Open Access

Differential protein expression in chicken macrophages and heterophils in vivo following infection with Salmonella Enteritidis

  • Zuzana Sekelova1,
  • Hana Stepanova1,
  • Ondrej Polansky1,
  • Karolina Varmuzova1,
  • Marcela Faldynova1,
  • Radek Fedr2, 3,
  • Ivan Rychlik1Email author and
  • Lenka Vlasatikova1
Veterinary Research201748:35

DOI: 10.1186/s13567-017-0439-0

Received: 15 January 2017

Accepted: 23 May 2017

Published: 17 June 2017

Abstract

In this study we compared the proteomes of macrophages and heterophils isolated from the spleen 4 days after intravenous infection of chickens with Salmonella Enteritidis. Heterophils were characterized by expression of MMP9, MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR. Macrophages specifically expressed receptor proteins, e.g. MRC1L, LRP1, LGALS1, LRPAP1 and a DMBT1L. Following infection, heterophils decreased ALB and FN1, and released MMP9 to enable their translocation to the site of infection. In addition, the endoplasmic reticulum proteins increased in heterophils which resulted in the release of granular proteins. Since transcription of genes encoding granular proteins did not decrease, these genes remained continuously transcribed and translated even after initial degranulation. Macrophages increased amounts of fatty acid elongation pathway proteins, lysosomal and phagosomal proteins. Macrophages were less responsive to acute infection than heterophils and an increase in proteins like CATHL1, CATHL2, RSFR, LECT2 and GAL1 in the absence of any change in their expression at RNA level could even be explained by capturing these proteins from the external environment into which these could have been released by heterophils.

Introduction

Macrophages and heterophils represent professional phagocytes acting as effectors and modulators of innate immunity as well as orchestrators of adaptive immunity [1]. Heterophils, the avian counterparts of mammalian neutrophils, belong among the first responders to bacterial infections and sensing of pathogen associated molecular patterns (PAMPs) stimulates heterophils for phagocytosis as well as release of bactericidal proteins stored in heterophil granules into the extracellular environment [2]. In agreement with their general function in host protection against pathogens, heterophils play a crucial role in the protection of chickens against Salmonella infection and chickens with heterophil depletion are not protected against colonization of systemic sites [35]. However, although there are several reports on specific heterophil functions during infection of chickens with Salmonella enterica, their genome-wide response to infection has not been characterized so far.

Macrophages are professional phagocytes responsible for the destruction and clearance of pathogens. When activated, macrophages increase their antibacterial activity by the expression of antimicrobial peptides like cathepsins B, C, D and S, avidin, ferritin or ovotransferrin [6], and production of NO radicals from arginine by inducible NO synthase. The antimicrobial proteins expressed by macrophages are commonly produced also by heterophils though it is not known to what extent these may differ in their immediate availability and total amount produced by both cell types. Macrophages can also regulate the immune response by the expression of cytokines e.g. IL1β, IL6, IL8, IL18 or LITAF [7] and are capable of antigen presentation [810]. However, similar to heterophils, an unbiased report on total proteome expressed by chicken macrophages is absent.

In our previous study we showed that heterophils and macrophages increase in the spleen of chickens when intravenously infected with Salmonella Enteritidis (S. Enteritidis) [7]. Next we characterized the gene expression at the tissue level in the whole spleen and expression of selected transcripts was tested in sorted leukocyte subpopulations [6]. However, none of this provided general data on the protein expression in chicken heterophils and macrophages. Although intravenous infection of chickens only partially represents specific Salmonella—chicken interactions which are mixed up with a general response to bacteremia caused by Gram negative bacterium, this way of infection represents a model for the understanding heterophil and macrophage functions during early response to infection. In the current study we therefore isolated heterophils and macrophages from chicken spleens by fluorescence-activated cell sorting (FACS), purified proteins from these cells and identified them by mass spectrometry. This allowed us to (1) characterize the total proteome of heterophils and macrophages, (2) define proteins which exhibited differential abundance in chicken heterophils compared to macrophages and (3) identify proteins that changed in abundance following the intravenous infection with S. Enteritidis in either of these populations. Since we also included a group of chickens which was vaccinated prior to challenge, we also addressed whether there are any proteins specifically expressed by the macrophages or heterophils from the vaccinated chickens. Using this approach we identified over one hundred proteins characteristic of either chicken heterophils or macrophages which allowed us to further refine their function in chickens.

Materials and methods

Ethics statement

The handling of animals in this study was performed in accordance with current Czech legislation (Animal protection and welfare Act No. 246/1992 Coll. of the Government of the Czech Republic). The specific experiments were approved by the Ethics Committee of the Veterinary Research Institute (permit number 5/2013) followed by the Committee for Animal Welfare of the Ministry of Agriculture of the Czech Republic (permit number MZe 1480).

Bacterial strains and chicken line

Newly hatched ISA Brown chickens from an egg laying line (Hendrix Genetics, Netherlands) were used in this study. Chickens were reared in perforated plastic boxes with free access to water and feed and each experimental or control group was kept in a separate room. The chickens were vaccinated with S. Enteritidis mutant completely lacking Salmonella pathogenicity island 1 (SPI-1) constructed as described earlier [11] and infected with isogenic wild type S. Enteritidis 147 spontaneously resistant to nalidixic acid. The strains were grown in LB broth at 37 °C for 18 h followed by pelleting bacteria at 10 000 × g for 1 min and re-suspending the pellet in the same volume of PBS as was the original volume of LB broth.

