Download Host immune responses during Brucella infection

pISSN 2287-7991, eISSN 2287-8009
J. Prev. Vet. Med. Vol. 38, No. 1: 26-34, March 2014
http://dx.doi.org/10.13041/jpvm.2014.38.1.26
Host immune responses during Brucella infection :
A brief review
Kyung Yong Sung, Han Sang Yoo
Department of Infectious Diseases, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea
Abstract: Brucellosis is a zoonotic infectious disease of domestic animals, wild animals and humans. Innate immunity is a
rapid and non-specific immune response that occurs during the early stages of Brucella invasion. Physical barriers such as
epithelial cells and gastric juice secretions form the first line of defense. Humoral components such as complement and lysozyme
can remove microorganisms by opsonization and bactericidal actions. Cellular components of the immune system, including
macrophages, dendritic cells, neutrophils and innate T cells, have major roles in innate immunity. They recognize invading
Brucella spp. by various cell surface receptors and then kill both the invading microorganisms and infected cells owing to
their phagocytic or cytotoxic activity. In addition, they present Brucella antigens or produce cytokines to trigger adaptive
immunity. Activated adaptive immunity consists of T helper cells, cytotoxic T cells and antigen-specific antibody-producing B
cells. These can eliminate Brucella spp. effectively via antigen-specific mechanisms and by immunological memory. T cells
activate bactericidal functions in macrophages by producing cytokines such as IFN-γ and by exerting cytotoxic effects on the
infected cells. B cells produce antigen-specific antibodies that neutralize or opsonize the antigen. Because Brucella spp. can
survive in macrophages and other host cells, Th-1 cellular immunity that enhances the bactericidal effects of phagocytic cells
and the cytotoxic effects of lymphocytes is more important than humoral immunity in Brucella infection.
Key words: Brucella spp., innate immunity, adaptive immunity, cytokines, lymphocytes
INTRODUCTION
Brucellosis is a zoonotic infectious disease of domestic
animals, wild animals, and humans. It was first identified
in Malta by David Bruce in the 1860s and became known
as Malta fever [3]. The causative organisms Brucella spp.
are small, gram‐negative, aerobic, facultative intracellular,
coccobacilli. They can invade, multiply, and survive for long
periods within host cells [53, 59]. Unlike other pathogenic
bacteria, Brucella spp. do not have well‐known bacterial
virulence factors such as exotoxins, cytolysins, capsules,
fimbriae or plasmids [16, 58]. The genus Brucella consists
of 10 species that are differentiated on the basis of antigen
variation and their primary hosts: Brucella abortus (cattle),
B. melitensis (sheep and goats), B. ovis (sheep), B. suis (pigs),
B. canis (dogs), B. neotomae (desert wood rats), B. ceti
(cetaceans), B. pinnipedialis (seals), B. microti (common
voles) and B. inopinata [6, 71].
Brucellosis is transmitted via the ingestion or inhalation
Received 15 February 2014, Revised 17 March 2014, Accepted 24 March, 2014
Corresponding Author. Han Sang Yoo, Phone: +82-2-880-1263, Fax: +82-2-874-2738,
E-mail: [email protected]
Copyright © 2013 The Korean Society of Preventive Veterinary Medicine.
The full text is freely available on the web at http://www.jpvm.kr/.
of organisms from an infected animal. The organism can
penetrate mucosal epithelium in the gastrointestinal and
respiratory tracts and then spread via macrophages, further
multiplying in the lymph nodes, spleen, liver, bone marrow,
mammary glands and sex organs [53, 59, 71]. Bovine bru‐
cellosis caused by B. abortus is the most widespread form of the infection, and it causes devastating economic effects
on livestock production due to abortion and infertility [2].
B. melitensis, B. abortus, and B. suis are the major zoonotic
pathogens of human brucellosis, causing various clinical
signs such as undulant fever, endocarditis, meningitis, arth‐
ritis and osteomyelitis [71]. Direct contact with infected
animals and the consumption of unpasteurized dairy pro‐
ducts obtained from infected animals are major causes of
human brucellosis [33].
The control of brucellosis in livestock is crucial for the
prevention of human brucellosis. An ideal control program should consist of prevention, monitoring, elimination of
infected animals via testing and slaughter programs, and
vaccination. The general diagnosis for brucellosis is based
on serological tests in both animals and humans [43].
