This information is current as
of June 18, 2017.
Differential Activation of Inflammatory
Pathways in Testicular Macrophages
Provides a Rationale for Their Subdued
Inflammatory Capacity
Sudhanshu Bhushan, Svetlin Tchatalbachev, Yongning Lu,
Suada Fröhlich, Monika Fijak, Vijith Vijayan, Trinad
Chakraborty and Andreas Meinhardt
Supplementary
Material
http://www.jimmunol.org/content/suppl/2015/04/25/jimmunol.140113
2.DCSupplemental
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Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 27 April 2015
http://www.jimmunol.org/content/early/2015/04/25/jimmun
ol.1401132
Published April 27, 2015, doi:10.4049/jimmunol.1401132
The Journal of Immunology
Differential Activation of Inflammatory Pathways in
Testicular Macrophages Provides a Rationale for Their
Subdued Inflammatory Capacity
Sudhanshu Bhushan,* Svetlin Tchatalbachev,† Yongning Lu,* Suada Fröhlich,*
Monika Fijak,* Vijith Vijayan,* Trinad Chakraborty,† and Andreas Meinhardt*
I
mmune privilege is defined as a special status of tissues in the
body that tolerate allo- and autoantigens. Beside the testis,
immune-privileged sites include the anterior chamber of the
eye, the brain, and temporarily the placenta/uterus during pregnancy as well as tumor-draining lymph nodes (1, 2). The rat testis
was identified as an immune-privileged organ when allografts
transplanted into the male gonad survived prolonged periods of
time without evidence of rejection (3). Testicular immune privilege seems logical, as the first appearance of a large number of
neoantigens in developing germ cells occurs only after establishment of self-tolerance and thus requires special adaptations of the
immune system to avoid rejection and ultimately infertility by
chronic orchitis. Tolerance is conferred by the testis itself, as
autoimmunity is rapidly elicited when testicular autoantigens are
*Department of Anatomy and Cell Biology, Justus Liebig University Giessen, 35392
Giessen, Germany; and †Department of Medical Microbiology, Justus Liebig University Giessen, 35392 Giessen, Germany
Received for publication May 2, 2014. Accepted for publication April 1, 2015.
This work was supported by Deutsche Forschungsgemeinschaft Grants KFO 181/2
and BH 93/1-1.
Address correspondence and reprint requests to Prof. Andreas Meinhardt, Department of Anatomy and Cell Biology, Justus Liebig University Giessen, Aulweg 123,
35392 Giessen, Germany. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CAPE, caffeic acid phenethyl ester; CSN, COP9
signalosome; PM, peritoneal macrophage; poly(I:C), polyinosinic-polycytidylic acid;
qRT-PCR, quantitative RT-PCR; SCF-bTrCP, Skp1–cullin–F-box/b-transducin repeat–containing protein; TM, testicular macrophage; TRAM, Toll/IL-1R domain–
containing adaptor-inducing IFN-b–related adaptor molecule; Treg, regulatory
T cell; TRIF, Toll/IL-1R domain–containing adapter-inducing IFN-b.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401132
injected under the skin (4). Data accumulated during the last decade now imply that local active immunosuppression rather than
simple sequestration of autoantigens by the blood/testis barrier is
central in the establishment and maintenance of testicular immune
privilege. Mechanisms implicated in the control of immune privilege include high levels of intratesticular androgens, and particularly immunosuppressive properties of local cells such as Sertoli
cells in the seminiferous epithelium as well as regulatory T cells
(Tregs) and macrophages in the interstitial space (1, 5, 6). One
way how Sertoli cells induce testicular immune tolerance is by
generation of Tregs (Foxp3+ Tregs) in a mechanism that triggers
Notch/Jagged signaling pathways in these cells (7, 8).
Testicular macrophages (TM) constitute the principal population
of immune cells in the testis of most species (1, 9). TM are morphologically and functionally closely linked to the androgenproducing Leydig cells in the testicular interstitial space (10).
There is strong evidence that TM play a vital role in testis development and adult function, as the absence of macrophages from the
testis leads to disordered testicular development. Most notably,
op/op mice deficient in the macrophage growth factor M-CSF have
poorly developed testes, with very few TM, aberrant steroidogenesis, and deficient sperm production, whereas the development of
Leydig cells and androgen production in testes depleted of macrophages by various other means is similarly inhibited (11).
TM, similar to other macrophages, show normal phagocytic and
bactericidal function. However, by contributing to immune privilege they display a clearly diminished innate immune response
following challenge with LPS compared with macrophages in other
tissues (for review, see Refs. 12, 13). Recently, Winnall et al. (14)
reported that the immune response of TM is skewed toward an
alternatively activated macrophage type similar to the M2 type in
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Spermatogenic cells express cell-specific molecules with the potential to be seen as “foreign” by the immune system. Owing to the
time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations
of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM)
play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with
other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway–specific genes compared with peritoneal macrophages.
Moreover, in TM stimulated with LPS, the NF-kB signaling pathway is blocked due to lack of IkBa ubiquitination and, hence,
degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling
pathways, which leads to production of proinflammatory cytokines such as TNF-a, although at much lower levels than in
peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and
CREB signaling pathways and attenuates LPS-induced TNF-a and IL-10 secretion. High levels of IL-10 production and activation
of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain
testicular immune privilege by inhibiting NF-kB signaling through impairment of IkBa ubiquitination and a general reduction
of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB
signaling pathways. The Journal of Immunology, 2015, 194: 000–000.
