Article

Modulation of Innate Host Factors by Mycobacterium avium Complex in Human Macrophages Includes Interleukin 17

NCI-Frederick, Фредерик, Maryland, United States
The Journal of Infectious Diseases (Impact Factor: 6). 08/2012; 206(8):1206-17. DOI: 10.1093/infdis/jis492
Source: PubMed

ABSTRACT

Background:
Although opportunistic infections due to Mycobacterium avium complex (MAC) have been less common since the introduction of highly active antiretroviral therapy, globally, human immunodeficiency virus-1 (HIV-1)-positive patients remain predisposed to these infections. Absence of a properly functioning acquired immune response allows MAC persistence within macrophages localized in lymph nodes coinfected with HIV and MAC. Although a deficiency in interferon γ appears to play a part in the ability of MAC to deflect the macrophage-associated antimicrobial attack, questions about this process remain. Our study examines the ability of MAC to regulate interleukin 17 (IL-17), a proinflammatory cytokine involved in host cell recruitment.

Methods:
Coinfected lymph nodes were examined for IL-17 by immunohistochemical analysis. In vitro, macrophages exposed to mycobacteria were evaluated for transcription activities, proteins, and signaling pathways responsible for IL-17 expression. Infected macrophages were also analyzed for expression of interleukin 21 (IL-21) and negative regulators of immune responses.

Results:
Infection of macrophages triggered synthesis of IL-17, correlating with IL-17 expression by macrophages in coinfected lymph nodes. Infected macrophages exposed to exogenous IL-17 expressed CXCL10, which favors recruitment of new macrophages as targets for infection. Blockade of nuclear factor κ-light-chain-enhancer of activated B cells and mitogen-activated protein kinase pathways suppressed mycobacteria-induced IL-17 expression. MAC triggered expression of IL-21, IRF4, and STAT3 genes related to IL-17 regulation, as well as expression of the negative immunoregulators CD274(PD-L1) and suppressors of cytokine signaling.

Conclusions:
MAC-infected macrophages can provide an alternative source for IL-17 that favors accumulation of new targets for perpetuating bacterial and viral infection while suppressing host antimicrobial immune responses.

Full-text

Available from: Jan Orenstein, Feb 02, 2016
MAJOR ARTICLE
Modulation of Innate Host Factors by
Mycobacterium avium Complex in Human
Macrophages Includes Interleukin 17
Nancy Vázquez,
1
Soa Rekka,
1
Maria Gliozzi,
1
Carl G. Feng,
2
Shoba Amarnath,
3
Jan M. Orenstein,
4
and Sharon M. Wahl
1
1
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research,
2
Laboratory of Parasitic Diseases, National Institute of
Allergy and Infectious Diseases, and
3
Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland; and
4
Department of Pathology, George Washington University, Washington, D.C
Background. Although opportunistic infections due to Mycobacterium avium complex (MAC) have been less
common since the introduction of highly active antiretroviral therapy, globally, human immunodeciency virus-1
(HIV-1)positive patients remain predisposed to these infections. Absence of a properly functioning acquired
immune response allows MAC persistence within macrophages localized in lymph nodes coinfected with HIV
and MAC. Although a deciency in interferon γ appears to play a part in the ability of MAC to deect the
macrophage-associated antimicrobial attack, questions about this process remain. Our study examines the ability
of MAC to regulate interleukin 17 (IL-17), a proinammatory cytokine involved in host cell recruitment.
Methods. Coinfected lymph nodes were examined for IL-17 by immunohistochemical analysis. In vitro, mac-
rophages exposed to mycobacteria were evaluated for transcription activities, proteins, and signaling pathways
responsible for IL-17 expression. Infected macrophages were also analyzed for expression of interleukin 21 (IL-21)
and negative regulators of immune responses.
Results. Infection of macrophages triggered synthesis of IL-17, correlating with IL-17 expression by macro-
phages in coinfected lymph nodes. Infected macrophages exposed to exogenous IL-17 expressed CXCL10, which
favors recruitment of new macrophages as targets for infection. Blockade of nuclear factor κ-light-chain-enhancer
of activated B cells and mitogen-activated protein kinase pathways suppressed mycobacteria-indu ced IL-17 expres-
sion. MAC triggered expression of IL-21, IRF4, and STAT3 genes related to IL-17 regulation, as well as expression
of the negative immunoregulators CD274(PD-L1) and suppressors of cytokine signaling.
Conclusions. MAC-infected macrophages can provide an alternative source for IL-17 that favors accumulation
of new targets for perpetuating bacterial and viral infection while suppressing host antimicrobial immune
responses.
The immunocompromised individual remains at risk
for opportunistic infections, including Mycobacterium
avium complex (MAC), most evident in individuals
infected with human immunodeciency virus-1
(HIV-1) [14]. Although infection with opportunistic
pathogens represented an early diagnostic feature of
AIDS, the nature of such opportunistic infections has
changed over the past 2 decades with the use of highly
active antiretroviral therapy (HAART) [5]. However,
viral resistance and noncompliance with HAART can
contribute to the prevalence of opportunistic infec-
tions that are associated with morbidity and mortality
in patients with advanced AIDS [3]. In these patients.
MAC has a predilection for the gastrointestinal tract
and for lymphoid tissu es and may disse minate via the
bloodstream [1, 3, 4]. Of interest, immune reconstitu-
tion inammatory syndrome, a transient focal mani-
festation of variable duration that begins after the
initiation of HAART and reactivates preexisting infec-
tions, such as those due to MAC, has been increasingly
reported in HIV-infected individuals [4, 6].
Received 6 December 2011; accepted 4 May 2012; electronically published 28
August 2012.
Presented in part: 2011 Annual Meeting of The Society for Leukocyte Biology,
Kansas City, MO; July 24, 2011.
Correspondence: N. Vázquez, PhD, Bldg 30, 30 Convent Dr, MSC 4352, NIDCR,
NIH, Bethesda, MD 20892-4352 (nvazquez@mail.nih.gov).
The Journal of Infectious Diseases 2012;206:120617
Published by Oxford University Press on behalf of the Infectious Diseases Society of
America 2012.
DOI: 10.1093/infdis/jis492
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The introduction of tumor necrosis factor α (TNF-α) block-
ers in the treatment of autoimmune diseases has also led to an
increased risk of infection and reactivation of infection due to
various mycobacterial species, with MAC responsible for most
pulmonary nontuberculous mycobacterial and disseminated
infections [7]. In some patients receiving antiTNF-α therapy,
pulmonary nontuberculous mycobacterial disease developed
even when therapy was administered with antimycobacterial
drugs. Individuals with genetic defects in interferon γ (IFN-γ)
and interleukin 12 (IL-12) signaling pathways, as well as
elderly individuals, are also susceptible to MAC [8, 9]. Two
severe cases of MAC infection, one of which was fatal, have
been reported in a new immunodeciency syndrome associat-
ed with CXCR4 dysfunction [10]. More recently, and for
reasons that are still being studied, an increase in the number
of nontuberculous MAC infections in nonHIV-infected indi-
viduals has become more evident [11].
