Clinical and Experimental Immunology R EVI EW A RTI CL E doi:10.1111/cei.

12674

Permissive and protective roles for neutrophils in leishmaniasis

E. D. Carlsen,*† Y. Liang,† Summary

T. R. Shelite,‡ D. H. Walker,‡
P. C. Melby†‡§ and L. Soong†‡
*Department of Internal Medicine, Division of
Infectious Diseases, MD–PhD Combined Degree
Program, †Department of Microbiology and
Immunology, ‡Department of Pathology, and
§
Department of Internal Medicine, University
of Texas Medical Branch, Galveston, TX, USA
Accepted for publication 10 June 2015
Correspondence: L. Soong, Department of
Microbiology and Immunology, University of
Texas Medical Branch, Medical Research
Building 3.132, 301 University Boulevard,
Galveston, TX 77555-1070, USA.
E-mail: lysoong@utmb.edu
Leishmania parasites are the causative agents of leishmaniasis, a neglected
tropical disease that causes substantial morbidity and considerable mortality
in many developing areas of the world. Recent estimates suggest that
roughly 10 million people suffer from cutaneous leishmaniasis (CL), and
approximately 76 000 are afflicted with visceral leishmaniasis (VL), which is
universally fatal without treatment. Efforts to develop therapeutics and
vaccines have been greatly hampered by an incomplete understanding of the
parasite’s biology and a lack of clear protective correlates that must be met
in order to achieve immunity. Although parasites grow and divide
preferentially in macrophages, a number of other cell types interact with
and internalize Leishmania parasites, including monocytes, dendritic cells
and neutrophils. Neutrophils appear to be especially important shortly after
parasites are introduced into the skin, and may serve a dual protective and
permissive role during the establishment of infection. Curiously, neutrophil
recruitment to the site of infection appears to continue into the chronic
phase of disease, which may persist for many years. The immunological
impact of these cells during chronic leishmaniasis is unclear at this time. In
this review we discuss the ways in which neutrophils have been observed to
prevent and promote the establishment of infection, examine the role of
anti-neutrophil antibodies in mouse models of leishmaniasis and consider
recent findings that neutrophils may play a previously unrecognized role in
influencing chronic parasite persistence.
Keywords: Leishmania, monoclonal antibody, mouse model, neutrophil

Introduction fission in macrophages before escaping into the extracellular

Leishmania spp. are a group of obligately intracellular proto-
zoan parasites belonging to the Trypanosomitida order.
Leishmania parasites are distributed widely in the tropics and
subtropics and are the causative agents of a cluster of clinical
diseases known as leishmaniases. Leishmania parasites alter-
nate between two life-cycle stages that are highly adapted to
the distinct environments of the parasite’s life cycle. Flagel-
lated promastigotes reside in the midgut of infected female
sandflies and gain access to the skin of vertebrate hosts when
sandflies take a bloodmeal. In the skin, promastigotes are
internalized rapidly by residential and recruited cell popula-
tions, with macrophages being the parasite’s primary target
cells. Within macrophages, promastigotes convert into amas-
tigotes, which actively replicate and cause disease in mam-
malian hosts. Amastigotes undergo several rounds of binary
space and seeking new host phagocytes. The parasite’s life
cycle is completed when sandflies consume blood containing
parasitized cells. Subsequently, sandfly-ingested amastigotes
rapidly convert back into promastigotes.
In humans, symptomatic leishmaniasis is highly variable
in its clinical presentation due to differences in the infect-
ing Leishmania species and the patient’s immune status.
Most Leishmania species that cause illness in humans ini-
tially induce a disease manifestation known as localized
cutaneous leishmaniasis (CL), which is characterized by
singular or multiple well-demarcated ulcerations of the
skin that correspond to sandfly bite sites. The time between
parasite inoculation and the development of ulcerated
lesions is highly variable, but typically takes weeks to
months [1]. In many cases, localized CL is self-limiting

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E. D. Carlsen et al.
after patients develop protective adaptive immunity,
although extensive scarification at the site of resolved
lesions is typical. However, lesion resolution may be hin-
dered in immunocompromised patients and in those
infected with particular parasite species (such as those
belonging to the L. mexicana complex) [2]. It is important
to emphasize that in the absence of pharmacotherapy, pro-
tective immunity and lesion resolution are not synony-
mous with sterile cure (i.e. the healing of ulcers does not
correlate with complete parasite elimination from the site
of infection) [3]. Therefore, factors that alter host immune
status (such as corticosteroids or HIV) can potentially trig-
ger disease reactivation in clinically resolved patients [4,5].
Additionally, resolution of localized CL does not preclude
patients from experiencing several secondary forms of
leishmaniasis, including diffuse cutaneous leishmaniasis
and mucocutaneous leishmaniasis. These sequelae are par-
ticularly alarming because they can be severely disfiguring.
For more information pertaining to these more rare forms
of disease, readers are directed to an excellent clinical
review of human leishmaniasis in its many forms [1].
Of note, Leishmania species in the L. donovani complex
(including L. donovani, L. infantum and L. chagasi) cause
little to no pathology upon entering the skin, but instead
disseminate to the bone marrow, spleen and liver to cause
visceral leishmaniasis (VL) [6]. VL is the most severe form
of leishmaniasis, characterized by fever, cachexia, hepatos-
plenomegaly and pancytopenia. In the absence of medical
management, VL is fatal due to secondary infection and/or
coagulopathy [7,8]. Patients who survive VL are at risk for
a unique form of secondary leishmaniasis known as post-
kala-azar cutaneous leishmaniasis [1].
Because neutrophils recruit rapidly to the site of infec-
tion and engage in early contact with Leishmania promasti-
gotes [9,10], these cells (and their role in early parasite
recognition and clearance) have been the subject of active
research in recent years [11–22]. Neutrophils play a critical
role as first-line defenders against invading microbes and
utilize a number of innate techniques to aid in pathogen
recognition and clearance. Importantly, they can exert
direct microbicidal activity in the form of proteolytic
enzymes [23,24], reactive oxygen species [25] and neutro-
phil extracellular traps [26]. In addition, neutrophils can
secrete a wide array of cytokines and chemokines, which
helps in the recruitment and education of more specialized
cells to the site of insult [27]. Even upon dying, neutrophils
can impact upon their surroundings to encourage the
amplification of inflammatory cascades via necrosis [28],
or facilitate immune response resolution and wound heal-
ing via apoptosis [29]. Finally, roles for neutrophils in anti-
gen presentation [30], regulating T cell proliferation [31]
and driving B cell class-switching and antibody production
[32] have also been identified, suggesting that neutrophils
may exert far more diverse functions than suspected
initially.
