Role of the chemokine decoy receptor D6 in balancing inflammation, immune activation, and antimicrobial resistance in Mycobacterium tuberculosis infection

Abstract

D6 is a decoy and scavenger receptor for inflammatory CC chemokines. D6-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis. The death of D6(-/-) mice was associated with a dramatic local and systemic inflammatory response with levels of M. tuberculosis colony-forming units similar to control D6-proficient mice. D6-deficient mice showed an increased numbers of mononuclear cells (macrophages, dendritic cells, and CD4 and CD8 T lymphocytes) infiltrating inflamed tissues and lymph nodes, as well as abnormal increased concentrations of CC chemokines (CCL2, CCL3, CCL4, and CCL5) and proinflammatory cytokines (tumor necrosis factor alpha, interleukin 1beta, and interferon gamma) in bronchoalveolar lavage and serum. High levels of inflammatory cytokines in D6(-/-) infected mice were associated with liver and kidney damage, resulting in both liver and renal failure. Blocking inflammatory CC chemokines with a cocktail of antibodies reversed the inflammatory phenotype of D6(-/-) mice but led to less controlled growth of M. tuberculosis. Thus, the D6 decoy receptor plays a key role in setting the balance between antimicrobial resistance, immune activation, and inflammation in M. tuberculosis infection.

Full text

The Journal of Experimental Medicine
ARTICLE
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 9 2075-2084
www.jem.org/cgi/doi/10.1084/jem.20070608
2075
The formation of granulomas at the site of my-
cobacterial infection is an essential component
of host immunity for controlling infection. This
process is dependent on the activation of myco-
bacteria-reactive T lymphocytes ( 1 ), particularly
IFN- ␥ – secreting CD4 and CD8 T cells ( 2, 3 ).
Granuloma formation, however, is a complex
process that requires not only the activation of
lymphocytes, but also their recruitment with
monocytes to the site of the infection, migra-
tion into the tissues, and juxtaposition around
mycobacteria-infected macrophages ( 4 ). This
colocalization facilitates the activation of bacte-
ricidal mechanisms in infected macrophages by
T cell – derived cytokines ( 1 ). Some mycobacte-
ria, however, survive within macrophages, and
persistent antigenic stimulation perpetuates the
process, leading to chronic granuloma forma-
tion characterized by dense accumulations of
infected macrophages, epithelioid cells, and T
lymphocytes ( 5 ). These granulomas contain the
mycobacterial infection and prevent dissemina-
tion to other organs, but they are also responsi-
ble for lung immunopathology, as the granulomas
displace and destroy parenchymal tissue ( 6 ). One
of the major roles of the granuloma is to local-
ize and contain not only the bacteria but also
the in ammatory response to the bacteria itself.
CORRESPONDENCE
Francesco Dieli:
dieli@unipa.it
Abbreviations used: ALT, alanine
aminotransferase; AST, aspartate
aminotransferase; BAL, bron-
choalveolar lavage; BCG, bacille
Calmette-Gu é rin; BUN, blood
urea nitrogen; i.n., intranasal;
MCP, monocyte chemotactic
protein; MIP, macrophage in-
 ammatory protein; RANTES,
regulated on activation, normal
T cell expressed and secreted.
D. Di Liberto and M. Locati contributed equally to this paper.
The online version of this article contains supplemental material.
Role of the chemokine decoy receptor D6
in balancing in ammation, immune
activation, and antimicrobial resistance
in Mycobacterium tuberculosis infection
Diana Di Liberto ,
1 Massimo Locati ,
2,3 Nadia Caccamo ,
1
Annunciata Vecchi ,
2 Serena Meraviglia ,
1 Alfredo Salerno ,
1 Guido Sireci ,
1
Manuela Nebuloni ,
4 Neus Caceres ,
5 Pere-Joan Cardona ,
5 Francesco Dieli ,
1
and Alberto Mantovani
2,3
1 Dipartimento di Biopatologia e Metodologie Biomediche, Universit à di Palermo, 90134 Palermo, Italy
2 Istituto Clinico Humanitas IRCCS, 20089 Rozzano, Italy
3 Istituto di Patologia Generale and
4 Pathology Unit, L. Sacco Institute of Medical Sciences, University of Milan,
20133 Milan, Italy
5 Unitat de Tuberculosi Experimental, Department of Microbiology, Fundaci ó Institut per a la Investigaci ó en Ci è ncies de la
Salut Germans Trias i Pujol and Universitat Aut ò noma de Barcelona, 08916 Badalona, Spain
D6 is a decoy and scavenger receptor for in ammatory CC chemokines. D6-de cient mice
were rapidly killed by intranasal administration of low doses of
Mycobacterium tuberculosis
.
The death of D6
ⴚ / ⴚ
mice was associated with a dramatic local and systemic in ammatory
response with levels of
M. tuberculosis
colony-forming units similar to control D6-pro cient
mice. D6-de cient mice showed an increased numbers of mononuclear cells (macrophages,
dendritic cells, and CD4 and CD8 T lymphocytes) in ltrating in amed tissues and lymph
nodes, as well as abnormal increased concentrations of CC chemokines (CCL2, CCL3, CCL4,
and CCL5) and proin ammatory cytokines (tumor necrosis factor ␣ , interleukin 1 ␤ , and
interferon ␥ ) in bronchoalveolar lavage and serum. High levels of in ammatory cytokines in
D6
ⴚ / ⴚ
infected mice were associated with liver and kidney damage, resulting in both liver
and renal failure. Blocking in ammatory CC chemokines with a cocktail of antibodies re-
versed the in ammatory phenotype of D6
ⴚ / ⴚ
mice but led to less controlled growth of
M. tuberculosis
. Thus, the D6 decoy receptor plays a key role in setting the balance between
antimicrobial resistance, immune activation, and in ammation in
M. tuberculosis
infection.
