BASIC RESEARCH
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IL-17C/IL-17 Receptor E Signaling in CD4+ T Cells
Promotes TH17 Cell-Driven Glomerular Inflammation
Sonja Krohn, Jasper F. Nies, Sonja Kapffer, Tilman Schmidt, Jan-Hendrik Riedel,
Anna Kaffke, Anett Peters, Alina Borchers, Oliver M. Steinmetz, Christian F. Krebs,
Jan-Eric Turner, Silke R. Brix, Hans-Joachim Paust, Rolf A. K. Stahl, and Ulf Panzer
III. Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
ABSTRACT
The IL-17 cytokine family and the cognate receptors thereof have a unique role in organ-specific autoimmunity. Most studies have focused on the founding member of the IL-17 family, IL-17A, as the central
mediator of diseases. Indeed, although pathogenic functions have been ascribed to IL-17A and IL-17F in
the context of immune-mediated glomerular diseases, the specific functions of the other IL-17 family
members in immunity and inflammatory kidney diseases is largely unknown. Here, we report that compared with healthy controls, patients with acute Anti-neutrophil cytoplasmatic antibody
(ANCA)-associated crescentic glomerulonephritis (GN) had significantly elevated serum levels of IL-17C
(but not IL-17A, F, or E). In mouse models of crescentic GN (nephrotoxic nephritis) and pristane-induced
lupus nephritis, deficiency in IL-17C significantly ameliorated the course of GN in terms of renal tissue
injury and kidney function. Deficiency of the unique IL-17C receptor IL-17 receptor E (IL-17RE) provided
similar protection against crescentic GN. These protective effects associated with a reduced TH17 response. Bone marrow transplantation experiments revealed that IL-17C is produced by tissue-resident
cells, but not by lymphocytes. Finally, IL-17RE was highly expressed by CD4+ TH17 cells, and loss of this
expression prevented the TH17 responses and subsequent tissue injury in crescentic GN. Our findings
indicate that IL-17C promotes TH17 cell responses and immune-mediated kidney disease via IL-17RE
expressed on CD4+ TH17 cells. Targeting the IL-17C/IL-17RE pathway may present an intriguing therapeutic strategy for TH17-induced autoimmune disorders.
J Am Soc Nephrol 29: 1210–1222, 2018. doi: https://doi.org/10.1681/ASN.2017090949
The identification and characterization of IL-17A–
producing CD4+ T cells, referred to as TH17 cells,1,2
have substantially advanced our understanding of
organ-specific autoimmunity and are a topic of intense ongoing clinical and basic research. TH17
cells, characterized by the expression of the
Received September 1, 2017. Accepted January 3, 2018.
S.Krohn and J.F.N. contributed equally to this work.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Ulf Panzer, Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, III. Martinistraße 52,
20246 Hamburg, Germany. Email: panzer@uke.uni-hamburg.de
Copyright © 2018 by the American Society of Nephrology
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ISSN : 1046-6673/2904-1210
Significance Statement
Recent studies have established the crucial role of the
TH17/IL-17A pathway in the pathogenesis of autoimmune diseases, such as human and experimental crescentic GN. So far, the focus of most studies has been on
the IL-17 founding member IL-17A as the central mediator of diseases. Here, we report that serum IL-17C
levels were significantly elevated in patients with
ANCA-associated crescentic GN. Moreover, using a
murine model of crescentic GN, we provide direct evidence that IL-17C promotes TH17 cell responses and
kidney injury via IL-17 receptor E, expressed on CD4+
TH17 cells. These findings have significant implications
for the understanding of how the IL-17 cytokine family
contributes to organ-specific TH17 autoimmunity and
potentially for the development of novel treatment
strategies.
J Am Soc Nephrol 29: 1210–1222, 2018
www.jasn.org
RESULTS
Serum IL-17C Levels Are Elevated in Patients with
ANCA-Associated Crescentic GN
Using an ultrasensitive electrochemiluminescence immunoassay, we analyzed the serum levels of IL-17A, C, E, and F in 70
patients with biopsy-confirmed, acute ANCA-associated crescentic GN and in 20 healthy controls. Unexpectedly, we
J Am Soc Nephrol 29: 1210–1222, 2018
IL-17F
IL-17A
Protein [pg/mL]
Protein [pg/mL]
30
10
4
2
1500
500
150
100
50
0
0
Controls
Controls
ANCA GN
ANCA GN
IL-17E
IL-17C
***
Protein [pg/mL]
Protein [pg/mL]
20
10
6
4
2
0
2.0
1.0
0.4
0.2
0.0
Controls
ANCA GN
Controls
TNF-α
ANCA GN
IL-6
***
***
20
60
Protein [pg/mL]
Protein [pg/mL]
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transcription factor RORgt3 and the expression of CCR6,4,5
play a pivotal role in crescentic GN by producing proinflammatory cytokines.6–9 In particular, IL-17A and IL-17F may
mediate tissue injury by the induction of renal CXCL1 and
CXCL5 expression.10–12 These chemokines, in turn, recruit
neutrophils into the kidney, which ultimately contributes to
the organ damage.13
The IL-17 cytokine family consists of six members (IL-17A–
F), of which IL-17A and F are the most closely related and best
characterized ones.14 Their biologic effects are mediated by
binding to heterodimeric receptors of the IL-17 receptor family (IL-17RA–RE). Except for the receptor for IL-17B
(IL-17RB), all receptor complexes contain the ubiquitously
expressed subunit IL-17RA and a second, ligand-specific receptor. So far, only the roles of IL-17A and IL-17F have been
analyzed in the pathogenesis of immune-mediated glomerular
diseases.10,11,15–17 The potential effect of the other IL-17 family members in renal autoimmunity and inflammation is unknown.
First, we assessed the serum levels of the IL-17 family members in patients with ANCA-associated crescentic GN and analyzed the systemic and renal expression patterns of these
cytokines and their receptors during the time course of an
experimental model of crescentic GN (nephrotoxic nephritis
[NTN]).18 Unexpectedly, these analyses demonstrated an upregulation of IL-17C and its specific receptor IL-17RE,
suggesting a functional role for the IL-17C/IL-17RE axis in
immune-mediated kidney diseases, and represented the rationale for our study.