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V
Although neutrophils are frequently the first-responders
during acute infection or tissue damage, it is important to
consider that these cells are also encountered commonly in
chronic inflammatory and infectious conditions, including
rheumatoid arthritis [33], systemic lupus erythematosis
[34], leprosy [35] and tuberculosis [36]. Recent studies
have also observed neutrophils in the chronic lesions of
animals that have been infected naturally or experimentally
with Leishmania [37–39], and several reports have identi-
fied neutrophils as a prevalent component of the chronic
inflammatory infiltrate in patients with CL [40–42], diffuse
cutaneous leishmaniasis [43] and mucocutaneous leishma-
niasis [44]. In this review, we will discuss the role of neu-
trophils in acute and chronic leishmaniasis, with a
particular focus on neutrophil–parasite interactions and
anti-neutrophil antibody treatments in experimental mod-
els of leishmaniasis.
Direct neutrophil–promastigote interactions
Several elegant studies have demonstrated the rapid
recruitment of neutrophils to the site of promastigote
infection in the skin [9,45–47]. However, the role of neu-
trophils during the early stages of infection is complex and
multi-faceted. We and others have demonstrated that neu-
trophils possess some direct leishmanicidal activity in vitro,
suggesting that these cells may represent an important
component of the early immune response against incoming
promastigotes [48,49]. However, as evidenced by parasite
persistence at the site of infection, direct neutrophil killing
of promastigotes is clearly insufficient in controlling the
establishment of infection and the development of clinical
disease.
Reports that a subset of promastigotes can survive for
prolonged periods in neutrophils without sustaining lethal
damage also highlight additional, non-microbicidal roles
for these cells during Leishmania infection [14,15,17]. Neu-
trophils that contact (but do not kill) promastigotes may
act as ‘Trojan horses’ or ‘Trojan rabbits’ [50], whereby par-
asitized cells or free parasites that have escaped from neu-
trophils, respectively, can be internalized readily by
neighbouring phagocytes (such as macrophages or dendri-
tic cells) in a manner that enhances parasite infectivity and
persistence [9,18,51]. Therefore, it is likely that neutrophils
play a dual protective and permissive role shortly after pro-
mastigote infection by reducing incoming parasite burden
and subsequently facilitating safe passage of surviving para-
sites to naive host cells.
Curiously, neutrophil recruitment and function at the site
of experimental infection may also differ depending on the
site and route of parasite inoculation. Ribeiro-Gomes et al.
recently compared neutrophil recruitment and early parasite
clearance in C57BL/6 mice that received L. major promasti-
gotes in the pinnae intradermally (i.d.) or in the footpad sub-
cutaneously (s.c.) [52]. Mice receiving intradermal parasites

exhibited a greater influx of neutrophils to the site of infec-
tion, and these cells were better able to internalize promasti-
gotes when compared to mice infected subcutaneously. This
early neutrophilic response corresponded with a 10-fold
higher parasite burden in intradermally infected mice versus
subcutaneously infected mice, and underscores the potential
for distinct immunological outcomes when comparing infec-
tion in different tissue compartments [52]. Comparing
L. major infection via needle inoculation and natural insect
vector has also yielded interesting results related to neutro-
phil function. Specifically, Peters et al. demonstrated that
neutrophil infiltration was more prolonged in response to
L. major promastigotes administered via natural sandfly bite
[53], and neutrophil depletion in this model improved para-
site clearance and amplified the immune response generated
by an experimental killed vaccine [9,53].
Impact of neutrophils upon macrophage infection
In addition to their role in direct pathogen clearance, neu-
trophils can also influence the local inflammatory environ-
ment and the development of protective immunity by
interacting with a number of immune cell types, such as
dendritic cells, macrophages, natural killer (NK) cells, T
cells and B cells [32,54–59]. Because of the important role
of macrophages during Leishmania infection, several
groups have investigated the cross-talk between neutrophils
and infected macrophages [60–64]. Collectively, these stud-
ies indicate that neutrophils can either promote parasite
clearance or facilitate parasite growth and survival in mac-
rophages, depending on several critical factors. Afonso
et al. reported that apoptotic and necrotic neutrophils had
opposing effects on L. amazonensis promastigote survival
and growth in human macrophages [60]. Neutrophils that
had undergone apoptosis in response to ultraviolet (UV)
irradiation strongly promoted parasite growth in macro-
phages in a transforming growth factor (TGF)-b1 and
prostaglandin E2 (PGE2)-dependent manner. In contrast,
freeze/thawed necrotic neutrophils facilitated parasite clear-
ance in macrophages via a tumour necrosis factor (TNF)-a
and neutrophil elastase-dependent mechanism. Surpris-
ingly, live neutrophils had little effect on the percentage of
infected macrophages or total parasite burden [60].
Historically, mouse strain-specific differences in suscep-
tibility to L. major have been attributed largely to differen-
ces in T helper cell polarization, with susceptible BALB/c
mice preferentially adopting a T helper type 2 (Th2)
response and resistant C57BL/6 mice adopting a Th1-type
response [65]. More recently, Ribeiro-Gomes et al. demon-
strated that the anti-parasite responses of these two strains
of mice also differ at the innate immune level [61]. In
L. major-infected BALB/c mice or BALB/c-derived macro-
phages, adding syngeneic exudate neutrophils (whether
intact, necrotic or apoptotic) increased parasite growth
substantially. In contrast, equivalent experiments in
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Complex roles for neutrophils in leishmaniasis
C57BL/6 mice or macrophages revealed that neutrophils
could facilitate parasite clearance [61]. However, host back-
ground may be less important with regard to New World
Leishmania infections, as neutrophils enhanced the ability
of macrophages to kill L. amazonensis [62] and L. brazilien-
sis [63] in vitro, regardless of which mouse strain was used.
Neutrophils in visceral leishmaniasis
VL-induced neutropenia has been a clinically described
entity for decades [66], and appears to be a risk factor for
death due to secondary infection in VL patients [67]. It has
long been proposed that neutropenia is probably related
to increased granulocyte clearance in the spleen [68],
although more recent reports suggest that anti-neutrophil
antibodies may also play a role [69]. Interestingly, a small
clinical trial demonstrated that the addition of granulo-
cyte–macrophage colony-stimulating factor (GM-CSF)
to standard pentavalent antimonial therapy improved neu-
tropenia in VL patients and reduced their risk of acquiring
secondary infection. However, adding GM-CSF failed to
directly accelerate the resolution of VL [70]. Almeida and
colleagues reported recently that derangements in neutro-
phil numbers and function also appear to be important
characteristics of canine VL [71]. In dogs with VL, the neu-
trophil phenotype appears to vary depending on the sever-
ity of disease. Neutrophils from severely affected dogs
undergo oxidative burst poorly and are more prone to apo-
ptosis. In contrast, neutrophils from dogs with less severe
disease undergo oxidative burst more readily and appear to
have improved half-life [71].
