# Lymphotoxin-α Plays Only a Minor Role in Host  Resistance to Respiratory Infection with Virulent  Type A Francisella tularensis in Mice

Authors: Deng Zhang; Rhonda KuoLee; Greg Harris; Qinxian Zhang; J. Wayne Conlan; Wangxue Chen
Journal: Mediators of Inflammation
 (2008)

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## Abstract

This study examined the role of lymphotoxin (LT)-α in host defense against airborne infection with Francisella tularensis, a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Following a low-dose aerosol infection with the highly virulent type A strain of F. tularensis, mice deficient in LTα (LTα−/−) consistently harbored approximately 10-fold fewer bacteria in their spleens at day 2 and 10-fold more bacteria in their lungs at day 4 than LTα+/+ mice. However, the mortality and median time to death were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were generally similar between LTα−/− and LTα+/+ mice. These data suggest that although LTα does not contribute significantly to the resistance and host responses of mice to airborne type A F. tularensis infection, it does play a subtle role in the multiplication/dissemination of F. tularensis.


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## Body

## 1. Introduction

Francisella
tularensis is  a gram-negative facultative intracellular bacterium and the
causative agent of tularemia, a systemic infection of many mammals including
humans.  Left untreated, the virulent type A
subspecies of F. tularensis routinely
caused lethal infection in people particularly after aerosol exposure to the
pathogen; as few as 10 virulent type A bacilli can initiate severe disease [1]. Consequently, F.
tularensis is considered a Category A biological warfare agent. Despite its clinical and biosecurity
importance, the molecular basis for the immunopathogenesis of F. tularensis infection,
particularly when initiated through the respiratory tract, remains largely
unknown.Lymphotoxin-α (LTα) is a member of the tumor
necrosis factor (TNF) superfamily of cytokines and has two distinct roles: as a membrane-bound heterotrimer in
combination with LTβ, it
binds the LTβ
receptor and is critical in the development and maintenance of organized secondary
lymphoid organs [2], and as a soluble homotrimer, it  signals through the TNF receptor pathway and leads
to activation of various inflammatory cytokines and chemokines [3, 4]. Indeed, LTs (LTα and LTβ), together with TNF and LIGHT (LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus
entry mediator on T cells), form an integrated signaling network which
is important for the regulation of both innate and adaptive immune responses [4]. In this regard, LTα has been implicated in the host defense
against several different bacterial, viral, and parasitic pathogens (reviewed in [3]). For instance, several studies
have shown that after infection with Mycobacterium tuberculosis,  mice genetically defective in LTα (LTα−/− mice) harbour increased bacterial
burdens and exhibited a shorter median time to death when compared to LTα+/+ mice [5–7]. Similarly, mice deficient in
LTα, LTβ, and the LTβ receptor (LTβR) are more susceptible to Listeria
monocytogenes infection than wild-type mice [6, 7]. Given that LTα is important in the control of these
intracellular bacterial pathogens, in the present study we sought to determine
whether it also plays a role in host defense against low-dose aerosol infection
with a virulent type A strain of F.
tularensis.


## 2. Materials and Methods

### 2.1. Mice

Eight- to twelve-week old,
age-matched B6.129S2-Ltαtm1Dch/J (LTα−/−), and wild-type C57BL/6J (LTα+/+) mice were used in this study. The
foundation breeding pairs of LTα−/− mice were purchased from
Jackson Laboratories (Bar Harbor, Me, USA). Mice were bred and housed
under specific-pathogen-free conditions in a federally licensed animal
biosafety level-3 facility and given free access to sterile water and certified
mouse chow. The animals were maintained and used in accordance with the
recommendations of the Canadian Council on Animal Care Guide to the Care and
Use of Experimental Animals.


### 2.2. F. tularensis and Experimental Infections

Stocks of type AF. tularensis strain FSC33/snMF,
originally isolated from a squirrel in Georgia, USA [8]. For low-dose aerosol exposure, thawed F. tularensis stocks were diluted in Mueller Hinton broth
containing 20% (v/v) glycerol to maintain infectivity at the high-relative
humidity employed. Aerosols of F.
tularensis strains were generated with a Lovelace nebulizer operating at a
pressure of 40 p.s.i. to produce particles in the 4–6 μm range required for inhalation and retention
in the alveoli [8]. Mice were exposed to these aerosols for 7
minutes (inhaled dose of ~10 organisms) using a customized commercial nose only
exposure apparatus (In-tox Products, Albuquerque, NM) resulting in the
implantation of 10–20 organisms into the lungs [8].


