Lupus (2012) 21, 656–661

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CONCISE REPORT

Administration of fasudil, a ROCK inhibitor, attenuates disease in

lupus-prone NZB/W F1 female mice
RA Stirzaker1, PS Biswas1, S Gupta1, L Song1, G Bhagat2 and AB Pernis1
1
Autoimmunity and Inflammation Research Program, Hospital for Special Surgery, New York, USA; and 2Department of Pathology
and Cell Biology, Columbia University, New York, USA

Accumulating evidence from murine studies suggests that the RhoA/ROCK pathway plays an
important role in the development of autoimmune disorders. We previously demonstrated that
ROCK inhibition ameliorates disease in MRL/lpr mice, a spontaneous model of lupus. This
study aimed to explore the protective effects of the ROCK inhibitor fasudil in a distinct model
of lupus, NZB/W F1 female mice, to assess the broad applicability of ROCK inhibition for the
treatment of lupus. NZB/W F1 female mice were administered fasudil continuously in their
drinking water starting at 18 or 24 weeks of age up until 44 weeks of age, or remained
untreated. Fasudil treatment significantly improved survival and decreased proteinuria, par-
ticularly when treatment was started at 18 weeks. There was also a significant decrease in
serum anti-dsDNA autoantibody production, glomerular IgG and C3 deposition, and glo-
merulonephritis. Analysis of the splenic lymphocyte compartment revealed reduced effector/
memory CD4þ T cell and plasma cell numbers in fasudil treated mice while the frequency of
other B cell and T cell subsets was unchanged. These results thus indicate that fasudil can
ameliorate disease in NZB/W F1 female mice, suggesting that ROCK inhibition might be
broadly effective for the treatment of lupus. Lupus (2012) 21, 656–661.

Key words: Systemic lupus erythematosus; anti-DNA antibodies; nephritis

Introduction the pathogenesis of autoimmunity via its capacity

to amplify the differentiation of Th17 cells and, par-
Systemic lupus erythematosus (SLE) is a prototypi- ticularly, to drive T-dependent humoral responses.6
cal systemic autoimmune disorder characterized Production of IL-17 and IL-21 is absolutely
by hypergammaglobulinemia, autoantibody pro- dependent on the transcription factor Interferon
duction and multi-organ involvement.1-3 Aberrant Regulatory Factor 4 (IRF4).7-9 In addition to the
T and B cell function is known to play a crucial induction of IL-17 and IL-21, IRF4 is also a critical
role in disease development and progression by driv- regulator of B cell function. In particular, IRF4
ing autoantibody production, a hallmark of SLE. controls Ig isotype switching and the differentiation
Recently, human and murine studies have provided of mature B cells into antibody-secreting plasma
evidence for a role for interleukin-17 (IL-17) and cells.10 Consistent with a critical role of IRF4 in
IL-21, two key cytokines produced by T helper 17 these processes, a recent study has demonstrated
(Th17) cells, in the pathogenesis of SLE and that deficiency of IRF4 can ameliorate lupus
other autoimmune disorders.4,5 While IL-17 has nephritis in a murine model, and that this effect
pro-inflammatory functions during host defense was accompanied by decreased production of
against extracellular pathogens, it can mediate IL-17 and IL-21 and marked reductions in Ig
inflammation and excessive tissue damage via its levels and autoantibodies.11 We have previously
ability to promote the secretion of cytokines and demonstrated that the activity of IRF4 can be con-
chemokines. IL-21 is also a critical contributor to trolled by the serine-threonine kinase ROCK2,
which phosphorylates IRF4, thereby promoting
IRF4 binding to the IL-17 and IL-21 promoters.12
Correspondence to: Alessandra Pernis, Caspary Research Building, Importantly, we have found that treatment of
Rm 318, 535 East 70th Street, New York, NY 10021, USA
Email: pernisa@hss.edu
MRL/lpr mice with the ROCK inhibitor fasudil
Received 30 September 2011; accepted 30 December 2011 reduced IL-17 and IL-21 production and
! The Author(s), 2012. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav 10.1177/0961203312436862