Experimental infection

There were 3 groups of chickens. Six chickens from the control group were sacrificed on day 48 of life. An additional 6 chickens (group 2) were infected intravenously with 107 CFU of wild type S. Enteritidis in 0.1 mL PBS on day 44 of life. The last 6 chickens (group 3) were orally vaccinated on day 1, revaccinated on day 21 of life with 107 CFU of S. Enteritidis SPI-1 mutant in 0.1 mL of inoculum and challenged intravenously with 107 CFU of wild type S. Enteritidis on day 44 of life. Intravenous mode of infection was used mainly to stimulate macrophage and heterophil response rather than to model natural infection of chickens with S. Enteritidis. All chickens in groups 2 and 3 were sacrificed 4 days post infection, i.e. when aged 48 days. The spleens from the chickens from all three groups were collected into PBS during necropsy. To confirm S. Enteritidis infection, approximately 0.5 g of liver tissue was homogenised in 5 mL of peptone water, tenfold serially diluted and plated in XLD agar, as described previously [11].

Collecting heterophil and macrophage subpopulations by flow cytometry

The cell suspensions were prepared by pressing the spleen tissue through a fine nylon mesh followed by 2 washes with 30 mL of cold PBS. After the last washing step, the splenic leukocytes were re-suspended in 1 mL of PBS and used for surface marker staining.

In total 108 of cells were incubated for 20 min with anti-monocyte/macrophage:FITC (clone KUL01 from Southern Biotech) and CD45:APC (clone LT40 from Southern Biotech), followed by wash with PBS. Monocytes/macrophages (CD45+KUL01+) and heterophils (identified based on FSC/SSC characteristics within CD45+ cells) were sorted using a FACSFusion flow cytometer operated by FACSDiva software (BD Biosciences). Only for simplicity, the monocytes/macrophages population will be called as “macrophage (Ma)” in the rest of this paper. Sorted cells were collected in PBS and immediately processed as described below. A small aliquot from each sample was subjected to immediate purity analysis. The purity of macrophages was 88.6 ± 5.3% and of heterophils 88.1 ± 4.2% when counting cell of expected staining, and FSC and SSC parameters out of all particles. When we gated at the area with live cells, the purity of macrophages and heterophils was between 97 and 98%. Majority of contaminants therefore represented cellular debris and only around 2.5% of contaminants were formed by non-target cells.

Protein and RNA isolation from sorted cells, reverse transcription of mRNA and quantitative real time PCR (qPCR)

Sorted leukocyte subpopulations were lysed in 500 µL of Tri Reagent (MRC) for parallel isolation of RNA and proteins. Upon addition of 4-bromoanisole and 15 min centrifugation at 14 000 × g, proteins were precipitated with acetone from the lower organic phase. RNA present in upper aqueous phase was further purified using RNeasy purification columns according to the instructions of the manufacturer (Qiagen). The concentration of RNA was determined spectrophotometrically (Nanodrop, Thermo Scientific) and 1 µg of RNA was immediately reverse transcribed into cDNA using MuMLV reverse transcriptase (Invitrogen) and oligo dT primers. After reverse transcription, the cDNA was diluted 10 times with sterile water and stored at −20 °C prior qPCR. qPCR was performed in 3 µL volumes in 384-well microplates using QuantiTect SYBR Green PCR Master Mix (Qiagen) and a Nanodrop pipetting station from Innovadyne for PCR mix dispensing following MIQE recommendations [12]. Amplification of PCR products and signal detection were performed using a LightCycler II (Roche) with an initial denaturation at 95 °C for 15 min followed by 40 cycles of 95 °C for 20 s, 60 °C for 30 s and 72 °C for 30 s, followed by the determination of melting temperature of resulting PCR products to exclude false positive amplification. Each sample was subjected to qPCR in duplicate and the mean values of the Cq values of genes of interest were normalized (ΔCt) to an average Cq value of three reference genes (GAPDH, TBP and UB). The relative expression of each gene of interest was finally calculated as 2−ΔCq. Statistical analysis using a two sample t test for means equality was performed when comparing levels of mRNA expression between chicken groups and results with p value ≤ 0.05 were considered as significantly different in expression. Sequence of reference genes GAPDH, TBP and UB have been published elsewhere [13, 14]. Sequences of all newly designed primers used in this study including their location within different exons and sizes of PCR products are listed in Additional file 1.

Sample preparation for LC–MS/MS analysis

Precipitated proteins were washed with acetone and dried. The pellets were dissolved in 300 µL of 8 M urea and processed by the filter aided sample preparation method [15] using Vivacon 10 kDa MWCO filter (Sartorius Stedim Biotech). Proteins were washed twice with 100 µL of 8 M urea and reduced by 100 µL of 10 mM DTT. After reduction, proteins were incubated with 100 µL of 50 mM IAA and washed twice with 100 µL of 25 mM TEAB. Trypsin (Promega) was used at 1:50 ratio (w/w) and the digestion proceeded for 16 h at 30 °C.

