Article

Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5

Medical Policlinic, University of Munich, Germany.
American Journal Of Pathology (Impact Factor: 4.59). 11/2009; 175(5):2014-22. DOI: 10.2353/ajpath.2009.080585
Source: PubMed
ABSTRACT
Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.

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Available from: Bruce Beutler
Immunopathology and Infectious Diseases
Viral RNA Induces Type I Interferon-Dependent
Cytokine Release and Cell Death in Mesangial Cells
via Melanoma-Differentiation-Associated Gene-5
Implications for Viral Infection-Associated Glomerulonephritis
Katharina Flu¨r,* Ramanjaneyulu Allam,*
Daniel Zecher,* Onkar P. Kulkarni,*
Julia Lichtnekert,* Martin Schwarz,*
Bruce Beutler,
Volker Vielhauer,*
and Hans-Joachim Anders*
From the Medical Policlinic,* University of Munich, Munich,
Germany; and the Department of Genetics,
The Scripps Research
Institute, La Jolla, California
Viral RNA can trigger interferon signaling in dendritic
cells via the innate recognition receptors melanoma-
differentiation-associated gene (MDA)-5 and retinod-
inducible gene (RIG)-I in the cytosol or via Toll-like
receptors (TLRs) in intracellular endosomes. We hy-
pothesized that viral RNA would also activate glomer-
ular mesangial cells to produce type I interferon (IFN)
via TLR-dependent and TLR-independent pathways.
To test this hypothesis, we examined Toll/Interleu-
kin-1 receptor domain-containing adaptor-inducing in-
terferon-
(TRIF)-deficient mice , which lack a key adap-
tor for TLR3 signaling. In primary mesangial cells, poly
I:C RNA-mediated IFN-
induction was partially TRIF
dependent; however, when poly I:C RNA was com-
plexed with cationic lipids to enhance cytosolic uptake,
mesangial cells produced large amounts of IFN-
and
IFN-
independent of TRIF. Mesangial cells expressed
RIG-I and MDA-5 and their mitochondrial adaptor IFN-
promoter stimulator-1 as well, and small interfering
RNA studies revealed that MDA5 but not RIG-I was re-
quired for cytosolic poly I:C RNA signaling. In addition,
mesangial cells produced Il-6 on stimulation with IFN-
and IFN-
, suggesting an autocrine proinflammatory
effect. Indeed, blockade of IFN-
␣␤
or lack of the IFNA
receptor reduced viral RNA-induced Il-6 production and
apoptotic cell death in mesangial cells. Furthermore, viral
RNA/cationic lipid complexes increased focal necrosis in
murine nephrotoxic serum nephritis in association with
increased renal mRNA expression of IFN-related genes.
Thus, TLR-independent recognition of viral RNA is a
potent inducer of type I interferon in mesangial cells,
which can be an important mediator of virally induced
glomerulonephritis.
(Am J Pathol 2009, 175:2014 –2022;
DOI: 10.2353/ajpath.2009.080585)
Chronic viral infections can trigger de novo immune com-
plex glomerulonephritis, eg, hepatitis C virus-associated
glomerulonephritis, but more frequently, acute viral infec-
tions trigger disease activity of pre-existing glomerulone-
phritis, like in IgA nephropathy, lupus nephritis, or renal
vasculitis.
1
Viral infections activate systemic antiviral immu-
nity, which may contribute to disease flares of glomerulone-
phritis by enhancing autoantibody production, immune
complex formation, or by systemic interferon (IFN) release.
2
In fact, rapid production of type I IFN is a central element of
antiviral immunity because type I IFNs inhibit viral replication in
the infected cells and have pleiotrophic immunomodulatory
effects on macrophages, T cells, and natural killer cells.
2,3
The main source of type I IFNs is plasmacytoid den-
dritic cells (pDCs) in the intravascular compartment.
4
Several viral components can induce type I IFNs in pDCs.
For example, viral proteins activate Toll-like receptor
(TLR) 2 and 4 signaling at the cell surface.
5
By contrast,
different shapes of viral nucleic acids can activate TLRs
in intracellular endosomes, ie, TLR3 (double-stranded
(dsRNA)), TLR7/8 (single-stranded (ssRNA)), and TLR9
Supported by the Deutsche Forschungsgemeinschaft (AN372/9-1 and
GRK 1202; to H.-J.A.). K.F., R.A., and J.L. were graduate fellows of the
Deutsche Forschungsgemeinschaft GRK 1202.
Parts of this project were prepared as a doctoral thesis at the Faculty of
Medicine, University of Munich, by K.F.
Accepted for publication July 13, 2009.
Address reprint requests to Hans-Joachim Anders, Medizinische Po-
liklinik, Universita¨t Mu¨nchen, Pettenkoferstr. 8a, 80336 Mu¨nchen, Ger-
many. E-mail: hjanders@med.uni-muenchen.de.
The American Journal of Pathology, Vol. 175, No. 5, November 2009
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.080585
2014
Page 1
(CpG-DNA).
5
Furthermore, viral RNA can also be
detected in the intracellular cytosol, eg, via melanoma-
differentiation-associated gene (MDA)-5 (dsRNA) and
retinoic-acid-inducible protein (RIG) (dsRNA and 5-
triphosphate RNA).
5– 8
Ligation of any of these innate
RNA recognition receptors rapidly triggers the produc-
tion of type I IFN in pDCs.
It is thought that most cells can produce type I IFNs
when they are infected with a DNA or RNA virus. For
example, TLR3-mediated recognition of viral dsRNA in
pancreatic islet cells can trigger autoimmune pancreatic
islet destruction via local production of IFN-
.
9
But
whether locally produced type I IFNs contribute to viral
infection-induced glomerulonephritis is not known.
10
In
fact, to our best knowledge, IFN release by glomerular
cells, including mesangial cells, has not been reported.
The only nucleic acid-specific TLR expressed by mesan-
gial cells is TLR3, and mesangial cells produce Il-6 and
CCL2 on exposure to viral dsRNA.
11
However, whether
this effect is mediated via endosomal TLR3 or by cytoso-
lic dsRNA receptors is not known. We hypothesized that
viral RNA will trigger an innate antiviral response program in
glomerular mesangial cells, including the release of type I
IFN, and that this effect is mediated by TLR-dependent as
well as TLR-independent RNA recognition.
Materials and Methods
Studies with Primary Mesangial Cells
Six-week-old female C57BL/6 mice were obtained from
Charles River Laboratories (Sulzfeld, Germany). Trif-mu-
tant mice in the C57BL/6 background were generated
as described previously.
