Immunity to Francisella

Center for Biologics Evaluation and Research, U.S. Food and Drug Administration Bethesda, MD, USA.
Frontiers in Microbiology (Impact Factor: 3.99). 02/2011; 2(26):26. DOI: 10.3389/fmicb.2011.00026
Source: PubMed
ABSTRACT
In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified, generating a wealth of new information on the interaction of this organism with the immune system. Here we review the basic elements of the innate and adaptive immune responses that contribute to protective immunity against Francisella species, with special emphasis on new data that has emerged in the last 5 years. Most studies have utilized the mouse model of infection, although there has been an expansion of work on human cells and other new animal models. In mice, basic immune parameters that operate in defense against other intracellular pathogen infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for control of Francisella infection. However, new important immune mediators have been revealed, including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in addition to macrophages are now recognized to support Francisella growth, including epithelial cells and dendritic cells. CD4(+) and CD8(+) T cells are clearly important for control of primary infection and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by T cells are only beginning to be defined. A significant role for B cells and specific antibodies has been established, although their contribution varies greatly between bacterial strains of lower and higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune responses, but susceptible to many elements of the immune system's antimicrobial arsenal.

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Available from: Siobhán C Cowley
www.frontiersin.org February 2011 | Volume 2 | Article 26 | 1
Review ARticle
published: 16 February 2011
doi: 10.3389/fmicb.2011.00026
Immunity to Francisella
Siobhán C. Cowley* and Karen L. Elkins
Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA
In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified,
generating a wealth of new information on the interaction of this organism with the immune
system. Here we review the basic elements of the innate and adaptive immune responses that
contribute to protective immunity against Francisella species, with special emphasis on new data
that has emerged in the last 5 years. Most studies have utilized the mouse model of infection,
although there has been an expansion of work on human cells and other new animal models.
In mice, basic immune parameters that operate in defense against other intracellular pathogen
infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for
control of Francisella infection. However, new important immune mediators have been revealed,
including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in
addition to macrophages are now recognized to support Francisella growth, including epithelial
cells and dendritic cells. CD4
+
and CD8
+
T cells are clearly important for control of primary infection
and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by
T cells are only beginning to be defined. A significant role for B cells and specic antibodies has
been established, although their contribution varies greatly between bacterial strains of lower and
higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune
responses, but susceptible to many elements of the immune system’s antimicrobial arsenal.
Keywords: Francisella, immunity, innate, adaptive, lymphocyte, cytokine
subsp. tularensis. These are now denoted A1a, A1b, and A2, in addi-
tion to Type B F. tularensis subsp. holarctica (Kugeler et al., 2009).
Type A1 infections of humans tend to have a fulminant course with
a high mortality rate, while Type A2 and Type B infections are rarely
if ever lethal in humans (Staples et al., 2006; Kugeler et al., 2008). In
studies in the U.S., differences in the attack rates of immunocom-
promised people for the various clades and subspecies have been
described; 11 of 108 (10%) of Type B infections, 6 of 133 (6%) of
A1 infections (both subtypes), and none of 68 A2 infections were
diagnosed in people with an underlying immunocompromising
condition, including medical conditions as diverse as end stage renal
disease (Staples et al., 2006) and chronic granulomatous disease
(CGD; Maranan et al., 1997). Nonetheless, the collective evidence
suggests that the differences in virulence are largely due to intrin-
sic properties of the bacterial strains, and not directly related to
host gender, susceptibility, genetics, or otherwise failed immune
responses (Kugeler et al., 2009).
Because infection with Francisella is relatively infrequent in
nature, informative examples of infection of people with primary
or acquired immunodeficiencies subjects are rare. The handful of
such cases prior to 2006, including infection of AIDS patients with
reduced CD4
+
T cell counts, has been reviewed elsewhere (Elkins
et al., 2007), and thus will not be repeated here. More recently,
an interesting case involving a 58-year-old man with refractory
rheumatoid arthritis was described. The patient had been treated
for about a year with methotrexate and an anti-TNF-α therapeutic
(Humira
®
, adalimumab), when he presented with fever, a leg wound,
enlarged lymph nodes, and eventually skin fistula. Tuberculosis was
suspected initially; the lesion was surgically removed, and found
IntroductIon and overvIew
Although Francisella tularensis is highly infectious and readily estab-
lishes disease at low doses in both humans and animals, it has long
been recognized that human tularemia victims rarely if ever suffer
a second episode of disease. The collective human infection expe-
rience therefore strongly suggests that natural infection engenders
strong immune responses that are usually protective. The older lit-
erature contains a wealth of information on the pathogenesis and
host response to Francisella drawn from studies in both humans
and animals, including vaccination and challenge studies in humans
that would be difficult if not impossible to replicate today. These
studies have been recently reviewed extensively elsewhere (Conlan
and Oyston, 2007; Elkins et al., 2007). But the history of studies
on Francisella includes its development as a biowarfare pathogen
(Dennis et al., 2001); as a result, the recent heightened interest in
biodefense has produced a flood of new and exciting data, particu-
larly on respiratory infection and mucosal immune responses to this
pathogen. The upsurge in studies is impressive: from 1900 to 2005,
a search of PubMed for Tularemia or Francisella yielded a total of
2921 references, but 858 citations from 2006 to 2011. Thus this review
will focus primarily on developments in about the last 5 years.
Human in vivo Immune responses to Francisella
InfectIon and vaccInatIon
Important components of Human Immune responses
revealed by Francisella InfectIon
The most recent studies of the epidemiology of infection with
F. tularensis subsp. tularensis, as well as subsp. holarctica, clearly
indicate differences in virulence among clades of Type A F. tularensis
Edited by:
Anders Sjostedt,
Umeå University, Sweden
Reviewed by:
Dennis Metzger,
Albany Medical College, USA
Shabaana Khader,
University of Pittsburgh, USA
*Correspondence:
Siobhán C. Cowley, Laboratory of
Mycobacterial Diseases and Cellular
Immunology, Division of Bacterial,
Parasitic and Allergenic Products/
Office of Vaccines Research and
Review/Center for Biologics Evaluation
and Research, U.S. Food and Drug
Administration, HFM-431, 29 Lincoln
Drive, Room 516, Bethesda, MD
20892, USA.
e-mail: siobhan.cowley@fda.hhs.gov
Page 1
Frontiers in Microbiology | Cellular and Infection Microbiology February 2011 | Volume 2 | Article 26 | 2
Cowley and Elkins Immunity to Francisella
and nascent specific T cells. As a result, recent biological assays being
developed for diagnosis have been based on ex vivo stimulation of
whole peripheral blood leukocytes with bacteria, followed by assess-
ment of IFN-γ secretion (Eliasson et al., 2008).
A curious, and still unexplained, feature of natural infection is
the large expansion of phosphoantigen-responsive Vγ9/Vδ2 T cells
in peripheral blood within the first 7 days after onset of symptoms
in tularemia patients (Poquet et al., 1998). This intriguing phe-
nomenon, which can result in as many as 30% of all CD3
+
cells
in human peripheral blood being Vγ9/Vδ2 T cells, is discussed in
more detail below.
Human Immune responses revealed by vaccInatIon agaInst
Francisella
As noted above, and discussed in detail in another chapter in this
issue, there is a long history of using various live attenuated strains
of Francisella as vaccines, particularly in the former Soviet Union
(ndstrom, 1994). As a reference point for further discussion of
immune responses generally, here we include a brief summary of
human immune responses to the LVS strain. Usually administered
by scarification, LVS has been studied in western countries and par-
ticularly in the U.S. These studies have mostly used investigational
lots of LVS produced in the 1970s by the Department of the Army;
more recently, LVS was re-derived from the original lots, and newly
manufactured under modern GMP conditions (El Sahly et al., 2009).
Microarray studies of gene changes in PBLs from five volunteers
obtained between 1 and 14 days after vaccination with LVS indi-
cated a robust up-regulation of expression of pro-inflammatory
mediators and genes involved in dendritic cell (DC) function, which
peaked within 2 days (Fuller et al., 2006, 2007). Similar to natural
infection, humans vaccinated with LVS develop specific IgM, IgA,
and IgG antibodies in serum about 2 weeks after vaccination that
persist for at least 1.5 years (Waag et al., 1995; El Sahly et al., 2009).
However, as noted at the outset, anti-Francisella serum antibodies
titers in vaccinated individuals have not been predictive of protec-
tion against virulent tularemia, and vaccination with killed bacteria
(that elicit anti-Francisella antibodies but no detectable cell-medi-
ated immune responses) has provided little or no benefits in human
studies (Francis and Felton, 1942; Foshay, 1950; Overholt et al., 1961;
Saslaw et al., 1961a,b; Hambleton et al., 1974; Burke, 1977; Tärnvik,
1989). In addition to serum antibodies, stimulated PBLs, or enriched
CD4
+
and CD8
+
T cells, obtained from volunteers 2–4 weeks or more
after LVS vaccination proliferated and produced typical Th1-type
cytokines, especially IFN-γ, ex vivo (Waag et al., 1995; El Sahly et al.,
2009). In one study, the responding cells from both LVS vaccines
and patients after natural infection were characterized as traditional
CD4 and CD8 memory T cells, mostly with an effector memory
phenotype (CD45RA
/+
, CD62
; Salerno-Goncalves et al., 2009).
anImal models of Human ImmunIty to Francisella
Detailed consideration of animal models of Francisella infection is
well beyond the scope of this article, and a comprehensive recent
review is available elsewhere (Lyons and Wu, 2007). Nonetheless,
because the bulk of data on immune responses is currently being
generated using animal models of infection, here we include brief
summaries of recent developments as they relate to modeling the
immune responses of humans (for summary, see Table 1).
to contain necrotic epithelioid granulomas. Surprisingly, both
serology and PCR of a biopsied lymph node diagnosed F. tularen-
sis infection (Konstantinou et al., 2009). It is tempting to speculate
that TNF-α deficiency, provided in this case by drug treatment,
increased susceptibility to Francisella infection, similar to observa-
tions in animal models (Cowley et al., 2008) and in mycobacteria
infections of humans (Gardam et al., 2003).
Of note, although only about 20 cases of human infection with
F. philomiragia have been reported in the literature, disease caused
by this species is usually associated with immune defects. These
include corticosteroid treatment, CGD, and near-drowning epi-
sodes (Hollis et al., 1989; Sicherer et al., 1997). The association
between Francisella and CGD, in which neutrophils fail to produce
fully functional NADPH oxidase and thus reactive oxygen radicals,
obviously suggests a role for these mediators in human resistance
to infection.
The experimental literature now frequently refers to the immu-
nosuppressive nature of infection with virulent Francisella (the topic
of another chapter in this issue), but naturally infected patients as
well as vaccines eventually develop robust and readily measurable
T and B cell responses to the bacterium. These responses have been
studied for many years, particularly in regions such as Scandinavia
which have appreciable amounts of disease. An outbreak of ulcero-
glandular tularemia in Sweden in 2003–2004 provided a unique
opportunity to obtain peripheral blood cells from patients within
days of infection, and perform large scale transcriptional profiling
of human gene expression by microarray (Andersson et al., 2006a).
Within 2–3 days, there was clear evidence for increased expression
of genes regulated by interferon gamma (IFN-γ) and related to
apoptosis, but also indications of down-regulation of many genes
related to both innate and adaptive immune responses. Thus infec-
tion in humans may be characterized by both appropriate and
subversive types of host reactions.
The major means to diagnose tularemia is still based on four-fold
increase of serum antibodies to the bacterium between acute and
convalescent sera. Robust specific IgM, IgG, and IgA serum antibod-
ies, much of them directed against Francisella lipopolysaccharide
(LPS), can be detected roughly simultaneously about 6–10 days
after the onset of symptoms, or about 2 weeks after infection.
Antibody responses peak between 1 and 2 months after infection,
and persist for about a decade before diminishing (Koskela and
Salminen, 1985). Large scale efforts using 2-D gel blotting (Janovska
et al., 2007) or protein chips with a library of Francisella proteins
(Sundaresh et al., 2007), reacted with patient sera, are beginning to
identify and catalog the bacterial proteins recognized. No obvious
immunodominant B cell epitopes have been revealed, however, and
as a result panels of antigens have been proposed for diagnostic
purposes (Sundaresh et al., 2007).