The host immune response can be functionally classified
into innate and adaptive immunity. The innate immune
Host responses in immune cells of Brucella
response is the first line of defense against invading patho‐
gens. Its elements include physical barriers (skin and
internal epithelial layers), humoral components (various
chemokines, complement system and opsonins) and cellular
components such as phagocytes (neutrophils, monocytes,
macrophages and dendritic cells) and innate lymphocyte
subsets (natural killer cells and γδ T cells) [36, 41, 44, 56].
Adaptive immunity can be classified into cell‐mediated
immunity and humoral immunity. T lymphocytes play a
major role in cell‐mediated immunity via cytokine produc‐
tion and by exerting cytotoxic effects. On the other hand,
antibody‐producing B lymphocytes are the major cells in
adaptive humoral immunity [56].
Because Brucella spp. have the ability to survive and
replicate within host cells, especially macrophages, host pro‐
tection against Brucella spp. is dependent on activated
antigen‐presenting cells (APCs) in innate immunity and on
activated T helper cells and cytotoxic T cells in adaptive
immunity, to remove the organisms and infected cells [4,
65].
INNATE IMMUNITY
Innate immunity is the rapid, non‐specific, and non‐me‐
mory immune response against invading pathogens. It
consists of physical barriers at the surface of the body,
humoral components such as complement proteins, and
cellular components that include macrophages, dendritic
cells (DCs), granulocytes (basophils, eosinophils, and neu‐
trophils) and natural killer (NK) cells [17].
PHYSICAL BARRIERS
Epithelial cells, the physical barrier that provides the
first line of defense, is located at the mucosal surfaces of
the intestine, genitourinary and respiratory tracts of the
host. Intestinal epithelial cells not only block invading
enteric pathogens but also trigger immune responses by
professional immune cells. Epithelial cells express receptors
of the innate immune system and can recognize microbial
pathogens and subsequently produce proinflammatory me‐
diators [1]. Brucella spp. induces only a weak proinflamma‐
tory response in intestinal epithelial cells but produces a
significant response via chemokine (C‐C motif) ligand 20
(CCL20) [37].
Gastric juices of the intestinal cavity expose enteric Bru‐
27
cella spp. to an extreme environment of low pH and digestive
enzymes [15]. Microfold cells (M cells) are found in the
follicle‐associated epithelium of Peyer’s patches and the
bronchus‐associated lymphoid tissue. They transport inva‐
ding Brucella spp. from the lumen to the immune cells
beneath the epithelial cells to stimulate innate immune
cells by phagocytosis [55].
CELLULAR COMPONENTS
Cellular components of the immune system, including
macrophages, DCs, neutrophils, and innate T cells, have major
roles in innate immunity [5, 61, 69, 73]. They recognize
invading Brucella spp. and kill these invading microorga‐
nisms or the infected host cells by phagocytic or cytotoxic
activity [4, 53, 73]. In addition, they can induce an adaptive
immune response through antigen presentation to adaptive
immune cells and by cytokine production [4, 69, 73].
ANTIGEN-PRESENTING CELLS
Macrophages and DCs are major APCs in the innate
immune response to Brucella infection. Both cell types
have various inducible mechanisms to eliminate bacteria
(Table 1). APCs are the first cells that react to invading
microbes and are responsible for induction of the adaptive
immune response by the presentation of antigen epitopes
to T helper (Th) cells. Pathogen recognition is achieved
by pattern recognition receptors (PRRs) expressed by the
APCs that recognize pathogen‐associated molecular patterns
(PAMPs) of the invading microbes. Major PAMPs of Brucella,
including bacterial lipopolysaccharide (LPS), DNA and lipo‐
protein, are recognized by PRRs of APCs. PRRs consist of
Toll‐like receptor 4 (TLR4), TLR9 and TLR2 [32, 57]. Re‐
cently, TLR6 was found to be essential to trigger the innate
immune response against B. abortus in vivo (Fig. 1) [13].
Binding of PRRs with their target PAMPs activates prin‐
cipal transcription factors such as nuclear factor (NF)‐κB,
activator protein (AP)‐1, and interferon (IFN) regulatory
factor (IRF) 3/7. As a result, APCs produce several cytokines
such as tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β,
IL‐12 and IL‐6, as well as express costimulatory molecules
such as cluster of differentiation (CD)80 and CD86, which
form part of the innate and adaptive immune system (Fig.
1) [7, 14, 45].