12
IFN-
␣␤
-R-deficient 129Sv/Ev
control mice were obtained from B&K Universal Group
(North Humberside, UK). For the preparation of primary
mesangial cells, capsule and medulla of the kidney were
removed, and the renal cortices were diced in cold PBS
and subsequently passed through a series of stainless
steel sieves (150, 103, 63, 50, and 45
m) and treated
with a 1 mg/ml solution of type IV collagenase (Worthington,
Lakewood, NY) for 15 minutes at 37°C. After five passages,
the glomerular cell isolates were 99% positive for smooth
muscle actin and 99% negative for cytokeratin 18, ie,
glomerular mesangial cells. Primary mesangial cells were
stimulated with endotoxin-free poly I:C RNA (InvivoGen,
Toulouse, France), poly I:C RNA transfected with the cat-
ionic lipid Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or
ultrapure LPS (InvivoGen) for 24 hours in RPMI 1640 con-
taining 5% FCS in the presence or absence of murine IFN-
(AbD Serotec, Oxford, UK), IFN-
(PBL, Piscataway, NJ), or
IFN-
(PeproTech, Rocky Hill, NJ). Poly I:C RNA and Lipo-
fectamine were preincubated with polymyxin B (Invivogen)
before use to block any residual LPS contamination. Rat
monoclonal antibodies against mouse IFN-
and IFN-
(PBL) were used to neutralize these IFNs in vitro. Cytokine
levels were measured in cell supernatants using commer-
cial ELISA kits for Il-6 (detection limit: 2 pg/ml, OptEiA; BD
Pharmingen), IFN-
(detection limit: 15 pg/ml; PBL), IFN-
(detection limit 10 pg/ml; PBL), or IFN-
(detection limit 10
pg/ml; BD Pharmingen). Proliferation of mesangial cells was
assessed using CellTiter 96 Proliferation Assay (Promega,
Mannheim, Germany) with and without inhibitors against
caspase-2, -8, -9, and -10 (all from Calbiochem, Gibbstown,
NJ).
Western Blot
Proteins from mesangial cells and unstimulated J774
macrophages were harvested using 120
l of radioim-
munoprecipitation assay buffer (50 mmol/L Tris, pH 8.0,
150 mmol/L NaCl, 0,1% SDS, 0.5% sodium deoxycholat,
and 1% Nonidet P-40) plus the protease inhibitor tablets
Complete (Roche), incubated on ice for 10 minutes, and
centrifuged for another 10 minutes at 15,000 g. Ex-
tracted proteins were incubated in 2 loading buffer for
30 minutes at 65°C, resolved by 10% SDS-PAGE, and
transferred to an Immobilon-P membrane (Millipore, Es-
chborn, Germany). After blocking with 3% skim milk, the
filter was incubated with a rabbit polyclonal antibody
against TLR3 (Abcam, Cambridge, UK), Rig-I (2
g/ml;
ProSci, Poway, CA), rabbit polyclonal antibody to Mda5
(AL 180) (1/1000; Alexis), or to
-actin (Abcam) over-
night. Binding was visualized using a peroxidase-conju-
gated donkey anti-rabbit IgG antibody (1/10,000; Amer-
sham Biosciences, Freiburg, Germany) and processed
for detection by enhanced chemiluminescence (NEN Life
Science Products, Boston, MA).
RNA Silencing Studies
Rig-I (DDX58) small interfering RNA (siRNA) ON-TARGET-
plus SMART pool oligonucleotides (Dharmacon), Mda5
siRNA and negative control siRNA (Ambion/Applied Biosys-
tems, Darmstadt, Germany) sequences were as follows:
DDX58 (Rig-1), 5-CAAGAAGAGUACCACUUAAUU-3,5-
GUUAGAGGAACACAGAUUAUU-3,5-GUUCGAGAUU-
CCAGUCAUAUU-3,5-GAAGAGCACGAGAUAGCAAUU-
3; and Mda5, 5-GAACGUAGACGACAUAUUA-3,5-
CAACGAAGCCCUACAAAUC-3,5-CUUGAUGCCU-
UUACCAUUA-3,5-GGGAGAUUGUUAAUGAUUU-3.
Mesangial cells (1 10
5
) were plated in 12-well plates in
antibiotic free 2% fetal calf serum-Dulbecco’s modified Ea-
gle’s medium. siRNA (40 nmol/L) was transfected twice with
cationic lipid as mentioned above. After 24 hours of second
transfection, cells were stimulated with 3
g of poly I:C RNA
with and without cationic lipids for 6 hours. Knockdown
efficacy of Rig-1, Mda5, and primary mesangial cell mRNA
expression of Cxcl10 and Il-6 mRNA expression were de-
termined by real-time RT-PCR after 6 hours.
Flow Cytometry
Primary mesangial cells were stimulated with poly I:C RNA
as before in the presence or absence of the caspase-8
inhibitor Ac-IETD-CHO (5
g/ml; Biomol, Hamburg, Ger-
many). Propidium iodide staining (Miltenyi Biotec, Bergisch
Gladbach, Germany) was used to identify death cells.
Viral RNA Aggravates Glomerulonephritis 2015
AJP November 2009, Vol. 175, No. 5
Page 2
Autologous Nephrotoxic Serum Nephritis
Nephritis was induced in groups of C57BL/6 wild-type
mice using a rabbit antiserum and protocol as previously
described in detail.
13
Mice were sacrificed by cervical
dislocation 10 days after injection of the antiserum. Uri-
nary albumin and creatinine concentrations were de-
termined as described previously.
13
Two-micrometer
renal sections for periodic acid-Schiff stains were pre-
pared following routine protocols. Glomerular focal ne-
crosis score defined by desintegrated glomerular loops
with glomerular cell necrosis was assessed on 15 cor-
tical glomerular sections each graded as follows: 0
intact glomerulus, 1 necrotic lesion 25% of glomer-
ular tuft, 2 25–50% of glomerular tuft, and 3 ⫽⬎50%
of glomerular tuft.
Real-Time PCR and Reverse Transcription
The mRNA expression in cultured cells or renal tissue
real-time PCR was quantified using TaqMan as de-
scribed previously.
11
Controls consisting of double-dis
-
tilled H
2
O were negative for target and housekeeper
genes. Oligonucleotide primer (300 nmol/L) and probes
(100 nmol/L) used were from PE Biosystems (Weiterstadt,
Germany) and are listed in Table 1.