For comparative analysis, peptide concentration was determined spectrophotometrically (Nanodrop, Thermo Scientific) and samples from the same group of chickens were pooled. Pooled samples were then labelled using the stable isotope dimethyl labelling protocol as described previously [16]. Labeled samples were mixed and 3 subfractions were prepared using Oasis MCX Extraction Cartridges (Waters). The samples were desalted on SPE C18 Extraction Cartridges (Empore) and concentrated in a SpeedVac (Thermo Scientific) prior to LC–MS/MS.

LC–MS/MS analysis

Protein samples were analysed on LC–MS/MS system using an UltiMate 3000 RSLCnano liquid chromatograph (Dionex) connected to LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific). Chromatographic separation was performed on EASY-Spray C18 separation column (25 cm × 75 µm, 3 µm particles, Thermo Scientific) with 2 h long (label free) or 3 h long (label based) 3–36% acetonitrile gradient.

High resolution (30 000 FWHM at 400 m/z) MS spectra were acquired for the 390–1700 m/z interval in an Orbitrap analyser with an AGC target value of 1 × 106 ions and maximal injection time of 100 ms. Low resolution MS/MS spectra were acquired in Linear Ion Trap in a data-dependent manner and the top 10 precursors exceeding a threshold of 10 000 counts and having a charge state of +2 or +3 were isolated within a 2 Da window and fragmented using CID.

Data processing, protein identification and quantification

Raw data were analysed using the Proteome Discoverer (v.1.4). MS/MS spectra identification was performed by SEQUEST using the Gallus gallus protein sequences obtained from Uniprot database. Precursor and fragment mass tolerance were 10 ppm and 0.6 Da, respectively. Carbamidomethylation (C) and oxidation (M) were set as static and dynamic modifications, respectively. Dimethylation (N-term and K) was set as static modification in the label-based analysis. Only peptides with a false discovery rate FDR ≤ 5% were used for protein identification.

Spectral counting, the protocol in which abundance of a protein is expressed as the total number of tandem mass spectra matching its peptides (peptide spectrum matches, PSM), was used for comparative label-free analysis of heterophil and macrophage proteomes [17]. For a general comparison of protein abundance between heterophils and macrophages, PSMs belonging to a particular protein from all three groups of chickens, i.e. 18 samples, were summed up. The identification of at least two distinct peptides belonging to the particular protein and the threshold of at least 5 PSMs in at least one sample was required for its reliable identification [18, 19]. All data were normalized to the total number of PSMs in individual samples. Statistical analysis using a t test was performed and the proteins with p value ≤ 0.05 and with at least four fold differences in its amounts were considered as significantly different in their abundance between the subpopulations.

In the label-based quantification, only unique peptide sequences with at least 20 PSMs were considered for peptide ratio calculations. Subsequent analysis of label-based data was performed in R (https://www.R-project.org). For each protein, its individual peptide ratios were log2 transformed, mean values were calculated and tested with a one sample t test. Benjamini-Hochberg correction for multiple testing was then applied to the obtained p values. Only proteins having ≥ twofold change and adjusted p value ≤ 0.05 were considered as being significantly different in abundance.

Bioinformatic analysis

Protein interaction networks were built using the online database resource Search Tool for the Retrieval of Interacting Genes (STRING). Proteins were further analyzed using Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) for their classification into specific pathways. PCA plots were calculated and created in R (https://www.R-project.org).

Results

S. Enteritidis infection

Intravenous S. Enteritidis infection resulted in a high colonization of systemic sites. Average log10 S. Enteritidis counts were 5.03 ± 0.54 and 3.06 ± 0.99 CFU/g of liver in the infected chickens and the vaccinated and infected chickens, respectively. Despite this, no fatalities were observed among infected chickens. No S. Enteritidis was detected in any of the control non-infected chickens.

Identification of heterophil and macrophage specific proteins

Proteins specific for chicken heterophils or macrophages were determined irrespective whether these were obtained from the infected or non-infected chickens.

Altogether, 858 proteins from heterophils and 1032 proteins from macrophages were detected. Out of these, 654 proteins were expressed both in heterophils and macrophages. Two-hundred and eight proteins were detected in macrophages only and an additional 126 proteins were 4 times or more abundant in macrophages than in heterophils. On the other hand, 34 proteins were detected in heterophils only and an additional 44 proteins were 4 times or more abundant in heterophils than in macrophages (Additional file 2).

Proteins characteristic for heterophils

Out of 78 proteins characteristic for heterophils (Additional file 2), 20 with the highest PSM difference between heterophils and macrophages are listed in Table 1. These included MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR proteins, all with antibacterial functions. STOM and RAB27A proteins controlling storage and release of granular proteins in neutrophils also belonged among the characteristic and highly expressed proteins in heterophils. Two serine protease inhibitors, SERPINB10 and SERPINB1, were also found among the 20 most characteristic heterophil proteins (Table 1). Only a single KEGG pathway was specifically enriched in heterophils and this was the starch and sucrose metabolism pathway comprising PYGL, PGM1 and PGM2 proteins (p = 1.7E−4). Despite the KEGG pathway designation, all these proteins represent enzymes involved in glycogen metabolism [20].
Table 1

Twenty most characteristic proteins of heterophils (Het) compared to macrophages (Ma)

Acc. no.