Within about 2 weeks after infection, ex vivo production of typical
Th1-type cytokines such as IFN-γ, TNF-α, and IL-2 by CD4
+
and CD8
+
T cells is readily detectable in human peripheral blood lymphocytes
(PBLs) obtained from tularemia patients (Koskela and Herva, 1980;
Surcel et al., 1991). Unlike antibody responses, however, human CD4
+
and CD8
+
T cell PBL responses persist for as long as 30 years after
documented infection (Ericsson et al., 1994). The development of
more sensitive assays has facilitated even earlier detection of cytokines
such as IFN-γ, which may be produced by both innate immune cells
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Cowley and Elkins Immunity to Francisella
Table 1 | Characteristics of human Francisella infections compared to current experimental animal models.
Experimental animal models
Strain Human infection/vaccination Non-mammalian Mammalian
+
Insect (Drosophila) Fish (zebrafish) Mice (C57BL/6J) Rats (Fisher 344) Marmosets (common)
Type A1a/b Ft Aggressive infection, Not tested Not tested LD
50
< 10, all LD
50
IT 5 × 10
2
LD
50
by aerosol <10
highly virulent, can be lethal routes tested
Type A2 Ft Causes morbidity, Not tested Not tested LD
50
< 10, all Not tested Not tested
but rarely lethal routes tested
Type B Ft Causes morbidity, Not tested Not tested LD
50
< 10, LD
50
IT 10
5
, IP < 10 Lethal natural
but rarely lethal all routes tested infections observed
LVS Attenuated Type B; skin or Productive infection; Not tested LD
50
10
6
ID, 10
3
IN, LD
50
IT 10
5
, IP >10
8
; Not tested
respiratory inoculation establishes intra/extracellular <10 IP or IV;vaccinates vaccinates when
productive infection, vaccinates* replication when sublethal* sublethal*
F. novicida Infections rare, never lethal; some Productive infection; Not tested LD
50
10
3
ID
#
, LD
50
IT 5 × 10
6
Not tested
association with immunodeficiencies intra/extra cellular <10 IP, IV, or IN
replication
F. philomiragia Infections rare, never lethal; associated Not tested Not tested Not tested Not tested Not tested
with CGD, other immunodeficiencies
Francisella spp. No known human pathogenicity Not tested Infects; Not tested Not tested Not tested
from fish cytokine mRNA
*Stimulates production of pro-inflammatory cytokines, serum anti-Francisella antibodies, Th1 T cells, and Th1-related cytokines; extent of protection against secondary challenge infection varies with route of
vaccination, route of challenge, and challenge strain.
+
All LD
50
s are approximate and expressed in terms of CFU.
#
Survival of primary and secondary infections are heavily B cell dependent, unlike other strains.
For references, see corresponding text.
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Cowley and Elkins Immunity to Francisella
Over the years, other small animal models for Francisella
infection have been developed using rats, guinea pigs, hamsters,
voles, and rabbits. Several recent reports, coupled with older
literature, indicate that infection of Fisher 344 rats with differ-
ent Francisella strains may better approximate the phenotype of
human infections that these other models. Both Fisher 344 and
Lewis rats are much more resistant to F. novicida infection than
mice, due at least in part to rapid production of nitric oxide
from macrophages following recognition of the F. novicida LPS
chemotype (Cowley et al., 1997; Ray et al., 2010). In a direct
comparison, the intratracheal (IT) LD
50
of Fisher 344 rats was
recently reported to be 5 × 10
2
for Type A F. tularensis subsp. tula-
rensis (SchuS4); 1 × 10
5
for Type B F. tularensis subsp. holarctica
(OR960246); 5 × 10
6
for F. novicida; and greater than 10
7
for LVS
(Ray et al., 2010). Of note, however, Fisher 344 rats were highly
susceptible to IP infection with a different Type B F. tularensis
subsp. holarctica strain (FSC108), although still quite resistant
to IP LVS infection (Raymond and Conlan, 2009). Nonetheless,
overall the hierarchy of susceptibility of rats appears to gener-
ally reflect human Francisella infections, and provide a more
satisfying profile than that of mice. Equally important, Fisher
344 rats vaccinated IT, intradermally (ID), or subcutaneously
(ID) with 10
7
LVS survived IT challenge with at least 100 LD
50
s
of challenge with Type A F. tularensis subsp. tularensis (SchuS4;
Wu et al., 2009; Ray et al., 2010). Thus this rat strain appears to
provide both a useful model for both infection and immunologi-
cal studies for further analyses.
Despite the practical appeal of small animal models, studies
using non-human primates remain important not only for basic
studies of host–pathogen interactions but for testing of drugs,
vaccines, and therapeutics. Historically, monkeys have been con-
sidered to be even more susceptible than humans to Francisella
infection. Outbreaks of tularemia in various species of mon-
keys in both zoo and experimental colonies have been reported
repeatedly (Splettstoesser et al., 2007). There is an extensive older
literature using monkeys, particularly Rhesus monkeys, for both
natural history and vaccination studies (Lyons and Wu, 2007;
Kugeler et al., 2008). Most recently, interest in product develop-
ment has spurred renewed efforts to establish non-human pri-
mate models using species currently available for experimental
studies. Marmosets (Callithrix jacchus) suffer lethal infection
with as few as 10 CFU of Type A F. tularensis subsp. tularensis
(SchuS4) administered by aerosol, with pathology that appears
similar to that of humans. Infected marmosets further exhibited
production of pro-inflammatory cytokines, as well as increased
numbers of the major lymphoid and myeloid cell subpopulations
in lungs and blood (Nelson et al., 2009, 2010). Similarly, the pro-
file of aerosol infection of African green monkeys (Chlorocebus
aethiops) given 700 CFU of SchuS4 was described recently
(Twenhafel et al., 2009), and studies in cynomolgus monkeys as
well as comparisons between species are underway (Wilder and
Gelhaus, 2009). While it is premature to draw conclusions about
the relative strengths and weaknesses of each of these approaches,
further studies will no doubt provide data that informs the value
of different non-human primate models for both pathogenesis
and immunological studies.
non-mammalIan InfectIon models
Two groups have explored insect models of Francisella infec-
tion. Both LVS (Vonkavaara et al., 2008) and F. novicida (Ahlund
et al., 2010; Moule et al., 2010) productively infect and replicate
in Drosophila melanogaster, both by infection of phagocytic fly
hemocytes and by extracellular replication. Both efforts focused
on the ability of the model to identify bacterial virulence factors;
mutations in bacterial genes important to virulence in mouse
models, notably pathogenicity island genes including mglA and
others regulated by mglA, clearly contributed to virulence in flies.
Given that deer flies are vectors of Francisella infection, however,
it remains to be revealed whether the fly host biology discovered
is applicable only to the vector, or also helpful in modeling human
responses.
Initial efforts to establish Francisella infection of zebrafish,
another genetically tractable system that has a more complex
immune system than Drosophila, have recently been reported as
well. In the last 3–4 years, new Francisella spp. have been isolated
from diseased fish, both wild and cultivated. The zebrafish study
used one of these strains, and demonstrated productive experi-
mental infection followed by up-regulated expression of IL-1β,
IFN-γ, and TNF-α, analogous to mammalian pro-inflammatory
responses. There was no increase, and perhaps transient down-
regulation, of iNOS, however (Vojtech et al., 2009).
mammalIan InfectIon models
For all the obvious reasons, the majority of immunological studies
of Francisella have used mouse models. The available data indicate
that mice are a reasonable model of human immune responses,
at least at a first approximation, but less satisfying as a model for
pathogenicity. Inbred laboratory strains are readily susceptible
to infection with all Francisella isolates tested to date; bacteria
disseminate to the same target organs of the reticuloendothelial
system, and infected tissues develop granulomatous pathologies
that appear roughly comparable to lesions described in tissues of
infected people. There is a major discrepancy between humans and
mice in virulence and lethality, however. In mice, the LVS strain
establishes a sublethal vaccinating infection when administered
via skin inoculation, but kills mice at low doses when introduced
by other routes, including intravenous (IV), intramuscular, or
intraperitoneal (IP), and is intermediate for respiratory infections
(Elkins et al., 2003). Importantly, infection with both Type A and
Type B F. tularensis, as well as F. novicida, kills mice within a week
with essentially any dose and when introduced by any route of
infection. In contrast, as noted above, human Type B infections in
particular are rarely lethal (Staples et al., 2006; Kugeler et al., 2008).
Human F. novicida infections are quite rare, and when detected
are sometimes associated with immunocompromised individuals
(Hollis et al., 1989; Whipp et al., 2003; Leelaporn et al., 2008). To
date, there is only one report of Francisella infection of HLA-DR4
transgenic mice, with the goal of uncovering antigens recognized
by human T cells (Yu et al., 2010); but as expected, these mice were
equally susceptible to intranasal LVS infection as wild type mice. It
remains to be determined whether “humanized” mice created by
engraftment of human stem cells exhibit susceptibilities that better
approximate human infection profiles.
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Cowley and Elkins Immunity to Francisella
in vivo Immune responses to Francisella novicida
and mutants based on F. novicida
Although a comprehensive discussion of the relative virtues of
conducting experimental studies using F. tularensis strains versus
F. novicida strains is outside of the topic of this review, some general
comments regarding immunological responses are pertinent here.
For many years, only F. novicida was amenable to genetic manipula-
tion; efforts to transform F. tularensis strains with transposons or
develop mutants via allelic exchange using traditional techniques
either failed, or were of such low efficiency as to be impractical.
Following recent concerted efforts to develop better genetic tools
for manipulation of F. tularensis, this situation has changed greatly.
A number of transposon banks and deletion mutants of F. tula-
rensis Type A, Type B, and LVS strains have been developed in the
last 5 years. Nonetheless, manipulation of F. novicida remains
considerably easier; further, this strain is exempt from Select Agent
registration in the U.S., and generally used under BSL-2 laboratory
conditions. Thus F. novicida remains popular for genetic studies,
and mutants have frequently been used for in vivo infection studies,
particularly those seeking virulence factors. However, when reading
this literature, it is important to note that F. novicidas name has
been written as both F. novicida and later F. tularensis subsp. novic-
ida, and the appropriate current designation remains controversial
(Busse et al., 2010; Huber et al., 2010; Johansson et al., 2010).
Francisella tularensis subsp. tularensis and holarctica clearly have
strong genetic homologies with F. novicida, and findings regarding
pathogenesis and cell biology made using F. novicida and its mutants
have often been applicable to the biology of F. tularensis. However,
there are important biological differences between F. novicida and
F. tularensis strains that appear particularly problematic for immu-
nological studies. As discussed above, the virulence of F. novicida
in both humans and various animal models is quite distinct from
that of the F. tularensis subsp. In mice given F. novicida intranasally,
the cell tropism in lungs is noticeably different from that for LVS
and SchuS4. The latter two strains preferentially infect alveolar
macrophages and later expand in macrophages, DCs, and neu-
trophils; in contrast, F. novicida starts in neutrophils and alveolar
macrophages, and then expands in neutrophils while macrophages
and DCs are lesser targets (Hall et al., 2008). Importantly, F. novicida
expresses a structurally distinct chemotype of LPS that is more pro-
inflammatory in mice than the dominant LPS chemotype expressed
by F. tularensis strains (Cowley et al., 1996; Kieffer et al., 2003;
Gunn and Ernst, 2007). Although not yet examined directly, the LPS
bioactivity may contribute to the observed sepsis-like syndrome
that follows intranasal infection of mice with F. novicida (Mares
et al., 2008). Each chemotype of LPS also appears to play distinct
roles in virulence of the respective bacteria and in contributing to
protection in mouse models (Thomas et al., 2007).
Studies of immune responses to F. novicida in mice consistently
reveal a prominent role for B cells and antibodies that is consider-
ably more dramatic than LVS or fully virulent Francisella strains.
The LD
50
of F. novicida administered to inbred mice ID is about 10
3
.
The ID LD
50
of F. novicida administered to B cell knockout mice is
less than 5 × 10
1
, and those that do survive vaccination are severely
compromised compared to wild type mice for survival of second-
ary lethal IP challenge with LVS (Chou and Elkins, manuscript
in preparation). Similarly, mice vaccinated IN with attenuated
mutants in iglB or iglC of F. novicida have large amounts of both
IgG
1
and IgG
2a
serum anti-Francisella antibodies, which adoptively
transfer protection against F. novicida challenge to recipients in the
absence of primed T cells (Pammit et al., 2006; Powell et al., 2008).