© 2008 Di Liberto et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the  rst six months
after the publication date (see http://www.jem.org/misc/terms.shtml). After six
months it is available under a Creative Commons License (Attribution–Noncom-
mercial–Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).

2076 ROLE OF THE CHEMOKINE RECEPTOR D6 IN TUBERCULOSIS | Di Liberto et al.
and has suggested a role for D6 in controlling the resolution
of CC chemokine – driven in ammatory responses. Accord-
ingly, D6
⫺ / ⫺
mice display exaggerated in ammatory responses
after skin application of phorbol esters ( 15 ) and subcutaneous
injection of complete Freund ’ s adjuvant ( 16 ). In this last
model, D6
⫺ / ⫺
mice developed granulomas characterized by
angiogenesis, extensive leukocyte in ltration, and prominent
areas of necrosis more rapidly and severely than WT coun-
terparts. However, whether D6 plays a similar role in in am-
matory responses caused by infectious microorganisms, or
has a role in the induction of a protective response to infec-
tious agents, is presently unknown. To test this possibility,
we have studied the course of M. tuberculosis infection in
D6
⫺ / ⫺
mice.
RESULTS
Expression of the D6 receptor during
M. tuberculosis
infection
Preliminarily, we investigated the expression of the D6 receptor
during M. tuberculosis infection. In human lungs and lymph nodes
from a patient with pulmonary tuberculosis, D6 expression
was observed in lymphatic endothelial cells and only occasion-
ally in scattered leukocytes (Fig. S1, available at http://www
.jem.org/cgi/content/full/jem.20070608/DC1). In the mouse,
qualitative RT-PCR experiments indicated that D6 transcripts
in the liver, spleen, and lungs do not undergo signi cant
changes at 4, 8, and 12 wk after infection with M. tuberculosis
(Fig. S2). These data suggest that D6 is mainly expressed in
lymphatic endothelial cells, in agreement with previous re-
ports ( 17 ), and that its expression does not change appreciably
during M. tuberculosis infection.
M. tuberculosis
infection in D6
ⴚ / ⴚ
mice
To determine whether the D6 receptor plays a role in the
control of M. tuberculosis infection, WT and D6
⫺ / ⫺
mice
were infected via the intranasal (i.n.) route with 2,000 CFU.
This dose did not cause mortality in C57BL/6 mice in our
study ( Fig. 1 A ). However, M. tuberculosis infection in D6
⫺ / ⫺
mice resulted in increased mortality, with ⵑ 20% of mice suc-
cumbing by week 8, 50% of mice succumbing by week 12,
and 100% of mice succumbing at week 16, at which time the
experiments were terminated ( Fig. 1 A ). The phenotype of
D6
⫺ / ⫺
was dramatic and was found highly reproducible over
a period of 3 yr in two additional experiments performed on
a total of 17 mice per group. Despite the exaggerated suscep-
tibility of D6
⫺ / ⫺
mice to M. tuberculosis infection, there was
no di erence between the two groups of mice in bacterial
loads, as determined by CFU counts in the lung, liver, and
spleen at 2, 4, 8, and 12 wk after infection ( Fig. 1 B ).
Of note, CFU data at 4, 8, and 12 wk after M. tuberculosis
infection were rechecked in two additional independent
experiments with three mice per group performed over a
period of 2 yr, con rming the results reported in Fig. 1 B .
We then examined by histology the e ect of D6 de ciency
on the in ammatory response in the lungs, liver, and spleen.
Although in WT mice a moderate cellular in ltration was
Indeed, if immune cells are not tightly controlled within the
lungs, this could lead to excess in ammation. Thus, rigorous
control of the organization of granulomas is likely necessary to
prevent immunopathology. In most cases, after the repair of
an in amed or damaged tissue, in ammation subsides and
the tissue returns to its homeostatic norm ( 7 ). However, if the
resolution phase of in ammation is ine ective, chronic in-
 ammatory pathologies may develop ( 7, 8 ). Failure to resolve
ongoing in ammation is an invariable key feature of pathol-
ogies, which are typically characterized by the high-level
expression of in ammatory cytokines and chemokines ( 9 ).
Therefore, understanding the resolution phase of transient in-
 ammatory responses will probably yield insights into some of
these chronic in ammatory pathologies.
TNF- ␣ and the related cytokine lymphotoxin- ␣ ( 10 – 13 )
are potent proin ammatory cytokines with a wide range
of activities in both the in ammatory and immune re-
sponses, and they play an essential role in host resistance
against infection with Mycobacterium tuberculosis and other
mycobacteria ( 10 – 12 ). TNF- ␣ – de cient mice infected by
aerosol with M. tuberculosis develop normal T cell responses
to mycobacterial antigens, but because of the failure of gran-
uloma formation in the infected organs they are profoundly
susceptible to the infection, succumbing with extensive ne-
crosis in the lungs and infected organs ( 12 ). Other than
TNF- ␣ , the role of other soluble mediators in regulating
granuloma formation and persistence is poorly understood.