Statistical Analysis
Data were expressed as mean SEM. Comparison be-
tween single groups were performed using Student’s
t-test. Otherwise analysis of variance using posthoc Bon-
ferroni‘s correction was used. A value of P 0.05 indi-
cated statistical significance.
Results
Poly I:C RNA Induces the Production of Il-6 and
IFN-
in Glomerular Mesangial Cells
Murine and human mesangial cell lines express TLR3
and produce Il-6 on exposure to poly I:C RNA, a synthetic
mimic of viral double-stranded RNA.
11,14
To verify this
observation in primary mesangial cells, we used 99%
pure mesangial cell cultures isolated from glomerular
preparations of C57BL/6 mice as described in Materials
and Methods. As expected, incubation with increasing
doses of poly I:C RNA induced Il-6 mRNA expression in
primary mesangial cells, which peaked at 3 hours after
poly I:C RNA stimulation and was maximal at a concen-
tration of 10
g/ml. (Figure 1). At 6 hours, Il-6 mRNA
levels declined below baseline expression (Figure 1).
Mesangial Cells Produce High Amounts of
IFN-
and IFN-
when Exposed to Poly I:C
RNA Complexed with Cationic Lipids in an
Tlr3/Trif-Independent Pathway
The recognition of viral RNA in DCs can involve Tlr3/Trif-
dependent and Tlr3/Trif-independent pathways.
5–7,15
To
assess the contribution of the Tlr3-dependent pathway
for RNA recognition in mesangial cells, we compared the
poly I:C RNA-induced Il-6 production in mesangial cells
from wild-type and Trif-mutant mice. In wild-type mesan-
gial cells, poly I:C RNA induced the secretion of low
amounts but still doubles Il-6 levels as compared with
medium (Figure 2A). Lack of functional Trif completely
blocked this effect (Figure 2A). At higher doses of poly
I:C RNA, Il-6 secretion declined below medium levels
consistent with mesangial cell death. In DCs, the Tlr3/Trif-
independent signaling pathway was shown to be specif-
ically activated when the viral RNA is complexed with
cationic lipids.
7
In fact, complexes of cationic lipids with
very small doses of poly I:C RNA activated mesangial
cells to produce high levels of Il-6 independent of the
presence of functional Trif (Figure 2A). Furthermore, com-
plexes of cationic lipids with poly I:C RNA activated mes-
angial cells to produce high levels of IFN-
and IFN-
independent of Trif (Figure 2, B and C). Mesangial cells
did not produce IFN-
on either of the stimuli tested (data
not shown). These data show that mesangial cells can
produce large amounts of type I IFN when exposed to
poly I:C RNA complexed with cationic lipids via a Trif-
independent pathway.
Table 1. Murine Probes Used for Real-Time RT-PCR
Gene Sequence
Il-6 FAM 5-AAATGAGAAAAGAGTTGTGCAATGG-3
IFN-
FAM 5-CTATTTTAACTCAAGTGGCATAGAT-3
Ifih/ Mda-5 FAM 5-GACACCAGAGAAAATCCATTTAAAG-3
Ips-1/Visa FAM 5-AGTGACCAGGATCGACTGCGGGCTT-3
Ddx58/Rig-I FAM 5-CCAAACCAGAGGCCGAGGAAGAGGCA-3
Tlr3 FAM 5-CACTTAAAGAGTTCTCCC-3
Tlr7 FAM 5-CCAAGAAAATGATTTTAATAAC-3
Predeveloped TaqMan assay reagents from Applied Biosystems
Cxcl10, Gapdh, 18srRNA
Il-6 mRNA/18S rRNA
0
5E-07
1E-06
1,5E-06
2E-06
2,5E-06
1h
3h
6h
1.0x10
-6
Medium 1μg 3μg 10μg 30μg 100μg
poly(I:C) RNA
0.5x10
-6
0
*
*
*
*
*
*
*
Figure 1. Mesangial cells produce Il-6 and IFN-
on exposure to poly I:C
RNA. Mesangial cells were prepared from C57BL/6 mice as described in
Materials and Methods. Cultured mesangial cells were stimulated with in-
creasing doses of poly I:C RNA, and Il-6 mRNA expression was determined
by real-time RT-PCR after 1, 3, and 6 hours of stimulation with increasing
doses of poly I:C RNA as indicated. Data are expressed as the ratio of Il-6
mRNA per respective 18S rRNA expression. Data are means SEM from
three experiments, each analyzed in duplicate.
P 0.05 versus medium
control.
2016 Flu¨r et al
AJP November 2009, Vol. 175, No. 5
Page 3
Mesangial Cells Express the Cytosolic Viral RNA
Recognition Receptors Mda5 and Rig-I but Only
Mda5 Mediates poly I:C RNA Recognition
Trif-independent recognition of poly I:C RNA in DCs in-
volves the cytosolic Rig-like helicases Rig-I and Mda5.
6,7
Primary mesangial cells expressed Rig-I and its mito-
chondrial adaptor IFN-
promoter stimulator-1 but not
Mda5 mRNA under basal culture conditions (Figure
3A). Rig-I and Mda5 mRNA expression markedly in-
crease on stimulation with IFN-
like Tlr3 within 6 hours
while that of IFN-
promoter stimulator-1 was rather
down-regulated (Figure 3A). By contrast, exposure to
increasing doses of Il-6 rather down-regulated Rig-I
and Mda5 (Figure 3B). At the protein level, mesangial
cells showed basal expression of Tlr3, Rig-I, and Mda5
(Figure 3C). Stimulation with poly I:C RNA/cationic lipid
complexes transiently reduced Tlr3 and increased
Mda5, whereas Rig-I expression was rather stable over
a period of 24 hours (Figure 3C). Which of the two viral
RNA recognition receptors mediates poly I:C RNA rec-
ognition in the cytosol? We used Mda-5- and Rig-I-
specific siRNA to answer this question. Each of the
specific siRNAs significantly reduced the mRNA levels
of its target in mesangial cells but only knockdown of
Mda5 impaired the Il-6 or Cxcl10 response on stimu-
lation with poly I:C RNA/cationic lipid complexes as
compared with transfection with nonspecific control
RNA (Figure 3, D and E). Knockdown of Mda5 did not
affect Il-6 or Cxcl10 induction on exposure to uncom-
plexed poly I:C RNA further demonstrating that cyto-
solic uptake is mandatory for Mda5 signaling. Taken
Figure 3. Cytosolic RNA receptors in mesangial cells. A: Primary mesangial
cells were cultured in the presence or absence of 2000 U/ml IFN-
(A)or
increasing doses of rmIl-6 (B). After 6 hours, mRNA was harvested, and
real-time RT-PCR was performed for various RNA recognition molecules, as
indicated. Data are expressed as the ratio of the specific mRNA per respective
18S rRNA expression. n.d., not detected;
P 0.05 versus medium. C:
Primary mesangial cells were cultured in the presence or absence of poly I:C
(pI:C) RNA/CL complexes over various time periods, as indicated. Unstimu-
lated J774 macrophages served as a positive control. Protein expression of
Tlr3, Rig-I, and Mda5 was determined by Western blot.