IL‐12 plays a critical role in downstream events, including
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KY Sung, HS Yoo
Table 1. Basic mechanisms of action of antigen-presenting
cells against Brucella
Effect mechanism
Mode of action
Phagocytosis and
autophagy
Degradation by hydrolytic enzymes
of phagolysosomes/autolysosomes
Antimicrobial cationic
peptides (defensins)
Direct killing
Oxidative burst
Direct killing by ROS
Cytokine production
TNF-α
Strong enhancement of bactericidal
activity of phagocytes
IL-12
Priming Th1 immune response, leading to production of IFN-γ
Chemokine secretion
(MCP-1, RANTES,
MIP1a/MIP1b)
Migration immune cells and maintenance of inflammation to limit
infection
Antigen presenting
Priming of specific immune response, leading to IFN-γ production and
cytotoxicity
CTL : cytotoxic lymphocyte.
MIP : microphage inflammatory protein.
MCP : monocyte chemoattracted protein.
ROS : reactive oxygen species.
RANTES : regulated and normal T cell expressed and secreted.
Source: Skendros & Boura (2013) [64].
the activation of innate immune cells and T and B cells,
which then differentiate into antigen‐specific effector cells
(Fig. 2) [48, 54]. APCs can process phagocytosed bacteria
into peptides that can be loaded into major histocompati‐
bility complex (MHC) class I and II molecules located on
the cell surface. The MHC‐peptide complexes stimulate T
NF : nucleus factor
IRAK : interluekine 1 receptor associated kinase
IGRE : interferon response elements
IRF : interleukin related factor
MyD88, TRAP, TRA, TRAM, TRIF : TLR-signaling adapt or proteins
Fig. 1. Overview of innate immunity signaling pathways in Brucella infection. Source: de Almeida LA et al. (2013) [13].
cells via T cell receptors (TCRs) and the costimulatory mole‐
cules B7.1/2 that trigger CD28 on the T cells (Fig. 3) [36].
In addition to antigen‐presenting functions, macrophages
can kill invading bacteria via phagocytic activity. Stimulating
macrophages with microbial products, IFN‐γ and TNF‐α
trigger multiple antimicrobial activities, including the pro‐
duction of reactive oxygen intermediates (ROIs) and reac‐
tive nitrogen intermediates (RNIs) [40, 41]. Both ROIs and
RNIs play a role in controlling Brucella infection in the
early stages [43]. The importance of the Th1 cytokine IFN‐γ
in the activation of macrophages and the limitation of
Fig. 2. Differentiation of immune systems in Brucella infection. Brucella triggers APCs to release IL-12, which causes Th0 cells to
differentiate into Th1 cells which secrete IFN-γ or IL-2. The Th1 cytokines enhance anti-Brucella mechanism of macrophage or induce
the CD8+ cytotoxicity. The Th2 response activates B cell for antibody production, facilitating the phagocytosis of Brucella through opsonization. The Th2 cytokines inhibit the action of Th1 cytokines. Source : Goldings et al. (2001) [36].
Host responses in immune cells of Brucella
29
brucellosis [42, 67].
Activated γδ T cells inhibit the growth of Brucella spp.
in macrophages through a combination of mechanisms that
include granule‐ and Fas ligand‐mediated cytotoxicity, mac‐
rophage activation via IFN‐γ production, and secretion of
the potent bactericidal factors granulysin and cathelicidin
[18, 51]. NK cells activated by IL‐12 and TNF‐α that are
released by infected macrophages kill the Brucella‐infected
cells through their cytotoxic effects. Additionally, NK cells
have a role in the regulation of the antibody response to
Brucella spp. [5. 29].
Fig. 3. Diagram of the interactions between antigen presenting
cell and Th cell. APCs deliver at least two signals to Th cells.
One signal is via MHC-peptide on the APC that activates the TCR.
The other is mediated by B7 molecules on the APCs which interact with CD28 on Th cells. Source : Goldings et al. (2001) [36].
Brucella infections has been proved both in vitro and in
vivo [70]. Conversely, the Th2 cytokine IL‐10 can suppress
macrophage function and increase the susceptibility to
infection by Brucella spp. [23, 62].
Neutrophils
Neutrophils are rapidly recruited to the infection site
and can kill microbes by phagocytosis, extracellular release
of granule contents, cytokine secretion and the formation
of neutrophil extracellular traps [12]. Neutrophils can ingest
Brucella spp. by opsonization [74]. Brucella lipoproteins,
including lipidated outer membrane protein 19 (L‐Omp 19),
can activate human neutrophil functions such as oxidative
burst, neutrophil migration and neutrophil survival [77].
In contrast to macrophages, brucellae cannot replicate within
neutrophils, although it seems to resist neutrophil‐mediated
killing [8].
