The iNOS Activity During an Immune Response Controls the CNS Pathology in Experimental Autoimmune EncephalomyelitisImage_1.TIFFImage_2.TIFF

Abstract

Inducible nitric oxide synthase (iNOS) plays a critical role in the regulation of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that iNOS plays pathogenic as well as regulatory roles in MS and EAE. However, how does iNOS alters the pathophysiology of the central nervous system (CNS) in neuronal autoimmunity is not clearly understood. In the present work, we show that treatment of mice with L-NAME, an iNOS inhibitor, during the antigen-priming phase primarily alters brain pathology, while in the subsequent effector phase of the immune response, the spinal cord is involved. Inhibition of iNOS during the priming phase of the immune response promotes the infiltration of pathogenic CD11b+F4/80−Gr-1+ cells, but there is low recruitment of regulatory CD11b+F4/80+ cells in the brain. Inhibition of iNOS during the effector phase shows similar pathogenic alterations in the spinal cord, instead of in the brain. Treatment of wild-type mice with L-NAME or mice having genetic deficiency of iNOS show lower MHC-II expression on the dendritic cells, but not on macrophages. Our data suggest that iNOS has a critical regulatory role during antigen-priming as well as in the effector phase of EAE, and inhibition iNOS at different stages of the immune response can differentially alter either the brain or spinal cord pathology. Understanding the cellular and molecular mechanisms through which iNOS functions could help to design a better strategies for the clinical management of neuroinflammation and neuronal autoimmunity.

Full text

ORIGINAL RESEARCH
published: 04 April 2019
doi: 10.3389/fimmu.2019.00710
Frontiers in Immunology | www.frontiersin.org 1April 2019 | Volume 10 | Article 710
Edited by:
Antje Kroner,
Medical College of Wisconsin,
United States
Reviewed by:
Stefan Bittner,
Johannes Gutenberg University
Mainz, Germany
Stella Tsirka,
Stony Brook University, United States
*Correspondence:
Girdhari Lal
glal@nccs.res.in
Specialty section:
This article was submitted to
Multiple Sclerosis and
Neuroimmunology,
a section of the journal
Frontiers in Immunology
Received: 16 January 2019
Accepted: 15 March 2019
Published: 04 April 2019
Citation:
Sonar SA and Lal G (2019) The iNOS
Activity During an Immune Response
Controls the CNS Pathology in
Experimental Autoimmune
Encephalomyelitis.
Front. Immunol. 10:710.
doi: 10.3389/fimmu.2019.00710
The iNOS Activity During an Immune
Response Controls the CNS
Pathology in Experimental
Autoimmune Encephalomyelitis
Sandip Ashok Sonar and Girdhari Lal*
National Center for Cell Science, Pune, India
Inducible nitric oxide synthase (iNOS) plays a critical role in the regulation of multiple
sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies
have shown that iNOS plays pathogenic as well as regulatory roles in MS and EAE.
However, how does iNOS alters the pathophysiology of the central nervous system (CNS)
in neuronal autoimmunity is not clearly understood. In the present work, we show that
treatment of mice with L-NAME, an iNOS inhibitor, during the antigen-priming phase
primarily alters brain pathology, while in the subsequent effector phase of the immune
response, the spinal cord is involved. Inhibition of iNOS during the priming phase of the
immune response promotes the infiltration of pathogenic CD11b+F4/80−Gr-1+cells,
but there is low recruitment of regulatory CD11b+F4/80+cells in the brain. Inhibition of
iNOS during the effector phase shows similar pathogenic alterations in the spinal cord,
instead of in the brain. Treatment of wild-type mice with L-NAME or mice having genetic
deficiency of iNOS show lower MHC-II expression on the dendritic cells, but not on
macrophages. Our data suggest that iNOS has a critical regulatory role during antigen-
priming as well as in the effector phase of EAE, and inhibition iNOS at different stages
of the immune response can differentially alter either the brain or spinal cord pathology.
Understanding the cellular and molecular mechanisms through which iNOS functions
could help to design a better strategies for the clinical management of neuroinflammation
and neuronal autoimmunity.
Keywords: experimental autoimmune encephalomyelitis, inducible nitric oxide synthase, NOS2−/−
neuroinflammation, central nervous system, autoimmunity
INTRODUCTION
Nitric oxide (NO) is a small bioactive lipophilic molecule that diffuse across the cell membrane
and controls many physiological functions of the body (1). NO production requires nitric oxide
synthase (NOS), which catalyze the oxidation of L-arginine to L-citrulline (2,3). In mammals,
there are three different isoforms of NOS - endothelial NOS (eNOS), neuronal NOS (nNOS) and
inducible NOS (iNOS) (4). Constitutive expression of eNOS and nNOS controls the vasodilation
of vessels and neuronal functions, respectively (5). Several inflammatory stimuli can induce
the expression of iNOS in various cell types such as macrophages, dendritic cells, neutrophils,
epithelial cells in the gut and lung mucosa, smooth muscle cells, and stromal cells of secondary

Sonar and Lal iNOS Controls the Pathology of CNS
lymphoid organs (6–9). iNOS is also expressed in microglial
cells, astrocytes, neurons in the central nervous system (CNS),
and endothelial cells at the blood-brain barrier (BBB) (7,10,
11). A clinical association between iNOS and pathogenesis has
been reported in many organ-specific autoimmune inflammatory
diseases, including multiple sclerosis (MS) and experimental
autoimmune encephalomyelitis (EAE) (12–15).
