min at 1 MHz (2 W/cm2, 10% duty cycle, ~250 kPa) and was repeated once a day from days 4 to 10. Mice received 2% DSS in drinking water from days 0 to 7.

Fig. 2

Clinical symptoms of 2% DSS colitis mice under TUS treatment. Experimental colitis was induced by DSS for 7 days in drinking water and TUS treatment was administered from day 4 to 10 over the abdomen. TUS attenuated clinical symptoms from day 9 and forward when measuring the (A) disease activity index (DAI), including (B) amelioration of stool consistency, (C) weight loss and (D) blood in the stool at different time points. *p < .05 compared to 2% DSS + TUS. Two-way ANOVA followed by Sidak post-hoc test. N = 15/group.

Fig. 3

Colon and spleen changes in 2% DSS colitis mice under TUS treatment. TUS attenuated (A) colon shortening and (D, E) histological damage at day 14 only, increasing (B) colon weight at days 7 and 9, while decreasing (C) spleen weight at day 11. Histological analysis demonstrated reduced tissue damage under TUS treatment at 14 days by partial preservation of the crypts, epithelial layer and goblet cells, diminishing immune cells infiltration. *p N = 5/group at each time point. Results are presented as mean ± SD. Images were taken with a 10× objective.

Fig. 4

Proteomic colon changes in 2% DSS colitis mice under TUS treatment. Experimental colitis was induced by DSS for 7 days in drinking water and TUS treatment was administered from day 4 to 10 over the abdomen. The colons were collected at days 5, 7, 9, 11 and 14, homogenized and later analysed by multiplex ELISA assay. Results demonstrate downregulation of colonic IL-5, IL-17 and Eotaxin, at different time points during TUS treatment (days 5 to 9); and upregulation of colonic IL-1β, IL-2, IL-4, IL-5, IL-7, IL-9, IL-12(p70) and G-CSF at different time points starting at day 5 with TUS treatment. *p < .05 compared to 2% DSS. *p < .05 compared to 2% DSS. Two-way ANOVA followed by Sidak post-hoc test. N = 5–6/group at each time point. Results are presented as mean ± SD. Heat maps of these results are presented in Supplementary Fig. 4.

Fig. 5

Proteomic colon changes in 2% DSS colitis mice under TUS treatment. Experimental colitis was induced by DSS for 7 days in drinking water and TUS treatment was administered from day 4 to 10 over the abdomen. The colons were collected at days 5, 7, 9, 11 and 14, homogenized and later analysed by multiplex ELISA assay. Results demonstrate downregulation of colonic MCP-1, M-CSF, MIG, RANTES and TNFα at different time points during TUS treatment (days 5 to 9); and upregulation of colonic MIP-1α, MIP-1β and MIP-2 at different time points starting at day 5 with TUS treatment. In addition, results show reduction in colonic levels of TGFβ and HSP70 at days 7 and 14, respectively, under TUS treatment. *p < .05 compared to 2% DSS. cp < .05 compared to control. Two-way ANOVA followed by Sidak post-hoc test for multiplex ELISA and one-way ANOVA followed by Tukey post-hoc test for ELISA Streptavidin-HRP assay. N = 5–6/group at each time point. Results are presented as mean ± SD. Heat maps of these results are presented in Supplementary Fig. 3.

Fig. 6

Immune cell population changes in colon, spleen and MLN in 2% DSS colitis mice under TUS. (A-E) Colon IHC analysis revealed no differences amongst all groups regarding B220+ B cells, increased levels of CD4+, CD8+ and F4/80+ cells in comparison to controls, while CD8+ levels were decreased when comparing TUS treated animals to 2% DSS group and CD25+ T cells were increased in 2% DSS only group. (F-G) Spleen FACS analysis demonstrated no changes for CD4+, CD25+ and F4/80+ cells. Increase percentage was seen for CD8+ T cells and decrease in B220+ B cells when comparing 2% DSS to control. In addition, TUS treatment normalized CD8+ T cells and B220+ B cells when compared to 2% DSS. (K—O) MLN IHC analysis demonstrated no difference amongst all groups for CD8+, CD25+ and F4/80+ cells. TUS treatment increased CD4+ and B220+ levels compared to control. *p < .05 compared to control. #p < .05 compared to 2% DSS. One-way ANOVA followed by Tukey post-hoc test. N = 4/group for IHC analysis and N = 6/group for FACS analysis. Results are presented as mean ± SD.