Innate lymphocytes
Innate lymphocytes, including NK cells, natural killer T
(NKT) cells and γδ T cells, are at the interface between
innate and adaptive immunity. Compared with antigen‐
specific T cells, these lymphocytes comprise a smaller
proportion of the blood cell population and recognize non‐
peptide antigens without MHC restrictions. The major role
of innate lymphocytes is to produce IFN‐γ before the ex‐
pansion of specific Th1 responses [44, 50]. γδ T cells show protective activity in the early stages of human and bovine
Humoral components
Complement is a systemic plasma protein with a variety
of functions that include opsonization by binding to anti‐
bodies or bacterial surfaces or direct killing of pathogens
by the formation of a membrane attack complex, causing
bacterial lysis [61, 69]. Both the classical pathway and the
lectin pathway of complement activation are involved in
complement deposition and complement‐mediated killing
of Brucella spp. Complement activity closely correlates to
antibody levels in the serum at the early stages of Brucella
infection. Later in the infection, complement is activated
by the binding of a mannose‐binding lectin to carbohyd‐
rates found on the surface of Brucella spp., rather than by
antibody binding. However, at the later stages of Brucella
infections, the increased concentration of immunoglobulin
causes prozone effects with complement and cannot kill
extracellular Brucella spp. [27, 39]. The interaction between
Brucella spp. and complement is mediated by LPS, which
is a major cell surface component of the organism. Because
B. abortus LPS protects the organism from complement
attacks, smooth strains of B. abortus are more resistant to
complement than are LPS‐deficient rough strains [21].
The lysosome plays an accessory role in the bactericidal
actions of antibodies and complement or directly acts through
bacteriolysis and opsonization. However, comparing antibody
titers of B. abortus‐positive bovine serum and bacteriolytic
power, the lytic activity of the lysosome slightly decreased
in the serum with high antibody titers. Therefore, the ly‐
sosome is not an essential protection factor in Brucella
infection [38].
ADAPTIVE IMMUNITY
Adaptive immunity is activated after the presentation
30
KY Sung, HS Yoo
of antigen epitopes by APCs and innate immunity. Its anti‐
gen‐specific effect and immunological memory can eliminate
pathogens rapidly and effectively. The adaptive immunity
consists of T helper cells, cytotoxic T cells and antigen‐speci‐
fic antibody‐producing B cells. T cells activate bactericidal
functions in macrophages by producing cytokines such as
IFN‐γ or by exerting cytotoxic effects on infected cells. B
cells produce antigen‐specific antibodies that neutralize or
opsonize the pathogen [36].
Cell-mediated immunity
+
The major immune cells in cellular immunity are CD4
T helper cells (Th) and CD8+ cytotoxic T cells (Tc). Following
activation by APCs, naïve Th0 cells differentiate into Th1
and Th2 subsets via stimulation by IL‐12 or IL‐4, respec‐
tively. Tc cells become cytotoxic lymphocytes. Th1 cells
primarily produce IFN‐γ and IL‐2 to activate cell‐mediated
immunity, where as Th2 cells mainly produce IL‐4, IL‐5
and IL‐10 to activate humoral immunity (Fig. 2).
Th cells help B cells in immunoglobulin production. The
Th1 response can activate IgG2 and IgG3 isotype switching,
whereas the Th2 response can promote IgG1 and IgE swit‐
ching [30]. These activities of T cells can limit or kill the
invading Brucella spp. both directly and indirectly [25, 75].
Th1 cells predominantly produce IFN‐γ, activating the
bactericidal activity of macrophages to limit Brucella infec‐
tion, and TNF‐α maximizes this activating function [11, 69,
76]. In addition, IFN‐γ promotes the expression of antigen
presentation and costimulatory molecules on APCs and
potentiates the apoptotic death of Brucella‐infected macro‐
phages [47, 73].
IL‐12 produced by Th cells and APCs induces a Th1 res‐
ponse to activate macrophages, whereas IL‐10 produced
by T and B cells suppresses IFN‐γ‐activated macrophage
function and increases susceptibility to infection [24, 43,
75]. B. abortus infection induces IL‐10 production to mo‐
dulate macrophage activity as a negative feedback control
of IL‐12 secretion [24, 26, 72].
+
Activated CD8 cytotoxic T lymphocytes (CTLs) can also
produce IFN‐γ and henumber of Brucella spp. and infected
macrophages through Fas‐ or perforin‐mediated cytotoxi‐
city [19, 52]. CTL‐mediated cytotoxicity can be activated
by IFN‐γ produced by other T lymphocytes [73]. Although
B. abortus can induce CTLs even in the absence of CD4 +
+
function, CD4 cells, especially Th1, are more important for
the control of Brucella infection than CD8 + cells [31, 60].