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ameliorated autoimmune symptoms, including anti-
dsDNA antibody production, immune-complex
deposition and proteinuria.12
Given the heterogeneity of SLE and the fact that
no single murine model emulates the complexity of
human lupus, particularly since multiple genes and
mechanistic pathways can trigger lupus-like
phenotypes, we investigated whether ROCK inhi-
bition could exert protective effects in another well-
established murine model of SLE, the NZB/W F1
mouse. We demonstrate that administration of the
ROCK inhibitor fasudil significantly prolongs sur-
vival and ameliorates SLE disease in NZB/W F1
female mice. This effect was associated with a sig-
nificant reduction in serum anti-dsDNA autoanti-
body titers. Analysis of the splenic lymphocyte
compartment revealed reduced effector/memory
CD4þ T cell and plasma cell numbers in fasudil-
treated mice, while the frequency of other B cell
and T cell subsets was unchanged between
groups. These studies thus suggest that ROCK
inhibition could be broadly effective for the treat-
ment of lupus.
Materials and methods
Mice
NZB/W F1 mice were obtained from the Jackson
Laboratory and were maintained under specific
pathogen-free conditions. The experimental proto-
cols were approved by the Institutional Animal
Care and Use Committee of Columbia University
and of the Hospital for Special Surgery.
In vivo fasudil studies
NZB/W F1 female mice were assigned to one of
three groups: 1) untreated controls, 2) fasudil
treatment commencing at 18 weeks of age and 3)
fasudil treatment commencing at 24 weeks of age
(n ¼ 10 per group). Mice in the three different
groups had similar serum anti-dsDNA antibody
titers. Mice were administered 100 mg/kg fasudil,
monohydrochloride salt (LC Laboratories) contin-
uously in their drinking water, and were monitored
regularly during the course of the study for urine
proteinuria (Chemstrip 2 GP, Roche), weight gain/
loss and water intake. Surviving mice were killed at
44 weeks of age, and blood, urine, kidney and
spleen specimens were collected at the time of sac-
rifice. Proteinuria was assessed semi-quantitatively
by using albumin reagent strips (Chemstrip 2 GP,
Roche). Grades of proteinuria were expressed as 0
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Administration of fasudil, a ROCK inhibitor
RA Stirzaker et al.
657
(none), trace (0–30 mg/dl), mild (30–100 mg/dl),
moderate (100–500 mg/dl) and severe (500 or
more mg/dl).
Histological and immunofluorescence analysis of
kidneys and spleens
Kidney and spleen samples were fixed in 10%
formalin and embedded in paraffin and the various
histopathologic alterations in different compart-
ments of the kidney, including the severity of
inflammation, were graded in a semi-quantitative
manner using hematoxylin and eosin (H and E)
stained sections (3 mm thickness). The severity of
glomerulonephritis (GN) was graded in a four
tiered fashion, normal, mild, moderate, or severe
(score 0–3), by assessing the size of glomeruli, glo-
merular cellularity (mesangial, capillary and pres-
ence of crescents), immune complex deposition
associated changes (capillary loop/glomerular base-
ment membrane thickening, mesangial widening/
nodularity) and extent of glomerulosclerosis.
A score of 3 (severe GN) was assigned to cases
that displayed cellular crescents, even in the pres-
ence of other less severe glomerular changes. The
severity of interstitial and perivascular chronic
inflammation (lymphocytes and plasma cells) was
also graded from normal to severe (score 0–3) in a
semiquantitative manner. Presence of vasculitis
led to the assignment of a score of 1 (and absence,
a score of 0). The severity of GN was tabulated
separately and scores of each component were
also added to generate a global ‘injury score’. For
immunofluorescence, kidneys were snap-frozen in
OCT compound and 6 -mm-thick sections stained
with FITC-conjugated goat anti-mouse IgG
(Jackson ImmunoResearch) or FITC-conjugated
goat anti-mouse complement C3 (MP Cappel).
Fluorescence intensity, representing IgG and C3
deposition, was measured as the mean fluorescence
in 10–15 glomeruli per mouse (Adobe Photoshop
CS4, Adobe).
Flow cytometry
Single cell suspensions from spleens were obtained
and stained with different fluorophore-conjugated
antibodies as previously described,12 and immuno-
fluorescence was measured using a BD LSR II
Flow Cytometer and data analyzed with FlowJo
software (Tree Star).
Serological assays
Anti-dsDNA IgG autoantibodies were measured
by ELISA (Alpha Diagnostic International).
Lupus
 Administration of fasudil, a ROCK inhibitor
RA Stirzaker et al.
658

Statistical analysis with 80% of untreated mice (Figure 1(b)).