Pioneer studies indicated that IL-17C might share similar
biologic features with IL-17A, as it stimulates inflammation by
upregulating cytokines and chemokines that are characteristic
of a TH17 response in epithelial cells.19 Moreover, the involvement of IL-17C in the immunopathogenesis of murine models
for multiple sclerosis and arthritis,20,21 potentially by direct
effects on the TH17 response, has been reported.20
The goal of this study was to elucidate the role of the IL-17C/
IL-17RE axis in immune-mediated glomerular diseases. We
therefore induced an experimental model of crescentic GN
(NTN) to (1) define the effect of IL-17C and its receptor IL17RE on the clinical outcome of the GN, (2) determine the
significance of this ligand receptor pair for the T-cell immune
response, and (3) identify the mechanisms by which the IL17C/IL-17RE pathway might drive renal tissue injury in experimental crescentic GN.
BASIC RESEARCH
10
6
4
2
0
20
10
5
0
Controls
ANCA GN
Controls
ANCA GN
Figure 1. IL-17C levels are significantly elevated in patients with
ANCA-associated crescentic GN. Serum level of IL-17 cytokines,
TNF-a, and IL-6 in patients with ANCA-associated GN. Quantitative measurement of IL-17A, C, E, F, TNF-a, and IL-6 in the
serum of patients with biopsy-proven ANCA GN (n=70) and
healthy controls (n=20). Symbols represent individual data points
with the mean as a horizontal line (***P,0.001).
observed a marked elevation (P,0.001) in IL-17C protein
levels in the ANCA GN patient group compared with healthy
controls (Figure 1). No significant differences were detected in
the levels of the remaining IL-17 family members (Figure 1).
We also analyzed the levels of TNF-a and IL-6 as general
markers for inflammation, which were significantly higher
in the patient population compared with the control group
(Figure 1). The clinical and demographic baseline characteristics of the patient group are shown in Supplemental Figure 1.
IL-17C and IL-17RE Expression Is Upregulated in
Experimental Crescentic GN
Next, we aimed at investigating the expression pattern of the IL17 family members and their receptors during the time course
of NTN. Renal quantitative PCR (qPCR) analyses during different periods of time (day 0 to day 30) of nephritic mice
revealed the regulation of Il-17a mRNA expression, which is
in agreement with our previous data.22 In addition, Il-17c
expression was upregulated 12 hours after NTN induction
(Figure 2A). A similar mRNA expression pattern of Il-17a
and Il-17c was detected in the spleen (Figure 2B). We also
IL-17C in Renal Inflammation
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A
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Kidney
30
20
10
30
20
10
5d 10d 20d 30d
Con 12h 24h 3d
20
10
0
30
20
10
5d 10d 20d 30d
30
20
10
0
40
30
20
10
24h
10d
10
5
40
30
20
10
4
3
2
1
0
Con
Il-17e
24h
10d
30d
Con 12h 24h 3d
Il-17f
30
20
10
0
5
40
30
20
10
0
30d
5d 10d 20d 30d
Il-17re
100
60
50
mRNA expression
[x-fold of control]
mRNA expression
[x-fold of control]
40
5d 10d 20d 30d
Il-17rc
100
60
50
30d
100
60
50
10d
1
Con 12h 24h 3d
0
0
24h
2
30d
mRNA expression
[x-fold of control]
20
Con
3
Il-17d
30
10d
4
0
Con
mRNA expression
[x-fold of control]
mRNA expression
[x-fold of control]
Kidney
5
Il-17c
40
5d 10d 20d 30d
Il-17ra
100
60
50
30d
100
60
50
24h
10
Con 12h 24h 3d
0
Con
20
5d 10d 20d 30d
mRNA expression
[x-fold of control]
40
10d
30
Il-17b
mRNA expression
[x-fold of control]
mRNA expression
[x-fold of control]
Il-17a
24h
80
60
40
0
Con 12h 24h 3d
C
100
60
50
5d 10d 20d 30d
Il-17f
80
60
40
Spleen
Con
10
Con 12h 24h 3d
0
Con 12h 24h 3d
20
5d 10d 20d 30d
mRNA expression
[x-fold of control]
30
30
Il-17e
mRNA expression
[x-fold of control]
mRNA expression
[x-fold of control]
Il-17d
80
60
40
80
60
40
0
0
Con 12h 24h 3d
B
Il-17c
80
60
40
mRNA expression
[x-fold of control]
mRNA expression
[x-fold of control]
Il-17b
0
mRNA expression
[x-fold of control]
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mRNA expression
[x-fold of control]
Il-17a
80
60
40
4
3
2
1
0
Con
24h
10d
30d
Con 12h 24h 3d
5d 10d 20d 30d
Figure 2. Expression of IL-17C and IL-17RE is upregulated in experimental crescentic GN. Renal expression of the IL-17 family and
their receptors in crescentic GN. (A) qPCR analysis of Il-17a–f mRNA expression levels in the kidney, (B) the spleen, and (C) renal
expression levels of Il-17ra, Il-17rc, and Il-17re at indicated time points after NTN induction. Controls (Con; n=5) and nephritic wild-type
mice (n=4–7). mRNA levels are expressed as x-fold of controls. Symbols represent individual data points with the mean as a horizontal
line.
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Journal of the American Society of Nephrology
J Am Soc Nephrol 29: 1210–1222, 2018
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model. The histologic and functional parameters were
analyzed to compare the clinical outcome in wild-type and
Il-17c2/2 mice upon NTN induction (Figure 3). Quantification of the renal tissue damage, in terms of glomerular crescent
formation and tubulointerstitial injury 10 days after NTN induction, revealed a reduction in Il-17c2/2 mice (Figure 3, A
and B). In line with these results, we also found a significant
reduction in BUN levels, and to a lesser degree in serum creatinine levels, whereas we did not find any changes in the
urinary albumin-to-creatinine ratio (ACR) (Figure 3C).
Assessment of renal leukocyte recruitment by immunohistochemical staining revealed that the tubulointerstitial infiltration
of F4/80+ macrophages and CD3+ T cells was significantly
IL-17C Promotes Renal Tissue Injury in Crescentic GN
On the basis of the expression pattern of Il-17c in human and
experimental GN described above, we examined whether IL17C signaling is involved in the pathogenesis of crescentic GN.
