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IN-DEPTH: RHODOCOCCUS EQUI
Immunity to Rhodococcus equi
M. Julia B. Felippe Flaminio, DVM, MS, PhD, Diplomate ACVIM
The development of disease involves pathogen, host, and environmental factors. When looking
specifically at the foal and its susceptibility to Rhodococcus equi infection and disease, age-dependent
limited function of the immune system and its naïve state at birth seem to be plausible explanations. Nevertheless, natural and experimental infections of the foal have been shown to induce
robust humoral and Th1-type immune responses superior or at least comparable with the adult horse,
which is considered protective. The challenge is that these types of responses require time and
effective signaling interactions between antigen-presenting cells, lymphocytes, phagocytes, and antibodies within a positive-feedback loop model. Perhaps some foals do require a longer warm-up
period of the immune system, and this time creates a window of greater susceptibility. A better
understanding of the pathogenic mechanisms in the early stages of infection in the airways could
bring new information to light about how the immune system of the foal coordinates these interactions for effective bacterial removal and killing before disease establishment and how this elaborates
into long-life immunity. Author’s address: College of Veterinary Medicine, Cornell University,
Ithaca, New York 14882; e-mail: [email protected]. © 2010 AAEP.
1.
Introduction
Foals are exposed to Rhodococcus equi in the first
few days to weeks of life.1,2 R. equi disease manifests before 4 – 6 mo of life and not in older foals and
adult horses, unless immunodeficiency is present.3,4
Several studies using experimental infection of foals
revealed that the respiratory tract is the most likely
route of exposure for infections resulting in pulmonary disease, whereas intragastric inoculation with
large numbers of organisms does not induce the
typical pulmonary lesions.5–7 During the establishment of infection, hypercellularity of the alveolar
septa develops, and the alveoli and terminal bronchioles become infiltrated with neutrophils, macrophages, giant cells, and numerous cell-associated
bacteria.8 Subsequently, pyogranulomatous inflammation and necrosis progress into the destruction of lung parenchyma. Experimental infections
NOTES
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of piglets and mice with doses greater than those
lethal to foals fail to produce pulmonary abscesses, unless immunosuppressive therapy is instituted.9 –11 Therefore, incompletely understood
host-specific natural conditions exclusive to the
foal are necessary for R. equi to establish disease.
The unique susceptibility of young foals to R. equi
disease is still puzzling, despite many studies investigating their innate and acquired immune systems.
Although age-dependent developmental limitations
of the immune system of the foal are suspected to be
involved, recent studies have shown that foals can
elaborate a Th1-type immunity in response to R.
equi infection. This is the type of protective immunity developed by adult horses on experimental infection. However, acquired immunity takes time to
establish, and this window of susceptibility could
favor pathogen replication to overwhelming levels
IN-DEPTH: RHODOCOCCUS EQUI
and tissue damage. Perhaps effective phagocytic
function could control pathogen expansion and disease development in the foal before acquired immunity is developed. However, the mechanisms of
appropriate activation of phagocytes against R. equi,
a key process to control infection in the airway environment, are poorly understood.
2.
The Pathogen
R. equi is a Gram-positive, obligate aerobic, facultative intracellular coccobacillus capable of multiplying
in macrophages, including alveolar macrophages.12
Of the 40 genera in the actinomycete group, Rhodococcus is placed among the mycolata taxon, along
with Mycobacterium, Corynebacterium, Nocardia,
Gordonia, Dietzia, Turicella, Tsukamurella, Williamsia, and Skermania.13 Mycolic acids are long
fatty acids found in the lipid-rich cell-wall envelope
of these bacteria, which form a protective barrier
that increases resistance to chemical damage, dehydration, oxidative stress, and low pH.14 Ellenberger
et al.15 suggested that a component of the R. equi
cell wall was capable of inhibiting bactericidal mechanisms of phagocytes and allowing intracellular survival. Surprisingly, recent studies by Sydor et al.16
have shown that the bacterial capsule of R. equi is
not an essential virulence factor. Nevertheless, R.
equi has evolved a mechanism to escape bactericidal
activity in macrophages. Inside macrophages,
maturation of phagosomes containing virulent R.
equi does not progress into a late endocytic organelle, which does not acidify and lacks the protonpumping vacuolar adenosine triphosphate (ATP)ase.12,17,18 The phagosome membrane remains
intact during the period of bacterial replication.