Data was analyzed using GraphPad Prism
(GraphPad Prism Software, Inc.) and results were
expressed as the mean  SD. Survival and protein-
uria curves were generated using the Kaplan–Meier
method and statistical relevance determined using
the log-rank (Mantel–Cox) test, and other data
analyzed using one-way analysis of variance fol-
lowed by Dunnett’s multiple comparison test.
p values < 0.05 were considered to be significant.
Results
Administration of fasudil to female NZB/W F1 mice
alleviates lupus-like disease
Since fasudil was effective in ameliorating disease in
MRL/lpr mice,12 we investigated whether fasudil
could also provide a protective effect against the
development of lupus in female NZB/W F1 mice.
Fasudil (100 mg/kg) was administered orally to
female NZB/W F1 mice starting at either 18 or 24
weeks of age, up to 44 weeks of age. Consistent
with previous reports, fasudil treatment was well
tolerated.12-14 Administration of fasudil resulted
in a marked decrease in overall mortality in
female NZB/W F1 mice (Figure 1(a)). While only
20% of untreated NZB/W F1 mice survived to 44
weeks of age, 70% of fasudil treated mice survived
to that age regardless of whether treatment was ini-
tiated at 18 weeks or 24 weeks of age. Consistent
with these findings only 30% of fasudil-treated
NZB/W F1 mice in the 18-week treatment
group developed severe proteinuria, compared
Although improvements in the development of pro-
teinuria were also observed when fasudil was admin-
istered starting at 24 weeks of age, these results did
not reach statistical significance (p ¼ 0.0588). Thus,
fasudil can be effective in ameliorating the develop-
ment of proteinuria and prolonging survival in
NZB/W F1 female mice, particularly when treat-
ment is initiated during the earlier stages of disease.
Effects of fasudil on autoantibody production,
immune-complex deposition and glomerulonephritis
SLE is characterized by autoantibody production
including antibodies to dsDNA, leading to immune
complex formation and deposition in multiple
organs particularly in the kidney, which
subsequently drives a local inflammatory response
that can lead to tissue damage and clinical dis-
ease.1–3 Fasudil treatment significantly lowered
anti-dsDNA IgG autoantibody titers in both the
18 and 24 weeks treatment groups (Figure 2(a)).
Consistent with the improved survival and reduced
proteinuria, furthermore, fasudil treatment resulted
in reduced renal IgG and C3 deposition as assessed
by quantitation of IgG and C3 staining intensity in the
glomeruli of mice in the different treatment groups
(Figure 2(b) to (e)). Histopathological analysis sup-
ported the idea that fasudil treatment lessened the
severity of glomerulonephritis (Figure 2(f), (g)).
Effects of fasudil treatment on immune cell subsets
To gain further insights into the effects of fasudil on
the immune abnormalities underlying development
of lupus in NZB/W F1 mice, flow cytometry

Figure 1 Fasudil treatment reduces mortality and protects against the development of lupus nephritis. NZB/W F1 female mice
were left untreated (No TX) or were administered fasudil (100 mg/kg) daily in their drinking water commencing at 18 (18w) or 24
(24w) weeks of age (n ¼ 10 per group). Arrows on the X-axis denote the 18 and 24 week time points. Mice were followed for up to
26 weeks and the development of nephritis was monitored by regular screening for the presence of urine proteinuria. Kaplan–Meier
plots depict incidence of survival (a) and severe proteinuria (  500 mg/dl) (b).

