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Annals of Microbiology, 59 (3) 587-592 (2009) Immune response of peripheral blood mononuclear cells to avian pathogenic Escherichia coli 1,2 1 1 1 1* Hassan H. MUSA , Sheng L. WU , Chun H. ZHU , Jun ZHU , Guo Q. ZHU College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China Faculty of Veterinary Science, University of Nyala, Nyala, 155, Sudan Received 6 April 2009 / Accepted 10 July 2009 Abstract - The exposure of chicken peripheral blood mononuclear cells (PBMCs) to pathogen can induce a rapid change in both pro- inflammatory and anti-inflammatory cytokine gene expression. Chicken PBMCs were infected with avian pathogenic Escherichia coli (APEC) and the isogenic fimH mutants of APEC, PBMCs immune response was determined by semi-quantitative RT-PCR. The results demonstrated that PBMCs were expressed TLR1/6/10, TLR3, TLR4, TLR5 and TLR7. APEC and non pathogenic E. coli strains were significantly (P < 0.05) increased the expression of IL-1β, IL-8, IL-18 and TGF-β4 cytokines compared to the isogenic fimH mutants. In addition, the opsonized avian pathogenic E. coli strains were significantly (P < 0.05) increased the expression of cytokines com- pared to the non-opsonized strains, whereas the opsonized avian non-pathogenic E. coli strain was significantly (P < 0.05) decreased the expression of cytokines compared to the non-opsonized strain. PBMCs are important in APEC infection, the increase in cytokines expression may play important role in the regulation of immune response. Key words: cytokines; PBMCs; Escherichia coli; chickens. INTRODUCTION like receptors are a family of germline-encoded innate immune receptors that recognize pathogen-associated molecular pat- Avian pathogenic Escherichia coli (APEC) infections are responsi- terns, such as bacterial flagellin, LPS and lipopeptides induce ble for significant economic losses in the poultry industry world- signaling through TLR5, TLR4 and TLR1/2/6, respectively (Akira wide. Serotypes O1, O2 and O78, are commonly implicated et al., 2006). Double strand RNA stimulates TLR3 (Alexopoulou in avian colibacillosis, and most are elaborate type 1 fimbriae et al., 2001), and single stranded viruses stimulates TLR7 (Heil (Orskov and Orskow, 1992; La Ragione and Woodword, 2002), et al., 2004). The ability of TLRs to recognize a broad spectrum which characterized by their ability to bind D-mannose and thus of microbial molecules enables the host to detect the presence bind to many types of eukaryotic cells and various inflamma- of pathogens rapidly, before a more widespread infection occurs tory cells (Hornick et al., 1991). Binding is conferred by the (Harris et al., 2006). Escherichia coli is recognized by several adhesin FimH a minor component of the fimbriae (Krogfelt et TLRs, including TLRs 2, 4, 5, and 11, and likely TLR9 (Andersen- al., 1990). Lymphocytes are an important part of the adap- Nissen et al., 2007). In human and mice eleven TLRs have been tive immunity against infections (Gronlund et al., 2006), they identified with each member recognizing and responding to dif- produce cytokines which stimulate antibody production, induce ferent microbial components (Kogut et al., 2005a). In chicken local inflammation and enhance the phagocytic and microbicidal TLR 1/6/10, TLR2 type 1, TLR2 type2, TLR3, TLR4, TLR5 and activities of macrophage (Abbas and Lichtman, 2003). Peripheral TLR7 were similarly identified (Fukui et al., 2003; Igbal et al., blood mononuclear cells (PBMCs) act as the main effectors via 2005; Kogut et al., 2005a). Heterophils expressed IL-18, IL-1β, the functional regulation of cytokines such as interleukins and IL-6, IL-8 and TGF-β4 upon stimulation that is initiated by recep- interferon-γ. The important role of PBMCs is well established, tor-mediated phagocytosis (Kogut et al., 2005b; Swaggerty et and has been demonstrated in experimental models (Kim et al., al., 2004). 