In foal alveolar macrophages, viable intracellular
bacteria were observed 24 h after infection.19 In
vitro macrophage infection with virulent R. equi revealed intracellular bacterial replication starting
beyond 6 –12 h in culture, reaching a five-fold increase in bacterial numbers by 48 h.20,21 R. equi
infection of macrophages is cytotoxic, and cell degeneration occurs because of necrosis.20
The virulent R. equi depends on the presence of a
large plasmid, which encodes a family of eight thermoregulated virulence-associated proteins (VapA
and VapC to VapI).22–24 This plasmid is critical for
intracellular replication within macrophages and
the development of disease in the foal.22,23,25 R.
equi depleted of the virulent plasmid replicates
poorly inside macrophages and does not induce disease.8,20 In vitro, R. equi binding to and invasion of
macrophages can be mediated by complement receptor CR3 (CD11b/CD18 or MAC-1), mannose receptor
(which binds lipoarabinomannan), and Toll-like receptor 2.4,12,26,27 When opsonized with specific antibodies, R. equi is internalized through Fc␥receptor on both neutrophils and macrophages, and
this mechanism increases phagolysosome formation
and bacterial killing.12
3.
The Innate Immune System
Early events in the pathogenicity and R. equi removal in the airways are poorly described, and most
of our understanding comes from studies in vitro
(using infected macrophages or neutrophils alone)
and mouse models. Phagocytes participate in the
early stages of immune response by removing and
killing pathogens and play a critical role in the protection of the immunologically naïve neonate.
Neutrophils are important cells of the innate immune system and inhabitants of the upper airways
of horses. They are the first cells to be recruited to
a site of infection in response to tumor necrosis
factor (TNF)␣ and interleukin (IL)-6 cytokines and
the chemokines IL-8, macrophage inflammatory
protein (MIP-2), and keratinocyte chemoattractant.28 Immunocompetent mice are resistant to intrabronchial challenge with R. equi; high doses of
inoculation result in slower but effective pulmonary
clearance, starting at 24 h with neutrophilic influx,
followed by macrophages at 48 –72 h, and peribronchial and perivascular mononuclear infiltrate.10
Indeed, using an anti-neutrophil monoclonal antibody, mice deficient in neutrophils developed more
severe disease and greater R. equi replication than
control mice.29 In contrast to macrophages, foal
neutrophils have been shown to be bactericidal to
the opsonized bacteria.12,30,31 Despite the presence
of large numbers of neutrophils in the airways in
response to R. equi infection, bacterial replication
and disease may still progress in the foal.32 In
contrast, adult horses have not shown a significant
increase in bronchoalveolar lavage neutrophils 7
days post-experimental infection.33 Opsonization
of bacteria with R. equi-specific antibodies is critical
for the efficient bacterial killing in neutrophils.12,31
In vitro, neutrophils of foals less than 7 days of life
revealed similar R. equi killing capacity in comparison to 35-day-old foals and adult horses, particularly in the presence of opsonic elements.29,30,31,34,35
Yet, some neonates in those studies showed decreased efficiency, which could partially explain
individual susceptibility to disease. Although
neutrophils are competent in the neonate, disease
process can alter their function.36,37 In addition,
R. equi preparations (pellet or supernatant fractions) have been shown to inhibit oxidative metabolism of neutrophils, including myeloperoxidasedependent peroxide production.12,15,30 Besides
bacterial removing and killing, neutrophils may
influence airway immunomodulation and the type
of adaptive immune response to R. equi, because
they serve as a source of many pro-inflammatory
cytokines and chemokines.38,39 Importantly, in
foals, the expression of certain pro-inflammatory
cytokines by neutrophils in response to R. equi is
influenced by age, which may limit important cellular signaling and activation during R. equi
infection.40
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IN-DEPTH: RHODOCOCCUS EQUI
R. equi evolved a mechanism to escape bactericidal activity in macrophages, which, paradoxically,
are important cells of the immune system that perform surveillance, removal, and killing of microorganisms. R. equi readily invades alveolar macrophages, multiplies, and causes their destruction,
favoring its replication and spreading in the respiratory tract.19 Virulent R. equi replicated efficiently in macrophages, starting beyond 6 –12 h of
culture and affecting macrophage viability by 48 h;
in contrast, bacterial numbers of avirulent (plasmidcured) R. equi decreased during the same period.20
Although macrophages fail to impede intracellular
