The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
Host control of Mycobacterium tuberculosis
is regulated by 5-lipoxygenase–dependent
Andre Bafica,1,2 Charles A. Scanga,1 Charles Serhan,3 Fabiana Machado,4 Sandy White,1
Alan Sher,1 and Julio Aliberti4
1Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland, USA.
2Laboratorio de Imunorregulacao e Microbiologia, Centro de Pesquisas Goncalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil.
3Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine,
Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Immunology,
Duke University Medical School, Durham, North Carolina, USA.
Th1 type cytokine responses are critical in the control of Mycobacterium tuberculosis infection. Recent findings
indicate that 5-lipoxygenase–dependent (5-LO–dependent) lipoxins regulate host IL-12 production in vivo.
Here, we establish lipoxins as key chemical mediators in resistance to M. tuberculosis infection. High levels of
lipoxin A4 (LXA4) were detected in sera from infected WT but not infected 5-LO–deficient mice. Moreover, lungs
from M. tuberculosis–infected 5-lo–/– animals showed increased IL-12, IFN-γ, and NO synthase 2 (NOS2) mRNA
levels compared with the same tissues in WT mice. Similarly, splenocyte recall responses were enhanced in
mycobacteria-infected 5-lo–/– versus WT mice. Importantly, bacterial burdens in 5-lo–/– lungs were significantly
lower than those from WT mice, and this enhancement in the resistance of the 5-lo–/– animals to M. tuberculosis
was completely prevented by administration of a stable LXA4 analog. Together our results demonstrate that
lipoxins negatively regulate protective Th1 responses against mycobacterial infection in vivo and suggest that
the inhibition of lipoxin biosynthesis could serve as a strategy for enhancing host resistance to M. tuberculosis.
Th1-mediated immunity plays a crucial role in host defense
against Mycobacterium tuberculosis. Cytokines such as IL-12, IFN-γ,
and TNF are essential for protection against this pathogen in the
mouse model (1–3). Additional evidence suggests that the same
cytokines are important resistance factors in the human immune
response against mycobacterial infection (4–7). In addition to the
Th1 type response mounted over the course of infection, down-
regulatory mediators may be important players in controlling
excessive synthesis of proinflammatory cytokines and subsequent
tissue damage and could contribute to the promotion of bacte-
rial survival. Nevertheless, Th2 cytokines such as IL-4 and IL-13
have been described as playing no or only a limited role in in vivo
M. tuberculosis infection (8–10). Similarly, although in vitro IL-10
production is associated with reduced human disease (11), mice
deficient in this important downregulatory cytokine show nearly
normal control of M. tuberculosis infection (9, 10).
There is a growing body of evidence indicating that a class of lipox-
ygenase-derived eicosanoids known as lipoxins plays an important
role in the immunoregulation of inflammation-associated disease
(12). We have previously shown that lipoxin A4 (LXA4), a lipid media-
tor derived locally from 5-lipoxygenase (5-LO) biosynthetic pathways,
acts in vitro as a negative regulator of DC IL-12 production triggered
by the intracellular protozoan parasite Toxoplasma gondii (13). An in
vivo role for this pathway in host resistance to the same pathogen
was suggested by the observation that T. gondii–infected 5-LO–defi-
cient mice succumb as a result of exacerbated proinflammatory
responses despite diminished parasite numbers (14).
In the present report, we asked whether 5-LO–dependent mecha-
nisms, and in particular those mediated by lipoxins, also play a
role in regulating host resistance to M. tuberculosis. To do so, we
examined the course of infection- and pathogen-induced cellu-
lar immune responses in 5-LO–deficient mice exposed to myco-
bacteria by aerosol exposure. Our results reveal a major role for
5-LO–dependent lipoxin synthesis in the immune modulation of
M. tuberculosis infection in vivo and suggest that this pathway may
be a potential target for therapeutic intervention in tuberculosis.