2
DIFFERENTIAL ACTIVATION OF PATHWAYS IN TESTIS MACROPHAGES
30 min at 32˚C, TM adhered to the culture dish and contaminating cells
were removed by extensive washing. Peritoneal macrophages (PM) were
isolated by peritoneal lavage as previously described (20). Briefly, PM
were seeded into culture plates at 37˚C for 30 min and washed vigorously
to remove the contaminating cells. Purity of TM and PM was ∼85–90% as
determined by immunofluorescence staining using the rat macrophagespecific Abs ED1 (CD68) and ED2 (CD163) (Serotec, Oxford, U.K.).
ELISA
Shortly after isolation, TM and PM were treated with 10 mg/ml LPS and
10 mg poly(I:C) for the indicated time points in the figures. Supernatants were
collected and measured by ELISA specific for TNF-a (eBioscience, San
Diego, CA), IL-10 (BD Biosciences, San Jose, CA), and IL-6 (DuoSet; R&D
Systems, Wiesbaden, Germany) following the manufacturers’ instructions.
Western blot
After treatment, TM and PM were washed with ice-cold PBS and lysed with
SDS-PAGE sample buffer. Lysed cells were gently sonicated on ice (10 s,
one pulse; Bandelin Sonopuls, Berlin, Germany) and heated at 90˚C for 10
min. Equal amounts of protein were separated on 10% SDS-PAGE and
transferred to a nitrocellulose membrane (Hybond ECL, 0.2 mm; GE
Healthcare, Buckinghamshire, U.K.). Blocking of unspecific binding, incubation with primary and secondary Abs, and ECL detection (GE Healthcare) occurred as previously described (20). Membranes were stripped and
reprobed with an anti-actin Ab to assess equal loading.
RNA isolation
Total RNA was isolated using the RNeasy mini kit and the RNase-free
DNase I set (Qiagen, Hilden, Germany) following the manufacturer’s
protocol. The RNA was recovered in RNase-free water, heat denatured for
10 min at 65˚C; quantified with the NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Rockland, DE), and a quality
profile with the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) was collected.
Real time RT-PCR
Adult male Wistar rats (249–270 g) were purchased from Harlan (Borchen,
Germany) and kept under standard conditions (22˚C, 12-h light/dark cycle)
with pelleted food and water ad libitum. The experiments were performed
according to the guidelines of the local authority (Regierungspraesidium,
Giessen, Germany) and conform to the Code of Practice for the Care and Use
of Animals for Experimental Purposes (permission GI 20/23 no. A 31/2012).
First-strand cDNA was synthesized with 400 ng purified RNA using
SuperScript II (Invitrogen) and a mixture of T21 and random nonamer
primers (Metabion, Martinsried, Germany) following the instructions for
the reverse transcription reaction recommended for the QuantiTect SYBR
Green kit (Qiagen). Real-time quantitative PCR was performed with
a QuantiTect SYBR Green Kit (Qiagen) on an ABI Prism 7700 real-time
cycler (Invitrogen). The relative expression of target genes was normalized
to that of a pool of four reference genes. PCR primer information is provided
in Table I. Reactions were done in triplicates using the QuantiTect SYBR
Green kit (Qiagen). For every target and reference gene a standard dilution
curve with a reference RNA sample was done and the linear equation was
used to transform threshold cycle values into nanograms of total RNA. The
relative fold change of target genes in the PM samples versus the TM
samples was normalized by the relative expression of a pool of the following four reference genes: HMBS, PPIA, RPLP2, and SHDA (see
Table I). Normalized fold change for a target gene versus every reference
gene was calculated and a mean fold change of these four was the final
value. In Fig. 6 mRNA expression analysis was performed as described
earlier using b2-microglobulin (15).
Abs and chemicals
Immunofluorescence
Abs directed against p38 (no. 9212), phospho-p38 (no. 9211), ERK1/2 (no.
9102), phospho-ERK1/2 (no. 9106), JNK1/2 (no. 9252), phospho-JNK1/2
(no. 9251), IĸBa (no. 4814), phospho-IĸBa (no. 9246), p65 (no. 3034),
phospho-p65 (no. 3036), phospho-CREB (no. 9198), phospho-STAT3 (no.
9145), and phospho–c-Jun (no. 3270) were purchased all from Cell Signaling Technology (Danvers, MA). The Ab directed against IĸBb was
purchased from Santa Cruz Biotechnology (Dallas, TX). The p38 inhibitor
(SB 203580) and ERK1/2 inhibitor (U0126) were purchased from Cell
Signaling Technology. Mouse monoclonal b-actin Abs (A5441), poly(I:C),
NF-ĸB inhibitor, caffeic acid phenethyl ester (CAPE), MG132, and LPS
(from E. coli 0127:B8) were purchased from Sigma-Aldrich (Steinheim,
Germany).
TM and PM were cultured on glass coverslips in 24-well plates (Sarstedt,
N€umbrecht, Germany) and treated with 10 mg/ml LPS or poly(I:C) for 1 h.
After treatment cells were washed three times with PBS and fixed with 4%
paraformaldehyde in PBS for 30 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 for 10 min. Subsequently, unspecific
binding sites were blocked by incubation for 1 h in PBS containing 5%
normal goat serum and 5% BSA, washed, and incubated with the primary
anti-rabbit p65 Ab (1:50) at 4˚C overnight. After washing, slides were incubated with the corresponding Cy3-conjugated secondary Ab (Dianova,
Hamburg, Germany) for 1 h at room temperature in the dark. Nuclei were
stained with TO-PRO-3 dye (Molecular Probes, Darmstadt, Germany) for
10 min at room temperature, coverslipped, and visualized with a confocal
laser scanning microscope (Leica Microsystems, Wetzlar, Germany).