Protein name

Gene ID

∆PSMa

Fold ratio Het:Ma

Response to the infection

Function

P28318

MRP126, calprotectin

MRP126

7170

9.07

No

Calcium and zinc binding

P08940

Myeloid protein 1

LECT2

5532

6.32

Decrease

Chemotactic factor for Het

P02789

Ovotransferrin

OTFB

2351

4.87

Decrease

Iron binding, immune response

O73790

Heterochromatin-associated protein MENT

SERPINB10

1760

6.00

No

DNA condensation, cysteine protease inhibitor

E1C0K1

Extracellular fatty acid-binding protein

ExFABP

1742

4.94

No

Fatty acid and bacterial siderophores binding

F1NG13

Transglutaminase 3

TGM3

1572

19.94

No

Transglutaminase

Q2IAL7

Cathelicidin 2

CATHL2

1402

7.49

Decrease

Antimicrobial peptide

P27042

Lysozyme G

LYG2

989

4.57

Decrease

Antimicrobial peptide

Q2IAL6

Cathelicidin 3

CATHL3

936

5.37

No

Antimicrobial peptide

P00698

Lysozyme C

LYZ

839

5.17

Decrease

Antimicrobial peptide

Q6QLQ5

Cathelicidin 1

CATHL1

833

4.62

Decrease

Antimicrobial peptide

E1BTH1

Leukocyte elastase inhibitor

SERPINB1

627

Only Het

Decrease

Protection against own proteases

F1P284

Leukotriene A(4) hydrolase

LTA4H

603

5.78

Decrease

Epoxide hydrolase and aminopeptidase

F1NGT3

Matrix metallopeptidase 9

MMP9

600

Only Het

Decrease

Degradation of the extracellular matrix

F2Z4L6

Serum albumin

ALB

557

4.79

Decrease

Plasma carrier

P30374

Ribonuclease homolog

RSFR

548

6.89

Decrease

Lysosomal cysteine protease

R9PXN7

Hematopoietic prostaglandin D synthase

HPGDS

504

17.79

No

Cytosolic glutathione S-transferases

E1BTV1

Stomatin

STOM

502

23.82

No

Integral membrane protein

D2D3P4

Rab27a

Rab27a

435

88.08

No

Small GTPase, exocytosis

R4GI24

Integrin alpha-D

ITGAD

379

7.73

No

Adhesion of leukocytes

aThe difference in PSM counts of particular protein in Het and Ma.

Proteins characteristic for macrophages

Out of 334 proteins specific for macrophages (Additional file 2), 20 with the highest PSM difference between macrophages and heterophils are listed in Table 2. Five of these represented receptor proteins MRC1L, LRP1, LGALS1, LRPAP1 and DMBT1L, the last one containing the scavenger receptor cysteine-rich (SRCR) domain. CTSB, CKB, MECR, PHB2, H9KZK0 and p41/Li are involved in phagocytosis and antigen presentation. An additional 4 proteins UQCR, UQCRC1, ACO2 and HADHB are localized to the mitochondria. Only 3 proteins, MRC1L, HSP70 and p41/Li, were already recorded in chicken macrophages [2123] although except for NAT3, PLB and SSB, the expression of the remaining proteins (out of the most abundant listed in Table 2) has been already recorded in murine or human macrophages. Proteins enriched in macrophages belonged to oxidative phosphorylation (p = 4.7E−8), fatty acid metabolism (p = 1.73E−6), citrate cycle (p = 4.2E−6), arginine and proline metabolism (p = 8.5E−8) and proteasome (p = 4.5E−4).
Table 2

Twenty most characteristic proteins for macrophages (Ma) compared to heterophils (Het)

Acc. no.

