Elsevier

Fish & Shellfish Immunology

Volume 84, January 2019, Pages 8-19
Fish & Shellfish Immunology

Full length article
N-terminal domain of EcC1INH in Epinephelus coioides can antagonize the LPS-stimulated inflammatory response

https://doi.org/10.1016/j.fsi.2018.09.063Get rights and content

Highlights

  • Full-length sequence of EcC1INH was identified from the orange-spotted grouper.

  • The up-regulation of EcC1INH was observed following vibrio challenge.

  • N-terminal domain of EcC1INH can attenuate the LPS-induced inflammation.

Abstract

Complement 1 inhibitor (C1INH) serving as a multifunctional factor can participate in the regulation of complement cascades and attenuate the activation of various proteases. In this study, we obtained EcC1INH cDNA and the tissue-specific analysis indicate that the highest expression level of EcC1INH mRNA was detected in liver. Moreover, Vibrio alginolyticus challenge can significantly increase EcC1INH mRNA expression in liver and kidney. N-terminal domain of EcC1INH could decrease LPS binding activity to cell surface, while loss of positively charged residues (PCRs) Arg21, His22, Lys50, Arg61 in N-terminal domain of EcC1INH can significantly reduce its interaction with LPS. Furthermore, LPS injection experiment indicated that the binding of EcC1INH N-terminal domain to LPS can antagonize LPS-induced inflammatory signaling pathway and attenuate the production of proinflammatory cytokines in vivo, indicating that EcC1INH was involved in negative regulation of inflammatory response.

Introduction

In mammal, cell-killing mechanisms are the most important components of host immune defense against invading microorganisms, infectious tissues and malignant cells via a cytotoxic manner by activating specific proteins such as complement proteins [1]. Complement cascade is one of the central constitutes in mammalian macrophage-activating immune system [2] and serves as professional effectors capable of increasing the phagocytosis [3], generating significant amounts of superoxide production [4] as well as synchronizing nature immune system with acquired immunity [5]. Additionally, classic pathway, alternative pathway and Mannose binding lectin (MBL) pathway can produce active opsonins or recruiters of other proteins and form membrane attack complexes (MAC), thus defending against the invasion of microorganism [6]. Recent studies indicate that mammalian complement system activated by bacterial lipopolysaccharide (LPS) stimulation can effectively confer protection against the subsequent bacterial infection by enhancing phagocytic activity, whereas a destroyed complement system can significantly reduce the in vivo clearance of virulent strains, implying that complement activation participates in the host killing mechanism of microorganism [7]. In addition, complement deficiency may relate to a reduced resistance to the microorganism invasion, leading to an increasing prevalence of pyogenic infections and immune complex diseases [8].

Complement 1 inhibitor (C1INH) plays an indispensable role in modulating complement pathways as a negative modulator that inhibits the classical pathway by depressing the activities of complement 1r (C1r) and complement 1s (C1s) [9], exhibits a downregulatory effect in the lectin complement pathway by inhibiting mannose binding lectin associated serum protease (MASP) [10], as well as blocks the alternative complement cascade to decrease complement 3 (C3) cleavage by directly inhibiting the binding activity [11]. Recent findings indicate that C1INH may also inactivate surface-mediated pathway [12,13], which may appear to be associated with the regulation of inflammatory response [14]. In addition, C1INH, a key inhibitor of plasma serine proteases, harboring the characteristic structure of serpin superfamily, may serve as a major downregulator of inflammatory processes in mammal [15].

Mounting evidences indicate that the innate immune defense system in invertebrates harbors pathogen recognition units, but it is only a teleostean basic defense mechanism [16]. In contrast, bony fish contain developed complement cascades [17], and the structures of teleostean complements are homologous to those of mammals [18]. Despite previous researches pay more attention to complement cascades in mammals, only a few reports study on the teleostean complement system. In recent years, teleostean C1INHs have been identified in several fish species, including Oreochromis niloticus [19,20], Pseudosciaena croceav [21], Oplegnathus fasciatus [22] and Sebastes schlegelii [23]. Most of these studies focus on the gene structure and gene analysis of teleostean C1INHs, but the possible function of teleostean C1INHs in LPS-mediated inflammatory response is still unclear.