Macrophages are essential in controlling MAC infection but
can become infected with substantial numbers of MAC organ-
isms when the level of IFN-γproducing CD4
+
T cells decreas-
es, which is typical in patients with AIDS [1]. Moreover,
macrophages infected with mycobacteria can become refracto-
ry to IFN-γ in vitro, and evidence suggests that therapeutic
administration of exogenous IFN-γ may not always resolve
MAC coinfections, even in the presence of HAA RT [12]. We
recently showed that macrophage IFN-γ unresponsiveness is
due, at least in part, to the ability of MAC to induce suppres-
sors of cytokine signaling (SOCS) and that coinfected lymph
nodes express high levels of SOCS1 and SOCS3 proteins [13].
To delineate factors that may inuence recruitment of mac-
rophage hosts to the site of mycobacterial replication, we ex-
amined the potential role of interleukin 17A (IL-17A), which
is recognized as pivotal, particularly in the early response to
infection [14]. IL-17 has been mostly linked to the CD4
+
helper T-cell 17 (Th17) lineage and is also produced by γδ
T cells, natural killer cells, neutrophils, and Paneth cells [ 14].
IL-17 is not only involved in initiating and sustaining the
inammatory response, it also plays critical roles in chronic
inammation and autoimmunity [15]. The IL-17 family of
cytokines consists of 6 members, IL-17AIL-17F, but their
individual roles in infectious diseases are poorly dened [14].
Here, we provide evidence that IL-17 is involved in the host
immune response to MAC but that increased IL-17 originates
in macrophages localized in coinfected lymph nodes of pa-
tients with AIDS and is detected in macrophages infected in
vitro. MAC-induced IL-17, in turn, may recruit new bacterial
hosts, even in the relative absence of IFN-producing T cells, by
inducing chemokines, such as CXCL10, associated with disease
progressioninMAC-infectedpatients[16]. Our data demon-
strate involvement of the nuclear factor κ-light-chain-enhancer
of activated B cells (NF-κB) and mitogen-activated protein
kinase (MAPK) signaling pathways in the regulation of
MAC-induced IL-17 transcription. Exposure of macro-
phages to mycobacteria resulted in modulation of additional
factors involved in regulation of IL-17 expression. On the
other hand, enhanced expression of SOCS and CD274/PD-L1
may support an immunosuppressive environment favoring
bacterial survival. MAC-induced IL-17 apparently triggers and
sustains inltration during the early and chronic immune re-
sponse to mycobacteria, ensuring abundant target cells for
both viral and mycobacterial replication, while dampening
protective host-pathogen responses. Collectively, our data im-
plicate MAC as modulating the immune response for its own
benet, thereby contributing to persistence of this opportunis-
tic pathogen in the immunocompromised host.
MATERIALS AND METHODS
Ethics Statement
Parafn-embedded lympho id tissues from uninfected individ-
uals, patients with HIV infection, and patients with HIV/
MAC coinfection were obtained through the AIDS/Cancer
Specimen Resource (ACSR; available at: http://acsr.ucsf.edu).
The ACSR is a National Cancer Institutefunded tissue-
banking program that obtains tissues from patients after ap-
propriate consent and a deidentication procedure before
sending tissues to ACSR-approved investigators. The ACSR is
recognized by the Ofce of Biorepositories and Biospecimen
Research at the National Institutes of Health (NIH) as being
HIPAA (Health Insurance Portability and Accountability Act
of 1996) compliant in accordance with ethical standards of the
Declaration of Helsinki. All material was obtained under ap-
proval from the UCSF Committee on Human Research.
Purication of Human Monocytes
Human peripher al blo od mononuclear cells obtained by leuka-
pheresis from normal volunteers in the Department of Tr ansfu-
sion Medicine a t the NIH (Bethesda, MD) w er e diluted in
endotoxin-free phosphat e-buffer ed saline without Ca2
+
and
Mg2
+
(BioWhittaker) for density sedimentation. Monocytes in
the mononuclear cell lay er and T lymphocytes were puried by
counterow centrifugal elutria tion within 4 hours after leukaphe-
resis [13, 17]. Fr esh ly elutriate d monocytes were r esuspen ded in
DMEM (2 mM/L glutamine, 50 μg/mL gentamicin; BioWhit-
taker), pla ted in 6-well plates at 6 × 10
6
cells/w ell, and allo w ed to
adher e for 24 hours, after which 10% f etal bo vine serum was
added. Cells were allow ed to differentia te into monocyte-derived
macrophages(MDMs)byculturingfor67daysat37°Cin5%
CO
2
. T cells were expo sed to anti-CD3/CD28 antibodies (e-
Bioscience) or phytohemagglutinin (Sigma).
Infection of MDMs
Macrophages were infected with M. avium strain 2-151, which
is virulent and has a smooth, transparent morphotype, or with
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HIV-1 [18]. MAC was added at a ratio of 5:1 or 10:1 to mac-
rophages or monocytes from 1 to 18 hours. Some cultures
were exposed to the NF- κB inhibitor Bay-11-7082, the MAPK
p38 inhibitor SB203580, or the Erk1/2 inhibitor U0126 (Cal-
biochem) for 60 minutes before mycobacteria were added.
Real-Time Reverse-Transcription Po ly merase Chain Reaction
(RT-PCR)
Total cellular RNA was extracted from adherent control or in-
fected macrophages by use of the RNeasy mini kit and were
exposed to DNase (Qiagen). For real-time RT-PCR, 1 μgof
total RNA was used for reverse transcription by oligodeoxy-
thymilic acid primer, and resulting complementary DNA was
amplied by PCR, using the ABI 7500 sequence detector
(Applied Biosystems). Amplication was performed using
Taqman expression gene assays for IL-17A (Hs_00174383_
m1), IL-17F (Hs_00369400_m1), CXCL10 (Hs_00171042_
m1), IL-21 (Hs_00222337_m1), CXCR3 (Hs_00171041_m1),
SOCS1 (Hs_00705164_s1), SOCS3 (Hs_00171041_m1), IRF4
(Hs_01056533_m1), STAT3 (Hs_01047580_m1), CD274
(Hs_01125301-m1), and GAPDH (Hs_99999905_m1) as nor-
malization control (Applied Biosystems). Data were examined
using the 2
ΔΔ
CT method [19], and results are expressed as
fold increases. Conventional PCR was performed on RNA
samples for IL-17 (forward: 5-GTGAAGGCAGGAATCA
CAATC-3; reverse: 5-ACCAGGATCTCTTGCTGGAT-3).