Chemokines and their receptors are involved in cell migra-
tion and are logical candidates for a role in granuloma for-
mation, although their expression has been studied to a limited
degree in M. tuberculosis infection (for review see reference
14 ). In general, the production of chemokines at a certain
level may be a factor in preventing cell movement out of
the granuloma.
Recently, studies of the D6 chemokine receptor (for re-
view see reference 9 ) have provided novel insights regarding
the mechanism of chemokine removal from in amed sites.
D6 is structurally similar to the other chemokine receptors,
and is most homologous to CCR4 and CCR5. However,
several properties set D6 apart from other CCRs. First, D6 is
extremely promiscuous, recognizing 15 chemokines. Inter-
estingly, all D6 ligands are in ammatory CC chemokines,
whereas constitutive CC chemokines, as well as chemokines
of other subfamilies, are not recognized. Thus, D6 is a pro-
miscuous receptor with selectivity for in ammatory CC che-
mokines. Second, the D6 expression pattern is unusual, with
the predominant expression sites being trophoblasts in the
placenta and lymphatic endothelial cells in the skin, gut, and
lung. Third, and most crucially, D6 does not signal in re-
sponse to the binding of any of its ligands. Conversely, the
only apparent function of D6 is to convey the entire ligand –
receptor complex intracellularly, target the chemokine for
degradation, and recycle the receptor to the membrane. This
observation, together with the ability of D6 to bind most in-
 ammatory CC chemokines, has resulted in the proposal that
D6 functions as a “ scavenging ” or “ decoy ” receptor ( 9, 15 ),

JEM VOL. 205, September 1, 2008
ARTICLE
2077
showed a di use liver necrosis in D6
⫺ / ⫺
mice when com-
pared with WT mice ( Fig. 1 C ). In agreement with the his-
tology results, at 12 wk after M. tuberculosis infection, serum
seen at 12 wk after infection and most of the lung airspace
remained intact, an exaggerated response was evident in sur-
viving D6
⫺ / ⫺
mice (see Fig. 5 A ). Histological analysis also
Figure 1.
M. tuberculosis
infection in D6
ⴚ / ⴚ
mice. (A) Survival curve of WT (closed squares) and D6
⫺ / ⫺
(open squares) mice infected i.n. with
2 × 10 3 CFU M. tuberculosis ( n = 10). Data indicate the percentage of surviving mice over time. The difference in the survival rates of WT and D6
⫺ / ⫺
mice was statistically signi cant (P < 0.001). (B) The course of M. tuberculosis infection in WT (closed squares) and D6
⫺ / ⫺
(open squares) mice in-
fected i.n. with 2 × 10
3 CFU M. tuberculosis and followed against time in the lungs, liver, and spleen. Data shown are the mean bacterial counts ± SD.
Differences between WT and D6
⫺ / ⫺
mice were not statistically signi cant at any time point. (C) Liver and kidney sections from WT and D6
⫺ / ⫺
mice at
12 wk after infection with M. tuberculosis . Hematoxylin and eosin staining is shown. Bars, 250 μ m. Insets show magni ed structures. Original magni-
 cation was 100 × for the main images and 400 × for the insets. (D) Transaminase (ALT and AST) levels are shown. (E) BUN and proteinuria values are
shown. Similar results were obtained in  ve different experiments (shaded bars, WT mice; open bars, D6
⫺ / ⫺
mice). Differences between WT and D6
⫺ / ⫺
mice
were highly statistically signi cant (P < 0.001). Data are the mean ± the SD.

2078 ROLE OF THE CHEMOKINE RECEPTOR D6 IN TUBERCULOSIS | Di Liberto et al.
35% of CD8 T cells expressed the same activation markers in
WT mice, although di erences were not statistically signi -
cant (unpublished data). Similar  ndings were also detected
in the liver, although the di erences between D6
⫺ / ⫺
and WT
mice were lower than in the lungs and generally reached sig-
ni cance at 8 and 12 wk after infection ( Fig. 2 B ).
Cell migration to lymph nodes
Because of the  nding that the lungs of D6
⫺ / ⫺
mice con-
tained more macrophages and T lymphocytes than the lungs
of WT mice, we asked whether a greater number of these
cells migrated from the lungs to the draining lymph nodes.
To investigate this possibility,  ow cytometric analysis was
performed on the mediastinal lymph nodes of D6
⫺ / ⫺
and
WT mice after M. tuberculosis infection. As shown in Fig. 2 C ,
the number of macrophages showed a signi cant increase in
the lymph nodes of D6
⫺ / ⫺
mice at 4 wk after infection. At
4 and 8 but not 12 wk after infection, there were also statisti-
cally signi cant di erences in the total number of DCs in the
lymph nodes of D6
⫺ / ⫺
mice. The numbers of CD4 and CD8
T lymphocytes were signi cantly higher in the lymph nodes
of D6
⫺ / ⫺
mice at 4 and 8 wk after infection, but although the
latter constantly increased, the former peaked at week 4 and
then decreased. The increased migration of DCs and T lym-
phocytes to the lymph nodes was expected to result in increased
numbers of primed T lymphocytes, followed by migration to
the lungs. ELISPOT analysis con rmed this hypothesis, show-
ing that signi cantly more CD4 and CD8 T lymphocytes
from the lungs of D6
⫺ / ⫺
mice were primed to produce IFN- ␥
when stimulated by bacille Calmette-Gu é rin (BCG) – infected
DCs ( Fig. 3 ).
transaminases were abnormally increased in D6
⫺ / ⫺
compared
with WT mice ( Fig. 1 D ). Areas of focal necrosis and mono-
nuclear cell in ltrates were also evident in the kidneys ( Fig.