Proper neuronal function requires the presence of the
minimal physiological concentrations of NO in the CNS, with
sustained high NO levels leading to detrimental effects (16). The
active MS patients show high NO levels in the cerebrospinal
fluid (CSF), and high concentrations of NO, peroxynitrite, and
other reactive nitrogen species have been found to correlate with
greater severity and chances of relapse of clinical symptoms
(15,17). The expression of iNOS in the CNS is very tightly
regulated, and several intrinsic and extrinsic stimuli can induce
its expression in immune cells (14,18). The T cell-derived
cytokine, IFN-γinduces the expression of iNOS in macrophages
and microglial cells which leads to the generation of higher NO
and peroxynitrite productions, and cause tissue destruction in the
CNS (13,19,20). However, iNOS−/−mice are hypersusceptible
to EAE, suggesting that iNOS may have a regulatory function
during CNS inflammation and autoimmunity (21,22). Several
studies have shown that iNOS can regulate the function of
regulatory dendritic cells (regulatory DCs) which in turn can
induce apoptosis of inflammatory CD4+T cells and help in
controlling the development of EAE (23–25). Furthermore,
iNOS expression in macrophages is linked with the suppression
of inflammasome activation-induced IL-1βproduction (26),
as well as a reduction in the frequency of M1 macrophages
(27). Myeloid cell-derived iNOS is also known to control the
CD4+T cell response (28,29). High levels of APCs-derived
NO suppresses CD4+T cell response, while low levels favor
the generation of a Th1 response (30). Th17-intrinsic iNOS
has been shown to suppress Th17 response through nitration
of the tyrosine residues of RORγt, and limiting its promoter
binding capacity (31). While it is known that Th17 cells in the
CNS express iNOS during EAE, the functional importance of
iNOS production by Th17 cells in the inflamed CNS is not
clearly understood. During chronic demyelination, a pathogenic
phenotype of microglial cells has been found to be associated
with iNOS expression (32,33). Since both lesion-associated
and non-associated astrocytes express iNOS, the contribution of
astrocyte-derived iNOS is still unclear. Some in vitro experiments
suggest that inflammatory cytokine-induced iNOS reduces the
expression of myelin proteins and causes oligodendrocyte death
in the mixed glial cultures (34). All these observations indicate
Abbreviations: APCs, Antigen-presenting cells; BMDCs, Bone marrow-
derived DCs; CNPase, 2’,3’-cyclic nucleotide 3’-phosphodiesterase; CNS, Central
nervous system; CFA, Complete Freund’s adjuvant; DCs, Dendritic cells; EAE,
Experimental autoimmune encephalomyelitis; eNOS, Endothelial NOS; GFAP,
Glial fibrillary acidic protein; iNOS, inducible NOS; L-NAME, N-ω-nitro-l-
arginine methyl ester; MDSCs, Myeloid-derived suppressor cells; MOG, Myelin
oligodendrocyte glycoprotein 35-55 peptide; Mo-MDSCs, Monocytic myeloid-
derived suppressor cells; MS, Multiple sclerosis; NO, Nitric oxide; NOS, Nitric
oxide synthase; nNOS, neuronal NOS.
that iNOS plays a dual role during neuronal autoimmunity. Anti-
IFN-γtreatment and IFN-γR−/−mice show hypersusceptibility
to the development of EAE, with preferential involvement
of the brain stem and cerebellum, resulting in the atypical
EAE symptoms with the critical participation of neutrophil
effector function (35–37). Given that IFN-γregulates the iNOS
expression in several immune cells, how does iNOS controls
the inflammation in the brain and the spinal cord, and whether
iNOS performs different functions during the antigen-priming
and effector phases of EAE is not known.
In the present study, we assessed the role of iNOS using L-
NAME-mediated inhibition of its activity during various stages
of the immune response in EAE, including the antigen-priming
phase and the effector phases, accompanied by monitoring
of cellular pathology in the CNS. Our results showed that
inhibition of iNOS during the antigen-priming as well as effector
phases of EAE worsened the disease, and histology indicated
differential regulation of infiltration of CD11b+F4/80−GR-
1+and CD11b+F4/80+cells in the brain and spinal cord.
iNOS inhibition during the antigen-priming phase selectively
promoted the infiltration of inflammatory CD11b+F4/80−GR-
1+cells, while lowering the frequency of infiltration of
CD11b+F4/80+cells into the brain. Conversely, inhibiting iNOS
during the effector phase led to mostly CD11b+F4/80−GR-
1+cells migrating into the spinal cord. A similar phenotype
with higher infiltration of CD11b+F4/80−GR-1+cells and
reduced infiltration of CD11b+F4/80+cells in the CNS was
observed in iNOS−/−mice or wild-type mice in which IFN-
γ, a known inducer of iNOS, was blocked. We show that
iNOS plays a regulatory role in promoting the infiltration
of CD11b+F4/80+suppressor cells, while at the same time
inhibiting the mobilization of pathogenic CD11b+F4/80−GR-1+
cells into the CNS.
RESULTS
Inhibition of NO Production in the Priming
Phase Promotes Granulocytic Myeloid
Cells Infiltration Specifically in the Brain
Active EAE was induced in C57BL/6 mice and given an
intraperitoneal injection of NOS inhibitor L-NAME (100 mg/kg/
every day) in the antigen-priming phase (one injection/day for
seven days). The inhibition of NO production by L-NAME at
the antigen-priming phase significantly increased the severity of
EAE mice, compared to control mice (Figure 1A). Interestingly,
the severity of the clinical symptoms of EAE increased with
time in mice that received L-NAME (Figure 1A). Analysis of
the brain and spinal cord tissues showed enhanced infiltration
of CD45+leukocytes, including CD4+T cells, in the brain but
not in the spinal cord (Figure 1B). CD11b+F4/80+cells include
mainly macrophages and monocytic myeloid-derived suppressor
cells (Mo-MDSCs). The F4/80-expressing MDSCs are known to
have a suppressive function in EAE (38), whereas CD11b+GR-
1+cells show a pathogenic phenotype (39). L-NAME-treated
mice showed a significant reduction in the infiltration of
CD11b+F4/80+GR-1−cells selectively in the brain, but not
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Sonar and Lal iNOS Controls the Pathology of CNS
in the spinal cord (Figure 1C). However, L-NAME treatment
significantly increased infiltration of CD11b+F4/80−GR-1+cells
(mainly neutrophils) in the brain but not in the spinal cord
(Figure 1C). Together, our results showed that inhibition of NOS
in the priming phase of EAE mostly affected the infiltration of
inflammatory CD4+T cells and GR-1+neutrophils in the brain,
and reduced the frequency of CD11b+F4/80+cells, which might
account for the severity of EAE in the later phase of the disease.
Inhibition or Deficiency of iNOS in the
Antigen-Priming Phase Does Not Alter the
Differentiation of Effector CD4+T Cells
Since L-NAME injections at day 0-7 coincided with the antigen-
specific priming, activation, and differentiation of effector CD4+
T cells, we measured the frequency of Th1 cytokine (IFN-γ) and
Th17 cytokine (IL-17A) producing cells, and Foxp3+regulatory
CD4+T cells in the spleen and lymph nodes. Our results
showed no significant alteration in the intracellular expression
of IL-17A and IFN-γin CD4+T cells (Figures 2A,B) or γδ T
cells (Figure S1A). To further confirm the role of iNOS in the
priming-phase of EAE, we immunized wild-type and iNOS−/−
mice with MOG in complete Freund’s adjuvant (CFA). On day
8, we compared the differentiation of effector immune cells in
their spleen and lymph nodes with those of L-NAME (d0-7)-
treated and untreated wild-type C57BL/6 mice. iNOS−/−mice
also did not show any significant change in the expression of the
Th lineage-specific transcription factors, T-bet, RORγt, Foxp3,
and Eomes in CD4+T cells, as compared to the control group
(Figures 2C,D and Figures S1B,C). However, L-NAME treated
mice showed a lower frequency of T-bet expressing CD4+T cells
in the draining lymph nodes, as compared to the control group
(Figures 2C,D). We did not observe any significant changes in
the expression of intracellular IL-17A, IFN-γ, and GM-CSF in
the CD4+T cells or γδ T cells (data not shown). On day 28, L-
NAME treated mice showed severe EAE symptoms but did not
show a significant change in the percentages of Th1, Th17, and
Tregs or IL-17A+and IFN-γ+or IL-17A+IFN-γ+γδ T cells in
the secondary lymphoid organs (Figures 2A,B and Figure S1A).
