Identification and characterization of C1 inhibitor in Nile tilapia (Oreochromis niloticus) in response to pathogenic bacteria

Accepted Manuscript Identification and characterization of C1 inhibitor in Nile tilapia (Oreochromis niloticus) in response to pathogenic bacteria Mingmei Ding, Meng Chen, Xiaofang Zhong, Yuhong Wang, Shengli Fu, Xiaoxue Yin, Zheng Guo, Jianmin Ye PII:

S1050-4648(16)30776-8

DOI:

10.1016/j.fsi.2016.12.014

Reference:

YFSIM 4355

To appear in:

Fish and Shellfish Immunology

Received Date: 24 July 2016 Revised Date:

10 December 2016

Accepted Date: 11 December 2016

Please cite this article as: Ding M, Chen M, Zhong X, Wang Y, Fu S, Yin X, Guo Z, Ye J, Identification and characterization of C1 inhibitor in Nile tilapia (Oreochromis niloticus) in response to pathogenic bacteria, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2016.12.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

ABSTRACT C1 inhibitor (C1INH) is a multi-functional serine protease inhibitor in plasmatic

3

cascades, not only inactivating various proteases, but also regulating both complement

4

and contact system activation. In this study, we described the identification and

5

characterization of a C1INH ortholog from Nile tilapia (Oreochromis niloticus) at

6

molecular, protein and cellular levels. The full-length cDNA of Oreochromis niloticus

7

C1INH (OnC1INH) consisted of 1788 bp of nucleotide sequence encoding

8

polypeptides of 596 amino acids. The deduced protein possessed a serpin domain at

9

the C-terminal domain, and two Ig-like domains in the N-terminal domain with

10

significant homology to teleost. Expression analysis revealed that the OnC1INH was

11

extremely highly expressed in the liver; however, much weakly exhibited in other

12

tissues including spleen, kidney, blood and heart. After the in vivo challenges of the

13

lipopolysaccharide (LPS) and Streptococcus agalactiae, the expression of OnC1INH

14

was significantly up-regulated in liver and spleen at the late phase, which was

15

confirmed at the protein level with immunohistochemical analysis. The up-regulation

16

of

17

monocytes/macrophages in vitro stimulated with LPS, Aeromonas hydrophila and

18

Streptococcus agalactiae, which was positively correlated with the protein expression

19

pattern in the culture media. Taken together, the results of this study indicated that

20

OnC1INH might be involved in the immune response of Nile tilapia against to

21

bacterial challenge.

22

Key words: Oreochromis niloticus; C1 inhibitor; Immune response; Streptococcus

23

agalactiae; Monocytes/macrophages

24

expression

was

also

demonstrated

in

head

kidney

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1. Introduction Complement is an essential system for both innate and adaptive immunity against

28

microbial infection within the vertebrate host [1-4]. In the vertebrate system, there are

29

three distinct pathways: the classical pathway, the lectin pathway, and the alternative

30

pathway. Because the complement system is nonspecific and thus capable of attacking

31

microorganisms as well as host cells, regulatory mechanisms have evolved to restrict

32

its activity, including regulatory proteins such as complement component 1 inhibitor

33

(C1INH) to inactivate complement components. C1INH, a member of the serine

34

protease inhibitor (serpin) [5], is the only known inhibitor of C1s and C1r of the

35

classical pathway [6-8]. C1INH either binds reversibly to pro-enzymic C1r and C1s

36

within C1 to prevent self-active [9] or binds to activated C1r and C1s or dissociates

37

them from C1q [8,10]. Besides, C1INH was also reported to inhibit the alternative

38

pathway [11] and lectin pathway [12] in which it could control a wide variety of

39

inflammatory process.

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In addition to regulate the complement system, C1INH, a multi-functional serine

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protease inhibitor, exerts its inhibition function in different plasmatic cascades

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including the contact (Factor XII and kallikrein), coagulation (Factor XI and thrombin)

43

and fibrinolytic (tPA and plasmin) systems [13-15]. Besides its protease inhibitory

44

role, C1INH possesses other important functions resulting from interaction of C1INH

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with leukocytes, endothelial cells, extracellular matrix components, bacterial

ACCEPTED MANUSCRIPT endotoxins, and various infectious agents [16-18]. Another significant important

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biological function of C1INH is to regulate vascular permeability, which has been

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exemplified in patients with hereditary angioedema (HAE) caused by either a

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decreased level of C1INH or a dysfunctional one [19-21].

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To date, the molecular information of C1INH has been reported in four teleosts

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including rainbow trout (Oncorhynchus mykiss) [22], large yellow croaker

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(Pseudosciaena crocea) [23], Nile tilapia (Oreochromis niloticus) [24] and rock

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bream (Oplegnathus fasciatus) [25]; however, only two species have been explored

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the immune response to pathogenic bacteria, rock bream and large yellow croaker. In

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mammals, the identification and characterization of C1INH has been well

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documented, including human [26] and mouse [27]. Hepatocytes, fibroblasts,

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monocytes, macrophages and endothelial cells are capable of C1INH synthesis

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[28-30]. In rats [31], Kupffer cells constitutively synthesize and secrete the protease

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inhibitor C1INH. And in vitro, IFN-γ treatment of monocytes in culture from patients

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with HAE is effective in increasing protein secretion of C1INH [32]. However, until

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now,

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monocytes/macrophages in teleost.

