Comparative analysis of the early transcriptome of Brucella abortus – Infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis

https://doi.org/10.1016/j.rvsc.2010.09.002Get rights and content

Abstract

Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.

Introduction

Infectious diseases are usually controlled by traditional interventions such as antibiotics or vaccines. However, these interventions are not completely effective, as diseases persist in animal populations. Repeated observations over time in domestic livestock have demonstrated that clinical manifestations of infectious disease rarely occur in all members of the population exposed to the same pathogen under similar conditions. Genetic implications of these observations were initially ignored until association of natural resistance to pathogens with genetic markers in animal species, breeds or families was established (Carmichael, 1941, Cameron et al., 1942, Bumstead and Barrow, 1993, Xu et al., 1993). The genetic regulation of natural resistance to infectious disease is variable and usually complex, and includes both immune and non-immune mechanisms, although sometimes expression of an allele at one locus can significantly modify the disease pathogenesis in individuals (Adams and Templeton, 1998).

Brucellae are the etiological agents of brucellosis, a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health (Corbel, 1997). Among animal species, most mammals are susceptible to brucellosis. Bovine brucellosis is mainly caused by Brucella abortus which is clinically characterized by abortion and infertility in cows, and orchitis and inflammation of the accessory sex organs in bulls (Enright, 1990). Natural B. abortus infection in cattle occurs primarily through penetration of the mucosa membrane of the oropharynx followed by uptake by macrophages (MØ) and transport to the regional lymph nodes (Adams, 2002, Olsen et al., 2004). Successful initial establishment is due to the stealthy strategy employed by Brucella to modulate activation of the innate immune system, while persistent infection resides in the ability of the pathogen to modify trafficking to survive and replicate inside MØ by overcoming bactericidal mechanisms (Roop et al., 2004, Barquero-Calvo et al., 2007).

The presence of invading microbes is detected by sentinel cells such as MØ and dendritic cells (DC). After contact with the pathogen, sentinel cells secret a mixture of cytokines and process and link the exogenous antigen to MHC-II molecules to activate T-helper (Th0) cells in secondary lymphoid organs. According to the stimulus received, Th0 cells differentiate into Th1 and Th2 subsets, which polarize the immune response (Salyers and Whitt, 2002). Th1 subset of cells develop in response of Th0 to IL-12, inducing a Th1-oriented immune response, mostly involved in protection against intracellular pathogens through cell-mediated immunity and characterized preferentially by secretion of interferon-gamma (IFN-γ) and interleukin 2 (IL-2) cytokines. On the other hand, sentinel cells that secrete IL-4 induce a Th2 subset of cells development and a Th2-oriented immune response. Th2 immunity is characterized by secretion of IL-4, IL-5, IL-10 and IL-13 and is mainly responsible for protection against extracellular pathogens by mediating antibody production (Tizard, 2004).

Previous studies have reported that Th1 immune response is particularly involved in host protection against Brucella infection through cell-mediated immunity (Oliveira et al., 2002). When Brucella invade naïve hosts non-activated professional phagocytes uptake the pathogen and release interleukin-12 (IL-12). Subsequently, IL-12 induce Th0 cells to differentiate into IFNγ-secreting Th1 cells that are capable of activating MØ for increased anti-microbial mechanisms, and thus promote clearance of the bacteria (Zaitseva et al., 1995, Dornand et al., 2002). However, virulent Brucella have developed active strategies to interfere with innate immunity and consequently avoid being eliminated. For instance, Brucella impair apoptosis in human MØ (Gross et al., 2000, Fernández-Prada et al., 2003) and inhibit or delay dendritic cells maturation and antigen presentation (Billard et al., 2008). Moreover, Brucella alter the production and secretion of cytokines of infected host cells (Caron et al., 1994), modify the intracellular trafficking (Rittig et al., 2003), inhibit degranulation of neutrophils (Bertram et al., 1986, Orduna et al., 1991), and impair NK cell activity (Salmerón et al., 1992).