Materials and Methods
Animals
Cell isolation
TM were isolated as described previously (20). Briefly, two adult rat testes
were decapsulated and the seminiferous tubules gently separated. Tubular
fragments were allowed to sediment for 5 min, and cells in the supernatant
were pelleted, resuspended, and then seeded into culture plates. After
Luciferase reporter assay
RAW264.7 macrophages and primary rat testicular macrophages were
seeded on 12-well plates. After overnight culture, the cells were transfected
with 1 mg NF-kB luciferase reporter plasmid or the empty vector pGL3
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mice upon treatment with both classical (LPS and IFN-g) or alternative (IL-4) activation ligands, although still a reduced capacity
for proinflammatory gene expression was observed. In support of
these observations, incubation with uropathogenic Escherichia coli
instead of LPS increased mRNA expression of the proinflammatory
cytokines IL-6 and TNF-a; however, protein levels were not elevated (15). In the same study, activation of the NF-kB signaling
pathway is prevented by maintaining stable levels of the NF-kB
inhibitor IkBa (15).
Generally, recognition of microbes by macrophages depends on
binding of conserved molecular patterns such as LPS to members of
the TLR family. All TLRs activate a common signaling pathway
that culminates in the activation of NF-kB and AP-1. In resting
cells, NF-kB p50/p65 heterodimers are retained in the cytoplasm
by the inhibitory molecule IkBa. Upon binding to the ligand (e.g.,
LPS to TLR4 or polyinosinic-polycytidylic acid [poly(I:C)] to
TLR3) IkBa is phosphorylated by IKK kinases, polyubiquitinated
by Skp1–cullin–F-box/b-transducin repeat–containing protein
(SCF-bTrCP), which finally destines IkBa for degradation by the
26S proteasomal complex. Degradation of IkBa exposes the nuclear localization sequence in NF-kB and leads to translocation of
NF-kB to the nucleus (16). Alternatively, activation of the AP-1
signaling pathways is mainly mediated by MAPKs such as p38,
ERK, and JNK. Activation of both NF-kB and AP-1 signaling
pathways leads to increased expression of proinflammatory cytokines such as IL-6 and TNF-a (17).
Expression of TLR1–11 was identified in total rat testis, epididymis, and vas deferens (18), whereas TLR1–9 were found in
human testis samples (19). Besides the occurrence of all TLRs,
isolated rat TM also synthesize CD14, MD2, and the adaptor
protein MyD88, all of which are required for the initiation of
TLR4-mediated signaling (20). Although TM are equipped with
the necessary machinery to sense pathogens and mount proinflammatory immune responses, there is little understanding at
what molecular level the immune response of TM is discerned
from that of other macrophages and how this mechanistically
contributes to immune privilege by reduced capacity for proinflammatory gene expression.
The Journal of Immunology
3
basic and 100 ng internal control vector (Renilla luciferase vector) using
the transfection reagent GeneCellin HTC (RAW264.7) and ViaFect (testicular macrophages) according to the manufacturer’s protocol. The plasmids were a generous gift from Dr. Stephan Immenschuh (Institute for
Transfusion, Hannover Medical School, Hannover, Germany). Twentyfour hours after transfection, the cells were treated with LPS (10 mg/ml)
for an additional 24 h, after which the cells were lysed and the luciferase
activity was measured using the Dual-Luciferase kit (Promega) according
to the manufacturer’s protocol. The firefly luciferase values were normalized with the respective Renilla luciferase values.
Flow cytometric analysis
After lysis of RBCs, freshly isolated rat TM and PM were processed for flow
cytometric analysis of macrophage-specific markers by using anti-rat CD45-
PE/Cy7 (BioLegend, no. 202213), anti-rat CD163–Alexa Fluor 647 (AbD
Serotec, no. MCA342A647), and anti-rat CD68-allophycocyanin-Vio770
(Miltenyi Biotec, Bergisch Gladbach, Germany, no. 130-103-366). Background staining was evaluated using appropriate isotype controls, that is,
PE/Cy7 mouse IgG1 (BioLegend, no. 400125), REA control-allophycocyaninVio770 (Miltenyi Biotec, no. 130-104-634), and Alexa Fluor 647 mouse
IgG1 (BioLegend, no. 406617). All incubation steps were conducted at 4˚C
for 30 min. Briefly, 1 3 106 cells were incubated in HBSS with anti-CD16/
32 (Fc block; BD Biosciences), after which CD45 and CD163 Abs were
added. Cells were washed with HBSS buffer. For intracellular staining of
CD68, cells were fixed and permeabilized using an intracellular fixation and
permeabilization buffer set (eBioscience). Data were collected for 20,000
events using a MACSQuant analyzer 10 flow cytometer (Miltenyi Biotec)
and analyzed with FlowJo software version X (Tree Star, Ashland, OR).