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study

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secretion

of

Nile tilapia (Oreochromis niloticus) is one of the economically important cultured

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fish in the world, and China is the largest tilapia farming country, with 1.6 million

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tonnes in 2013 [33]. Since 2009, a Gram-positive bacterium Streptococcus agalactiae

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has been reported to cause the mass mortality in Nile tilapia [34-36] in China,

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resulting in huge economic loss. Further, there is another pathogenic bacteria to Nile

ACCEPTED MANUSCRIPT tilapia, Aeromonas hydrophila, a Gram-negative bacterium [37], which has also led to

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the mortality. Therefore, it is demand urgently for the understanding the defense

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mechanisms against infectious bacterial diseases in order to prevent the disease

71

outbreak and establish the economic viability of tilapia industry. Although the

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molecular information of C1INH from Nile tilapia has been reported, the defense

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mechanisms against pathogenic bacteria in term of host-pathogen relationships is still

74

unclear. In this study, we reported the identification and characterization of OnC1INH

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at mRNA expressional, cellular and protein levels: (1) The temporal OnC1INH

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mRNA expression in vivo upon bacterial infection was investigated, (2) The temporal

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of OnC1INH expression in vitro after being immune challenged was determined and

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compared, (3) The protein level of OnC1INH in vivo challenges was detected by

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immunohistochemical analysis, (4) The stimulus inducibility of OnC1INH protein by

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monocytes/macrophages was examined in vitro using a competitive-inhibition

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ELISA.

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2. Materials and methods

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2.1. Cloning OnC1INH genes and bioinformatics analysis

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The gene of OnC1INH with complete ORF was cloned based on the published

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sequence

of

Oreochromis

niloticus

C1INH

mRNA

(GenBank

Accession

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NM_001279558.1). Primers OnC1INH 1F and 1R (Table 1) were designed by using

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Primer Premier 5.0. The PCR segment was suffered to electrophoresis on a 1.0%

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agarose gel (BIOWEST, Spain) to test and then cloned into the pMD-18T vector

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(TaKaRa, Japan), and transformed into competent E. coli cells. Then the positive

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clones were sequenced by BGI. The potential open reading frame (ORF) was analyzed with the ORF Finder

93

program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The protein analysis was

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conducted with ExPASy tools (http://expasy.org/tools/). The putative ORF was

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analyzed for the presence of N-linked glycosylation sites with the NetNGlyc 1.0

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Server (http://www.cbs.dtu.dk/services/NetNGlyc/).The glycosylation sites prediction

97

was conducted with CBS Prediction Servers (http://www.cbs.dtu.dk/services/).

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Multiple alignments of OnC1INH amino acid sequences were performed with the

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Clustal (http://clustalW.ddbj.nig.ac.jp/). The similarity analyses of the determined

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nucleotide sequences and deduced amino acid sequences were performed by BLAST

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programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenic trees were constructed

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by the neighbor-joining method using MEGA 6 software with 1000 bootstrap

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replications.

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2.2. Fish, challenge, and tissue sampling

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Healthy Nile tilapias with mean body weight of 100±10 g were acquired by

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Guangdong Tilapia Breeding farm (Guangdong, China). They were maintained in 300

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L tanks of a recirculating system at 28±3 °C for two weeks prior to challenge and

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RNA isolation. All animal protocols were reviewed and approved by the University

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Animal Care and Use Committee of the South China Normal University.

ACCEPTED MANUSCRIPT In order to study OnC1INH expression in healthy Nile tilapias, ten samples

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including spleen, gill, head kidney, thymus, liver, heart, intestine, skin, muscle, blood

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were collected from three unchallenged fishes and all samples were immediately

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stored at -80 °C until RNA extraction.

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Each Nile tilapia was injected intraperitoneally with 100 µL LPS (1 µg/µL) (E. coli

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055:B5, Sigma, USA) or 100 µL S. agalactiae (1×108 CFU/mL) [38, 39]. They were

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all re-suspended in sterile phosphate buffered saline (PBS). S. agalactiae was

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obtained from The College of Aquaculture at the Guangdong Ocean University

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(China). A control group was injected with 100 µL sterile PBS alone. Four fishes

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were collected from all groups at 0, 3, 6, 12, 24, 48 and 72 h post injection, tissue

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samples were harvested and frozen by liquid nitrogen for further analysis.

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2.3. Total RNA isolation and cDNA synthesis

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Total RNA was isolated from samples by Trizol Reagent (Invitrogen, USA)

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following manufacturer’s suggestions. The quantity and quality of the total RNA were

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measured using a NanoDrop2000 spectrophotometer (Thermo, USA) at 260 nm and

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260/280 nm absorbance ratios, respectively. The first-strand cDNAs were synthesized

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from 1µg total RNA according to guidelines of PrimeScriptTM RT reagent Kit with

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gDNA Eraser (TaKaRa, Japan). The cDNA was diluted 10-fold and then stored at –

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80 °C until used in quantitative real time PCR.

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2.4. Spatial and temporal expressional analysis of OnC1INH by qPCR

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In order to determine the OnC1INH mRNA expression, qPCR was performed on a

ACCEPTED MANUSCRIPT 7500 Real Time PCR System (Life Technologies, USA) using the SYBR green dye

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method. A pair of primers (3F and 3R; Table 1) were employed. The O. niloticus

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β-actin (Accession No. KJ126772.1) was chosen as internal standardization and

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amplified with primers of β-actin qF and β-actin qR (Table 1). The reactions were in a

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total volume of 20 µL: 3 µL of diluted cDNA template, 10 µL of 2 × TaKaRa Ex

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Taq™ SYBR premix, 2 µL of each primer, 0.5 µL DyeⅡand 2.5 µL dH2O. The PCR

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program was: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1

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min.

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2.5. Expression and purification of recombinant OnC1INH [(r) OnC1INH] protein

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The expression primers OnC1INH 2F and 2R (Table 1) were synthesized by BGI.