Interestingly, during chronic/relapsing brucellosis in both
murine models and human clinical cases, the Th1 response
decreased and CTL activity increased in a compensatory
manner [28, 66]. Other recent data from murine chronic
brucellosis models indicate that CTLs also decrease with
Th1 cytokines [20].
In addition to a primary role in innate immunity, poly‐
morphonuclear leukocytes (PMNs), including neutrophils,
may have roles in adaptive immunity. The absence of PMNs
in B. abortus‐infected neutropenic mice, named Genista,
displayed a more active adaptive immune response, reveal‐
ing the unexpected negative influence of PMNs on the
adaptive immune response [9, 46, 63].
Humoral immunity
B lymphocytes produce antigen‐specific antibodies th‐
rough direct stimulation with antigens and costimulation
by T cells and cytokines. However, B cells can produce anti‐
gen‐specific antibodies via direct stimulation with B. abortus
in the absence of T cell costimulation [36]. These antibo‐
dies can neutralize the antigens and act as opsonins that
facilitate the phagocytosis of bacteria. Additionally, antibo‐
dies can activate complement to bind the microbes and
promote antibody‐dependent cell‐mediated cytotoxicity
(ADCC) by macrophages, neutrohphils and NK cells [64].
Sera from B. abortus‐infected cattle generally contain
antibodies against the LPS that covers the outer surface of
the bacteria. Antibody isotype switching requires the colla‐
boration between B cells and Th cells. Because the Th1
immune response is predominant in Brucella infection, the
dominant IgG isotype is IgG2. The antibody response to B.
abortus in cattle consists of the early production of IgM,
and it almost immediately progresses to the production
of IgG2, and later produces small amounts of IgG1 and IgA
[22, 34].
Although passive transfer of B. abortus‐specific antibo‐
dies can protect mice from B. abortus infection [49], in
bovine brucellosis, humoral immunity only limits Brucella
spp. at the initial stage of infection and has little protective
effects on the intracellular course of infection [4, 10].
Moreover, the high concentration of IgG disturbs extra‐
cellular bactericidal effects mediated by complement and
enhances intracellular localization of bacteria, resulting in
the extension of brucellosis [39].
B cells also play an immunoregulatory role in brucellosis,
producing IL‐10 and transforming growth factor (TGF) β,
Host responses in immune cells of Brucella
which attenuates the IFN‐γ‐mediated Th1 response. In
addition, B. abortus expresses the virulence factor proline
racemase (PrpA) that induces IL‐10 secretion from B cells
[35, 68].
31
and Rural Affairs (iPET112012‐3) and Research Institute
of Veterinary Science, Seoul National University, Republic
of Korea.
REFERENCES
CONCLUSION
Brucellae are intracellular bacteria that cause brucellosis.
Physical barriers against Brucella infection include epithelial
cells in the skin, respiratory and intestinal tracts, and gas‐
tric juice secretions. After the brucellae pass these physical
barriers, the bacteria activate innate immunity. APCs, inclu‐
ding macrophages and DCs, can detect invading organisms
by several TLR signaling pathways and produce an array
of inflammatory cytokines that activate both the innate and
adaptive immune responses. In addition, the signal activates
macrophages to kill the invading brucellae via ROIs or RNIs.
Together with the macrophages, neutrophils and innate T
cells can reduce the infection by phagocytosis, cytotoxic
effects and the production of inflammatory cytokines prior
to the initiation of the adaptive immune response. Additionally,
the complement system can be activated via antibody‐
dependent or independent mechanisms for the opsonization
of Brucella spp.
The APCs phagocytose Brucella spp. to prepare antigen
epitopes for presentation to CD4 + cells and activate CD8 +
cells for adaptive immunity. The activated APCs then induce
differentiation of CD4 + cells to Th1 cells, result in cellular
immune responses by IL‐12 and Th2 cells, and humoral
immune response by IL‐4. The Th1 response leads to IFN‐γ
production, further enhancing the clearance of infected cells
and Brucella spp. by macrophages and CTLs. Antibodies
produced in response to Brucella infection mainly consist
of IgG2, the production of which is activated by the Th1
immune response. Although antigen‐specific antibodies limit
the number of extracellular Brucella spp. via enhancement
of complement activity at early stages, the humoral immune
response is not protective during the intracellular phase of
Brucella infection. Understanding the interactions between
Brucella spp. and the host immune system is crucial for
the development of preventive and diagnostic methods
against brucellosis.
ACKNOWLEDGEMENTS
This study was supported by Ministry of Agriculture, Food
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