M. tuberculosis–infected mice produce LXA4 in a 5-LO–dependent man-
ner. To assess whether 5-LO plays a role in M. tuberculosis infection
in vivo, we first measured its products leukotriene B4 (LTB4) and
LXA4 in sera from B6, 129S F2/J mice at different time points after
aerosol infection (300 CFU/animal). As shown in Figure 1, these
eicosanoids were detected at high levels as early as 1 week after
M. tuberculosis infection. LXA4, but not LTB4, synthesis was main-
tained during chronic infection. Importantly, neither eicosanoid
was detected above background levels in M. tuberculosis–infected
5-lo–/– animals, which confirmed the dependence of LTB4 and LXA4
on 5-LO in vivo (Figure 1, A and B). To address the issue of which
cell population is responsible for 5-LO activity, we performed
immunostaining for the enzyme in lung sections of M. tuberculosis–
infected WT mice. 5-LO–positive staining was found to colocalize
with endothelium (Figure 1C) and F4/80+ cells (Figure 1D). Taken
together, these results indicate that LTB4 and LXA4 are strongly
induced during M. tuberculosis infection in vivo and suggest that
Nonstandard abbreviations used: LTB4, leukotriene B4; 5-LO, 5-lipoxygenase; LXA4,
lipoxin A4; NOS2, NO synthase 2.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 115:1601–1606 (2005).
Related Commentary, page 1473
1602 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
endothelial cells and macrophages provide the source of the 5-LO
required for the synthesis of these eicosanoids.
5-LO–deficient mice display enhanced control of M. tuberculosis infec-
tion. To investigate the role of 5-LO in M. tuberculosis infection in
vivo, we assessed bacterial burdens and tissue histopathology in
5-lo–/– and control animals. Lungs from 5-lo–/– mice displayed sig-
nificant reductions in mycobacterial load at both 21 and 42 days
after infection when compared with similarly infected WT control
animals (Figure 2, A and B). Similar reductions in bacterial counts
were observed in spleens from the same animals (data not shown).
Acid-fast staining confirmed that fewer mycobacteria were present
in the lungs of 5-LO–deficient mice compared with B6, 129S F2/J
control mice (Figure 2, C and D, respectively). In addition, lungs
from 5-LO–deficient mice infected for 50 days with M. tuberculo-
sis showed dramatically reduced tissue inflammation compared
with lungs from infected WT animals. Consistent with their high
mycobacterial burden 50 days after infection, lungs from WT mice
exhibited severe, widespread alveolitis and interstitial pneumonitis
as well as areas of necrosis (Figure 3, A and B). In contrast, lungs
from similarly infected 5-lo–/– mice displayed much less inflamma-
tion and little evidence of tissue necrosis (Figure 3, C and D).
Consistent with their reduced bacterial load, 5-lo–/– mice infected
with 300 CFU/mouse displayed enhanced survival compared with
similarly infected B6, 129S F2/J mice (Figure 2E). In these experi-
ments, WT mice succumbed to aerogenic M. tuberculosis infection
more rapidly than has been reported previously in mice of this
genetic strain. We reasoned that this was likely a result of the fact
that the inoculum (300 CFU) was larger than that (50 CFU) used
in prior studies (15). Although the extended survival of 5-lo–/– mice
infected at high dose argues for their enhanced resistance, we also
examined mortality at the more conventional low infectious dose
(50 CFU/mouse). In this setting, both 5-LO–deficient and control
mice survived at similar rates until 300 days after infection (Figure
2E). Nevertheless, a highly significant reduction in bacterial bur-
den similar to that observed at the higher dose of infection was
evident in the lungs of these animals at days 21 and 42 (Figure 2D).
Taken together, these results demonstrate that 5-LO promotes
both mycobacterial growth and host susceptibility to infection.
5-LO–deficient mice infected with M. tuberculosis show increased expres-
sion of proinflammatory mediators. To determine whether the absence
of 5-LO affects the proinflammatory responses induced by myco-
bacteria, we studied the time course of expression of the genes
Increased resistance of 5-LO–deficient mice to M. tuberculosis infec-
tion. Lungs from WT (black bars) and 5-lo–/– (gray bars) mice were har-
vested at several time points after infection with an average of 300 (A)
or 50 (B) CFU/mouse and mycobacterial burdens determined. Results
are mean ± SE of measurements from 4 animals. *P < 0.05. Represen-
tative acid-fast bacilli–stained sections from lungs of 50-day–infected
WT (C) and 5-lo–/– (D) mice (300 CFU/animal) illustrate the reduction in
acid-fast bacilli (red staining) in the KO animals. Original magnification,
×63. (E) WT B6, 129S F2/J (filled symbols) and 5-LO–deficient (open
symbols) animals were aerogenically infected with an average of 300
CFU/mouse (high dose [HiD]; squares) or with 50 CFU/mouse (low
dose [LoD]; circles) (n = 10 animals per group) and survival monitored.