1 C ), and consistent with this, at 12 wk after M. tuberculosis
infection renal function was also severely compromised
in D6
⫺ / ⫺
mice, which had signi cantly higher blood urea
nitrogen (BUN) levels and frequency of severe proteinuria
as compared with WT mice ( Fig. 1 E ). Of note, although
M. tuberculosis growth occurred in the liver ( Fig. 1 B ), we
failed to detect M. tuberculosis CFU in kidneys of both WT
and D6
⫺ / ⫺
mice over a 12-wk period (not depicted). Thus,
despite the successful control of the growth of the infect-
ing mycobacteria, D6 de ciency led to increased mortality
associated with lung in ammation and combined renal and
liver failure.
Leukocyte in ltration in tissues during
M. tuberculosis
infection in D6
ⴚ / ⴚ
mice
We examined the cellular in ltrate in the lungs and liver of
D6
⫺ / ⫺
mice after M. tuberculosis infection. At predetermined
time points, organs were removed, the total number of cells
was calculated, and  ow cytometric analysis was performed to
determine cell populations ( Fig. 2 ). Numbers of DCs (F4/80
⫺
and CD11c
+ ) in the lungs ( Fig. 2 A ) were not signi cantly
di erent between D6
⫺ / ⫺
and WT mice. The numbers of
macrophages and CD4 and CD8 lymphocytes were signi -
cantly higher in D6
⫺ / ⫺
mice at all time points after infec-
tion. Approximately 45% of CD4 and CD8 T cells in ltrating
the lungs of M. tuberculosis – infected D6
⫺ / ⫺
mice at 8 and
12 wk after infection expressed activation markers (CD25
high ,
CD44
high , and CD62L
low ), whereas only 30% of CD4 and
Figure 2. Leukocyte recruitment to tissues and lymph nodes after infection with
M. tuberculosis
. Leukocytes were isolated from the lungs
(A), livers (B), and mediastinal lymph nodes (C) of WT (closed squares) and D6
⫺ / ⫺
(open squares) mice and analyzed by  ow cytometry at different
times after infection with M. tuberculosis . The cells were stained with  uorescent antibodies to F4/80 (macrophages), CD11c (bright; DCs), CD4,
and CD8. Shown are the mean cell numbers ± SD for each cell type. Similar results were obtained in  ve different experiments. *, P < 0.001;
**, P < 0.01.

JEM VOL. 205, September 1, 2008
ARTICLE
2079
and CCL5/regulated on activation, normal T cell expressed
and secreted (RANTES) were also signi cantly increased in
the serum of D6
⫺ / ⫺
mice at 12 wk after infection ( Fig. 4 B ).
Control of M. tuberculosis infection depends on the activation
of macrophages by IFN- ␥ and TNF- ␣ , leading to the induc-
tion of inducible NO synthase and the production of reactive
nitrogen intermediates such as NO. TNF- ␣ is reported to be
essential for the early expression of mRNA encoding CC and
CXC chemokines, the initial recruitment of CD4 T lympho-
cytes, and the formation and maintenance of the granulomas
( 12, 19, 20 ). To determine whether these components of the
Chemokine and cytokine levels in D6
ⴚ / ⴚ
mice
Previous studies have shown that exacerbated in ammation in
mice lacking the D6 receptor is associated with higher levels
of chemokines in blood and tissues ( 15, 16, 18 ). We therefore
examined chemokine levels in the bronchoalveolar lavage
(BAL) and serum of M. tuberculosis – infected mice. As shown
in Fig. 4 A , the concentrations of all tested chemokines were
signi cantly higher in the BAL of D6
⫺ / ⫺
than of WT mice at
12 wk after infection. Similarly, concentrations of CCL2/
monocyte chemotactic protein 1 (MCP-1), CCL3/macro-
phage in ammatory protein 1 ␣ (MIP-1 ␣ ), CCL4/MIP-1 ␤ ,
Figure 3. IFN- ␥ – producing CD4 and CD8 T cells in the lungs after infection with
M. tuberculosis
. Lung leukocytes were isolated from WT
(closed squares) and D6
⫺ / ⫺
(open squares) mice at the indicated time points after infection and were restimulated in vitro with irradiated BCG-infected
WT DCs for 36 – 40 h at 37 ° C. The numbers of IFN- ␥ – producing CD4 and CD8 T cells were quanti ed by ELISPOT at the indicated weeks. The mean
numbers ± SD of IFN- ␥ – producing CD4 and CD8 T cells from WT and D6
⫺ / ⫺
mice at each time point are shown. Similar results were obtained in  ve
different experiments. *, P < 0.005; **, P < 0.01.
Figure 4. Chemokine and cytokine levels in the BAL and serum of
M. tuberculosis
– infected WT and D6
ⴚ / ⴚ
mice. WT (shaded bars) and D6
⫺ / ⫺
(open bars) mice were infected i.n. with M. tuberculosis , and BAL or sera were obtained 12 wk later. The levels of CC chemokines (A and B) and cytokines
(C and D) were measured by ELISA. Shown are the mean values ± SD. Similar results were obtained in three different experiments. *, P < 0.001; **, P < 0.01.