Therefore, we analyzed Il-17c2/2 mice using the NTN mouse
A
Il-17c-/- NTN
WT NTN
PAS
Control
C
20
Con
NTN
40
20
0
Con
p=0.06
**
80
BUN [mg/dL]
40
0
60
Interstitial Area [%]
Crescents [%]
60
***
60
40
20
0
NTN
Con
WT
NTN
0.8
250
0.6
200
ACR [g/g]
***
Serum Creatinine [mg/dL]
B
0.4
0.2
150
100
50
0
0.0
Con
NTN
Con
60
40
20
0
Con
NTN
CD3+ cells / gcs
MAC-2+ cells / gcs
F4/80+ cells / hpf
80
6
10
8
6
4
2
0
Con
NTN
E
4
2
0
***
100
CD3+ cells / hpf
*
100
NTN
Il-17c-/-
D
Con
80
60
40
20
0
NTN
Con
NTN
F
WT NTN
Il-17c-/- NTN
***
GR1+ cells / lpf
20
GR1+
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observed a regulation of the mRNA expression of Il-17rc and
Il-17re, which are the specific receptor subunits of IL-17A and
IL-17C, respectively (Figure 2C). The receptor chain IL-17RA,
which serves as a common receptor chain for all IL-17 ligands
(except for IL-17B), was highly expressed throughout the
course of crescentic GN, but did not show any changes in its
expression pattern (Figure 2C).
BASIC RESEARCH
15
10
5
0
Con
NTN
Figure 3. IL-17C promotes renal tissue injury in crescentic GN. (A) Representative photographs of PAS-stained kidney sections from
control (Con), nephritic wild-type (WT), and nephritic Il-17c2/2 mice at day 10 of NTN (original magnification, 3400). (B) Quantification
of glomerular crescent formation and tubulointerstitial damage. A combination of results from two independent experiments is shown.
Controls (n=5), nephritic wild-type (n=11), and nephritic Il-17c2/2 mice (n=15). (C) BUN, serum creatinine, and ACR determined in the
aforementioned groups. (D) Quantification of tubulointerstitial F4/80+, glomerular MAC-2+ and CD3+, and tubulointerstitial CD3+ cells
in the aforementioned groups. (E) Representative photographs of GR-1–stained kidney sections from nephritic wild-type and nephritic
Il-17c2/2 mice. Original magnification, 3400. (F) Quantification of tubulointerstitial GR-1+ cells in the aforementioned groups. Symbols
represent individual data points with the mean as a horizontal line (*P,0.05; **P,0.01; ***P,0.001).
J Am Soc Nephrol 29: 1210–1222, 2018
IL-17C in Renal Inflammation
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Figure 4. IL-17C-driven tissue injury in crescentic GN is IL-17A-dependent. (A) Representative FACS plots of intracellular cytokine
staining for IL-17A and IFN-g in renal T cells (pregated on singlets, live, CD45+, CD3+, and CD4+ cells). (B) Quantification of intracellular
cytokine FACS analysis for IL-17A, IL-17F, and IFN-g in renal CD4+ T cells. Data are representative of two independent experiments
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reduced in nephritic Il-17c2/2 mice (Figure 3D). Staining of
tubulointerstitial GR-1+ neutrophils showed a significant reduction in these cells in nephritic Il-17c2/2 mice (Figure 3, E and F).
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Amelioration of the GN Disease Course in Il-17c2/2
Mice Is a Consequence of a Reduced TH17/IL-17A
Response
To elucidate the immunologic mechanisms that lead to a less
severe course of GN in Il-17c2/2 mice, we investigated the
renal and systemic immune responses in these animals in
more detail. Flow cytometry studies revealed a significant reduction in renal TH17 cells, whereas the TH1 cell response in
the kidney was unaffected (Figure 4, A and B). To determine
whether IL-17C might also modulate systemic immunity in
the NTN model, we measured the serum concentration of
several cytokines, including IL-17C. The concentration of
IL-17C in the serum of nephritic mice was increased at day
10 and, as expected, was not detectable in nephritic Il-17c2/2
mice (Figure 4C). The analysis of serum IL-17A levels
revealed a significant reduction in nephritic Il-17c2/2 mice
compared with nephritic wild-type mice. Other effector cytokines related to TH17 immune responses did not show significant differences. The IFN-g serum level was slightly, but significantly, increased in Il-17c2/2 mice.
IL-17C did not have major effects on the humoral immune
response because nephritic wild-type and nephritic Il-17c2/2
mice had similar serum titers of anti-sheep total IgG, IgG1,
IgG2a/c, IgG2b, and IgG3 antibodies directed against the
nephritogenic antigen (Supplemental Figure 2, A and B).
To further investigate the influence of IL-17C on the local
immune/inflammatory response in the kidney, expression analyses of the renal cortex using a cytokine and chemokine pathwayfocused PCR array (RT2 Profiler) were performed. These analyses
revealed a predominant downregulation of TH17/IL-17A–specific
target genes in nephritic Il-17c2/2 mice (Figure 4D). Accordingly,
qPCR analysis confirmed significantly reduced Cxcl2 and Cxcl5
mRNA expression in nephritic Il-17c2/2 mice. Likewise, we
found a reduction in the neutrophil-attracting chemokine Cxcl1
as well as Ccl20, which interacts with the chemokine receptor
CCR6 expressed on TH17 cells (Figure 4E). Moreover, IL-17C
deficiency resulted in a reduction in the renal expression of IL1b and IL-6, whereas IL-23 expression was not affected (Figure
4E). This suggests that the downregulated TH17/IL-17A immune
BASIC RESEARCH
response in nephritic Il-17c2/2 mice might be responsible for the
less severe crescentic GN. To test this hypothesis, we treated nephritic wild-type and nephritic Il-17c2/2 mice with a neutralizing
anti–IL-17A antibody. IL-17A neutralization diminished the differences in kidney damage between the wild-type and knockout
groups (Figure 4F). In line with these results, we did not observe a
difference in the functional parameters of BUN, serum creatinine,
or ACR (Figure 4G). Taken together, these results strongly indicate
that the ameliorated GN disease course in Il-17c2/2 mice is a
consequence of a reduced TH17/IL-17A response.