Together, these results suggest that inhibition of iNOS during the
priming phase of EAE does not affect the differentiation of Th1,
Th17, and Treg cells in the secondary lymphoid tissues.
Inhibition of iNOS in the Effector Phase
Shows Increase Cellular Infiltration in the
Spinal Cord
To understand how inhibition of NO production during the
effector phase of EAE changes the pathophysiological phenotype
and transmigration of effector immune cells in the CNS, L-
NAME was administered (100 mg/kg/day; i.p.; day 8–15 of MOG
injection) in C57BL/6 mice. Inhibition of NO production during
the effector phase resulted in exacerbated EAE in these mice,
as compared to control mice (Figure 3A). Interestingly, while
increased infiltration of CD45+leukocytes and CD4+T cells was
observed in the spinal cord in mice treated with L-NAME in the
effector phase, infiltration of these cells in the brain was similar in
L-NAME-treated and control mice (Figure 3B). This suggested
that inhibition of NO production during the effector phase of
EAE differentially diverts the inflammatory cells to the spinal
cord and worsens the disease. Both meningeal and parenchymal
regions showed higher infiltration in L-NAME-treated mice,
as compared to control group (data not shown). Surprisingly,
immunohistological analysis showed higher expression of iNOS
in the brain-infiltrating CD45+leukocytes in the priming
phase, as compared to that observed upon treatment with L-
NAME in the effector phase (Figure 3C). However, inhibition
of NO production during the effector phase showed a higher
frequency of iNOS-expressing CD45+leukocytes in the spinal
cord as compared to control group, or the mice that received
iNOS inhibitor in the priming phase (Figure 3D). Analysis
of iNOS expression in the brain and spinal cord astrocytes
further revealed that the frequency of iNOS-expressing GFAP+
astrocytes was significantly lower in the brains of mice treated
with L-NAME during the antigen-priming phase as compared
to control, whereas the spinal cord resident astrocytes did
not show alteration in the number of iNOS-expressing cells
(Figures 3C,D). These results indicate that L-NAME-mediated
transient inhibition of NOS during the antigen-priming and
effector phase of EAE leads to the reappearance of iNOS
expression selectively in the infiltrated CD45+leukocytes and
possibly in the CD45int CNS-resident microglial cells, in the
brain and spinal cord, respectively. Consistent with the previous
reports (21,22), our results also showed that the a genetic
deficiency of iNOS in mice leads to the development of more
severe EAE than that observed in wild-type animals (Figure 3E).
Immunohistological analysis of the brain and spinal cord in
iNOS−/−mice showed significantly increased infiltration of
CD45+leukocytes and CD4+T cells in the brain but not in
the spinal cord (Figure 3F). These observations were consistent
with those we obtained with the L-NAME injections in the early
priming phase of the EAE, suggesting that lack of NO production
in the initial stage of EAE directs pathological mechanisms
specifically to the brain but not the spinal cord.
Furthermore, inhibition of NO production during the
effector phase showed significantly reduced infiltration of
CD11b+F4/80+GR1−cells in the brain and spinal cord
as compared to control animals (Figure S2A). Interestingly,
substantially higher infiltration of CD11b+F4/80−GR-1+cells
was observed in the spinal cord but not in the brain tissue during
effector phase iNOS inhibition as compared to control mice
(Figure S2A).
To further confirm the role of iNOS in the infiltration of the
CD11b+F4/80+GR-1−cells and CD11b+F4/80−GR-1+cells in
the CNS, we induced active EAE in iNOS−/−mice and analyzed
myeloid cell infiltration in the brain and spinal cord. Consistent
with our observations with L-NAME treatment during the
priming or effector phase, our results showed that deficiency
of iNOS leads to reduced infiltration of CD11b+F4/80+GR-1−
cells and increased infiltration of CD11b+F4/80−GR-1+cells
in the brain and spinal cord, as compared to wild-type mice
(Figure S2B). These results suggest that inhibition of NOS or a
genetic deficiency of iNOS selectively promotes the neutrophilic
infiltration and inhibits the infiltration of CD11b+F4/80+GR-1−
cells in the CNS.
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Sonar and Lal iNOS Controls the Pathology of CNS
FIGURE 1 | Inhibition of iNOS during the antigen-priming phase of EAE lead to increased infiltration of myeloid cells in the brain. C57BL/6 mice were s.c. injected with
200 µg MOG35−55 (MOG) in CFA emulsion, and two doses of i.v. pertussis toxin (PTx, 200 ng/mouse) at day 0 and 2. Mice were administered i.p. with L-NAME (100
mg/kg) from day 0 to 7 daily. Control groups were given i.p. PBS. (A) EAE clinical score was monitored and plotted. Arrow shows the day of L-NAME injection. Error
bars represent ±standard error of mean (SEM). (B) Mice were sacrificed on day 28, and the brain and spinal cord were analyzed by immunofluorescence staining.
Representative images of CD4 (red), CD45 (gray), CD31 (green), and nuclear stain DAPI (blue) stained brain and spinal cord tissues are shown (left). The mean number
of infiltrated CD4+T cells and CD45+leukocytes were quantified from at least 25–30 sections of the brain and spinal cord and plotted (right). (C) Representative
images of the brain and spinal cord tissues stained with CD11b (gray), GR-1 (red). F4/80 (green), and nuclear stain DAPI (blue) are shown (left). The mean number of
infiltrated CD11b+F4/80−GR-1−, CD11b+F4/80+GR-1−and CD11b+F4/80−GR-1+cells from at least 25–30 sections of the brain and spinal cord are plotted
(right). Error bars represent ±standard error of mean (SEM) (A–C). Original magnification 400x (B,C). Scale bar 100 µm(B,C). *p<0.05, **p<0.01, ****p<0.0001;
two way ANOVA followed by Tukey’s test (B), Student t-test (A,C).n=5 mice/group.
Together, our results show that lack of iNOS during EAE
development facilitates inflammation in both the brain and spinal
cord, with enhanced inflammatory cell infiltrations specifically
in the brain but not the spinal cord as compared to wild-type
mice. However, differential preference observed with L-NAME-
mediated inhibition of NOS during the priming versus effector
phase of EAE. These results suggest that inhibition of NOS in the
different stages of EAE differentially regulates the infiltration of
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Sonar and Lal iNOS Controls the Pathology of CNS
FIGURE 2 | Inhibition of iNOS during the antigen-priming phase of EAE does not affect Th1, Th17, and Tregs in the secondary lymphoid organs. Active EAE was
induced as given in Figure 1.(A,B) On day 28, single cell suspensions were prepared from the spleen and draining lymph nodes. Intracellular IL-17A and IFN-γwere
analyzed in the CD4+T cells using flow cytometry and plotted. (A) FACS plots show the intracellular expression of IL-17A and IFN-γin the CD4+T cells (left).
