Interleukin-22 protects from endotoxemia by inducing suppressive F4/80+Ly6GhiLy6Chi cells population

Yu, Chang; Ling, Qihua; Jiao, Junzhe; Liu, Juhong; Huang, Zhihua; Wang, Fang; Sun, Xuehua; Kong, **aoni

doi:10.1186/s12865-022-00511-6

Interleukin-22 protects from endotoxemia by inducing suppressive F4/80⁺Ly6G^hiLy6C^hi cells population

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Published: 19 September 2022

Volume 23, article number 45, (2022)
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Interleukin-22 protects from endotoxemia by inducing suppressive F4/80⁺Ly6G^hiLy6C^hi cells population

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Chang Yu¹^na1,
Qihua Ling²^na1,
Junzhe Jiao¹^na1,
Juhong Liu³,
Zhihua Huang³,
Fang Wang¹,
Xuehua Sun¹ &
…
**aoni Kong¹

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Abstract

Background

Excessive inflammatory response is the primary cause of early death in patients with endotoxemia. Interleukin 22 (IL-22) has been shown to play critical roles in the modulation of infectious diseases, but its function in regulating immune responses during endotoxemia remains unclear.

Methods

Lipopolysaccharide (LPS) was used to induce endotoxemia mouse model with or without a recombinant fusion protein containing human IL-22 (F-652). IL-6, TNF-α, IL-1β, and MCP-1 were measured by ELISA assays. The type of macrophage was assessed by flow cytometry. Real-time PCR was used to detect the expression of S100A9.

Results

We found that F-652 injection significantly improved the survival rates and reduced pro-inflammatory cytokines (IL-6, TNF-a, IL-1β, MCP-1) in LPS-induced endotoxemia mice. However, the mice injected with F-652 had a higher number of infiltrated immune cells after LPS treatment, suggesting an impaired immune response. Flow cytometry analysis showed a higher number of F4/80⁺Ly6G^hiLy6C^hi cells that highly expressed M2-like macrophage markers (Ym1, Arg, CCL17) in the peritoneal cavity of the F-652-treated endotoxemia mice. Further investigation found that these suppressive M2 macrophages might be induced by F-652 since the F-652 treatment could increase S100A9 in vitro.

Conclusions

Our study suggests that IL-22 has a protective role against endotoxemia by inducing the development of immunosuppressive cells through S100A9.

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Background

Endotoxemia remains a serious problem in hospitals, especially in ICUs. It has been considered as a severe clinical problem and a global public health threat. According to the epidemiology of endotoxemia in Chinese ICUs, endotoxemia affects 20% of ICU patients, its incidence in ICUs is about 20.6%, and the 90-day mortality rate is around 35.5% [Full size image

We summarized our study in Fig. 5. IL-22 (F-652) protects against LPS-induced cytokines storm in the LPS-induced endotoxemia model by inducing the expression of APPs from the liver, like S100A9, and thereby inducing F4/80⁺Ly6G^hiLy6C^hi M2-like suppressor cells differentiation.

Discussion

There are many types of lymphocyte subpopulations, such as Th17, Th22, and ILC3s, that could produce IL-22 [24]. Multiple studies have illustrated that IL-22R, one of the IL-22 receptors, is preferentially expressed in epithelial cells, and IL-22 acts mainly on tissue epithelial and stromal cells to induce tissue repair and innate host responses [25]. Numerous reports have shown that IL-22 modulates systemic immunity by acting indirectly on immune cells, and the liver plays an essential role in this process [7]. For example, in bacteria-induced lung infections, IL-22 acts on the liver to induce the expression of C3 and, thereby, modulates immune responses to control the infection [26]. These studies have demonstrated that IL-22 plays important functions in systemic and local immunity by indirectly acting on immune cells.