Anti-neutrophil treatment in mouse models
Anti-neutrophil antibody treatment is a popular method
for assessing the role of neutrophils in various infectious
and inflammatory diseases in vivo [72–75]. A handful of
studies have utilized this approach to determine the role of
neutrophils after Leishmania promastigote infection, but
conflicting results and considerable differences in experi-
mental design between studies make it difficult to draw
concrete conclusions (Table 1). For example, Chen et al.
and Tacchini-Cottier et al. examined the effects of neutro-
phil depletion on L. major burden and lesion progression
in BALB/c mice, but reported contradictory findings
[12,20]. It is possible that the use of different parasite
strains and dissimilar neutrophil depletion antibodies/dos-
ing schedules contributed partially to the discrepancies in
these studies. Notably, contrary observations have also
been published regarding the role of neutrophils in experi-
mental VL. Several studies reported that anti-neutrophil
treatment during L. donovani and L. infantum infection
exacerbated visceralization [16,77,78], while Thalhofer
et al. noted that antibody treatment reduced parasite
carriage transiently in the draining lymph nodes of
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Table 1. Anti-neutrophil treatment in mouse models of cutaneous and visceral leishmaniasis. Studies utilizing anti-neutrophil antibodies in mouse models of Leishmania major, L. donovani, L. infan-
tum, L. braziliensis and L. amazonensis infection suggest that these cells can play an important and complex role in the anti-parasite immune response and can impact disease progression.
Parasite species/strain Antibody treatment Host strain Effects of antibody treatment
E. D. Carlsen et al.

L. major LV39 NIMP-R14 1 mg 26 h BALB/c Delayed lesion progression, reduced parasite burden
C57BL/6 Accelerated early lesion progression, time required for lesion resolution
unaffected, no differences in parasite burden [20]
L. major 5ASKH RB6-8C5 250 lg Day 21 BALB/c Accelerated lesion progression and enhanced parasite burden
C3H/HeJ No change in lesion progression [12]
L. major Bokkara RB6-8C5 2 mg Days 23, 0, 3 BALB/c Accelerated lesion progression between weeks 1 and 6, chronic progression
unaltered
C57BL/6 Accelerated lesion progression at weeks 3 and 4, higher dLN burden at days

V
16 and 29, chronic progression unaltered [76]
L. major Friedlin V1 (sandfly) NIMP-R14 0.5 mg Days 3.5, 9, and 14 C57BL/6 Enhanced parasite clearance and stronger IFN-g and TNF-a responses
induced by a killed Leishmania vaccine in neutrophil-depleted animals
[53]
L. major Friedlin V1 RB6-8C5 500 lg 216 h C57BL/6 Depletion prior to sandfly-mediated infection reduced parasite burden at
the site of infection at 1 and 4 weeks [9]
L. donovani LV82 NIMP-R14 250 lg Days 0, 3, 6, 9, 12 BALB/c Increased burden in the spleen and bone marrow, hepatosplenomegaly [16]
L. donovani LV9 RB6-8C5 200 lg Days 21, 2, 5, 8, 11, 14 BALB/c Enhanced parasite burden in the liver and spleen
C57BL/6 Enhanced parasite burden in the liver and spleen [77]
L. infantum MON1 RB6-8C5 200 lg 248 h, 25 h, 72 h, 168 h BALB/c Increased parasite burden in the spleen, but not the liver
RB6-8C5 200 lg starting at 3 months, No effect [78]
2 doses/week for 4 weeks
L. chagasi MHOM/BR/00/1669 RB6-8C5 25 lg Day 21 BALB/c Impaired extracellular parasite clearance and reduced parasite carriage in
draining lymph node cells on day 1. At day 3 and onwards, parasite bur-
den was similar in control and antibody-treated animals.
1A8 25 lg Day 21 Impaired extracellular parasite clearance and reduced parasite carriage in
draining lymph node cells on day 1 [47]
L. braziliensis BA788 RB6-8C5 500 lg Days 21, 3, 6, 9, 12 BALB/c Increased parasite burden at the site of infection and in the dLNs at 2
weeks, increased IL-2, IL-4, IL-5, IFN-g, TNF-a, faster early lesion pro-
gression [63]
L. amazonensis PH8 RB6-8C5 500 lg 216 h BALB/c Increased lesion size and parasite burden at 1 week
C57BL/6 No effect
RB6-8C5 500 lg Days 21, 1, 3, 5, 7 BALB/c Increased lesion size at weeks 4 and 8
C57BL/6 No effect [79]
L. mexicana MYNC/BZ/62/M379 1A8 150 lg 26 h, 36 h C57BL/6 Decreased carriage of parasites in phagocytes and lesion resolution [80]
IL 5 interleukin; IFN 5 interferon; TNF 5 tumour necrosis factor.

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L. chagasi-infected mice at day 1 post-inoculation without
impacting the parasite burden at day 3 [47].
Although the effects of anti-neutrophil treatment vary
greatly in these published reports, most of them indicate
that neutrophils can play some sort of a protective role
shortly after parasite infection. However, several studies
highlight an opposing regulatory role for neutrophils,
implying that these cells may possess considerably more
plasticity than appreciated previously. Regardless, these
studies demonstrate co-operatively that neutrophils are
involved in the early anti-parasite response, and suggest
that disrupting normal neutrophil functions at the site of
infection can influence parasite growth and disease pathol-
ogy potently.
One possible confounding factor in the previous reports is
the anti-neutrophil antibodies utilized. Many studies used
RB6-8C5, but in recent years the specificity of this clone for
neutrophils has been questioned [19]. Importantly, RB6-8C5
also appears to target non-neutrophil myeloid cell popula-
tions, including some plasmacytoid dendritic cells [81],
monocytes [82], eosinophils [83] and T lymphocytes [84].
Additionally, the findings of a compensatory increase in neu-
trophil numbers above the controls in the footpad [79] and
blood [76,78] of Leishmania-infected mice after completing
RB6-8C5 treatments make it difficult to determine whether
the observed phenotypes were due to initial neutrophil deple-
tion or subsequent neutrophil influx. With the advent of
newer anti-neutrophil antibody clones (such as 1A8), it has
become possible to target these cells with greater specificity.
Interestingly, Wang et al. demonstrated that very low doses of
1A8 (as little as 5 lg per animal) can block neutrophil recruit-
ment to sites of inflammation without inducing protracted
periods of systemic neutropenia [75], which may help to pre-
serve unrelated neutrophil functions at distal sites, such as
immunoglobulin production in the spleen [32]. Therefore,
additional studies to investigate the impact of low-dose 1A8
treatment on promastigote infection will improve our under-
standing of neutrophils in acute leishmaniasis.