For the iglC mutant of F. novicida, vaccinated mice depleted of CD4
+
T cells at the time of challenge with F. novicida survived, and thus
protection depended on the presence of antibodies but not effector
T cells (Powell et al., 2008). Collectively, the mechanistic data to
date paints a picture that is quite distinct from studies using LVS
as a vaccine or challenge with virulent Francisella.
systemIc versus mucosal ImmunIty to F. tularensis
Although Francisella infection can be initiated via multiple routes,
historically the majority of studies in the Francisella murine model
have focused on ID or SC exposure to the pathogen. In partic-
ular, ID infection with LVS has been utilized because this route
approximates the most likely method of vaccination in humans
(scarification or SC inoculation), and also conveniently allows
for mechanistic studies of immunity after a sublethal infection
in mice. More recent studies have focused on respiratory infec-
tions, given the interest in biodefense applications. Mice are much
more susceptible to Francisella infections initiated via pulmonary
routes as compared to the ID/SC route. For LVS, the IN LD
50
is
10
2
–10
4
bacteria, whereas infection via the ID/SC route exhibits
an LD
50
of 10
6
–10
7
bacteria. For the more virulent Type A and
Type B F. tularensis strains, both routes of primary infection are
rapidly lethal, although protection against secondary respiratory
challenge is much more difficult to achieve than protection against
ID/SC challenge (Chen et al., 2003). Indeed, to date reasonable
protection against virulent Type A respiratory challenge has only
been achieved following mucosal (but not parenteral) LVS vac-
cination (Conlan et al., 2005; Wu et al., 2005; KuoLee et al., 2007).
These observations recall theories of the compartmentalization
of the mucosal immune system (Gill et al., 2010). In support of this
concept, respiratory vaccination is not the only mucosal immuni-
zation route that is protective against respiratory challenge: LVS
immunization via the oral route also results in survival of Type
A pulmonary challenge, whereas ID/SC immunization does not
(KuoLee et al., 2007). The immune mechanisms that are uniquely
induced by mucosal – but not parenteral – vaccination remain to
be identified. Recent data indicates that important cytokines such as
IL-17A are preferentially produced in LVS-infected lungs following
respiratory – but not parenteral – infection (Woolard et al., 2008;
Cowley et al., 2010). Thus certain immune mediators may be of
greater importance depending on the initial tissue encountered
during vaccination and/or challenge; this possibility remains an
interesting avenue of future investigation.
As noted above and in detail in another article in this issue, one
theme that has emerged in recent years is the ability of Francisella to
initially suppress or avoid induction of early immune responses fol-
lowing primary respiratory infection. Although multiple cell func-
tions are clearly suppressed by both LVS and Type A F. tularensis,
virulent Type A strains execute a broader range of immunosup-
pression that likely contributes to their increased virulence (Bosio
et al., 2007). Further, some immunosuppressive functions such
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Cowley and Elkins Immunity to Francisella
another article in this issue, here we focus our discussion on the
current knowledge of the contribution of TLRs to the initiation of
in vivo host responses to Francisella.
Current in vitro and in vivo evidence indicates that TLR2
and MyD88 are critical mediators of inflammatory responses to
Francisella. In vitro studies have demonstrated that TLR2 is required
for murine DCs to activate NF-KB, produce TNF-α, and up-regulate
maturation markers in response to LVS (Katz et al., 2006). Similarly,
TLR2 was required for induction of a range of pro-inflammatory
cytokine genes (e.g., TNF-α, IL-1β, KC, p40, RANTES, IFN-γ, IL-6,
IFN-β, MCP-1, and iNOS) by peritoneal macrophages in response
to LVS (Cole et al., 2007; Abplanalp et al., 2009). Full signaling
via TLR2 in LVS-infected macrophages required the adaptor mol-
ecules MyD88 and TIRAP (Cole et al., 2010). Consistent with the
membrane-bound nature of TLR2, LVS co-localized intracellularly
within a TLR2/MyD88-containing phagosome (Cole et al., 2007).
Interestingly, an LVS mutant that cannot escape the phagosome
induced greatly increased expression of TLR2-dependent pro-
inflammatory genes (such as TNF-α), and decreased expression
of genes that rely on cytosolic recognition of bacteria (such as
IFN-β; Cole et al., 2008). These data underscore the existence of
the two separate arms of pathogen detection and early cytokine
responses those cytokines whose production is initiated solely
via TLR signaling (TNF-α, IL-6), and those cytokines that require
both TLR and NLR signaling (IFN-β, IL-1β).
In vitro data are supported by numerous in vivo studies that
demonstrate increased susceptibility of TLR2 and MyD88 KO mice
to LVS infection (Collazo et al., 2006; Malik et al., 2006; Abplanalp
et al., 2009). Intranasal LVS infection of TLR2 KO mice resulted
in increased mortality and decreased survival times as compared
to their WT counterparts, accompanied by higher bacterial organ
burdens and lower levels of TNF-α and IL-6 in the lungs (Malik
et al., 2006; Abplanalp et al., 2009). Although TLR2 KO mice were
significantly more susceptible to LVS infection than wild type mice
via both the IN and ID routes, mice were consistently able to sur-
vive lower doses of LVS. This was in stark contrast to MyD88 KO
mice, which were exquisitely susceptible to even the smallest doses
of LVS delivered via both routes. This indicates a role for MyD88
in LVS infection that extends beyond its function as an adaptor
for TLR2 signaling (Collazo et al., 2006; Abplanalp et al., 2009).
Other MyD88-dependent molecules that have been tested in the
LVS infection model include IL-18, IL-1R, TLR4, TLR1, TLR6, and
TLR9 (for summary, see Table 2). Although most have not been
exhaustively studied, mice singly deficient for these molecules did
not exhibit notably increased susceptibility to LVS infection as com-
pared to their WT counterparts (Collazo et al., 2006; Abplanalp
et al., 2009). Similarly, TLR2/9 double KO mice readily survived
doses of up to 10
5
LVS given ID (Chou and Elkins, manuscript in
preparation). Further studies in mice deficient in multiple MyD88-
dependent receptors would be needed to rule out the possibility of
redundancy and/or compensatory effects.
Investigations of the bacterial ligands responsible for TLR rec-
ognition of Francisella indicate that LVS expresses lipoproteins that
can activate HeLa cells transfected with either TLR2/TLR1 or TLR2/
TLR6 heterodimers. Specifically, the lipoproteins Tul4 and FTT1103
stimulated activity via the TLR2/1 heterodimer, and induce expres-
sion of a panel of chemokines in both human peripheral blood
as prostaglandin-E2 (PGE2) production or reduced levels of
CD14 may be operative primarily in certain tissues such as the
lung (Woolard et al., 2008). Thus, both the route of inoculation and
the strain of Francisella can have a widely different impact on the
quantity and quality of immune responses measured. Differences
in these factors can often make it difficult to draw comparisons
between different studies.
medIators of Innate Immune responses
complement
Recent studies documenting that F. tularensis and F. novicida can
survive extracellularly in whole blood in vitro and in vivo dur-
ing mouse infections (Forestal et al., 2007; Yu et al., 2008) have
generated new perspectives on the role of extracellular mediators
of immunity during infection. F. tularensis is clearly resistant to
the bactericidal effects of sera from a variety of species, a feature
that was initially associated with cell surface carbohydrate struc-
tures described as a capsule (Hood, 1977). More recently, however,
studies using Francisella strains with targeted mutations in LPS
biosynthesis genes have demonstrated that complement resist-
ance is critically dependant upon LPS O antigen (Thomas et al.,
2007; Clay et al., 2008). Although resistant to its bactericidal effects,
Francisella clearly binds by complement components: complement-
derived opsonins and complement receptors enhance phagocytic
uptake of F. tularensis by a variety of cell types, including human
and mouse monocytes and macrophages (Clemens et al., 2005;
Pierini, 2006; Schulert and Allen, 2006), and human monocyte-
derived DCs (Ben Nasr et al., 2006; Ben Nasr and Klimpel, 2008).
Indeed, C3 complement components – but not the lethal C5b-C9
membrane attack complex were shown to be deposited on the
cell surface of F. tularensis after incubation in human sera. These
C3-derived opsonins enhanced phagocytic uptake by human DCs
via a process that promoted intracellular survival, bacterial growth,
and DC death (Ben Nasr et al., 2006; Ben Nasr and Klimpel, 2008).
This strategy of resistance to complement killing, and the use of
complement opsonins to gain entry into an intracellular niche that
supports bacterial growth, is likely an important virulence deter-
minant of Francisella. In support of this hypothesis, Francisella O
antigen mutants are attenuated in the mouse model of infection
(Thomas et al., 2007).
pattern-recognItIon receptors and early cytokIne
productIon
Host cells express a variety of germline-encoded “pattern-
recognition receptors (PRRs), which recognize a number of
evolutionarily conserved molecular patterns expressed only by
pathogens. PRRs include the membrane-anchored Toll-like recep-
tors (TLRs) and the cytosolic NOD-like receptors (NLRs). Because
Francisella initially resides in a membrane-bound phagosome prior
to escape into the cytosol, the bacterium has the potential to interact
with both membrane and cytosolic PRRs. Indeed, recent evidence
has shown that bacterial DNA engaged the cytoplasmic NLR sen-
sor “absent in melanoma 2” (AIM2) within Francisella-infected
macrophages, a process that was necessary to initiate inflamma-
some assembly, caspase-1 activation, and IL-1β release (Jones et al.,
2010; Rathinam et al., 2010; Ulland et al., 2010). Since the role of
the inflammasome in Francisella infection is reviewed in detail in
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Cowley and Elkins Immunity to Francisella
demonstrated that it is uniquely tetra-acylated and monophospho-
rylated, both structural properties that can reduce the endotoxicity
of LPS (Gunn and Ernst, 2007). Indeed, the only biological activ-
ity ascribed to F. tularensis LPS (Ft-LPS) thus far is its ability to
bind and stimulate rapid antibody production by the “innate B
lymphocyte subset B1a cells, and this function was clearly TLR4-
independent (Cole et al., 2009); see below. Although Francisella
LPS clearly has little if any TLR4-dependent stimulatory activities
(Duenas et al., 2006), one recent study revealed that the F. tularensis
heat shock protein DnaK can initiate cytokine production by DCs
through TLR4 in vitro (Ashtekar et al., 2008). Despite the potential
for Francisella to stimulate TLR4 through non-LPS ligands, TLR4-
deficient mice do not exhibit increased susceptibility to Francisella
infection, indicating that this TLR is not critical for in vivo defense
(Chen et al., 2004, 2005b; Collazo et al., 2006).
More recently, the downstream effects of TLR2/MyD88 signaling
have been studied in more detail. LVS infection of bone marrow-
derived macrophages (BMDMs) induced TLR2-dependent splicing
of mRNA for XBP-1, a transcription factor necessary for production
of TNF-α and IL-6 mRNA. The importance of XBP-1 in resistance
to LVS infection was confirmed by the presence of higher levels of
mononuclear cell (PBMC) and mouse bone marrow-derived DCs
(Thakran et al., 2008). A role for TLR6 in recognition of LVS was
similarly demonstrated by the inability of bone marrow-derived
DCs harvested from TLR6 KO mice to produce TNF-α in response
to LVS (Katz et al., 2006). Conversely, in other studies, macrophages
from TLR6 KO mice infected with LVS expressed higher levels of
TNF-α, IL-6, and MCP-1 than their WT counterparts, and, as men-
tioned above, TLR6 KO mice exhibit no increased susceptibility to
LVS infection (Abplanalp et al., 2009). Thus, it is possible that in
some circumstances TLR6 and TLR1 are redundant in their abilities
to recognize LVS ligands in concert with TLR2.
Bacterial LPS is usually one of the first Gram negative
pathogen-associated molecular patterns to be detected by the
immune system, specifically targeted by the PRR TLR4. However,
studies of Francisella LPS over the years have found it to be bio-
logically inactive, unable to induce production of pro-inflamma-
tory cytokines from all cell types tested. Subsequent studies have
shown that Francisella LPS is not recognized by either human or
murine TLR4 or TLR2, and further cannot act as an antagonist
to block binding of Salmonella LPS to TLR4 (Duenas et al., 2006;
Hajjar et al., 2006). Elucidation of the structure of Francisella LPS
Table 2 | The contribution of cytokine receptors, toll-like receptors, and other effector components of immune responses to in vivo murine infection
with Francisella tularensis*.