IL-17C Is Produced by Resident Tissue Cells
To study whether IL-17C in the kidney is mainly produced by
resident renal tissue cells or by hematopoietic cells, we performed
bone marrow (BM) transplantation studies using wild-type and
Il-17c2/2 mice. Twenty-four hours after induction of nephritis
(the time point of highest renal Il-17c mRNA expression), we
obtained high Il-17c expression levels in animals with wild-type
renal tissue cells. By contrast, Il-17c mRNA expression in Il17c2/2 mice receiving wild-type BM or Il-17c2/2 BM was not
elevated (Supplemental Figure 3). These data indicate that Il17c is mainly expressed by resident kidney cells.
IL-17C Does Not Induce Chemokine Expression in
Resident Kidney Cells
In order to investigate whether IL-17C has direct effects on
resident kidney cells, murine proximal tubular and murine
mesangial cells were cultured and stimulated with IL-17C
(1–100 ng/ml), or TNF-a and IL-17A as positive control.
RT-PCR analysis revealed the mRNA expression of IL-17RA
and IL-17RE in both cell lines (data not shown). Stimulation
with IL-17C alone had no effect on Cxcl1 and Cxcl5 mRNA
expression and protein formation (Supplemental Figure 4, A–
D), but a moderate, synergistic effect was observed after stimulation together with a IL-22 or TNF-a (data not shown).
Taken together, these results argue against major effects of
IL-17C on resident kidney cells, which is in contrast to the
pathophysiologic concept of IL-17A and IL-17F.
IL-17RE Deficiency Ameliorates Renal Injury in
Crescentic GN
To analyze whether deficiency in IL-17RE, the specific receptor of
IL-17C, also protects against immune-mediated glomerular
with nephritic wild-type (WT; n=6) and nephritic Il-17c2/2 mice (n=10). (C) Serum cytokine levels measured by electrochemiluminescence
immunoassay analysis. Data are representative of two independent experiments with controls (Con; n=3), nephritic wild-type (n=7), and
nephritic Il-17c2/2 mice (n=11). (D) Cytokine and chemokine expression profiles of nephritic Il-17c2/2 and nephritic wild-type control
mice. The expression levels are normalized to the housekeeping gene b-actin (Actb). (E) qPCR analysis of renal Cxcl1, Cxcl2, Cxcl5, and
Cxcl9, as well as Ccl5 and Ccl20 mRNA expression. A combination of results from two independent experiments is shown, from controls
(n=5), nephritic wild-type (n=11), and nephritic Il-17c2/2 mice (n=15). mRNA levels are expressed as x-fold of controls. (F) Quantification of
glomerular crescent formation and tubulointerstitial damage of controls (n=5), nephritic wild-type (n=8), and nephritic Il-17c2/2 mice
(n=6) treated with a neutralizing anti–IL-17A antibody (400 mg administered intraperitoneally at days 2, 4, and 8 after NTN induction). (G)
Levels of BUN and serum creatinine, and ACR determined in the aforementioned groups. Data are presented as bar graphs with the
mean6SEM or symbols, which represent individual data points with the mean as a horizontal line (*P,0.05; **P,0.01).
J Am Soc Nephrol 29: 1210–1222, 2018
IL-17C in Renal Inflammation
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significantly reduced in nephritic Il-17re2/2 mice (Figure 5,
E and F).
Control
IL-17RE Is Required for the Promotion of Renal TH17
Cell Responses
In line with the observed immunologic phenotype in Il-17c2/2
mice, IL-17RE deficiency resulted in a significant reduction in
the renal TH17 cell response in GN, whereas no effect was seen
on the TH1 cell population (Figure 6, A and B). Analyses of the
mRNA expression profiles of whole renal cortices revealed
that the absence of IL-17RE had a predominant effect on
TH17/IL-17A target genes because the neutrophil-attracting
chemokines Cxcl1 and Cxcl5 showed a decreased upregulation
in nephritic Il-17re2/2 mice compared with their nephritic
wild-type counterparts (Figure 6C). In contrast, TH1 target
Il17-re-/- NTN
WT NTN
PAS
A
C
40
20
0
40
20
0
Con
NTN
*
150
BUN [mg/dL]
Interstitial Area [%]
Crescents [%]
60
***
60
100
50
0
Con
NTN
Con
*
0.8
500
400
0.6
ACR [g/g]
***
80
Serum Creatinine [mg/dL]
B
0.4
0.2
0.0
NTN
300
200
100
Con
0
NTN
Con
NTN
D
*
10
0
6
4
2
0
Con
NTN
WT NTN
3
2
1
40
20
0
Con
NTN
0
Con
WT
E
60
CD3+ cells / hpf
20
4
CD3+ cells / gcs
8
MAC-2+ cells / gcs
F4/80+ cells / hpf
30
Il-17re-/- NTN
NTN
Con
NTN
Il-17re-/-
F
*
GR1+ cells / lpf
15
GR1+
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disease, we compared the clinical course of experimental GN
between wild-type mice and Il-17re2/2 mice. The evaluation of
kidney sections stained with periodic acid–Schiff (PAS) for the
presence of crescents and tubulointerstitial injury revealed a significant reduction in the destruction of regular tissue structures
in nephritic Il-17re2/2 mice (Figure 5, A and B). BUN and serum
creatinine levels of nephritic Il-17re2/2 mice were significantly
reduced as compared with nephritic wild-type mice (Figure 5C),
but no effect on ACR was detectable (Figure 5C).
The migration of F4/80+ macrophages and CD3+ T cells
into the kidney revealed no difference among nephritic wildtype and nephritic Il-17re2/2 mice (Figure 5D), whereas the
number of glomerular Mac-2+ mononuclear phagocytes was
increased in nephritic Il-17re2/2 mice (Figure 5D). Most importantly, the tubulointerstitial GR-1+ cell infiltration was
10
5
0
Con
NTN
Figure 5. IL-17RE–mediated signaling aggravates crescentic GN. (A) Representative photographs of PAS-stained kidney sections from
control (Con), nephritic wild-type (WT), and nephritic Il-17re2/2 mice at day 9 of NTN (original magnification, 3400). (B) Quantification
of glomerular crescent formation and tubulointerstitial damage. Data are representative of two independent experiments. Controls
(n=2), nephritic wild-type (n=6), and nephritic Il-17re2/2 mice (n=5). (C) BUN, serum creatinine, and ACR were determined in the
aforementioned groups. (D) Quantification of tubulointerstitial F4/80+, glomerular MAC-2+, glomerular CD3+, and tubulointerstitial
CD3+ cells in the aforementioned groups. (E) Representative photographs of GR-1–stained kidney sections from nephritic wild-type
and nephritic Il-17re2/2 mice. Original magnification, 3400. (F) Quantification of tubulointerstitial GR-1+ cells in the aforementioned
groups. Symbols represent individual data points with the mean as a horizontal line (*P,0.05; ***P,0.001).