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The pMD-18T/OnC1INH and pET-32a were digested by the same restriction enzyme,

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Bam HⅠand Hind Ⅲ, and then ligated using T4 DNA Ligase (Thermo). The

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recombinant plasmid pET32a-OnC1INH was verified by sequencing (BGI) and

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transformed to E. coli BL21 (DE3) (TianGen, China).

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Transforming into the competent cells of Escherichia coli BL21 (DE3) and then

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was cultured in LB medium at 37 °C until O.D.600 reached 0.4-0.6. The expression of

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the objective protein was induced by 1 mM Isopropyl β-D-1-Thiogalactopyranoside

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(IPTG) for an additional 4 h at 37 °C. Cells were collected by centrifugation at 13,000

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× g for 10 min at 4 °C. The precipitation was re-suspended in 1×PBS with lysozyme

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(1 mg/mL) and disrupted by ultrasonic on ice. The lysate was suffered to centrifuge at

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13,000 × g for 30 min at 4 °C. The pellet was dissolved in binding buffer (20 mM

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ACCEPTED MANUSCRIPT Tris-HCl, 0.5 M NaCl)，the supernatant was harvested after centrifugation at 13,000 ×

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g for 30 min at 4 °C . Purification of the recombinant protein was completed with His

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Band Resin (Novagen, Germany). The imidazole concentrations of the elution buffer

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were: 20 mM, 40 mM, 60 mM, 100 mM and 250 mM, respectively. The purified

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protein was dialyzed into 1×PBS. Eventually, the resulting product was enriched by

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PEG 20,000 and measured by NanoDrop2000 spectrophotometer to determine

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concentration.

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2.6. Production of polyclonal antibodies against recombinant OnC1INH

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For preparation of polyclonal antibody, the purified recombinant protein was used

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as antigen to immune six-week-old female BALB/c mice. Six mice were injected

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intraperitoneally with the antigen emulsified with an equal volume of Freund’s

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adjuvant (Sigma) for four times (the 1st immunization with 100 µg in FCA, and the

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2nd-4th with 50 µg in FIA) according to a standard procedure. Five days after the

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fourth immunization when the titer achieve 500,000 units/ml, the mice were bled, and

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the antiserum was prepared and stored at -80 °C.

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2.7. Immunohistochemical identification of OnC1INH

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Liver and spleen were dissected out from Nile tilapias immunized with S.

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agalactiae, then dewaxed in xylene and rehydrated in a series of graded ethyl alcohol.

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Followed normal immunohistochemistrical procedures, after the antigen retrieval

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using Proteinase K, primary antibody (1:500 working dilution, mouse polyclonal

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antibody to Nile tilapia C1INH) was incubated overnight at 4 °C. After 1 h incubation

ACCEPTED MANUSCRIPT with a HRP-labelled secondary antibody (Southern Biotech, USA) at room

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temperature, the peroxidase reaction was developed with DAB (BOSTER, China).

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The reaction was terminated by distilled water when desired signal was seen as a

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brown reaction. After counterstaining with hematoxylin, sections were dehydrated

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through a graded ethanol series to observe and preservation.

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2.8. Primary Nile tilapia monocytes/macrophages isolation and cultures

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The head kidney phagocytic cells of the Nile tilapia were isolated according to the

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modified method as previously described [40]. Briefly, head kidney leukocytes were

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separated from the cell suspension by density gradient centrifugation: 10 mL of

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homogenate was layered over equal volume of Histopaque® 1077 (Sigma) and

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centrifuged at 500 × g for 40 min at 4 °C [40, 41]. The leukocytes at the interface

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were collected and washed three times in L-15 medium (Gibco, USA). Cell quantity

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and viability were assessed using trypan blue. The cells were re-suspended in L-15

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medium

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penicillin/streptomycin (Hyclone, USA). The cells were added to 96-well microplates

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(Corning, USA) (1×106 cells/well) and incubated at 25 °C for 2 h. Then non-adherent

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cells were removed [42].

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2%

fetal

bovine

serum

(Gibco),

1%

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The adherent cells were supplied with 100 µL fresh L-15 medium including 5%

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fetal bovine serum, 1% penicillin/streptomycin. These 96-well microplates were

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incubated at 25 °C for 5 h. The treatment group was challenged with LPS (40 µg/mL),

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S. agalactiae (1×106 CFU/µL) and 10µL A. hydrophila (1×106 CFU/µL), which have

ACCEPTED MANUSCRIPT been inactived, a group with the equal 1×PBS and the one without challenged

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represented control. Each group was maintained at 25 °C and cells from one 96-well

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microplate from each group were lysed with TRI Reagent at 0, 3, 6, 12, 24, 48 and 72

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h post-stress. The cell culture supernatant was harvested for further experimentation.

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2.9. Competitive-inhibition ELISA

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A competitive-inhibition ELISA was adopted to estimate the concentration of

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C1INH in the culture medium of Nile tilapia monocytes/macrophages [43].

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Recombinant protein OnC1INH was added to the 96-well plates at 37 °C for 2 h and

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then blocked at 37 °C for 2 h. After washing with 1×TTBS, the culture supernatant

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and anti-(r)OnC1INH pAb (1:1600, the optimal dilution determined ahead) were put

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into each well. After incubating for 1 h at 37 °C，the plates were washed three times

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with 1×TTBS and horseradish peroxidase-conjugated goat anti-mouse secondary

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antibody (1:2500) (SouthernBiotech) was added. And incubating for 1 h at 37 °C, the

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plates were washed three times with 1×TTBS. In the end, ABTS (Sigma) was added.