Table I. Information of primer sequence, annealing temperature, and amplicon size
Gene
HMBS
IKB-ε
IKK-b
IKK-g
IKK-a
IL-1b
IL-1a
IL-6
iNOS
IP-10
IRAK1
IRAK3
IRAK4
IRF3
IkB-a
LBP
MAL
MCP-1
MD2
MyD88
NIK
PPIA
RIP1
Ro52
RPLP2
SARM
SCF-bTrCp
SDHA
SOCS1
SOCS3
b2M
TAK1
TLR3
TLR4
TNF-a
TRAM
TRIF
Ube2n
QT00402857
QT00394275
Forward: 59-GAACAACATTCCCTTCCTTCG-39
Reverse: 59-TGGTGGGCTGTCAATCAAAT-39
QT00179123
QT01802178
QT00188622
QT00437465
QT01627724
Forward: 59-TGCCTCGTGCTGTCTGACCCA-39
Reverse: 59-AGGCCCAAGGCCACAGGGAT-39
Forward: 59-CCGGGTGGTGGTGTCAGCAA-39
Reverse: 59-GCTGTGAGGTGCTGATCTGGGT-39
Forward: 59-TCCTACCCCAACTTCCAATGCTC-39
Reverse: 59-TTGGATGGTCTTGGTCCTTAGCC-39
Forward: 59-GTTTCCCCCAGTTCCTCACT-39
Reverse: 59-ATCTCTCCATTGCCCCAGTT-39
Forward: 59-GAAGCACCATGAACCCAAGT-39
Reverse: 59-CATGGAAGTCGATGCAGGT-39
QT01571969
QT02542428
QT00415037
QT00366898
QT00383103
QT01080786
Forward: 59-GGTTTGGAGGCCTGTGCTAT-39
Reverse: 59-GGAGGGTGAGAGACCATCCT-39
Forward: 59-TGTTCACAGTTGCTGCCTGT-39
Reverse: 59-CCGACTCATTGGGATCATCT-39
Forward: 59-GGATCTGCAACTCCTCCGATGCA-39
Reverse: 59-TGCTGTGTTGACAGCCTCCCCT-39
QT01569274
QT01620591
QT00177394
QT00372827
QT01592535
QT00369635
QT00428855
QT01595356
QT00195958
QT00425656
QT00450513
Forward: 59-CCGTGATCTTTCTGGTGCTT-39
Reverse: 59-AAGTTGGGCTTCCCATTCTC-39
QT00385315
QT00377300
QT00387184
Forward: 59-GCCTCTTCTCATTCCTGCTC-39
Reverse: 59-CCCATTTGGGAACTTCTCCT-39
Forward: 59-CAGTGGGAGAGCAAGACCAA-39
Reverse: 59-CGGGCTCTGTGACACCTTA-39
Forward: 59-CTCAAGACCCCTACAGCCAG-39
Reverse: 59-GCACCTAGAATGCCAAAGGC-39
QT00417291
Annealing
Temperature (˚C)
Accession No.
Amplicon Size (bp)
55
55
60
NM_001007798
NM_021744
XM_006249971
127
78
77
55
55
55
55
55
61.8
NM_013168
NM_001108854
NM_053355
NM_199103
NM_001107588
NM_031512
109
78
107
142
92
137
61.8
NM_017019
148
59.6
NM_012589
79
60
XM_003750865
121
60
NM_139089
117
55
55
55
55
55
55
60
NM_001127555
NM_001108101
NM_001106791
NM_001006969
NM_001105720
NM_017208
XM_001055833
98
109
100
78
67
105
195
60
NM_031530
110
60
NM_001024279
271
55
55
55
55
55
55
55
55
55
55
55
60
NM_198130
NM_001108301
NM_017101
NM_001107350
NM_001082572
NM_001030021
NM_001105817
NM_001007148
NM_130428
NM_145879
NM_053565
NM_012512
61
138
106
64
66
143
70
135
72
67
143
109
55
55
55
59.6
NM_001107920
NM_198791
NM_019178
NM_012675
109
73
107
101
60
NM_001108890
133
60
NM_174989
194
55
NM_053928
107
HMBS, hydroxymethylbilane synthase; iNOS, inducible NO synthase; IP-10, IFN-g–inducible protein 10; IRAK, IL-1R–associated kinase; IRF3, IFN regulatory factor 3; b2M,
b2-macroglobulin; PPIA, peptidylprolyl isomerase A; RPLP2, ribosomal protein, large P2; SDHA, succinate dehydrogenase a subunit; SOCS, suppressor of cytokine signaling.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
BTK1
CD14
Cox-2
Primer Sequence
4
DIFFERENTIAL ACTIVATION OF PATHWAYS IN TESTIS MACROPHAGES
Measurement of NF-kB (p50 and p65) transcription activity
The transcription activity of NF-kB subunits p50 and p65 was measured
with a transcription factor kit for NF-kB p50 and p65 (Thermo Scientific,
Waltham, MA) following the manufacturer’s instructions. Briefly, TM and
PM were treated with LPS (10 mg/ml) for 1 h and total proteins were
isolated by RIPA extraction buffer. Binding of NF-kB subunits to their
respective consensus sequences was measured by using luminometry.
NO measurement
TM and PM were treated with LPS (10 mg/ml) for 24 h. Subsequently, cell
culture supernatants were collected and NO was quantified using a Griess
reagent system (Promega) according to the manufacturer’s instructions.
