Heme oxygenase-1 ameliorates endotoxin-induced acute lung injury by modulating macrophage polarization via inhibiting TXNIP/NLRP3 inflammasome activation

https://doi.org/10.1016/j.freeradbiomed.2022.11.032Get rights and content

Highlights

  • HO-1 could ameliorate LPS-induced ALI by regulating macrophage polarization.

  • HO-1 could inhibit the activation of NLRP3 inflammasome in ALI induced by LPS.

  • TXNIP/NLRP3 signaling pathway might be involved in the HO-1-induced modulating macrophage polarization.

Abstract

Acute lung injury (ALI) remains a global public health issue without specific and effective treatment options available in the clinic. Alveolar macrophage polarization is involved in the initiation, development and progression of ALI; however, the underlying mechanism remains poorly understood. Heme oxygenase-1 (HO-1) acts as an antioxidant in pulmonary inflammation and has been demonstrated to be linked with the severity and prognosis of ALI. In this study, the therapeutic effects of HO-1 were examined, along with the mechanisms involved, mainly focusing on alveolar macrophage polarization. HO-1 depletion induced higher iNOS and CD86 (M1 phenotype) expression but was significantly decreased in Arg-1 and CD206 (M2 phenotype) expression in BALF alveolar macrophages after equivalent LPS stimulation. We also found that HO-1 deletion distinctly accelerated the expression of inflammasome-associated components NLRP3, ASC and caspase-1 in vivo and in vivo and in vitro. Moreover, on the basis of LPS for MH-S cells, levels of TXNIP, NLRP3, ASC and caspase-1 were increased and HO-1 depletion exacerbated these changes, whereas double depletion of HO-1 and TXNIP partially mitigated these elevations. Also, HO-1 knockdown induced more M1 phenotype and less M2 phenotype compared with LPS alone, whereas double silence of HO-1 and TXNIP partially changed the polarization state. Taken together, we demonstrated that HO-1 could modulate macrophage polarization via TXNIP/NLRP3 signaling pathway, which could be a potential therapeutic target for ALI treatment.

Introduction

Acute lung injury (ALI) is a clinical syndrome caused by a variety of endo- and exogenous factors including acute pneumonia, sepsis and severe trauma [1,2]. Due to the pandemic of coronavirus disease, lung, the most vulnerable organ to inflammation during sepsis, is especially at risk of acute injury. Currently, the therapeutic methods to treat ALI, while helpful, are extremely limited [3,4]. Although mechanical ventilation improves outcomes, the fatality rate of ALI is still as high as 30%–45% [5]. Therefore, it is vital that treatments be developed to ameliorate pulmonary inflammation and damage.

Alveolar macrophages (AMs) make up approximately 70% of alveolar immune cells and form the first line of lung defense, which plays a critical role in the development of sepsis-associated ALI via modulating the release of various inflammatory mediators [6]. AMs display remarkable plasticity and can trigger different activation states in response to alterations in surrounding contexts [7,8]. There are two main phenotypes of AMs: M1 and M2. Under physiological conditions, the predominant phenotype of the resident is M2 [9]. During infections, the resident M2, as well as the recruited macrophages, switch to M1, releasing pro-inflammatory mediators and recruiting neutrophils to kill the pathogens [10,11]. In sepsis, excessive M1 polarization exacerbates uncontrolled inflammation, further aggravating lung damage, whereas M2 polarization restricts inflammatory reactions to aid tissue healing [9,12]. Utilizing this property of AMs has vast potential for treating ALI by suppressing inflammatory response or facilitating the anti-inflammatory response. At present, how to effectively modulate the polarization of AMs and depress inflammatory cascades is a topical issue in the treatment of ALI.