bacterial replication, R. equi infection still induces
an immune response in macrophages characterized
by translocation of nuclear factor ␬B (NF-␬B) into
the nucleus and subsequent secretion of cytokines.41,42 Both virulent and avirulent R. equi
strains induce IL-1␤, IL-10, IL-12p40, and TNF␣
cytokine response in murine macrophages by 4 h of
infection.43 Opsonization of R. equi with antibody
against capsular components increases phagocytosis
and intracellular killing in foal alveolar macrophages.12 Macrophages also become activated
when cell-surface receptors (e.g., Toll-like receptors
[TLRs]) recognize highly conserved structural motifs
only expressed by microbial pathogens (so-called
pathogen-associated molecular patterns [PAMPS]).
Darrah et al.42 and Aderem and Ulevitch44 suggested that TLR-2 is involved in R. equi recognition,
similar to the response to Mycobacterium tuberculosis.45 In addition, macrophage activation is also
enhanced by cytokines, in particular interferon ␥
(IFN␥) and TNF␣.46,47 It is possible that macrophage activation before intracellular infection results in efficient R. equi killing. Kanaly et al.48,49
showed that mice treated with IFN␥ monoclonal
antibodies (to block IFN␥ activity) failed to clear
pulmonary infection. Therefore, this cytokine
plays an important role in resistance to R. equi
pneumonia. Indeed, macrophages from R. equi-exposed foals showed a 100% increase in their killing
capacity when co-cultured with autologous lymphocyte factors (pre-conditioned medium derived by in
vitro co-culture of lymphocytes and R. equi surface
antigens), whereas macrophages from non-exposed
foals showed only a 50% increase.12 The exposure
of macrophages to cytokines, importantly IFN␥, results in enhanced microbicidal activity through the
secretion of both reactive oxygen intermediates
(ROIs) and reactive nitrogen intermediates (RNIs).
Interferon regulatory factors (IRFs) augment the
production of ROIs, RNIs, and cytokines (e.g.,
TNF␣). Individually, ROIs and RNIs promote negligible microbicidal activity; in fact, hydrogen peroxide alone has been shown to induce the expression of
R. equi virulence-associated genes.50 However,
their combination (e.g., peroxynitrite ONOO⫺)
brings greater efficiency in killing of R. equi.51,52
Antigen-presenting cells including macrophages
and dendritic cells potentially direct the type of im134
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mune response (Th1 or Th2) on encounter with R.
equi. Similar to macrophages, dendritic cells encounter pathogens in the tissues through TLRs,
phagocytose, and process them for presentation to
effector cells belonging to the acquired immune system (lymphocytes) in the regional lymph nodes.
Ultimately, a Th1-type immune response with IFN␥
production and the development of cytotoxic T cells
(CTLs) specific for R. equi would have to derive from
antigen-presenting signals (i.e., IL-12 production) in
the initial stages of infection. Therefore, in immunocompetent individuals, R. equi induces a protective response that starts from the innate immune
system. The effect of R. equi on dendritic cells has
been poorly studied in vivo. In vitro, Darrah et
al.42 showed that purified R. equi virulence-associated protein VapA induced murine dendritic cell
maturation. Additionally, the newborn foal monocyte-derived dendritic cells have been shown to respond to R. equi infection comparably with adult
horse cells, with the expression of IL-12.53 Although TLR-mediated immune response to ligands
has been implicated in immature immunity in neonates, dendritic cells from the foal seem to recognize,
process, and respond to R. equi.54 One concern is
the age-dependent limited expression of major histocompatibility complex class II (MHC class II) molecules on the surface of foal dendritic cells, which
are used as markers of maturation and competence
for antigen presentation to lymphocytes.53,55,56
Although both macrophages and neutrophils patrol the airway surface to eradicate bacteria, recent
studies of lung inflammation have suggested that
airway epithelial cells are the first cell type to respond to environmental stimuli. Respiratory epithelial cells play a key role in the recruitment and
activation of inflammatory cells and perhaps, coordinate the type of inflammatory process.57 Consequently, airway epithelial cells not only form a
barrier for protection against inhaled pathogens and
toxins but also are an integral part of the innate
immune system for inflammatory response. Bronchial epithelial cells of the horse express TLR-2 and
can potentially recognize and bind to R. equi.58,59
Mathew et al.60 showed that NF-kB activation in
mouse tracheal epithelial cells generated signals
that contributed to activation of macrophages.