The results shown are representative of 2 independent experiments
performed at each dose.
5-LO–dependent LXA4 and LTB4 production and 5-LO expression dur-
ing M. tuberculosis infection. WT (B6, 129J F2; filled squares) and
5-LO–deficient (B6, 129J Alox-5; open circles) animals were infected by
aerosol exposure with an average of 300 CFU/mouse of M. tuberculo-
sis H37Rv and LXA4 (A) and LTB4 (B) assessed by ELISA in serum at
8, 21 and 42 days after infection. Results are mean ± SE of measure-
ments from 5 animals. *P < 0.05 between experimental groups. Results
shown are representative of 2 independent experiments. (C and D) WT
lung sections were stained with anti–5-LO (red) and costained with anti-
F4/80 (green), followed by counterstaining with DAPI (blue). (C) 5-LO+
endothelium. (D) Several F4/80+5-LO+ cells infiltrating pulmonary tissue
during M. tuberculosis infection. Original magnification, ×63.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
encoding IL-12, IFN-γ, TNF, and NO synthase 2 (NOS2) in the
lungs of M. tuberculosis–infected animals. Levels of both IL-12p40
and IFN-γ mRNA were found to be significantly elevated in infected
5-lo–/– mice compared with their WT counterparts (Figure 4, A and
D), consistent with their diminished mycobacterial burdens. Despite
the marked differences in pulmonary histopathology (Figure 3, A
and B versus C and D), the levels of TNF expression in lungs did
not differ significantly between infected 5-lo–/– and WT mice (Figure
4E). Importantly, expression of the gene encoding NOS2, an enzyme
required for host resistance to M. tuberculosis in mice (16), was found
to be dramatically elevated in the absence of 5-LO at both 21 and 42
days after infection (Figure 4B). Nevertheless, no differences in IL-10
expression were observed in the lungs of 5-lo–/–
versus control animals at the time intervals
examined (data not shown). To confirm this
gene expression data, we assayed IL-12p40
and TNF protein levels in lung homogenates
from the same infected animals at day 21
after infection. As shown in Figure 4, C and F,
5-lo–/– mice displayed significantly increased
levels of IL-12p40 but not TNF. In addition,
immunostaining of lung sections was per-
formed in an attempt to identify the cellular
source of the increased proinflammatory
cytokine. Few CD11c+ cells were found to
coexpress IL-12p40 in lungs of WT animals
(Figure 5A and Table 1), while a higher fre-
quency of CD11c+IL-12p40+ cells was found infiltrating the lungs
of 5-LO–deficient mice (Figure 5D and Table 1). However, not all of
IL-12p40 staining was associated with CD11c+ cells (green), which
suggests that 5-LO regulates IL-12 expression in both DCs and
other leukocytes in the tissue infiltrates. In contrast, the expression
of TNF and NOS2 was found to be restricted to the F4/80+ (macro-
phage) cell population, and although TNF expression was similar in
the 2 animal groups (Figures 5, B and E and Table 1), the frequency
of NOS2+F4/80+ cells was dramatically enhanced in the lungs of the
infected 5-LO–deficient mice (Figures 5, C and F and Table 1). These
findings both agree and contrast with our previous observations
on cell-associated cytokine and NOS2 expression during T. gondii
infection in 5-LO–deficient mice (14). In that study, we observed
enhanced IL-12 production by both CD11c+ and CD11c– cells in
brain tissue of infected 5-LO–deficient mice but failed to detect sig-
nificant changes in NOS2 as reported here.