Component Effect of depletion/knockout
§
Cellular source(s)
1° infection 2° infection Innate Acquired (T cells)
CYTOKINE RECEPTORS
IL-4R Slightly more susceptible N/A Ms N/A
at high dose (3 × 10
3
IN)
IL-4Rα Less susceptible N/A N/A N/A
to lethal dose (10
4
IP)
IFNαR1 Less susceptible
(ID) N/A N/A N/A
IL-1R No change (ID) Survive lethal 10
6
IP challenge N/A N/A
TNFR1/2 Lethal at low doses N/A Ms CD4
+
, CD8
+
(10
2
aerosol and 20 ID)
TOLL-LIKE RECEPTOR-RELATED
TLR2 More susceptible at high/moderate Survive lethal 10
6
IP challenge Ms, DCs N/A
doses (4 × 10
3
IN and 4 × 10
4
ID)
MyD88 Lethal at all doses (IN and ID) N/A Ms, DCs N/A
TLR4 No change (IN and ID) Survive lethal 10
6
IP challenge Ms, DCs N/A
TLR9 No change (ID) Survive lethal 10
6
IP challenge N/A N/A
TLR6 No change (IN) N/A Ms, DCs N/A
TLR1 No change (IN) N/A N/A N/A
OTHER
STAT1 Lethal at high dose
(10
3
aerosol) N/A Ms N/A
iNOS Lethal at all doses (ID and aerosol) N/A Ms N/A
p47
phox
Sublethal at low doses; lethal N/A Ms N/A
at moderate doses (LD
50
4 × 10
3
ID)
MMP-9 Less susceptible (IN) N/A N/A N/A
*Data presented are for LVS unless otherwise noted.
Data for F. novicida infection.
§
Data presented describe changes in organ CFUs or survival differences as compared to WT mice. All bacterial doses are expressed in terms of CFU.
Only the cell types for which there is direct or indirect evidence of expression during Francisella infection (in vitro or in vivo) are listed.
N/A, data not available; ID, intradermal; IN, intranasal; IP, intraperitoneal; Ms, macrophages; DCs, dendritic cells.
For references, see corresponding text.
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Cowley and Elkins Immunity to Francisella
LVS in the organs of XBP-1-deficient mice as compared to WT mice
after aerosol infection (Martinon et al., 2010). In a different study,
F. novicida infection of human PBMCs and murine macrophages
resulted in up-regulation of the micro-RNA miR-155 in a manner
that was dependent on TLR2 and MyD88. This micro-RNA blocks
translation of SHIP, a negative regulator of the PI3K/Akt pathway
(Cremer et al., 2009). The net effect of the release of Akt from
inhibition was an increase in pro-inflammatory gene expression.
Indeed, mice that express a constitutively active form of Akt survive
an otherwise lethal F. novicida infection (Rajaram et al., 2009).
Interestingly, experiments using the virulent Type A F. tularensis
SchuS4 indicated that SchuS4 had the opposite effect on miR-155
induction, namely down-regulating the pro-inflammatory cytokine
response. These results thus reveal one mechanism by which viru-
lent Francisella may inhibit early immune responses (Cremer et al.,
2009). These data are also in agreement with Affymetrix microarray
and Western blot analyses of human PBMCs that indicated down-
regulation of the PI3K/Akt pathway and TLR2 after SchuS4, but not
F. novicida, infection (Butchar et al., 2008a; Melillo et al., 2010).
cytokInes and cHemokInes In prImary InfectIon wItH
Francisella
Cytokine production during Francisella infection is a very active
area of research, and our understanding of the important cytokine
and chemokine mediators continues to evolve (for an overview,
refer to Figure 1). Similar to other intracellular pathogens, rapid
production of pro-inflammatory and Th1-type cytokines is critical
for initial control of Francisella infection in all settings examined to
date. However, as noted earlier, there is a striking lack of crucial pro-
inflammatory cytokines during the first 48 h of murine pulmonary
Francisella infection. It is not until after the first 48–72 h of murine
infection that key cytokines and chemokines become readily detecta-
ble. During virulent F. tularensis respiratory infection of mice, mRNA
levels of essential antimicrobial cytokines such as IFN-γ and TNF-α
rose in the lungs between days 2 and 4 (Andersson et al., 2006b), and
serum/distal organ levels of pro-inflammatory mediators such as
RANTES, IL-6, and IL-1β became detectable on days 3–4 (Conlan
et al., 2008). However, after 2 days of unrestricted bacterial growth,
key organs such as the lungs and liver harbor extremely high bacte-
rial burdens. Thus the relatively late up-regulation of antimicrobial
host immune mechanisms appears to be too late to prevent death.
Indeed, a number of studies support the hypothesis that augment-
ing production of pro-inflammatory cytokines very early during
infection can be beneficial. For example, mice administered either a
synthetic TLR4 agonist, the TLR3 agonist poly I:C, or recombinant
IL-12 shortly before inhalation of Francisella exhibited diminished
organ bacterial burdens, and enhanced survival (Duckett et al., 2005;
Lembo et al., 2008; Pyles et al., 2010).
The widespread up-regulation of multiple cytokines and
chemokines by day 3 after murine F. novicida pulmonary infec-
tion resembles a cytokine storm that is associated with severe
sepsis – a condition characterized by excessive production of pro-
inflammatory cytokines that culminates in capillary leakage, tissue
injury, and organ failure. One mediator of severe sepsis, the nuclear
DNA-binding protein HMGB-1, was strongly up-regulated and
localized extracellularly in mouse lungs by day 3 after F. novicida
intranasal infection (Mares et al., 2008). Thus damage-associated
molecular pattern molecules such as HMGB-1 are shed from
dying host cells, and may be responsible for lethal severe sepsis.
This striking outcome was age-dependent: older mice given a pul-
monary F. novicida infection had significantly increased survival
and an associated reduction in development of hypercytokinemia
and cell death (Mares et al., 2008). Whether a similar intriguing
phenomenon is responsible for mortality during LVS or virulent
F. tularensis infection in vivo remains to be determined, but macro-
phages infected in vitro with SchuS4 also release HMGB-1 (Mares
et al., 2008).
Consistent with the ability of virulent F. tularensis strains to
initially suppress and then ultimately overwhelm the murine
immune response, most studies examining the role of various
cytokines and chemokines in this infection model have shown
that mice deficient for such key cytokines as IFN-γ, TNF-α,
lymphotoxin-α, and iNOS exhibit the same extreme susceptibil-
ity to infection via the pulmonary and parenteral routes as fully
immunocompetent mice (Chen et al., 2004; Zhang et al., 2008). In
contrast, the lower virulence of LVS allows for the determination of
a spectrum of susceptibility for the different cytokines and chem-
okines that contribute to immunity. Thus far, the only cytokine-
deficient mice that are exquisitely susceptible to all doses of LVS
delivered via any route are those that lack either of the canonical
Th1 cytokines, IFN-γ or TNF-α (for summary, see Table 3; Elkins
et al., 2007). Mice deficient for either cytokine usually die within
a week after inoculation, suggesting that early innate immune cell
production of IFN-γ and TNF-α is critical for survival. A recent
study of the cell types that produce IFN-γ after primary sublethal
LVS ID infection revealed that a wide variety of liver and spleen
innate immune cells produce IFN-γ during the first 7 days after
infection, including NK cells, neutrophils, DCs, and cells that match
the staining profile of “NK DCs” (De Pascalis et al., 2008). Given
that both IFN-γ and TNF-α are important for macrophage produc-
tion of RNI, and that RNI are effective mediators of inhibition of
LVS intramacrophage growth in vitro, it is likely that induction of
iNOS-derived products is one critical early role for these cytokines
in vivo (Anthony et al., 1992; Lindgren et al., 2004, 2005). Indeed,
iNOS-deficient mice die following sublethal LVS infections initiated
via a variety of routes (Lindgren et al., 2004), although are not as
dramatically impaired as IFN-γ or TNF-α knockout mice. Although
the cells that produce TNF-α in response to ID LVS infection have
not yet been systematically identified, the role of TNF-α during
in vivo primary ID LVS infection clearly includes induction of reac-
tive nitrogen species (RNS; Cowley et al., 2008). Further, membrane
TNF-α is sufficient to partially mediate resistance to primary LVS
ID infection, since mice that express only the membrane-bound
form of TNF-α (and not the soluble form) exhibited an intermedi-
ate level of susceptibility to LVS infection, as well as intermediate
levels of RNS (Cowley et al., 2008).
In contrast to the prototypic Th1 cytokines, Th2 mediators have
not been examined in much detail during in vivo infection. Mice
treated with anti-IL-4 antibodies and then infected with LVS ID
exhibited an ID LD
50
that was comparable to wild type mice, if
not higher (Leiby et al., 1992), while IL-4 knockout mice were
found to be only slightly more susceptible to IN LVS pulmonary
infection (Ketavarapu et al., 2008), and IL-10 knockout mice were
considerably more susceptible to IN LVS pulmonary infection
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Cowley and Elkins Immunity to Francisella
occurs in mice given LVS infection via other routes awaits compre-
hensive characterization: mice deficient for p35, p40, p19, and/or
their associated receptors were more susceptible to pulmonary LVS
infection, but the studies to date only used doses approaching the
LD
50
(Duckett et al., 2005; Lin et al., 2009).
IL-17A has an unexpected and critical role in primary LVS pul-
monary murine infection. First detectable in mouse lungs by day 3
after infection, by days 6–7 the IL-17A-producing T cells identified
in mouse lungs included CD4
+
T cells, CD8
+
T cells, double nega-
tive (DN) T cells, and γ/δ T cells (Lin et al., 2009; Cowley et al.,
2010; Markel et al., 2010). The role of IL-17A in LVS pulmonary
infection appears to be multi-fold; IL-17A stimulates LVS-infected
DCs to up-regulate production of IL-12 and IFN-γ in vitro, and
stimulates IFN-γ production by ovalbumin-specific transgenic T
cells. Thus IL-17A appears poised to augment early IFN-γ produc-
tion and aid in polarization of Th1 cells (Lin et al., 2009). However,
IL-17A also has a role in the later stages of in vivo infection (days
10–21), when T cell-mediated immunity is critical for clearance
of the infection: IL-17A knockout mice given a sublethal LVS pul-
monary infection exhibit significantly increased bacterial organ
burdens at these late time points (Cowley et al., 2010). Higher
numbers of IFN-γ-producing CD4
+
and DN T cells were present in
the lungs of IL-17A KO mice than their WT counterparts at these
later time points, indicating that although IL-17A has a role in
inducing early Th1 immunity, IFN-γ-producing T cells are capable
of responding to the infection at later time points. Importantly, the
discovery that IL-17A can act in concert with IFN-γ to inhibit LVS
intracellular growth in macrophages and alveolar type II epithelial
(Metzger et al., 2007). Interestingly, as discussed in more detail
below, IL-4 receptor α chain knockout mice were less susceptible
to lethal IP LVS challenges (Shirey et al., 2008).
Unlike IFN-γ and TNF-α, the role for IL-12 in primary in vivo
LVS infection is more nuanced. IL-12 is a heterodimer that consists
of two distinct proteins, the p35 and p40 subunits. In addition to
constituting one component of IL-12, the p40 subunit can also pair
with another protein, denoted p19, to produce the IL-23 heterodimer.
Thus, mice deficient for p35 lack IL-12, whereas mice deficient for
p40 lack both IL-12 and IL-23. Although both p35- and p40-deficient
mice can survive sublethal LVS ID infection, they are both clearly
compromised. Whereas 35-deficient mice exhibit higher bacterial
organ burdens and cleared the infection more slowly than WT
mice, p40 KO mice were unable to clear ID LVS infection, exhibiting
chronic high liver and spleen bacterial numbers (Elkins et al., 2002).
Both types of LVS-infected knockout mice exhibit reduced levels of
serum IFN-γ (Elkins et al., 2002), a finding that is consistent with
the role of IL-12 in positive feedback regulation of IFN-γ production
by T cells and NK cells. Interestingly, p40-deficient mice exhibited
a greater defect in IFN-γ-production than their p35 counterparts,
indicating that IL-23 may have an additional role in inducing IFN-γ
production (Elkins et al., 2002). Indeed, recent studies found that
IL-23 produced by Francisella-infected human monocytes induced
NK cell production of IFN-γ, indicating that both IL-23 and IL-12
can positively regulate IFN-γ production (Butchar et al., 2007, 2008b).