2008). In vitro and in vivo studies have shown that APEC adheres to Recognition of potential pathogenic microbes by the immune tracheal and pharyngeal cells, whereas their FimH mutants are system is the function of a class of cellular receptors known as unable to adhere to the cells (Marc et al., 1998). Mellata et al. the pattern recognition receptors (PRRs), which include Toll-like (2003) indicated that type1 fimbriae can protect bacteria against receptors (TLRs) (Igbal et al., 2005; Harris et al., 2006). Toll- the bactericidal effect of phagocytes. Kaiser et al. (2006) dem- onstrate that exposure of chicken PBMC to Salmonella enteri- tidis can induce a rapid change in both pro-inflammatory (IL-6, * Corresponding Author. E-mails: hassan_hm30@yahoo.com; CXCLi2) and anti-inflammatory (TGF-β4) cytokine gene expres- yzgqzhu@yzu.edu.cn 588 H.H. MUSA et al. sion. In this study we determined the expression of cytokines in bacteria strains (wild type 1 fimbriae and isogenic fimH mutant) peripheral blood mononuclear cells (PBMCs) after infection with at a multiplicity of infection (MOI) of 100 per cell in 1.5 ml wild type APEC and their isogenics fimH mutants. Understanding Eppendorf tube at 37 °C for 1 h. the distribution patterns of chicken TLR will enable more defined interpretation of immune induction and the host-pathogen rela- Primers. Chicken TLRs primers used for semi-quantitative tionships that define infectious disease biology in the chicken. In RT-PCR were designed by Kogut et al. (2005a), while TLR5 and addition, PBMCs functional efficiency and cytokines production cytokines primers were designed based on their gene sequence may be useful biomarkers for development of immunocompetent in gene bank (Tables 1 and 2). chickens. RNA extraction and semi-quantitative RT-PCR analysis. The untreated PBMCs and treated PBMCs were pelleted by MATERIALS AND METHODS centrifugation at 8000 x g for 2 min. Total RNA was extracted in the presence of buffer containing β-mercaptoethanol and Experimental animals. Green shell egg fowl chicks were guanidine using RNAiso plus kit (Takara Biotechnology Dalian, obtained on the day-of-hatch from the hatchery of Poultry Co. LTD, China) following manufacturer’s instructions eluted with Institute, Chinese Academy of Agricultural Sciences, Yangzhou, 40 μl RNase-free water. Total RNA was measured using a Nano China and placed in floor pens. Birds were provided supplemental Drop ND-1000 Spectrophotometer (Nano Drop Technologies, heat, water and a balanced, unmedicated chick based ration ad Wilmington, USA) and a purity (A /A ) of > 1.8 was used. 260 280 libitum. Two hundred and fifty ng of total RNA from each sample was transcribed into cDNA using the Takara reverse transcription kit Bacterial strains and growth condition. The avian pathogenic (Takara Biotechnology Dalian, Co. LTD) according to manufac- Escherichia coli (APEC) strains serotype (O78:K89, O1:K89 and turer’s instructions. Briefly, oligo dT Primer (50 μM) was used to O2:K89) and non-APEC strain serotype (O24:K89) were used reverse transcribe 250 ng/μg of respective RNA in the presence TM as a prototypes of E. coli type 1 fimbriae. Their isogenic FimH of dNTPs mixture (10 mM each), 5X PrimeScript buffer, RNase TM mutants (O78:K89ΔfimH, O1:K89 ΔfimH, O2:K89ΔfimH and Inhibitor (40 U/μl) and PrimeScript RTase (200 U/μl) at 42 O24:K89ΔfimH) were constructed by λ Red-mediated recombina- °C for 60 min following inactivation at 95 °C for 5 min, all RNA tion system (He et al., 2008). Expressed FimH from