Immunohistochemical Analysis
Biopsy specimens from lymph node tissues were obtained
from 3 patients with AIDS-dening opportunistic infection or
from HIV-1seropositive subjects without evidence of oppor-
tunistic infection and were xed in 10% neutral-buffered for-
malin, parafn embedded, and sectioned. Tissue sections were
dewaxed with xylene, rehydrated through graded alcohol solu-
tions, and processed for antigen retrieval in a decloaking
chamber (Biocare Medical) in unmasking solution (Vector
Laboratories), followed by cooling at room temperature. En-
dogenous peroxidase activity was blocked with 3% H
2
O
2
in
50% methanol (for 15 minutes). Prior to adding primary anti-
body, tissue sections were incubated with blocking serum for
30 minutes, followed by incubation with antiIL-17A (5 μg/mL;
Santa Cruz Biotechnology), antiIL-21 (0.5 μg/mL; eBioscience),
anti-CD3 (25 μg/mL; Abcam), or anti-CD68 antibody (Invi-
trogen) overnight at 4°C. Sections were incubated for 30
minutes with biotinylated secondary antibo dy. IL-17Aimmu-
noreactive staining was performed using ABC reagent from
Vectastain Elite Kit (Vector Laboratories) for 30 minutes.
Bound antibodies were visualized using 3,3-diaminobenzi-
dine-tetrahydrochloride substrate chromogen (Zymed). Slides
were counterstained with Meyer hematoxylin, dehydrated, and
mounted with Permount (Fisher Scientic). Immunohisto-
chemical staining was also performed using isotype-matched
control primary antibody (Jackson ImmunoResearch Labora-
tories). Alexa-Fluor-488 and Alexa-Fluor-546 secondary anti-
bodies and Hoechst stain (Invitrogen) were used for
immunouorescence analysis. Staining of mycobacteria was
performed as described elsewhere [1, 17, 18].
Weste rn Blot
Macrophages were exposed to MAC, and protein lysates were
harvested at indicated time points. Lysates were prepared
using a protein-extraction reagent, and cell debris was
removed [13]. Protein concentration was determined using the
Bio-Rad D
C
Protein Assay (Bio-Rad). Samples were analyzed
by sodium dodecyl sulfatepolyacrylamide gel electrophoresis,
followed by Western blot [13 ], for p38, P-p38, Erk1/2, P-
Erk1/2, Iκbα, and P-Iκbα (Cell-Signaling Technology); IL-21
(eBioscience); and tubulin (Sigma). Signal was developed
using the SuperSignal West Pico Chemiluminescent Substrate
(Pierce).
IL-17 and Viral p24 Antigen Enzyme-Linked Immunosorbent
Assay (ELISA)
Macrophage culture supernatants were collected after exposure
to mycobacteria and evaluated for IL-17A by ELISA (R&D
Systems). Culture supernatants were also examined for p24
viral antigen (PerkinElmer).
Flow Cytometry
Cells were washed with phosphate-buffered saline containing
0.1% bovine serum albumin and 0.01% sodium azide and
were stained with CD14-FITC (clone:M5E2), PD1-PE (clone:
MH4), and PDL1-PEcy7 (clone:MIH1) (BD Bioscience s) with
predetermined concentrations, according to the manufactur-
ers instructions. Data acquisition was performed using
LSR-II, and data were analyzed using FlowJo Software. Data
are expressed as the percentage of positive cells.
Statistical Analysis
Data are presented as means ± standard error of the mean and
were analyzed using the Student t test, with P values .05 con-
sidered statistically signicant.
RESULTS
Expression of IL-17 in HIV/MAC-Coinfected Lymph Nodes
Coinfection with MAC and HIV provides an environment
propitious for replication of both pathogens [ 13, 18]. In previ-
ous studies, we demonstrated that massive inltration of host
macrophages is evident in coinfected lymph nodes of patients
with AIDS [1, 13]. To investigate mechanisms contributing to
an inux of host cells to the site of infection despite a paucity
of T cells [1], we examined lymph node tissues for IL-17, a
proinammatory cytokine. Immunohistochemical analysis of
tissue from coinfected lymph nodes revealed enhanced IL-17
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expression, compared with tissues infected with HIV alone, in
which IL-17 was minimally expressed (Figure 1A and 1B).
The majority of the cells positive for IL-17 were determined to
be macrophages (Figure 1A) on the basis of size, morphology,
macrophage-specic CD68 immunohistochemical staining
(Figure 1C), and presence of intracellular mycobacteria
(Figure 1C). By comparison, inltration of monocytes into the
lymph nodes occurs typically after an unsuccessful attempt to
control mycobacterial infection [20]. Macrophages appeared to
be minimally represented in lymph nodes lacking MAC
(Figure 1B), as did detection of IL-17, further implicating
macrophages as the originating cell population for this cyto-
kine in the context of coinfection. Staining was not observed
in isotype-matched antibody-stained coinfected tissues
(Figure 1D). To further document macrophages as the IL-17
producing cell population, we used serial tissue sections to
stain for IL-17, CD68, and CD3
+
T cells (Figure 2). First, the
distribution of T cells and macrophages in uninfected and
HIV/MAC-coinfected lymph nodes was strikingly different.
CD3
+
T cells are the predominant population in uninfected
lymph nodes (Figure 2A), whereas in coinfected tissues this
population is depleted (Figure 2B). By comparison, relatively
modest numbers of CD68
+
cells were detected in uninfected
lymph nodes but accumulated in large numbers in infected
tissues (Figure 2C and 2D). Staining of serial sections of
uninfected and infected lymph nodes revealed minimal IL-17
in uninfected, CD3
+
T-cellenriched tissues but dramatically
positive staining in infected lymph nodes inltrated by CD68
+
macrophages (Figure 2E and 2F). Similar IL-17 staining pat-
terns were observed in 2 additiona l coinfected specimens ob-
tained from patients with AIDS, further suggesting that this
was a shared response to MAC in HIV-positive populations
(Figure 2G and 2H). Finally, we performed colocalization
studies using immunouorescence with antibodies to CD68
and IL-17 and documented that CD68
+
macrophages were
indeed also positive for IL-17 (Figure 3).