Fig. 7

Clinical and histological analysis of splenectomised mice. There was no difference between the groups in (A) disease activity index, (B) stool consistency, (D) weight loss, (E) histological colonic damage, (F) colon length and (G) colon weight at day 14. TUS decreased the amount of (C) blood in the stools on days 6 and 7. H&E staining of the colons demonstrated destruction of the crypts, loss of the epithelial barrier, loss of goblet cells and high immune cell infiltration for both groups. *p t-test for histological scores and macroscopic measurements. N = 10/group. Results are presented as mean ± SD for histological scores. Images were taken with a 10× objective.

Fig. 8

Colon and MLN immune cell changes in splenectomised mice. There was no difference between the groups when analysing the colons for (A) CD4+, (B) CD8+, (D) F4/80+ and (E) B220+ cells. TUS induced an increase in (C) colonic CD25+ T cells while decreasing the percentage of (H) CD25+ T cells in MLN. Furthermore, (F) CD4+ T cell levels were decreased in the MLN, while no difference was seen regarding (G) CD8+, (I) F4/80+ and (J) B220+ cells. *p < .05 compared to 2% DSS. Student's t-test. N = 4/group. Results are presented as mean ± SD.

Fig. 9

Clinical and histological analysis of α7nAChR KO mice. The absence of α7nAChR induced worsening of the disease in both 2% DSS and 2% DSS + TUS groups (compared to 2% DSS WT mice) at different time points regarding (A) disease activity index, (B) stool consistency, (C) blood in the stools and (D) weight loss. 2% DSS + TUS α7nAChR KO group resulted in worsening of (D) weight loss when compared to 2% DSS α7nAChR KO mice at days 9 and 11. There was no difference amongst all groups when analysing the colons for (E) histological damage and (G) colon weight. Both KO groups presented with (H) increased spleen size and TUS (F) worsened colon shortening, when compared to 2% DSS WT mice. H&E images of the colons reveal partial destruction of the crypts, partial loss of goblet cells and infiltration of immune cells. *p 

Fig. 10

Colon, spleen and MLN immune cell changes in α7nAChR KO mice. There was no difference amongst all groups when analysing the colons for (A-E) CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Splenic levels of (F) CD4+ were decreased (compared to 2% DSS) and (H) CD25+ T cells levels were not different from controls with TUS treatment. (G) CD8+ T cells were increased in both 2% DSS and 2% DSS + TUS KO groups. MLN levels of (M) CD25+ T cells were increased in both 2% DSS and 2% DSS + TUS KO groups, while decreased (O) B220+ levels were seen in the 2% DSS KO group only. There was no difference regarding splenic (I) F4/80+ and (J) B220+ cells, and no difference in MLN (K) CD4+, (L) CD8+ and (N) F4/80+ cells. *p < .05 compared to 2% DSS WT. #p < .05 compared to 2% DSS KO. One-way ANOVA followed by Tukey post-hoc test. N = 4/group for IHC analysis. Results are presented as mean ± SD.

Fig. 11

Colon, spleen and MLN analysis for F4/80+α7nAChR+ cells. Photomicrographic images revealed (A) increased levels of F4/80+α7nAChR+ cells in the colons of 2% DSS and 2% DSS + TUS, and even higher levels at the 2% DSS + TUS mice. No difference was seen across all groups in the (B) spleen and (C) MLN, in addition to the colons from (D) splenectomised animals. However, (E) TUS increased the levels of F4/80+α7nAChR+ cells in the MLN of splenectomised animals. Images show staining for the nuclei (blue), F4/80 macrophages (green) and 〈7nAChR (red). Merged images demonstrate co-staining of F4/80+α7nAChR+ macrophages in orange (insert). *p < .05 compared to control. #p < .05 compared to 2% DSS. One-way ANOVA followed by Tukey post-hoc test for WT C57BL/6 groups. Student's test for splenectomised animals. N = 3/group. Results are presented as mean ± SD. Images were taken with a 20× objective, whereas inserts were taken with a 63× objective. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 12

Colon analysis for GFAP+ enteric glial cells. IHC analysis revealed (A-C) no difference amongst all groups when comparing GFAP levels. Images show staining for the nuclei (blue) and GFAP (red). One-way ANOVA followed by Tukey post-hoc test or Student's t-test. N = 3–4/group. Results are presented as mean ± SD. Images were taken with a 10× and a 20× objective. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 13

Schematics of therapeutic ultrasound (TUS) effects on Dextran Sulphate Sodium (DSS)-induced acute colitis. TUS administration to the mouse abdomen attenuated DSS colitis through stimulation of the splenic nerve, activating the cholinergic anti-inflammatory pathway (CAIP). The release of norepinephrine (NE) in the spleen stimulates CD4+ T cells to release acetylcholine (ACh), which binds to α7 nicotinic acetylcholine receptors (α7nAChR) on macrophages and inhibits the release of pro-inflammatory cytokines. The vagus nerve (VN) could be the one initially stimulated by TUS, thus carrying out a therapeutic effect through the splenic nerve and, to a lesser extent, possibly the colon and mesenteric lymph node (MLN). In particular, enteric neurons release ACh to the muscularis macrophages in the gut, consequently carrying an anti-inflammatory effect and contributing to attenuation of experimental colitis. Further studies are needed to clarify the involvement of the vagus nerve. β2AR = β2 Adrenergic Receptor.