Treatment with an LXA4 analog reverses resistance in M. tuberculosis–
infected 5-LO–deficient mice. 5-LO is involved in the biosynthesis of
several eicosanoids, including LXA4, that are known to mediate local
control of inflammation. Therefore, it was critical to formally estab-
lish whether the enhanced protection against M. tuberculosis infec-
tion, elevated level of type 1 cytokines, and lower mycobacterial bur-
Decreased inflammation in lungs of M. tuberculosis–infected 5-LO–
deficient mice. Representative H&E-stained sections of lungs from
50-day–infected (300 CFU inoculum) B6, 129S F2/J control (A and B)
and 5-LO–deficient (C and D) animals. Note the reduction in inflamma-
tory infiltration and greatly increased alveolar space in 5-lo–/– animals
(C and D). Original magnification, ×5 (A and C) and ×40 (B and D).
5-LO–deficient mice infected with M. tuber-
culosis display increased expression of
proinflammatory mediators. WT and 5-lo–/–
mice were aerogenically infected (300 CFU
inoculum), and relative expression of mRNAs
for IL-12p40 (A), NOS2 (B), IFN-γ (D), and TNF
(E) was determined in the lungs at 8, 21 and 42
days after M. tuberculosis infection. To further
confirm these observations, we prepared lung
homogenates from the same animal groups
shown above and determined IL-12 (C) and
TNF (F) levels by ELISA. *P < 0.05. The results
shown are representative of 2 independent
experiments. Uninf., uninfected.
1604 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
den in 5-LO–KO mice are indeed related to the absence of lipoxin
generation during infection. To address this issue, we administered
a stable lipoxin analog, ATLa2, to both WT controls and 5-LO–defi-
cient mice during the first 21 days after infection. This eicosanoid
analog, created by design modification of the ω end of LXA4, was
shown to have increased half-life in vivo and to inhibit inflamma-
tion in several disease models (17–19). As shown in Figure 6, A and
B, ATLa2 treatment abrogated the enhanced control of bacterial
growth in both lungs and spleens of 5-LO–deficient mice. No altera-
tion of in vivo resistance was noted in M. tuberculosis–infected WT
controls treated with the LXA4 analog at this dose. These findings
suggest that the levels of endogenous lipoxin present in the infected
5-LO–competent mice are already optimal and that additional lipox-
in does not alter the host response to M. tuberculosis infection. When
examined at 21 days after infection, the M. tuberculosis–infected
5-lo–/– mice treated with the LXA4 analog displayed a weakened Th1
response, as evidenced by reduced IFN-γ production by splenocytes
restimulated ex vivo with M. tuberculosis antigen (Figure 6C) but
unaltered TNF levels (Figure 6D). Importantly, ATLa2 had no direct
effects on mycobacterial proliferation in vitro, which argues against
the possibility that the in vivo activity of this eicosanoid is due to a
direct antibiotic effect (data not shown).
Proinflammatory cytokines such as IL-12
play critical roles in the induction of
host resistance to M. tuberculosis as well
as other intracellular pathogens. These
responses must be carefully regulat-
ed to avoid host tissue damage. The
antiinflammatory cytokines IL-10 and
TGF-β have been implicated as key
protein mediators that prevent excess
IL-12, TNF-α, and IFN-γ production in
intracellular infections. Nevertheless,
these downregulatory cytokines appear
to have only limited effects in controlling M. tuberculosis replication
during infection in animal models (8, 20). In the present study, we
report evidence for the role of a novel pathway involved in damp-
ening M. tuberculosis–driven proinflammatory immune responses
and regulating bacterial growth that involves the 5-LO–dependent
production of lipoxins.
Lipoxins such as LXA4 are biosynthesized by different cell types,
including leukocytes, endothelial cells, and platelets by means of tran-
scellular pathways (21). Recently, LXA4 was shown to have downreg-
ulatory actions on several proinflammatory mechanisms including
NK cell cytotoxicity (22), leukocyte responses to proinflammatory
cytokines (23), and microbial stimulation (14) as well as migration
of both neutrophils (18) and eosinophils (24). Interestingly, stimu-
lation of mucosal epithelial cells with lipoxin analogs induced the
expression of a bactericidal/permeability-increasing protein, which
exhibits antimicrobial activities and enables epithelial cells to engage
in active microbial host defense (25, 26).