Bronchial epithelial cells incubated in the presence
of endotoxin revealed a significant increase in the
expression and production of TNF␣, IL-8, integrin
CD11b/CD18 (Mac-1), and the adhesion molecules
ICAM-1, which are important activation signals to
phagocytes.28 In addition, the presence of bacteria
directly induces mucin production by the airway
epithelial cells.61 Mucins are the key structural
components of mucus, and they form a barrier to
protect underlying cells from inhaled microorganisms and irritants. A wide variety of infectious and
non-infectious equine respiratory diseases are associated with increased volumes of respiratory secretions and a change in the physical and chemical
IN-DEPTH: RHODOCOCCUS EQUI
62,63
nature of the mucus.
Whereas the mucus gel
in the respiratory tract is vital for the protection and
normal function of the airways, the overproduction
and change in its composition significantly affects
the pathologic feature of these conditions. Indeed,
phagocytic function may be reduced in mucus secretions.64 Limited information is available about
these gel-forming glycoproteins in the horse airway
mucus, and much less is known about their role in R.
equi pneumonia. Our laboratory is currently examining how airway epithelial cells interact with
phagocytes and R. equi in a biologically relevant in
vitro three-dimensional culture system of equine
bronchial epithelium that fully differentiates into
ciliary beating and mucus-producing cells.65
4.
The Acquired Immune System—Cellular Immunity
Specific host factors that determine protection or
susceptibility in R. equi infection in foals have not
been fully identified. In mice, protective immune
response against R. equi is mediated by CD4⫹
T cells, and the role of CD8⫹ T cell is disputed.48,49,66,67 The clearance of R. equi in mice
depends mainly on IFN␥ production by lymphocytes,
whereas biased production of IL-4 (Th2-biased) increases risk of disease.48,49 Experimentally infected adult horses rapidly clear the organism
without developing disease; the immune response
includes an increase in both CD4⫹ and CD8⫹ T
lymphocyte counts in the bronchoalveolar lavage
fluid, an increase in the number of CD4⫹ and CD8⫹
T cells producing IFN␥, and a greater post-infection
proliferative response to R. equi antigen or recombinant VapA in vitro.15,33,68,69 The latter suggests an
antigen-specific recall response in the lungs of adult
horse. Therefore, it seems that, in the horse, both
CD4⫹ and CD8⫹ T cells are present for clearance of
the organism. Although R. equi pneumonia is observed in human patients with immunodeficiency
and CD4⫹ T cell lymphopenia, foals that develop R.
equi pneumonia have comparable CD4⫹ and CD8⫹
T cell distribution in peripheral blood with healthy
foals.70 Importantly, T lymphocytes and plasma
cells are virtually absent in lung tissues in the first
week of life, and bronchus-associated lymphoid tissue (BALT) is only observed by 12 wk of age.71
In addition, the concentration of leukocytes in bronchoalveolar lavage fluid in foals less than 3 wk of age
is one-half the value for adult horses.70 The percentage of CD4⫹ T cells increases markedly in the
first 3 wk of life, and B cell counts in bronchoalveolar
lavage fluid (BALF) are almost undetectable in the
first 4 wk of life, reaching values comparable with
adult horses by 8 wk.72 Therefore, the pulmonary
leukocyte population is quite limited during the first
several weeks of life, and it is unlikely that these
cells would control substantial airway infection during this period. Adult horse and ⬎6-wk-old foal
CTLs efficiently kill R. equi-infected cells; however,
the same is not observed with CTLs from foals
younger than 3 wk.73 Patton et al.74 have shown
that CD8⫹ T cells recognize and kill R. equi-infected
macrophages in a major histocompatibility complex
(MHC class I) unrestricted fashion in adult horses,
perhaps involving the CD1 molecule. Importantly,
foal monocyte-derived macrophages express lower
levels of CD1 than adult horse cells, and R. equi
induces down-regulation of CD1b on equine monocyte-derived macrophages.75
Despite a reported age-dependent limited IFN␥
production in foals in response to mitogens,76,77 R.