Results
TM express low levels of mRNAs for TLR and NF-kB signaling
pathway genes
IkBa degradation is blocked in LPS- and poly(I:C)-treated TM
Activation of the classical NF-kB signaling pathway requires the
phosphorylation and subsequent degradation of IkBa, the negative
regulator NF-kB. Treatment with LPS and poly(I:C) induced rapid
phosphorylation of IkBa in a similar pattern in both TM and PM
with the phosphorylation being detected as early as 15 min and
sustained up to 120 min (Fig. 3A, 3B). However, degradation of
IkBa was observed only in PM, but not in TM, at all time points
investigated. Additionally, we have examined the degradation of
IkBb after treatment with LPS in TM. The kinetics of ubiquitinmediated degradation of IkBb are similar to those of IkBa, albeit
occurring at a slower rate. Similar to IkBa, degradation of IkBb
was not observed in LPS-treated TM (Supplemental Fig. 3A).
Despite the lack of IkBa degradation, TM showed an increase in
p65 phosphorylation as was evident for PM when treated with
FIGURE 1. (A–C) Gene expression profiles for TLR signaling pathway genes in TM and PM. (D) Basal mRNA expression of TLR signaling pathways
genes and proinflammatory cytokines IL-1a, IL-1b, IL-6, and TNF-a were quantified in TM and PM by using qRT-PCR. The expression of each target gene
is normalized by four reference genes in TM and PM samples. For each gene the mean of expression level of PM is divided by the respective mean of TM
samples and results were presented as the PM/TM ratio and expressed as mean 6 SD (n = 3). The unpaired Student t test was employed for statistical
analysis. *p , 0.05, **p , 0.01, ***p , 0.001.
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In rats, TM are characterized by the expression of surface markers
CD68 (ED1) and CD163 (ED2). Analysis of macrophage-specific
markers on freshly isolated TM and PM revealed substantial differences in the protein levels of CD68 and CD163 between different cell subtypes. PM showed significantly higher levels of
CD68 (ED1). In contrast, TM displayed significantly higher expression of CD163 (ED2) as compared with PM (Supplemental
Fig. 1).
To determine whether the subdued inflammatory response observed in rat TM (21) is due to an aberrant expression of TLR
signaling pathway genes, the mRNA expression of representative
genes (Table I) was compared in TM and PM by quantitative
RT-PCR (qRT-PCR). In contrast to PM, TM displayed profoundly lower basal expression of TLR pathway–specific genes such
as CD14, LBP, MD2, MyD88, Toll/IL-1R domain–containing
adaptor-inducing IFN-b (TRIF), TRAM (TRIF-related adaptor
molecule), MAL, TNFR-associated factor 6, TAK1, RIP1, and
NIK (Fig. 1, Table I). Expression of SARM (negative regulator of
TLR3 signaling) and RP105 (negative regulator of MD2/TLR4
signaling) were significantly higher in TM (Fig. 1B, Table I).
Additionally, we have checked the expression of TLR3 and TLR4
protein by Western blot analysis. Levels of TLR3 protein are
comparable in both cell types, whereas TLR4 protein was abundantly expressed in PM and only faintly detectable in TM
(Supplemental Fig. 2). The basal expression of TLR4-triggered
proinflammatory cytokines genes TNF-a and IL-6 were significantly lower in TM than PM (Fig. 1D, Table I). Increasing concentrations of LPS and poly(I:C) induced the secretion of TNF-a
and IL-6 protein in a dose-dependent manner in both TM and PM
in vitro (Fig. 2). However, levels of secreted TNF-a and IL-6 were
much lower in TM.
The Journal of Immunology
5
LPS or poly(I:C) (Fig. 3A, 3B). However, immunofluorescence
revealed that translocation of p65 was observed only in PM, but
not in TM (Fig. 3C). Next, we investigated the transcriptional
activity of NF-kB subunits following treatment with LPS in TM
and PM. The NF-kB p65 transcriptional activity significantly increased in PM, whereas transcriptional activity of p65 did not
FIGURE 3. Downstream elements of the NF-kB signaling pathway are not activated in testicular macrophages. TM and PM (A–C) were stimulated with (A)
LPS (10 mg/ml) and (B) poly(I:C) for the indicated time points. Lysed cells were subjected to Western immunoblotting analysis using Abs specific for the
phospho-IkBa, IkBa, phospho-p65, p65, and b-actin (loading control). Each experiment has been performed at least three times and a representative experiment
is shown. Immunoblots of phospho-IkBa, IkBa, and phospho-p65 were quantified and normalized by loading control b-actin and p65 by using Adobe Photoshop
7.0. Quantified value is indicated on the top of the respective blots. (C) TM and PM treated with LPS and poly(I:C) for 1 h were analyzed for nuclear translocation
of the NF-kB p65 subunit using confocal microscopy (red, p65; blue, nuclei). p65 localized predominantly in the cytoplasm of untreated PM and translocated into
the nucleus after LPS and poly(I:C) treatment. In contrast, in TM the NF-kB subunit p65 remained exclusively in the cytoplasm regardless of the experimental
condition. Scale bars, 15 mm. (D) NF-kB transcriptional activity was determined in total cell lysates by using an ELISA-based NF-kB p50 and p65 transcription
factor kit. Data are presented as means 6 SD of three independent experiments. The unpaired Student t test was used for statistical analysis. **p , 0.01.
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FIGURE 2. TM and PM secrete proinflammatory cytokines upon stimulation with LPS and poly(I:C). TM and PM were treated with different doses of
LPS and poly(I:C) for 24 h. Conditioned media from cells were analyzed for (A and C) TNF-a and (B and D) IL-6 levels by specific sandwich ELISA. Data
are presented as the means 6 SD of three to four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
6
DIFFERENTIAL ACTIVATION OF PATHWAYS IN TESTIS MACROPHAGES
change in TM (Fig. 3D). Inactivation of NF-kB signaling pathways in TM was further confirmed by NF-kB luciferase reporter
assay. In agreement with our above results, no significant changes
in luciferase activity of p65 transcription factor were evident
(Supplemental Fig. 3B). Interestingly, transcriptional activity of
the p50 subunit was not observed in both TM and PM. In summary, these results indicate that TM are unable to activate the NFkB signaling pathway.