LXA4 dramatically reduces T. gondii–induced IL-12 produc-
tion by DCs in vitro and by DCs as well as other cells in vivo (13,
14), which indicates a role for LXA4 in preventing uncontrolled
proinflammatory responses. T. gondii triggered high levels of LXA4
(∼100 ng/ml) in sera of WT mice, while infected 5-LO–deficient
mice produced elevated amounts of IL-12p40. Interestingly, it was
recently shown that T. gondii synthesizes its own LO that may play
a role in increasing local concentrations of LXA4 (27). In the pres-
ent report, using an aerosol model of infection with M. tuberculo-
sis, we also detected the induction of high levels of LXA4 as well
as the leukotriene LTB4 in the sera of WT mice. However, LXA4
and LTB4 were induced with different kinetics, and only LXA4
persisted at high levels during chronic infection. Furthermore, we
observed high levels of expression of 5-LO in lung endothelium
and macrophages during infection. The latter results suggest that
these cell populations participate in lipoxin generation in vivo
and may be specifically involved in regulating local inflammatory
responses during chronic experimental tuberculosis.
Lung-infiltrating DCs and macrophages express high levels of IL-12
and NOS2 in 5-LO–deficient hosts infected with M. tuberculosis. Fro-
zen sections of lungs from infected (300 CFU inoculum) WT (A, B,
and C) and 5-lo–/– (D, E, and F) mice were double stained with anti-
CD11c (A and D) or anti-F4/80 (B, C, E, and F) (green) and with anti–
IL-12p40 (A and D), anti-TNF (B and E), or anti-NOS2 (C and F) (red),
then counterstained with DAPI (blue). Note the presence of many more
CD11c+IL-12p40+ cells and F4/80+NOS2+ cells in the tissue sections
from 5-lo–/– animals. The asterisk in A indicates the presence of mul-
tinucleated cells at the center of a granuloma. Representative micro-
graphs (magnification, ×63) from 4 animals per group are shown.
Higher frequency of CD11c+IL-12p40+ and F4/80+NOS2+ cells in lung sections from M. tuber-
culosis–infected 5-LO–deficient versus WT animalsA
39 ± 6.11
100.16 ± 6.63B
28.66 ± 6.4
39.5 ± 4.81 182.66 ± 13.96B 15.33 ± 1.01 135.66 ± 16.44
101 ± 19.05
16.33 ± 4.8
157 ± 15.77
AMicroscopic quantitation of fluorochrome-positive cells was performed on the lung sections described
in Figure 5. Results are expressed as the number of positive cells per field ± SEM determined from 10
observation fields per slide and 3 slides per mouse (3 animals/group). BP < 0.05 vs. WT.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
Whereas T. gondii–exposed 5-lo–/– mice succumbed rapidly to
infection, the reduced lipoxin generation in 5-lo–/– mice infected
with M. tuberculosis was associated with enhanced survival at high-
dose aerosol challenge, although there were no apparent differenc-
es in mortality at the lower inoculum infections. These contrasting
outcomes of the 2 infection models may stem from differences in
the intrinsic virulence and immune-stimulatory properties of the
pathogens in question. T. gondii is a highly virulent and fast-rep-
licating microorganism that requires the induction of a potent
immune response to protect the host and produce chronic per-
sistent infections necessary for promoting its transmission.
M. tuberculosis, while also inducing latent infections, replicates slow-
ly and, at least in the mouse model, induces a weaker Th1 response
than does T. gondii. Hence, in the absence of lipoxin-mediated
counterregulation, the ensuing cellular responses are enhanced,
triggering immunopathology and mortality in T. gondii infection,
whereas in M. tuberculosis infection, this enhancement results in
increased control of bacterial replication. The observed restriction
in mycobacterial growth does not appear to be complete, howev-
er, since high-dose M. tuberculosis–infected 5-lo–/– mice eventually
began to succumb at 150 days after infection, and this mortality is
associated with an approximate log increase in mycobacterial load
compared with earlier time points (e.g., day 42) (data not shown).
Whether the late death of the infected 5-LO–deficient animals is
due solely to increased bacterial burden remains unclear.