Stressors such as various pathogenic diseases may exert a harmful effect on the immune system in grouper [24,25]. The recent emergence of global climate anomaly arising a wide public concern may be one of the abnormal phenomena, which can aggravate the expansion of vibrio infection via the long-distance geographical transportation [26]. Vibrio alginolyticus is one of vibrio strains can produce a extracellular toxin [27]. In addition, fecal population may facilitate the increasing level of vibrio population [28]. Vibrio strains also contain a TonB/ExbB/ExbD complex that can regulate the iron uptake processes, thus rendering the invading bacteria less susceptible to a microenvironmental condition of limited iron availability [29]. Recent findings indicate that in vivo injection of V. alginolyticus or its extracellular products can significantly induce toxicological effect in grouper, leading to a slight exophthalmia with corneal opaqueness in moribund/dead fish [27]. Thus, a better understanding of the immunity-related mechanism underlying the change of EcC1INH expression to vibrio stimulation is of great importance and may contribute to the sustainable aquaculture. In this research, we mainly investigated the binding activity of N-terminal domain of EcC1INH to LPS and its protective effect on LPS-induced inflammatory responses.

Section snippets

Fish preparation

Healthy groupers (approximately 12.50 g) were obtained from a fish farm (Guangdong, China). Groupers were fed twice and acclimatized in aerated seawater before vibrio challenge.

Cloning

Cloning methods and protocols of full-length EcC1INH cDNA was based on our previous studies using a rapid amplification of cDNA ends (Clontech, China) [30] and the primers for gene cloning were shown in Table 1.

Bioinformatics analysis

Based on our pervious studies, the domain structures of predicted EcC1INH amino acid sequences were analyzed by

Bioinformatics analysis of EcC1INH cDNA

In Supplementary Fig. 1A, EcC1INH cDNA of 2219 bp comprised an ORF of 1797 bp with a poly(A) tail. In addition, the molecular mass of the deduced EcC1INH polypeptide (598 amino acids) was about 66.46 KDa.

In Supplementary Fig. 1B, EcC1INH was a member of serpin superfamily and the deduced amino acid sequence of EcC1INH contained two potential functional domains: Ig domain (amino acids 28–106 and amino acids 116–208, respectively) and serpin domain (amino acids 234–598). GenBank analysis

Discussion

The activation of mammalian complement cascades is involved in the host defense mechanism against microorganism infection [49]. Generally speaking, microorganism infection can activate the human alternative pathway by targeting to C3 receptor-bearing cells [50]. Among the known complement cascades, alternative pathway is an antibody-independent complement cascade, which can be directly activated by LPS stimulation [[51], [52], [53]]. Recent findings demonstrate that mammalian C1INH plays a

Acknowledgements

This project was supported by Guangzhou science and technology plan project (grant nos. 201707010135) and National Natural Science Foundation of China (Grant No.31670423).

References (71)

  • R. Harikrishnan et al.

    Molecular studies, disease status and prophylactic measures in grouper aquaculture: economic importance, diseases and immunology

    Aquaculture

    (2010)
  • K.-K. Lee

    Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus, Bloch et Schneider

    Microb. Pathog.

    (1995)
  • S. Luo et al.

    Molecular cloning, characterization and expression analysis of PPAR gamma in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge

    Fish Shellfish Immunol.

    (2015)
  • S.-W. Luo et al.

    Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2) Bcl-2 in the orange-spotted grouper (Epinephelus coioides)

    Dev. Comp. Immunol.

    (2017)
  • S.-W. Luo et al.

    Effects of a recombinant complement component C3b functional fragment α 2 MR (α 2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge

    Fish Shellfish Immunol.

    (2015)
  • S.-W. Luo et al.

    Molecular cloning and characterization of PTEN in the orange-spotted grouper (Epinephelus coioides)

    Fish Shellfish Immunol.

    (2016)
  • G.-H. Cha et al.