Protein name

Gene ID

∆PSMa

Fold ratio Ma:Het

Response to the infection

Function

M1XGZ4

Macrophage mannose receptor 1 like

MRC1L

993

Only Ma

No

C-Type lectin

P98157

Low-density lipoprotein receptor-related protein 1

LRP1

810

Only Ma

No

Endocytic receptor

P07583

Galectin 1

LGALS1

607

Only Ma

No

Beta-galactoside-binding lectin

P43233

Cathepsin B

CTSB

538

8.42

Increase

Cysteine protease

F1NZ86

Heat shock 70 protein, mortalin

HSP70

508

5.30

No

Chaperon

P05122

Creatine kinase B-type

CKB

467

34.77

No

Energy transduction

F1NDD6

LDL receptor related protein associated protein 1

LRPAP1

374

Only Ma

No

LDL receptors trafficking

F1NIX4

Trans-2-enoyl-CoA reductase

MECR

356

33.16

Increase

Fatty acid elongation

F1P180

Aspartate aminotransferase

GOT2

350

7.27

No

Transaminase

P13914

Arylamine N-acetyltransferase

NAT3

350

23.92

No

Conjugating enzyme

H9KZK0

Protein containing the scavenger receptor cysteine-rich (SRCR) domain

DMBT1L

318

Only Ma

No

Scavenger receptor

E1BZF7

Putative phospholipase B

PLB

317

6.23

No

Removing fatty acids from phospholipids

Q6J613

Invariant chain isoform p41

Li

312

6.87

No

Chaperone

F1P582

Mitochondrial ubiquinol-cytochrome-c reductase complex core protein 2

UQCR

309

4.36

No

Oxidative phosphorylation

Q5ZMW1

Aconitate hydratase, mitochondrial

ACO2

306

6.17

No

TCA cycle

F1NAC6

Cytochrome b-c1 complex subunit 1

UQCRC1

289

6.42

No

Oxidative phosphorylation

F6R1X6

Lupus la protein

SSB

288

6.90

No

Protecting of 3′ poly(U) terminus of transcribed RNA

E1BTT4

Trifunctional enzyme subunit beta, mitochondrial

HADHB

287

30.61

Increase

β-Oxidation of fatty acids

Q5ZMN3

Prohibitin-2

PHB2

282

10.52

No

Not clear

F1NJD6

Guanine deaminase, cypin

GDA

275

Only Ma

No

Oxidizes hypoxanthine to xanthine

aThe difference in PSM counts of particular protein in Ma and Het.

Heterophil proteins responding to in vivo infection with S. Enteritidis

Altogether, 153 proteins were present in different abundance in the heterophils before and after S. Enteritidis infection. Of these, 109 proteins increased and 44 proteins decreased in abundance (Additional files 3 and 4 for all quantified heterophil proteins). Proteins belonging to 2 KEGG categories were enriched in heterophils following S. Enteritidis infection. These included the category translation with 39 proteins (p = 2.58E−62) and protein processing in endoplasmic reticulum (12 proteins, p = 1.74E−11). Twenty proteins with the highest increase in abundance, except for those belonging to the category translation, are listed in Table 3. Among others, these included AVD, F13A, ANXA2, ANXA7 or CTSC.
Table 3

Proteins which increased in abundance in heterophils in response to S. Enteritidis infection

Acc. no.

Protein name

Gene ID

Fold ratio Inf: noninf

Fold ratio vac: noninf

Function

P02701

Avidin

AVD

55.57*

32.06*

Biotin binding

F1P4F4

Translocon-associated protein

SSR1

9.22*

6.36

Protein translocase

P17785

Annexin A2

ANXA2

6.44*

2.11

Activates macrophages for cytokine production

E1BWG1

Coagulation factor XIIIA

F13A

5.63*

2.60*

Crosslinking of fibrin chains, entrapment of bacteria

R4GJX3

Interferon-induced transmembrane protein

IFITM

4.99*

1.73

Acidification of the endosomal compartments, mediator of the host antiviral response

F1NK96

Protein disulfide-isomerase A6

PDIA6

4.33*

2.66*

Protein foldase

F1NVA4

Nucleophosmin

NPM1

3.68*

1.87

Alarmin, nuclear chaperon

F1NT28

Inorganic pyrophosphatase

PPA1

3.52*

1.67

Hydrolysis of inorganic pyrophosphate (PPi)

Q90593

78 kDa glucose-regulated protein

BiP

3.44*

1.94

Chaperon

F1NWB7

Endoplasmin

HSP90B1

3.33*

1.99

Chaperon

E1C1D1

Annexin 7

ANXA7

3.27*

2.68*

Granular membranes fusion and degranulation

P24367

Peptidyl-prolyl cis–trans isomerase B

PPIB

3.26*

2.23*

Regulation of protein folding and maturation

E1C2S1

Talin-1

TLN1

3.12*

2.56*

Activation of neutrophils

Q49B65

EF hand-containing protein 1

EFHD1

3.12*

1.72

Calcium binding

F1NWG2

Cathepsin C

CTSC

3.10*

1.99

Activates serine proteases (elastase, cathepsin G and granzymes)

F1NDY9

Protein disulfide-isomerase A4

PDIA4

2.93*

1.86

Protein foldase

E1C8M9

Calnexin

CANX

2.88*

1.75

Integral protein of the endoplasmic reticulum

E1BQN9

Calcyclin-binding protein

CACYBP

2.88*

2.38*

Calcium-dependent ubiquitination

H9L340

ATP synthase subunit beta

ATP5B

2.82*

1.56

Energy metabolism

F1NB92

Endoplasmic reticulum aminopeptidase 1

ERAP1

2.78*

0.89

Antigen processing and presentation of endogenous peptide via MHC class I

* Significantly different from the expression in heterophils from the non-infected chickens.

Forty-four proteins decreased in abundance in heterophils following S. Enteritidis infection and 20 of these with the highest decrease are listed in Table 4. Proteins with decreased abundance were those found in heterophil granules such as MPO, LYZ, LYG2, CTSG, CTSL1, CATHL1, CATHL2, RSFR, MMP9 and LECT2. Another set of proteins which decreased in heterophils following S. Enteritidis infection included ALB, FN1 and OTFB (Table 4).
Table 4

List of proteins which decreased in abundance in heterophils in response to S. Enteritidis infection

Acc. no.