IkBa is not ubiquitinated in TM
LPS and poly(I:C) activate MAPKs in TM
To investigate whether poly(I:C) and LPS activate TLR3 and TLR4
downstream signaling pathways in TM and PM, we examined the
LPS and poly(I:C) activate the AP-1 and CREB signaling
pathways in TM
Despite the lack of activation of the NF-kB signaling pathway, TM
were able to respond with the production of a number of cytokines
and chemokines following challenge with LPS or poly(I:C)
(Fig. 6A, Table I). Gene expression of IL-1a, IL-1b, IL-6, TNF-a,
COX-2, and inducible NO synthase was strongest in LPS-treated
TM, whereas IFN-g–inducible protein 10 mRNA levels were
substantially elevated in poly(I:C)-treated TM. MCP-1 mRNA
expression was similar in both LPS- and poly(I:C)-treated cells
(Fig. 6A). Pending on upstream activation of the MAPK p38,
regulation of proinflammatory cytokine production can also be
mediated by AP-1 and CREB transcription factors besides NF-kB
(17, 22, 23). Phosphorylation of CREB was found in LPS- and
poly(I:C)-stimulated TM, whereas c-Jun was phosphorylated only
in LPS-treated cells (Fig. 6B).
LPS and poly(I:C) secrete proinflammatory cytokines through
the AP-1 and CREB signaling pathways in TM
In support of the above-mentioned data, the p38 inhibitor SB203580
dose-dependently diminished activation of c-Jun and CREB
(Fig. 7A) and suppressed TNF-a secretion in LPS-stimulated TM
(Fig. 7B). In contrast, CAPE, a specific inhibitor of NF-kB, did
not cause inhibition of TNF-a secretion. Notably, treatment of PM
with CAPE significantly suppressed TNF-a release (Fig. 7B).
Similar results were observed for the ERK1/2 inhibitor U0126
(data not shown). These findings indicate that in TM, MAPKs play
a key role in inducing the secretion of proinflammatory cytokines
through the AP-1 and CREB signaling pathways.
TM show characteristics of regulatory macrophages
FIGURE 4. Phospho-IkBa is not ubiquitinated in stimulated TM. TM
and PM (A and B) were stimulated with 10 mg/ml (A) LPS or (B) poly(I:C) for
60 min and then treated with proteasome inhibitor MG132 (50 mM) for 90 min.
Lysed cells were analyzed by Western blotting using Abs specific for phosphoIkBa, IkBa, and b-actin (loading control). Each experiment has been performed
at least three times and a representative experiment is shown. (C) Basal mRNA
expression of ubiquitinating enzymes in TM and PM were quantified by using
qRT-PCR. The expression of each target gene is normalized by the four reference
genes in TM and PM samples. For each gene the mean of expression level of PM
is divided by the respective mean of TM samples and results are presented as the
PM/TM ratio and expressed as means 6 SD (n = 3). The unpaired Student t test
was used for statistical analysis. *p , 0.05, **p , 0.01, ***p , 0.001.
Contrary to PM, treatment of TM with LPS resulted in a significant upregulation of the mRNA and protein secretion of the
anti-inflammatory/regulatory cytokine IL-10 (Fig. 8A, 8B).
Moreover, LPS-stimulated production of NO as a marker of
classically activated M1 macrophages was substantially higher
in PM than in TM (Fig. 8C). Similarly, secretion of the proinflammatory cytokine IL-12, a marker for M1 macrophages,
was significantly increased in PM (Fig. 8D). To further examine an alternative phenotype of TM, we performed immunoblot analysis with a phospho-specific STAT3 Ab on LPStreated TM and PM. In contrast to PM, treatment with LPS
causes phosphorylation of STAT3 (Supplemental Fig. 4). In
Fig. 7B, we have observed that secretion of TNF-a is mediated
through MAPK signaling pathways. To determine the role of
these signaling pathways in secretion of IL-10, TM were pretreated with the p38 inhibitor SB203580 followed by LPS
challenge. Inhibition of p38 MAPK significantly abrogates the
secretion of IL-10. Likewise, IL-10 secretion also was attenuated by the EKR inhibitor. In contrast, CAPE, a NF-kB–specific
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In the usual sequence of events on the molecular level, IkBa
requires phosphorylation as a prerequisite for ubiquitination
and final degradation by the 26S proteasome. Whereas LPS- and
poly(I:C)-challenged PM showed strong polyubiquitination of phospho-IkBa in the presence of the proteasome inhibitor MG132, the
same effect was barely detected in TM (Fig. 4A, 4B).