### 2.3. Quantitative Bacteriology and Histopathology

Groups of five mice of each strain
were sacrificed at 2 and 4 days after inoculation (dpi) by CO2 asphyxiation.
Blood samples were collected for the determination of serum cytokine levels. The
phenotype of the LTα−/− mice was confirmed by visual
inspection at necropsy to confirm the absence of peripheral lymph nodes. The
lungs and spleens were removed aseptically, cut into small pieces, and then
homogenized using an aerosol-proof homogenizer for quantitative bacteriology or
fixed immediately by immersion in 10%
neutral buffered formalin for histopathology. For quantitative
bacteriology, ten-fold serial dilutions of the tissue homogenates were plated
on cysteine heart agar supplemented with 1% (w/v) hemoglobin and sulfamethoxazole
and trimethoprim. Colonies were counted after 72 hours of incubation at 37°C [8]. For histopathology, the tissues were processed by standard
paraffin embedding methods (Department of Pathology and Laboratory Medicine, University of Ottawa,
Ottawa, Ontario).
Sections were cut 4 μm thick, stained with haematoxylin-eosin (HE)
and examined by light microscopy.


### 2.4. Bronchoalveolar Lavage (BAL) and Cytokine Measurements

In some experiments, the
lungs were lavaged with five 1.0-ml aliquots of PBS supplemented with 3 mM EDTA
[9], and the total lavage cell numbers
were counted on a haemocytometer, and differential cell counts were carried out
on cytospin preparations stained with Hema3 Stain Set (Fisher Scientific,
Middletown, Va, USA). The bronchoalveolar lavage (BAL)
fluid was centrifuged at 3000 xg for 7 minutes, supernatants were removed, sterile-filtered, and
stored at –80°C. Serum
and BAL levels of cytokines and
chemokines were determined using Beadlyte Mouse 21-Plex Cytokine Detection
System (Upstate, Temecuta, Calif, USA) on a Luminex 100IS system (Luminex,
Austin, Tex, USA) [10]. Undiluted BAL and 1:2 diluted serum samples (50 μl) were analyzed as specified by the manufacturer (http://www.millipore.com/userguides/tech1/ proto_mpxmcyto-70k).
The analysis was done in duplicate, and the cytokine/chemokine concentrations
were calculated against the standards using Beadview software (ver 1.03,
Upstate).


### 2.5. Statistical Analysis

All data are presented as mean± standard deviation (SD)
for each group. Differences between groups were analyzed by two-way ANOVA followed
by the Bonferroni multiple comparison test (GraphPad Prism 4.0, GraphPad Software, Inc., San Diego, USA). P < .05 was considered to be statistically significant.


## 2.1. Mice

Eight- to twelve-week old,
age-matched B6.129S2-Ltαtm1Dch/J (LTα−/−), and wild-type C57BL/6J (LTα+/+) mice were used in this study. The
foundation breeding pairs of LTα−/− mice were purchased from
Jackson Laboratories (Bar Harbor, Me, USA). Mice were bred and housed
under specific-pathogen-free conditions in a federally licensed animal
biosafety level-3 facility and given free access to sterile water and certified
mouse chow. The animals were maintained and used in accordance with the
recommendations of the Canadian Council on Animal Care Guide to the Care and
Use of Experimental Animals.


## 2.2. F. tularensis and Experimental Infections

Stocks of type AF. tularensis strain FSC33/snMF,
originally isolated from a squirrel in Georgia, USA [8]. For low-dose aerosol exposure, thawed F. tularensis stocks were diluted in Mueller Hinton broth
containing 20% (v/v) glycerol to maintain infectivity at the high-relative
humidity employed. Aerosols of F.
tularensis strains were generated with a Lovelace nebulizer operating at a
pressure of 40 p.s.i. to produce particles in the 4–6 μm range required for inhalation and retention
in the alveoli [8]. Mice were exposed to these aerosols for 7
minutes (inhaled dose of ~10 organisms) using a customized commercial nose only
exposure apparatus (In-tox Products, Albuquerque, NM) resulting in the
implantation of 10–20 organisms into the lungs [8].