The unique phenotype of p40 knockout mice indicates that p40 and
by extension, IL-23 has an as-yet unidentified role in the clearance
of sublethal murine LVS ID infection. Whether a similar phenotype
Table 3 | The contribution of cytokines and chemokines to in vivo murine infection with Francisella tularensis*.
Component Effect of depletion/knockout
§
Cellular source(s)
1° infection 2° infection Innate Acquired (T cells)
IFN-γ Lethal at all Sublethal at some challenge Ms, DCs, NK CD4
+
, CD8
+
, and DN
doses (ID, IV, and IN) doses (2 × 10
6
IV and 10
2
IP) cells, “NK DCs”
TNF-α Lethal at all doses (ID, IV, and IN); Sublethal at lower challenge doses (9 × 10
3
IV); Ms, DCs CD4
+
, CD8
+
memTNF survive only low doses (10
2
ID) memTNF survive high lethal 5 × 10
5
IP challenge
IL-17A Sublethal at moderate doses (10
4
ID and N/A γδ T cells CD4
+
, CD8
+
, and DN
10
2
IN); lethal at high doses (10
3
IN and IT)
IL-18 No change (ID) Survive high lethal 10
6
IP challenge N/A N/A
IL-4 No change (ID) N/A Mast cells N/A
IL-10 More susceptible at high dose (10
3
IN) N/A N/A N/A
IL-12p35 Sublethal at moderate doses, infection cleared Survive high lethal 5 × 10
6
IP challenge Ms, DCs N/A
(10
6
ID); lethal at high doses (10
3
IN and IT)
IL-12p40 Sublethal at moderate doses, chronic infection Survive high lethal 10
6
IP challenge Ms, DCs N/A
(10
6
ID); lethal at high doses (10
3
IN and IT)
IL-23p19 Lethal at high doses (10
3
IT) N/A N/A N/A
IL-22 No change (IT) N/A N/A N/A
Mig/CXCR3 No change (ID) Survive high lethal IP challenge N/A N/A
CX3CR1 No change (IN) N/A N/A N/A
*Data presented are for LVS unless otherwise noted.
§
Data presented are for survival (or organ CFUs) as compared to WT mice.
Only the cell types for which there is direct or indirect evidence of expression during Francisella infection (in vitro or in vivo) are listed.
N/A, data not available; IN, intranasal; IT, intratracheal; ID, intradermal; IV, intravenous; IP, intraperitoneal; MTD, mean time to death; Ms, macrophages; DCs,
dendritic cells.
For references, see corresponding text.
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Cowley and Elkins Immunity to Francisella
(Anthony et al., 1991; Ben Nasr et al., 2006; Katz et al., 2006; Chase
et al., 2009; Chase and Bosio, 2010). Further, Francisella can grow
in vitro in murine phagocytes harvested from a variety of differ-
ent tissues, including elicited peritoneal macrophages, alveolar
macrophages, BMDMs, bone marrow-derived DCs, and alveolar
DCs (Anthony et al., 1991; Polsinelli et al., 1994; Bosio and Elkins,
2001; Bosio et al., 2007). In the absence of exogenous factors that
induce activation, intracellular bacterial growth continues unre-
stricted until death of the host cell; at high multiplicities of LVS
infection, this process results in caspase 3-dependent apoptosis
of macrophages in vitro (Lai et al., 2001; Lai and Sjostedt, 2003).
Although this phenomenon has not been demonstrated in vivo
with LVS, virulent Type A Francisella induced widespread cas-
pase 3-dependent apoptosis of macrophages in the organs of mice
infected IN. Thus unrestricted growth of Francisella can induce
apoptotic death of infected host cells in vivo as well as in vitro
(Wickstrum et al., 2009).
cells (ATII) in vitro indicates that IL-17A can be a potent effector
cytokine with more than just regulatory properties (Lin et al., 2009;
Cowley et al., 2010).
IL-17A is perhaps best known for its ability to recruit neutrophils
to the site of infection. In LVS pulmonary infection, IL-17A-deficient
mice exhibited decreased levels of lung G-CSF that was accompanied
by a reduction in the proportion of lung neutrophils at early time
points (days 4 and 6) after infection (Lin et al., 2009; Cowley et al.,
2010). Interestingly, type 1 Interferon receptor knockout mice exhibit
increased resistance to ID F. novicida infection, a phenomenon that
was associated with the ability of Type I IFNs to down-regulate the
number of IL-17A
+
γ/δ T cells and diminished recruitment of neu-
trophils to infected spleens (Henry et al., 2010). A similar Type I IFN-
mediated negative regulation of γ/δ T cell IL-17A production was also
observed in mice infected IN with SchuS4, as well as mice given IV L.
monocytogenes infection. Thus, low levels of IL-17A production by γ/δ
T cells during Francisella infection may be a consequence of negative
regulation effected by Type I IFNs. This outcome was at least partially
attributed to the ability of type I IFNs to induce IL-27 production by
Francisella-infected macrophages (Henry et al., 2010).
The role of chemokines in host responses to Francisella infec-
tion is only beginning to be elucidated. Potent mediators of cell
trafficking, chemokines are responsible for drawing critical cell
types to the site of infection. Indeed, multiple cell types have been
shown to produce chemokines in response to Francisella infection,
including DCs, endothelial cells, alveolar type II cells, and macro-
phages. Despite the abundant production of many chemokines
in response to Francisella infection, only a few have been directly
examined to date. CCR2 knockout mice, which lack responses to
MCP-1/3/5 group of macrophage chemotactic proteins, are quite
susceptible to ID LVS infection compared to knockout mice, and
fail to exhibit increases in numbers of responding lymphoid and
myeloid cells typically found in the spleens of LVS-infected wild
type mice (Meierovics and Elkins, manuscript in preparation).
However, mice lacking Mig or its receptor CXCR3 did not exhibit
increased susceptibility to primary sublethal ID or secondary lethal
IP LVS challenges (Park et al., 2002). Similarly, mice deficient for
the chemokine receptor CX3CR1 were not more susceptible to IN
LVS infection, although they did exhibit modest but significant
dysregulation in recruitment of monocytes, neutrophils, and DCs
to the lungs (Hall et al., 2009). Chemokines and/or their receptors
are functionally redundant, so future studies utilizing mice that
are multiply deficient for these factors will no doubt be needed
to better define the critical roles of chemokines during in vivo
Francisella infection.
Innate Immune responses followIng InteractIons
of Francisella wItH Host cells
macropHages and dendrItIc cells
The virulence of Francisella has long been associated with its abil-
ity to exploit host phagocytic cells to support its own growth. In
particular, the ability of macrophages and DCs from a variety of
different host tissues to act as a replicative niche, as well as pro-
vide antimicrobial effector functions, has been intensively studied.
Resident peritoneal macrophages from rats, mice, and guinea pigs,
as well as human peripheral blood monocytes and monocyte-
derived DCs, support Francisella growth when infected in vitro
FIGURE 1 | Components of murine innate and adaptive immune
responses to Francisella. (1) The initial interaction of Francisella with host
cells, such as macrophages, dendritic cells, epithelial cells, and endothelial
cells, stimulates production of pro-inflammatory cytokines and chemokines
(2a) in a manner that is dependent upon MyD88, TLR2, and other unidentified
receptors that signal through MyD88. Bacterial DNA engagement of the
NOD-like receptor (NLR) AIM2 may also be critical for inflammasome
assembly and release of IL-1β. Simultaneously, important innate immune cells
recruited to the area of infection produce effector cytokines such as IL-12p40,
TNF-α, IFN-γ, and IL-17A (2b) that influence T cell development (3), and induce
host cell production of antimicrobial molecules (4). In addition to the classic
TH1-type cytokines, other mediators include mast cell production of IL-4,
which can directly inhibit Francisella intramacrophage growth, and B-1a B cell
production of anti-LPS antibodies that limit intraperitoneal infection. PMNs are
essential for survival of Francisella infections initiated via some routes, but fail
to eradicate intracellular organisms in vitro, so their contribution to infection
remains unclear. After several days, activation and expansion of Francisella-
specific T cells and B cells occurs (3). αβ T cells are essential for clearance of
primary infection, and produce effector cytokines such as IL-17A and IFN-γ, as
well as the membrane-bound and soluble forms of TNF-α. These factors
presumably amplify and extend activation of infected host cells to limit
Francisella intracellular growth through production of reactive oxygen and
nitrogen intermediates, as well as other unidentified antimicrobials (4).
Asterisks (*) indicate host cells that have been shown to harbor intracellular
Francisella. The blue box indicates cell types that are neither fully innate nor
adaptive, based on classical definitions.
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Cowley and Elkins Immunity to Francisella
pro-inflammatory cytokine production and reduced expression of
alternative activation markers (Shirey et al., 2008). Similarly, lethal
pulmonary F. novicida infection resulted in the development of
alternatively activated macrophages in mouse lungs (Mares et al.,
2010). Alternative macrophage activation in the F. novicida model
was exacerbated by the accumulation of dead cell debris in vitro and
the poor ability of F. novicida-infected macrophages to perform effe-
rocytosis (the uptake and clearing of dead cells; Mares et al., 2010).
Thus, it appears that during lethal Francisella infections, alternative
activation of macrophages diminishes pro-inflammatory cytokine
production and the generation of macrophage effector mechanisms,
and may contribute to the progression of lethal disease.
Using several different F. tularensis strains in the mouse model
of pulmonary infection, studies have demonstrated that both DCs
and macrophages in the lung were infected within 1 h after inocula-
tion; these remained the predominant infected cell types by 24 h
after infection (Bosio and Dow, 2005; Hall et al., 2008). Indeed,
Francisella grows unrestricted within both human and mouse
DCs in vitro until the host cells die (Ben Nasr et al., 2006; Bosio
et al., 2007). In vitro and in vivo evidence indicates that Francisella-
infected human and mouse DCs are actively immunosuppressed
by the bacterium. Both are impaired in the ability to produce pro-
inflammatory cytokines, and are refractory to stimulation with
potent immunomodulators such as E. coli LPS (Bosio et al., 2007;
Chase et al., 2009). This immunosuppression has been attributed to
a variety of factors, including the ability of SchuS4 to increase pro-
duction of the immunosuppressive cytokine TGF-β (an indicator
of alternative macrophage activation, as described above), as well
as the absence of CD14 expression by pulmonary DCs (Bosio et al.,
2007; Chase and Bosio, 2010). IN administration of soluble CD14
to SchuS4-infected mice increased lung cytokine production, and
reduced SchuS4 replication in the lungs and dissemination to the
spleen (Chase and Bosio, 2010). Thus CD14 increased the capacity
of DCs to detect” SchuS4 infection, although paradoxically CD14
is not normally abundant in pulmonary tissues and thus its paucity
may contribute to SchuS4 immune evasion. Interestingly, recent
data has shown that DCs not only serve as a silent replicative niche
for Francisella after respiratory infection, but they also transport
the pathogen to the mediastinal lymph nodes, and therefore may
play a prominent role in early pathogen dissemination (Bar-Haim
et al., 2008).
non-pHagocytIc cells
In recent years the spectrum of cell types infected by Francisella
has broadened to include non-phagocytic cells, such as kidney
epithelial cells, ATII, hepatocytes, and fibroblasts. The contribu-
tion of non-phagocytic cells to Francisella virulence was revealed
by a recent study using a pyrF SchuS4 mutant. Although this
mutant grew less vigorously than WT bacteria in macrophages, it
exhibited close to WT levels of growth in some non-macrophage
cell types, and retained full virulence in the murine model of IT
infection (Horzempa et al., 2010). Although not conclusive, this
study suggests that Francisella growth in non-macrophage cell types
contributes substantially to the virulence of the organism.
Alveolar epithelial cells, which form the interface between the
outside environment and the host lung interior, are well positioned
to interact with Francisella very early during respiratory infection.
Similar to many other intracellular pathogens, treatment of
infected macrophages ex vivo with IFN-γ inhibits Francisella growth.