Quantifications of the percentage of intracellular cytokines in the CD4+T cells are shown (right). (B) Foxp3 expression in the CD4+T cells (left). Quantifications of
Foxp3+CD4+T cells (right). (C,D) C57BL/6 and iNOS−/−mice were s.c. injected with 200 µg MOG35−55 (MOG) in CFA emulsion, and two doses of i.v. pertussis
toxin (PTx, 200 ng/mouse) at day 0 and 2. Mice were administered i.p. L-NAME (100 mg/kg) from day 0 to 7 daily. Control groups were given i.p. PBS. On day 8,
single cell suspensions were prepared, and expression of Eomes, T-bet, Foxp3, and RORγt were analyzed in CD4+T cells. (C) Spleen and (D) draining lymph nodes
were analyzed using flow cytometry and plotted. Numbers in the dot plots show the percentages of CD4+T cells (A,B). Each dot represents an individual mouse, and
the horizontal line denotes mean and error bars represent ±SEM (A–D). (A–D). Student’s t-test (A,B), **p<0.01, ***p<0.001; ANOVA followed by Tukey’s test
(C,D).n=4–5 mice/group.
effector CD45+leukocytes, CD4+T cells, CD11b+F4/80−GR-1+
cells, and CD11b+F4/80+GR1−cells in the CNS, and this may, in
turn, account for the severity of the disease.
Inhibition of iNOS During the
Priming-Phase Reduces MHC Class II
Expression on DCs and Reduces the
Frequency of Monocytic-MDSCs in
Secondary Lymphoid Tissues.
Myeloid cells like macrophages and dendritic cells serve as
antigen presenting cells (APCs) and significant producers of
iNOS, and play a crucial role in the pathogenesis of EAE (23,24).
Our result showed that EAE induced in iNOS−/−mice, as well
as L-NAME-treated mice (day 0–7), had significantly reduced
frequency of CD11c+I-Ab+DCs (Figure 4A), and significantly
lower median fluorescence intensity (MFI) of the class II MHC
molecule (I-Ab) on the DCs, as compared to control mice
(Figure 4B).Further analysis showed that among the various
subsets of DCs, the CD11b+CD11c+and CD11b−CD11c+
subsets showed reduced expression of class II MHC in L-NAME-
treated mice and iNOS−/−mice, as compared to control EAE
mice (Figure 4C). L-NAME-treated and iNOS−/−mice also
showed a reduced percentage of CD11b+F4/80+macrophages
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Sonar and Lal iNOS Controls the Pathology of CNS
FIGURE 3 | The Inhibition of iNOS during the effector phase exacerbates inflammation in the spinal cord, leading to the development of severe EAE. Active EAE was
induced as in Figure 1. Mice were administered i.p. L-NAME from day 8 to 15 daily. Control groups were given i.p. PBS. (A) EAE clinical scores were monitored and
plotted. (B) On day 28, brain and spinal cord cryo-sections were analyzed by immunofluorescence staining. Representative images of the brain and spinal cord tissue
sections stained with CD45 (gray), CD4 (red), and nuclear stain DAPI (dark blue) are shown (left). Mean numbers of infiltrating CD45+and CD4+cells from 16 to 25
images of the brain and spinal cord were quantitated and plotted (right). (C,D) Representative images of the (C) brain and (D) spinal cord sections stained with GFAP
(gray), iNOS (red), CD45 (green), and nuclear stain DAPI (dark blue) are shown (left). Magnified images of the regions marked with the dotted square and are shown at
the bottom (D). Mean numbers of GFAP+iNOS+and CD45+iNOS+cells from 15 to 35 images of the brain and spinal cord were quantitated and plotted (right). (E)
C57BL/6 and iNOS−/−mice were s.c. injected with 200 µg MOG35−55 (MOG) in CFA emulsion, and two doses of i.v. pertussis toxin (PTx, 200 ng/mouse) at day 0
and 2. Mice were monitored for the development of clinical symptoms of EAE and the mean clinical scores were plotted (5 mice/group). (F) Mice in (E) were sacrificed
on day 20, and brain and spinal cord cryo-sections were analyzed by immunofluorescence staining. Representative images of the brain and spinal cord tissue
sections stained with CD45 (gray), CD4 (red), and nuclear stain DAPI (dark blue) and analyzed are shown (left). Mean numbers of infiltrating CD45+and CD4+cells
from 25 to 35 images of the brain and spinal cord were quantitated and plotted (right). Error bars represent ±SEM (A–F). Original magnification 400x (Brain; B,C,F);
200x (spinal cord; B,D,F). Scale bar, 100 µm (Brain; B,C,F), 300 µm (spinal cord; B,D,F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Student’s t-test
(A,B,F), two way ANOVA followed by Tukey’s test (C,D).n=5 mice/group.
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Sonar and Lal iNOS Controls the Pathology of CNS
in the spleen (Figure 4D), which may correspond to the lower
infiltration of CD11b+F4/80+cells observed in the brain of L-
NAME-treated mice, as compared to untreated mice (Figure 1C
and Figure S2B). L-NAME treatment did not have any effect
on the MFI of I-Abon non-DCs, whereas a lack of iNOS in
iNOS−/−mice showed a significant decrease in the MFI of
I-Abon non-DCs, as compared to control mice (Figure 4E).
The F4/80+CD11b+Ly6ChiLy6G−cells mainly represent Mo-
MDSCs, and are potent suppressors of effector CD4+and
CD8+T cells, and known to express their suppressive effect
via an iNOS-dependent mechanism (38). Our results showed a
significantly reduced frequency of F4/80-expressing Mo-MDSCs
in the iNOS−/−mice spleen, and to some extent in the L-
NAME-treated mice, as compared to control mice (Figure 4F).
These results suggest that inhibition or lack of iNOS can alter
the generation of CD11b+F4/80+cells, including suppressive
F4/80+Mo-MDSCs and mature antigen-presenting DCs in the
spleen, leading to reduced infiltration of CD11b+F4/80+GR-1−
cells and increased infiltration of CD11b+F4/80−GR-1+cells in
the brain.