1216
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WT NTN
IL-17A
Il-17re-/- NTN
IL-17RE Is Expressed by CD4+ TH17
Cells and Is Instrumental for TH17
Responses and Subsequent Tissue
Injury in Crescentic GN
IFN-γ
*
20
15
10
5
0
8
6
4
2
0
NTN
Cxcl1
**
mRNAexpression
[x-fold of control]
500
50
40
30
20
10
0
NTN
WT
C
% IFN-γ+ of CD4+ cells
25
% IL-17F+ of CD4+ cells
% IL-17A+ of CD4+ cells
B
400
NTN
Il-17re-/Cxcl5
Cxcl2
**
800
10000
8000
600
300
6000
400
200
4000
200
100
0
2000
0
Con
NTN
0
Con
Ccl20
Ccl5
NTN
Cxcl9
8
8
1500
Con
NTN
10
2000
mRNAexpression
[x-fold of control]
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genes such as Ccl5 and Cxcl9 were not affected in nephritic Il-17re2/2 mice. The
serum titers of anti-sheep total IgG, IgG1,
IgG2a/c, IgG2b, and IgG3 antibodies of nephritic wild-type and nephritic Il-17re2/2
mice showed no effect of IL-17RE–mediated
signaling on the humoral immune
response (Supplemental Figure 2, C and D).
Kidney
A
BASIC RESEARCH
6
6
4
1000
4
500
2
2
0
0
Con
NTN
0
Con
WT
NTN
Con
NTN
Il-17re-/-
Figure 6. IL-17RE promotes renal TH17 cell responses in crescentic GN. (A) Representative FACS plots of intracellular cytokine staining for IL-17A and IFN-g in renal T
cells (pregated on singlets, live, CD45+, CD3+, and CD4+ cells). (B) Quantification of
intracellular cytokine FACS analysis for IL-17A, IL-17F, and IFN-g in renal CD4+ T cells.
Nephritic wild-type (WT; n=6) and nephritic Il-17re2/2 mice (n=5). (C) Renal qPCR
J Am Soc Nephrol 29: 1210–1222, 2018
To investigate the cellular expression pattern of IL-17RE, NK1.1 + NK cells, CD11b
+
/CD11c + /MHC2 + monocytes/macrophages, CD19+ B cells, and CD3+/CD4+
T cells from the kidneys of control and
nephritic mice were sorted by FACS. Subsequent RT-PCR analysis revealed that Il17re expression is primarily upregulated
by CD4 + T cells (Figure 7A). Using IL17A YFP+ fate reporter mice,23 we isolated CD4+ TH17 cells from the kidney and,
by qPCR, compared their Il-17re mRNA
expression with CD4 + T H 17 2 cells. As
shown in Figure 7B, Il-17re expression
was increased in the T H 17 cell population. Finally, we polarized CD4+ T cells
in vitro in TH1, TH17, regulatory T cells,
and T H 0 cells and assessed the Il-17re
expression. In line with the other experiments, TH17 cells demonstrated the highest expression level of the receptor
(Figure 7C). Because of the lack of suitable IL-17RE antibodies, it is currently
not feasible to confirm these data by
flow cytometry.
To study the potential function of IL17RE in the TH17 response, we isolated
CD4+ T cells from the spleen of CD45.1
wild-type and CD45.2 Il-17re 2 /2 mice
and transferred them into Rag12/2 mice
(competitive adoptive transfer, 1:1 ratio),
mRNA expression analysis of Cxcl1, Cxcl2,
Cxcl5, and Cxcl9, as well as Ccl5 and Ccl20.
mRNA levels are expressed as x-fold of controls (Con). Data are representative of two independent experiments with controls (n=2),
nephritic wild-type (n=6), and nephritic Il17re2/2 mice (n=5). Data are presented as
individual data points with the mean as a
horizontal line (*P,0.05; **P,0.01).
IL-17C in Renal Inflammation
1217
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B
+
Mo/MФ (CD11b )
B cells (CD19 )
T cells (CD3+)
0.0
0.5
1.0
1.5
2.0
50
10
5
0
2.5
% of IL-17A+ CD4+ cells
CD45.1 (WT)
CD4
100
TH0
TH1
TH17 Treg
**
WT
Il-17re-/-
60
40
20
0
Rag1-/- NTN
+ Il-17re-/- CD4+ cells
F
**
80
PAS
Crescents [%]
Rag1 Control
10
NTN
Rag1-/- NTN
+ WT CD4+ cells
-/-
20
80
CD45.2 (KO)
E
30
0
D
IL-17A
40
IL-17A- IL-17A+
YFP- YFP+
Il-17re mRNA expression
[x-fold of non-nephritic group]
60
40
20
***
80
0
60
40
20
0
Con
NTN
Con
NTN
G
**
10
5
0
H
100
50
0
Con
NTN
80
150
60
100
40
50
20
NTN
1.0
0.5
Con
NTN
Cxcl5
**
150
100
Ccl20
Cxcl9
ns
400
1000
300
800
600
200
400
50
0
NTN
Rag1-/Rag1-/- + WT CD4+ cells
Rag1-/- + Il-17re-/- CD4+ T cells
1.5
Cxcl2
200
Con
2.0
0.0
Con
Cxcl1
0
2.5
MAC2+ cells / gcs
15
**
150
F4/80+ cells / hpf
GR1+ cells / lpf
20
mRNA expression
[x-fold of control]
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+
15
mRNA expression
[x-fold of TH0]
NK cells (NK1.1+)
Il-17re
C
Il-17re
mRNA expression
[x-fold of IL-17A- YFP-]
A
Interstitial Area [%]
BASIC RESEARCH
100
0
Con
NTN
200
0
Con
NTN
0
Con
NTN
Con
NTN
Figure 7. IL-17RE is highly expressed by CD4+ T cells and promotes renal tissue injury in crescentic GN. (A) Regulation of Il-17re mRNA
expression on selected FACS sorted renal leukocyte subsets under nephritic conditions (n=4). (B) Il-17re mRNA expression profile of IL17A2, YFP2, and IL-17A+, YFP+ sorted renal CD4+ T cells of nephritic IL-17A YFP+ fate reporter mice. (C) Il-17re mRNA expression
profile of cells isolated from naïve mice and cultured under TH0-, TH1-, TH17-, or Treg-polarizing conditions for 65 hours (n=5). mRNA
levels are expressed as x-fold of controls. (D) Representative FACS plots and quantification of IL-17A+ cells from nephritic Rag12/2
mice repopulated with CD45.1 wild-type (WT) and CD45.2 Il-17re2/2 CD4+ T cells. (n=7). (E) Representative photographs of PASstained kidney sections of Rag12/2 controls (Con; n=3), nephritic Rag12/2 mice repopulated with 1.53106 wild-type CD4+ T cells
(n=6), and nephritic Rag12/2 mice repopulated with 1.53106 Il-17re2/2 CD4+ T cells (n=8) at day 10 of NTN (original magnification,
3400). (F) Quantification of glomerular crescent formation and tubulointerstitial damage of the aforementioned groups.