IL-17 Gene Expression in MAC-Infected Macrophages In Vitro
Although macrophages infected with MAC in lymph nodes
stained positively for IL-17, they could have been the source of
the cytokine and/or could have acquired IL-17 from another
source. To determine whether MAC triggered IL-17 synthesis
in this population, we examined the effect of MAC on IL-17
production in vitro, using human MDMs [17 , 18, 21]. Induc-
tion of IL-17 messenger RNA (mRNA) was evident early, with
a 15-fold increase in production detected 12 hours after
MAC exposure (Figure 4A), as demonstrated by conventional
PCR (Figure 4A) or real-time RT-PCR, and a 3050-fold in-
crease detected by 34 hours (P .05; Figure 4A). Incubation
of macrophages with MAC triggered transcription of the
Figure 1. Interleukin 17 (IL-17) expression in macrophages from patients with AIDS who were coinfected with human immunodeciency virus-1
(HIV-1) and Mycobacterium avium complex (MAC). A, Representative lymph node tissue sections from 3 individuals coinfected with MAC and HIV-1
show IL-17 protein detected by immunohistochemical analysis. B, Lymph node tissue sections for 2 individuals positive only for HIV show minimal IL-17
protein. C, Lymph node tissue sections from 3 individuals coinfected with MAC and HIV-1 show massive numbers of CD68
+
macrophages, reecting
similar pattern as IL-17 staining. The inset shows detection of MAC (red) in coinfected lymph nodes, as described in Materials and Methods. D,No
staining was observed with an isotype-matched control antibody (coinfected tissue shown). Original magnication in all panels is 63×.
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closely related cytokine IL-17F at levels relatively similar to
those of IL-17 (Figure 4B). IL-17 isoforms such as IL-17C/B/D
that were examined in parallel were minimally induced (ap-
proximately 2-fold for IL-17C) or not induced (data not
shown). In contrast, MAC did not directly trigger IL-17 tran-
scription in T lymphocytes, compared with CD3/CD28 or
phytohemagglutinin (Figure 4C). Culture supernatants from
MAC-exposed macrophages revealed increased IL-17 expres-
sion, as demonstrated by ELISA (Figure 4D). Macrophages co-
infected in vitro with HIV/MAC revealed higher IL-17
expression, compared with cultures exposed to MAC alone
(P .05), and no transcriptional induction occurred as a
result of HIV infection (Figure 4E). Although IL-17 may itself
trigger myeloid cell recruitment, it is also known to increase
CXCL10 levels [22]. Infected macrophages express elevated
mRNA levels of CXCL10, a chemokine known to participate
in recruitment of T lymphocytes and monocytes/macrophages
[23, 24] and to be associated with poor response to treatment
in patients with nontuberculous mycobacteria [16]. Moreover,
concomitant exposure to MAC and IL-17 resulted in higher
Figure 2. CD68-positive macrophages are also positive for interleukin 17 (IL-17). Serial lymph node tissue sections from a representative uninfected
individual (A, C, and E ) and a patient with AIDS coinfected with human immunodeciency virus-1 (HIV-1) and Mycobacterium avium complex (MAC; B,
D, and FH) were stained for CD3 (A and B ), CD68 (C and D ), and IL-17 (EF ) by immunohistochemical assay. Overlapping CD68 and IL-17 staining was
detected using serial sections of tissue from patients with AIDS who were coinfected with HIV-1 and MAC (D and F ) with minimal CD3
+
cells (B ). G
and H, Additional tissues from coinfected patients that are positive for IL-17 staining, demonstrating that this is a typical presentation in patients with
AIDS who are infected with MAC. Original magnication is 20×.
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levels of CXCL10, compared with cells exposed to either MAC
or cytokines alone (P < .05) (Figure 4F). Of interest, exposure
of blood-derived monocytes to MAC resulted in an increase in
CXCR3 mRNA, the CXCL10 receptor (Figure 4F).
MAC- Induced Signal Transduction Leading to IL-17
Interaction of mycobacteria with Toll-like receptor 2 (TLR2)
on macrophages engages several signaling pathways, including
the NF-κB signaling pathway [17, 25] (Figure 5A). Activation of
this pathway by MAC occurred within 1530 minutes, as
detected by phosphorylation of IkB (Figure 5A). Evaluation
of NF-κB after using the NF-κB inhibitor Bay-11-7082
(administered 1 hour prior to MAC exposure) to inhibit bacte-
ria-induced phosphorylation of IκBα (Figure 5A) showed sig-
nicant reduction in IL-17 transcription (Figure 5B). Since
interaction of MAC with macrophages also triggers activation
of MAPK pathway (Figure 5C), preexposure of cultures to a
p38 or Erk1/2 MAPK inhibitor resulted in diminished induc-
tion of IL-17, compared with cells exposed to MAC alone
(P .01; Figure 5D). Our ndings indicate that NF-κB and
MAPK signaling pathways contribute to MAC-induced IL-17
transcription in macrophages and that disruption of these
pathways blunts IL-17 expression.
MAC Regulation of IL-17 Transcription
Further analyses of MAC-infected macrophages revealed that
these cells express sustained elevated levels of IL-17 mRNA,
with levels remaining high 18 hours after infection (Figure 6A),
suggesting that additional elements may participate in sup-
porting IL-17. We analyzed gene expression of RORc, which
has been described as playing a role in IL-17 expression in
lymphoid cells [26], but we did not detect modulation of this
transcription factor (data not shown). Of interest, IRF4, which
has been reported to regulate IL-17 transcription [27], was sig-
nicantly increased in MAC-infected macrophages, with
optimal gene expression evident at 47 hours and declining
but still signicantly elevated expression 18 hours after infec-
tion (Figure 6B). The level of STAT3, another transcription
factor that regulates IL-17 transcription, was enhanced in
macrophages exposed to MAC (Figure 6C). Despite a 1020-
fold increase in IL-17A/F expression 1 hour after exposure to
MAC, IRF4 and STAT3 levels were not signicantly elevated
within 34 hours, potentially sustaining IL-17 expression in
infected cells.
Another potential regulatory molecule is IL-21, known to
participate in IL-17 regulation in Th17 cells by mechanisms
involving IRF4 and STAT3 [27, 28]. MAC induced
Figure 3. CD68 and interleukin 17 (IL-17) colocalization in macrophages in lymph node tissues from patients coinfected with human immunodeciency
virus-1 (HIV-1) and Mycobacterium avium complex (MAC). Lymph node tissue sections were stained by indirect immunouorescence, using both mouse
anti-CD68 and rabbit antiIL-17 antibodies, followed by corresponding secondary antibodies conjugated to either Alexa-488 or Alexa-546. Sections were
also stained with Hoechst. A, Nuclear detection as determined by Hoechst staining (blue). B, Macrophages in coinfected lymph nodes show positive
immunouorescence staining for CD68 (green). C, The same section shown in A and B is also positive for IL-17 (red). D, Overlay images (A, B, and C )
demonstrate colocalization of CD68 and IL-17 in macrophages of coinfected lymph nodes. Original magnication is 100×.