## 2.3. Quantitative Bacteriology and Histopathology

Groups of five mice of each strain
were sacrificed at 2 and 4 days after inoculation (dpi) by CO2 asphyxiation.
Blood samples were collected for the determination of serum cytokine levels. The
phenotype of the LTα−/− mice was confirmed by visual
inspection at necropsy to confirm the absence of peripheral lymph nodes. The
lungs and spleens were removed aseptically, cut into small pieces, and then
homogenized using an aerosol-proof homogenizer for quantitative bacteriology or
fixed immediately by immersion in 10%
neutral buffered formalin for histopathology. For quantitative
bacteriology, ten-fold serial dilutions of the tissue homogenates were plated
on cysteine heart agar supplemented with 1% (w/v) hemoglobin and sulfamethoxazole
and trimethoprim. Colonies were counted after 72 hours of incubation at 37°C [8]. For histopathology, the tissues were processed by standard
paraffin embedding methods (Department of Pathology and Laboratory Medicine, University of Ottawa,
Ottawa, Ontario).
Sections were cut 4 μm thick, stained with haematoxylin-eosin (HE)
and examined by light microscopy.


## 2.4. Bronchoalveolar Lavage (BAL) and Cytokine Measurements

In some experiments, the
lungs were lavaged with five 1.0-ml aliquots of PBS supplemented with 3 mM EDTA
[9], and the total lavage cell numbers
were counted on a haemocytometer, and differential cell counts were carried out
on cytospin preparations stained with Hema3 Stain Set (Fisher Scientific,
Middletown, Va, USA). The bronchoalveolar lavage (BAL)
fluid was centrifuged at 3000 xg for 7 minutes, supernatants were removed, sterile-filtered, and
stored at –80°C. Serum
and BAL levels of cytokines and
chemokines were determined using Beadlyte Mouse 21-Plex Cytokine Detection
System (Upstate, Temecuta, Calif, USA) on a Luminex 100IS system (Luminex,
Austin, Tex, USA) [10]. Undiluted BAL and 1:2 diluted serum samples (50 μl) were analyzed as specified by the manufacturer (http://www.millipore.com/userguides/tech1/ proto_mpxmcyto-70k).
The analysis was done in duplicate, and the cytokine/chemokine concentrations
were calculated against the standards using Beadview software (ver 1.03,
Upstate).


## 2.5. Statistical Analysis

All data are presented as mean± standard deviation (SD)
for each group. Differences between groups were analyzed by two-way ANOVA followed
by the Bonferroni multiple comparison test (GraphPad Prism 4.0, GraphPad Software, Inc., San Diego, USA). P < .05 was considered to be statistically significant.


