Comparative analysis of the early transcriptome of Brucella abortus – Infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis
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.
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2012, Veterinary Immunology and ImmunopathologyCitation 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).