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transcriptional IL-21 activation in macrophages, but, of inter-
est, this occurred in a somewhat delayed fashion. Rather than
preceding detection of IL-17, IL-21 was detected after 34
hours of bacterial exposure (Figure 6D), and the level was de-
clining by 8 hours (data not shown), which suggests mainte-
nance rather than induction of IL-17. Blockade of IL-21 in
infected cultures resulted in a 40%50% reduction in MAC-
induced levels of IL-17 (Figure 6D). In this regard, exogenous
IL-21, modestly (by 23-fold) increased IL-17 levels in control
macrophages (Figure 6E), whereas higher IL-17 transcription
was evident when IL-21 was added concomitantly with MAC
(Figure 6E). These results may be explained, at least in part,
by the ability of IL-21 to enhance transcription of IRF4 (by
approximately 2-fold) (Figure 6E), which may support not
only IL-17 transcription, but also its own expression [27].
Moreover, IL-21 was detected in coinfected lymph nodes
(Figure 6F) in populations corresponding with CD68 staining
and also in infected macrophages in vitro (Figure 6G).
MAC Induces Immunosuppressive Molecules
Recent evidence suggests that IL-21 may also be of signicance
in regulating dendritic cell expression of SOCS [29]. Here, we
show that addition of exogenous IL-21 to human macrophages
in vitro signicantly augmented SOCS1 (data not shown) and
SOCS3 transcription (Figure 7A). As we previously showed,
SOCS is also induced by MAC, with maximal levels detectable
within 34 hours (Figure 7B)[13], and collectively, these path-
ways leading to SOCS could dampen host responses to the
protective cytokine IFN-γ. Reportedly, IL-17 and IL-21 can in-
uence programmed death 1 (PD1) and its ligands (PD-L1/
PD-L2) to further exert suppressive roles on myeloid cells re-
stricting T-lymphocy te function [30, 31]. Of importance, PD1/
PD-L1 play key roles in chronic viral infections, including
those due to HIV [32]. In our studies, exposure of macrophag-
es to IL-21 resulted in an increase in CD274/PD-L1 mRNA
(Figure 7C), while exposure of macrophages to MAC led to
striking levels of CD274/(PD-L1) mRNA and cell surface
Figure 4. Induction of interleukin 17 (IL-17) messenger RNA (mRNA) and protein by Mycobacterium avium complex (MAC). A, IL-17 mRNA expression
in unexposed and MAC-exposed (ratios, 10:1 or 5:1) adherent macrophage cultures after 14 hours, as determined by conventional (inset) or real-time
reverse-transcription polymerase chain reaction (RT-PCR) analysis of total mRNA from cultures (*P .05; n = 4). B, Transcriptional analysis of levels of
IL-17F mRNA in unexposed and MAC-exposed macrophages, as determined by real-time RT-PCR (*P .05; n = 3). C, Levels of IL-17 transcription after
exposure of T lymphocytes to medium alone, MAC (ratio, 10:1), anti-CD3/CD28 antibodies, or phytohemagglutinin for 4 hours (*P .01; n = 3). D,
Increased IL-17 protein in culture supernatants of macrophages that or were not exposed to MAC (ratio, 10:1) for 48 hours in vitro (*P < .01; n = 3). E,
IL-17 transcription and viral p24 antigen levels (inset) in macrophages exposed to MAC for 4 hours at periods of 2 hours or 7 days after HIV infection
(*P .05, **P < .01; n = 3). F, CXCL10 gene expression after macrophages were incubated with MAC and/or IL-17 for 4 hours, showing modulation of
CXCL0 by MAC and IL-17, as determined by real-time RT-PCR (*P < .01 for no exposure vs MAC exposure, **P < .05 for MAC- or IL-17-alone exposure vs
both IL-17 and MAC exposure; n = 3). The inset shows levels of CXCR3 mRNA in monocytes exposed to MAC for 4 hours (*P < .05; n = 3). Representative
donor data are shown.
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expression (60% increase), compared with values in unexposed
macrophages. Moreover, a 24-fold increase in mean uores-
cence intensity was noted in MAC-exposed cultures (Figure 7D
and 7E) but not for PD1 (Figure 7E). An increased CD274/
PD-L1 level was evident 1 hour after infection, and signi-
cantly elevated transcription was still seen 8 hours after infec-
tion. Even higher levels of CD274/PD-L1 mRNA were
detected following concomitant exposure of monocytes or
macrophages to IL-17 and MAC (Figure 7F and 7G). Aug-
mentation of CD274/(PD-L1) appeared to be relatively specic
in that gene expression for PD-L2 and PD-1 was variable or
unaltered in multiple donors. These data shed light on novel
MAC-induced immunosuppressive mechanisms that may
permit this opportunistic pathogen to prevail in immunocom-
promised hosts.
DISCUSSION
Our studies reveal the involvement of IL-17 in the host innate
immune response to the opportunistic bacterium MAC.
However, unanticipated was the nding that macrophages rather
than T cells were the cellular source of IL-17. We provide data
supporting the notion that IL-17 plays a role during early host
immune responses against mycobacteria, evident shortly after in
vitro exposure of macrophages to MAC. Moreover, detection of
IL-17 in macrophages from coinfected lymph nodes of patients
with AIDS suggests that IL-17 contributes to the immune re-
sponse and persistence of MAC infection in the immunocom-
promised host, where IL-17 can lead to recruitment of new
hosts and support IFN nonresponsiveness. IL-17F, one of the
members of the IL-17 family with the most homology to
Figure 5. Mycobacterium avium complex (MAC)induced interleukin 17 (IL-17) is mediated by NF-κB and mitogen-activated protein kinase (MAPK)
pathways. A, Human macrophages were incubated with MAC, and whole protein cell lysates were generated after 15 and 30 minutes and examined for
P-IκB activation and IκB by Western blot. B, Preexposure of macrophages to the NF-κB inhibitor Bay-11-7082 (for 1 hour) suppressed MAC-induced IL-
17A, as shown by real-time polymerase chain reaction (*P < .01 for no exposure vs MAC exposure, **P < .05 for both MAC and inhibitor exposure vs
MAC-only exposure; n = 3). C, Whole protein cell lysates (30 minutes) from unexposed and MAC-exposed macrophages were analyzed for phosphoryla-
tion of p38 and Erk1/2 MAPK and total MAPK (n = 3). D, IL-17 mRNA expression in macrophage cultures preexposed to a p38 or Erk1/Erk2 MAPK
inhibitor for 1 hour prior to exposure to MAC for 4 hours (*P < .01 for no exposure vs MAC-only exposure, ** P .01 for both MAC and inhibitor exposure
vs MAC-only exposure; n = 3). Representative donor data are shown.
M. avium Complex Enhances IL-17 in Macrophages
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IL-17A induced in activated monocytes [33], was also elevated
in MAC-infected macrophages.