Amastigote–neutrophil interactions: implications
for chronic cutaneous leishmaniasis
Despite clear evidence of sustained neutrophil recruitment
during chronic leishmaniasis [37–44], and the finding that
reduced neutrophil recruitment correlates with decreased
lesion ulceration in infected mice [85], the contribution of
these cells to amastigote clearance and the maintenance
of a low-grade inflammatory response and tissue damage
is incompletely understood. Currently, there is a scarcity
of studies examining neutrophil–amastigote interactions
in vitro, and anti-neutrophil studies assessing the impact
of these cells during the chronic phase of cutaneous leish-
maniasis are largely absent from the literature. To deter-
mine whether neutrophils respond to amastigotes and
promastigotes differently, we recently compared neutrophil
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Complex roles for neutrophils in leishmaniasis
activation and microbicidal activity following infection
with L. amazonensis [48], a species responsible for diffuse
CL in humans [86] and non-healing CL in mice [2]. We
found that murine neutrophils internalized L. amazonensis
promastigotes and amastigotes efficiently after 4 h, and
both parasite forms triggered neutrophil activation, oxida-
tive burst and early neutrophil death. However, amastigotes
and promastigotes were very different in their susceptibility
to neutrophil microbicidal mechanisms. Notably, while
more than half of promastigotes were cleared by cells
within 6 h, nearly 100% of amastigotes survived prolonged
co-culture with neutrophils [48]. These findings corrobo-
rated and expanded upon our previous observations that
L. amazonensis amastigotes were highly resistant to purified
human histone proteins, which are an important anti-
microbial component of neutrophil extracellular traps
[22]. Additionally, the consensus findings from these
in-vitro studies highlight the superior resistance of L. ama-
zonensis amastigotes against neutrophil microbicidal
defence, even in the face of amastigote-mediated activation
and oxidative burst [48].
In an effort to understand neutrophil interactions with
different Leishmania species more clearly, we recently
assessed neutrophil activation, cytokine production and
microbicidal activity in response to amastigotes of L. brazil-
iensis, the species responsible for mucocutaneous leishmani-
asis in humans [87] and self-healing CL in mice [88].
We were surprised to find that amastigotes of L. amazonensis
and L. braziliensis triggered distinct neutrophil responses.
L. braziliensis amastigotes were a far stronger stimulus for
neutrophil activation, oxidative burst, degranulation and
proinflammatory cytokine production than L. amazonensis
amastigotes [64]. Additionally, neutrophils exerted measura-
ble leishmanicidal activity against L. braziliensis amastigotes,
while L. amazonensis amastigotes were highly resistant to
neutrophil microbicidal mechanisms [64]. These in-vitro
findings support the notion that these two Leishmania spe-
cies differ in their ability to trigger (or evade) immune rec-
ognition upon entering a host [89,90] and emphasize that
neutrophils may interact with amastigotes from different
Leishmania species in vastly different ways. Currently, it is
unclear whether neutrophils also respond to L. amazonensis
and L. braziliensis amastigotes differently in vivo and how
such differential responses impact on the chronic phase of
leishmaniasis.
Reports identifying neutrophils in the chronic lesions of
patients [42–44] and animals [37–39,91] prompted us to
analyse these cells in detail using a persistent model of
L. amazonensis infection. We found that C57BL/6 mice
infected with L. amazonensis for as long as 14 weeks had
notable accumulations of neutrophils in infected, non-
ulcerated footpads (Fig. 1a), indicating that neutrophil
recruitment to the site of infection persists during chronic
disease. Additionally, we detected a small but appreciable
population (0.2%) of lymphocyte antigen 6 complex G
E. D. Carlsen et al.
locus/lymphocyte antigen 6 complex C locus (Ly6G1/
Ly6C1) neutrophils in the draining lymph nodes of chroni-
cally infected mice (Fig. 1b). To assess the impact of neutro-
phils during the chronic stage of L. amazonensis infection
without altering early host–parasite interactions, we treated
preinfected mice with anti-neutrophil antibodies. To
achieve this, mice were infected with 2 3 105 metacyclic
promastigotes and infections were allowed to proceed until
the onset of visible footpad swelling at 4 weeks. Subse-
quently, mice received intraperitoneal injections of 300 lg
of anti-Ly6G (1A8) or isotype control (2A3) every 6 days.
Repeated 1A8 treatment efficiently blocked Ly6G on circu-
lating neutrophils, as assessed by reduced binding of allo-
phycocyanin (APC)-conjugated anti-Ly6G (Fig. 2a). As our
treatment antibody and APC-conjugated antibody both
114 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 109–118
V
Fig. 1. Neutrophil detection in the tissues of a
mouse model of chronic cutaneous leishmaniasis.
(a) At 14 weeks of infection with Leishmania
amazonensis, frozen footpad sections from
C57BL/6 mice were stained for myeloperoxidase
(MPO, red). Shown is a representative section
containing MPO-positive cells with distinct
polymorphonuclear phenotype. (b) Flow
cytometric detection of Ly6C1Ly6G1 neutrophils
in the draining lymph nodes of similarly treated
mice.
utilize the 1A8 clone, it was unclear whether 1A8 treatment
resulted in neutrophil depletion or whether Ly6G was sim-
ply blocked on circulating cells. To address this issue, we
developed a Ly6G-independent flow cytometry panel, in
which cells were co-stained for myeloperoxidase (MPO, a
marker highly expressed in neutrophils, but also in rela-
tively low levels in macrophages and monocytes [92,93])
and 7/4 (a marker for macrophages, monocytes, neutro-
phils and myeloid progenitor cells [94]). We found that
co-staining for MPO and 7/4 could be used in lieu of Ly6G
staining to identify the neutrophil population in circulating
CD11b1 cells, and that MPOhi 7/41 cells were equivalent to
Ly6G1 cells (Fig. 2b). This panel revealed that repeat anti-
neutrophil treatment did not substantially alter the fre-
quency of CD11b1MPOhi 7/41 neutrophils in the blood at
Fig. 2. Effect of anti-neutrophil treatment on
circulating neutrophils and impact of treatment
on progression of chronic leishmaniasis. Four
weeks post-infection with Leishmania
amazonensis promastigotes, C57BL/6 mice were
treated with 1A8 antibody or 2A3 isotype control
at 6-day intervals for a total of 6 weeks. (a) Flow
cytometry of blood samples collected at 6 weeks
post-antibody treatment, indicating efficient
binding of lymphocyte antigen 6 complex G
locus (Ly6G) in circulating leucocytes. (b)
Surrogate flow cytometry panel utilizing
myeloperoxidase (MPO) and 7/4 to identify
neutrophils in lieu of Ly6G staining. Ly6G– cells
are shown in grey, while Ly6G1 cells are shown
in blue. (c) Comparison of neutrophil
percentages in circulating CD11b1 leucocytes in
response to serial antibody treatments. (d) Effect
of repeated antibody treatment on lesion
progression in preinfected mice (10 animals were
included in each antibody treatment group).