A recent increase in attention has been directed at the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, due to its close involvement with ALI [2,13]. There are three components of the NLRP3 inflammasome: sensor molecule NLRP3, adaptor protein ASC, and pro-caspase-1, which can be activated by various exogenous and endogenous substances [14]. Activated NLRP3 promotes pro-caspase-1 self-cleavage into active caspase-1, which subsequently induces pro-IL-1β and pro-IL-18 maturation and secretion of IL-1β and IL-18 [15]. Recent studies have shown that adverse stimuli may contribute to M1 macrophage polarization by activating the NLRP3 inflammasome and the conversion of procaspase-1 to caspase-1 during the development of inflammation [16,17]. Thioredoxin-interacting protein (TXNIP) has been demonstrated to bind to NLRP3 in a redox-dependent manner and to play a critical role in activating the NLRP3 inflammasome [[18], [19], [20]]. Several studies have indicated that TXNIP regulates inflammation in macrophages, and elevated TXNIP in macrophages promotes M1 polarization and inhibits M2 polarization [[21], [22], [23], [24], [25]]. To some extent, therapeutic strategies based on the modulation of TXNIP/NLRP3 activation may be suitable for ALI.

Heme oxygenase-1 (HO-1), a stress-responsive enzyme, has been reported to be anti-inflammatory and antioxidative and to promote the return to homeostasis after the onset of many pathologic conditions [[26], [27], [28]]. Our previous studies demonstrated that HO-1-derived carbon monoxide provides cytoprotection against sepsis-induced ALI [[29], [30], [31]]. The role of HO-1 in LPS-induced TXNIP/NLRP3 activation leading to alveolar macrophage polarization and protection remains to be investigated.

In this research, we hypothesized that HO-1 regulates alveolar macrophage polarization and attenuates LPS-induced lung injury by inhibiting TXNIP/NLRP3 activation.

Section snippets

Animals and treatment

According to laboratory animal legislation, the study was approved by the Animal Care and Use Committee of Tianjin Nankai Hospital (Approval No. NKYY-DWLL-2021-049). Wild-type (WT) C57BL/6 male mice (age 6–8 weeks, weight 20–22g) were obtained from Vital River Laboratories (Beijing, China). HO-1 conditional knockout (HO-1-/-) mice on a C57BL/6 background (HO-1fl/fl/CAG-CreERT2) were purchased from Beijing Biocytogen Co., Ltd. To evaluate the effect of HO-1 on LPS-induced ALI, mice were divided

HO-1 ameliorated LPS-induced acute lung injury in mice

Our previous studies reported that HO-1 expression enhanced in lung tissue during LPS-induced acute lung injury [33]. In this research, conditional HO-1fl/fl/CAG-CreERT2 mice and mice injected with hemin, a potent inducer of HO-1, were used to further explore the effect of HO-1 on the development of LPS-induced ALI. HO-1 was detected and quantified by Western blot. As shown in Fig. 1A and B, compared with the control group, HO-1 expression in the LPS group and hemin group was significantly

Discussion

The pathogenesis and possible treatments of septic ALI have been extensively studied. However, optimal strategies to improve inflammation and patient outcomes are yet to be identified. The present study demonstrated that HO-1, a critical endogenous protecting factor, mitigated endotoxin-induced ALI by regulating M1/M2 of alveolar macrophage polarization during inflammation. Additionally, HO-1 effectively modulated NLRP3 inflammasome activation to inhibit M1 macrophage polarization but promote

Conclusion

Taken together, we demonstrated that HO-1 could effectively ameliorate sepsis-induced ALI. Mechanically, HO-1 modulates macrophage polarization via TXNIP/NLRP3 signaling pathway, which could be a potential therapeutic target for ALI treatment.

Author contributions

XY Wu, LL Wu and Y Wu designed the study, analyzed and interpreted data. W Chen and JK Chen performed statistical analysis. LR Gong supervised the study. XY Wu drafted the manuscript. LR Gong and JB Yu critically reviewed the manuscript. All the authors did final approval of the version to be submitted.

Declaration of competing interest

All authors declare no conflicts of proprietary or interest.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 82172121 and 82074153) and Key project of Tianjin Natural Science Foundation (No. 20JCZDJC00480) and Tianjin Health Science and Technology Foundation (No. TJWJ2021MS028).

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