IL-17A/F are potent inducers of inammatory mediators,
including ch emokines such as CXCL10 [22]. CXCL10 partici-
pates in recruitment of T lymphocytes, monocytes, and mac-
rophages [23, 24], and elevated levels of CXCL10 correlate
with poor response to treatment in patients with MAC infec-
tions [16]. IL-17 may further sustain inammation by enhanc-
ing the stability of chemokine mRNA transcripts [34].
Therefore, enhanced CXCL10 induction by MAC and IL-17
suggests that this mechanism may be operational and contri-
bute to continuous recruitment of new targets for bacterial
and viral infections.
Our study shows that MAC induces IL-17 production
through a mechanism involving the MAPK and NF-κB
pathways. Recent reports have shown that chitin, a ubiquitous
polysaccharide in fungi, insects, and parasites, regulates IL-17
and acute tissue inammation in macrophages [35] and that
p38MAPK inuences IL-17 [36]. In addition, TLR2 stimula-
tion in combination with T-cell receptor activation can
promote Th17 differentiation [37].
Prolonged IL-17 expression in mycobacteria-exposed mac-
rophages correlated with high IRF4 and STAT3 levels, which
are known to support IL-17 [14, 26]. These ndings correlated
with enhanced expression of IL-21, a regulator of IL-17 that is
important for Th17 polarization and differentiation, represent-
ing an alternative pathway for generation of the Th17 subset
[38], which we considered to be a potential regulator of mac-
rophage IL-17. The synergistic effect of MAC and IL-21 on
IL-17 transcription can be explained, at least in part, by the
Figure 6. Mycobacterium avium complex (MAC) induces interleukin 17 (IL-17)related transcription factors IRF4, STAT3, and interleukin 21 (IL-21). A,
IL-17 messenger RNA (mRNA) levels in total mRNA from macrophages exposed to MAC (ratio, 10:1) for 418 hours (*P < .01; n = 3). B, Findings of
kinetic transcriptional analysis of IRF4 mRNA in macrophage cultures incubated with MAC for various intervals (*P < .05; n = 3). C, STAT3 mRNA levels
in macrophages that were or were not exposed to MAC (*P < .05; n = 3). D, Findings of kinetic transcriptional analysis of IL-21 and IL-17 by real-time
reverse-transcription polymerase chain reaction in macrophage cultures incubated with MAC for 14 hours (*P < .01; n = 3). The inset shows ndings for
macrophages that were incubated with MAC overnight in the presence or absence of neutralizing IL-21 antibodies and evaluated for IL-17 transcription.
Results are expressed as percentage response, compared with cultures that did not receive antibodies (*P < .05). E, IL-17 mRNA levels in macrophages
exposed to MAC and/or IL-21 (10 ng/mL) for 4 hours (*P .05, **P < .01; n = 3). The inset shows IRF4 mRNA levels in macrophages that were exposed
to IL-21 for 4 hours (*P < .05; n = 3). Data are for a representative donor. F, Immunohistochemical staining revealed IL-21positive cells in lymph node
tissue from an individual with AIDS who was coinfected with MAC and HIV-1. G, Whole cell protein extracts of macrophages exposed to MAC showed
enhanced IL-21 protein expression, compared with unexposed cultures, as determined by Western blot (n = 3).
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fact that macrophages cultured with IL-21 or MAC show en-
hanced gene expression of IRF4, which is known to participate
in the transcriptional regulation of IL-17 and IL-21 [27, 28].
IL-21, in turn, activates the MAPK pathway [39] and inuenc-
es phosphorylation of IRF4 [27]. Whi le we cannot rule out the
participation of additional factors in the regulation of IL-17 in
macrophages [14], RORγt gene expression linked to regulation
of IL-17 downstream of IRF4 [14] was not signicantly in-
creased in macrophages by MAC (data not shown).
Despite the IL-17propagated inammatory response, mac-
rophages in the immunocompromised host do not clear MAC,
in part because of enhanced SOCS1/3 interference with IFN-γ
signaling [13]. Here, we show that abrogation of IFN-γ
protective innate imm une responses may be further
exacerbated by IL-21driven SOCS, consistent with its effects
in dendritic cells [29]. Higher SOCS expression is found in
patients with tuberculosis and recurrent tuberculosis, and re-
duction of SOCS expression has been considered a plausible
approach to improve host protective responses [40]. SOCS
may also interfere with protective antiviral activity of type I
and type II inte rferons [13, 41]. Further complicating this sce-
nario is the fact that infection of macrophages with MAC in
the presence or absence of IL-21 enhanced levels of immuno-
suppressive CD274/PD-L1, as did coexposure to IL-17 and
mycobacteria, likely via MAPK [30, 42]. This can be further
aggravated by the ability of HIV-1 to enhance CD274/PD-L1
on macrophages, mediated via TLR [43]. CD274/PD-L1, con-
sidered a marker for disease progression, and its receptor,
Figure 7. Expression of suppressors of cytokine signaling (SOCS) and CD274 by Mycobacterium avium complex (MAC) and MAC-induced cytokines.
A, SOCS3 gene expression with and without exposure to exogenous interleukin 21 (IL-21) (*P .01; n = 3). B, SOCS messenger RNA (mRNA) levels in
macrophages after no exposure or 14 hours of exposure to MAC, as determined by analysis of total mRNA levels (*P .01; n = 3). C, CD274 mRNA
levels in macrophages with or without exposure to IL-21, as determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) detection
of CD274/PD-L1 (*P < .05; n = 3). D, CD274 mRNA levels in macrophage cultures with or without exposure to MAC alone for 18 hours, as determined
by real-time RT-PCR analysis of total mRNA for detection of CD274/(PD-L1) (*P .05, **P .01 for no exposure vs MAC exposure; n = 3). E, Flow
cytometry analyses of unexposed (blue line) and MAC-exposed macrophages (red line) for CD274 or PD1 (inset). The gray line denotes isotype. F, CD274
mRNA levels in monocytes with or without exposure to MAC and interleukin 17 (IL-17) for 4 hours, as determined by real-time RT-PCR (*P < .005 for no
exposure vs MAC-only exposure, **P < .05 for no exposure vs IL-17only exposure, ***P = .03 for both MAC and IL-17 exposure vs MAC-only exposure;
n=3) G, CD274 mRNA levels in macrophage cultures exposed to MAC and/or IL-17, as determined by real-time RT-PCR (*P = .002 for no exposure vs
MAC exposure, *P = .002 for no exposure vs both MAC and IL-17 exposure, ** P < .05 for no exposure vs IL-17 exposure, ***P < .05 for MAC-only
exposure vs both MAC and IL-17 exposure; n = 3). Representative donor data are shown (n = 3).