Protein name

Gene ID

Fold ratio inf: noninf

Fold ratio vac: noninf

Function

F1P1U6

Myeloperoxidase

MPO

0.013*

0.071*

Oxidative burst

E1C677

Natural killer cell activator

Gga.18306

0.026*

0.21*

GO prediction: regulation of cytokine biosynthetic process

F1NJT3

Fibronectin

FN1

0.11*

0.56

Binds components of extracellular matrix

F1NFQ7

Serine protease 57

PRSSL1

0.15*

0.37*

Serine-type endopeptidase activity

P00698

Lysozyme C

LYZ

0.16*

0.37*

Antimicrobial peptide

H9L027

Cathepsin G

CTSG

0.19*

0.30*

Lysosomal cysteine protease

Q6QLQ5

Cathelicidin-1

CATHL1

0.20*

0.51

Bactericidal, fungicidal and immunomodulatory activity

F1NZ37

Cathepsin L1

CTSL1

0.22*

0.48*

Controlling element of neutrophil elastase activity

P30374

Ribonuclease homolog

RSFR

0.23*

0.51

Lysosomal cysteine protease

P27042

Lysozyme G

LYG2

0.24*

0.60

Antimicrobial peptide

F2Z4L6

Serum albumin

ALB

0.24*

0.67

Plasma carrier

P02789

Ovotransferrin

OTFB

0.26*

0.55

Iron binding, immune response

F1NGT3

Matrix metallopeptidase 9

MMP9

0.26*

0.77

Degradation of the extracellular matrix

F1NVM1

G-protein coupled receptor 97

GPR97

0.27*

0.66

Regulates migration

Q2IAL7

Cathelicidin-2

CATHL2

0.31*

0.78

Antimicrobial peptide

Q2UZR2

Phosphoglucomutase 1

PGM1

0.35*

0.43*

Glucose metabolic process

E1BZS2

Nucleosome assembly protein 1-like

NAP1L1

0.36*

0.22*

Chaperone for the linker histone

P08940

Myeloid protein 1

LECT2

0.37*

0.62

Chemotactic factor

R4GH86

Glutathione peroxidase

GPX

0.41*

0.57

Protects organism from oxidative damage

F1NYH8

Ena/VASP-like protein

EVL

0.42*

0.70

Regulators of the actin cytoskeleton and cell migration

* Significantly different from the expression in heterophils from the non-infected chickens.

Macrophage proteins responding to in vivo infection with S. Enteritidis

Four KEGG pathways were specifically enriched when testing proteins of increased abundance in macrophages following S. Enteritidis infection. These included fatty acid elongation pathway (MECR and HADHB proteins, p = 2.49E−4), lysosomal proteins CTSB and CTSC (p = 6.98E−3), phagosomal proteins RAB7A and STX7 (p = 9.23E−3) and LDHA and HADHB from the microbial metabolism in diverse environments pathway (p = 9.4E−3). Other proteins with increased abundance in macrophages following S. Enteritidis infection were MRP126, CATHL1, CATHL2, GAL1, CTSB, CTSC, RSFR, SOD1, LECT2, LY86 and FTH, all with antibacterial functions (Table 5). Proteins which decreased in abundance in macrophages following S. Enteritidis infection included RBMX, NDUFA4, FNBP1, FAM107, STMN1, GLOD4 and OLA1 (Table 5; Additional files 5, 6 for all quantified macrophage proteins).
Table 5

Proteins of increased or decreased abundance in macrophages in response to S. Enteritidis infection

Acc. no.