-Actin expression
was used as a loading control. D and E: Mesangial cells were transfected
twice with Mda-5-specific siRNA (D), Rig-I-specific siRNA (E), or nonspecific
control RNA as described in Materials and Methods. Knockdown efficacy
was tested by real-time PCR for the respective target (left panels). Impact of
knockdown on pI:C recognition was determined by real-time PCR for Il-6 or
Cxcl10 after 6 hours of incubation with 3
g of pI:C RNA either complexed
with cationic lipid (CL) or uncomplexed. Data represent means from three
experiments SEM;
P 0.05 versus nonspecific control RNA (n.c.).
Figure 2. Viral dsRNA complexed with cationic lipids activates mesangial
cells to produce high amounts of Il-6 and type I IFN via a Trif-indepen-
dent pathway. Primary mesangial cells were isolated from wild-type
C56BL/6 mice and Trif-mutant mice as described in Materials and Meth-
ods. Cells were stimulated with increasing doses of poly I:C (pI:C) RNA
alone or complexed with the cationic lipid (CL) Lipofectamine. Superna-
tants were harvested after 24 hours and analyzed by ELISA for the
following mediators: Il-6 (A), IFN-
(B), and IFN-
(C). Data are means
SEM from three experiments each analyzed in duplicate and presented in
a logarithmic scale.
P 0.05 wild-type versus medium;
ⴱⴱ
P 0.05
Trif-mutant versus wild type.
Viral RNA Aggravates Glomerulonephritis 2017
AJP November 2009, Vol. 175, No. 5
Page 4
together, type I IFN induces the expression of Rig-I and
Mda5 in mesangial cells, but only Mda-5 is required for
the triggering cyokine and chemokine release on cyto-
solic poly I:C RNA.
Poly I:C RNA/Cationic Lipid-Induced Activation
of Mesangial Cells Involves a Type I IFN
Autocrine-Paracrine Activation Loop
In DCs, the recognition of viral RNA induces the release
of type I IFN, which enhances subsequent Tlr signaling
via autocrine-paracrine recognition of type I IFN involving
the type I IFN receptor (IFNR) and Stat1 phosphoryla-
tion.
16
Therefore, we questioned whether this autocrine-
paracrine activation loop does also apply to mesangial
cells. Exposing mesangial cells to increasing doses of
IFN-
, IFN-
, or IFN-
revealed that only IFN-
or IFN-
induced Il-6 release in a dose-dependent manner (Figure
4A). Does IFN enhance viral dsRNA-mediated activation
of mesangial cells? Prestimulation of mesangial cells with
1000 units of either IFN-
and IFN-
for 24 hours mark-
edly enhanced the Il-6 response induced by poly I:C RNA
(Figure 4B). IFN-
had no significant effect on the Il-6
response induced by poly I:C RNA. Obviously, type I but
not type II IFN enhanced the poly I:C RNA-mediated
activation of mesangial cells in association with an induc-
tion of the RNA recognition machinery. To demonstrate
that type I IFN produced by the mesangial cells can
mediate this autocrine mechanism, we stimulated pri-
mary mesangial cells with poly I:C RNA/cationic lipid
complexes in the absence or presence of increasing
doses on antibodies that neutralize the functions of IFN-
and IFN-
. By blocking both type I IFN, the poly I:C
RNA/cationic lipid-induced Il-6 production was almost
completely prevented (Figure 5A). Furthermore, primary
mesangial cells from IFNR-deficient mice produced
much less Il-6 on poly I:C RNA/cationic lipid exposure as
mesangial cells from wild-type mice, respectively (Figure
5B). These data show that poly I:C RNA-induced activa-
tion of mesangial cells depends on the production and
recognition of type I IFN, ie, an autocrine-paracrine acti-
vation loop.
Poly I:C RNA-Cationic Lipid Complexes Induce
Mesangial Cell Death
Viral recognition often triggers a signal for apoptotic cell
death that is thought to contribute to the control of viral
replication and spreading, which is supported by our
finding that exposure to higher doses of poly I:C RNA is
Figure 4. IFN-
and -
enhance Il-6 release and RNA recognition in mesan-
gial cells. A: Primary mesangial cells were stimulated with increasing doses of
IFN-
-
as indicated. Supernatants were harvested after 24 hours and ana-
lyzed by ELISA for Il-6. Data represent means SEM from three independent
experiments.
P 0.05 versus medium. B: Mesangial cells were prestimu
-
lated with 1000 U/ml IFN-
,-
,or-
for 24 hours. The prestimulated cells
were then exposed to increasing doses of pI:C RNA as indicated. Superna-
tants were harvested after 24 hours and analyzed by ELISA for Il-6. Data
represent means SEM;
P 0.05 versus medium.
A
B
0
200
400
600
muideM pIC0.3+ opiL pIC0.3+ opiL+ gnikcolBAntydobia,ß1 pIC0.3+ opiL+ gnikcolBAntydobia,ß30 pIC1 + opiL pIC1+ opiL gnikcolB+Antydobia,ß1 pIC1 + opiL gnikcolB+Antydobia,ß30
pI:C - 0.3 0.3 0.3 1 1 1 μg/ml
CL - + + + + + +
Anti Ifn-α/β - - 1 30 - 1 30 μg/ml
IL-6 (pg/ml)
1000
800
600
400
200
0
*
*
0,1
1
10
100
1000
pI:C - 1 3 10 30 100 0.1 0.3 0.7 1 3 10 μg/ml
CL + - - - - - + + + + + +
10000
1000
100
10
1
0.1
Ifna-R +/+
Ifna-R -/-
IL-6 (ratio vs. unstimulated cells)
Figure 5. Poly I:C (pI:C) RNA-induced Il-6 release in mesangial cells de-
pends on type I IFNs. A: Primary mesangial cells were stimulated with
increasing doses of pI:C RNA/cationic lipid (CL) complexes as indicated. At
given concentrations of such complexes, increasing doses of neutralizing
antibodies against IFN-
and IFN-
were added. B: Mesangial cells were
prepared from wild-type or IFNa-R
/
mice, and cells were stimulated as
before. In A and B, supernatants were harvested after 24 hours and analyzed
by ELISA for Il-6. Data are means SEM from four experiments each
analyzed in duplicate.