2080 ROLE OF THE CHEMOKINE RECEPTOR D6 IN TUBERCULOSIS | Di Liberto et al.
ing the e cacy of the blocking. Conversely, D6
⫺ / ⫺
mice
treated with control antibody had very high levels of serum
chemokines, as did untreated D6
⫺ / ⫺
mice. TNF- ␣ , IL-1 ␤ ,
and IFN- ␥ levels in serum were also consistently reduced by
treatment with antibodies to CC chemokines (Table S1), and
cellular in ux to the infected tissues signi cantly decreased
(unpublished data). Additionally, both renal and liver func-
tion appeared improved by this treatment, as suggested by
the low levels of serum transaminases and BUN, and by the
low proteinuria detected in these mice (Table S1). Over-
all, the most impressive consequence of the neutralization
of CC chemokines in D6
⫺ / ⫺
mice was a signi cantly pro-
longed survival, as demonstrated by the only 10% mortality
at 12 wk and 30% mortality at 16 wk after infection with
M. tuberculosis ( Table I ). However, in spite of the ability of
chemokine-blocking antibodies to reduce the overall in am-
matory response ( Fig. 5 A ), the ability of D6
⫺ / ⫺
mice treated
in this manner to control the growth of M. tuberculosis in the
lungs was impaired, with higher CFU counts found at 8 and
12 wk ( Fig. 5 B ).
This result therefore indicates that the D6
receptor plays a crucial role in M. tuberculosis infection, and in
the balance between immunopathology and protective im-
mune responses.
DISCUSSION
A better understanding of the immunological mechanisms of
pathogenesis and protection is of essential importance for the
design of novel vaccines and immunotherapies against tuber-
culosis. It has been proposed that the protective response to
M. tuberculosis infection requires CD4 and CD8 lymphocytes,
the Th1-type cytokines IFN- ␥ and TNF- ␣ , and activated
macrophages (for review see reference 21 ). The cooperation
between the cells and cytokines requires close interaction,
which is achieved after migration and granuloma formation
in the lungs. The hallmark of infection in the lung is granuloma
formation, consisting of clusters of macrophages, lympho-
cytes, and DCs, which physically contains the mycobacteria
and creates a microenvironment for immune cell interaction,
limiting M. tuberculosis growth and dissemination. Chemo-
kines are potent leukocyte activators and chemoattractants
aiding granuloma formation and thought to be critical for the
immune response to M. tuberculosis ( 14 ). In vitro experiments
demonstrated that M. tuberculosis infection of bone marrow –
derived mouse macrophages results in the expression of
TNF- ␣ , as well as several chemokines, including ligands for
the chemokine receptors CXCR3, CCR5, and CCR2. Neu-
tralization of TNF- ␣ by using antibody or TNFR1-de cient
macrophages demonstrated that expression of certain chemo-
kines (CXCL9/monokine induced by IFN- ␥ , CXCL10/
IFN-inducible protein 10, CXCL11/IFN-inducible T cell ␣
chemoattractant, CCL5/RANTES, and CCL2/MCP-1) af-
ter M. tuberculosis infection was dependent, at least in part, on
TNF- ␣ . However, the lack of TNF- ␣ did not completely
abrogate chemokine expression, indicating that there are
other factors, induced as a result of infection, that stimulate
chemokine production ( 22 ).
antimycobacterial immune response were a ected by D6 de-
 ciency, ELISA was performed on the BAL and serum of
M. tuberculosis – infected mice at 8 (unpublished data) and 12 wk
after infection. Concentrations of TNF- ␣ , IL-1 ␤ , and IFN- ␥
were signi cantly higher in the BAL ( Fig. 4 C ) and serum
( Fig. 4 D ) of M. tuberculosis – infected D6
⫺ / ⫺
mice at all tested
time points, as compared with infected WT mice. Thus,
M. tuberculosis infection in the presence of D6 de ciency
causes a local and systemic in ammatory response charac-
terized by increased levels of proin ammatory chemokines
and cytokines.
Neutralization of chemokines in D6
ⴚ / ⴚ
mice in vivo reverses
pathology but increases susceptibility to infection
The results in the previous paragraph clearly show that
M. tuberculosis infection in the absence of the D6 receptor is
characterized by an exaggerated in ammatory response and
increased mortality, despite the successful control of bacterial
growth. To investigate whether the highly increased levels
of chemokines observed in D6
⫺ / ⫺
mice were involved in
the pathogenesis, we attempted to block in ammatory che-
mokines in vivo. For this purpose, mice were treated with a
mixture of antibodies to the CC chemokines CCL2/MCP-
1, CCL3/MIP-1 ␣ , CCL4/MIP-1 ␤ , and CCL5/RANTES,
as previously described ( 15, 18 ), or with antibodies to each
individual chemokine. On the same days, control mice re-
ceived i.p. injections of an equivalent amount of irrelevant
antibodies. Mice were injected weekly with M. tuberculosis ,
starting from the third week after infection. Following this
treatment schedule, CCL2/MCP-1, CCL3/MIP-1 ␣ , CCL4/
MIP-1 ␤ , and CCL5/RANTES production in the serum of
D6
⫺ / ⫺
mice was consistently reduced (Table S1, available at
http://www.jem.org/cgi/content/full/jem.20070608/DC1),
even below levels detected in WT mice, thus demonstrat-
Table I. Effects of treatment with anti – CC chemokine
antibodies on the survival of D6
⫺ / ⫺
mice
to M. tuberculosis infection
Weeks after infection
Mice Treatment 8 12 16
WT Nil 0/10 0/10 0/10
D6
⫺ / ⫺
Nil 3/10 5/10 9/10
D6
⫺ / ⫺
Control antibody 2/10 4/10 9/10
D6
⫺ / ⫺
Anti-CCL2/MCP-1 3/10 3/10 8/10
D6
⫺ / ⫺
Anti-CCL3/MIP-1 ␣ 4/10 6/10 9/10
D6
⫺ / ⫺
Anti-CCL4/MIP-1 ␤ 2/10 4/10 8/10
D6
⫺ / ⫺
Anti-CCL5/RANTES 2/10 4/10 9/10
D6
⫺ / ⫺
Mix antibody 0/10* 1/10* 3/10**
WT mice and D6
⫺ / ⫺
mice either untreated or treated with monoclonal antibodies
to single individual CC chemokines, or with a cocktail of anti – CC chemokines (mix
antibody) or irrelevant control antibodies, were infected with M. tuberculosis .