Two-way analysis of variance (ANOVA) indicates
that the progressions are statistically different.
**P < 0
01 and ***P < 0
001 indicate statistically
significant differences between groups at
designated time-points using the Bonferroni
method. Data shown are from one of two
conducted experiments with similar trends.

the time of killing (Fig. 2c). Nevertheless, mice receiving
prolonged anti-neutrophil treatment (between 4 and 10
weeks post-infection) exhibited lesions with a significantly
greater rate of growth than mice receiving isotype control
(Fig. 2d). Surprisingly, anti-neutrophil treatment did little
to alter parasite burden in infected footpads (data not
shown), suggesting that neutrophils may limit detrimental
tissue damage during chronic disease without exerting
extensive leishmanicidal activity during L. amazonensis
infection. The results of this study identify neutrophils as a
component of the chronic inflammatory infiltrate at the
site of infection and in the draining lymph nodes, and sug-
gest that neutrophils may impact the inflammatory milieu
to modulate lesion progression. While exciting, the results
of this initial study highlight the necessity of investigating
the detailed in-vivo function of neutrophils during chronic
leishmaniasis, as well as the contribution of these cells to
the pathophysiology of other chronic infectious and inflam-
matory disorders.
Animal care and use
All animals were maintained under specific pathogen-free
conditions and used in accordance with protocols approved
by the Animal Care and Use Committee of the University
of Texas Medical Branch (Galveston, TX, USA).
Conclusions
Animal models of leishmaniasis indicate that neutrophils
are a prominent component of the inflammatory infiltrate
throughout the course of parasite infection, and several
clinical reports confirm that neutrophils are also present in
the persistent lesions of human patients. Despite recent
suggestions that neutrophils may play an important role in
maintaining the inflammatory environment in patients
with cutaneous and mucocutaneous leishmaniasis [40,44],
few studies have investigated the role of these cells in the
chronic disease process. Mounting evidence suggests that
neutrophils are not passive bystanders in disease pathoge-
nesis and immunopathology; rather, it is probable that
neutrophils play a complex and active role in parasite rec-
ognition and the anti-parasite response in both acute and
chronic leishmaniasis.
In-vivo anti-neutrophil treatment is a powerful method
for ascertaining the role of neutrophils in animal models
of infectious and inflammatory diseases, especially when
focusing on their function during a specific phase or
period of disease progression. However, poor neutrophil
specificity and observations of compensatory neutrophilia
make past murine leishmaniasis studies difficult to inter-
pret. Despite these limitations, recent studies help to
establish that neutrophils are important regulators of the
anti-Leishmania immune response and aid in demon-
strating that the presence of these cells has important
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 109–118
V 115
Complex roles for neutrophils in leishmaniasis
implications on the outcome of infection. In order to
assess reliably the impact of neutrophils in the context of
infection in the future, it will be helpful for researchers
to reach a consensus regarding the use of prototype para-
site strains, highly neutrophil-specific antibodies and sim-
ilar dosing regimens that minimize compensatory and/or
off-target effects.
Importantly, despite decades of research, there is no
anti-Leishmania vaccine approved for human use, which is
due partially to the fact that clear correlates of immune
protection are lacking for certain parasite species. For
example, although a strong Th1 response is sufficient for
control of L. major [95,96], effector Th1 cells are patho-
genic during experimental L. amazonensis infection [97],
and stimulating L. amazonensis-infected macrophages with
interferon (IFN)-g can actually accelerate parasite growth
[98]. Consequently, the development of future vaccines
and therapeutics for leishmaniasis may benefit greatly from
unconventional strategies that focus on amplifying innate
immune functions (particularly those functions that can
exert direct leishmanicidal activity). We suspect that
obtaining a clearer understanding of how neutrophils con-
tribute to anti-parasite immunity and inflammation will be
critical to this tactic.
Acknowledgements
The authors would like to thank Dr Hui Wang for technical
immunohistochemistry assistance and Victor Hugo Carpio
for helpful flow cytometry suggestions. The authors also
wish to express gratitude to other members of the UTMB
Joint Immunology Working Group (Drs Y. Cong, R. Ste-
phens, R. Rajsbaum, J. Sun and their trainees) for helpful
discussions. This work was supported in part by NIAID
grants (R56 AI043003; R03 AI103812) to L.S., NIAID grant
(R21 AI107419) to P.M. and the James W. McLaughlin
Predoctoral Fellowship and Blocker Scholar Fellowship in
Biomedical Research to E.C.
Disclosure
The authors declare no competing interests.
References
1 Paniz Mondolfi AE, Duffey GB, Horton LE et al. Intermediate/
borderline disseminated cutaneous leishmaniasis. Int J Dermatol
2013; 52:446–55.
2 Soong L. Subversion and utilization of host innate defense by
Leishmania amazonensis. Front Immunol 2012; 3:58.
3 Mendonca MG, de Brito ME, Rodrigues EH, Bandeira V,
Jardim ML, Abath FG. Persistence of Leishmania parasites in
scars after clinical cure of American cutaneous leishmaniasis: is
there a sterile cure? J Infect Dis 2004; 189:1018–23.
4 Tuon FF, Sabbaga Amato V, Floeter-Winter LM et al. Cutaneous
leishmaniasis reactivation 2 years after treatment caused by
E. D. Carlsen et al.
systemic corticosteroids – first report. Int J Dermatol 2007; 46:
628–30.
5 Okwor I, Uzonna JE. The immunology of Leishmania/HIV co-
infection. Immunol Res 2013; 56:163–71.
6 McGwire BS, Satoskar AR. Leishmaniasis: clinical syndromes
and treatment. Q J Med 2014; 107:7–14.
7 Belo VS, Werneck GL, Barbosa DS et al. Factors associated with
visceral leishmaniasis in the Americas: a systematic review and
meta-analysis. PLOS Negl Trop Dis 2013; 7:e2182.
8 Werneck GL, Batista MS, Gomes JR, Costa DL, Costa CH. Prog-
nostic factors for death from visceral leishmaniasis in Teresina,
Brazil. Infection 2003; 31:174–7.