P 0.05 versus no IFN-
and IFN-
antibody group.
2018 Flu¨r et al
AJP November 2009, Vol. 175, No. 5
Page 5
associated with a decline in Il-6 and IFN production. In
fact, the number of proliferating mesangial cells was
reduced on exposure to increasing doses of poly I:C RNA
(Figure 6A). The dose effect was enhanced up to a 100-
fold when pI:C RNA was complexed to cationic lipids
(Figure 6A). Poly I:C RNA-induced cell death involved the
extrinsic and intrinsic apoptosis pathways because poly
I:C RNA-induced cell death was prevented by caspase-8
or -10 and caspase-9 or -2 inhibitors, respectively (Figure
6B). Again, poly I:C RNA-induced cell death largely de-
pended on the autocrine-paracrine type I IFN activation
loop because the impact of poly I:C RNA/cationic lipid
complexes on cell proliferation was blunted in IFNa-R-
deficient mesangial cells (Figure 6C). Thus, triggering a
death pathway is part of the innate antiviral response
program of mesangial cells on exposure to higher con-
centrations of viral RNA, especially when it reaches the
intracellular cytosol.
Poly I:C RNA-Cationic Lipid Complexes
Induce Diffuse Focal Glomerular Necrosis
in Nephrotoxic Serum Nephritis in Mice
To test the in vivo relevance of the proposed mechanism,
we injected C57BL/6 mice with autologous nephrotoxic
serum nephritis with either vehicle, cationic lipids, poly
I:C RNA or poly I:C RNA/cationic lipid complexes. The
doses of nephrotoxic serum used for these experiments
usually cause robust immune complex glomerulonephri-
tis and massive albuminuria after 21 days. Injection of
poly I:C RNA and poly I:C RNA/cationic lipid complexes
induced proteinuria at day 7, which progressed to mas-
sive albuminuria at day 10 so that the mice had to be
sacrificed because of massive ascites as a marker of
nephrotic syndrome (Figure 7, A–C). However, only poly
I:C RNA/cationic lipid complexes led to severe global
segmental necrotic glomerular lesions at 10 days (Fig-
ures 7). These histopathological changes were associ-
ated with increased renal mRNA expression of Rig-I,
Mda5, and Cxcl10 in poly I:C RNA-injected mice (Figure
8, A and B).
Discussion
The glomerular filtration process exposes mesangial cells
to circulating micro- and macromolecules of the intravas-
cular compartment, including viral particles during viral
infections. RNA can be protected from RNase digestion
when being complexed to proteins such as Igs or nucleo-
proteins.
17–19
In glomerulonephritis, such immune com
-
plexes are often deposited in the glomerular mesangium
where they are taken up by glomerular mesangial cells.
20
Studies from our group showed that poly I:C RNA acti-
vates human and murine mesangial cells to produce Il-6
and Ccl2 in vitro and in vivo, which we confirmed here at
the mRNA level for Il-6. Interestingly, Il-6 mRNA expres-
sion fell below the baseline level after 6 hours, but on the
protein level, Il-6 production was sustained at 24 hours.
The effect of poly I:C RNA was previously attributed to
Tlr3, ie, the only RNA-specific Tlr expressed by mesan-
gial cells.
11,16
In contrast to our previous studies with a
murine mesangial cell line, primary mesangial cells do
not constitutively express Tlr3.
11
Furthermore, primary
Figure 6. Cytosolic recognition of viral dsRNA-induced mesangial cells
death. A: Primary mesangial cells were stimulated with increasing doses of
poly I:C (pI:C) RNA with or without cationic lipid (CL) as indicated. After 72
hours, the number of proliferating cells was determined by a biolumines-
cence assay as described in Materials and Methods.
P 0.05 versus me
-
dium;
ⴱⴱ
P 0.05 versus sine CL. B: Primary mesangial cells were stimulated
with poly I:C RNACL (pIC) in the presence or absence of the caspase-2
inhibitor Z-VDVAD-FMK (CI, 5
g/ml), the caspase-8 inhibitor Ac-IETD-CHO
(CI, 5
g/ml), the caspase-9 inhibitor Z-LEHD-FMK (CI, 5
g/ml), and the
caspase-10 inhibitor Z-AEVD-FMK (CI, 5
g/ml). After 72 hours, the number
of proliferating cells was determined by a bioluminescence assay.
P 0.05
versus pI:C RNA. Note that all caspase inhibitors prevented pI:C RNACL-
induced inhibition of cell growth. C: In similar experiments, mesangial cells
from IFNa-R
/
(black bars) and IFNa-R
/
cells (white bars) were stimu
-
lated with pI:C/cationic lipid complexes.
P 0.05 versus IFNa-R
/
.
Viral RNA Aggravates Glomerulonephritis 2019
AJP November 2009, Vol. 175, No. 5
Page 6
mesangial cells produce only small amounts of Il-6 on
poly I:C RNA challenge unless activation by IFN-
. How-
ever, viral dsRNA can induce the maturation of DCs
independent of the Tlr3/Trif pathway.
8,21
The discovery of
Rig-I and Mda5 as cytosolic viral dsRNA receptors
7
sug
-
gests an alternative pathway how viral dsRNA could ac-
tivate mesangial cells. In fact, in nonimmune cells, viral
dsRNA appears to preferentially activate the cytosolic
Rig-I route rather than the Tlr3 pathway.
8,21
Here we provide experimental evidence that Tlr3/Trif-
dependent and Tlr-independent recognition of viral dsRNA
both can trigger specific antiviral immunity in mesangial
cells. Viral dsRNA induced low levels of IFN-
in mesangial
cells, a response that was only in part mediated through the
Tlr3/Trif-signaling pathway in intracellular endosomes. To
test the significance of Tlr-independent RNA recognition
requires transfection of viral dsRNA into the intracellular
cytosol, which might also be the predominant route of RNA
delivery on direct viral infection of cells. Experimentally, we
complexed the poly I:C RNA to cationic lipids, which is as
effective as other ways of transfection.
7,22,23
Interestingly,
transfecting poly I:C RNA triggers the production of large
amounts of IFN-
and IFN-
as well as cell death of mes-
angial cells, which involves the apoptosis pathway. Re-
markably, poly I:C RNA/cationic lipid complexes induced
high levels of Il-6 and type I IFN despite mesangial cell
apoptosis unless high doses of poly I:C RNA were applied.