Since 5-LO is required for both leukotriene and lipoxin biosyn-
thesis, reconstitution experiments were performed to more directly
assess the role of the latter group of eicosanoids in the regulation
of mycobacterial growth in vivo. Importantly, administration of
the stable lipoxin analog ATLa2 to M. tuberculosis-infected 5-lo–/–
mice, restored both pulmonary mycobacterial loads and IFN-γ
production by purified protein derivative–stimulated splenocytes
to levels comparable to those observed in infected WT animals.
Although this observation does not rule out the possible partici-
pation of other 5-LO–dependent mediators, it demonstrates that
a deficiency in lipoxins is sufficient to explain the effects on bacte-
rial growth and host response seen in the infected 5-LO–deficient
animals. ATLa2 treatment has previously been shown to reduce
inflammatory cell infiltration in a number of different disease
models (17–19). Although this subject is not directly addressed in
the present article, it is probable that the observed effects of ATLa2
reconstitution in our experiments result from decreased effector
cell recruitment into infected lung.
In summary, our findings demonstrate the existence of a novel
pathway involved in controlling proinflammatory and Th1 immune
responses against M. tuberculosis infection in vivo via the generation
of 5-LO–dependent lipoxin formation. These observations suggest
that the regulation of lipoxin biosynthesis merits further inves-
tigation as a potential immunopharmacologic intervention for
enhancing the control of mycobacterial replication in tuberculosis
patients. In this regard, it should be noted that 5-LO inhibitors are
already in clinical trial for asthma, and therefore it may be possible
to rapidly design and implement a study testing the efficacy of this
strategy for intervention in tuberculosis (28–30).
Mice. WT controls (B6, 129S F2/J) and 5-LO–deficient (B6, 129S Alox-5,
F2/J) mice were obtained from The Jackson Laboratory and were bred and
maintained in an NIAID Association for the Assessment and Accreditation
of Laboratory Animal Care–accredited animal facility. Female animals 5–8
weeks old were used in all experiments. All experiments were approved by
the NIAID Institutional Animal Care and Use Committee.
M. tuberculosis infection. The M. tuberculosis H37Rv strain was passaged
through mice, grown in culture once, and frozen in aliquots. Prior to infec-
tion, an aliquot was thawed, diluted in PBS, and briefly sonicated in a cup-
horn sonicator. Mice were placed in a closed, nose-only aerosolization system
(CH Technologies) and exposed for 15 minutes to nebulized M. tuberculosis.
Two different bacterial doses were employed: 50 and 300 CFU per mouse. To
assess mycobacterial load, we harvested lungs and spleens at different times
after infection, and tissue homogenates were diluted in buffered saline and
cultured on 7H11 agar plates. Colony counts were determined 21 days later.
Histopathology. Lung and spleen tissues were harvested, fixed with neu-
tral buffered formalin, and paraffin embedded. Serial sections were stained
with H&E for histopathologic analysis or with Kinyoun’s acid fast stain for
in situ detection of mycobacteria.
Eicosanoid and cytokine determinations. Serum levels of eicosanoids and
cytokines were measured using commercial ELISA kits obtained from
Neogen Corp. (LXA4), Cayman Chemical Co. (LTB4), and R&D Systems,
(IL-12p40, TNF, IFN-γ). For cytokine detection in lungs, tissue was homog-
enized and centrifuged at 300 g for 7 minutes. Supernatants aliquots were
frozen at –80°C for later analysis by ELISA.
Measurement of gene expression in lung. Total RNA was isolated from lungs and
real-time RT-PCR was performed on an ABI Prism 7900 sequence detection
system (Applied Biosystems) using SYBR Green PCR Master Mix (Applied
Biosystems) after reverse transcription of 1 μg RNA using Superscript II
reverse transcriptase (Invitrogen Corp.). The relative level of gene expression
was determined by the comparative Ct method as described by the manu-
facturer, whereby data for each sample were normalized to hypoxanthine
phospho-ribosyl-transferase (hprt) and expressed as a fold change compared
In vivo administration of a stable analog of LXA4 in M. tuberculosis–
infected mice. WT or 5-lo–/– mice were infected by aerosol exposure
with M. tuberculosis (300 CFU inoculum) and treated 3 times a week
by gavage from days 2 to 20 with vehicle or ATLa2 at 100 ng/ani-
mal/treatment. Mice were sacrificed, and mycobacterial burdens were
assessed in lungs (A) and spleens (B), 21 days after infection. Spleen
cell cultures from the same animals were restimulated with purified
protein derivative, and 72 hours later, IFN-γ (C) and TNF (D) levels
were measured in supernatants by ELISA. Results are the mean ± SD
from 5 animals per group. *P < 0.05 between groups.