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which are deficient in T cells and B cells. Subsequently, we
induced NTN and analyzed the “origin” of renal TH17 cells
at day 10, using the congenic markers CD45.1 and CD45.2. As
shown in Figure 7D, the deficiency in IL-17RE on CD4+ T cells
led to a significantly reduced renal TH17 cell response compared with the wild-type group.
In addition, we performed adoptive transfer experiments
to investigate the effect of IL-17RE–expressing CD4+ T cells
on the clinical course of crescentic GN. Therefore, CD4+
T cells from the spleen of wild-type and Il-17re2/2 mice
were isolated and separately transferred into Rag12/2 mice.
Six days after the adoptive cell transfer, NTN was induced.
Rag12/2 mice reconstituted with Il-17re2/2 CD4+ T cells
developed a less severe course of the GN with respect to glomerular crescent formation and tubulointerstitial injury
(Figure 7, E and F). In agreement with the reduced TH17
response, the recruitment of neutrophils into the kidney
was decreased (Figure 7G), as was the expression of Cxcl5
(Figure 7H).
IL-17C Plays a Role in a Chronic Model of Lupus
Nephritis
We then assessed the effect of IL-17C deficiency in the
chronic model of pristane-induced lupus nephritis. The application of pristane resulted in early mortality because of the
development of severe pulmonary vasculitis,24 which was
comparable between wild-type and Il-17c2/2 mice (Supplemental Figure 5A). Twelve months after pristane injection
(the point of time when lupus nephritis was established),
renal analysis was performed. PAS staining of renal tissue
sections showed glomerular alterations, including hypercellularity, segmental proliferation, hyalinosis, and capillary
wall thickening, as well as an increase of glomerular area
as a sign of immune-mediated kidney damage (Supplemental Figure 5, B and C). A comparison of pristane-treated wildtype and Il-17c2/2 mice revealed that the percentage of abnormal
glomeruli tended to be reduced in Il-17c2/2 mice (Supplemental
Figure 5C), and the glomerular area of pristane-treated Il-17c2/2
mice was significantly decreased compared with pristane-treated
wild-type mice (Supplemental Figure 5C). Moreover, BUN levels
were significantly reduced in Il-17c2/2 lupus mice (Supplemental
Figure 5D).
Humoral immunity and immunologic reactivity were
determined by analyzing glomerular IgG deposition and
anti-dsDNA and anti–U1-snRNP autoantibody production, respectively. No difference was observed between
wild-type and Il-17c 2/2 lupus mice (Supplemental Figure
5, E–G).
BASIC RESEARCH
DISCUSSION
Here, we demonstrate that the IL-17C/IL-17RE axis significantly contributes to renal tissue injury in a murine model of
crescentic GN. Our findings indicate that IL-17C promotes
TH17 cell responses via IL-17RE, expressed on CD4+ TH17 cells.
This results in increased renal expression of IL-17 target genes,
such as the chemokines CXCL1 and CXCL5, and further recruitment of neutrophils, ultimately leading to tissue injury.
In recent years IL-17A and, to lesser degree, IL-17F and IL17RA have been identified as important proinflammatory regulators and potential therapeutic targets in autoimmune and
inflammatory kidney diseases, including human and experimental crescentic GN.6–8,25–28 However, the potential function
of other IL-17 cytokines and IL-17 receptors is still unclear. We
measured serum protein levels of the IL-17 cytokine family
members in 70 patients with ANCA-associated GN, the most
common form of crescentic GN. Surprisingly, we found no
significant differences in IL-17A, E, and F levels in the ANCA
GN patient group compared with healthy controls, but a
highly significant increased level of IL-17C. An upregulated
expression of IL-17C was also detectable in a model of crescentic GN (NTN), suggesting a potential role of this cytokine
in crescentic GN. We were not able to detect significant serum
levels of IL-17B and D.