equi infection in foals is characterized by a significant increase in the percentage of CD4⫹ T lymphocytes in bronchoalveolar lavage fluid and a robust
IFN␥ response.78,79 Compared with adult horses,
experimentally infected foals are capable of mounting equivalent or superior lymphoproliferative responses and high IFN␥ expression and IFN␥/IL-4
ratio by regional bronchial lymph-node lymphocytes.80 Altogether, these data suggest that foals
can elaborate a Th1-type immune response to R.
equi at least equivalent to adult horses, which is
considered protective.
5.
The Acquired Immune System—Humoral Immunity
Antigen-specific antibodies can directly neutralize
pathogens before they reach the intracellular space.
Antibodies also play an essential role as opsonins for
efficient phagocytosis. In the foal, humoral protection would be useful during the initial exposure to
the pathogen, before R. equi reaches the intracellular environment. Therefore, in the neonate, the
passive transfer of immunoglobulins through colostrum or plasma transfusion could be helpful in
providing the initial protection in the airways until
its own cellular and humoral immunity are developed. Few studies have investigated the immunization of dams with R. equi and the efficiency of
antibodies transferred through colostrum in preventing R. equi pneumonia in foals.81– 83 Although
results were somewhat favorable in controlled conditions, individual differences in humoral response
to vaccines, quality of colostrum (antibody concentration), and factors involved in adequate passive
transfer of immunoglobulins would put a percentage
of foals at risk of disease. A more effective means
to standardize the transfer of R. equi-specific antibodies to each foal is through intravenous transfusion of hyperimmune plasma; however, some studies
also indicate disparity in the efficiency of intravenous hyperimmune plasma transfusion in the prevention of disease. In equine neonates infected
experimentally with R. equi, intravenous plasma
transfusion containing specific antibodies against R.
equi or VapA administered before infectious challenge aided significantly in the recovery of disease
compared with placebo.35,84,85 Perkins et al.86 suggested that survival and severity of disease in foals
experimentally infected with R. equi was comparable when foals were pre-treated with intravenous
transfusion of plasma with low R. equi antibody
titers or hyperimmune plasma; however, this study
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IN-DEPTH: RHODOCOCCUS EQUI
did not use a placebo control group to evaluate spontaneous recovery from infection. In naturally infected foals and herd studies, intravenous administration of hyperimmune plasma showed a decrease
in the incidence of pneumonia,81 a reduction in the
risk of developing disease,87 or no significant effect,88,89 which suggests the participation of other
risk factors (e.g., pathogen exposure or competence
of different arms of the immune system) in the development of disease in natural conditions. It is
possible that R. equi-specific antibodies and other
non-specific immunomodulators present in plasma
may synergistically enhance bacterial removal and
killing.
When instituted as prophylaxis and as a means to
standardize humoral protection, intravenous transfusion of hyperimmune plasma would be the most
beneficial in the first few hours of life before the foal
is exposed to R. equi. The mechanism of protection
of the hyperimmune plasma has not been fully described. In vitro experiments using a commercially
available hyperimmune plasma enriched with R.
equi-specific antibody for the opsonization of R. equi
revealed an increase in the oxidative burst activity
of neutrophils and macrophages and the release of
the pro-inflammatory cytokine TNF-␣ from macrophages compared with non-opsonized bacteria.90
In addition, bacterial viability and growth in broth
was reduced in the presence of the hyperimmune
plasma. The viability of intracellular bacteria may
be affected by the opsonization, but results suggest
the need for additional activation of macrophages to
elicit effective bacterial killing. If R. equi-specific
antibodies transferred through intravenous administration of hyperimmune plasma effectively diffuse
into the airways of the foal, it is possible that they
improve bacterial clearance at least before R. equi
reaches the intracellular space.