Mice were scored for survival over time. Data shown are the numbers of deaths
out of the number of treated mice ( n = 10 per group). *, P < 0.001; and **, P <
0.01 when compared with values in D6
⫺ / ⫺
mice either untreated (Nil) or treated
with control antibody.

JEM VOL. 205, September 1, 2008
ARTICLE
2081
The action of chemokines is tightly controlled by decoy
receptors ( 9 ), which internalize and target chemokines for
intracellular degradation, thus avoiding excessive in amma-
tory responses and the consequent tissue damage. D6 is a
decoy receptor that recognizes most in ammatory CC che-
mokines, implying that it may play a key role in resolving
CC chemokine – driven in ammatory responses. The results
reported in this paper con rm this possibility in a mouse
model of M. tuberculosis infection through the mucosal (i.n.)
route and show that D6
⫺ / ⫺
mice have an exaggerated in am-
matory response leading to death, despite their ability to e -
ciently control bacterial load. Upon i.n. delivery of low-dose
M. tuberculosis , 20% of mice succumbed at week 8, 50% of
mice succumbed at week 12, and 100% of mice succumbed
at week 16, despite the successful control of the growth of
M. tuberculosis bacilli. The phenotype of D6
⫺ / ⫺
was dramatic and
was found highly reproducible over a period of 3 yr in two
additional experiments performed on a total of 17 mice per
group. Moreover, CFU data at 4, 8, and 12 wk after M. tu-
berculosis infection were found highly reproducible in two ad-
ditional independent experiments performed over a period of
2 yr. Mortality in D6
⫺ / ⫺
mice coincided with an overwhelm-
ing local and systemic in ammatory response that mainly
compromised liver and kidney functions in the host, charac-
terized by increased and sustained numbers of macrophages,
DCs, and CD4 and CD8 T lymphocytes, and increased
production of in ammatory CC chemokines and cytokines
(TNF- ␣ , IL-1 ␤ , and IFN- ␥ ). However, we failed to detect
M. tuberculosis CFU in kidneys of both WT and D6
⫺ / ⫺
mice
over a 12-wk period. This might re ect either M. tuberculosis
growth below the CFU detection limit or the development
of kidney damage in the absence of kidney infection. A simi-
lar increase in macrophage, DC, and T cell numbers was
detected in the mediastinal lymph nodes, suggesting the
possibility that more T lymphocytes were being primed and
migrating to the lungs. Such increased priming likely ac-
counts for the substantially increased numbers of T lympho-
cytes migrating to the lungs of D6
⫺ / ⫺
mice after M. tuberculosis
infection, as con rmed by ELISPOT analysis showing that
signi cantly more CD4 and CD8 T lymphocytes from the
lungs of D6
⫺ / ⫺
mice were primed to produce IFN- ␥ when
stimulated in vitro by BCG-infected DCs. The  nding that
D6
⫺ / ⫺
mice had more in ammation is intriguing and con-
vincingly supports an immunoregulatory role for the D6 re-
ceptor. Recent data in other systems have shown that D6
⫺ / ⫺
mice display exaggerated responses to in ammatory stimuli,
resulting in an aggressive cutaneous in ammatory pathology
after the application of phorbol esters ( 15 ). In another study
( 16 ), D6
⫺ / ⫺
mice displayed an exaggerated response to the
subcutaneous injection of complete Freund ’ s adjuvant. Un-
fortunately, none of these studies has been able to de ne
a mechanism for the heightened immune response in the
D6
⫺ / ⫺
mice. Although there are several di erent possibilities
for the increased in ammatory response in M. tuberculosis –
infected D6
⫺ / ⫺
mice, the one we favor is that lack of the D6
receptor fails to remove CC chemokines from many di erent
In mouse models, gene expression of CXC and CC che-
mokines has been detected in the lungs after M. tuberculosis
infection ( 22 – 24 ). CXCR3-de cient mice ( 25 ) have an im-
paired granuloma formation after aerosol infection with M.
tuberculosis , although this e ect is transient, occurring at the
early stages of infection. CCR2-de cient mice are extraordi-
narily susceptible to moderate- or high-dose M. tuberculosis
administered i.v. ( 26 ), and susceptibility is dose-dependent
( 27 ). Conversely, CCR5 ( 28 ) may not be essential to the de-
velopment of a protective response to M. tuberculosis infection.