M. avium Complex Enhances IL-17 in Macrophages
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PD1, are augmented on monocytes/macrophages and T cells
during HIV-1 infection, suggesting dampening of the HIV-
specic effector T-cell function [32, 43, 44]. Although IL-21
producing CD4
+
T cells have been associated with retroviral
control by affecting CD8
+
T lymphocytes in patients with a
low viral load [45], in immunocompromised hosts, IL-21 may
deect innate and adaptive protective imm une responses
by inducing SOCS and CD274 expression. In addition, the
PD-1/PD-L1/PD-L2 pathway has been connected to sup-
pression of effector T-cell function against Mycobacterium
tuberculosis [46].
Although appropriate levels of IL-17 are thought to have a
protective role in response to infection, especially during the
early stages of infection with HIV-1 and M. tuberculosis, per-
sistent IL-17 is also associated with tissue-damaging inam-
mation and negative outcomes [22, 47]. For instance, IL-17
has been reported to have immunopathological roles during
infection with multidrug-resistant M. tuberculosis and persis-
tently elevated levels of antigen [48]. IL-17 contributes to the
pathogenesis of autoimmune/inammatory conditions such
as rheumatoid arthritis, systemic lupus erythematosis, multiple
sclerosis, Sjögren syndrome, asthma, and Crohn disease
[15, 22]. IL-17 in CD68
+
monocytes/macrophages found in in-
amed mucosa of patients with inammatory bowel disease
has been linked to induction and persistence of mucosal in-
ammation [49]. Recently, tissue macrophages expressing IL-17
have been described in breast tumors, where they promote
invasiveness [50].
MAC regulates multiple host molecules in macrophages in
vitro, corresponding to evidence of their dysregulation in vivo
[13, 18]. Here, we exposed a new strategy used by this opportu-
nistic pathogen to promote persistence within the macrophage.
It is possible that, initially, IL-17 production by MAC-infected
macrophages may aid in recruiting cells and thereby mediate
resistance/protection activities that are important during the
early innate immune response. In immunocompromised indi-
viduals and during advanced disease, dysregulated production
of IL-17 in the absence of Th1 lymphocytes, IFN-γ, or ap-
propriate counterregulatory mechanisms to disengage IL-17
responses could drive pathogenesis.
Notes
Acknowledgments. We are grateful to Dr Ke-jian Lei, Wenwen Jin,
and Vichit Lorn, for technical assistance; to Calley Grace, for editorial
assistance; and Drs Alfredo Molinolo and Ramiro Iglesias-Bartolome, for
help with immunoourescence staining of tissue.
Financial support. This work was supported by the Intramural
Research Program of the National Institute of Dental and Craniofacial
Research, NIH.
Potential conicts of interests. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the
content of the manuscript have been disclosed.
References
1. Orenstein JM, Fox C, Wahl SM. Macrophages as a source of HIV
during opportunistic infections. Science 1997; 276:185761.
2. Wahl SM, Greenwell-Wild T, Hale-Donze H, Moutsopoulos N, Oren-
stein JM. Permissive factors for HIV-1 infection of macrophages.
J Leukoc Biol 2000; 68:30310.
3. Karakousis PC, Moore RD, Chaisson RE. Mycobacterium avium
complex in patients with HIV infection in the era of highly active
antiretroviral therapy. Lancet Infect Dis 2004; 4:55765.
4. Tuon FF, Mulatti GC, Pinto WP, de Siqueira Franca FO, Gryschek
RC. Immune reconstitution inammatory syndrome associated with
disseminated mycobacterial infection in patients with AIDS. AIDS
Patient Care STDS 2007; 21:52732.
5. Palella FJ Jr, Delaney KM, Moorman AC, et al. Declining morbidity
and mortality among patients with advanced human immunodecien-
cy virus infection. HIV Outpatient Study Investigators. N Engl J Med
1998; 338:85360.
6. Lawn SD, Bekker LG, Miller RF. Immune reconstitution disease asso-
ciated with mycobacterial infections in HIV-infected individuals
receiving antiretrovirals. Lancet Infect Dis 2005; 5:36173.
7. Winthrop KL, Chang E, Yamashita S, Iademarco MF, LoBue PA.
Nontuberculous mycobacteria infections and anti-tumor necrosis
factor-alpha therapy. Emerg Infect Dis 2009; 15:155661.
8. Dorman SE, Holland SM. Mutation in the signal-transducing chain of
the interferon-gamma receptor and susceptibility to mycobacterial in-
fection. J Clin Invest 1998; 101:23649.
9. Haerynck F, Holland SM, Rosenzweig SD, Casanova JL, Schelstraete P,
De Baets F. Disseminated Mycobacterium avium infection in a patient
with a novel mutation in the interleukin-12 receptor-beta1 chain.
J Pediatr 2008; 153:7212.
10. Doncker AV, Balabanian K, Bellanne-Chantelot C, et al. Two cases of
disseminated Mycobacterium avium infection associated with a new
immunodeciency syndrome related to CXCR4 dysfunctions. Clin Mi-
crobiol Infect 2011; 17:1359.
11. Parrish SC, Myers J, Lazarus A. Nontuberculous mycobacterial
pulmonary infections in Non-HIV patients. Postgrad Med 2008;
120:7886.
12. Lauw FN, van Der Meer JT, de Metz J, Danner SA, van Der Poll T.
No benecial effect of interferon-gamma treatment in 2 human im-
munodeciency virus-infected patients with Mycobacterium avium
complex infection. Clin Infect Dis 2001; 32:e81
2.
13. Vazquez N, Greenwell-Wild T, Rekka S, Orenstein JM, Wahl SM.
Mycobacterium avium-induced SOCS contributes to resistance to
IFN-gamma-mediated mycobactericidal activity in human macro-
phages. J Leukoc Biol 2006; 80:113644.
14. Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of the
immune system. Nat Rev Immunol 2010; 10:47989.
15. Katsis GE, Rekka S, Moutsopoulos NM, Pillemer S, Wahl SM. Sys-
temic and local interleukin-17 and linked cytokines associated with
Sjogrens syndrome immunopathogenesis. Am J Pathol 2009;
175:116777.
16. Lim A, Allison C, Tan DB, Oliver B, Price P, Waterer G. Immuno-
logical markers of lung disease due to non-tuberculous mycobacteria.
Dis Markers 2010; 29:1039.
17. Greenwell-Wild T, Vazquez N, Sim D, et al. Mycobacterium avium
infection and modulation of human macrophage gene expression.
J Immunol 2002; 169:628697.
18. Wahl SM, Greenwell-Wild T, Peng G, et al. Mycobacterium avium
complex augments macrophage HIV-1 production and increases
CCR5 expression. Proc Natl Acad Sci U S A 1998; 95:125749.
19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-Delta Delta C(T))
Method. Methods 2001; 25:4028.
20. Hale-Donze H, Greenwell-Wild T, Mizel D, et al. Mycobacterium
avium complex promotes recruitment of monocyte hosts for HIV-1
and bacteria. J Immunol 2002; 169:385462.