To determine whether the failed polyubiquitination of phosphoIkBa in TM was due to aberrant expression of ubiquitinating and
deubiquitinating enzymes, the gene expression of various members of the ubiquitination/deubiquitination complex involved in
the activation and repression of IkBa and the IKK complex was
examined. In PM, basal expression of IKK-a, IKK-b, IKB-ε,
NEMO (IKK-g), and RO52 was significantly higher than in TM
(Fig. 4C). Interestingly, expression of SCF-bTrCP, an E3 ligase
whose binding to phospho-IkBa is leading to recognition by the
26S proteasome and degradation, was 25-fold higher in TM than
PM (Fig. 4C).
activation of the three MAPKs (p38, ERK1/2, and JNK) by
detecting their dually phosphorylated (Tyr/Thr) forms by Western
blot analysis by using phospho-specific Abs (Fig. 5). LPS strongly
stimulated a rapid but persistent activation of both p38 and
ERK1/2 MAPKs in both TM and PM. In contrast to PM, poly(I:C)
activates both p38 and ERK1/2 in TM, although the level of activation was much weaker than for LPS (Fig. 5A, 5B). In poly(I:C)treated TM, activation of p38 was observed at 30 min, whereas
increased phosphorylation of ERK1/2 was seen at 60 min of
treatment. The levels of phosphorylated JNK compared with total
JNK remained largely the same in both LPS- and poly(I:C)-treated
TM (Fig. 5). However, in PM, activation of JNK was observed after
15 min of treatment.
The Journal of Immunology
7
FIGURE 5. The MAPK signaling pathway is
activated in stimulated TM and PM. TM and PM
were stimulated with (A and C) 10 mg/ml LPS and
(B and D) 10 mg/ml poly(I:C) for the indicated time
points. Lysed cells were examined by Western blot
analysis using Abs specific for phospho-p38, p38,
phospho-ERK1/2, ERK1/2, phospho-JNK, and JNK.
Each experiment has been performed at least three
times with a representative experiment shown.
FIGURE 6. Proinflammatory mediators are produced in stimulated TM.
(A) TM were stimulated with 10 mg/ml LPS for 6 h. Expression of
proinflammatory cytokines and chemokines were quantified by using qRTPCR. Results were normalized for b2-macroglobulin and expressed as
means 6 SD as compared with untreated control. (B) TM were stimulated
with 10 mg/ml LPS and 10 mg/ml poly(I:C) for the indicated time points.
Cell lysates were subjected to Western blotting using Abs specific for
phospho–c-Jun, phospho-CREB, and b-actin (loading control). Each experiment has been performed at least three times with a representative
experiment documented.
Discussion
TM contribute to the immune privilege of the testis by diminished
secretion of proinflammatory cytokines (1, 9). However, the molecular mechanisms involved in this phenomenon are largely unknown. Their understanding could contribute to better knowledge
of organ-specific immune suppression in the testis that when
disturbed leads to inflammation/infection-based male infertility,
which paradoxically represents the second most prevalent etiology in the male (24, 25). It is therefore of importance to unravel
the mechanisms by which TM display a diminished inflammatory response to protect the developing germ cells from cytotoxic effects of excessive proinflammatory cytokine production,
but still maintain an adequate responsiveness to infection and
inflammation.
In this study, we provide two lines of evidence for the subdued
inflammatory response of TM at the molecular level. First, TM
show a significantly reduced expression of CD14, MD2, and of
adaptor molecules of TLR signaling pathways such as MyD88,
MAL, TRAM, and TRIF, which could explain decreased expression
of proinflammatory cytokines by inefficient signal transduction. In
agreement, TM expressed increased levels of mRNA for SARM and
RP105, negative regulators for TLR3 and TLR4 signaling pathways,
respectively (26, 27). Similar mechanisms have been reported in
intestinal macrophages, where reduced expression of TLR signaling
molecules and proinflammatory cytokine genes contribute to intestinal homeostasis needed to tolerate the commensal microbes of
the gut (28).
Second, a striking contrast of TM in comparison with PM is
their inability to activate the NF-kB signaling pathway upon
treatment with the classical TLR ligands LPS and poly(I:C). In
this study, we have shown that inhibition of NF-kB signaling
principally takes place at the level of IkBa processing. Although
TLR ligands induce the phosphorylation of IkBa, the subsequent
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inhibitor, did not cause any significant changes in the secretion
of IL-10 (Fig. 8E). Taken together, these results suggest that
under inflammatory conditions, TM display the phenotype of
a regulatory macrophage, a characteristic that helps protecting
the developing germ cells from cytotoxic proinflammatory
mediators.
8
DIFFERENTIAL ACTIVATION OF PATHWAYS IN TESTIS MACROPHAGES
degradation was not observed in TM. Normally, phosphorylated
IkBa is polyubiquitinated by SCF-bTrCP, which finally destines
IkBa for degradation by the 26S proteosomal complex (16). Our
data suggest that intrinsic mediators in TM may interfere at the
level of ubiquitination of phosphorylated IkBa. Because degradation of IkBa is prevented at the level of ubiquitination, we
hypothesize that the COP9 signalosome (CSN) (29) may play
a role in IkBa ubiquitination in TM. The association between
CSN and IkBa suggests that the turnover of IkBa protein is
dependent on CSN, leading to increased amounts of stable cytoplasmic IkBa and thereby inhibiting NF-kB activation (30).
Thus far, transfection experiments in delineating the role of the
COP9 signalosome in stabilizing IkBa have been hindered in
TM by their limited numbers, the short period of survival in
culture, and the multimeric protein composition of the CSN, but
the data in this study encourage challenging experiments along
this line.