In natural conditions, serum R. equi-specific immunoglobulin G (IgG) is detected in all foals, and
concentrations correlate with quantitative changes
of fecal R. equi, suggesting a role for oral antigenic
stimulation.91 Thus, foals are capable of producing
a robust humoral response against R. equi antigens;
therefore, the presence of R. equi-specific antibodies
in serum reflects exposure to the pathogen and not
exclusively disease. In another study, the antibody
response to R. equi virulence proteins VapA and
VapC was comparable among healthy foals, foals
with clinical pneumonia, and healthy adult horses,
with higher concentrations of IgG4/7 isotype in pneumonic foals.84 R. equi-specific IgA is also detected
in tracheal aspirates of foals with pneumonia.92
Experimental infection induces rapid humoral response in foals and adult horses.15 Three doses of
oral administration of virulent R. equi at 2, 7, and 14
days of life induced protection against intratracheal
challenge and specific humoral response characterized by higher concentrations of IgG3/5 isotype.93
In foals ⬍10 days of life, intratracheal experimental
infection induced a marked increase in serum IgG1136
2010 Ⲑ Vol. 56 Ⲑ AAEP PROCEEDINGS
and IgG4/7-specific antibody concentrations against
antigens of R. equi, with concentrations higher than
control adult horses.78 Interestingly, the size of inoculum modulates the immunoglobulin isotype response and possibly, the cytokine profile of foals.94
R. equi IgG1- and IgG4/7-specific antibodies are effective in opsonization of the pathogen and complement activation, which favor bacterial removal and
killing. Altogether, these data indicate the foal’s
competence in elaborating a robust humoral response to R. equi antigens.
6.
Summary
The development of disease involves pathogen, host,
and environmental factors. R. equi evolved resistance to escape mechanisms of bacterial killing.
Naïve foals are exposed to high numbers of virulent
bacteria on farms endemic with R. equi. However,
most foals do not develop disease, suggesting individual risk factors. Taken together, these data suggest that susceptibility to R. equi infection and
disease in the foal is not because of their inability to
produce a Th1-type immune response, although this
type of response requires time and effective signaling interaction between antigen-presenting cells
and lymphocytes. Perhaps susceptibility to disease
relates to an early phase of infection when phagocytes fail to remove and kill the pathogens during
the establishment of infection. Although foal
phagocytes are competent at birth, R. equi is killed
more efficiently by phagocytes after primary exposure to the organism, which suggests the involvement of a recall response and activation of
macrophages, perhaps involving IFN␥ from lymphocytes. Therefore, a positive-feedback interaction
between the innate and acquired immune system is
required for protection of the foal against R. equi.
The effect of colostrum- or hyperimmune plasmaderived R. equi-specific antibodies in the neutralization and opsonization of the bacterium before it
reaches the intracellular space is not clear, and passive transfer of immunoglobulins through intravenous plasma transfusion helps but does not prevent
disease completely. Passive transfer of R. equi-specific antibodies through colostrum may vary according to individual colostrum quality and foal factors
that result in efficient or deficient antibody absorption. The administration of hyperimmune plasma
may add to a positive outcome in the prophylaxis of
R. equi, but its effectiveness may depend on the
timing of administration (before pathogen exposure)
and quality of the product (standardized concentration and quality of antibodies relevant to virulent
strains). Many current studies investigate the
pathogenic mechanisms in the early stages of infection to design better prophylactic methods, including the development of immunomodulators and
vaccines and improved herd-management practices.
IN-DEPTH: RHODOCOCCUS EQUI
Acknowledgments
The author would like to thank Dr. Noah Cohen
and Dr. Steeve Giguère for their comments on this
manuscript and the Harry M. Zweig Memorial Fund
for their research financial support throughout the
years.
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