There are also many controversial studies about the effects of IL-22 in modulating the immune response. IL-22 has been reported to be involved in both pro- and anti-inflammatory effects. For example, Interleukin-22 deficiency alleviates doxorubicin-induced oxidative stress and cardiac injury via the p38 MAPK/macrophage/Fizz3 axis in mice [27]. In another study about the rIL-22-treated ApoE^−/− mice, the levels of IL-6 were significantly increased. This increase in IL-6 concentration promoted the maturation of DC and, thereby, induced Th17 cell proliferation and exacerbated inflammation [28]. However, most studies on the anti-inflammatory effects of IL-22 have focused on its ability to maintain the integrity of the intestinal barrier and eliminate bacteria [10, 29].

There were also several studies about the direct effects of IL-22 in modulating immune cells. In 2019, Eun-Young Kim and his colleagues discovered that IL-22 treatment could directly promote the polarization of M2 macrophages [30]. Subsequently, in 2020, Si-BiaoSu et al. found that IL-22 could promote the differentiation of Kupffer cells into the M2-like macrophages [31]. As one of the primary innate immune cells, macrophages play an important role in endotoxemia. However, there are very few studies on the regulation of macrophages by IL-22. Therefore, it remains unclear whether IL-22 plays an important role in endotoxemia and whether it can regulate the polarization of M2 macrophages in the LPS-induced endotoxemia model. In the current study, we adopted a recombinant fusion protein containing human IL-22 called F-652. According to the results of clinical research, F-652 is a safe and well-tolerated drug that demonstrates favorable PK and pharmacodynamic properties [17]. In addition, F-652 can significantly reduce inflammation and increase hepatic regeneration in patients with alcohol-associated hepatitis compared to the placebo [17, 18]. Therefore, F-652 has the potential to treat inflammatory diseases, and we thereby investigated the role and potential mechanisms of F-652 in endotoxemia.

In this study, we found that LPS + F-652-treated mice had improved mortality rates and decreased the production of pro-inflammatory cytokines (IL-6, TNF-α, Mcp-1, and IL-1β) compared to the mice treated with LPS only (Fig. 1). In contrast, the number of immune cells in the lung, peritoneal lavage fluid, and bronchoalveolar lavage fluid was significantly increased in the LPS + F-652-treated mice (Fig. 2). More immune cells with lower cytokine production implied that these immune cells exhibited a suppressive immune response. Since IL-22 could promote the polarization of the M2-like macrophages, we hypothesized that F-652 might promote the polarization of the M2-like macrophages and thus suppress the inflammatory response in the LPS-induced endotoxemia model. Subsequently, our results demonstrated that the addition of F-652 in the LPS-induced endotoxemia mice resulted in more F4/80⁺Ly6G^hiLy6C^hi cells in the peritoneal cavity, showing a generic signature of M2-type macrophage (Fig. 3). To investigate whether IL-22 directly induces M2 macrophage polarization, we isolated macrophages and treated them with LPS or LPS + F-652 in vitro. Results showed that F-652 treatment did not increase Ly6C^hi cells, but the treatment with serum from F-652-treated mice induced macrophages to differentiate into F4/80⁺Ly6G^hiLy6C^hi cells (Fig. 4d–f). Thus, our results suggest that IL-22 promotes M2 macrophage polarization not by acting directly on macrophages but through hepatic APPs in the serum.

The liver is the main target organ of IL-22. Studies have shown that hepatocytes are the primary target of IL-22, since it can induce hepatic production of acute-phase proteins [32]. IL-22 can affect hepatic gene expression 30 min after injection [22]. By using serum from LPS + F-652-treated mice, Ly6C^hi cells were significantly enhanced compared to other groups (Fig. 4e, f). This suggests that APPs in the serum are involved in the induction of Ly6C^hi cells. In addition, IL-22 can dose-dependently induce the expression of multiple S100 family proteins (S100A7, S100A8, and S100A9) [14]. Both SAA and S100A9 play important roles in inflammatory diseases and can promote the differentiation of M2-type macrophages [15, 23, 33]. Therefore, we examined the serum levels of SAA and S100A9 in the LPS- and the LPS + F-652-treated mice. We found that there is no significant difference in the level of SAA between LPS- and LPS + F-652-treated mice (Fig. 4a–c). However, the level of S100A9 in LPS + F-652-treated mice was higher than that of the LPS-treated mice (Fig. 4b). Then, we found that S100A9 was significantly increased in the liver of F-652-treated mice (Fig. 4c). More importantly, the blockage of S100A9 decreased the polarization of the F4/80⁺Ly6G^hiLy6C^hi cells (Fig. 4e, f). Therefore, IL-22 induced more suppressive Ly6Chi cells in the LPS-induced endotoxemia model, probably by inducing S100A9 production in the liver.