Neutralization of IFN-γShows iNOS
Expression and Apoptosis of CNPase+
Oligodendrocytes in the CNS
IFN-γis a potent inducer of class II MHC molecules on APCs. It
can render either inflammatory or tolerogenic function to DCs,
depending on the presence or absence of a Toll-like receptor
(TLR) and CD40L signaling, and is essential for the development
of allograft tolerance. IFN-γis also a known inducer of iNOS
in a variety of cell types, including neutrophils, monocytes,
macrophages, dendritic cells, microglial cells, and astrocytes
(28). The IFN-γ−/−or IFN-γR−/−mice or antibody-mediated
neutralization of IFN-γsignaling, confers hyper-susceptibility to
the EAE. Similarly, our findings revealed that L-NAME-mediated
inhibition of NOS in the early and effector phase of EAE causes
severe EAE in wild-type mice.
Since our results showed that lack of iNOS reduced the
expression of class II MHC molecule on DCs, we further
investigated the link between IFN-γand iNOS during EAE. We
analyzed the influence of IFN-γon iNOS-expression in astrocytes
and CNS-infiltrated immune cells, and on apoptosis of the
myelin-synthesizing cells, oligodendrocytes. For this purpose, we
neutralized the IFN-γwith the anti-IFN-γ(100 µg/mouse; i.v.)
mAb during MOG-induced active EAE. Consistent with several
published reports (35,36), our results showed exacerbation of
the clinical symptoms of EAE with the neutralization of IFN-
γ(Figure 5A). We also observed an increased infiltration of
CD45+leukocytes and CD4+T cells in the brain as well as
spinal cord (Figure 5B). Both the meninges and parenchyma
showed increased infiltration of immune cells in the anti-
IFN-γ-treated group, as compared to the isotype control IgG-
treated mice (data not shown). While anti-IFN-γ-treated mice
showed significantly low infiltration of CD11b+F4/80+GR-1−
cells, however, the numbers of CD11b+F4/80−GR-1+cells
were dramatically increased in the brain and spinal cord, as
compared to control group (Figure 5C). The neutralization of
IFN-γusing mAb did not induce iNOS expression in the
astrocytes (Figure 5D). However, with anti-IFN-γ-treatment,
iNOS expression was significantly induced in CD45+leukocytes
in both brain and spinal cord, as compared to isotype IgG-
treated control mice (Figure 5D), suggesting that neutralization
of IFN-γleads to the reappearance of iNOS-expression on
CD45+leukocytes and possibly in microglia, similar to the effects
observed with L-NAME treatment.
We then asked whether increased infiltration of
CD11b+F4/80−GR-1+cells could have an effect on the survival
of oligodendrocytes during EAE. Our results showed that during
EAE, the cleaved-caspase 3 signal co-localizes with CNPase+
oligodendrocytes, suggesting that they are undergoing apoptosis
(Figure 5E). The anti-IFN-γ-treated mice showed significantly
higher co-localization of cleaved-caspase 3 with CNPase+
oligodendrocytes in the brain, as compared to the isotype control
group (Figure 5E), suggesting that the neutralization of IFN-γ
signaling lead to increased apoptosis of oligodendrocytes during
EAE. Furthermore, treatment with anti-IFN-γmAb increased
the infiltration of effector CD4+T cells, CD11b+F4/80−GR-
1+cells, and iNOS-expressing inflammatory immune cells,
and reduced infiltration of suppressive CD11b+F4/80+cells
(includes F4/80-expressing suppressive Mo-MDSCs) in the CNS.
These could have contributed to increased oligodendrocyte and
neuronal damage, consequently exacerbating the EAE pathology.
DISCUSSION
In the present study, we show that iNOS critically regulates
neuroinflammation at different phases of EAE by controlling
the infiltration of immune cells in the brain and spinal
cord. Inhibition of iNOS in the antigen-priming phase or
effector phase of EAE exacerbates the symptoms of the
disease via selectively increasing the infiltration of inflammatory
CD11b+F4/80−GR-1+cells, while at the same time reducing the
infiltration of CD11b+F4/80+GR-1−cells in the brain and spinal
cord, respectively. Similarly, with anti-IFN-γmAb treatment,
we showed differential infiltration of CD11b+F4/80+GR-1−
and CD11b+F4/80−GR-1+cells in the CNS during EAE.
Furthermore, lack of iNOS or neutralization of IFN-γpromoted
the infiltration of CD11b+F4/80−GR-1+cells in the brain or
spinal cord and enhanced the inflammation and apoptosis of
CNPase+oligodendrocytes.
Compelling evidence has suggested that various types of
myeloid cells do infiltrate into inflamed CNS along with myelin-
reactive Th1 and Th17 cells (40). These cells contribute to the
various inflammatory pathways leading to demyelination and
axonal damage in the CNS. iNOS controls the function of a
variety of myeloid and lymphoid cell populations in both a
cell-intrinsic and extrinsic manner (41). iNOS is also critically
involved in the pathogenesis of EAE and MS (17). However,
how it affects neuroinflammation in the various phases of EAE
was not clear. In the present work, we showed that inhibition
of iNOS during the antigen-priming phase leads to a selectively
increased infiltration of CD45+leukocytes and CD4+T cells
selectively in the brain but not in the spinal cord. Together with
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Sonar and Lal iNOS Controls the Pathology of CNS
FIGURE 4 | Inhibition of iNOS in the priming phase inhibits MHC class II expression on DCs and reduces the frequency of monocytic-MDSCs in the secondary
lymphoid tissues. C57BL/6 and iNOS−/−mice were s.c. injected with 200 µg MOG35−55 (MOG) in CFA emulsion, and two doses of i.v. pertussis toxin (PTx, 200
ng/mouse) at day 0 and 2. Mice were administered i.p. with L-NAME (100 mg/kg) from day 0 to 7 daily. Control groups were given i.p. PBS. On day 8, single cell
suspensions were prepared, and myeloid cell populations in the secondary lymphoid organs were analyzed using flow cytometry. (A) The percentages of
I-Ab+CD11c+cells and (B) mean fluorescence intensity (MFI) of I-Abexpression on CD11c+cells are shown (C) The MFI of I-Abon CD11b+CD11c+,
CD11b−CD11c+
,and CD11b−CD11c+myeloid cells were quantitated and plotted. (D) Data show the percentage of CD11b+F4/80+cells in the spleen. (E) The MFI
of I-Abon CD11b+CD11c−cells were analyzed and plotted. (F) The percentage of F4/80+of CD11b+Ly6ChiLy6G−cells in the spleen were analyzed and plotted.
Each dot represents an individual mouse, and the horizontal line denotes mean and error bars represent ±SEM. (A–F). Student’s t-test (A–F). *p<0.05, **p<0.01,
***p<0.001; n=5 mice/group.
data from iNOS−/−mice, our results suggest that a lack of iNOS
in the priming phase induces inflammation in the secondary
lymphoid organs in a manner that causes pathogenic cells to be
mobilized into the brain. The molecular details of trafficking of
these cells across the blood-brain barrier to CNS and the effect
of iNOS and possibly other isoform of NOS, such as eNOS in
their transmigration need further investigation. Previous studies
have shown that while iNOS does not affect the differentiation of
Th1, Th2 and Tregs, it does influence Th17 differentiation (31).