Previously, our laboratory identified cattle naturally resistant (R) and susceptible (S) to B. abortus infection (Harmon et al., 1985, Templeton et al., 1988). In these studies, the R cattle developed low transient serologic titers and were negative for Brucella isolation, while S infected cows developed high titers, aborted and Brucella was isolated from secretions. Later experiments focused on innate immunity found that mammary gland MØ from R cows produced significantly higher oxidative burst activity and had significantly greater in vitro bacteriostatic activity than MØ from S cows, when both were stimulated with opsonized B. abortus (Harmon et al., 1989). Furthermore, B. abortus were demonstrated to bind differentially to the peripheral blood monocyte-derived MØ (MDMs) from R or S cattle and also the cells from R animals were significantly superior in their ability to control the in vitro intracellular replication of B. abortus than those derived from S cattle (Price et al., 1990, Campbell and Adams, 1992, Campbell et al., 1994, Qureshi et al., 1996). These findings further substantiate the importance of the mononuclear phagocyte system in natural resistance to bovine brucellosis. In order to associate natural resistance with genetic markers, later studies identified the bovine SLC11A1 gene (formerly NRAMP1) as one of the major elements in controlling of intracellular replication of B. abortus in MØ (Feng et al., 1996, Adams and Templeton, 1998, Barthel et al., 2001). To better understand the differences in the phenotype and identifying novel cattle candidate genes and pathways involved in innate resistance to brucellosis, we characterized the expression profile of B. abortus-infected MDMs from naïve cattle naturally R or S to brucellosis using a cDNA microarray technology. In concordance with previous knowledge, our results demonstrated that R MDMs were superior controlling B. abortus infection due to the ability to polarize an immune response toward Th1, while the innate immune system of S MDMs failed to generate appropriate signals to mount an effective immune response against invading bacteria.

Section snippets

Bacterial strain, media and culture conditions

The smooth virulent Brucella abortus S2308 (gift of Dr. Billy Devoe, USDA, Agricultural Research Service, National Animal Disease Center, Ames, IA) was maintained as frozen glycerol stocks and re-suspended in fresh complete RPMI (C-RPMI) 1640 medium (RPMI 1640 medium supplemented with 4 mM l-glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate and 2.9 mM 7.5% sodium bicarbonate) (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (HI-FBS) (American Type

Invasion and intracellular growth of B. abortus S2308 revealed different patterns in MDMs from R and S cattle

At T0, the CFU of Brucella recovered from wells with R MDMs was significantly lower (P < 0.05) than those from wells with S MDMs. At 12 h p.i. (T12), the number of intracellular B. abortus was 18% lower in MDMs from R animal, and 27% higher in MDMs from S animal, compared to their own T0 value (in both cases P < 0.05) (Fig. 1). The number of B. abortus S2308 CFU present in growth control wells increased more than 1 log in the first 12 h p.i. compared with the original inoculum and the number of

Discussion

Our initial results indicate that B. abortus attach and internalize less efficiently in R than S MDMs. This result is in line with those obtained by Campbell et al. (1994), who found that MDMs from R cattle were less permissive to invasion by B. abortus 2308 than MDMs from S animals. These authors also found that the pathogen was bound to different surface’s molecules on R or S MDMs, influencing the intracellular fate of Brucella. It is well known that Brucella is an intracellular pathogen that

Conflict of interest

Authors declare no conflict of interest.

Acknowledgements

Mr. Alan Patranella for taking care of the animals and blood recollection, and Mrs. Roberta Pugh and Mrs. Doris Hunter for their technical assistance. This study was supported by US Department of Homeland Security – National Center of Excellence for Foreign Animal and Zoonotic Disease (FAZD) Defense Grant ONR-N00014-04-1-0 and a NIH grant 2U54AI057156-06. CAR was sponsored by Fulbright-INTA scholarship from Argentina.

References (77)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method

    Methods

    (2001)
  • C. Lundin et al.

    RAD51 is involved in repair of damage associated with DNA replication in mammalian cells

    Journal of Molecular Biology

    (2003)
  • R. Manetti et al.

    Interleukin-I favors the in vitro development of type 2 T helper (Th2) human T-cell clones

    Research in Immunology

    (1994)
  • R.A. Meter et al.

    Secretion of monocyte chemotactic protein-1 by human uterine epithelium directs monocyte migration in culture

    Fertility and Sterility

    (2005)
  • B. Mosley et al.

    Dual oncostatin M (OSM) receptors

    Journal of Biological Chemistry

    (1996)
  • D. Novick et al.

    Interleukin-18 binding protein: A novel modulator of the Th1 cytokine response

    Immunity

    (1999)
  • T. Qureshi et al.

    Intracellular survival of Brucella abortus, Mycobacterium bovis BCG, Salmonella dublin and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus

    Veterinary Immunology and Immunopathology

    (1996)
  • N. Utku et al.

    Prevention of acute allograft rejection by antibody targeting of TIRC7, a novel T cell membrane protein

    Immunity

    (1998)
  • T. Warger et al.

    Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses

    Journal of Biological Chemistry

    (2006)
  • L.G. Adams et al.

    Genetic resistance to bacterial diseases of animals

    Revue Scientifique et Technique (International Office of Epizootic)

    (1998)
  • N.M. Ampel et al.

    Resistance to infection in murine B-thalassemia

    Infection and Immunity

    (1989)
  • B.M. Babior

    The respiratory burst of phagocytes

    Journal of Clinical Investigation

    (1984)
  • C.L. Baldwin et al.

    Host immune responses to the intracellular bacteria Brucella: does the bacteria instruct the host to facilitate chronic infection?

    Critical Review in Microbiology

    (2006)
  • E. Barquero-Calvo et al.

    Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection

    Plos One

    (2007)
  • R. Barthel et al.

    Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival

    Infection and Immunity

    (2001)
  • T.A. Bertram et al.

    Preferential inhibition of primary granule release from bovine neutrophils by a Brucella abortus extract

    Infection and Immunity

    (1986)
  • E. Billard et al.

    Vir B type IV secretory system does not contribute to Brucella suis avoidance of human dendritic cell maturation

    FEMS Immunology and Medical Microbiology

    (2008)
  • N. Bumstead et al.

    Resistance to Salmonella gallinarum, S. Pollurum and S. enteritidis in inbred lines of chickens

    Avian Disease

    (1993)
  • H.S. Cameron et al.

    Genetic resistance to brucellosis in swine

    Journal of Animal Science

    (1942)
  • E. Caron et al.

    Live Brucella spp. fail to induce tumor necrosis factor alpha excretion upon infection of U937-derived phagocytes

    Infection and Immunity

    (1994)
  • D.J. Comerci et al.

    Brucella abortus synthesizes phophatidilcoline from coline provided by the host

    Journal of Bacteriology

    (2006)
  • M.J. Corbel

    Brucellosis: an overview

    Emerging Infectious Disease

    (1997)
  • T.F. Deuel et al.

    Platelet factor 4 is chemotactic for neutrophils and monocytes

    Proceedings of the National Academy of Sciences of the United States of America

    (1981)
  • B.G. Dorner et al.

    MIP-1alpha, MIP-1 beta, RANTES, and ATAC/lymphotactin function together with IFN gamma as type 1 cytokines

    Proceedings of the National Academy of Sciences of the United States of America

    (2002)
  • F.M. Enright

    The pathogenesis and pathobiology of Brucella infection in domestic animals

  • L. Eskra et al.

    Microarray analysis of mRNA levels from RAW264.7 macrophages infected with Brucella abortus

    Infection and Immunity

    (2003)
  • E. Everts et al.

    A 7872 cDNA microarray and its use in bovine functional genomics

    Veterinary Immunology and Immunopathology

    (2005)
  • J. Feng et al.

    Bovine natural resistance associated macrophage protein 1 (Nramp1) gene

    Genome Research

    (1996)
  • Cited by (22)

    • Brucellosis vaccines for livestock

      2016, Veterinary Immunology and Immunopathology
    • Polymorphisms at the 3' untranslated region of SLC11A1 gene are associated with protection to Brucella infection in goats

      2014, Veterinary Immunology and Immunopathology
      Citation Excerpt :

      It is well known that Th1 cellular immune response is effective to promote Brucella clearance from the host (Dornand et al., 2002; Oliveira et al., 2002; Rolán and Tsolis, 2008) while Th2 humoral immune response is detrimental for controlling B. abortus infection (Fernández and Baldwin, 1995). In agreement with these observations, Rossetti et al. (2011) showed that resistant animal to B. abortus infection had the ability to mount a Th1 immune response against this pathogen that was impaired in susceptible animal. In the same direction, Harmon et al. (1985) demonstrated that after an experimental challenge with B. abortus, cattle resistant to B. abortus infection developed low transient serologic titers and were negative for Brucella isolation, while susceptible infected cows developed high serologic titers, aborted and Brucella was isolated from secretions.

    • Monocyte-derived macrophages from Zebu (Bos taurus indicus) are more efficient to control Brucella abortus intracellular survival than macrophages from European cattle (Bos taurus taurus)

      2013, Veterinary Immunology and Immunopathology
      Citation Excerpt :

      Thus, bactericidal activity of macrophages is a surrogate of natural resistance against bovine brucellosis (Qureshi et al., 1996; Price et al., 1990; Martínez et al., 2010). Indeed, the ability of bovine macrophages to control B. abortus intracellular replication in vitro has been demonstrated to be a reliable phenotypic marker of natural resistance against brucellosis (Harmon et al., 1989; Price et al., 1990; Campbell and Adams, 1992; Campbell et al., 1994; Qureshi et al., 1996; Rossetti et al., 2011). However there is no evidence that natural resistance to brucelose linked to bovine breeds.

    • Characterization of Brucella abortus infection of bovine monocyte-derived dendritic cells

      2012, Veterinary Immunology and Immunopathology
      Citation Excerpt :

      Resistance to B. abortus in cattle appears to be hereditary and may be attributed to variations in the gene NRAMP (natural resistance associated macrophage protein) (Price et al., 1990). Macrophages from resistant cattle bind B. abortus less efficiently, internalize fewer bacteria and are less permissive for intracellular growth of B. abortus (Price et al., 1990; Rossetti et al., 2011). Findings from one study suggest that it may be unlikely for the Holsteins in this study to be of the susceptible genotype, as the susceptible genotype was only found in Zebu breeds (Martinez et al., 2008).

    View all citing articles on Scopus
    View full text