CCL2/MCP-1 – de cient mice did not demonstrate an in-
creased susceptibility to M. tuberculosis infection, but whether
cell in ltration or histology was a ected in these mice was not
reported ( 29, 30 ). However, transgenic mice overexpressing
CCL2/MCP-1 were more susceptible to tuberculosis ( 31 ).
Figure 5.
M. tuberculosis
growth in D6
ⴚ / ⴚ
mice treated with anti –
CC chemokine antibodies. (A) WT mice and D6
⫺ / ⫺
mice either untreated
(Nil), or treated with a cocktail of anti – CC chemokine (Anti-CC Ab) or irrel-
evant control antibodies (Control Ab), were infected with M. tuberculosis .
Granulomatous in ltration in the lungs of M. tuberculosis – infected mice
was assessed by analyzing two lung lobes from each mouse. Data are ex-
pressed as the percentage ± SD of the lung area involved, calculated by
dividing the granuloma-involved area by the total tissue area. A representa-
tive of two different experiments is shown. (B) The course of M. tuberculosis
infection followed against time in lungs. WT mice and D6
⫺ / ⫺
mice either
untreated (Nil), or treated with antibodies against individual chemokines,
with a cocktail of anti – CC chemokine (Mix Ab), or with irrelevant control
antibodies (Control Ab), were infected with M. tuberculosis . Data shown are
the mean bacterial counts ± SD. *, P < 0.01 when compared with CFU val-
ues in D6
⫺ / ⫺
mice either untreated (Nil) or treated with control antibody.

2082 ROLE OF THE CHEMOKINE RECEPTOR D6 IN TUBERCULOSIS | Di Liberto et al.
in amed tissue, may favor resolution of chronic in amma-
tory responses and overall provide a  ne mechanism for the
control of the balance between protective immune responses
and immunopathology.
MATERIALS AND METHODS
Mice. D6
⫺ / ⫺
mice were generated as previously described ( 15 ). Homoge-
neous populations were established by backcrossing heterozygous mice to
C57BL/6 mice for more than eight generations. The resultant heterozygous
mice were bred to obtain homozygotes. WT C57BL/6J and D6
⫺ / ⫺
mice
were bred in a speci c pathogen-free/viral antibody – free barrier facility and
obtained from Charles River Laboratories. 8 – 14-wk-old male and female
mice were used in accordance with institutional guidelines in compliance
with national (D.L. N.116, Gazzetta U ciale della Repubblica Italiana, sup-
plement 40, 18-2-1992) and international law and policies (European Eco-
nomic Community Council, 1987, Directive 86/609, O cial Journal of
European Communites L 358,1; and Institute of Laboratory Animal Re-
sources, Committee on Life Sciences, National Research Council, 1996,
Guide for the Care and Use of Laboratory Animals). Each experimental
group consisted of 7 – 10 mice. All infected mice were maintained under
germ-free conditions and were routinely monitored for mouse pathogens.
Experiments were performed in speci c pathogen-free facilities.
Chemicals and reagents. All chemicals were purchased from Sigma-
Aldrich, unless otherwise noted. Middlebrook 7H9 liquid medium and 7H10
agar were obtained from Difco Laboratories. Antibodies used in  ow cytom-
etry were obtained from BD Bioscience or R & D Systems.
M. tuberculosis and infection of mice. Mice were infected via the i.n.
route with M. tuberculosis H37Rv (2 × 10
3 CFU in 20 μ l). This resulted in
reproducible delivery of 50 – 100 viable CFU M. tuberculosis , as con rmed
by CFU determination on the lungs of infected mice 1 d after infection. In
fact, in a total of 10 mice per group from four independent experiments,
day 1 M. tuberculosis CFU ranged from 50 to 100 (with only one exception
in one WT mouse of 110 CFU), and in any case no statistical signi cant
di erences between day 1 CFU in WT (72.5 ± 21) and D6
⫺ / ⫺
(75 ± 19)
mice were detected (P = 0.9658 by the Student ’ s t test). Moreover, using
the i.n. infection route, we failed to detect M. tuberculosis CFU in nasal tis-
sues in two di erent experiments. The tissue bacillary load was quanti ed
by plating serial dilutions of the lung, liver, and spleen homogenates into
7H10 agar, as described previously ( 34 ).
Flow cytometric analysis of tissue cells. To determine cellular in ltrate,
the lungs, liver, and mediastinal lymph nodes were removed at di erent
weeks after infection and digested in the presence of 200 U/ml of collage-
nase, and mononuclear cell suspensions were obtained through Lympholyte
M (Cederlane Laboratories) gradient centrifugation. The viability of cells, as
determined by Trypan blue exclusion, was > 90%. A single-cell suspension
was prepared by pushing the tissue through a cell strainer. In some experi-
ments, lung mononuclear cells were enriched in T cells by passage through
a nylon wool column, and CD4 and CD8 T cells were then sorted by anti-
CD4 or -CD8 immunomagnetic beads (Miltenyi Biotec), according to the
manufacturer ’ s instructions ( 34 ). The cells were incubated for 24 h at 37 ° C
in complete medium to allow cells and beads to dissociate. Single-cell sus-
pension were counted. The samples were triple stained with  uorochrome-
conjugated anti-CD3, -CD4, -CD8, -CD44, -CD62L, -CD25, -CD11c,
and -F4/80, in FACS bu er (0.1% Na azide, 0.1% BSA, and 20% mouse se-
rum). Puri ed or PE-, FITC-, CyChrome-, or allophycocyanin-conjugated
isotype control Igs were used as controls. After washes, the cells were  xed
in 4% paraformaldehyde for 1 h and collected on a FACSCalibur (BD Bio-
sciences). Analysis was performed on CellQuest software (BD Biosciences).