    Optimal conditions for expressing a complement component 3b functional fragment (α2-macroglobulin receptor) gene from Epinephelus coioides in Pichia pastoris

    Protein Expr. Purif.

    (2015)
  • S.-W. Luo et al.

    Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge

    Dev. Comp. Immunol.

    (2016)
  • S.-W. Luo et al.

    Functional analysis of a dietary recombinant Fatty acid binding protein 10 (FABP10) on the Epinephelus coioides in response to acute low temperature challenge

    Fish Shellfish Immunol.

    (2014)
  • I.G. Wagenaar-Bos et al.

    Structure and function of C1-inhibitor

    Immunol. Allergy Clin.

    (2006)
  • I.G. Bos et al.

    The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant

    J. Biol. Chem.

    (2003)
  • I.G. Bos et al.

    Structural and functional aspects of C1-inhibitor

    Immunobiology

    (2002)
  • Z.-H. Qi et al.

    Molecular characterization and functional analysis of a complement C3 molecule in the orange-spotted grouper (Epinephelus coioides)

    Fish Shellfish Immunol.

    (2011)
  • A. Ellis

    Innate host defense mechanisms of fish against viruses and bacteria

    Dev. Comp. Immunol.

    (2001)
  • A. Ellis

    Immunity to bacteria in fish

    Fish Shellfish Immunol.

    (1999)
  • D. Liu et al.

    C1 inhibitor prevents Gram-negative bacterial lipopolysaccharide-induced vascular permeability

    Blood

    (2005)
  • M.Z. Djie et al.

    Intrinsic specificity of the reactive site loop of α1-antitrypsin, α1-antichymotrypsin, antithrombin III, and protease nexin I

    J. Biol. Chem.

    (1997)
  • T. Kawai et al.

    Unresponsiveness of MyD88-deficient mice to endotoxin

    Immunity

    (1999)
  • H. Wesche et al.

    MyD88: an adapter that recruits IRAK to the IL-1 receptor complex

    Immunity

    (1997)
  • K. Takeda et al.

    TLR signaling pathways

  • E. Lye et al.

    The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling

    J. Biol. Chem.

    (2004)
  • Y.-C. Lu et al.

    LPS/TLR4 signal transduction pathway

    Cytokine

    (2008)
  • P.E. Wakefield et al.

    Tumor necrosis factor

    J. Am. Acad. Dermatol.

    (1991)
  • H.J. Muller-Eberhard

    The membrane attack complex of complement

    Annu. Rev. Immunol.

    (1986)
  • H.L. Collins et al.

    Cytokine enhancement of complement‐dependent phagocytosis by macrophages: synergy of tumor necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor for phagocytosis of Cryptococcus neoformans

    Eur. J. Immunol.

    (1992)
  • Cited by (12)

    • An overview of complement systems in teleosts

      2022, Developmental and Comparative Immunology
      Citation Excerpt :

      In black rockfish and rock bream, C1INH exhibited protease inhibiting activity against C1 esterase and thrombin (Fig. 1) (Umasuthan et al., 2014; Nilojan et al., 2018). In Epinephelus coioides, the N-terminal domain of C1INH can antagonize the LPS-induced inflammatory signaling pathway and attenuate the production of proinflammatory cytokines (Luo et al., 2019). In Nile tilapia, C1INH may be involved in the immune response against bacterial challenge (Ding et al., 2017).

    • Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection

      2021, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
    • Effect of Lipopolysaccharide (LPS) stimulation on apoptotic process and oxidative stress in fibroblast cell of hybrid crucian carp compared with those of Carassius cuvieri and Carassius auratus red var

      2021, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
      Citation Excerpt :

      Total RNA was extracted from fish cells by using HiPure Total RNA Mini kit. Concentration and purity quotient of total RNA was determined by measurement of 260/280 nm absorbance, while its integrity was determined by 1% agarose gel electrophoresis (Luo et al., 2019a). According to manufacturer's protocols, purified total RNA was used to synthesize cDNA by using RevertAid RT Reverse Transcription kit.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text