Our in vivo studies support the concept that poly I:C RNA/
cationic lipid complexes more potently aggravate glomeru-
lonephritis in association with increased renal IFN-related
cytokine expression as well as enhanced glomerular cell
death. However, only a mesangial cell-specific knockout of
Trif could ultimately prove that the Trif-independent activa-
tion of mesangial cells proposed by our in vitro studies
applies in vivo.
Cytokine secretion as well as apoptosis both are con-
sidered to be important local mechanisms to control viral
infection. These Tlr3/Trif-independent effects are sugges-
tive for Rig-I- or Mda5-dependent RNA recognition.
7
In
fact, Rig-I and Mda5 are both expressed in mesangial
cells, and Rig-I was recently shown to be expressed in
human lupus nephritis,
24
a disease state closely linked to
type I IFN signaling.
25,26
However, our studies with Rig-I-
and Mda5-specific siRNA clearly identified Mda5 and not
Rig-I as the cytosolic poly I:C RNA receptor in murine
mesangial cells. It is of note that mesangial cells produce
IFN-
and IFN-
but not IFN-
, ie, type II IFN, which
should relate to the different functions of type I and type
II IFN in immunity.
2
Hence, poly I:C RNA stimulates glo
-
merular mesangial cells to produce type I IFN, especially
when activating Mda5 as part of the Tlr-independent RNA
recognition pathway in the cytosol.
When pDCs produce type I IFN, these modulate the
function of other cells as well as of the pDC itself, ie, an
autocrine-paracrine regulatory mechanism.
2,27
For exam
-
Figure 7. Nephrotoxic serum nephritis in C57BL/6 mice. A: Urinary albumin/
creatinine ratios were determined from all groups of mice at days 7 and 10 as
described in Materials and Methods. Data represent means SEM;
P 0.05
versus day 0,
ⴱⴱ
P 0.05 versus vehicle on day 10. B: Focal glomerular
necrosis was quantified on periodic acid-Schiff (PAS)-stained renal sections
from all groups on day 10 applying a semiquantitative score from 0 to 3 in 15
glomeruli per section. Data represent means SEM;
ⴱⴱ
P 0.05 pI:C RNA/
cationic lipid versus pI:C;
P 0.05 versus vehicle. C: Renal sections from
mice of all groups were stained with PAS. Representative images were taken
at an original magnification of 100 and 400. Focal necrosis is indicated by
asterisk.
0
6E-05
0,0001
s enilayendik yendikPATOD PI:Cyendik PI:Cdotap yendik
0
2E-06
4E-06
s enila IPyendikPATODyendik :C IPyendik :Cdotap yendik
0
5E-05
1E-04
s enila IPyendikPATODyendik :CIPyendik:Cdotap yendik
6x10
-5
12x10
-5
0
Rig-I
Vehicle CL pI:C pI:C/CL
10x10
-5
5x10
-5
0
Mda5
2x10
-6
4x10
-6
0
Tlr3
Vehicle CL pI:C pI:C/CL Vehicle CL pI:C pI:C/CL
*
*
*
*
*
mRNA/18s rRNA
0
2E-04
4E-04
6E-04
8E-04
0,001
0,001
12x10
-4
Cxcl10
Vehicle CL pI:C pI:C/CL
*
*
6x10
-4
0
mRNA/18s rRNA
A
B
Figure 8. Intrarenal gene expression in nephro-
toxic serum nephritis. Real-time RT-PCR was
performed for RNA recognition receptors (A)
and IFN-related genes (B) on total kidney RNA
isolates from four mice of each group. Data are
expressed as the ratio of the specific mRNA per
respective 18S rRNA expression;
P 0.05 ver
-
sus vehicle group.
2020 Flu¨r et al
AJP November 2009, Vol. 175, No. 5
Page 7
ple, in type I IFNR-deficient pDCs, the challenge with poly
I:C RNA results in much lower cytokine responses as
compared with wild-type pDCs.
16
Obviously, the secre
-
tion of type-I IFN enhances Tlr signaling in an autocrine-
paracrine manner in pDCs, the professional IFN-produc-
ing cells. This mechanism was also shown to apply for
endothelial and epithelial cells.
28
We therefore hypothe
-
sized that the same mechanism enhances antiviral re-
sponses in glomerular mesangial cells exposed to poly
I:C RNA. In fact, mesangial cells produce Il-6 on expo-
sure to IFN-
and IFN-
. The presence of either IFN-
or -
enhanced the poly I:C RNA-induced production of
Il-6. The ultimate evidence for an autocrine loop came
from blocking intrinsic type I IFN with neutralizing anti-
bodies or by genetic deletion of the type I IFNR in mes-
angial cells, which dramatically reduced the amount of
IL-6 produced on exposure to viral dsRNA.
Hence, viral dsRNA activates mesangial cells to pro-
duce type I IFN, which enhances the production of proin-
flammatory cytokines like Il-6 in an autocrine-paracrine
manner via the type I IFNR. In this process, IFN-
induces
innate viral RNA recognition receptors in primary mesan-
gial cells just like Tnf/IFN-
previously shown for cell lines
of murine and human mesangial cells.
14,29
The patho
-
genic relevance of this mechanism for glomerulonephritis
was recently demonstrated by Jørgensen et al
30
autoim
-
mune B6.Nba2 mice and (B6Nba2 NZW)F
1
mice defi
-
cient for the type I IFNR failed to develop glomerulone-
phritis, although the mice had substantial glomerular
immune complex deposits.
29
In summary, poly I:C RNA stimulates glomerular mesan-
gial cells to produce large amounts of type I IFN, especially
when being delivered into the intracellular cytosol where it
can interact with Mda5. Type I IFN production enhances the
dsRNA-induced production of proinflammatory mediators
such as Il-6 as well as cell death in a positive autocrine
amplification loop. Thus, the recognition of viral RNA trig-
gers type I IFN in glomerular mesangial cells, which we
propose as a novel pathomechanism for glomerular pathol-
ogy in viral infection-associated glomerulonephritis.
Acknowledgments
We thank Dr. Heike Weighardt (Department of Surgery,
Technische Universita¨t, Munich, Germany) for providing the
IFNa-R-deficient and control 129 Sv/Ev mice. The expert
technical assistance of Ewa Radomska, Iana Mandelbaum,
and Dan Draganovic is gratefully acknowledged.