In quiescent murine and human macrophages, Francisella survives
in these cells by rapidly escaping the phagosome to replicate in the
cytosol, thereby circumventing phagosome–lysosome fusion and
the associated bactericidal effects. LVS phagosomal escape was par-
tially diminished in IFN-γ-treated murine peritoneal exudate cells
(PECs; Lindgren et al., 2004). Similarly, control of F. novicida growth
by IFN-γ-activated human monocyte-derived macrophages was
attributed to its inability to disrupt phagosome–lysosome fusion in
the activated cells (Santic et al., 2005). These data suggest that pre-
vention of efficient escape from the phagosome is one mechanism
by which IFN-γ inhibits Francisella intracellular growth. However,
more recent studies examining LVS and SchuS4 growth in murine
BMDMs and the murine macrophage-like cell line J774.1 demon-
strated that IFN-γ-activation did not disrupt phagosomal escape
(Bonquist et al., 2008; Edwards et al., 2010). These discrepancies
are likely to be related to the different Francisella subsp. and host
cells used for the different studies; regardless, although disruption
of phagosomal escape is one potential mechanism by which IFN-
γ-activation could diminish Francisella intracellular growth, it is
certainly not the only mechanism at work.
The generation of reactive oxygen species (ROS) and RNS are
two well-known mechanisms elicited by IFN-γ that can inhibit the
growth of intracellular pathogens. Several studies have focused on
defining the role of these two classic mechanisms in the control of
Francisella growth both in vitro and in vivo. Optimal IFN-γ-induced
inhibition of LVS growth in murine PECs required the combined
action of Phox and iNOS, and was dependent upon the generation
of peroxynitrite (Lindgren et al., 2005). These data are supported by
the increased susceptibility of mice deficient for either iNOS or the
phagocyte oxidase p47
phox
to ID LVS infection as compared to WT
mice (Lindgren et al., 2004). In contrast, killing of virulent SchuS4
by IFN-γ-activated murine PECs was only partially dependent on
iNOS, and correspondingly, SchuS4 was very resistant to killing by
H
2
O
2
and peroxynitrite compared to LVS (Lindgren et al., 2007).
Further, a study using murine gp91
phox
/iNOS double-deficient
BMDMs determined that control of SchuS4 growth by IFN-γ was
independent of ROS and RNS, iron sequestration, and tryptophan
depletion by indoleamine 2,3-dioxygenase (Edwards et al., 2010).
Therefore, the IFN-γ-elicited bactericidal mechanism(s) involved
in inhibition of SchuS4 growth in activated macrophages remain to
be fully elucidated, while the less virulent LVS is more susceptible
to the well-known bactericidal effects of ROS and RNS.
Interestingly, although LVS-infected murine macrophages ini-
tially produce pro-inflammatory cytokines in vitro, after several
hours they begin to exhibit an “alternatively activated” phenotype
a condition characterized by mitigation of the pro-inflammatory
response (including iNOS expression) and up-regulation of anti-
inflammatory cytokines such as IL-4, IL-13, and TGF-β (Shirey
et al., 2008). Differentiation of LVS-infected macrophages into the
alternatively activated phenotype required TLR2, IL-4, and IL-13.
These alternatively activated macrophages are evident in the peri-
toneum upon in vivo IP LVS infection, and mice deficient for the
IL-4 receptor α chain (used by both IL-4 and IL-13 for signaling)
exhibited increased survival following a normally lethal IP LVS
infection. This increased resistance was associated with prolonged
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Cowley and Elkins Immunity to Francisella
their critical contribution to early survival of systemic infection may
lie in their ability to secrete cytokines and chemokines that recruit
other important effector cells to the site of infection.
In contrast, the effect of neutrophil depletion on pulmonary
LVS infection was much less striking than that noted for parenter-
ally infected mice, with only a minor increase in bacterial burdens
in the livers when using a dose just above the LD
50
(Conlan et al.,
2002a). Regardless, neutrophils are clearly actively recruited to
Francisella-infected lungs: recent studies show that neutrophils
constitute as much as 50% of the Francisella-infected cells in the
lungs of mice 3 days after pulmonary LVS and SchuS4 infection
(Hall et al., 2008), and depend at least partially on IL-17A for their
early recruitment to the lungs (Lin et al., 2009; Cowley et al., 2010).
However, excessive recruitment of neutrophils to the lung appears
to contribute to Francisella pathogenesis: mice deficient for matrix
metalloproteinase 9 which generates neutrophil chemoattractants
via cleavage of the extracellular matrix exhibited reduced neu-
trophil numbers in the lungs after LVS pulmonary infection, and an
associated reduction in bacterial burden that was accompanied by
increased survival (Malik et al., 2007). Thus, the role of neutrophils
in Francisella infection is likely to be a fine balance between aiding
in control of infection and exacerbation of pathology.
mast cells
Mast cells are classically known for their involvement in allergic con-
ditions associated with type I hypersensitivity reactions (Geha, 2003).
However, recent data has revealed an unexpected and intriguing role
for mast cells and IL-4 in control of respiratory Francisella infection.
Mast cell-deficient mice were much more susceptible to pulmonary
LVS infection than WT mice, exhibiting diminished production of
IL-4 in the lungs (Ketavarapu et al., 2008). Further, IL-4-deficient mice
were slightly more susceptible to LVS pulmonary infection. In vitro,
mast cells inhibited LVS intramacrophage growth in a manner that
was dependent on IL-4 (Ketavarapu et al., 2008). Overall, these data
suggest that mast cells are capable of IL-4-dependent inhibition of
LVS growth in macrophages, a phenomenon that was associated with
increased macrophage cellular ATP levels and co-localization of LVS
with acidified organelles (Rodriguez et al., 2010).
nk cells
Numerous studies have shown that NK cell-deficient mice are not
more susceptible to IN or ID LVS infection than fully immunocom-
petent mice (Lopez et al., 2004; Duckett et al., 2005; Bokhari et al.,
2008), although clear conclusions from these studies are hampered
by the inability to fully deplete mice of NK cells in vivo. Despite
this limitation, it is clear that NK cells are important producers of
IFN-γ during primary LVS infection initiated via both the ID and
IN routes. During the first 96 h after an IN LVS infection, a subset
of CD11b
+
DX5
+
NK1.1
+
cells were primarily responsible for IFN-γ
production in the lungs and livers (Lopez et al., 2004; Bokhari et al.,
2008), while NK1.1
+
cells were a large proportion of the IFN-γ
producers in the spleens and livers of mice for the first 5–7 days
after an ID LVS infection (De Pascalis et al., 2008). The NK1.1
+
cell types that produce IFN-γ in response to LVS infection were
quite heterogeneous, and could be sub-divided into populations
phenotypically reminiscent of NK T cells and “NK DCs” (Bokhari
et al., 2008; De Pascalis et al., 2008).
TEM micrographs showed LVS in contact with ATII cells in the
airways of mice 2 h after an IN infection (Gentry et al., 2007).
In vitro cultures of primary human ATII cells stimulated with
SchuS4 and LVS produced high levels of IL-8, MCP-1, GRO-α, and
GM-CSF (Gentry et al., 2007). Further, these conditioned culture
supernatants induced transmigration of PMNs and DCs through
cultured primary human pulmonary microvasculature endothelial
cells (HVECs), a cell type that lines blood vessels and is found in
close juxtaposition to ATII cells in vivo (Gentry et al., 2007). Studies
of the direct interaction of Francisella with HVECs showed that
LVS could be internalized – but did not replicate in these cells,
inducing a blunted pro-inflammatory response and transmigra-
tion of PMNs with a suppressed phenotype (Forestal et al., 2003;
Moreland et al., 2009; Bublitz et al., 2010). It is not yet known
whether PMNs that transmigrate across the endothelial layer in
response to chemokines produced by Francisella-infected ATII cells
are also functionally inhibited. Regardless, it is clear that, in addi-
tion to providing an early replicative niche for Francisella, ATII cells
have the potential to play a vital role in initiation of inflammatory
immune responses and recruitment of key immune cells.
The mechanisms exploited by Francisella for uptake and growth
in these non-macrophage cell types as well as the immune mecha-
nisms that ultimately control this growth – are only beginning to
be understood. Thus far it is clear that LVS uses host processes for
invasion of murine ATII cells, and similar to its growth in macro-
phages escapes the initial phagosome to replicate in the cytoplasm
(Craven et al., 2008). LVS infection of the human ATII cell line A549
resulted in up-regulation of the antimicrobial β-defensin molecule
hBD-2, but not hBD-3, the β-defensin that had the most potent
anti-Francisella activity in a cell-free system (Han et al., 2008).
Therefore, Francisella avoids eliciting detrimental antimicrobial
mechanisms in resting ATII cells. However, the combined action
of recombinant IFN-γ and IL-17A limited LVS growth in a murine
ATII cell line in vitro (Cowley et al., 2010), and thus ATII cells can
be activated to produce antimicrobial activity against Francisella.
neutropHIls
Another important cell type that responds early to Francisella infec-
tion is the neutrophil. Neutrophils are key innate immune cells
that use toxic ROS, cationic peptides, and degradative enzymes to
kill ingested pathogens. In particular, the multicomponent enzyme
NADPH oxidase, which catalyzes the conversion of molecular oxy-
gen to superoxide anions, is a primary antimicrobial weapon in the
neutrophil arsenal. Interestingly, although LVS and SchuS4 were
readily phagocytosed by human neutrophils in vitro, they inhibited
NADPH oxidase assembly and the associated production of ROS
(McCaffrey and Allen, 2006; McCaffrey et al., 2010). Further, instead
of being killed by neutrophils, Francisella escaped from the phago-
some and persisted in the neutrophil cytosol (McCaffrey and Allen,
2006). Thus, instead of killing Francisella, neutrophils appear to pro-
vide a safe if not replicative niche for the organism. Nonetheless,
in mice the in vivo importance of neutrophils in defense against
systemic Francisella infection is clear: neutrophil-depleted mice are
highly susceptible to otherwise sublethal parenteral LVS infections,
succumbing quickly to an overwhelming disseminated infection
(Sjostedt et al., 1994; Elkins et al., 1996; Conlan et al., 2002a). The
inability of neutrophils to kill intracellular Francisella suggests that
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Cowley and Elkins Immunity to Francisella
although transfer of anti-LVS or anti-Francisella LPS antibodies
conferred partial protection against LVS and the less virulent Type
B Francisella challenges (Fortier et al., 1991; Fulop et al., 1995, 2001;
Conlan et al., 2002b; Stenmark et al., 2003; Kirimanjeswara et al.,
2007; Lavine et al., 2007). Similarly, efforts to stimulate protective
immunity in mice through vaccination with Francisella LPS or
its protein-conjugated derivatives have only met with success fol-
lowing challenge with LVS or the less virulent Type B Francisella
(Fulop et al., 1995; Conlan et al., 2002b, 2003; Kieffer et al., 2003;
Cole et al., 2009).
Although administration of inactivated preparations of
Francisella or its components have historically been unable to confer
protective immunity to virulent Francisella challenge, recent studies
have challenged this dogma; further, some of these strategies are
antibody-dependent. Vaccination with inactivated Francisella prep-
arations augmented by co-administration with immunostimula-
tory compounds can induce antibody-dependent protection against
challenge with the less virulent LVS. Mice given an IN vaccination of
killed LVS and IL-12 survived a subsequent lethal IN LVS challenge
in an IgA-dependent manner (Baron et al., 2007). Similarly, mice
administered heat-killed LVS IP alongside an IL-12 expressing viral
vector survived a high dose lethal IP LVS challenge in a manner that
was mediated by antibodies (Lavine et al., 2007). The ability of IL-12
administration to augment humoral immunity and protect against
virulent Francisella challenge remains to be explored. Further, mice
vaccinated several times IP with Francisella outer membrane pro-
teins or ethanol-inactivated LVS were significantly protected against
subsequent IN Type A F. tularensis challenge (40–50% survival;
Huntley et al., 2008). Intramuscular immunization of mice with
killed LVS in conjunction with immunostimulatory complexes
(ISCOMs) admixed with CpG protected 40% of mice against a
low dose SC challenge with SchuS4 (Eyles et al., 2008). Further,
intranasal immunization of mice with inactivated LVS and the
mucosal adjuvant cholera toxin B subunit resulted in substantial
protection against lethal IN LVS challenge, and partial protection
against IN SchuS4 challenge (Bitsaktsis et al., 2009). However, in
this case the presence of a mucosal adjuvant augmented Th1-type
responses, such that the protection mediated against LVS challenge
was fully operative in BKO mice; thus, the observed protection was
not actually mediated by B cells or antibodies. Overall, it appears
that antibody-mediated immune responses generated by immuni-
zation with live or killed LVS or antigenic Francisella preparations
are, at best, partially protective against virulent strains of Francisella,
and that T cell functions are necessary to achieve optimal pro-
tection. Interestingly, however, antisera from mice immunized IN
with virulent SchuS4 and subsequently rescued by levofloxacin
treatment was able to protect 90% of naive mice against a lethal
SchuS4 IN challenge (Klimpel et al., 2008).