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Finally, the reaction was measured at O.D.405 using a microplate reader

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(MULTISKAN FC, Thermo Scientific).

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2.10. Data interpretation and statistical analysis

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The 2-△△Ct method (Livak method) was used to analyze the mRNA expression level

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of OnC1Inh. All data were presented as the quantity of OnC1INH mRNA relative to

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that of β-actin mRNA and expressed as mean ± standard deviation (SD). In tissue

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mRNA expressional analysis, expression of OnC1INH in gill was set as the basal line,

ACCEPTED MANUSCRIPT and relative level of OnC1INH in each tissue was calculated with respect to that of

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gill. In challenge test, expression of OnC1INH in treated at 0 h was set as control to

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compare significance level and post-challenge expression at each time point was

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further compared with the corresponding PBS-injected controls. All experiments were

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performed at least three times, and statistical analyses were carried out by one-way

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ANOVA followed by LSD multiple group comparisons using SPSS19.0 software

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(SPSS, Chicago, USA). Differences were considered significant at P < 0.05.

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3. Results

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3.1. Characterization of Nile tilapia C1INH and phylogeny analysis The ORF of OnC1INH was 1788 bp, encoding a 596-amino-acid peptide. The

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deduced amino acid sequence of OnC1INH ORF was examined using DNA star and

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found that the molecular mass was 65.254 kDa and an isoelectric point of 5.72 in

228

theory. In the amino acids of OnC1INH, a signal peptide was detected at the

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N-terminal between the residues 20 and 21 by Signal IP 4.1, from that we could

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speculate OnC1INH was a secreted protein. The mature OnC1INH contained two

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N-terminal Ig domains and a C-terminal serpin (serine protease inhibitor) domain

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were determined by Motif Scan server. Furthermore, glycosylation servers identified

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that OnC1INH has four putative N-linked glycosylation sites (N–X–S/T) located at

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the positions 199, 281, 328 and 350, respectively.

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Sequences alignment of C1INH genes were from several vertebrates include fish,

ACCEPTED MANUSCRIPT poultry and mammal by using Clustal (http://clustalW.ddbj.nig.ac.jp/). It showed that

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this gene in Nile tilapia was homologous to other teleost species and mammals, with

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sequence identities of 65%, 59%, 60%, 53%, 31%, and 33% with rock bream, large

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yellow croaker, rainbow trout, medaka, chicken, and human, respectively. Bony fishes

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shared highly conservation with OnC1INH (Fig. 1).

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In order to examine the phylogeny of OnC1INH, a phylogenetic tree was

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constructed with different species, by using the NJ method. As shown in Fig. 2,

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almost the non-fish vertebrate classes converged within one of the main branches and

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left the fish group independently as the second main cluster, hinted that fish C1INHs

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were somewhat unique from those of other vertebrates. The OnC1INH demonstrated

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an evolutionary relevance with rock bream’s similitude that shared highest homology

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with OnC1INH.

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3.2. Tissues distribution of OnC1INH expression

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In order to detect the tissue distribution of OnC1INH, the RNA of ten tissues from

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healthy fishes were extracted for qPCR and the β-actin gene was used as an internal

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control. Relative OnC1INH mRNA expression levels were calculated compared with

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the lowest mRNA expression in gill. As shown in Fig. 3, it illustrated that OnC1INH

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mRNA expression had wide distributions. The relative OnC1INH mRNA expression

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was higher in liver, spleen and heart than that in gill, muscle and thymus, which

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showed obvious tissue specific variation of OnC1INH. The most predominant

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expression of OnC1INH was detected in the liver (1×107 fold).

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3.3. Expression of C1INH gene after challenges Quantitative real-time PCR was used to detect the changes in the expression of

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OnC1INH mRNA in liver and spleen after injected with LPS and S. agalactiae. Upon

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LPS challenge, the highest mRNA expression of OnC1INH was detected at 72 h

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post-infection (p.i.) (P < 0.05) in liver tissue (Fig. 4A). The highest mRNA expression

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of OnC1INH was observed at 48 h p.i. following the LPS challenge in spleen (Fig.

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4B). Moreover, OnC1INH showed greater mRNA expression at 24 h p.i. following S.

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agalactiae challenge in liver, while in spleen, it was significantly upregulated at 48 h

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p.i. with S. agalactiae (Fig. 4A). The higher transcription level of OnC1INH was

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noticed at late phase of the experiment whether in liver or spleen. Interestingly, LPS

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(6.2-fold) and S. agalactiae (5.3-fold) significantly elevated the level of OnC1INH

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transcripts in spleen more than that in liver (1.3-fold, 1.6-fold).

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3.4. Recombinant OnC1INH protein expression, purification and Western blotting

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The ORF of mature OnC1INH was cloned into pET-32a vector, transformed into

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BL21 (DE3) and recombinant protein fused with His-tag was purified with Ni-affinity

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chromatography. Usually, recombinant protein forms inclusion bodies when they were

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induced in prokaryotic expression system. After sonication and centrifugation,

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(r)OnC1INH was separated as different fractions, supernatant and precipitation. Then

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the fractions were analyzed by SDS-PAGE (Fig. 5A). Most of the (r)OnC1INH were

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in insoluble fraction. As shown in Fig.5, a band (~90 kDa) corresponding to

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OnC1INH-His fusion protein could be found in the insoluble fraction. The

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ACCEPTED MANUSCRIPT (r)OnC1INH was purified and applied to produce polyclonal antibody. Mice were

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immunized with the purified recombinant OnC1INH for four times immunity. When

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the titers reached 500,000 units/ml to the (r)OnC1INH, the antiserum was collected

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from mice used as the primary antibody for Western blotting. The purified

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(r)OnC1INH was used to test the specificity of antiserum. As shown in Fig. 5B, the

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antiserum reacted strongly with (r)OnC1INH, a specific band about 90 kDa could be

284

detected. This result verified the polyclonal antibodies were prepared successfully.