However, studies on the mechanism through which iNOS and
NO influence Th17 differentiation have yielded mixed results.
Studies in mice have revealed that this inhibitory function of
endogenous iNOS is via the nitration of tyrosine residues in
RORγt, and the inhibition of aryl hydrocarbon receptor (Ahr)-
signaling (31,42). However, others have reported that iNOS
and NO support human Th17 differentiation via the cyclic
guanosine monophosphate (cGMP)-dependent protein kinase
pathway, and endogenous iNOS play an essential role in the
stability of human Th17 cells when differentiated in the presence
of IL-1β, IL-6, and IL-23 (43). Our data show that inhibition of
NOS or genetic deficiency of iNOS neither affect the generation
of pathogenic Th1, Th17 cells, and Foxp3+regulatory CD4+T
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Sonar and Lal iNOS Controls the Pathology of CNS
FIGURE 5 | Neutralization of IFN-γinduces the expression of iNOS in the effector immune cells and results in apoptosis of CNPase+oligodendrocytes in EAE. Active
EAE was induced in C57BL/6 mice. Anti-IFN-γmonoclonal antibody (XMG1.2, 100 µg/mouse) monoclonal antibody was i.v. injected at days 0, 4, 8, 12, 16 after
MOG-immunization. (A) The clinical symptoms of EAE were recorded and mean clinical scores are plotted (5 mice/group). (B) On day 21, the brain and spinal cord
cryo-sections were analyzed by immunofluorescence staining. Representative images of the brain and spinal cord tissue sections stained with CD45 (gray), CD4 (red),
and nuclear stain DAPI (dark blue) are shown (left). Mean numbers of infiltrating CD45+and CD4+cells from 25 to 35 images of the brain and spinal cord were
quantitated and plotted (right). (C) Representative images of the brain and spinal cord stained with CD11b (red), F4/80 (green), GR-1 (light blue), and nuclear stain
DAPI (dark blue) are shown (left). Infiltration by indicated cell populations was quantified and plotted (right). (D) Representative images stained with GFAP (green), iNOS
(red) and CD45 (blue) are shown (left). iNOS-expressing GFAF+cells and CD45+cells were quantified from 20 to 30 images of the brain and spinal cord and plotted
as mean number of cells/field (right). (E) Representative images stained for cleaved caspase 3 (green), CNPase (red), CD45 (gray), and DAPI (blue) are shown (left).
Cleaved-caspase 3 expressing CNPase+cells oligodendrocytes were quantified from 20 to 30 images of the brain and spinal cord and plotted as mean number of
cells/field (right). Regions marked with white dotted square (D,E) are shown as magnified images at the right. Error bars represent ±SEM (A–E). Original magnification
200x (B, spinal cord), 400x (B, brain)and (C), 630x (D,E). Scale bar 50 µm(D,E), 100 µm (B, brain) and (C) and 300 µm (B, spinal cord). *p<0.05, **p<0.01, ***p
<0.001, ****p<0.0001, ANOVA followed by Tukey’s test (A), ANOVA followed by Sidak’s test (C), Student’s t-test (B,D,E).n=5 mice/group.
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Sonar and Lal iNOS Controls the Pathology of CNS
cells nor IFN-γand IL-17A-expressing γδ T cells in the secondary
lymphoid organs.
iNOS regulates the generation and function of regulatory
DCs that control the effector function of CD4+and CD8+T
cells, and helps in amelioration of EAE (23–25). CD11b+F4/80+
cells and F4/80-expressing Mo-MDSCs are known to have a
regulatory role in EAE (38). We show that a lack of iNOS function
in the priming-phase results in a significant reduction in the
frequency of CD11b+F4/80+cells as well as F4/80-expressing
Mo-MDSCs in the spleen. L-NAME-treatment or lack of iNOS
in mice yielded significantly lower infiltration of these cells in the
inflamed CNS. In contrast, lack of iNOS significantly increased
the previously reported pathogenic CD11b+F4/80−GR-1+cells
in the brain (39). In contrast, inhibition of iNOS in the
effector phase of EAE, when ongoing inflammation in the CNS
causes demyelination and axonal damage, resulted in higher
infiltration of CD4+T cells and CD11b+F4/80−GR-1+cells
preferentially in the spinal cord, but not in the brain. These
results suggest that temporal inhibition of iNOS during EAE
modulates differential clinical pathology in the brain and spinal
cord. iNOS inhibition during the antigen-priming phase of EAE
showed a high frequency of iNOS-expressing CD45+leukocytes
selectively in the brain, but not in the spinal cord. Since
CNS-resident microglia express iNOS under inflammation and
microglia are also characterized by CD45int expression, our study
cannot exclude the potential role of iNOS-expressing microglia
in exacerbation of EAE symptoms. Instead, this further opens the
avenue to investigate the relative functions of iNOS-expressing
microglia and circulation-derived macrophages in the early and
late phases of neuroinflammation during EAE. The GFAP+
astrocytes are also an important source of iNOS-expression
during neuroinflammation (16). We also observed higher iNOS-
expressing astrocytes in the brain and spinal cord in EAE mice.
However, inhibition of iNOS in the priming phase selectively
reduced the frequency of iNOS-expressing GFAP+astrocytes in
the brain, whereas it was mostly unaffected upon inhibition of
iNOS during the effector phase.