## 3. Results and Discussion

Initially, a total of 22 LTα−/− and 14 LTα+/+ mice were challenged by
low-dose aerosol with virulent type A F. tularensis and their survival monitored.
With the exception of two LTα−/− mice and one LTα+/+ mouse, all mice succumbed to
infection between day
4 and 7 with a median time to death of 5 days (range 4–6 days for LTα−/− mice and 5–7 days for LTα+/+ mice, P > .05 by
Kaplan-Meier survival analysis) (Figure 1), indicating that LTα−/− mice are no more susceptible to
low-dose aerosol challenge with this strain of the pathogen than control LTα+/+ mice. It has been previously
reported that increased susceptibility of certain immunocompromised miceto
intradermal infection with the live vaccine strain (LVS) of F. tularensis [11] and oral type A F. tularensis infection [10] is only apparent when using a
very high inoculum. To
examine the possible effect of inoculum size on the need for LTα expression to control respiratory infection
with type A F. tularensis, groups of LTα−/− and LTα+/+ mice were intranasally
challenged with 10, 100, and 1000 cfu type A F. tularensis and their survival monitored. This study revealed
that the LD100 of type A F.
tularensis for LTα−/− and LTα+/+ mice was comparable in that all mice
died of the infection by dpi 5 (data not shown). These results indicate that LTα does not appear to play a significant
role in determining the clinical outcome of respiratory infection with various
doses of type A F. tularensis in mice.Figure 1
Comparison of the survival rates of LTα−/− (open circles) and LTα+/+ (closed circles) mice following
aerosol inoculation with a low-dose of virulent type A F. tularensis. Groups of LTα−/− (n = 22) and LTα+/+ (n = 14) mice were challenged by
aerosol with type A F. tularensis strain
FSC033 (inhaled dose of ~10 organisms) and their survival was monitored daily.Since type A strains ofF.
tularensis are extremely virulent for mice even at the minimum challenge
dose [8], it remained possible that
subtle effects of LTα expression were overlooked by the above
relatively crude survival experiments. Therefore, we next examined whether LTα contributes to the control of F.
tularensis replication and systemic dissemination by comparing the
bacterial burdens in the lungs and spleens of LTα−/− and LTα+/+ mice at dpi 2 and 4 following aerosol challenge (Figure 2). There was no difference in the bacterial burdens
in the lungs, the primary site of infection, between LTα−/− and LTα+/+ mice at dpi 2. However, the
bacterial burdens in the spleens  of LTα−/− mice were about 1 to 1.5 log, but
not statistically significant, lower than those in LTα+/+ mice at this time point. By dpi 4, LTα−/− mice had approximately 10-fold more
bacteria in their lungs than did LTα+/+ mice (P < .01), and the bacterial
burdens in the spleens of LTα−/− mice were also higher, although not
statistically significant, than those in LTα+/+ mice. The subtle differences in
bacterial burdens were consistently observed in three independent experiments. Hence,
these data imply that although LTα is not sufficient to control virulent F. tularensis infection, it may play a minor
role in the initial dissemination of F.
tularensis from the lung to spleen and subsequent multiplication of the
pathogen in the lungs and spleen.Bacterial burdens in the lungs and
spleens of LTα−/− (open bars) and LTα+/+ (filled bars) mice on days 2 and 4
after aerosol inoculation with a low-dose of type A F. tularensis strain, FSC033. The data shown are
compiled from two independent experiments with similar results and expressed as
mean ± standard deviation (n = 8). P**<.01 versus LTα+/+ mice.Histopathologically,
both LTα−/− and LTα+/+ mice showed moderate
inflammatory infiltrations in the livers and spleens and mild, focal
bronchopneumonia at dpi 2, and by dpi 4 moderately severe necrotic hepatitis,
lymphoid follicle destruction in the spleen, and bronchopneumonia.
However, as would be expected from the quantitative
bacteriology and survival data, no overt differences in tissue histopathology
or blood clinical chemistry (data not shown) were observed between LTα−/− and LTα+/+ mice following aerosol exposure
to type A F. tularensis.Early pulmonary recruitment of inflammatory cells and local and
systemic production of proinflammatory cytokines are considered important
characteristics of innate host responses against respiratory infections includingF. tularensis  [12]. Previous studies have shown that
respiratory infection of mice with type A and attenuated live vaccine strain of F. tularensis upregulates a number of
proinflammatory cytokines, which play important roles in host defense against F.
tularensis infection [13–15]. Therefore, we determined total and differential
leukocyte counts  in the BAL fluid to identify the inflammatory
cell influx into the lungs on dpi 2 and 4. As can be seen in Table 1, low-dose
aerosol infection of mice with type A F.
tularensis induced no significant difference in either the total
cell number or the composition of cell
populations (macrophage, neutrophil, and lymphocyte) in the lavage fluids of LTα+/+ and LTα
−/− mice with the exception ofa small but not significant
increase in lymphocytes in LTα−/− mice on both dpi 2 and 4. This is likely due to a higher baseline number of lymphocytes in the
lungs of LTα−/− mice [16] rather than a result of F.
tularensis infection. To
assess whether LTα deficiency alters F.
tularensis-induced cytokine responses following aerosol challenge with the
pathogen, levels of a panel of 21 cytokines and chemokines, including IFN-γ, IL-6, KC, and MCP-1, in the BAL and the sera of LTα−/− and LTα+/+ mice killed at dpi 2 and 4 were measured. Overall, there was little change in the levels of