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3.5. Immunohistochemical detection of OnC1INH

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Immunoreactivity against (r)OnC1INH antibody was detected in liver and spleen

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from fish immunized with S. agalactiae, as shown in Fig. 6. Compared with PBS, the

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fish infected with S. agalactiae at 48 h revealed a marked increase in positive of

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C1INH proteins in the spleen (Fig. 6C). Nevertheless, different specimens

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demonstrated jointly in the spleen, C1INH proteins diffusely distributed in the

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endochylema.

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In liver, at 24 h after immunized with S. agalactiae, the result showed a more

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intense immunoreaction compared to other liver samples (Fig. 6F). Further, C1INH

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proteins were also commonly observed in the endochylema.

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3.6. Effects of stimuli on OnC1INH expression of monocytes/macrophages

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In an attempt to investigate the biological activity of LPS, A. hydrophila and S.

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agalactiae in vitro, Nile tilapia head kidney monocytes/macrophages were incubated

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with different stimulus (40 µg/mL LPS, S. agalactiae 1×106 CFU/µL, and A.

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hydrophila 1×106 CFU/µL) from 3 to 72 h. In this case, when LPS, S. agalactiae

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and A. hydrophila stimulated, it resulted in a time dependent manner OnC1INH

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mRNA expression from 3 to 72 h, and exhibited peaks at 24 h (Fig. 7A). A competitive-inhibition ELISA was adopted to measure concentrations of the

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secreted OnC1INH in culture media, in order to elucidate the effect of LPS, A.

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hydrophila and S. agalactiae on the OnC1INH secretion of monocytes/macrophages.

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As shown in Fig. 7B, when treated with LPS (40 µg/mL), S. agalactiae (1×106

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CFU/µL) and A. hydrophila (1×106 CFU/µL) for 3–72 h, it had obvious effect on the

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C1INH release.

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4. Discussion

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In this study, we presented the identification and characterization of the

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complement component 1 inhibitor from Nile tilapia (Oreochromis niloticus) at

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molecular, protein and cellular levels, which indicated that OnC1INH might be

313

involved in the immune response of Nile tilapia against to bacterial challenge.

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Domain structural and phylogenetic evidence revealed that OnC1INH was a member

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of serpin family and shared highly similar with other teleost species. The constitutive

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expression of OnC1INH emphasized its importance in different tissues. Moreover,

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regulated OnC1INH mRNA expression in liver and spleen upon in vivo challenges as

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well as induced OnC1INH by pathogenic bacteria in vitro, both of which might

319

intimate that C1INH was a key molecule to balance the immune response of host

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ACCEPTED MANUSCRIPT 320

against pathogen bacteria. Furthermore, the dynamics of OnC1INH secretion was

321

detected in both in vitro and in vivo challenges suggested that the expression of

322

OnC1INH was coordinated in molecular, protein and cellular levels. C1INH is conserved, although with variable sequence identities and similarities, in

324

all analyzed vertebrates from teleost to mammalian, especially in the core serpin

325

domain. Comparison the amino acid sequence with other known vertebrate

326

counterparts revealed that the OnC1INH shared higher identities with fish

327

counterparts, particularly with rock bream [25]. In contrast, OnC1INH demonstrated

328

low identities with human C1INH. OnC1INH had the core serpin domain and two

329

immunoglobulin-like domains while human just possessed serpin domain (Fig. 1),

330

which directly resulted in the difference of molecular weight. Thus, this could explain

331

the lowest identities between Nile tilapia C1INH and human C1INH. The specific

332

Ig-like domains have been also observed in other teleost species including zebrafish

333

[44], Japanese flounder [22], large yellow croaker [23], rainbow trout [22] and rock

334

bream [25]. Since these Ig domain motifs were only reported in teleost, the C1INH

335

with Ig domains might be a common feature of fishes. Generally, these Ig domains are

336

approximately 100 residues long, with two “invariant” cysteines forming the

337

intrachain disulfide crosslinking, which are composed of a spatial structural topology

338

[27, 45]. The Ig domains are observed in many immunoglobulin superfamily, which

339

are involved in either protein-protein or protein-ligand interactions [46]. Thus, the

340

well-conserved Ig domains in the teleostean C1INH sequences imply that they may

341

have functional significance, probably enabling their function to be regulated by

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ACCEPTED MANUSCRIPT supplying extra binding sites for plasma proteins. Recently, one study on molecular

343

phylogenetic aspects of C1INH elucidated that the teleostean C1INH with two Ig

344

domains as the N-terminal extension is due to one specific intron insertion in the exon

345

Im2 after separation from zebrafish [44]. So far, the function of the Ig domains in the

346

fish C1INH, although merit further investigation, is still unclear.

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Glycosylation is an important feature of glycoproteins, which impacts their

348

biological function and structure, including the C1INH molecule involved in the

349

complement system. In human and mouse, C1INH is a heavily glycosylated protein

350

(approximately 30% by weight) [27]. It has been demonstrated that the

351

N-glycosylation may play a role in the clearance of mammalian C1INH, as shown the

352

regulation of its plasma half-life by altering glycosylation [47]. The recent study [48]

353

also revealed that deglycosylation of C1INH affected its function regarding protease

354

targets and autoantibody binding. Furthermore, the N-linked glycosylation has been

355

reported to be required for interaction of C1INH with LPS and lipid A [49, 50].