In summary, our findings suggest that F-652 (IL-22) protects mice against LPS-induced endotoxemia by promoting M2-like macrophage polarization, and this process may depend on S100A9 (Fig. 5).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ALI:: Acute liver injury
APPs:: Acute phase reaction proteins
DSS:: Dextran sodium sulfate
Elisa:: Enzyme-linked immunosorbent assay
HSCs:: Hepatic stellate cells
H&E:: Hematoxylin and eosin
IL-22:: Interleukin 22
IL-22BP:: IL-22-binding protein
IL-6:: Interleukin-6
ILCs:: Innate lymphoid cells
IgG2-Fc:: Immunoglobulin constant region
LPS:: Lipopolysaccharide
NASH:: Nonalcoholic steatohepatitis
OECD:: Organization for Economic Cooperation and Development
PK:: Pharmacokinetics
SAA:: Serum amyloid A
SCFA:: Short-chain-fatty acids
TNF-α:: Tumor necrosis factor α
TGF-β:: Transforming growth factor-β
TREM-1:: Triggering receptor expressed on myeloid cells-1
RFC:: Relative fold change

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Acknowledgements

Not Applicable.

Funding

This work was supported by the National Natural Science Foundation of China (82070633 and 81873582 to **aoni Kong, 82004321 to Qihua Ling, 82074336 to Xuehua Sun), Program of Shanghai Academic/Technology Research Leader (20XD1403700) to **aoni Kong.

Author information

Chang Yu, Qihua Ling and Junzhe Jiao have contributed equally to this study

Authors and Affiliations

Central Laboratory, Department of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, 528 Zhangheng Road, Shanghai, 201203, China
Chang Yu, Junzhe Jiao, Fang Wang, Xuehua Sun & **aoni Kong
Department of Emergency Internal Medicine, Shuguang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
Qihua Ling
Evive Biotechnology (Shanghai) Ltd, Shanghai, China
Juhong Liu & Zhihua Huang

Authors

Chang Yu
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Qihua Ling
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Junzhe Jiao
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Juhong Liu
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Zhihua Huang
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Fang Wang
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Xuehua Sun
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**aoni Kong
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Contributions

All authors contributed to the study conception and design. XK conceived and designed the study. CY, QL and JJ performed the research, data collection and analysis. The F-652 was provided by JL and ZH. Material preparation was performed by FW. XS drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to **aoni Kong.

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Ethics approval and consent to participate

The care and use of the mice were all approved by the Ethics committee of the Shanghai University of Chinese Traditional Medicine. All animal experiments were proceeded following the guidelines of the Shanghai University of Chinese Traditional Medicine Regulation for Administration of Laboratory Animals and were performed in compliance with the ARRIVE guidelines (https://arriveguidelines.org).

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Competing interests

Author Juhong Liu and Zhihua Huang were employed by the company Evive Biotechnology (Shanghai) Ltd, China. The remaining 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.

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Yu, C., Ling, Q., Jiao, J. et al. Interleukin-22 protects from endotoxemia by inducing suppressive F4/80⁺Ly6G^hiLy6C^hi cells population. BMC Immunol 23, 45 (2022). https://doi.org/10.1186/s12865-022-00511-6

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Received: 29 March 2022
Accepted: 13 July 2022
Published: 19 September 2022
DOI: https://doi.org/10.1186/s12865-022-00511-6

Interleukin-22 protects from endotoxemia by inducing suppressive F4/80⁺Ly6G^hiLy6C^hi cells population