1606 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 6 June 2005
with untreated controls. The following primer pairs were used: for hprt,
GTTGGTTACAGGCCAGACTTTGTTG (forward) and GAGGGTAGGCT-
GGCCTATAGGCT (reverse); il-12p40, CTCACATCTGCTGCTCCACAAG
(forward) and AATTTGGTGCTTCACACTTCAGG (reverse); ifn-γ, AGAGC-
CAGATTATCTCTTTCTACCTCAG (forward) and CTTTTTTCGCCTTGCT-
GCTG (reverse); nos2, TGCCCCTTCAATGGTTGGTA (forward) and ACTG-
GAGGGACCAGCCAAAT (reverse); tnf, AAAATTCGAGTGACAAGCCTGTAG
(forward) and CCCTTGAAGAGAACCTGGGAGTAG (reverse).
In situ staining. In situ immunostaining of CD11c, F4/80, 5-LO, IL-12p40,
TNF, and NOS2 was performed as previously described (14). In brief, ace-
tone-fixed, frozen sections were incubated with biotin-conjugated anti-
bodies against CD11c or F4/80 (BD). After washing, sections were exposed
to streptavidin-conjugated Alexa Fluor 486 (Invitrogen Corp.). Sections
were simultaneously double stained with rabbit anti–IL-12p40, anti-TNF,
anti–5-LO, or anti-NOS2 pAb, and the reaction was developed with anti-
rabbit IgG Alexa Fluor 594 (Invitrogen Corp.), followed by counterstaining
with DAPI (Invitrogen Corp.). After washing, the sections were examined
microscopically, and the images were recorded using the ApoTome system
(Carl Zeiss Microimaging, Inc.).
In vivo lipoxin analog treatment. ATLa2, 15-epi-16-phenoxy-parafluoro-
LXA4-methyl ester (a generous gift from J. Parkinson, Berlex Biosciences,
Richmond, California, USA), was used in vivo as a stable lipoxin analog as
previously reported (18). It was administered by gavage 3 times a week at
a dose of 0.2 ml (100 ng/animal/treatment) as previously described (19).
Treatment was started 2 days after infection and continued until day 20.
Similarly infected control mice were likewise treated with vehicle alone.
Spleen cell cultures. Spleens from M. tuberculosis–infected mice were
disaggregated through 40-μm cell strainers, and red blood cells were
lysed osmotically. Splenocytes (5 × 106 cells/ml) in RPMI 1640 medium
(Invitrogen Corp.) supplemented with 10% fetal calf serum (HyClone),
10 mM HEPES (Invitrogen Corp.), 2 mM glutamine (Invitrogen Corp.),
100 U/ml penicillin, 100 g/ml streptomycin (Invitrogen Corp.), and
5.5 × 10–5 M 2-mercaptoethanol (Invitrogen Corp.) were distributed in
96-well plates and stimulated with 10 μg/ml of purified protein derivative
(Statens Serum Institut). After 72 hours at 37°C with 5% CO2 atmosphere,
supernatants were collected for determination of IFN-γ levels.
Statistical analysis. Statistical significance was assessed by unpaired Stu-
dent’s t test (parametric) or Mann-Whitney U test (nonparametric), and
P < 0.05 was considered significant.
We are grateful to Jose Ribeiro and Warwick Britton for their help-
ful discussions and criticism.
Received for publication November 11, 2004, and accepted in
revised form March 29, 2005.
Address correspondence to: Julio Aliberti, Department of Immu-
nology, Duke University Medical School, 136 Jones Building, Box
3010, Duke University Medical Center, Durham, North Carolina
27710, USA. Phone: (919) 613-7833; Fax: (919) 684-8982; E-mail:
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