9 Peters NC, Egen JG, Secundino N et al. In vivo imaging reveals
an essential role for neutrophils in leishmaniasis transmitted by
sand flies. Science 2008; 321:970–4.
10 Laskay T, van Zandbergen G, Solbach W. Neutrophil granulo-
cytes – Trojan horses for Leishmania major and other intracellu-
lar microbes? Trends Microbiol 2003; 11:210–4.
11 Charmoy M, Megnekou R, Allenbach C et al. Leishmania major
induces distinct neutrophil phenotypes in mice that are resistant
or susceptible to infection. J Leukoc Biol 2007; 82:288–99.
12 Chen L, Zhang ZH, Watanabe T et al. The involvement of neu-
trophils in the resistance to Leishmania major infection in sus-
ceptible but not in resistant mice. Parasitol Int 2005; 54:109–18.
13 Gabriel C, McMaster WR, Girard D, Descoteaux A. Leishmania
donovani promastigotes evade the antimicrobial activity of neu-
trophil extracellular traps. J Immunol 2010; 185:4319–27.
14 Gueirard P, Laplante A, Rondeau C, Milon G, Desjardins M.
Trafficking of Leishmania donovani promastigotes in non-lytic
compartments in neutrophils enables the subsequent transfer of
parasites to macrophages. Cell Microbiol 2008; 10:100–11.
15 Laufs H, Muller K, Fleischer J et al. Intracellular survival of
Leishmania major in neutrophil granulocytes after uptake in the
absence of heat-labile serum factors. Infect Immun 2002; 70:
826–35.
16 McFarlane E, Perez C, Charmoy M et al. Neutrophils contribute
to development of a protective immune response during onset
of infection with Leishmania donovani. Infect Immun 2008; 76:
532–41.
17 Mollinedo F, Janssen H, de la Iglesia-Vicente J, Villa-Pulgarin
JA, Calafat J. Selective fusion of azurophilic granules with Leish-
mania-containing phagosomes in human neutrophils. J Biol
Chem 2010; 285:34528–36.
18 Ribeiro-Gomes FL, Peters NC, Debrabant A, Sacks DL. Efficient
capture of infected neutrophils by dendritic cells in the skin
inhibits the early anti-Leishmania response. PLOS Pathog 2012;
8:e1002536.
19 Ribeiro-Gomes FL, Sacks D. The influence of early neutrophil-
Leishmania interactions on the host immune response to infec-
tion. Front Cell Infect Microbiol 2012; 2:59.
20 Tacchini-Cottier F, Zweifel C, Belkaid Y et al. An immunomo-
dulatory function for neutrophils during the induction of a
CD41 Th2 response in BALB/c mice infected with Leishmania
major. J Immunol 2000; 165:2628–36.
21 Xin L, Vargas-Inchaustegui DA, Raimer SS et al. Type I IFN
receptor regulates neutrophil functions and innate immunity to
Leishmania parasites. J Immunol 2010; 184:7047–56.
22 Wang Y, Chen Y, Xin L et al. Differential microbicidal effects of
human histone proteins H2A and H2B on Leishmania promasti-
gotes and amastigotes. Infect Immun 2011; 79:1124–33.
116 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 109–118
V
23 Massberg S, Grahl L, von Bruehl ML et al. Reciprocal coupling
of coagulation and innate immunity via neutrophil serine pro-
teases. Nat Med 2010; 16:887–96.
24 Hahn I, Klaus A, Janze AK et al. Cathepsin G and neutrophil
elastase play critical and nonredundant roles in lung-protective
immunity against Streptococcus pneumoniae in mice. Infect
Immun 2011; 79:4893–901.
25 Bonfim-Mendonca Pde S, Ratti BA, Godoy Jda S et al. b-glucan
induces reactive oxygen species production in human neutro-
phils to improve the killing of Candida albicans and Candida
glabrata isolates from vulvovaginal candidiasis. PLOS ONE
2014; 9:e107805.
26 Lu T, Kobayashi SD, Quinn MT, Deleo FR. A NET Outcome.
Front Immunol 2012; 3:365.
27 Tecchio C, Micheletti A, Cassatella MA. Neutrophil-derived
cytokines: facts beyond expression. Front Immunol 2014; 5:508.
28 Heijink IH, Pouwels SD, Leijendekker C et al. Cigarette Smoke
Induced DAMP Release from Necrotic Neutrophils Triggers
Pro-inflammatory Mediator Release. Am J Respir Cell Mol Biol
2015; 52:554–62.
29 El Kebir D, Gjorstrup P, Filep JG. Resolvin E1 promotes
phagocytosis-induced neutrophil apoptosis and accelerates reso-
lution of pulmonary inflammation. Proc Natl Acad Sci USA
2012; 109:14983–8.
30 Kambayashi T, Laufer TM. Atypical MHC class II-expressing
antigen-presenting cells: can anything replace a dendritic cell?
Nat Rev Immunol 2014; 14:719–30.
31 Hock BD, Taylor KG, Cross NB, Kettle AJ, Hampton MB,
McKenzie JL. Effect of activated human polymorphonuclear leu-
cocytes on T lymphocyte proliferation and viability. Immunol-
ogy 2012; 137:249–58.
32 Puga I, Cols M, Barra CM et al. B cell-helper neutrophils stimu-
late the diversification and production of immunoglobulin in
the marginal zone of the spleen. Nat Immunol 2012; 13:170–80.
33 Verri WA Jr, Souto FO, Vieira SM et al. IL-33 induces neutro-
phil migration in rheumatoid arthritis and is a target of anti-
TNF therapy. Ann Rheum Dis 2010; 69:1697–703.
34 Leffler J, Martin M, Gullstrand B et al. Neutrophil extracellular
traps that are not degraded in systemic lupus erythematosus
activate complement exacerbating the disease. J Immunol 2012;
188:3522–31.
35 Lee DJ, Li H, Ochoa MT et al. Integrated pathways for neutro-
phil recruitment and inflammation in leprosy. J Infect Dis 2010;
201:558–69.
36 Lowe DM, Redford PS, Wilkinson RJ, O’Garra A, Martineau
AR. Neutrophils in tuberculosis: friend or foe? Trends Immunol
2012; 33:14–25.
37 Lopez Kostka S, Dinges S, Griewank K, Iwakura Y, Udey MC,
von Stebut E. IL-17 promotes progression of cutaneous leishma-
niasis in susceptible mice. J Immunol 2009; 182:3039–46.
38 Souza-Lemos C, de-Campos SN, Teva A et al. Dynamics of
immune granuloma formation in a Leishmania braziliensis-
induced self-limiting cutaneous infection in the primate Macaca
mulatta. J Pathol 2008; 216:375–86.