IFN-γis the upstream regulator of iNOS expression in
a variety of myeloid and lymphoid cells, and cells of non-
hematopoietic lineages (9,12). The neutralization of IFN-γusing
anti-IFN-γmAb during EAE in mice have been observed to
result in more severe symptoms and pathology (44). IFN-γ
is a known suppressor of Groα/KC (CXCL1) (45) and MIP-
2 (CXCL2) (46), both being neutrophil chemoattractants that
recruit neutrophils to the site of inflammation (47). In the
absence of IFN-γsignaling, Th17 cells predominate infiltrate
into the CNS, and predominantly recruit neutrophils in the CNS
(48,49), possibly via CXCL1- and CXCL2-mediated neutrophil
chemoattraction. Neutralization of IFN-γtherefore promotes
neutrophil trafficking to the inflamed CNS. Consequently,
we reasoned that a lack of iNOS function during anti-IFN-
γtreatment (12) may contribute for bias differentiation of
CD11b+F4/80+cells and F4/80-expressing Mo-MDSCs in the
lymphoid organs, and infiltration of these cells in CNS may
cause severe EAE. However, we also observed an increase in
the expression of iNOS in the CNS-infiltrating immune cells
and possibly CD45int microglia in anti-IFN-γ-treated mice,
suggesting that in the absence of IFN-γ, some other factors
can also induce the expression of iNOS in the inflamed CNS
(50). Furthermore, the enhanced expression of iNOS in the
CNS infiltrating immune cells was associated with increased
apoptosis of myelin-synthesizing oligodendrocytes in anti-IFN-
γ-treated mice, suggesting that high level of iNOS produced by
inflammatory CNS infiltrates can affect the remyelination process
and contribute to severity of the disease. A study with cuprizone-
induced demyelination model reported that infiltration and the
inflammatory function of CXCR2+neutrophils are required to
induce oligodendrocyte damage and demyelination in addition to
the toxic effect of cuprizone on mitochondrial function (51). The
increased apoptosis of CNPase+oligodendrocytes in anti-IFN-
γtreated mice, might be in part due to increased neutrophilic
infiltration in the CNS. A similar pathological mechanism may
exist in iNOS-deficient, or L-NAME treated wild-type mice and
warrants further investigation. We showed that MOG-induced
inflammation caused the induction of iNOS-expression in the
astrocytes, and that anti-IFN-γtreatment completely inhibits
the iNOS expression in astrocytes. The numbers of iNOS-
expressing astrocytes and the iNOS produced by them have
different consequences. While low levels of iNOS or NO are
beneficial, and their sustained high levels are detrimental to
the CNS homeostasis (52). Therefore, the high levels of iNOS
produced in the anti-IFN-γrecipients in our studies might have
caused apoptosis of oligodendrocytes in the brain, and thus
contributed to increased the clinical severity of the EAE. In
conclusion, we have shown here that immunoregulatory role of
iNOS safeguards the brain and spinal cord from inflammatory
granulocytic infiltration during the antigen-priming and effector
phase of EAE, respectively.
MATERIALS AND METHODS
Mice
Wild-type C57BL/6 and iNOS−/−(B6.129P2-Nos2tm1Lau/J) mice
were obtained from The Jackson Laboratory (Bar Harbor, ME).
Mice were maintained and bred in the Experimental Animal
Facility of the National Centre for Cell Science (NCCS), Pune,
India. All mice experiments were performed with the protocols
approved by the Institutional Animal Ethics Committee. (Project
Id: EAF/2016/B-257).
Antibodies and Reagents
Alexa Fluor 488-CD4 (GK1.5), APC-eFluor 780-CD4 (GK1.5),
FITC-γδTCR (GL3), APC-γδTCR (GL3), APC-CD45 (30F-
11), FITC-F4/80 (BM8), APC-Cy7-F4/80 (BM8), FITC-Ly6G
(1A8), Alexa Fluor 647-Ly6C (HK1.4), biotin-CD11b (M1/70),
APC-GR-1 (RB6-8C5), PE-GM-CSF (MP1-22E9), Brilliant violet
421-IL-17A (TC11-18H10.1), and purified anti-mouse GFAP
(MCA-5C10) were purchased from BioLegend (San Diego, CA).
Biotin-CD11c (N418), PE/Cy7-IFN-γ(XMG1.2), Pacific blue-
Foxp3 (FJK-16s), PE-Foxp3 (MF-14), PE-Eomes (Dan11mag),
eFluor 450-IAb(AF6-1201) were obtained from eBioscience
(San Diego, CA). Purified anti-IFN-γ(XMG1.2), and rat
IgG2b, k isotype control (LTF-2) were purchased from BioXcell
(West Lebanon, NH). PE-Cy7-CD11b (M1/70) antibody was
from BD Biosciences (San Diego, CA). Purified anti-iNOS
antibody (EPR16635) was purchased from Abcam (Cambridge,
Frontiers in Immunology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 710

Sonar and Lal iNOS Controls the Pathology of CNS
MA). Purified cleaved caspase 3 (5A1E) and purified anti-
CNPase (D83E10) was obtained from Cell Signaling Technology
(Danvers, MA). N-ω-nitro-l-arginine methyl ester (L-NAME)
was purchased from MP Biomedicals (Santa Ana, CA).
Induction of Active EAE
Wild-type C57BL/6 or iNOS−/−mice were given subcutaneous
(s.c.) injections of an emulsion of MOG35−55 (MOG) (200
µg/mouse) in complete Freund’s adjuvant (CFA) containing
Mycobacterium tuberculosis H37Ra (5 mg/ml), and also given
intravenous injections (i.v.) of pertussis toxin (PTx, 200
ng/mouse) at day 0 and 2. L-NAME was administered
intraperitoneally (i.p. 100 mg/kg) daily from day 0 to 7
(priming phase) or day 8 to 15 (effector phase). Control animals
received i.p. injections of PBS. For the neutralization of IFN-
γ, animals were given anti-mouse IFN-γmAb (clone XMG1.2;
100 µg/mouse) i.v. on days 0, 4, 8, 12, and 16. The animals
that received isotype control IgG were used as a controls. Mice
were followed for the development of clinical signs of EAE.
Scoring of clinical symptoms was performed as follows; score 0,
no symptoms; 1, limp tail or hind limb weakness but not both; 2,
limp tail and hind limb weakness; 3, partial hind limb paralysis;
4, complete hind limb paralysis; and 5, death by EAE.
Immunofluorescence Staining of the Brain
and Spinal Cord
Mice were sacrificed, ice-cold PBS was transcardially perfused,
and the brain and spinal cord were harvested. The tissues
were immediately snap-frozen in OCT compound (Sakura
Finetek, Torrance, CA). Seven-micrometer-thick cryosections
were prepared using a cryomicrotome. Sections were fixed
with chilled acetone for 5 min, air dried, followed by washing
with PBS. The tissue sections were blocked with 10% horse
serum (Jackson ImmunoResearch, West Grove, PA) in PBS at
room temperature (RT) for 30 min, followed by washing thrice
with ice-cold PBS. The sections were incubated with primary
antibodies overnight (12–14 h) at 4◦C, washed, further incubated
with secondary antibodies at RT for 60 min, and then washed
five times with PBS. The sections were stained with nuclear
stain DAPI at RT for 5 min and washed twice with ice-cold
PBS. Sections were mounted in mounting medium (Electron
Microscopy Sciences, Hatfield, PA). Images were acquired
on a Leica DMI6000 inverted fluorescent microscope (Leica
Microsystems, Germany) at 100, 400, and 630x magnifications.
Images were analyzed using Leica MMAF (Leica) or Image J
software (National Institute of Health, Bethesda, MD).
The quantification of cells from microscopic images was
performed using the MMAF software (Leica Microsystems,
Germany). The single channel and multi-channel overlay images
of sufficient magnification (original magnification, 400x) were
used for quantification of the number of cells. At least 10 different
microscopic fields per organ and more than three mice/group
were analyzed. Each cell was analyzed for its cellular morphology,
nuclear staining with DAPI, and specific markers. For evaluating
cellular infiltration into the CNS, complete brain tissues sections
were used.