wild types preparations were standardized by RT-PCR for β-actin. and non expressed from mutants were confirmed by the ability Polymerase chain reactions (PCR) were performed with the of agglutination reaction with both guinea pig erythrocytes and primers indicated in Table 1 and Table 2. Briefly, cDNA (2 μl) yeast cells. All strains were grown in LB broth at 37 °C for 48 h was reacted with 250 mM dNTPs, 1X reaction buffer (Takara to allow a high level of expression of type 1 fimbriae. Biotechnology Dalian, Co. LTD), forward and reverse primers (5 pM) and 0.4 units Taq polymerase in a 25 μl final reaction Opsonization of bacteria. The APEC bacteria were opsonized volume. PCR conditions were as follows, 1 cycle of 94 °C for 4 as previously described (Mellata et al., 2003). Briefly, APEC (10 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min CFU/ml) were suspended in 25% hyper-immune serum from and 72 °C for 1 min, followed by 1 cycle at 72 °C for 10 min. adult chickens immunized against inactivated APEC solution, Each PCR product (7 μl) was electrophoresed on a 2% agarose incubated at 37 °C for 30 min, washed twice with HBSS and gel (Gene Tech. Shanghai Company limited) in 1X TBE buffer at used immediately. 60 V for 45 min and visualized with ethidium bromide under UV light (BIORAD). Peripheral blood mononuclear cells isolation. Avian periph- eral blood mononuclear cells (PBMCs) were isolated from the Statistical analysis. Semi-quantitative analysis for cytokines peripheral blood of two day-old chickens following an established was performed by image Quant TL (Amersham Biosciences, protocol (Kaiser et al., 2006). Briefly blood from chickens was USA). Data was presented as mean and standard error of mean, collected in tube containing disodium ethylenediaminetetraacetic differences between wild type and their isogenic fimH mutants acid (EDTA) and mixed thoroughly. The blood and EDTA was and between opsonized and non-opsonized strains were deter- diluted 1.5:1 with RPMI-1640 media containing 1% methylcel- mined by student t-test, P < 0.05 was considered to be signifi- lulose (Sigma-Aldrich, USA) and centrifuged at 1000 rpm for cant, analysis was performed by SAS. 15 min. The serum and buffy coat layers were retained and 2+ 2+ suspended in an equal volume of Ca , Mg free Hanks bal- anced salt solution (HBSS). This suspension was layered over RESULTS a discontinuous Histo-paque 1077 gradient (Sigma-Aldrich).The gradient was then centrifuged at 2000 rpm for 45 min. After Cytokine expression in PBMCs centrifugation, the interfaces and band containing the PBMCs was Chickens PBMCs were expressed TLR1/6/10, TLR3, TLR4, TLR5 collected into a new tube and washed twice in HBSS and resus- and TLR7 mRNA (Fig. 1). The level of cytokines expression was pended in fresh RPMI 1640 media. Cell viability was determined significantly increased upon stimulation with avian pathogenic by trypan blue exclusion, and the purity of the cell was assessed and non-pathogenic E coli. by microscopic examination of Wright-Giemsa Stain smears. The cell concentration was adjusted to 1 x 10 cells of PBMCs/ml and IL-1β expression following stimulation stored on ice until used. IL-1β acts in many cells as an inflammatory mediator, the stimu- lation of PBMCs with APEC and fimH mutant of APEC increased Bacterial infection of peripheral blood mononuclear cells. quantitatively IL-1β expression compared to non-stimulated con- The PBMCs (1 x 10 cells/ml) suspended in RPMI-1640 medium trol PBMCs. The wild type of APEC O1:K89 and O2:K89 strains without antibiotics served as control to establish basal cytokine increased significantly (P < 0.05) the expression of IL-1β com- levels. PBMCs were infected with opsonized and non opsonized