*2.4. Ech A Could Modulate the Polarization of Resting Macrophages into M1 and M2 Type In Vitro*

As a key component of the innate immune system, macrophages play important roles in the host defense reaction by mediating acute inflammatory response against danger signals and promoting stimulation of the adaptive immune system [19]. Typically, two main subtypes of macrophages have been described after polarization: Classically activated M1 type (M1) and alternative M2 type (M2) [20]. In general, M1 macrophages tend to mediate the excessive and persistent pro-inflammatory responses, while M2 macrophages are known to contribute to tissue regeneration and resolution of inflammation. Given that chronic inflammation with the M1/M2 polarization balance skewed toward the M1 phenotype is the major characteristic of IBD progression [21,22], modulation of macrophage activity could be another therapeutic target. Thus, we conducted an in vitro experiment to evaluate the influence of Ech A on M1/M2 differentiation. For M1 type polarization, phorbol 12-myristate-13-acetate (PMA)-treated THP1 macrophages were further stimulated with LPS and IFN-γ. As expected, M1 macrophages produced a high level of tumor necrosis factor-alpha (TNF-α) upon stimulation (Figure 4A). It was noted that Ech A could reduce M1-derived TNF-α secretion in a dose-dependent manner (Figure 4A). On the contrary, the basal secretion level of interleukin 10 (IL-10) was increased in the presence of Ech A, indicating that Ech A could induce the spontaneous M2 polarization in vitro (Figure 4B).

**Figure 4.** Ech A impact on M1/M2 macrophage polarization in vitro. (**A**) After five days of M1 induction, TNF-α concentration in the culture supernatant was measured by ELISA to estimate the M1 polarization efficiency. It is noted that Ech A prevented M1 polarization in vitro in a dose-dependent manner. (**B**) The IL-10 secretion level was determined using ELISA to evaluate the spontaneous M2 induction efficiency. Ech A treated macrophages were differentiated into M2 macrophages more effectively compared to vehicle-treated cells; (–), no induction control; (+), polarization induced control. The p-value significance was calculated by comparing other groups against the (+) and (–) groups (marked as +) in A and B, respectively. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001. Results are shown as mean ± SD.

From the in vivo observation, Ech A provided significant protection against experimental colitis as it alleviated disease severity and increased the survival rate of mice. In the histomorphological analysis of the colon, marked loss of villus-crypt structure accompanied with edematous erosion and granulation tissue formation was attenuated by Ech A injection. Ech A also decreased the degree of inflammatory cell infiltrates in a dose-dependent manner, suggesting that Ech A could exhibit an anti-inflammatory capacity in the IBD status.

Importantly, following in vitro Th cell differentiation, the experiment revealed that Ech A treatment could promote Treg generation without affecting the Th1 or Th2 populations. Considering that Treg cells function as a key immunomodulator that regulates activation of other Th cells, Ech A could contribute to correct the immunological imbalance in vivo by inducing the Treg population, although it does not directly regulate Th1 or Th2 polarization. Indeed, Tsai et al. have reported that a member of the lectin family galectin-3 prevents colitis progression while galectin-3 knockout mice suffered from more severe symptoms than controls, and the therapeutic impact of galectin-3 was abrogated by CD25 neutralization, implying the importance of Treg cells in this context [23]. Here we also demonstrated that Ech A could regulate the direction of macrophagic polarization toward M2 differentiation. M1 suppression and/or M2 induction strategies have proven beneficial for IBD treatment. Abron et al. have reported that the soy bean-derived natural agent genistein reduced the severity of DSS-induced colitis by transforming M1 macrophages into M2 type followed by the reduction of pro-inflammatory cytokine levels [24]. In addition, adipose-tissue-derived mesenchymal stem cells could provide protection in colitis mice models via TNF-α-induced gene/protein 6-mediated M2 induction [25]. In particular, the TNF-α inhibitory effect of Ech A could be directly applicable for IBD therapeutics considering that TNF-α expressing immune cells are increased in the intestinal tract of IBD patients and TNF-α blockers, as well as anti-TNF-α agents, have been approved in clinic for IBD treatment [26].

To further highlight the benefits of Ech A application, the underlying mechanisms of Ech A mediated immune cell fate regulation should be explored for the fundamental understanding of the Ech A therapeutic function. According to a recent study, Ech A could stimulate ex vivo expansion of CD34<sup>+</sup> hematopoietic stem/progenitor cells (HSPC) with enhanced colony-forming capacity via suppression of intracellular ROS generation [27]. Mechanistically, ex vivo treatment of Ech A or anti-oxidant agent N-acetyl cysteine could not only inhibit oxidative stress-associated P38 MAPK activation but also increase Lyn/Src phosphorylation followed by activation of the PI3K-Akt signaling pathway, leading to high-quality HSPC expansion. Therefore, it would be worthwhile to investigate the therapeutic contribution of the anti-oxidative capacity of Ech A in IBD prevention in terms of HSPC-derived immune cell proliferation and differentiation. In addition, Ech A could bind directly to protein kinase C-iota (PKC i) and enhanced myocardial cell differentiation of mouse embryonic stem cells by antagonizing its activity [28]. Considering that the PKC family plays an important role in Th cell homeostasis [29,30], Ech A might regulate PKC activity to induce Treg population followed by M2 macrophagic activation.

In conclusion, we demonstrate the therapeutic potential of Ech A for IBD for the first time. We also emphasize that Ech A could induce immunomodulatory effector cell generation such as Treg cells and M2 macrophages along with suppression of pro-inflammatory M1 macrophage, leading to attenuation of excessive inflammation. These data suggest that Ech A could exert immune-cell type specific regulatory roles in vitro and possibly in vivo. Therefore, our findings not only suggest Ech A as a novel potent therapeutic agent for incurable IBD but also contribute to expanding the therapeutic utility of Ech A for various inflammatory diseases.