Protein name

Gene ID

Fold ratio inf:noninf

Fold ratio vac:noninf

Function

P28318

MRP126, calprotectin

MRP126

15.67*

5.01*

Calcium and zinc binding

Q6QLQ5

Cathelicidin-1

CATHL1

7.32*

2.95*

Antimicrobial peptide

P30374

Ribonuclease homolog

RSFR

5.84*

1.66

Lysosomal cysteine protease

F1NIX4

Trans-2-enoyl-CoA reductase

MECR

5.47*

3.99*

Fatty acid elongation

P46156

Gallinacin 1

GAL1

4.15*

1.12

Antimicrobial protein

F1N8Q1

Superoxide dismutase

SOD1

4.01*

2.58

Oxygen scavenger

P08940

Myeloid protein 1

LECT2

3.87*

1.35

Chemotactic factor for Het

F1P4F3

Lymphocyte antigen 86, MD-1

LY86

3.53*

3.03

Inhibits LPS response of immune cells

F1NS91

60S ribosomal protein L9

RPL9

3.51*

3.82

Structural part of ribosome

E1BTT4

Trifunctional enzyme subunit beta, mitochondrial

HADHB

3.38*

3.54*

β-Oxidation of fatty acids

P43233

Cathepsin B

CTSB

2.88*

2.57*

Lysosomal cysteine protease

B4X9P4

Microsomal glutathione S-transferase 1

MGST1

2.87*

1.46

Membrane protection from oxidative stress

Q5ZMP2

Syntaxin 7

STX7

2.72*

2.94*

Late endosome–lysosome fusion

E1C0F3

Ras-related protein Rab-7a

RAB7A

2.69*

2.38*

Involved in endocytosis, phagosome–lysosome fusion

F1N9J7

Tubulin alpha-3 chain

Tuba3a

2.63*

1.96

Major constituent of microtubules

P08267

Ferritin heavy chain

FTH

2.62*

2.33*

Storage of iron in a soluble, nontoxic state

P02263

Histone H2A-IV

H2A4

2.61*

3.64*

Formation of nucleosome

F1NWG2

Cathepsin C

CTSC

2.48*

2.46*

Activates serine proteases

Q2IAL7

Cathelicidin-2

CATHL2

2.45*

1.01

Antimicrobial peptide

Q6EE32

Calreticulin

CALR

2.33*

2.21*

Molecular chaperon

Q9I9D1

Voltage-dependent anion-selective channel protein 2

VDAC2

2.27*

2.07*

Inhibits mitochondrial way of apoptosis

P02607

Myosin light polypeptide 6

MYL6

2.7*

1.66

Found in phagosome

F1NB92

Endoplasmic reticulum aminopeptidase 1

ERAP1

2.21*

2.04

Antigen processing and presentation of endogenous peptide via MHC class I

E1BTT8

Lactate dehydrogenase A

LDHA

2.07*

1.71

Glycolysis

R4GM10

Fructose-bisphosphate aldolase C

ALDOC

2.07*

2.33

Glycolysis

P24367

Peptidyl-prolyl cis–trans isomerase B

PPIB

2.00*

0.97

Regulation of protein folding and maturation

Q5ZKQ9

RNA binding motif protein, X-linked

RBMX

0.49*

0.59

Regulation of pre- and post-transcriptional processes

R4GGZ2

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4

NDUFA4

0.38*

0.65

Oxidative phosphorylation

E1BYF8

Formin-binding protein 1

FNBP1

0.33*

0.47*

Role in late stage of clathrin-mediated endocytosis

R4GJP1

Family with sequence similarity 107, member B

FAM107

0.32*

0.30*

Candidate tumor suppressor gene

P31395

Stathmin 1

STMN1

0.27*

0.51

Promotes disassembly of microtubules

E1BQI4

Glyoxalase domain-containing protein 4

GLOD4

0.21*

0.18*

Unknown

Q5ZM25

Obg-like ATPase 1

OLA1

0.12*

0.11

Negative role in cell adhesion and spreading

* Significantly different from the expression in macrophages from the non-infected chickens.

RNA expression

Finally we verified the expression of 37 genes coding for selected proteins listed in Tables 1, 2, 3, 4 and 5. Expression of 4 genes, LRP1, MPO, PPIB and TUBA3A was too low and these genes were excluded from further consideration (Additional file 7).

Six genes (LGALS1, MRC1L, GDA, MECR, DMBT1, LRPAP1) out of 7 proteins selected as specific for macrophages were transcribed in macrophages at a higher level than in heterophils. Only HSP70 was transcribed in macrophages and heterophils at the same level though it was present in higher abundance at the protein level in macrophages. Nine genes (MRP126, OTFB, LYG2, LYZ, SERPINB1, CATHL1, CATHL2, MMP9, LECT2) out of 14 heterophil specific proteins were transcribed in heterophils at a higher level than in macrophages. Two genes of this group (GPX, CTSG) were transcribed in heterophils and macrophages at the same level and the remaining 2 genes (RSFR, LTA4H) were transcribed at a higher level in macrophages though protein mass spectrometry indicated their higher abundance in heterophils.

Expression of 11 proteins which increased in abundance in macrophages following infection of chickens with S. Enteritidis was also tested at the RNA level. Except for MRP126, 10 of these (MECR, CTSC, ERAP1, RSFR, SOD1, CALR, CATHL1, CATHL2, LECT2, GAL1) did not exhibit any difference at the transcriptional level. 6 of 7 proteins (ANXA2, F13A, CTSC, ERAP1, AVD, HSP90B1) exhibiting an increased abundance in heterophils following infection of chickens with S. Enteritidis, also increased their expression at the level of transcription. Only IFITM did not change its expression at the RNA level. Finally we verified the expression of 11 proteins which decreased in abundance in heterophils following infection of chickens with S. Enteritidis. Eight of them (FN1, ALB, CTSL1, OTFB, LYZ, CATHL1, MMP9, LECT2) did not change their expression at the level of transcription and transcripts of 3 of them (RSFR, LYG2, CSTC) even increased following infection.

Similar to the results of protein mass spectrometry, RNA levels of the tested genes in the heterophils or macrophages from the vaccinated chickens were in between the expression in non-infected chickens and chickens infected without previous vaccination. Only 3 genes in heterophils did not follow this scheme and CATHL1, CATHL2 and LECT2 were expressed in heterophils from the vaccinated chickens at significantly higher level than in the heterophils from infected chickens.

Discussion

Until now, chicken heterophils and macrophages have been characterized only by their specific characteristics like cytokine signaling or production of antimicrobial peptides [2, 6, 7, 24, 25] and an unbiased report characterizing their total proteome, before and after infection, has been missing. In the current study we therefore isolated proteins from heterophils and macrophages and quantified their abundance before and after infection with S. Enteritidis by mass spectrometry. We have to remind that mass spectrometry provides reliable data for approximately 800 the most abundant proteins. The lowly represented proteins, despite their potential specificity or responsiveness to infection, could not be therefore detected.