pared to the isogenic fimH mutants (Fig. 2A). The opsonized Ann. Microbiol., 59 (3), 587-592 (2009) 589 TABLE 1 - Sequence of TLR primers used in semi-quantitative RT-PCR Primer* Sequence Accession number TLR1/6/10 F 5 CGGAAAGCCTATCATTGTCA 3 BQ484541/BU471924 TLR1/6/10 R 5 TTTGTCTGCGTCCACTGC3 BQ484541/BU471924 TLR2 Type1 F 5 TTAAAAGGGTGTGCCAGGAG3 AB050005 TLR2 Type1 R 5 GTCCAAACCCATGAAAGAGC3 AB050005 TLR2 Type2 F 5 AGGCACTTGAGATGGAGCAC3 AB046533 TLR2 Type2 R 5 CCTGTTATGGGCCAGGTTTA3 AB046533 TLR3 F 5 CCACTCTGGAAGAAAATGAGC3 BI066273 TLR3 R 5 TCATTCTCACCGCTTTTCAG3 BI066273 TLR4 F 5 AGTCTGAAATTGCTGAGCTCAAAT3 AY064697 TLR4 R 5 GCGACGTTAAGCCATGGAAG 3 AY064697 TLR5 F 5 CCACATCTGACTTCTGCCTTT 3 AJ626848 TLR5 R 5 CAGCTAGGGTTACATTGGTTTC 3 AJ626848 TLR7 F 5 GCCTCAAGGAAGTCCCCAGA 3 AJ632302 TLR7 R 5 AAGAAACATTGCATGGATTACGG3 AJ632302 β-actin F 5 TGCTGTGTTCCCATCATCG 3 L08165 β-actin R 5 TTGGTGACAATACCGTGTTCA 3 L08165 * F, refer to forward; R, refer to reverse. TABLE 2 - Sequence of interleukins primers used in semi-quantitative RT-PCR Primer* Sequence Accession number IL-1 β F 5 GTG GCA CTG GGC ATC AAG GG 3 AJ245728 IL-1 β R 5 CAG GGA GGT GCA GAT GAA C 3 AJ245728 IL-8 F 5 GCC CTC CTC CTG GTT TCA 3 AJ009800 IL-8 R 5 TGC TGG CAT GTA TAA AGA AGA GAG 3 AJ009800 IL-18 F 5 CCA TGC ACA TAA TAC TGA G 3 AJ416937 IL-18 R 5 AGT CGA TTG CTA CAG AAA G 3 AJ416937 TGF-β4 F 5 AAG GAT CTG CAG TGG AAG TGG A 3 M31160 TGF-β4 F 5 CAT TCC GGC CCA CGT AGT AA 3 M31160 * F, refer to forward; R, refer to reverse. APEC O2:K89 and O78:K89 strains were significantly (P < 0.05) to the control. Only O2:K89 strain was found significantly (P < expressed higher IL-1β whiles the opsonized non-pathogenic E. 0.05) expressed higher IL-8 compared to fimH mutant strain coli O24:K89 strain was significantly (P < 0.05) expressed lower (Fig. 3A). Similarly, IL-8 expression was significantly (P < 0.05) IL-1β. Similarly the opsonized O1:K89, O2:K89 and O78:K89 increased when PBMCs was infected with opsonized APEC (wild mutant strains were significantly (P < 0.05) expressed higher and mutant) strains compared to non-opsonized strains, while in IL-1β, while the opsonized O24:K89 mutant strain was signifi- non-pathogenic (wild and mutant) strain the opsonization signifi- cantly (P < 0.05) expressed lower IL-1β (Fig. 2B). cantly (P < 0.05) decreased IL-8 expression (Fig. 3B). IL-8 expression following stimulation IL-18 expression following stimulation IL-8 is a chemokine involved in the recruitment of PBMCs to the IL-18 is critical in initiating an inflammatory response and involved site of infection. In this study all wild type and the isogenics in determining resistance or susceptibility to bacterial infection. fimH mutants strains were increased IL-8 expression compared The wild type APEC O1:K89, O2:K89 and O78:K89 strains signifi- FIG. 1 - TLRs mRNA expression in chicken peripheral blood mononuclear cells (PBMCs). Lane 1: DL2000 Marker, lane 2: Beta actin, lane 3: TLR1/6/10, lane 4: TLR2 type 1, lane 5: TLR2 type 2, lane 6: TLR3, lane 7: TLR4, lane 8: TLR5, lane 9: TLR7. 590 H.H. MUSA et al. FIG. 2 - IL-1β mRNA expression in chicken peripheral blood mononuclear cells (PBMCs) stimulated with avian pathogenic Escherichia coli and avian non pathogenic E. coli and their isogenic fimH mutants. A: compared the relative ratio of IL-1β/beta actin (%) between control PBMCs, APEC and isogenic fimH mutants. B: compared the relative ratio of IL-1β/beta actin (%) between opsonized and non-opsonized strains FIG. 3 - IL-8 mRNA expression in chicken peripheral blood mononuclear cells (PBMCs) stimulated with avian pathogenic Escherichia coli and avian non pathogenic E. coli and their isogenic fimH mutants. A: compared the relative ratio of IL-8/beta actin (%) between control PBMCs, APEC and isogenic fimH mutants. B: compared the relative ratio of IL-8/beta actin (%) between opsonized and non-opsonized strains. FIG. 4 - IL-18 mRNA expression in chicken peripheral blood mononuclear cells (PBMCs) stimulated with avian pathogenic Escherichia coli and avian non pathogenic E. coli and their isogenic fimH mutants. A: compared the relative ratio of IL-18/beta actin (%) between control PBMCs, APEC and isogenic fimH mutants. B: compared the relative ratio of IL-18/beta actin (%) between opsonized and non-opsonized strains. cantly (P < 0.05) expressed higher IL-18 compared to the isogen- TGF-β4 expression following stimulation ic fimH mutants (Fig. 4A).The opsonized APEC O1:K89, O2:K89 The wild type pathogenic and non-pathogenic strains were sig- and O78:K89 wild strains and the opsonized APEC O2:K89 and nificantly (P < 0.05) increased the expression of TGF-4 compared O78:K89 mutant strains significantly (P < 0.05) expressed higher to the isogenics fimH mutant strains (Fig. 5A). The opsonized IL-18 compared to non opsonized strains, whereas the opsonized APEC (wild and mutant) O1:K89 and O2:K89 strains and non- non pathogenic (wild and mutant) O24:K89 strain significantly (P pathogenic (wild and mutant) O24:K89 strains were significantly < 0.05) expressed lower IL-18 (Fig. 4B). (P < 0.05) expressed lower TGF-β4 compared to non-opsonized Ann. Microbiol., 59 (3), 587-592 (2009) 591 FIG. 5 - TGF-β4 mRNA expression in chicken peripheral blood mononuclear cells (PBMCs) stimulated with avian pathogenic Escherichia coli and avian non pathogenic E. coli and their isogenic fimH mutants. A: compared the relative ratio of TGF-β4/beta actin (%) between control PBMCs, APEC and isogenic fimH mutants. B: compared the relative ratio of TGF-β4/beta actin (%) between opsonized and non-opsonized strains. strains. The opsonized pathogenic (wild and mutant) O78:K89 specific mechanisms of the cellular immune response (Moser et strain expressed significantly (P < 0.05) higher TGF-β4 compared al., 2004). Kogut et al. (2005a) reported that the bacterial TLR to non-opsonized strains (Fig. 5B). agonists (peptidoglycan, lipopolysaccharide and flagellin) induced up-regulation of IL-1β, IL-6 and IL-8 mRNA when compared to the non-stimulated controls. Avian Influenza Virus infection to DISCUSSION PBMCs from chickens induced nearly 20-fold IL-1 β, whereas, from ducks IL-1 β was strongly suppressed (Adams et al., 2008). The interacting cellular and molecular pathways of inflam- In addition, Swaggerty et al. (2004) indicated that heterophils mation and immunity were evolved to protect the host from from Salmonella enteritidis-resistant chickens had significantly pathogens (Kogut, 2000). Both inflammatory responses and higher levels of IL-6, IL-8 and IL-18 mRNA expression, and lower specific immune responses to invasive microbes are controlled level of TGF-β4 upon treatment with all agonists compared to by a complex network of cytokines (Fresno et al., 1997). As heterophils from SE-susceptible lines. In neonatal chickens, an regulators of the initiation and maintenance of host defenses, IL-8-like chemokine is involved in heterophils recruitment to the cytokines ultimately determine the type of response generated site of infection in neonatal chickens following an intraperitoneal and the effector mechanisms generated to mediate resistance challenge with SE (Kaiser et al., 2000). Therefore, the increase (Kogut, 2000). In this study APEC strains and their isogenic fimH in IL-1β, IL-8 and IL-18 expression by immune cells is associated mutants were used to stimulate the immune response of PBMCs. with increased resistance to extraintestinal pathogen infections in The highly pathogenic