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. 2021 Jan 29:11:594297.
doi: 10.3389/fimmu.2020.594297. eCollection 2020.

Myosin Light Chain 9/12 Regulates the Pathogenesis of Inflammatory Bowel Disease

Masaya Yokoyama  1   2 , Motoko Y Kimura  1 , Toshihiro Ito  1 , Koji Hayashizaki  1 , Yukihiro Endo  1 , Yangsong Wang  1 , Ryoji Yagi  1 , Tomoo Nakagawa  3 , Naoya Kato  3 , Hisahiro Matsubara  2 , Toshinori Nakayama  1   4
Affiliations

Myosin Light Chain 9/12 Regulates the Pathogenesis of Inflammatory Bowel Disease

Masaya Yokoyama et al. Front Immunol. .

Abstract

The numbers of patients with inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), have been increasing over time, worldwide; however, the pathogenesis of IBD is multifactorial and has not been fully understood. Myosin light chain 9 and 12a and 12b (Myl9/12) are known as ligands of the CD69 molecule. They create "Myl9 nets" that are often detected in inflamed site, which play a crucial role in regulating the recruitment and retention of CD69-expressing effector cells in inflamed tissues. We demonstrated the strong expression of Myl9/12 in the inflamed gut of IBD patients and mice with DSS-induced colitis. The administration of anti-Myl9/12 Ab to mice with DSS-induced colitis ameliorated the inflammation and prolonged their survival. The plasma Myl9 levels in the patients with active UC and CD were significantly higher than those in patients with disease remission, and may depict the disease severity of IBD patients, especially those with UC. Thus, our results indicate that Myl9/12 are involved in the pathogenesis of IBD, and are likely to be a new therapeutic target for patients suffering from IBD.

Keywords: CD69; Crohn’s disease; Myl9; plasma biomarker; ulcerative colitis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The Myl9/12 expression in the colon after the administration of DSS. (A) Immunoblotting of the total amount of Myl9/12 protein in the colon at 0, 5, and 7 days after the administration of DSS. Tubulin-α protein was used as a loading control. (B–D) The immunohistological analysis of the colon at 0 and 7 days after the administration of DSS, stained as indicated. Red square indicates a blood vessel (C) or CD69-expressing cells close to Myl9/12-positive vessels (D). (E) The frequency of CD69-expressing cells located near to Myl9/12-positive vessels versus Myl9/12-negative vessels in inflamed colon specimens from mice with DSS-induced colitis. A total of nine fields, where Myl9/12-postive vessels were located in the center and six fields, where Myl9/12-negative vessels were located in the center were examined using three individual mice with DSS-induced colitis. *p < 0.05.
Figure 2
Figure 2
The administration of anti-Myl9/12 Ab ameliorated DSS-induced colitis. (A–C) The rate of survival (A), percent change of body weight on each day relative to those on day 0 (B), and disease activity index (DAI) (C) of the DSS-induced colitis model mice treated with either anti-Myl9/12 Ab or Control Ab (n = 10 per group); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001. (D) A representative image showing the gross appearance of the colon from the DSS-induced colitis model mice (upper) and the mice drinking normal water (bottom) treated with either anti-Myl9/12 Ab or Control Ab. Each image included the image from a healthy mouse (Normal colon). The colon length from the mice either at the time of death or on day 15. ***p < 0.001; ****p < 0.001. (E) Hematoxylin and eosin (HE) staining of the colon from a healthy mouse or DSS-induced colitis model mice treated with either anti-Myl9/12 Ab or Control Ab (left). The histological score is determined based on the changes described in Supplementary Table 2. *p < 0.05; **p < 0.01; ****p < 0.001. (F) IL-6, IL-1β, TNFα production in the plasma from DSS-induced colitis model mice treated with either anti-Myl9/12 Ab or Control Ab. *p < 0.05; ****p < 0.0001. ns, not significant.
Figure 3
Figure 3
The high expression of Myl9/12 in the inflamed colon in UC patients. (A) Immunohistological analyses of inflamed colon specimens of UC patients and non-inflamed colon specimens of colon cancer patients, stained as indicated (UC, n = 3; No inflammation, n = 3). Red squares indicate blood vessels, which are visualized by vWF staining (×400). (B) Immunohistological analyses of the inflamed colon of UC patients, stained as indicated. Red squares indicate blood vessels (×400). (C) The frequency of CD69-expressing cells close to Myl9/12-positive vessels versus Myl9/12-negative vessels in the inflamed colon. A total of 23 fields where Myl9/12-postive vessels were located in the center and 18 fields where Myl9/12-negative vessels were located in the center were examined using colon samples from five patients. **p < 0.01.
Figure 4
Figure 4
Plasma Myl9 levels depict the disease activity of UC. (A) Plasma Myl9 levels from UC patients with active UC (Mayo ≥3) or UC patients who were in remission (Mayo <2) and healthy volunteers (active UC, n = 16, remission, n = 29, healthy volunteers, n = 11). **p < 0.01; ***p < 0.001; ****p < 0.001. (B) The plasma Myl9 levels in the patients before (pre) and after (post) treatment. The Myl9 levels from the same patients are connected. (C–J) The correlations of plasma Myl9 with the Mayo Score (C), Mayo endoscopic score (D), WBC count (E), CRP (F), serum Alb (G), serum cholinesterase (H), and platelet count (I). (J) ROC curves for Myl9 and CRP in UC patients in remission and those with active UC. ns, not significant.
Figure 5
Figure 5
The Myl9/12 expression in CD patients. (A) Immunohistological analyses of inflamed gut specimens from CD patients, stained as indicated (n = 3). Red squares indicate blood vessels, which are identified based on the DIC image (×400). (B) The frequency of CD69-expressing cells close to Myl9/12-positive vessels versus Myl9/12-negative vessels in the inflamed gut. A total of 4 fields where Myl9/12-postive vessels were located in the center and 14 fields where Myl9/12-negative vessels were located in the center were examined in gut samples from three patients. (C) Plasma Myl9 levels from CD patients with active CD (HBI ≥5) or CD patients who were in remission (HBI <5) and healthy volunteers (active CD, n = 9, remission CD, n = 28, healthy volunteers, n = 11). **p < 0.01; ***p < 0.001; ****p < 0.001. (D) The plasma Myl9 levels in the patients before (pre) and after (post) treatment. The Myl9 levels from the same patients are connected. (E–J) Correlations of the plasma Myl9 level with the HBI (E), WBC count (F), CRP (G), serum Alb (H), serum cholinesterase (I), and platelet count (J). ns, not significant.
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