Chicken macrophages differed from heterophils in 3 specific features. First, macrophages specifically expressed receptors such as MRC1L, LRP1, LGALS1, LRPAP1 and DMBT1L. Second, macrophages exhibited higher mitochondrial activity including fatty acid degradation, TCA cycle and oxidative phosphorylation. And third, macrophages specifically expressed enzymes involved in arginine and proline metabolism (Figure 1). Receptors specifically expressed by macrophages indicate their potential to sense signals from the external environment which allows them to modulate immune response [6, 7] including their own polarization [26, 27]. The dependency of macrophages on oxidative phosphorylation and mitochondria functions was already described for human macrophages and neutrophils [28]. Macrophages were also enriched in arginine and proline metabolism since one of their bactericidal activities is the production of NO radicals by iNOS and arginine [29]. Following infection with S. Enteritidis, macrophages increased the expression of lysosomal and phagosomal proteins what could be associated not only with S. Enteritidis inactivation but also with macrophage ability of antigen presentation.
Figure 1

The most characteristic proteins and their functions in chicken heterophils and macrophages. Heterophils express MMP9, MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR proteins. Following S. Enteritidis infection, heterophils decreased fibrinogen FN1 and albumin ALB, and increased ribosomal proteins. In addition, endoplasmic reticulum proteins are activated which results in the release of granular proteins. Heterophils expressed glycogen (Gly) metabolism pathway which allows for rapid glucose (Glu) availability and anaerobic ATP generation via glycolysis while macrophages increased mitochondrial activity. Macrophages expressed receptor proteins MRC1, LGALS1, LRPAP1 and DMBT1L, mitochondria-localized proteins and arginine metabolism proteins. Following infection with S. Enteritidis, macrophages increased the expression of lysosomal and phagosomal proteins (CTSB, CTSC, RAB7A, CATHL1, RSFR, GAL1, SOD1).

Heterophils specifically expressed granular proteins MPO, LYZ, LYG2, RSFR, LECT2, CATHL1, CATHL2, CTSL1, CTSG, OTFB, SERPINB1 and MMP9, and endoplasmic reticulum proteins SSR1, PDIA4, PDIA6, PPIB, BiP, HSP90B1 and CANX. The latter group of proteins is activated when lumenal conditions in endoplasmic reticulum are altered or chaperone capacity is overwhelmed by unfolded or misfolded proteins [30]. Induction of an unfolded protein response leads to neutrophil degranulation in mice [31] and based on our results, a similar response can be predicted also in chicken heterophils.

Granular proteins decreased in heterophils in response to infection. Since transcription of genes encoding these proteins did not change and the number of ribosomal proteins increased, these genes must have remained continuously transcribed and translated even after initial degranulation [24, 3235]. However, not all proteins that decreased in heterophils following S. Enteritidis infection were assigned to pathogen inactivation. Matrix metalloproteinase MMP9 is used for degradation of the extracellular matrix to enable leukocyte infiltration to the site of inflammation [36], and ALB and FN1, are found at the surface of granulocytes and inhibit their migration [37, 38]. The decrease of ALB and FN1 together with the degradation of extracellular matrix by MMP9 leads to heterophil translocation from the blood circulation to the site of inflammation.

Comparing expression at the protein and RNA levels provided several unexpected results. Changes in expression at the RNA level in response to infection were more pronounced in heterophils than in macrophages. We can exclude any technical issues in macrophage gene expression analysis since there were at least 3 genes inducible at the RNA level also in macrophages (AVD, MRP126 and F13A). Unlike macrophages, there were also greater differences in the expression profiles of heterophils obtained from vaccinated chickens in comparison to those obtained from naive but infected animals and an increase in CATHL2 and LECT2 in the heterophils from the vaccinated chickens following S. Enteritidis challenge appeared as a specific positive marker of vaccination. Despite this, expression in heterophils and macrophages in naive but infected chickens tended to approach a similar expression profile (Figure 2).
Figure 2

PCA cluster analysis of chicken heterophils and macrophages using expression data from qPCR. Each spot represents heterophils (circles) or macrophages (triangles) isolated from non-infected (green color), infected (red color), and vaccinated and infected chickens (blue color), 6 chickens per group. Heterophils from vaccinated chickens responded to infection more than macrophages from the same chicken. Transcription of heterophils and macrophages from naive but infected chickens approached the same profile.

In this study we characterized protein expression in chicken heterophils and macrophages in response to intravenous infection with S. Enteritidis. Heterophils decreased ALB and FN1, and released MMP9 to enable their translocation to the site of infection. Secondly the endoplasmic reticulum proteins increased in heterophils which resulted in the release of granular proteins. On the other hand, macrophages were less responsive to acute infection and an increase in proteins like CATHL1, CATHL2, RSFR, LECT2 and GAL1 in the absence of any change in their expression at RNA level could even be explained by capturing these proteins from the external environment into which these could have been released by heterophils.

Declarations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

ZS and OP purified proteins and performed protein mass spectrometry. HS and RF sorted splenic leukocytes by flow cytometry. KV and MF were responsible for RNA purification and qPCR. IR and LV designed the study, analysed data and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Authors would like to thank Peter Eggenhuizen for language corrections and acknowledge the excellent technical assistance of Andrea Durisova.

Funding

This work has been supported by project from P502-13-31474P of the Czech Science Foundation, AdmireVet project CZ.1.005/2.1.00/01.0006–ED0006/01/01 from the Czech Ministry of Education and RO0516 project of the Czech Ministry of Agriculture. RF was supported by the project LQ1605 from the National Program of Sustainability II (MEYS CR). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors’ Affiliations

(1)
Veterinary Research Institute
(2)
Department of Cytokinetics, Institute of Biophysics of the CAS
(3)
Center of Biomolecular and Cellular Engineering, International, Clinical Research Center, St. Anne’s University Hospital Brno

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