A recent study reveals an interesting mechanism by which viru-
lent Francisella may evade the protective effects of anti-Francisella
antibodies. The highly virulent Type A strain SchuS4 but not
LVS can directly bind plasmin, a host serine protease that degrades
opsonizing antibodies, thus inhibiting antibody-mediated uptake of
SchuS4 by macrophages (Crane et al., 2009). Importantly, antibody-
opsonized SchuS4 elicited increased production of TNF-α and IL-6
from macrophages, an effect that was reduced by the addition of plas-
min. Thus, the ability of SchuS4 to bind plasmin likely contributes to
Further studies investigating the role of NK cell production of
IFN-γ in Francisella infection have revealed some of its downstream
impacts. For example, human monocyte production of IL-23 in
responses to Francisella infection induced NK cells to produce
IFN-γ; this IFN-γ subsequently up-regulated monocyte produc-
tion of IL-23 and IL-12p70, establishing NK cells at the center of
a positive feedback loop for IL-23 and IFN-γ production (Butchar
et al., 2007, 2008b). In addition, NK cell production of IFN-γ was
critical for hepatic granuloma formation during IN LVS infection;
depletion of NK cells although not lethal to the mice resulted in
“leaky” granulomas that poorly contained the infection (Bokhari
et al., 2008).
Overall, although NK1.1
+
cells clearly respond vigorously to early
Francisella infection, much of their contribution identified thus
far relates to IFN-γ-production; the role of the lytic activities of
NK cells has not been studied directly. Since NK1.1
+
cells are not
the only innate immune cells capable of producing IFN-γ during
these early critical time points (other cell types identified included
DCs, PMNs, and macrophages; De Pascalis et al., 2008), it is likely
that compensation by these other cells explains the dispensability
of NK cells during survival of primary sublethal LVS infection.
Indeed, mice depleted of NK cells during primary IN LVS infection
exhibited only a 50% decrease in serum IFN-γ as compared to their
WT counterparts (Bokhari et al., 2008).
elements of tHe adaptIve Immune response
Elucidation of the mechanisms involved in protection against intra-
cellular pathogens is critical for successful vaccine development.
Adaptive immune responses typically develop over a longer time
frame than that of innate immunity, first requiring activation and
clonal expansion of antigen-specific B and T cells. The resulting
memory T and B cells respond rapidly to a second exposure to their
cognate antigen, thus forming the basis of vaccine-induced immu-
nity. For many intracellular pathogens, cellular immune responses
have received the greatest attention; organisms are sequestered
within cells and relatively inaccessible to antibodies, and as dis-
cussed above, serum antibody levels often do not correlate with
protection. Thus T cell mechanisms including cytokine production
and cytotoxicity are logical candidates for effecting pathogen eradi-
cation. However, recent data demonstrating significant extracellular
phases for Francisella in vivo (Forestal et al., 2007; Yu et al., 2008), as
well as a clear contribution of B cell-mediated functions to protec-
tion against secondary infections with Francisella strains of lower
virulence, are leading to a new appreciation for how a combination
of both B and T cell responses contribute to protective immunity
against Francisella (for an overview, refer to Figure 1).
b lympHocytes and antIbodIes
As discussed above, while specific antibodies are clearly made dur-
ing Francisella infection and following vaccination, their contribu-
tion to protection may be relatively limited, at least in isolation.
Murine studies have used both passive transfer of immune sera and/
or purified antibodies, as well as genetically deficient mice, to elu-
cidate the role of B cells and antibodies in resistance to Francisella
infection. Older studies demonstrated that immune serum trans-
fer to naive animals does not confer protection against the highly
virulent Type A Francisella strains (Thorpe and Marcus, 1967),
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Cowley and Elkins Immunity to Francisella
t lympHocytes: t cell subpopulatIons
Although B cells and their products have significant roles in pro-
tective immunity to Francisella, optimal protection to Francisella
infection clearly requires T cell-mediated immunity. Mice defi-
cient in T cells (such as nu/nu or α/β TCR-deficient mice) initially
control a primary sublethal parenteral LVS infection, but the mice
maintain high bacterial organ burdens and ultimately succumb
after approximately a month. Both CD4
+
and CD8
+
T cells are suffi-
cient to resolve this infection, as mice depleted of either population
individually clear the bacteria from the tissues. In contrast, mice
simultaneously depleted of both CD4
+
and CD8
+
T cells survive –
but do not clear LVS infection, instead developing a long-term
chronic infection for many months that is characterized by steady
levels of bacteria in the organs of the reticuloendothelial system.
Control of LVS infection in these depleted mice has been attrib-
uted to an unusual CD4
CD8
NK1.1
TCRαβ
+
Thy1.2
+
“DN” T cell
population. Enriched populations of these cells potently control
LVS intramacrophage growth in vitro (Cowley et al., 2005). Thus,
a variety of T cells contribute to control of primary parenteral LVS
infection in mice, although only CD4
+
and CD8
+
αβ T cells have
the ability to fully resolve the infection.
The role of T cells in the control and clearance of murine pri-
mary sublethal respiratory LVS infection is considerably less well
studied than parenteral infection. Mice simultaneously depleted
of CD4
+
and CD8
+
T cells and infected with LVS IN also develop a
long-term chronic infection, indicating that one or both of these
T cell subsets are necessary for clearance of pulmonary infections
(Cowley et al., 2010). However, the requirement for the individual
T cell subsets, or αβ TCR
+
T cells as a group, has not been system-
atically studied in pulmonary LVS infection. Since fully immu-
nocompetent mice die following both parenteral and respiratory
primary infections with virulent Francisella, it is not surprising
that infected mice deficient in T cells die at the same time (Chen
et al., 2004; Wu et al., 2005). Interestingly, lethal murine pulmonary
infection with virulent type A F. tularensis has been shown to result
in thymic atrophy and loss of CD4
+
CD8
+
thymocytes (Chen et al.,
2005a), whereas mice given a lethal pulmonary F. novicida infec-
tion exhibited a profound depletion of αβ T cells in their lungs that
was associated with apoptosis (Sharma et al., 2009). Overall, these
data suggest that primary lethal pulmonary Francisella infections
share the ability to undermine the development and/or function
of T cell-mediated immune responses.
t lympHocytes: effector mecHanIsms
Efforts to determine the T cell mechanisms that contribute to
defense against Francisella infection have revealed a small number
of key cytokines that are produced by responding T cells. CD4
+
,
CD8
+
, and DN T cells in the lungs of mice given a primary sublethal
LVS pulmonary infection produce IFN-γ and IL-17A (Woolard
et al., 2008; Cowley et al., 2010). Correspondingly, mice deficient in
either IFN-γ or IL-17A exhibit increased susceptibility to primary
LVS pulmonary and ID infections (Lin et al., 2009; Cowley et al.,
2010; Markel et al., 2010). However, since both cytokines are also
produced by innate immune cells (see above), it is difficult to deter-
mine the relative importance of production of these factors by T
cells versus that of other cell types during primary in vivo infection.
Greater success on this front has been achieved through the use of
its capacity to evade the host antibodies response. Further, the in vivo
protective effects provided by adoptively transferred immune serum
against IN LVS challenge were dependent upon the Fcγ receptor and
alveolar macrophages, indicating a role for opsonophagocytosis in
antibody-mediated protection to LVS (Kirimanjeswara et al., 2007).
Control of LVS growth by IFN-γ-treated alveolar macrophages was
significantly greater when the bacteria had been serum opsonized
prior to uptake (Kirimanjeswara et al., 2007). Since LVS does not
bind plasmin, it is subject to opsonophagocytosis by macrophages
that are stimulated to increase their production of pro-inflammatory
mediators and control intracellular growth more potently. These
phenomena may at least partially explain the aforementioned abil-
ity of adoptively transferred sera to protect against LVS, but not
SchuS4, challenges.
A novel role for “innate immune B cells” in immunity to
Francisella has emerged in the last several years. Called B-1 lym-
phocytes, these cells are primarily located in the pleural cavity,
intestinal mucosa, and the spleen of mice, and rapidly produce
antibodies against T-independent antigens. In the Francisella
murine infection model, these cells mediate a very rapid antibody
response within 2–3 days of LVS or Ft-LPS vaccination. The B-1a B
cell response and resulting antibody secretion provides substantial
protection against moderate lethal LVS challenges administered IP,
and was not dependent on the presence of T cells (Dreisbach et al.,
2000; Cole et al., 2009). In the Ft-LPS-vaccinated mice, B1a cells
proliferated in the spleen, and differentiated into plasma cells that
produced Ft-LPS-specific antibodies that were detectable in the sera
by days 3–4 after immunization. This protection did not depend on
TLR4 and was not elicited by LVS lipid A, indicating that the epitope
recognized by B1a cells is LVS O antigen. Whether these rapid B1a
protective responses provide immunity to Francisella challenge via
other routes of infection remains to be determined. Nonetheless,
the discovery that the resultant anti-Ft-LPS antibodies were detect-
able at low levels in the serum for months after immunization
suggests that they have the capacity to contribute to longer-term
immune responses, in addition to rapid immunity.
It is important to note that the contributions of B cells to pri-
mary and secondary Francisella infections may be quite different.
B cell-deficient mice (BKO) given a primary ID LVS infection con-
trolled bacterial growth with kinetics similar to WT mice with a
minimal impact on overall susceptibility, and similarly, BKO mice
administered a primary low dose aerosol LVS infection also readily
survived (Elkins et al., 1999; Chen et al., 2004). In contrast, LVS-
vaccinated BKO mice were significantly more susceptible than their
WT counterparts to IP LVS secondary challenge, in a manner that
could be readily rescued by transfer of immune splenic B cells but
not immune sera (Elkins et al., 1999). Thus, in addition to revealing
a more significant role for B cells in secondary as compared to pri-
mary infection, these results further suggest that part but not all of
the contribution of B cells to protective immunity during secondary
LVS challenge is antibody-mediated. The role of other functions
of B cells, such as production of cytokines and chemokines and
antigen presentation, remains to be fully explored. Interestingly,
recent evidence indicates that Francisella can grow inside B cells,
ultimately inducing apoptosis of the infected cells (Krocova et al.,
2008). Thus the potential for Francisella to subvert B cell functions
from within remains an interesting avenue for future research.
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Cowley and Elkins Immunity to Francisella
an in vitro co-culture system, which directly measures the ability of
LVS-immune T cells to control LVS intramacrophage growth. Using
this system, it is apparent that IFN-γ and TNF-α contribute to the
ability of all three T cell subsets to control LVS intramacrophage
growth. However, the different T cell subsets utilize these two fac-
tors differentially: LVS-immune CD4
+
T cells exhibited the greatest
reliance on IFN-γ to control LVS growth, while LVS-immune CD8
+
and DN T cells displayed a greater degree of IFN-γ-independence
(Cowley and Elkins, 2003). In contrast, LVS-immune CD4
+
T cells
did not require TNF-α to control LVS intramacrophage growth,
while CD8
+
T cells were almost completely reliant on TNF-α pro-
duction (Cowley et al., 2007). Further, LVS-immune CD8
+
T cells
were capable of utilizing the membrane-bound form of TNF-α, in
the absence of soluble TNF-α, to execute full control of LVS growth
in vitro (Cowley et al., 2007).
Secondary immune responses to LVS vaccination in mice have
typically been studied by administering either IP, IV, or pulmonary
Francisella challenges at doses that are lethal for a naive mouse. For
secondary LVS IP challenges, all three αβ T cell subsets CD4
+
,
CD8
+
, and DN T cells are sufficient individually for survival
and clearance of an LVS infection. However, mice with only DN
T cells (i.e., simultaneously depleted of both CD4
+
and CD8
+
T cells)
eradicated the infection more slowly. In contrast, full resistance to
virulent F. tularensis pulmonary secondary challenges of vaccinated
mice requires both CD4
+
and CD8
+
T cells, as depletion of either T
cell subset significantly reduces survival (Conlan et al., 2005; Wu
et al., 2005; Bakshi et al., 2008).