39 Vercosa BL, Melo MN, Puerto HL, Mendonca IL, Vasconcelos
AC. Apoptosis, inflammatory response and parasite load in skin
of Leishmania (Leishmania) chagasi naturally infected dogs: a
histomorphometric analysis. Vet Parasitol 2012; 189:162–70.
40 Morgado FN, Schubach A, Rosalino CM et al. Is the in situ
inflammatory reaction an important tool to understand the

cellular immune response in American tegumentary leishmania-
sis? Br J Dermatol 2008; 158:50–8.
41 Dabiri S, Hayes MM, Meymandi SS, Basiri M, Soleimani F,
Mousavi MR. Cytologic features of ‘dry-type’ cutaneous leish-
maniasis. Diagn Cytopathol 1998; 19:182–5.
42 Daboul MW. Role of neutrophils in cutaneous leishmaniasis.
East Mediterr Health J 2010; 16:1055–8.
43 Dantas ML, Oliveira JM, Carvalho L et al. Comparative analysis of
the tissue inflammatory response in human cutaneous and disse-
minated leishmaniasis. Mem Inst Oswaldo Cruz 2014; 109:202–9.
44 Boaventura VS, Santos CS, Cardoso CR et al. Human mucosal
leishmaniasis: neutrophils infiltrate areas of tissue damage that
express high levels of Th17-related cytokines. Eur J Immunol
2010; 40:2830–6.
45 Beil WJ, Meinardus-Hager G, Neugebauer DC, Sorg C. Differen-
ces in the onset of the inflammatory response to cutaneous
leishmaniasis in resistant and susceptible mice. J Leukoc Biol
1992; 52:135–42.
46 Muller K, van Zandbergen G, Hansen B et al. Chemokines, nat-
ural killer cells and granulocytes in the early course of Leishma-
nia major infection in mice. Med Microbiol Immunol 2001;
190:73–6.
47 Thalhofer CJ, Chen Y, Sudan B, Love-Homan L, Wilson ME.
Leukocytes infiltrate the skin and draining lymph nodes in
response to the protozoan Leishmania infantum chagasi. Infect
Immun 2011; 79:108–17.
48 Carlsen ED, Hay C, Henard CA, Popov V, Garg NJ, Soong L.
Leishmania amazonensis amastigotes trigger neutrophil activa-
tion but resist neutrophil microbicidal mechanisms. Infect
Immun 2013; 81:3966–74.
49 Guimaraes-Costa AB, Nascimento MT, Froment GS et al. Leish-
mania amazonensis promastigotes induce and are killed by neu-
trophil extracellular traps. Proc Natl Acad Sci USA 2009; 106:
6748–53.
50 Ritter U, Frischknecht F, van Zandbergen G. Are neutrophils
important host cells for Leishmania parasites? Trends Parasitol
2009; 25:505–10.
51 van Zandbergen G, Klinger M, Mueller A et al. Cutting edge:
neutrophil granulocyte serves as a vector for Leishmania entry
into macrophages. J Immunol 2004; 173:6521–5.
52 Ribeiro-Gomes FL, Roma EH, Carneiro MB, Doria NA, Sacks
DL, Peters NC. Site-dependent recruitment of inflammatory
cells determines the effective dose of Leishmania major. Infect
Immun 2014; 82:2713–27.
53 Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P,
Sacks DL. Vector transmission of Leishmania abrogates vaccine-
induced protective immunity. PLOS Pathog 2009; 5:e1000484.
54 Schuster S, Hurrell B, Tacchini-Cottier F. Crosstalk between
neutrophils and dendritic cells: a context-dependent process.
J Leukoc Biol 2013; 94:671–5.
55 Silva MT. When two is better than one: macrophages and neu-
trophils work in concert in innate immunity as complementary
and cooperative partners of a myeloid phagocyte system.
J Leukoc Biol 2010; 87:93–106.
56 Costantini C, Cassatella MA. The defensive alliance between
neutrophils and NK cells as a novel arm of innate immunity.
J Leukoc Biol 2011; 89:221–33.
57 Jaeger BN, Donadieu J, Cognet C et al. Neutrophil depletion
impairs natural killer cell maturation, function, and homeosta-
sis. J Exp Med 2012; 209:565–80.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 109–118
V 117
Complex roles for neutrophils in leishmaniasis
58 Kalyan S, Kabelitz D. When neutrophils meet T cells: beginnings
of a tumultuous relationship with underappreciated potential.
Eur J Immunol 2014; 44:627–33.
59 Sabbione F, Gabelloni ML, Ernst G et al. Neutrophils suppress
gammadelta T-cell function. Eur J Immunol 2014; 44:819–30.
60 Afonso L, Borges VM, Cruz H et al. Interactions with apoptotic
but not with necrotic neutrophils increase parasite burden in
human macrophages infected with Leishmania amazonensis.
J Leukoc Biol 2008; 84:389–96.
61 Ribeiro-Gomes FL, Otero AC, Gomes NA et al. Macrophage
interactions with neutrophils regulate Leishmania major infec-
tion. J Immunol 2004; 172:4454–62.
62 de Souza Carmo EV, Katz S, Barbieri CL. Neutrophils reduce
the parasite burden in Leishmania (Leishmania) amazonensis-
infected macrophages. PLoS One 2010; 5:e13815.
63 Novais FO, Santiago RC, Bafica A et al. Neutrophils and
macrophages cooperate in host resistance against Leishmania
braziliensis infection. J Immunol 2009; 183:8088–98.
64 Carlsen ED, Jie Z, Liang Y et al. Interactions between neutrophils
and Leishmania braziliensis amastigotes facilitate cell activation
and parasite clearance. J Innate Immun 2015; 7:354–63.
65 Mougneau E, Bihl F, Glaichenhaus N. Cell biology and immu-
nology of Leishmania. Immunol Rev 2011; 240:286–96.
66 Bertrand A, Jourdan J, Prefaut C. Granulopenia of infectious
origin. Sem Hop 1975; 51:667–76.
67 Costa CH, Werneck GL, Costa DL et al. Is severe visceral leish-
maniasis a systemic inflammatory response syndrome? A case
control study. Rev Soc Bras Med Trop 2010; 43:386–92.
68 Musumeci S, D’Agata A, Schiliro G, Fischer A. Studies of the
neutropenia in kala-azar: results in two patients. Trans R Soc
Trop Med Hyg 1976; 70:500–3.
69 Pollack S, Nagler A, Liberman D et al. Immunological studies
of pancytopenia in visceral leishmaniasis. Isr J Med Sci 1988;
24:70–4.
70 Badaro R, Nascimento C, Carvalho JS et al. Recombinant
human granulocyte-macrophage colony-stimulating factor
reverses neutropenia and reduces secondary infections in vis-
ceral leishmaniasis. J Infect Dis 1994; 170:413–8.