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 Administration of fasudil, a ROCK inhibitor
RA Stirzaker et al.
659

(a) (b) (d)

90 *** 60

dsDNA IgG OD450

4 ***
*** *** ***
**

IgG (MFI)

C3 (MFI)
3 60 40
2
30 20
1
0 0 0
NoTx 18w 24w NoTx 18w 24w NoTx 18w 24w

(c) (e)

No Tx No Tx

18w 24w 18w 24w

(f) * (g)
4 ns NoTx
GN (score)

1
18w 24w
0
NoTx 18w 24w

Figure 2 Fasudil-treated NZB/W F1 mice exhibit decreased kidney pathology. (a) Levels of serum IgG antibodies specific for
double stranded DNA were determined by ELISA. Immunofluorescent staining and quantitation of kidney IgG ((b), (c)) and C3
((d), (e)) demonstrating reduced deposition in the kidneys of fasudil-treated mice. Representative IgG (c) and C3 (e) staining of
kidney sections from mice scored in (b) and (d), respectively. Untreated (top panels) and fasudil-treated (bottom panels) (  100
magnification). Mean fluorescence intensity (MFI) of individual glomeruli were quantitatively evaluated as described in Materials
and methods ((b), (d)). (f) Hematoxylin and eosin-stained kidney sections from untreated and fasudil-treated NZB/W F1 mice were
examined by light microscopy, and semi-quantitative grading of kidney mesangioproliferative glomerulonephritis (GN) was per-
formed in a blinded fashion using a four-tiered scale. (g) Representative histological analysis of kidneys from mice scored in (f)
demonstrating reduced severity of GN in mice administered fasudil. Untreated (top) and fasudil treated (bottom) mice (  200
magnification). No Tx: no treatment; 18w: fasudil treatment commencing at 18 weeks; 24w: fasudil treatment commencing at 24
weeks; ns: not significant. *p < 0.05, **p < 0.01, ***p < 0.0001.

analysis was conducted on surviving mice at 44 Consistent with the decrease in autoantibody pro-
weeks of age. Fasudil treatment did not affect duction, NZB/W F1 mice administered fasudil
splenomegaly, the frequency of total splenic exhibited a striking decrease in the number of
CD4þ T cells, or the numbers of splenic regulatory B220loCD138þ plasma cells (Figure 3(i)). Taken
T cells (CD4þCD25þFoxp3þ) (Figure 3(a) to (c)). all together these results suggest that fasudil treat-
There was, however, a reduction in the frequency of ment markedly diminishes the aberrant plasma cell
effector/memory CD4þ T cells in both 18 and 24 accumulation that characterizes NZB/W F1 mice.
weeks-treated mice as assessed by the expression of
CD44 and CD62L (Figure 3(d)). No alterations in
the frequency of B220þ B cells, marginal zone B Discussion
cells (B220þCD21hiCD23lo), follicular B cells
(B220þCD21loCD23hi) or germinal center B cells Previous studies from our lab have demonstrated
(B220þPNAþ) were observed (Figure 3(e) to (h)). that in vivo administration of the ROCK inhibitor
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 Administration of fasudil, a ROCK inhibitor
RA Stirzaker et al.
660

(a) (b) (c)

Total Tregs (x106)

Total CD4+ (x106)
Total splenocytes
160 40 6
120 30
4

(x106)
80 20
2
40 10
0 0 0
No Tx 18w 24w No Tx 18w 24w No Tx 18w 24w

(d)
***
Total activated CD4 +

30 ** NoTx 18w 24w

20
(x106)

10

CD44
0
NoTx 18w 24w CD62L

(e) (f) (g) (h)

Total follicular (x106)

Total B220 + (x106)

80 15 30 2.5

Total GC (x106)
Total MZ (x106)

60 20
10 1.5
40
5 10
20 0.5
0 0 0 0
NoTx 18w 24w NoTx 18w 24w NoTx 18w 24w NoTx 18w 24w

(i)
6 ***
106)