Elucidation of immune T cell effector mechanisms that pro-
mote survival of secondary challenges remains an ongoing area
of active investigation. Such information is likely to be critical for
derivation of protective correlates of immunity. Mice administered
neutralizing anti-TNF-α antibodies during lethal IV LVS second-
ary challenge were highly susceptible to the infection, exhibiting
bacterial burdens that exceeded those of mice similarly treated with
anti-IFN-γ antibodies (Sjostedt et al., 1996). However, mice geneti-
cally engineered to produce only the membrane-bound form of
TNF-α (and not the soluble form), were resistant to a high lethal
secondary IP LVS challenge and generated similar numbers of
CD44hi-responding T cells in their spleens. These data suggest
that membrane TNF-α is sufficient for the generation of activated
responding memory T cells (Cowley et al., 2007). In contrast, mice
treated with anti-IFN-γ Abs at the time of secondary IP LVS chal-
lenges were only able to survive the lowest challenge doses, and
T cells harvested from vaccinated IFN-γ-deficient mice failed to
control LVS intramacrophage growth in vitro, indicating that IFN-γ
is required for successful priming of LVS-immune T cells (Elkins
et al., 2010). In addition to clear roles for IFN-γ and TNF-α, one
recent study found that vaccine efficacy against SchuS4 challenge
best correlated with pulmonary levels of IL-17 (Shen et al., 2010).
Further, antibody neutralization of IL-17A during in vivo secondary
challenge slightly enhanced SchuS4 growth in mouse lungs, indi-
cating that IL-17A may participate in vaccine-induced immunity
to Francisella (Shen et al., 2010).
Classic cytotoxic T cell mechanisms do not appear to play a sig-
nificant role in survival of primary or secondary LVS infections in
mice, and similarly do not contribute to the ability of LVS-immune
murine T cells to control LVS intramacrophage growth in vitro
(Cowley, manuscript in preparation). However, the recent finding
that Francisella is susceptible to killing by granulysin, a lytic antimi-
crobial peptide component of human – but not mouse – cytolytic
granules, suggests that granule cytotoxicity may be an effective part
of the human T and NK cell arsenal that cannot be assessed in the
mouse model (Endsley et al., 2009).
The study of T cell responses during Francisella infection has
been hampered by a paucity of information on the precise epitopes
recognized by antigen-specific T cells, and a corresponding lack of
tools such as tetramers to probe T cell biology. Efforts to identify
protein epitopes recognized by T cells during Francisella infection
have only recently begun to come to fruition. To date, T cell hybrid-
omas, bioinformatics, proteomics, and “immunoinformatics” have
been applied to identify several Francisella T cell epitopes in both
mice and humans (McMurry et al., 2007; Valentino and Frelinger,
2009; Valentino et al., 2009; Yu et al., 2010). One murine CD4
+
T cell
epitope has been identified as amino acids 86–99 of the lipoprotein
Tul4 (Valentino et al., 2009). Constituting as much as 20% of the
responding splenic CD4
+
T cells at the height of an ID LVS infec-
tion, this epitope appears to be immunodominant in C57Bl/6 mice
(Valentino et al., 2009). Other murine studies have identified the
LVS proteins GroEl, KatG, and bacterioferritin (Bfr) as immunos-
timulatory during in vitro recall assays with splenocytes harvested
from LVS-vaccinated BALB/c mice, although the exact epitopes
present in these proteins have yet to be identified (Lee et al., 2006).
The identification of human epitopes has been approached largely
through bioinformatics analyses to select candidate promiscuous
or supertype CD4
+
and CD8
+
epitopes in the SchuS4 genome; this
effort yielded a large number of candidate epitopes that induced
low levels of IFN-γ production by PBMCs from former tularemia
patients (McMurry et al., 2007). Immunization with a pool of 13
of these epitopes provided some protective immunity against a two
or five LD
50
IT LVS challenge in HLA class II (DRB1*0401) trans-
genic mice (Gregory et al., 2009), but this effort utilized a difficult
vaccination regimen. Further studies to identify alternate vehicles
or routes of vaccination will no doubt be necessary to develop a
peptide-based vaccination approach that is feasible for humans.
Despite apparently productive interactions between T cells and
Francisella-infected macrophages in vitro, infected macrophages
also have the capacity to directly inhibit T cell responses in vitro.
PGE2 production by BMDMs infected with a high multiplicity
of infection of LVS inhibited proliferation of T cell hybridomas
specific for SIINFEKL and hen egg lysozyme antigens, skewing the
T cell responses from IL-2 to IL-5 production (Woolard et al., 2007).
In addition, macrophage production of a PGE2-dependent factor
modulated antigen presentation by LVS-infected macrophages via
ubiquitinization and degradation of macrophage MHC class II
molecules (Wilson et al., 2009). Finally, F. novicida-infected mac-
rophages inhibited expression of the IFN-γ receptor and dimin-
ished the ability of ovalbumin-specific T cells to produce IL-2 in
response to their cognate antigen (Roth et al., 2009). The in vivo
consequences of these phenomena remain to be fully explored,
although inhibition of the PGE-2-producing enzyme cyclooxygen-
ase in mice during LVS pulmonary infection increased the number
of IFN-γ-producing T cells and decreased the bacterial burden in
the lungs. Thus lung PGE2 production may have a significant
immunosuppressive impact in vivo.
Page 15
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Cowley and Elkins Immunity to Francisella
between dendritic cell trafficking
and Francisella tularensis dissemina-
tion following airway infection. PLoS
Pathog. 4, e1000211. doi: 10.1371/
journal.ppat.1000211
Baron, S. D., Singh, R., and Metzger, D. W.
(2007). Inactivated Francisella tularen-
sis live vaccine strain protects against
respiratory tularemia by intranasal
vaccination in an immunoglobulin
A-dependent fashion. Infect. Immun.
75, 2152–2162.
Ben Nasr, A., Haithcoat, J., Masterson, J. E.,
Gunn, J. S., Eaves-Pyles, T., and Klimpel,
G. R. (2006). Critical role for serum
opsonins and complement receptors
CR3 (CD11b/CD18) and CR4 (CD11c/
CD18) in phagocytosis of Francisella
tularensis by human dendritic cells
Ashtekar, A. R., Zhang, P., Katz, J.,
Deivanayagam, C. C., Rallabhandi,
P., Vogel, S. N., and Michalek, S. M.
(2008). TLR4-mediated activation of
dendritic cells by the heat shock pro-
tein DnaK from Francisella tularensis.
J. Leukoc. Biol. 84, 1434–1446.
Bakshi, C. S., Malik, M., Mahawar, M.,
Kirimanjeswara, G. S., Hazlett, K.
R., Palmer, L. E., Furie, M. B., Singh,
R., Melendez, J. A., Sellati, T. J., and
Metzger, D. W. (2008). An improved
vaccine for prevention of respira-
tory tularemia caused by Francisella
tularensis SchuS4 strain. Vaccine 26,
5276–5288.
Bar-Haim, E., Gat, O., Markel, G.,
Cohen, H., Shafferman, A., and
Velan, B. (2008). Interrelationship
Andersson, H., Hartmanova, B., Kuolee,
R., Ryden, P., Conlan, W., Chen, W., and
Sjostedt, A. (2006b). Transcriptional
profiling of host responses in mouse
lungs following aerosol infection with
type A Francisella tularensis. J. Med.
Microbiol. 55, 263–271.
Anthony, L. D., Burke, R. D., and Nano, F.
E. (1991). Growth of Francisella spp.
in rodent macrophages. Infect. Immun.
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Anthony, L. S., Morrissey, P. J., and
Nano, F. E. (1992). Growth inhibi-
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macrophages is mediated by reactive
nitrogen intermediates derived from
l-arginine metabolism. J. Immunol.
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infection, and only a small but detectable increase in susceptibility
was evident upon secondary IP challenge (Yee et al., 1996; Markel
et al., 2010). However, studies using the primary sublethal pul-
monary infection model revealed that these cells respond in the
lungs of LVS-infected mice by producing IL-17A (Lin et al., 2009;
Markel et al., 2010). Thus, overall it appears that any responses
provided by γδ T cells in the murine model are not essential for
survival of LVS infection.
conclusIons and perspectIves
In the last 5 years, abundant new findings have added to an already
rich literature regarding Francisella pathogenesis and immunity. Of
necessity, some of it has been more descriptive than hypothesis-
driven, in reporting new genetic systems, results of genomic mining,
advances in establishing and characterizing novel animal models,
and in cataloging responding host cell types and mediators. But
the tools developed are now poised to have payoffs not only for
understanding of human infection with Francisella per se, but for
insights into the biology of intracellular pathogens in general. As
an experimental model, Francisella is unique in establishing pro-
ductive infection in a huge variety of insects and mammals by
many routes. Between this convenient biology and the plethora
of experimental options now available, the field is well poised to
perform detailed mechanistic studies. To do so, immunologists,
long biased toward studies in mice, may need to make room in their
thinking and their animal colonies for species with advantages in
modeling human infection, such as rats. The Francisella models
seem to be particularly attractive for tackling some of the current
themes regarding immunity to pathogens: comparing similarities
and differences in systemic or mucosal immunity; understanding
the tension between pathogen-induced immunosuppression and
host immunoresponsiveness; appreciating the ongoing interplay
between rapid innate reactions and later specific immunity; and
getting past a tendency to think of adaptive immune responses as
either antibody-centric or T cell dominated, when instead both may
complement, augment, and regulate each other. Collectively, we
expect studies on immunity to Francisella to continue to be excit-
ing, novel, and perhaps pivotal in achieving a critical public health
goal: the search for predictive correlates of protective immunity to
intracellular pathogens.
Similar to murine Francisella infections and as discussed above,
human CD4
+
and CD8
+
T cell responses have been demonstrated.
PBMCs from LVS vaccines and naturally infected tularemia patients
produce typical Th1-type cytokines such as IL-2, IFN-γ, and TNF-α
upon ex vivo re-stimulation with LVS or its antigens (Karttunen
et al., 1991; Surcel et al., 1991; Salerno-Goncalves et al., 2009).
Further, memory CD4
+
and CD8
+
T cells in re-stimulated PBMCs
from LVS-vaccinated volunteers also produce the Th17 cytokines
IL-17 and IL-22 (Paranavitana et al., 2010). One recent study further
investigated the phenotype of LVS-induced recall responses in the
PBMCs of tularemia patients and LVS vaccines, revealing that CD4
+
and CD8
+
T cell responders (as defined by IFN-γ-production and/
or proliferation) largely exhibited an effector memory phenotype,
and expressed cell surface markers that were indicative of cytolytic
potential and the ability to home to both mucosal and non-mucosal
sites (Salerno-Goncalves et al., 2009). These effector memory cells
were long-lived and in some cases detected greater than 25 years
after primary pneumonic tularemia.
In addition to CD4
+
and CD8
+
T cell responses, it is interesting
to note that γ/δ T cells respond vigorously to Francisella infection
in humans. The Vγ9/Vδ2 γδ T cell subset is known for its ability
to specifically respond to pathogen-produced non-peptidic phos-
phoesters known collectively as “phosphoantigens. In tularemia
patients, the Vγ9/Vδ2 subset expanded to constitute as much as
30.5% of all CD3
+
cells in human peripheral blood within the
first 7 days after the onset of symptoms (Poquet et al., 1998).
These cells remained elevated for at least a month, and persisted
in some individuals for as long as a year after infection (Kroca
et al., 2000). Acellular extracts from a variety of Francisella strains,
including LVS, contained phosphoantigens that activated Vγ9/
Vδ2 T cell expansion and effector functions in human PBMCs.
Curiously, despite the existence of activating phosphoantigens
in LVS extracts, LVS vaccines do not exhibit the same Vγ9/Vδ2
T cell subset expansion as tularemia patients. Since mice do not
express a γδ T cell receptor that is homologous to Vγ9/Vδ2, it
has not been possible to further explore this phenomenon in
the murine model of infection. To date, only a small number of
studies have addressed the role of γδ T cells in murine tularemia.
Mice deficient for the γδ T cell receptor did not exhibit nota-
ble increased susceptibility to primary sublethal ID or IN LVS
Page 16
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Cowley and Elkins Immunity to Francisella
the live vaccine strain of Francisella
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virulent type A strain of the patho-
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