References
1. Lai AS, Lai KN: Viral nephropathy. Nat Clin Pract Nephrol 2006,
2:254 –262
2. Theofilopoulos AN, Baccala R, Beutler B, Kono DH: Type I inter-
ferons (
/
) in immunity and autoimmunity. Annu Rev Immunol
2005, 23:307–336
3. Stark GR, Kerr LM, Williams BR, Silvemann RH, Schreiber RD: How
cells respond to interferons. Ann Rev Biochem 1998, 67:227–264
4. Shortman K, Liu YJ: Mouse and human dendritic cell subtypes. Nat
Rev Immunol 2002, 2:151–161
5. Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate
immunity. Cell 2006, 124:783–801
6. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T,
Miyagishi M, Taira K, Akira S, Fujita T: The RNA helicase RIG-I has an
essential function in double-stranded RNA-induced innate antiviral re-
sponses. Nat Immunol 2004, 5:730 –737
7. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K,
Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K,
Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita T, Akira S:
Differential roles of MDA5 and RIG-I helicases in the recognition of RNA
viruses. Nature 2006, 441:101–105
8. Saito T, Owen DM, Jiang F, Marcotrigiano F, Gale Jr M: Innate
immunity induced by composition-dependent RIG-I recognition of
hepatitis C virus RNA. Nature 2008, 454:523–527
9. Lang KS, Recher M, Junt T, Navarini AA, Harris NL, Freigang S,
Odermatt B, Conrad C, Ittner LM, Bauer S, Luther SA, Uematsu S,
Akira S, Hengartner H, Zinkernagel RM: Toll-like receptor engage-
ment converts T cell autoreactivity into overt autoimmune disease.
Nat Med 2005, 11:138–145
10. Schroder M, Bowie AG: TLR3 in antiviral immunity: key player or
bystander? Trends Immunol 2005, 26:462– 468
11. Patole PS, Grone HJ, Segerer S, Ciubar R, Belemezova E, Henger A,
Kretzler M, Schlondorff D, Anders HJ: Viral double-stranded RNA
aggravates lupus nephritis through Toll-like receptor 3 on glomerular
mesangial cells and antigen-presenting cells. J Am Soc Nephrol
2005, 16:1326 –1338
12. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO, Goode J,
Lin P, Mann N, Mudd S, Crozat K, Sovath S, Han J, Beutler B:
Identification of Lps2 as a key transducer of MyD88-independent TIR
signalling. Nature 2003, 424:743–748
13. Vielhauer V, Stavrakis G, Mayadas TN: Renal cell-expressed TNF
receptor 2, not receptor 1, is essential for the development of glo-
merulonephritis. J Clin Invest 2005, 115:1199 –1209
14. Wo¨ rnle M, Schmid H, Banas B, Merkle M, Henger A, Roeder M,
Blattner S, Bock E, Kretzler M, Gro¨ ne HJ, Schlo¨ndorff D: Novel role of
Toll-like receptor 3 in hepatitis C-associated glomerulonephritis. Am J
Pathol 2006, 168:370–385
15. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA: Recognition of
double-stranded RNA and activation of NF-
B by Toll-like receptor 3.
Nature 2001, 413:732–738
16. Gautier G, Humbert M, Deauvieau F, Scuiller M, Hiscott J, Bates EE,
Trinchieri G, Caux C, Garrone P: A type I interferon autocrine-para-
crine loop is involved in Toll-like receptor-induced interleukin-12p70
secretion by dendritic cells. J Exp Med 2005, 201:1435–1446
17. Lau CM, Broughton C, Tabor AS, Akira S, Flavell RA, Mamula MJ,
Christensen SR, Shlomchik MJ, Viglianti GA, Rifkin IR, Marshak-
Rothstein A: RNA-associated autoantigens activate B cells by com-
bined B cell antigen receptor/Toll-like receptor 7 engagement. J Exp
Med 2005, 202:1171–1177
18. Yasuda K, Richez C, Maciaszek JW, Agrawal N, Akira S, Marshak-
Rothstein A, Rifkin IR: Murine dendritic cell type I IFN production
induced by human IgG-RNA immune complexes is IFN regulatory
factor (IRF)5 and IRF7 dependent and is required for IL-6 production.
J Immunol 2007, 178:68766885
19. Savarese E, Chae OW, Trowitzsch S, Weber G, Kastner B, Akira S,
Wagner H, Schmid RM, Bauer S, Krug A: U1 small nuclear ribonu-
cleoprotein immune complexes induce type I interferon in plasmacy-
toid dendritic cells through TLR7. Blood 2006, 107:3229 –3234
20. Gomez-Guerrero C, Lopez-Armada MJ, Gonzalez E, Egido J: Soluble
IgA and IgG aggregates are catabolized by cultured rat mesangial
cells and induce production of TNF-
and IL-6, and proliferation.
J Immunol 1994, 153:5247–5255
21. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K,
Tsujimura T, Takeda K, Fujita T, Takeuchi O, Akira S: Cell type-
specific involvement of RIG-I in antiviral response. Immunity 2005,
23:19 –28
22. Braun CS, Vetro JA, Tomalia DA, Koe GS, Koe JG, Middaugh CR: The
structure of DNA within cationic lipid/DNA complexes. Biophys J
2003, 84:1114 –1123
23. Stetson DB, Medzhitov R: Recognition of cytosolic DNA activates an
IRF3-dependent innate immune response. Immunity 2006, 24:93–103
24. Suzuki K, Imaizumi T, Tsugawa K, Ito E, Tanaka H: Expression of
retinoic acid-inducible gene-I in lupus nephritis. Nephrol Dial Trans-
plant 2007, 22:2407–2409
Viral RNA Aggravates Glomerulonephritis 2021
AJP November 2009, Vol. 175, No. 5
Page 8
25. Crow MK, Kirou KA: Interferon
in systemic lupus erythematosus.