E. coli have virulence factors that cannot neonatal chickens (Swaggerty et al., 2004). In the present study be found in commensal strains or strains with lower pathogenc- the opsonized pathogenic strains stimulated significantly (P < ity. Pourbakhsh et al. (1997) demonstrated that expression of F1 0.05) higher cytokines expression compared to non-opsonized. fimbriae lead to rapid bacterial killing by chicken macrophages in In contrast, the opsonized non-pathogenic strain decreased the the in vitro assay. Most studies examine the cellular expression level of cytokines expression. Mellata et al. (2003) indicated that of TLRs on immune cells have focused on neutrophils, monocytes the opsonized APEC was highly associated with heterophills when and dendritic cells, but there is little evidence of TLRs being compared with non-opsonized bacteria. Heterophils exposed to expressed on lymphocytes (Dasari et al., 2005). Our results opsonized and non-opsonized SE enhance both the innate and showed that PBMCs was expressed TLR1/6/10, TLR3, TLR4, TLR5 acquired immune response, by the increased transcription of both and TLR7. Iqbal et al. (2005) reported that the peripheral blood pro-inflammatory and Th 1 cytokine genes (Kogut et al., 2005b). monocyte-derived macrophages expressed considerable levels The expression of IL-8 and IL-18 cytokines was significantly higher of TLR4 and TLR1/6/10, moderate levels of TLR2 type2, low but in the opsonized SE compared to non-opsonized SE (Swaggerty et detectable levels of TLR5 and TLR7 and TLR3 or TLR2 type1 mes- al., 2004). The susceptibility of E. coli isolates to phagocytosis by sage. The patterns of chicken TLRs mRNA expression in immune PBMCs in the absence of opsonins correlated with the degree of F1 cell subsets are broadly similar to those detected in mammalian fimbriation (Poubakhsh et al., 1997). Mellata et al. (2003) noted species (Zarember and Godowski, 2002). Activation of the TLR that in the absence of type 1 fimbriae bacteria was poorly associ- signal transduction pathway leads to the induction of numerous ated with macrophages and opsonization had little effect on it. genes that function in host defence, including those for inflam- In conclusion, our finding demonstrated that PBMCs are matory cytokines, chemokines, antigen presenting molecules expressed Toll-like receptors, and after activation with pathogen and costimulatory molecules (Harris et al., 2006). increased significantly the levels IL-1β, IL-8, IL-18 and TGF-β4 FimH through specific ligand-receptor interactions can trigger expression. Therefore, PBMCs are important in APEC infection, various responses in cells involved in host defenses such as mac- and the increase in cytokines may play important role in the rophage, lymphocytes B and neutrophils (Arne et al., 2000). In regulation of immune response. the present study the expression of IL-1β, IL-8, IL-18 and TGF-β4 were significantly (P < 0.05) increased in response to wild type Acknowledgements and fimH mutant of avian pathogenic E. coli. The production of This study was supported by grants from China Postdoctoral chemokines stimulates migration of lymphocytes to the site of Science Foundation (No. 20070420201), the Chinese National inflammation and switches initial immune reactions to the antigen- Science Foundation Grant (No. 30571374 and No. 30771603), 592 H.H. MUSA et al. the Chinese National Science Grant 863 projects (No. Kaiser P., Rothwell L., Galyovl E.E., Barrow P.A., Burnside 2007AA10Z357), Jiangsu High Education Key Basic Science J., Wigley P. (2000). 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Annals of Microbiology – Springer Journals
Published: Nov 20, 2009
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