Treatment of collagen-induced arthritis by Natura-α via regulation of Th-1/Th-17 responses

Treatment of collagen-induced arthritis by Natura-avia regulation of Th-1/Th-17 responses

Simon Glatigny1,2, Marie-Agnes Blaton1,2, Simon K. Mencher3,

Sylvie Mistou1,2, Bruno Lucas1,2, Catherine Fournier1,2, Long G. Wang3

and Gilles Chiocchia1,2,4

1 Institut Cochin, Universite Paris Descartes CNRS (UMR 8104), Paris, France2 INSERM U567, Departement d’Immunologie, Paris, France3 Natrogen Therapeutics International Inc., NY, USA4 Hopital Ambroise Pare, Boulogne-Billancourt, France

Cytokines and CD41 Th cells play a crucial role in the pathogenesis of rheumatoid

arthritis. Among the Th populations, Th-1 and Th-17 have been described as pathogenic in

collagen-induced arthritis (CIA) whereas Th-2 and Treg were found to have protective

effects. The objective of this study was to examine the affect of Natura-a, a newly devel-

oped cytokine regulator, on CIA and on Th cell development. Natura-a treatment was

administered before or during arthritis induction. Anti-type II collagen antibodies and

cytokine expression were evaluated by ELISA. Emergence of CD41CD251Foxp31 T cells

was assessed by flow cytometry. Th-17 differentiation of naive CD4 T cells was assessed in

cultures with anti-CD3 and anti-CD28. We showed that Natura-a both prevented and

treated CIA. We further demonstrated that in vivo treatment with Natura-a inhibited IL-17

production and anti-type II collagen IgG development. We showed in vitro, using an APC-

free system, that Natura-a acted directly on differentiating T cells and inhibiting the

formation of Th-1 and Th-17 cells but did not affect Th-2 cells. Since Natura-a inhibits a

large spectrum of important pathogenic factors in CIA, it may provide a new and powerful

approach to the treatment of rheumatoid arthritis and other inflammatory diseases.

Key words: Arthritis . Cytokine . Th-1 . Th-17

Supporting Information available online

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized

by synovial inflammation and pannus formation, which lead to

irreversible cartilage and bone degradation. Although many

candidate autoantigens are suspected of initiating a harmful immune

response in RA, none of them have been formally identified as such.

Type II collagen (CII) is a plausible target autoantigen involved in

the pathogenesis of RA. This inference is supported by the detection

of CII-specific Ab in the serum of patients and the isolation of T cells

reactive to CII from affected synovial tissues [1]. In addition, an RA-

like disease can be induced in susceptible strains of rodents and non-

human primates, upon immunization with CII [2–6].

Over the past decade, collagen-induced arthritis (CIA) that

mimics the clinical symptoms and histopathological changes

observed in RA has been extensively used to investigate a variety of

approaches for the immunotherapy of autoimmune arthritides.

Considerable data implicate CD41 T cells as expressing the Th-1

phenotype in the pathogenesis of CIA [7–10]. Conversely, several

lines of evidence assign a protective role to Th-2 lymphocytes inCorrespondence: Dr. Gilles Chiocchiae-mail: [email protected]

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.200939566 Eur. J. Immunol. 2010. 40: 460–469Simon Glatigny et al.460

CIA via production of anti-inflammatory cytokines. Thus, admin-

istration of Th-2-type cytokines such as IL-4, IL-10 and IL-13 are

shown to reduce the incidence and the severity of clinical arthritis

[11–16]. We and others have previously addressed the role of IFN-

g, a prototypic Th-1 type cytokine, in CIA. From these previous

studies, both enhancing and suppressing influences have been

reported depending on the dose, timing of infusion and the model

used. IFN-g exerts a biphasic effect. Indeed, blocking IFN-g at the

early stages after immunization with CII resulted in decreased anti-

collagen Ab levels and severity of arthritis, whereas late treatment

had aggravating effects, and these effects were mirrored by IFN-gtreatment [17].

We and others reported puzzling results regarding the role of

IFN-g in CIA by showing that susceptible DBA/1 mice that lack

the IFN-g receptor developed an early and high incidence of

arthritis [18, 19]. Furthermore, it was seen that treatment of the

mice with anti-IL-12 or IL-18 Ab [20, 21] had a protective effect on

CIA and IFN-g was reduced. Moreover, mice deficient in the gene

product IL-12p40 or the proinflammatory cytokine IL-18 [22, 23]

had a reduced incidence of the disease. Because of IL-12 p40,

heterodimerizes with p35 to form bioactive IL-12, and hetero-

dimerizes with p19 to comprise the cytokine IL-23, it seems that

some of the results obtained with mice lacking IL-12p40 can be

attributable to decreased IL-23 [24]. Because IL-23 is involved in

Th-17 differentiation, it is now clear that the effects observed were

partially due to the effect on the Th-17 type of T-cell differentiation

In the light of recent findings, RA should now be viewed as both a

Th-17 and Th-1 regulated disease [25]. Therefore it is of interest to

seek molecular agents that have the ability to modify the differ-

entiation and/or cytokine pattern production by T cells.

Natura-a, a newly developed cytokine regulator, has been

shown to have potent therapeutic activities against dextran

sulfate sodium salt-induced chronic ulcerative colitis in Balb/c

Mice. This molecule is capable of rebalancing expression/

production of various cytokines, i.e. inhibiting expression of

pro-inflammatory cytokines, TNF-a, IL-1b, IL-6 and simulta-

neously stimulating regulatory cytokine IL-10 in LPS-stimulated

THP1 human monocytes and inhibit STAT3 phosphorylation

(unpublished data; US patents pending). In this study, so as to

analyze Natura-a’s anti-inflammatory activities in detail, we

examined its preventive and therapeutic effects in CIA mice, and

explored possible underlying mechanisms.