Curr Opin Rheumatol 2004, 16:541–547
26. Ronnblom L, Alm GV: An etiopathogenic role for the type I IFN system
in SLE. Trends Immunol 2001, 22:427– 431
27. Honda K, Sakaguchi S, Nakajima C, Watanabe A, Yanai H, Matsumoto
M, Ohteki T, Kaisho T, Takaoka A, Akira S, Seya T, Taniguchi T: Selective
contribution of IFN-
/
signaling to the maturation of dendritic cells
induced by double-stranded RNA or viral infection. Proc Natl Acad Sci
USA 2003, 100:10872–10877
28. Tissari J, Siren J, Meri S, Julkunen I, Matikainen S: IFN-
enhances
TLR3-mediated antiviral cytokine expression in human endothelial
and epithelial cells by up-regulating TLR3 expression. J Immunol
2005, 174:4289 4294
29. Patole PS, Pawar RD, Lech M, Zecher D, Schmidt H, Segerer S,
Ellwart A, Henger A, Kretzler M, Anders HJ: Expression and reg-
ulation of Toll-like receptors in lupus-like immune complex glomer-
ulonephritis of MRL-Fas
lpr
mice. Nephrol Dial Transplant 2006,
21:3062–73
30. Jørgensen TN, Roper E, Thurman JM, Marrack P, Kotzin BL: Type I
interferon signaling is involved in the spontaneous development of
lupus-like disease in B6.Nba2 and (B6.Nba2 NZW)F
1
mice. Genes
Immun 2007, 8:653–662
2022 Flu¨r et al
AJP November 2009, Vol. 175, No. 5
Page 9
  • Source
    • "Therefore, immunoglobulin therapy can also have systemic immunosuppressive effects and reduce immunopathology in lupus nephritis [66]. Finally, the nucleic acid component of immune complexes may also trigger glomerular injury, e.g., via viral nucleic acid receptors of the innate immune system in mesangial cells or intraglomerular macrophages and dendritic cells to produce large amounts of pro-inflammatory cytokines and interferon-α [67][68][69][70][71][72][73][74][75]. Depending on the capacity of glomerular autoantibody deposits to trigger cell injury, e.g., by activating complement, FcR, and TLRs, their deposition will cause no (class I), mild, or massive (class II) mesangial hypercellularity [76][77][78]. "
    [Show abstract] [Hide abstract] ABSTRACT: When patients with systemic lupus erythematosus (SLE) present with urinary abnormalities, a renal biopsy is usually needed to rule out or confirm lupus nephritis. Renal biopsy is also needed to define the type of renal manifestation as different entities are associated with different outcomes; hence, renal biopsy results shape lupus management. But why does lupus nephritis come in different shapes? Why do patients with SLE often show change over time in class of lupus nephritis or have mixed forms? How does autoimmunity in SLE evolve? Why does loss of tolerance against nuclear antigens preferentially affect the kidney? Why are immune complex deposits in different glomerular compartments associated with different outcomes? What determines crescent formation in lupus? In this review, we discuss these questions by linking the latest information on lupus pathogenesis into the context of the different classes of lupus nephritis. This should help the basic scientist, the pathologist, and the clinician to gain a more conceptual view on the immunopathology of lupus nephritis.
    Full-text · Article · Jan 2014 · Seminars in Immunopathology
  • Source
    • "Patients with viral infections or malignant tumors frequently develop SLE-like manifestations and anti-DNA antibodies following IFN-α treatment, thereby corroborating the importance of this cytokine in the development of lupus [79–81]. Although plasmacytoid dendritic cells are the primary source of type I IFNs in lupus patients, intrinsic renal cells such as mesangial cells and glomerular endothelial cells can also synthesize IFN-α following stimulation with viral components mediated through toll-like receptor dependent and independent pathways [82–84]. Synthesis of IFN-α by endothelial cells may contribute to the infiltration of inflammatory cells into the kidney parenchyma. "
    [Show abstract] [Hide abstract] ABSTRACT: Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is a major cause of morbidity and mortality. It is characterized by a breakdown of immune tolerance, production of autoantibodies, and deposition of immune complexes within the kidney parenchyma, resulting in local inflammation and subsequent organ damage. To date, numerous mediators of inflammation have been implicated in the development and progression of lupus nephritis, and these include cytokines, chemokines, and glycosaminoglycans. Of these, type I interferons (IFNs) can increase both gene and protein expression of cytokines and chemokines associated with lupus susceptibility, and interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α ) and hyaluronan have been shown to elicit both pro- and anti-inflammatory effects on infiltrating and resident renal cells depending on the status of their microenvironment. Expression of IL-6, TNF- α , type I IFNs, and hyaluronan are increased in the kidneys of patients and mice with active lupus nephritis and have been shown to contribute to disease pathogenesis. There is also evidence that despite clinical remission, ongoing inflammatory processes may occur within the glomerular and tubulointerstitial compartments of the kidney, which further promote kidney injury. In this review, we provide an overview of the synthesis and putative roles of IL-6, TNF- α , IFN- α , and hyaluronan in the pathogenesis of lupus nephritis focusing on their effects on human mesangial cells and proximal renal tubular epithelial cells.
    Full-text · Article · Sep 2013 · Clinical and Developmental Immunology
  • Source
    • "MDA5 and RIG-I were recently shown to function as pathogen recognition receptors of viral dsRNA in the cytostome, and both receptors may play an important role in innate immune reactions [4, 5]. Although the expression of MDA has been documented in human MCs [13] as well as murine MCs [15], the detailed implications of MDA5 expression in human MCs have not yet been clarified. Since C-X-C motif chemokine 10 (CXCL10, also known as IFN-γ-induced protein 10), a chemokine with chemotactic activity for leukocytes with CXCR3, is thought to be involved in the pathogenesis of glomerular diseases [23], we examined the effect of poly IC and the role of MDA5 in CXCL10 expression in cultured human MCs [13, 14]. "
    [Show abstract] [Hide abstract] ABSTRACT: The innate and adaptive immune systems have been reported to play an important role in the pathogenesis of glomerular diseases. Since viral infections may trigger the development of inflammatory renal disease or the worsening of preexisting renal disease, recent studies have focused on the involvement of toll-like receptors (TLRs) and their signaling pathways in the inflammatory processes of glomerular cells. Viral double-stranded RNA (dsRNA) can activate not only TLR3 located within intracellular endosomes but also retinoic-acid-inducible-gene-I- (RIG-I-) like helicase receptors located within the cytosol. RIG-I and melanoma differentiation-associated gene 5 (MDA5) are members of the RNA helicase family in the cytosol, and both act as pathogen recognition receptors. The activation of TLRs and their downstream immune responses can be induced by both infectious pathogens and noninfectious stimuli such as endogenous ligands, and this mechanism may be involved in the pathogenesis of autoimmune renal diseases. However, there are few data on the interaction between TLR3, MDA5, and RIG-I in autoimmune glomerular diseases. Based on our recent experimental studies using cultured normal human mesangial cells (MCs), we found that novel TLR3-mediated signaling pathways in MCs may be involved in the pathogenesis of glomerular diseases. In the present paper, we summarize our recent findings.
    Full-text · Article · Jun 2013 · Clinical and Developmental Immunology
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