Supplementary Fig. 1

Ultrasound transducer calibration and temperature changes in mice. (A) Calibration of the ultrasound transducer was performed by measuring the effective transducer output utilizing a needle-type hydrophone in degassed water, reported as Peak Negative Pressure (kPa) vs Input Power (W/cm2). (B) Temperature changes in mice under TUS treatment over the abdomen for 7 min at 1 MHz, 10% duty cycle and 2 w/cm2. Results demonstrate a change of ~2 °C over 7 min, with a decrease when TUS is turned off. N = 3. Results are presented as mean ± SD for body temperature changes.

Supplementary Fig. 2

FACS gating for Immune cell profiling of the spleen. Representative gating of flow cytometry performed at days 0 (control) and 14 (2% DSS and 2% DSS + TUS) in the spleen for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. F4/80+ macrophages and CD3+CD4+CD25+ T cells were enriched through magnetic separation before flow cytometry analysis.

Supplementary Fig. 3

Proteomic colon changes in 2% DSS colitis mice under TUS treatment. Experimental colitis was induced by DSS for 7 days in drinking water and TUS treatment was administered from day 4 to 10 over the abdomen. The colons were collected at days 5, 7, 9, 11 and 14, homogenized and later analysed by multiplex ELISA assay. Results demonstrate no changes of colonic IL-1α, IL-6, IL-10, IL-12(p40), IL-15, IFNγ, KC, LIF, LIX and VEGF, compared to 2% DSS. Two-way ANOVA followed by Sidak post-hoc test. N = 5–6/group at each time point. Results are presented as mean ± SD. Heat maps of these results are presented in Supplementary Fig. 4.

Supplementary Fig. 4

Colonic proteomic analysis in both 2% DSS and 2% DSS + TUS groups. Heat map of temporal proteomic analysis based on multiplex ELISA revealed mice receiving 2% DSS only demonstrated increased fold changes of IL-1α, IL-1β, IL-6, IL-17, Eotaxin, G-CSF, KC, MCP-1, LIF, LIX, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES and TNFα, and decreased fold changes of IL-2, IL-7, IL-10 and IL-15 at different time points, normalized to normal control colons (day 0). Mice receiving 2% DSS + TUS treatment demonstrated increased fold changes of IL-1α, IL-1β, IL-6, IL-9, IL12(p70), IL-17, Eotaxin, IFNγ, G-CSF, KC, MCP-1, LIF, LIX, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα and VEGF, and decreased proteomic levels of IL-2, IL-4, IL-7, IL-10 and IL-15 at different time points, compared to day 0 (control). *p < .05 fold increases compared to day 0 (control). #p < .05 fold decreases compared to day 0 (control). Two-way ANOVA followed by Sidak post-hoc test. N = 5–6/group at each time point.

Supplementary Fig. 5

IHC staining for Immune cell profiling of the colon. Representative images of IHC analysis performed at days 0 (control) and 14 (2% DSS and 2% DSS + TUS) in the colon of C57BL6 WT mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 6

IHC staining for Immune cell profiling of the MLN. Representative images of IHC analysis performed at days 0 (control) and 14 (2% DSS and 2% DSS + TUS) in the MLN of C57BL6 WT mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 7

IHC staining for Immune cell profiling of the colon of splenectomised mice. Representative images of IHC analysis performed at day 14 in the colon of splenectomised mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 8

IHC staining for Immune cell profiling of the MLN of splenectomised mice. Representative images of IHC analysis performed at day 14 in the MLN of splenectomised mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 9

IHC staining for Immune cell profiling of the colon of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the colon of WT or α7nAChR KO mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 10

IHC staining for Immune cell profiling of the MLN of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the MLN of WT or α7nAChR KO mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.

Supplementary Fig. 11

IHC staining for Immune cell profiling of the spleen of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the spleen of WT or α7nAChR KO mice for CD4+, CD8+, CD25+, F4/80+ and B220+ cells. Images were taken with a 20× objective.