71 Almeida BF, Narciso LG, Bosco AM et al. Neutrophil dysfunc-
tion varies with the stage of canine visceral leishmaniosis. Vet
Parasitol 2013; 196:6–12.
72 Shi C, Hohl TM, Leiner I, Equinda MJ, Fan X, Pamer EG.
Ly6G1 neutrophils are dispensable for defense against systemic
Listeria monocytogenes infection. J Immunol 2011; 187:5293–8.
73 Archer NK, Harro JM, Shirtliff ME. Clearance of Staphylococcus
aureus nasal carriage is T cell dependent and mediated through
interleukin-17A expression and neutrophil influx. Infect Immun
2013; 81:2070–5.
74 Ribes S, Regen T, Meister T et al. Resistance of the brain to
Escherichia coli K1 infection depends on MyD88 signaling and
the contribution of neutrophils and monocytes. Infect Immun
2013; 81:1810–9.
75 Wang JX, Bair AM, King SL et al. Ly6G ligation blocks recruit-
ment of neutrophils via a beta2-integrin-dependent mechanism.
Blood 2012; 120:1489–98.
76 Lima GM, Vallochi AL, Silva UR, Bevilacqua EM, Kiffer MM,
Abrahamsohn IA. The role of polymorphonuclear leukocytes in
the resistance to cutaneous Leishmaniasis. Immunol Lett 1998;
64:145–51.
E. D. Carlsen et al.
77 Smelt SC, Cotterell SE, Engwerda CR, Kaye PM. B cell-deficient
mice are highly resistant to Leishmania donovani infection, but
develop neutrophil-mediated tissue pathology. J Immunol 2000;
164:3681–8.
78 Rousseau D, Demartino S, Ferrua B et al. In vivo involvement
of polymorphonuclear neutrophils in Leishmania infantum
infection. BMC Microbiol 2001; 1:17.
79 Sousa LM, Carneiro MB, Resende ME et al. Neutrophils have a
protective role during early stages of Leishmania amazonensis
infection in BALB/c mice. Parasite Immunol 2014; 36:13–31.
80 Hurrell BP, Schuster S, Grun E et al. Rapid sequestration of
Leishmania mexicana by neutrophils contributes to the develop-
ment of chronic lesion. PLOS Pathog 2015; 11:e1004929.
81 Nakano H, Yanagita M, Gunn MD. CD11c1B2201Gr-11 cells in
mouse lymph nodes and spleen display characteristics of plas-
macytoid dendritic cells. J Exp Med 2001; 194:1171–8.
82 Geissmann F, Jung S, Littman DR. Blood monocytes consist of
two principal subsets with distinct migratory properties. Immu-
nity 2003; 19:71–82.
83 Tepper RI, Coffman RL, Leder P. An eosinophil-dependent
mechanism for the antitumor effect of interleukin-4. Science
1992; 257:548–51.
84 Matsuzaki J, Tsuji T, Chamoto K, Takeshima T, Sendo F,
Nishimura T. Successful elimination of memory-type CD81 T
cell subsets by the administration of anti-Gr-1 monoclonal anti-
body in vivo. Cell Immunol 2003; 224:98–105.
85 Tasew G, Nylen S, Lieke T et al. Systemic FasL and TRAIL neu-
tralisation reduce leishmaniasis induced skin ulceration. PLOS
Negl Trop Dis 2010; 4:e844.
86 Souza VL, Veras PS, Welby-Borges M et al. Immune and inflam-
matory responses to Leishmania amazonensis isolated from dif-
ferent clinical forms of human leishmaniasis in CBA mice. Mem
Inst Oswaldo Cruz 2011; 106:23–31.
87 Guerra JA, Prestes SR, Silveira H et al. Mucosal Leishmaniasis
caused by Leishmania (Viannia) braziliensis and Leishmania
(Viannia) guyanensis in the Brazilian Amazon. PLOS Negl Trop
Dis 2011; 5:e980.
88 Vargas-Inchaustegui DA, Tai W, Xin L, Hogg AE, Corry DB,
Soong L. Distinct roles for MyD88 and Toll-like receptor 2
118 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 109–118
V
during Leishmania braziliensis infection in mice. Infect Immun
2009; 77:2948–56.
89 Soong L, Henard CA, Melby PC. Immunopathogenesis of
non-healing American cutaneous leishmaniasis and progres-
sive visceral leishmaniasis. Semin Immunopathol 2012; 34:
735–51.
90 Vargas-Inchaustegui DA, Xin L, Soong L. Leishmania braziliensis
infection induces dendritic cell activation, ISG15 transcription,
and the generation of protective immune responses. J Immunol
2008; 180:7537–45.
91 Tenorio Mda S, Oliveira e Sousa L, Paixao Mdos S et al. Vis-
ceral leishmaniasis in a captive crab-eating fox Cerdocyon thous.
J Zoo Wildl Med 2011; 42:608–16.
92 Liu C, Desikan R, Ying Z et al. Effects of a novel pharmacologic
inhibitor of myeloperoxidase in a mouse atherosclerosis model.
PLOS ONE 2012; 7: e50767.
93 Hazen SL, Zhang R, Shen Z et al. Formation of nitric oxide-
derived oxidants by myeloperoxidase in monocytes: pathways
for monocyte-mediated protein nitration and lipid peroxidation
in vivo. Circ Res 1999; 85:950–8.
94 Rosas M, Thomas B, Stacey M, Gordon S, Taylor PR. The mye-
loid 7/4-antigen defines recently generated inflammatory macro-
phages and is synonymous with Ly-6B. J Leukoc Biol 2010; 88:
169–80.
95 Sadick MD, Locksley RM, Tubbs C, Raff HV. Murine cutaneous
leishmaniasis: resistance correlates with the capacity to generate
interferon-gamma in response to Leishmania antigens in vitro.
J Immunol 1986; 136:655–61.
96 Locksley RM, Heinzel FP, Sadick MD, Holaday BJ, Gardner KD
Jr. Murine cutaneous leishmaniasis: susceptibility correlates with
differential expansion of helper T-cell subsets. Ann Inst Pasteur
Immunol 1987; 138:744–9.
97 Soong L, Chang CH, Sun J et al. Role of CD41 T cells in
pathogenesis associated with Leishmania amazonensis infection.
J Immunol 1997; 158:5374–83.
98 Qi H, Ji J, Wanasen N, Soong L. Enhanced replication of Leish-
mania amazonensis amastigotes in gamma interferon-stimulated
murine macrophages: implications for the pathogenesis of cuta-
neous leishmaniasis. Infect Immun 2004; 72:988–95.