** NoTx 18w 24w

Total PCs (x

2
CD138

0
NoTx 18w 24w B220

Figure 3 Reduced effector/memory CD4þ T cell and plasma cell populations in the spleens of fasudil-treated mice. Total spleen
counts were performed and the frequency of various immune cell subsets were determined by flow cytometry. Administration of
fasudil did not alter splenomegaly in NZB/W F1 mice (a), nor did it change the frequency of total CD4þ T cells (b) or
CD4þCD25þFoxp3þ regulatory T cells (Tregs) (c). Fasudil treatment decreased the frequency of CD62LhiCD44lo effector/
memory CD4þ T cells (d). NZB/W F1 mice that received fasudil exhibited no changes in total B220þ B cells (e),
B220þCD21hiCD23lo/- marginal zone (MZ) B cells (f), B220þCD21intCD23hi follicular B cells (g) or B220þPNAþ germinal
center (GC) B cells (h). Administration of fasudil decreased the frequency of CD138þB220lo plasma cells (i). All of the data in
this figure was collected from n ¼ 2 mice in the untreated group and n ¼ 7 and 8 for the 18 and 24 weeks treatment groups,
respectively. For (d) and (i) representative FACS plots from one mouse per group are also depicted. No Tx: no treatment; 18w:
fasudil treatment commencing at 18 weeks; 24w: fasudil treatment at 24 weeks. *p < 0.05, **p < 0.01, ***p < 0.001.

fasudil to MRL/lpr mice leads to decreased IL-17 inhibition may be broadly effective for the treat-
and IL-21 expression, lowers the production of ment of lupus.
autoantibodies and ameliorates the development One of the most noticeable effects of fasudil
of lupus.12,13 Here we demonstrate that administra- treatment was the significant decrease in anti-
tion of fasudil lengthens the life span and delays the dsDNA autoantibody production. This was associ-
development of proteinuria in NZB/W F1 mice, a ated with a lower number of plasma cells in the
distinct murine model of lupus, particularly when spleens of fasudil-treated NZB/W F1 mice, a find-
treatment is commenced at 18 weeks of age. Taken ing also observed in fasudil-treated lupus-prone
all together these results suggest that ROCK MRL/lpr mice.12 Given that IL-21 is a critical
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5
regulator of humoral responses and that we previ-
ously demonstrated that aberrant activation of
ROCK2 in MRL/lpr T cells leads to elevated
levels of IL-21 production via its effects on
IRF4,12 it is likely that inhibition of the ROCK2-
mediated production of this cytokine plays a role in
the beneficial effects exerted by fasudil in NZB/W
F1 mice. In further support of this hypothesis both
murine and human studies support a role for dereg-
ulation of IL-21 production in lupus pathogenesis
and IL-21 producing T cells have been observed in
the acceleration of lupus disease in NZB/W F1 that
occurs upon IFNa administration.15
It is, however, likely that the protective effects of
fasudil also encompass actions on additional cellu-
lar compartments. Although we have not observed
any direct effects of ROCK inhibitors on the ability
of B cells to differentiate into plasma cells in vitro
upon stimulation with anti-CD40 and IL-21
(PB, unpublished observations), we cannot exclude
that ROCK inhibition may exert direct effects on
the B cell compartment. Furthermore, the improve-
ments in survival and proteinuria that we have
observed in fasudil-treated NZB/W F1 mice are
also likely to involve actions beyond its effects on
lymphocytes. Indeed, the RhoA/ROCK pathway
has previously been shown to play an important
role in inflammation, via its ability to regulate the
recruitment of macrophages and neutrophils as well
as their function, including the production of pro-
inflammatory cytokines and pro-fibrogenic factors
such as TGFb.16,17 Recent studies utilizing various
models of diabetic nephropathy have furthermore
highlighted the critical importance of the RhoA/
ROCK pathway in progressive kidney disease via
effects on glomerular endothelial cells, mesangial
cells, podocytes and tubular epithelial cells.16,17
Interestingly, although the protective effects of
fasudil on the development of proteinuria were
less marked when treatment was initiated at 24
weeks, we did see beneficial effects on survival
even when treatment was started at such a time.
Additional studies will thus be required to fully
assess the effectiveness of ROCK inhibition at
later stages of disease. In conjunction with our pre-
vious findings, these studies thus suggest that
ROCK inhibition might be broadly effective for
the treatment of lupus and other autoimmune dis-
orders, and thus warrants further investigation as a
potential therapy for the treatment of human auto-
immune disorders.
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Administration of fasudil, a ROCK inhibitor
RA Stirzaker et al.
661
Funding
This work was supported by the NIH (HL62215),
the Alliance for Lupus Research and the Mary
Kirkland Center for Lupus Research.
Conflict of interest statement
None declared.
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