the majority of assayed cytokines in either the BAL or the sera at dpi 2 or 4 in either mouse strain (data not shown). However, F. tularensis infection resulted in a substantial
increase of MCP-1 and a moderate increase of KC  in BAL fluid at dpi 2 (Figure 3(b)) and a substantial increase of IFN-γ, IL-6, KC, and MCP-1 in both BAL and sera at dpi 4 (Figures 3(a) and 3(b)), but again no differences were
observed between the two mouse strains with the exception of IL-6, which was
significantly higher in BAL fluid
of LTα−/− mice than that of LTα+/+ mice (Figure 3(b), P < .05).Table 1
Comparison of cell
populations in the bronchoalveolar lavage fluid of LTα−/− and LTα+/+ mice on day 2 and 4 following low-dose
aerosol inoculation with type A F.
tularensis.
Days post-inoculationMouse strainTotal cell count
(× 105)(a)Differential counts (%)MacrophagesLymphocytesNeutrophils2LTα−/−2.91±1.5997.60±0.892.00±1.000.40±0.55LTα+/+1.96±0.3198.80±0.840.40±0.550.80±0.844LTα−/−3.02±0.7695.20±3.272.40±1.522.40±1.82LTα+/+2.70±0.5594.80±6.760.20±0.455.00±6.82(a) The total leukocyte
counts are expressed as absolute numbers, and differential counts are expressed
as percentages. All data are mean ± standard deviation (n = 5) in each group at each time
point. No significant differences were observed between LTα−/− and LTα+/+ mice at any time point.Cytokine and chemokine levels in sera (a)
and bronchoalveolar lavage (BAL) fluid (b) of mice inoculated by aerosol with
type AF. tularensis. Groups of LTα−/− (open symbols) and LTα+/+ (closed symbols) mice (n = 5) were
challenged by aerosol with low-dose type A F.
tularensis strain, FSC033 (inhaled dose of ~10 organisms) on day 0, and blood
samples and bronchoalveolar lavage fluid samples were collected at dpi 0, 2,
and 4. Cytokine and chemokine levels in the serum and BAL fluid were determined
using the Beadlyte Mouse 21-Plex Cytokine Detection System on a Luminex 100 IS
instrument. Each symbol represents the corresponding cytokine concentration of
an individual mouse. Horizontal lines indicate the median of each group of mice
on the indicated post-inoculation days. The detection limits of the assays
were <5 pg/ml for both sera and BAL fluid. P*<.05 versus LTα+/+ mice.
(a)(b)Recent studies have established that, in addition to its role in
the organogenesis of secondary lymphoid organs, LTα plays an important role in host defenses against microbial
infections (reviewed in [3]). However, the role of LTα in host defenses against infection appears to be complex and
varies from pathogen to pathogen. In this study, we utilized LTα−/− mice and performed some preliminary studies to examine the
potential role of LTα in the host resistance to respiratory infection with virulent
type A F. tularensis. Our
results showed that LTα−/− mice had lower bacterial
burdens in their spleens on dpi 2 and higher bacterial burdens in their lungs
on dpi 4 when compared to LTα+/+ mice but showed no overt
differences in clinical outcome, tissue damage, or host immune responses to the
infection.Although our data suggest that LTα exerts some subtle influence over the
course of aerosol-initiated tularemia, its mechanism of action remains unknown.
The possible reasons are as
follows: (1) LT-α is not crucial in host defense against
this pathogen; (2) the role of LT-α can be compensated by other
cytokines/chemokines in this infection model; and (3) the pathogen is too
virulent and even immunocompetent hosts have little resistance to the
infection. In this regard, we have previously shown that a number of
immunodeficient mice show similar clinical outcome to the immunocompetent mice
[17]. The lower bacterial burdens in the spleen of LTα−/− mice at dpi 2 could be
explained by a delay/reduction in the dissemination of bacteria from the lung
since LTα−/− mice lack tracheobronchial
lymph nodes which normally are the major draining lymph nodes for the lung.
Once F. tularensis reached the spleens, however, bacteria quickly
multiplied and by dpi 4, bacterial burdens were no longer significantly
different in the spleens of LTα−/− and LTα+/+ mice. In fact, LTα−/− mice actually seem to have
slightly higher burdens in their spleens at this time point despite starting on
dpi 2 with substantially lower bacterial burdens (see Figure 2). Also by this
time, LTα−/− mice harbored significantly
more bacteria in their lungs than LTα+/+ mice (P < .01),
suggesting that LTα may play a role in host
defense against F. tularensis infection which is distinct from its role
in lymphoid organogenesis. Alternatively, the lack of draining lymph nodes in
LTα−/− mice may simply cause a
delay in antigen presentation leading to a delayed or otherwise impaired
antibacterial host response.In summary, following a low-dose aerosol
infection with the highly virulent type A strain ofF. tularensis, LTα−/− mice consistently harbored approximately 10-fold
fewer bacteria in their spleens at dpi 2- and 10-fold more bacteria in their
lungs at dpi 4 than LTα+/+ mice. However, the mortality and median time to death were indistinguishable
between the two mouse
strains. In addition, the inflammatory responses to the infection,
as reflected by the cytokine
levels and leukocyte influx in BAL fluid and histopathological analysis, were generally similar between LTα−/− and LTα+/+ mice. These data suggest that although LTα does not contribute significantly to
the resistance and host responses of mice to airborne type A F. tularensis infection; it does play a subtle role in the multiplication/dissemination of F.
tularensis.



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