356

Compared the high number of potential N-glycosylation sites in mammals, there are

357

four potential N-linked glycosylation sites predicted in OnC1INH, and the similar

358

situation was also observed in rainbow trout and rock bream with two sites [22, 25],

359

and in large yellow croaker with three sites [23]. Until now, whether the glycosylation

360

of teleost C1INH has the impact on its biological function is not known. Since C1INH

361

is an important multi-functional serine protease inhibitor involving many systems, the

362

mechanism of glycosylation in teleost C1INH associated with their functions should

363

be further investigated.

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ACCEPTED MANUSCRIPT The tissue distribution of the OnC1INH mRNAs in healthy fish showed that the

365

highest expression of OnC1INH was detected in liver (Fig. 3). It suggested that the

366

liver was a major organ for the expression of C1INH in normal conditions, in

367

accordance with other species, such as human [27], mouse [26], rainbow trout [22]

368

and rock bream [25]. In human, hepatic parenchymal cells are the primary site of

369

synthesis of C1INH [51]. In mice [26], northern blot analyses revealed higher mRNA

370

levels in liver, lung and heart. However, the western blot analyses showed that the

371

expression of large yellow croaker C1INH protein in liver was relatively low,

372

compared to the high levels in spleen, kidney and heart [23]. The variations in the

373

tissues distribution of C1INH expression observed within fishes may be due that

374

teleost species have the different tissue expression patterns. Another possibility is that

375

the tissue distribution profiles of lycC1INH were investigated in the protein level;

376

however, other species including rainbow trout and rock bream were evaluated in the

377

mRNA expression. Besides the strongest expression in the liver, OnC1INH was also

378

detected in a relatively high level in other tissues such as spleen and kidney,

379

suggesting OnC1INH may be involved in the immune response. The constitutive

380

expression of OnC1INH was observed in most of examined tissues, which may be

381

related to the multi-functional feature of C1INH contributing many systems.

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364

382

Complement system plays an important role in host defense by mediating acute

383

inflammatory reactions and killing of pathogenic microorganisms [52]. However, the

384

complement system is nonspecific and thus capable of attacking pathogens as well as

385

host cells, if without properly regulatory mechanisms to restrict its activity, which will

ACCEPTED MANUSCRIPT cause detrimental effects on healthy host cells. Since C1INH is the well-known

387

complement regulator of the classical and lectin pathways, the investigation of

388

expression profiles of this regulatory mediator in response of pathogen challenge will

389

provide potential clues on how C1INH regulate the response in fish. Temporal in

390

OnC1INH expression in liver and spleen tissues of S. agalactiae and LPS

391

administrated fishes were analyzed, since the liver is the major site to synthesize

392

C1INH and the spleen is an important immune organ in teleost and main target organs

393

attacked by S. agalactiae [53]. Streptococc agalactiae is a major pathogen of Nile

394

tilapia, which can cause an acute and rapid immune response, especially in liver [54,

395

55]. After challenges with S.agalactiae, the highly significant up-regulation in

396

expression of OnC1INH gene was appeared early at 24 h p.i. in liver. However,

397

following the LPS (a polyclonal activator) challenge, a modest up-regulation occurred

398

late during 48-72 h p.i. in liver (Fig.4), similar with the previous study conducted in

399

Rock bream [56]. In comparison with liver, OnC1INH expression in spleen showed

400

different patterns, as shown in Fig.4. After challenges with LPS, the transcript level of

401

OnC1INH was slightly increased early at 6 h p.i. in spleen, then a significant

402

up-regulation (about 6-fold) of OnC1INH expression was detected at the late response

403

(48 h p.i.) in spleen. The higher up-regulation in spleen than in liver in LPS challenge

404

may due that the spleen is a main immunological organ to catch LPS antigen with

405

ample antigen-presenting cells [57, 58]. Moreover, after injection with S.agalactiae,

406

there was also a significant up-regulation of OnC1INH expression at 48 h p.i. in

407

spleen. A comparable expression profile in response to S. iniae and LPS has been

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386

ACCEPTED MANUSCRIPT found in rock bream [25], with C1INH expression significantly induced at the late

409

phase in liver. In large yellow croaker, western blotting revealed that the expression

410

level of lycC1INH was gradually up-regulated during 20 days after stimulation [23].

411

Oppositely, upon the LPS challenge, the rainbow trout [22] C1INH expression

412

remained relatively stable, with no significant change. In current study, the

413

immunohistochemical analyses have confirmed the qPCR data, suggesting that the

414

protein expression profile of OnC1INH was similar to that of mRNA transcription

415

with significantly up-regulation at the late phase of response (Fig. 6).

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408

Since the liver exhibits an extremely strong OnC1INH mRNA expression which is

417

much higher than that in spleen (Fig. 3), it should be noted that a little change in the

418

magnitude of OnC1INH transcription in liver means a great increase with huge

419

quantity of C1INH mRNA (Fig. 4). Interestingly, in our preliminary experiment, we

420

observed that OnC1q reached peak quickly, then followed by OnC1INH, (for example,

421

OnC1INH reached peak at 24h while OnC1q attained at 12h (data not shown)), and

422

the similar circumstance was also shown in rock bream [25, 59]. All these evidences

423

collectively might imply clues that teleostean C1INH play a role on the regulation of

424

complement system in response of pathogen infection.