LPS triggers the TLR signaling pathway through MyD88
adaptor molecules ultimately resulting in the activation of the NFkB and MAPK (p38 and ERK) pathways (31, 32). Activated
MAPKs phosphorylate the transcription factors AP-1 (c-Jun) and
CREB, which subsequently leads to the secretion of proinflammatory cytokines such as IL-6, TNF-a, as well as the expression of the anti-inflammatory cytokine IL-10 (33, 34). In this
study, we have clearly demonstrated that LPS induces the activation of MAPKs (p38 and ERK1/2) and subsequent activation
of AP-1 and CREB observed by its phosphorylation. We hypothesize that in the absence of NF-kB signaling the residual
cytokine expression is regulated by AP-1 and CREB signaling
pathways. In line with this hypothesis, the treatment of TM with
the MAPK inhibitors SB203580 and U0126 blocks AP-1 and
CREB phosphorylation and more importantly the LPS-induced
secretion of TNF-a. MAPK-mediated activation of AP-1 and
CREB signaling pathways is also required for the production of
IL-10 (Fig. 8D). Hence, AP-1 and CREB signaling pathways
could play an important role in limiting excessive inflammation.
In addition to MAPK pathways, the expression of IL-10 is
regulated by the NF-kB signaling pathway. Upon stimulation
with inflammatory stimuli, NF-kB p50 subunit homodimerizes
and then translocates to the nucleus and exclusively binds to the
IL-10 promoter. Moreover, IL-1R–associated kinase 1 binding
protein 1 promotes translocation of p50/p50 homodimers over
to p50/p65 heterodimers and induces the production of IL-10 in
macrophages (35, 36). In this study, we have not observed the
activation of the NF-kB signaling pathway, and hence production of IL-10 is mediated mainly through the AP-1 and CREB
signaling pathways.
Macrophages are polarized to M1, M2, or regulatory macrophages upon stimulation with respective stimuli. Macrophages
develop toward M1 macrophages upon stimulation with IFN-g
and microbial stimuli such as LPS (37). Conversely, Th2 cytokines IL-4 and IL-13 promote polarization toward M2 macrophages, particularly during allergy and parasitic infections (38).
However, in the recent past a third macrophage subclass called
regulatory macrophages has been described with similar properties as those of M2 macrophages. Macrophages are polarized
to regulatory macrophages by stimulation with TLR ligands in
the presence of the immune complex and PGs and are characterized by secretion of immunosuppressive cytokines such as
IL-10 and the low-secretion proinflammatory cytokine IL-12
(39). Rat TM produce high basal levels of PGE2, and treatment with LPS significantly increases the concentration of
PGE2 (40). In this study, we have shown that upon treatment
with LPS, TM exhibit a regulatory macrophage phenotype by
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FIGURE 7. LPS-induced activation of AP-1 and
CREB is p38-dependent. (A) TM were pretreated for
30 min with the selective p38 inhibitor SB203580
with the indicated doses and then stimulated with
10 mg/ml LPS for 30 min. Cell lysates were subjected
to Western blot analysis using Abs specific for
phospho-p38, p38, phospho–c-JUN, phospho-CREB,
and b-actin (loading control). Each experiment has
been performed at least three times and a representative experiment is shown. (B) TM and PM were
pretreated for 30 min with CAPE and SB203580
with the indicated dose and then stimulated with
10 mg/ml LPS for 24 h. Conditioned media from
cells were analyzed for TNF-a protein levels by
specific sandwich ELISA. Data are presented as
the means 6 SD of two to three independent experiments. The one-way ANOVA test was employed for
statistical analysis. *p , 0.05, ***p , 0.001, ****p ,
0.0001.
The Journal of Immunology
9
producing a large amount of IL-10, low secretion of IL-12, and
failure to induce NO release. The main physiological function
of regulatory macrophages is to dampen proinflammatory responses by producing large quantities of IL-10, thus maintaining tissue homeostasis. IL-10 exerts anti-inflammatory activity
by activation of STAT3 signaling pathway and through heme
oxygenase-1 (41). In the present study, we have shown that LPS
induced the activation of STAT3 signaling pathways in TM and
hence provide evidence that the activation of IL-10/STAT3
signaling pathways could play a role in inhibiting the secretion of proinflammatory cytokines, at least partially. The role of
IL-10 in maintaining homeostasis of tissues is demonstrated by
the development of spontaneous colitis in IL-10–deficient mice
(34). Additionally, mutations in IL-10 and IL-10 receptors result
in severe infantile inflammatory bowel disease (34). Of note,
intestinal macrophages are potent producers of IL-10, which in
turn increases the Treg population by maintaining stable Foxp3
expression (33). It is tempting to speculate that IL-10 secreted
by TM in vivo helps to maintain the testicular population of
Tregs, which are known to be relevant for the establishment of
testicular immune privilege (42). In the future, it will be worthwhile to study the role of TM in the induction of Tregs and elucidate the molecular mechanism of immune tolerance in testis to
protect the developing germ cells from profound inflammatory
response.
In conclusion, the results obtained from this study suggest that
TM maintain immune privilege of testis by profound downregulation of inflammatory genes and as a unique mechanism by
suppression of the NF-kB signaling pathway at the level of IkBa
ubiquitination. Moreover, TM display characteristics of regulatory macrophages, a subset known to control and dampen inflammatory immune response. Although TM are unable to induce
NF-kB signaling, they maintain some capacity to respond to inflammatory stimuli by secreting proinflammatory cytokines through
the AP-1 and CREB signaling pathways. These properties of TM
provide them with the ability to adequately respond to microbial
challenge while protecting the sensitive germ cells from the negative consequences of a high-magnitude immune response.
Acknowledgments
The authors wish to acknowledge the excellent technical assistance of
Eva Wahle and English editing by Dr. Vera Michel.
Disclosures
The authors have no financial conflicts of interest.
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