ELISPOT analysis for IFN- ␥ . The ELISPOT method ( 35 ) was used to
detect IFN- ␥ secretion by individual CD4 or CD8 T cells from infected
organs and tissues, determining their increased concentration.
Such increased expression/circulation of CC chemokines
may, in turn, lead to signaling through their receptors and
could result in the in ux of immune cells and abundant, un-
controlled production of proin ammatory cytokines, which
ultimately mediate tissue damage. However, the increased
expression may also have been a reaction to the numbers of
cells in infected organs. Whatever the case, the concentra-
tions of CCL2/MCP-1, CCL3/MIP-1 ␣ , CCL4/MIP-1 ␤ ,
and CCL5/RANTES were found to be highly increased in
the BAL and serum of M. tuberculosis – infected D6
⫺ / ⫺
mice
and sustained over time; moreover, blocking all four of them
in D6
⫺ / ⫺
mice in vivo (CCL2/MCP-1, CCL3/MIP-1 ␣ ,
CCL4/MIP-1 ␤ , and CCL5/RANTES) by repeated injec-
tion of a cocktail of neutralizing antibodies signi cantly re-
duced cell recruitment, production of proin ammatory CC
chemokines and cytokines, liver and kidney damage, and,
most importantly, the mortality of D6
⫺ / ⫺
mice. However,
despite controlling in ammatory responses, these mice be-
came more susceptible to infection with M. tuberculosis , as
demonstrated by higher CFU counts in the lungs at 8 and 12
wk after infection. The data reported in Table I clearly show
that none of the neutralized chemokines alone accounts for
the reduced in ammatory response and mortality, and for the
increased susceptibility to infection.
CCL3/MIP-1 ␣ , CCL4/MIP-1 ␤ , and CCL5/RANTES
are all ligands of CCR5, which can attract and activate mac-
rophages and Th1 lymphocytes. However studies in CCR5
⫺ / ⫺
mice have found that although CCR5 plays a role in the mi-
gration of DCs to and from lymph nodes ( 28, 32 ), it is not in-
dispensable for granuloma formation and immune protection
against M. tuberculosis infection ( 32 ). CCL5/RANTES has
been associated with the generation of type 1 cytokine – pro-
ducing granulomas ( 33 ). CCR2-de cient mice are extraordi-
narily susceptible to moderate- or high-dose M. tuberculosis
administered i.v. but not to low aerosol doses ( 27 ), and CCL2/
MCP-1 – de cient mice do not demonstrate an increased sus-
ceptibility to M. tuberculosis infection ( 29 ). However, there is
redundancy in the chemokine system. Speci c to this study, the
chemokines CCL2/MCP-1, CCL3/MIP-1 ␣ , CCL4/MIP-1 ␤ ,
and CCL5/RANTES are ligands of CCR5, but CCL3/MIP-
1 ␣ and CCL5/RANTES can also signal through CCR1 and
CCR3. This  nding makes it di cult to dissect the precise
roles of individual chemokines and their receptors. In this
study, enhancing the type 1 T cell response or the ability of
T lymphocytes to enter the lungs over the WT level does not
have an obvious bene cial e ect on the control of infection
and in ammation, indicating that there are additional factors
that must be induced or enhanced to increase the ability of the
host to eliminate M. tuberculosis infection.
In summary, our study suggests an important role for
the D6 receptor in immune regulation. D6 controls chemo-
kine accumulation and activity, migration, and possibly, the
maintenance of DCs and T cells in infected organs, as well
as the production of in ammatory cytokines. Therefore D6,
through its ability to avoid an excess of chemokines in the

JEM VOL. 205, September 1, 2008
ARTICLE
2083
determine the statistical signi cance of the di erences in survival times of
WT and D6
⫺ / ⫺
mice. The signi cance of di erences of log
10 CFU counts
between groups was determined using one-way analysis of variance
(ANOVA), with Sche e tests for post-ANOVA individual comparisons. P <
0.05 was considered signi cant.
Online supplemental material. Fig. S1 shows lung and draining lymph
nodes sections from M. tuberculosis patients stained for D6 and the macro-
phage marker CD68. Fig. S2 shows expression levels of the D6 transcript in
mouse tissues during M. tuberculosis infection. Table S1 reports the e ects
of treatment of D6
⫺ / ⫺
mice with anti – CC chemokine antibodies. Online
supplemental material is available at http://www.jem.org/cgi/content/full/
jem.20070608/DC1.
This study was carried out with  nancial support from the European Union
FP6 contracts LSHP-CT-2003-503240 (MUVAPRED), LSHB-CT-2005-518167
(INNOCHEM), and LSHG-CT-2005-005203 (MUGEN); the CARIPLO Foundation
(NOBEL project); the Ministero dell ’ Istruzione, Universit à e Ricerca (FIRB project);
and the Universities of Palermo and Milan (FIRST projects). This work was
conducted with the support of the Fondazione Humanitas per la Ricerca and Italian
Association for Cancer Research.
The authors have no con icting  nancial interests.
Submitted: 26 March 2007
Accepted: 3 July 2008
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