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425

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416

Monocytes and macrophages are capable of expressing complement genes,

426

including C1INH synthesis, which have been demonstrated in human [31] and

427

rainbow trout [22]. To examine the effect of stimuli on the expression of OnC1INH in

428

cell level, the monocytes/macrophages isolated from head kidney were challenged in

ACCEPTED MANUSCRIPT vitro. In LPS challenge, the expression pattern revealed a quite stable transcription or

430

relatively small up-regulated expression of OnC1INH during the early phase; however,

431

there was significant up-regulation at the late phase (peak at 24h) of the challenge

432

(Fig. 7). In contrast, employing in vitro culture of a trout monocyte/macrophage cell

433

line RTS11, trout C1INH showed no obvious transcriptional changes upon LPS

434

stimulation [22]. The variance of the current study and trout work may be due to the

435

variety of LPS and/or the different cell source (isolated cells from kidney tissue vs. a

436

specific cell line). As the major pathogenic bacteria to Nile tilapia, S. agalactiae and

437

A.

438

monocytes/macrophages (Fig.7), suggesting that the expression of OnC1INH was

439

induced by pathogenic bacteria.

hydrophila

apparently

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429

increased

the

expression

of

OnC1INH

in

Taken together, the current study provided the evidence for the possible

441

involvement of OnC1INH in Nile tilapia against to pathogenic bacteria, and the

442

difference among three levels (molecular, protein and cellular) of C1INH response to

443

different stimuli. The findings may suggest potentially productive and intriguing

444

avenues for future research. What is the function of the special two Ig domains? Does

445

glycosylation occur in OnC1INH? If any, how the glycosylation affect OnC1INH

446

structure and function? The interaction of OnC1INH with OnC1 is another potential

447

interesting on. Herein, the regulatory mechanism of OnC1INH remains to be fully

448

elucidated.

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449 450

Acknowledgements

ACCEPTED MANUSCRIPT This project was supported by National Natural Science Foundation of China

452

(31472302, 31172432, 31402324), and Natural Science Foundation of Guangdong

453

Province, China (2014A030313437, 2014A030313790). The authors gratefully

454

acknowledge the critical reviews of Liangliang Mu and Yuan Li.

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451

455

References

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[43] Wei H, Yin L, Feng S, Wang X, Yang K, Zhang A, et al. Dual-parallel inhibition of IL-10 and TGF-β1 controls LPS-induced inflammatory response via NF-κB signaling in grass carp monocytes/macrophages, Fish Shellfish Immunol 2015;44:445-452. [44] Kumar A, Bhandari A, Sarde SJ, Goswami C. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish. Biochem Biophys Res Commun 2014;450:219-226. [45] Smith DK, Xue H. Sequence profiles of immunoglobulin and immunoglobulin-like domains. J Mol Biol 1997;274:530–545. [46] Williams AF, Barclay AN. The immunoglobulin superfamily–domains for cell surface recognition. Annu Rev Immunol 1988;6:381–405. [47] Wagenaar-Bos IGA, Hack CE. Structure and function of C1-inhibitor. Immunol Allergy Clin North Am 2006;26:615-632. [48] Ghannam A, Sellier P, Fain O, Martin L, Ponard D, Drouet C. C1 inhibitor as a glycoprotein: the influence of polysaccharides on its function and autoantibody target. Mol Immunol 2016;71:161-165. [49] Liu D, Cai S, Gu X, Scafidi J, Wu X, Davis AE. C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide. J Immunol 2003;171:2594–2601. [50] Liu D, Cai S, Gu X, Scafidi J, Davis AE. N-linked glycosylation is required for c1inhibitor-mediated protection from endotoxin shock in mice. Infect Immun 2004;72:1946–1955. [51] Johnson AM, Alper CA, Rosen FS, Craig JM. C1 Inhibitor: Evidence for Decreased Hepatic Synthesis in Hereditary Angioneurotic Edema. Science 1971;173:553-554. [52] Volanakis JE, Frank MM. The human complement system in health and disease. Crc Press 1998. [53] Li Y, Liu L, Huang P, Fang W, Luo Z, Peng H, et al. Chronic streptococcosis in Nile tilapia, Oreochromis niloticus (L.), caused by Streptococcus agalactiae. J Fish Dis 2014;37:757-763. [54] Alsaid M, Mohd H, Mohamed N, Khairani S, Mohamed Y, Farag A, Hayati, R. Pathological findings of experimental Streptococcus agalactiae infection in red hybrid tilapia (Oreochromis sp.). In: International Conference on Chemical. Agricultural and Medical Sciences (CAMS) 2013; pp70–73. [55] Asencios YO, Sánchez FB, Mendizábal HB, Pusari KH, Alfonso HO, et al. First report of Streptococcus agalactiae, isolated from Oreochromis niloticus, in Piura, Peru: Molecular identification and histopathological lesions. Aquaculture Reports 2016;4:74-79. [56] Godahewa GI, Bathige SDNK, Herath HMLPB, Jae Koo Noh, Jehee Lee. Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure. Fish and Shellfish Immunology 2015;46:656-668. [57] Press CM, Dannevig BH, Landsverk T. Immune and enzyme histochemical phenotypes of lymphoid and nonlymphoid cells within the spleen and head kidney of Atlantic salmon ( Salmo salar, L.). Fish and Shellfish Immunology 1994;4:79-93. [58] Iliev DB, Jørgensen SM, Rode M, Krasnov A, Harneshaug I, Jørgensen JB. CpG-induced secretion of MHCIIβ and exosomes from salmon (Salmo salar) APCs. Developmental and Comparative Immunology 2010;34:29-41. [59] Bathige SDNK, Whang I, Umasutha N, Wickramaarachchi WDN, Wan Q, Lim BS, et al. Three complement component 1q genes from rock bream, Oplegnathus fasciatus: Genome characterization and potential role in immune response against bacterial and viral infections. Fish Shellfish Immunol 2013;35:1442-1454.