The biologic role of IL-17C is not well characterized and its
function in renal autoimmune and inflammatory kidney diseases
is unknown. IL-17C was cloned in 2000 by large-scale screen of
expressed sequence tag databases for proteins homologous to IL17A.29 Six years later, the specific receptor subunit IL-17RE was
identified by a sequence-based homology search.30 The observation that IL-17C binds to IL-17RE was made in 2011.19,20
During the past decade, the interaction of IL-17C and IL17RE has been reported to have proinflammatory effects by
stimulating innate immune responses on epithelial cells in response to bacterial or sterile inflammatory stimuli.31,32 Toll-like
receptor signaling has been identified as one pathway to upregulate IL-17C in these disease entities.19 IL-17C then drives innate immune responses in an autocrine manner in epithelial
cells to fight off the infection. Yet little is known about the involvement of the IL-17C/IL-17RE axis in the pathogenesis of
autoimmune diseases. Recently, it was suggested that the axis
plays a role in autoimmune hepatitis by induction of IL-2 in CD4
+
T cells which, in turn, stimulate NK cells to cause hepatic
damage.33 Also, IL-17C had proinflammatory effects in an imiquimod-induced mouse model of autoimmune skin inflammation.19 Chang et al. suggested that IL-17RE is expressed on TH17
cells and that its signaling has an effect on the IL-17A production
(G) Quantification of tubulointerstitial GR-1+ cells, tubulointerstitial F4/80+ cells, and glomerular MAC-2+ cells in the aforementioned
groups. (H) Renal qPCR mRNA expression analysis of Cxcl1, Cxcl2, Cxcl5, and Cxcl9, as well as Ccl20. mRNA levels are expressed as x-fold
of controls. Data are presented as bar graphs with the mean6SEM or symbols, which represent individual data points with the mean as a
horizontal line (**P,0.01; ***P,0.001).
J Am Soc Nephrol 29: 1210–1222, 2018
IL-17C in Renal Inflammation
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of cultured TH17 cells overexpressing the receptor subunit.20
They also report that IL-17C is implicated in the pathogenesis
of experimental autoimmune encephalomyelitis. Moreover, IL17C serum levels were increased in patients with active inflammatory bowel diseases.34
We studied the function of IL-17C in a well established
model of crescentic GN (NTN). Compared with wild-type
mice, IL-17C–deficient mice developed less severe disease,
with significantly better renal function, and a reduced frequency of glomerular crescent formation and tubulointerstitial tissue injury. In line with this, the lack of IL-17RE (the
unique receptor for IL-17C) also resulted in an ameliorated
course of the GN in terms of renal function and tissue damage.
Additionally, studies in the chronic model of pristane-induced
lupus nephritis showed that IL-17C–deficient lupus mice
had a moderately improved course of the disease. In summary,
these results demonstrated for the first time the pathogenic
role of the IL-17C/IL-17RE axis in immune-mediated glomerular disease.
Mechanistically, we provide evidence that the IL-17C/IL17RE axis directly stimulates the nephritogenic TH17 response
and thereby promotes renal tissue injury in crescentic GN.
This hypothesis is on the basis of the finding that (1) renal
IL-17A–producing TH17 cells, the expression of IL-17 target
genes, and the infiltration of neutrophils were selectively
reduced in nephritic Il-17c2/2 and Il-17re2/2 mice; (2) application of a neutralizing IL-17A antibody diminished the
observed differences in kidney damage between nephritic
wild-type and Il-17c2/2 animals; (3) IL-17RE is highly expressed on renal CD4+ TH17 cells; and (4) the transfer of IL17RE–deficient CD4+ T cell into Rag12/2 mice resulted in a
reduced renal TH17 response and subsequent tissue injury in
crescentic GN.
Our attempts to study the IL-17C/IL-17RE–induced signaling pathways in TH17 cells that potentiate the TH17 cell response were hampered by the lack of suitable FACS antibodies
for IL-17RE and the lack of available IL-17RE reporter mice,
and remains to be fully elucidated. First experiments indicated
that IL-17C/IL-17RE did not influence the polarization of naïve
T cells toward TH17 cells in vitro (data not shown), suggesting
that other pathways such as TH17 cell maintenance, expansion,
or stabilization are involved in the process.
Our study highlighted major differences in the biologic
function of IL-17C compared with IL-17A and IL-17F. IL17C is predominantly expressed by resident cells in the kidney,
whereas in the spleen different cell populations, including
mononuclear phagocytes, expressed IL-17C mRNA. Further
studies are clearly needed to characterize in detail the cellular
source of IL-17C under inflammatory and homeostatic conditions. So far, we have not been able to establish reliable IL-17C
immunohistochemical staining in the kidney or spleen. In
contrast, IL-17A and F are derived from renal TH17 cells or
gd T cells in GN. Moreover, IL-17C has only weak effects on
resident kidney cells, in terms of proinflammatory chemokine
induction, but directly promotes systemic TH17 response via
1220
Journal of the American Society of Nephrology
IL-17RE signaling. This is different compared with IL-17A and
F, which are supposed to act locally on renal cells and might
explain why systemic levels of IL-17C reflect the local TH17
response in the kidney better than IL-17A or F. On the basis of
our finding that IL-17C stimulates the TH17 cell response upstream of IL-17A, targeting of the IL-17C/IL-17RE pathway
may present an intriguing therapeutic strategy for TH17driven autoimmune and inflammatory diseases, especially because neutralization of the TH17/IL-17A axis has already been
confirmed to be an effective concept of treating autoimmune
diseases such as psoriasis.35,36
CONCISE METHODS
Human Study Cohort
A total of 70 patients were recruited in this study (Hamburg
Glomerulonephritis Registry). The inclusion criterion was biopsyproven, ANCA-associated GN. The trial was performed in accordance
with the declaration of Helsinki. After informed consent was obtained,
patient data and renal biopsy samples were collected according to the
guidelines of the respective local ethics committees.
Animals
Il-17c2/2 mice were obtained from the Mutant Mouse Regional Resource Centers (University of California, Davis, CA). Il-17re2/2 mice
were kindly provided by Bristol-Myers Squibb Company (Princeton,
NJ). Rag12/2 mice were obtained from the Jackson Laboratory. All
mice were on the C57BL/6J background. Age-matched C57BL/6J
wild-type controls were bred in our facility. All mice were raised under specific pathogen-free conditions. All animal experiments were
performed according to national and institutional animal care and
ethical guidelines and were approved by the local authorities.
Induction of Experimental GN and Functional Studies
NTN was induced by intraperitoneal injection of 2.5 mg of nephrotoxic sheep serum per gram of body weight into 8–12-week-old male
mice.10 Lupus nephritis was induced by intraperitoneal injection of
0.5 ml pristane into 8–10-week-old male mice.37,38 Urine samples
were collected by housing the mice in metabolic cages for 3–5 hours.