1216
JID 2012:206 (15 October)
Vázquez et al
by guest on January 7, 2016http://jid.oxfordjournals.org/Downloaded from
Page 11
21. Wahl SM, Greenwell-Wild T, Peng G, Hale-Donze H, Orenstein JM.
Co-infection with opportunistic pathogens promotes human immuno-
deciency virus type 1 infection in macrophages. J Infect Dis 1999;
179(Suppl 3):S45760.
22. Gaffen SL. An overview of IL-17 function and signaling. Cytokine
2008; 43:4027.
23. Zhou J, Tang PC, Qin L, et al. CXCR3-dependent accumulation and
activation of perivascular macrophages is necessary for homeostatic arterial
remodeling to hemodynamic stresses. J Exp Med 2010; 207:195166.
24. Taub DD, Lloyd AR, Conlon K, et al. Recombinant human interfer-
on-inducible protein 10 is a chemoattractant for human monocytes
and T lymphocytes and promotes T cell adhesion to endothelial cells.
J Exp Med 1993; 177:180914.
25. Rocco JM, Irani VR. Mycobacterium avium and modulation of the
host macrophage immune mechanisms. Int J Tuberc Lung Dis 2011;
15:44752.
26. Hirahara K, Ghoreschi K, Laurence A, Yang XP, Kanno Y, OShea JJ.
Signal transduction pathways and transcriptional regulation in Th17
cell differentiation. Cytokine Growth Factor Rev 2010; 21:42534.
27. Biswas PS, Gupta S, Chang E, et al. Phosphorylation of IRF4 by
ROCK2 regulates IL-17 and IL-21 production and the development of
autoimmunity in mice. J Clin Invest 2010; 120:328095.
28. Huber M, Brustle A, Reinhard K, et al. IRF4 is essential for IL-21-
mediated induction, amplication, and stabilization of the Th17 phe-
notype. Proc Natl Acad Sci U S A 2008; 105:2084651.
29. Strengell M, Lehtonen A, Matikainen S, Julkunen I. IL-21 enhances
SOCS gene expression and inhibits LPS-induced cytokine production
in human monocyte-derived dendritic cells. J Leukoc Biol 2006;
79:127985.
30. Zhao Q, Xiao X, Wu Y, et al. Interleukin-17-educated monocytes sup-
press cytotoxic T-cell function through B7-H1 in hepatocellular carci-
noma patients. Eur J Immunol 2011; 41:231422.
31. Kinter AL, Godbout EJ, McNally JP, et al. The common gamma-chain
cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of pro-
grammed death-1 and its ligands. J Immunol 2008; 181:673846.
32. Kaufmann DE, Walker BD. Programmed death-1 as a factor in
immune exhaustion and activation in HIV infection. Curr Opin HIV
AIDS 2008; 3:3627.
33. Starnes T, Robertson MJ, Sledge G, et al. Cutting edge: IL-17F, a novel
cytokine selectively expressed in activated T cells and monocytes,
regulates angiogenesis and endothelial cell cytokine production.
J Immunol 2001; 167:413740.
34. Hartupee J, Liu C, Novotny M, Li X, Hamilton T. IL-17 enhances che-
mokine gene expression through mRNA stabilization. J Immunol
2007
; 179:413541.
35.
Da Silva CA, Hartl D, Liu W, Lee CG, Elias JA. TLR-2 and IL-17A in
chitin-induced macrophage activation and acute inammation.
J Immunol 2008; 181:427986.
36. Noubade R, Krementsov DN, Del Rio R, et al. Activation of p38
MAPK in CD4 T cells controls IL-17 production and autoimmune
encephalomyelitis. Blood 2011; 118:3290300.
37. Nyirenda MH, Sanvito L, Darlington PJ, et al. TLR2 stimulation
drives human naive and effector regulatory T cells into a Th17-like
phenotype with reduced suppressive function. J Immunol 2011;
187:227890.
38. Nurieva R, Yang XO, Martinez G, et al. Essential autocrine regulation
by IL-21 in the generation of inammatory T cells. Nature 2007;
448:4803.
39. Fuqua CF, Akomeah R, Price JO, Adunyah SE. Involvement of ERK-
1/2 in IL-21-induced cytokine production in leukemia cells and
human monocytes. Cytokine 2008; 44:1017.
40. Mistry R, Cliff JM, Clayton CL, et al. Gene-expression patterns in
whole blood identify subjects at risk for recurrent tuberculosis. J Infect
Dis 2007; 195:35765.
41. Fenner JE, Starr R, Cornish AL, et al. Suppressor of cytokine signaling
1 regulates the immune response to infection by a unique inhibition
of type I interferon activity. Nat Immunol 2006; 7:339.
42. Wole SJ, Strebovsky J, Bartz H, et al. PD-L1 expression on
tolerogenic APCs is controlled by STAT-3. Eur J Immunol 2011;
41:41324.
43. Rodriguez-Garcia M, Porichis F, de Jong OG, et al. Expression of
PD-L1 and PD-L2 on human macrophages is up-regulated by
HIV-1 and differentially modulated by IL-10. J Leukoc Biol 2011;
89:50715.
44. Boasso A, Hardy AW, Landay AL, et al. PDL-1 upregulation on
monocytes and T cells by HIV via type I interferon: restricted expres-
sion of type I interferon receptor by CCR5-expressing leukocytes. Clin
Immunol 2008; 129:13244.
45. Yue FY, Lo C, Sakhdari A, et al. HIV-specic IL-21 producing CD4+
T cells are induced in acute and chronic progressive HIV infection
and are associated with relative viral control. J Immunol 2010;
185:498506.
46. Jurado JO, Alvarez IB, Pasquinelli V, et al. Programmed death (PD)-1:
PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions
during human tuberculosis. J Immunol 2008; 181:11625.
47. Torrado E, Cooper AM. IL-17 and Th17 cells in tuberculosis. Cyto-
kine Growth Factor Rev 2010
; 21:45562.
48.
Basile JI, Geffner LJ, Romero MM, et al. Outbreaks of Mycobacterium
tuberculosis MDR strains induce high IL-17 T-cell response in patients
with MDR tuberculosis that is closely associated with high antigen
load. J Infect Dis 2011; 204:105464.
49. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin
17 in inammatory bowel disease. Gut 2003; 52:6570.
50. Zhu X, Mulcahy LA, Mohammed RA, et al. IL-17 expression by
breast-cancer-associated macrophages: IL-17 promotes invasiveness of
breast cancer cell lines. Breast Cancer Res 2008; 10:R95.
M. avium Complex Enhances IL-17 in Macrophages
JID 2012:206 (15 October)
1217
by guest on January 7, 2016http://jid.oxfordjournals.org/Downloaded from
Page 12
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