Flow Cytometry
Cells were harvested from spleen and draining lymph nodes of
the mice. RBCs were lysed using ACK lysis buffer, and single
cell suspensions were prepared. Cells were surface-stained with
PE/Cy7-anti-mouse CD11b, PE/Cy5-anti-mouse CD11c, Alexa
fluor 647-anti-mouse Ly6C, FITC-anti-mouse Ly6G, APC/Cy7-
anti-mouse F4/80 and pacific blue-anti-mouse I-Ab (MHC class
II) antibodies. Cells were incubated on ice in the dark for 30 min,
washed with ice-cold PBS, and fixed with 1% paraformaldehyde.
Cells were acquired on FACS Canto-II (BD Biosciences), and data
were analyzed using the FlowJo software.
Intracellular Cytokine Staining
Single cell suspensions were prepared from spleen and draining
lymph nodes of mice. For transcription factor analysis, 1 ×
106cells were stained for the surface molecules CD4 and γδ
TCR on ice for 30 min and washed with ice-cold PBS. Cells
were subjected to fixation, and permeabilization with the Foxp3
fixation/permeabilization buffer kit (Biolegend), and intracellular
staining for Foxp3, T-bet, RORγt, and Eomes was performed
as per the manufacturer’s instructions. For intracellular cytokine
analysis, cells (6 ×106cells/well) were stimulated with phorbol
myristate acetate (PMA; 50 ng/ml) and ionomycin (850 ng/ml)
in the presence of brefeldin-A (5 µg/ml) and monensin (2 µM)
in 500 µl/well complete RPMI 1,640 medium in 24-well plates at
37◦C in a humidified 5% CO2incubator for 6 hours. Cells were
collected, washed with PBS, and stained for the surface molecules
CD4 and γδ TCR on ice for 30 min, and washed with ice-
cold PBS. Cells were subjected to fixation and permeabilization
using the Foxp3 fixation/permeabilization buffer kit (Biolegend)
and intracellular staining for IL-17A, Foxp3, Eomes, and IFN-
γwas performed as per the manufacturer’s instructions. Cells
were acquired on FACS Canto II (BD Bioscience), and data were
analyzed using the FlowJo software.
Statistical Analysis
Statistical comparisons were performed using the GraphPad
Prism 6 software (GraphPad, San Diego, CA). Unpaired two-
tailed Student’s t-test was used to compare two variables. The
ANOVA test was used to compare the means of more than two
groups followed by appropriate multiple comparison tests. The
other statistical methods used are described in the figure legends.
Ap<0.05 was considered statistically significant.
ETHICS STATEMENT
All mice experiments were performed with the approved
protocols from the NCCS Institutional Animal Ethics Committee
(Project Id: EAF/2016/B-257).
AUTHOR CONTRIBUTIONS
SAS and GL designed the experiments, analyzed the data, and
wrote the manuscript. SAS performed all the experiments.
Frontiers in Immunology | www.frontiersin.org 11 April 2019 | Volume 10 | Article 710

Sonar and Lal iNOS Controls the Pathology of CNS
ACKNOWLEDGMENTS
We thank Dr. Ramanamurthy Boppana and Dr. Rahul Bankar
for help with animal experiments. We also thank Dr. Jyoti Rao
for critical suggestions and edits. SAS received senior research
fellowship from the Council of Scientific and Industrial Research,
Government of India. GL received NCCS intramural funding
and grants from the Department of Biotechnology, (Grants
numbers, BT/PR4610/MED/30/720/2012, BT/PR15533/MED/
30/1616/2015; BT/PR14156/BRB/10/1515/2016), Science and
Engineering Research Board (EMR/2016/007108), Ministry of
Science and Technology, Government of India.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu.
2019.00710/full#supplementary-material
Figure S1 | Inhibition of iNOS or lack of iNOS does not affect the generation of
Th1, Th17, and Tregs in the secondary lymphoid organs. (A) EAE was induced in
C57/BL/6 mice as shown in Figure 1A. The intracellular expression of IL-17A and
IFN-γin the γδ T cells of the spleen and draining lymph nodes of the mice was
monitored and plotted (left). The dot plot shown is gated on γδ T cells.
Quantifications of the percentage of intracellular cytokines in the γδ T cells are
shown (right). (B,C) C57BL/6 and iNOS−/−mice were s.c. injected with 200 µg
MOG35−55 (MOG) in CFA emulsion, and two doses of i.v. pertussis toxin (PTx,
200 ng/mouse) at day 0 and 2. Mice were administered i.p. L-NAME from day 0 to
7 daily. Control groups were given PBS. On the day 8, single cell suspensions
were prepared from spleen and draining lymph nodes, and expression of the
transcription factors, Eomes, Foxp3, RORγt, and T-bet in CD4+T cells from the
(B) spleen and (C) draining lymph nodes were analyzed using flow cytometry. Dot
plots show the expression of the indicated transcription factors in CD4+T cells.
Numbers in the dot plots show percentage of indicated molecules in γδ T cells (A)
and CD4+T cells (B,C). The horizontal line denotes mean and error bars
represents ±SEM (A). Student’s t-test (A).n=4 mice/group (A) and 5
mice/group (B).
Figure S2 | Inhibition of iNOS or its deficiency differentially regulates the infiltration
of myeloid cells in the CNS. The brain and spinal cord tissue sections of mice from
Figures 3A,E were stained with CD11b (red), F4/80 (green), GR-1 (light blue) and
nuclear stain DAPI (dark blue). (A) Representative images of the brain and spinal
cord of mice either untreated or treated with L-NAME in the effector phase of EAE
are shown (upper). Magnified views of the areas marked with the dotted squares
are shown next to the images. The mean number of infiltrated
CD11b+F4/80−GR-1−, CD11b+F4/80+GR-1−and CD11b+F4/80−GR-1+
cells from at least 11–14 fields of the brain and spinal cord sections were
quantitated and shown (lower). (B) Representative images of the brain and spinal
cord sections of wild-type and iNOS−/−mice with EAE at day 20 are shown
(upper). Magnified views of the areas marked with the dotted squares are shown
next to the images. The mean numbers of infiltrated CD11b+F4/80−GR-1−,
CD11b+F4/80+GR-1−and CD11b+F4/80−GR-1+cells from at least 19–29
fields of the brain and spinal cord were quantitated and shown (lower). Original
magnification, 400x (A,B). Scale bar, 100 µm(A,B).∗p<0.05, ∗∗p<0.01, ∗∗∗ p
<0.001, ∗∗∗∗p<0.0001. Student’s t-test (A,B). Error bars represents ±SEM
(A,B).n=5 mice/group.
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Conflict of Interest Statement: The authors declare that the research was
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