Urinary albumin excretion was determined by standard ELISA technology (Mice-Albumin Kit; Bethyl), and urinary creatinine, BUN,
and serum creatinine levels were measured using standard laboratory
methods.
qPCR Analyses
Total RNA of the renal cortex and cell subsets were prepared according
to standard laboratory methods. qPCR analyses were performed for 40
cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems,
Foster City, CA). The samples were run in duplicate and normalized to
18S rRNA. The cytokine and chemokine expression profiles were
determined using the Mouse cytokines & chemokines RT2 Profiler
PCR Array (Qiagen, Hilden, Germany). The samples were normalized to the housekeeping gene b-actin (Actb). Data were analyzed
using the StepOne Software v2.0.
J Am Soc Nephrol 29: 1210–1222, 2018
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Electrochemiluminescence Immunoassay
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Mouse cytokine serum levels as well as serum samples from patient
visits to our clinic, thawed on ice, were analyzed using a customized
Meso Scale Discovery immunoassay (U-Plex Th17 Combo 2). This
technology includes biotinylated and conjugated SULFO-TAG detection antibodies to simultaneously measure different analytes in a one
microtiter well. Unprocessed serum from each patient (50 ml) was
thawed and analyzed for the cytokines according to the manufacturer’s protocol (Meso Scale Discovery, Rockville, MD). Electrochemiluminescence was detected using the QuickPlex SQ120
instrument. Analyses were performed with the MSD Discovery
Workbench software v4.0.
Morphologic Analyses
Immunohistochemistry was performed using routine laboratory methods.
Glomerular crescent formation and tubulointerstitial injury were assessed
in PAS-stained paraffin sections in a blinded fashion, as described.11
BASIC RESEARCH
Assessment of the Humoral and Cellular Nephritogenic
Immune Responses
Mouse anti-sheep IgG antibody titers were determined in sera by ELISA
as previously described.42 Anti–dsDNA-IgG and anti–U1-snRNP-IgG
antibodies were analyzed by ELISA as previously described.11
Flow Cytometry
Cells were stained with fluorochrome-labeled antibodies directed
against CD45, CD3, CD4, CD8, gdTCR, NK1.1, IL-17A, IL-17F, IL22, IFN-g, CD11b, and Ly6G (BioLegend, BD Biosciences, eBioscience, or R&D Systems) as previously described.39 Flow cytometry
measurements were performed using the BD FACS LSR II. Data were
analyzed by using the FlowJo software (Tree Star).
Neutralization Experiments
The animals were treated with 400 mg of a neutralizing mouse antimurine IL-17A antibody (clone MM17F3; BioCell). The antibody
was injected intraperitoneally on days 2, 4, and 8 after induction of
NTN, as described.22
Isolation of Leukocytes from Murine Kidney
The leukocytes from murine kidneys were isolated as previously described.39 Briefly, kidneys were digested for 45 minutes at 37°C by
adding 0.4 mg/ml collagenase D (Roche, Mannheim, Germany) and
0.01 mg/ml DNase I (Roche) to RPMI 1640 (Life Technologies, Karlsruhe, Germany) medium supplemented with 10% heat-inactivated
FCS (Gibco, Eggenstein, Germany). Subsequently, kidneys were
finely minced using the gentle MACS Dissociator (Miltenyi Biotec,
Teterow, Germany). Single-cell suspensions were separated using
Percoll density gradient centrifugation.
Culturing and Stimulation of Mouse Kidney Tubular
and Mesangial Cells
Mouse kidney tubular cells40 and mouse kidney mesangial cells41
were cultured in DMEM medium (Life Technologies) containing
3%–10% FCS (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technologies) at 37°C with 5% CO2. The cells were
incubated in serum-free DMEM medium 24 hours before stimulation. Cells were stimulated with IL-17C (10 ng/ml), TNF-a
(10 ng/ml), and IL-22 (100 ng/ml), or with a combination. Cells
were harvested after 4 hours of stimulation and stored at 280°C for
mRNA analyses. The protein levels in the supernatant were determined after 24 hours of incubation, using a specific ELISA according to the manufacturer’s protocol (R&D Systems, Wiesbaden,
Germany).
CD4+ T Cell Polarization
For polarization of naïve CD4+ cells, splenocytes were isolated from
naïve wild-type mice using the MACS CD4+ T Cell Isolation Kit,
mouse (Miltenyi Biotec). A total of 23105 cells/well was plated in
anti-CD3 antibody-coated 96-well plates (eBioscience, San Diego,
CA) and incubated for 65 hours with soluble anti-CD28. The following cytokines and neutralizing antibodies were used: for TH0 conditions, anti–IFN-g and IL-2; for TH1-polarizing conditions, anti–IL-4
and recombinant IL-12; for TH17-polarizing conditions, anti–IL-4,
anti–IFN-g, IL-6, TGF-b, IL-1b, and IL-23; and for Treg-polarizing
conditions, anti–IFN-g, IL-2, and TGF-b (all BioLegend).
J Am Soc Nephrol 29: 1210–1222, 2018
BM Transplantation
BM transplantation was performed as previously described.12 Briefly,
age-matched Il-17c2/2 mice and C57BL/6J wild-type mice received
9.5 Gy total body irradiation. Each recipient mouse (Il-17c2/2 mice
or wild-type) obtained 303106 BM cells (Il-17c2/2 or wild-type
cells) by intravenous injection. NTN was induced 2 weeks after irradiation and BM transplantation.
Cell Transfer in Rag12/2 Mice
CD4+ cells were isolated by magnetic-activated cell sorting with the Mouse
CD4+ Cell Isolation Kit II (Miltenyi Biotec). We intravenously injected
1.53106 cells into Rag12/2 mice. NTN was induced 6 days after cell transfer.
Statistical Analyses
The results are shown as the mean6SEM when presented as a bar
graph or as single data points with the mean in a scatter dot plot.
Differences between two individual experimental groups were compared using a two-tailed t test. For survival analysis the Kaplan–Meier
plot with a log-rank test was used. P,0.05 was considered to be
statistically significant.
ACKNOWLEDGMENTS
FACS sorting was performed at the Universitätsklinikum HamburgEppendorf FACS sorting core facility.
This study was supported by grants from the Deutsche Forschungsgemeinschaft(Sonderforschungsbereich1192: toU.P. andR.A.K.S.).
DISCLOSURES
None.
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IL-17C in Renal Inflammation
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This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2017090949/-/DCSupplemental.
J Am Soc Nephrol 29: 1210–1222, 2018