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ACCEPTED MANUSCRIPT 603 604 605

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Table

616

Table 1: Primers used in this study.

617

Primer name

618

1F

619

1R

TTACGGCTCGGTCAACCTGC

620

2F

CCCGGATCCAAAATCAGAGT

621

2R

GGGAAGCTTCGGCTCGGTC

622

3F

AATTTAATGGTGATGTGGTAAGGGTG

EP

615

AC C

Nucleotide sequence (5’-3’)

ATGGGACAAAAGGCCACACTTT

Purpose

gene cloning

protein expression

real-time PCR

ACCEPTED MANUSCRIPT 3R

TCAATAGGCTGAGGAGATGATGTTT

624

β-actin qF

CAAAGCCAACAGGGAGAA

625

β-actin qR

CTTGATGTCACGCACGAT

RI PT

623

626

SC

627

628

M AN U

629

630

632

TE D

631

Figure Legends

634 635 636 637 638 639

Fig.1. Multiple alignment of the deduced amino acid sequence of Nile tilapia C1 inhibitor with homologs from other selected vertebrates. Abbreviations: On, Nile tilapia; Of, rock bream; Lc, large yellow croaker; Om, rainbow trout; Ol, medaka; Gg, chicken; Rn, Norway rat; Hs, human. Residues conserved among different species are shaded in black. The numbers on the right indicate the percent amino acid sequence identity and similarity of Nile tilapia of various species.

641 642 643 644 645 646 647 648

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640

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633

Fig.2. Phylogenetic tree of the C1INH among different species. The tree was constructed using the NJ method by MEGA 6 program based on the alignment of 18 members of the C1INH performed with Clustal W using their entire amino acid sequences. Numbers on branches are bootstrap values of 1,000 replicates supporting a given partitioning. The GenBank nucleotide sequence databases used here with the following accession number(s): Oplegnathus fasciatus, JN593100; Larimichthys crocea, FJ423636; Oncorhynchus mykiss, NM_001124379; Paralichthys olivaceus, BN000290; Danio rerio, NM_001123285; Oryzias latipes , XM_004065756; Gallus gallus, XM_003641376; Macaca mulatta, XM_001092271;

ACCEPTED MANUSCRIPT 649 650 651 652

Ailuropoda melanoleuca, XM_002922708; Sus scrofa, NM_001123194; Bos taurus, NM_174821; Rattus norvegicus, NM_199093; Mus musculus, AF010254; Homo sapiens,BC011171; Pan troglodytes, XM_003317978; Heterocephalus glaber, EHB10156.1; Cuculus canorus, XP_009562372.1.9.

653

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Fig.3. Tissue distribution of OnC1INH mRNA in normal Nile tilapia. The ten tissues were the gill (Gl), heart (Ht), liver (Lv), and intestine (It), head kidney (Hk), spleen (Sp), thymus (Ty), skin (Sk), blood (Bl) and muscle (Ms). The Nile tilapia β-actin was chosen as the internal reference gene. The calculation was performed using the Livak method and values were calibrated against mRNA level in gill.

SC

654 655 656 657 658

659

Fig.4. Temporal mRNA expression of Nile tilapia C1INH in the liver and spleen after LPS and S.agalactiae challenges. (A) liver, (B) spleen. The Nile tilapia β-actin was chosen as the internal reference gene and performed using Livak method. The values are shown as mean ± SEM (N=3). Significant difference was indicated by asterisks, *: 0.01
M AN U

660 661 662 663

664

671 672 673 674

675 676 677 678 679 680 681 682

TE D

EP

670

Fig.5. Purification of (r)OnC1INH and validation of polyclonal antibody for (r)OnC1INH. (A) SDS–PAGE analysis of recombinant Nile tilapia C1INH [(r)OnC1INH] fusion protein. The pET-OnC1INH was induced with 1 mM IPTG at 37 °C for 4 h. Lanes: (P) insoluble fractions after cell disruption; (F) purified (r)OnC1INH fusion protein; (M) marker. (B) Western blot analysis of anti-(r)OnC1INH Ab. Lanes: (F) (r)OnC1INH; (M) marker.

Fig.6. Immunohistochemical detection of Nile tilapia C1INH. The spleen (A) and the liver (D) as control in healthy fish. Distribution of OnC1INH positive cells (arrow) in the tissues by IHC after challenged with S.agalactiae at 6 h in the spleen (B) and in the liver (E), at 48 h in the spleen (C), and 24h in the liver (F), respectively.

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665 666 667 668 669

Fig.7. Effects of stimuli on mRNA expression and secretion of Nile tilapia C1INH in monocytes/macrophages. Nile tilapia head kidney monocytes/macrophages were challenged with LPS (40 ug/mL), A. hydrophila (1×106 CFU/µL), S. agalactiae (1×106 CFU/µL) or PBS for the indicated times. (A) OnC1INH mRNA expression was detected by qPCR and relative mRNA expression levels were analyzed using β-actin as an internal reference, (B) OnC1INH concentration was assayed by a competitive-inhibition ELISA. The results are shown as mean ± standard deviation of triplicates. Values are shown as means ± standard deviation (SD).

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Significant difference was indicated by asterisks, *: 0.01
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ACCEPTED MANUSCRIPT

Highlights C1INH homolog was identified from Nile tilapia (OnC1INH). OnC1INH was up-regulated by Streptococcus agalactiae challenge.

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stimulated with S. agalactiae.

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The up-regulation was demonstrated in monocytes/macrophages