Results

Natura-a markedly affected CIA clinical parameters

Blind scoring of macroscopic manifestations of CIA was used for

each mouse to determine the average course of CIA in the subject

CA

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Figure 1. Prevention of CIA development by Natura-a. (A–C) Treatment consisted of gavages three times per week with 50 mg/Kg of Natura-a (orvehicle) from day 0 (i.e. day of collagen immunization) to the day of sacrifice. Blinded clinical evaluation of CIA (reported as arthritic score) wasperformed as described in the Materials and methods at the indicated timepoints until day 56 post-immunization. (A) Severity of arthritis, mean1

SEM of ten mice/group.�po0.02; 7po0.01 versus vehicle-treated group (Student’s t-test). (B) Time course of CIA incidence in Natura-a fed mice. Dataare percent of affected mice in each group. (C) Treatment-induced differences in body weight in the different treatment groups, recorded weekly,mean1SEM of ten mice in each group. (D) Dose- and time-dependent treatment. Gavage of mice was performed twice for five consecutive days:firstly at immunization and secondly at priming with CII. The experiment included one control and two experimental groups (n 5 10/group) asfollows: CII-immunized mice treated with Natura’s vehicle; CII-immunized mice treated with Natura-a dose 1 (100 mg/Kg/dose); CII-immunizedmice treated with Natura-a dose 2 (50 mg/Kg/dose). ypo0.05; 7po0.01 versus vehicle-treated group (Student’s t-test).

Eur. J. Immunol. 2010. 40: 460–469 Immunomodulation 461


groups (Fig. 1A). Severe arthritis developed in CII-immunized

positive controls (fed with vehicle throughout the experiment).

The onset of disease was evidenced from day 26 post-priming,

and the maximal arthritis scores in mice were exhibited by day

50. In the vehicle-treated group, the incidence of arthritic mice

rapidly increased and reached a maximum of 90% by day 44,

thus exhibiting the classical features of a CIA model (Fig. 1B).

Notably, in the group of mice that were fed with 50 mg/Kg

Natura-a (three times a week for 8 wk; starting at priming), fewer

mice were affected at all times, and at the end of the treatment

time, the cumulative incidence was markedly lower (50%). It is

seen that arthritis occurred at a later time and the disease was

less severe in the treated mice (Table 1). Further detailed analysis

of data from the early treatment group revealed that two out of

the five affected mice had swelling of only one and three digits,

respectively. In the remaining three mice, the first clinical

symptoms consisted of moderate inflammation of some digits

that lasted for 1–2 wk before flareup of the disease extended to

other joints such as tarsus, ankles or carpus. This slow course of

severity at onset of CIA sharply contrasted with that of the mice in

the vehicle-treated group, in which the disease developed

aggressively shortly after it was diagnosed.

Figure 1C shows that, during the first week of treatment with

either Natura-a or the vehicle, the growth of mice was affected.

Weight increase was significantly lower in the treated group than

the untreated group (po0.02 and po0.05). These findings,

however, may result from the stress caused by the first gavages.

Thereafter, treated mice progressively recovered to reach weights

almost identical to those of the untreated group by day 21.

Though the differences are not statistically significant, it is

interesting to note that, in the group treated as from day 0 with

Natura-a, mice gained weight until day 42 while in the other

group, the growth was stopped and mice started to slim down by

day 28 due to CIA-induced systemic effects (Fig. 1C). This

observation indicates that Natura-a treatment may have

improved the overall health of the CIA mice.

Taken together, provided that the drug is administered during

the induction phase of the disease, these findings point to a

markedly protective effect of Natura-a on CIA.

Dose and time dependency of Natura-a treatment

Since treatment with Natura-a during the induction phase of the

disease induced a distinct protective effect on CIA, we then tested

for a similar protective effect when the treatment was delivered

only when the immune system was stimulated and boosted (the

five first consecutive days and from day 21–25). As expected,

naive control mice did not exhibit any sign of inflammatory

arthritis whether or not they were administered Natura-a (data

not shown). Conversely, CII-immunized positive controls (fed

with vehicle) developed severe arthritis beginning from day 28

post-priming (Fig. 1D). Treatment with either a 50 or 100 mg/Kg

dose of Natura-a induced a protective effect during early phases

of clinical symptoms (Fig. 1D). Significant differences (po0.05)

between ‘‘Natura 100’’-treated and control mice were observed

from day 30 to day 37 post-immunization. However, the

beneficial action of the drug was transient and fully abrogated

by day 51. Data obtained with the dose of 50 mg/Kg were very

similar but did not reach statistical significance.

Natura-a treatment reduces the severity of developingCIA

For the CIA treatment study, treatment started at day 35. At this

stage, 40% of the mice had already developed clinical signs of

disease. As shown in Fig. 2A, Natura-a treatment required one

week before realizing any efficacy and significantly diminished

the severity of the disease 2 wk thereafter. The later treatment of

the mice did not result in a difference compared with the controls

in terms of both kinetics and total number of mice that developed

CIA (Fig. 2B).

Effects of Natura-a treatment on the anti-CII B-cellresponse

Immunization with CII elicits a specific humoral response in all

mice. Although levels of anti-CII Ab are not strictly correlated to

arthritis scores, in sera from severely diseased mice they are usually

high. Thus, anti-CII IgM and anti-CII IgG were measured in all mice

on day 20 (before challenge with CII) and on day 40 (during CIA

course), and at sacrifice of the mice. On day 20, just before

antigenic boost, no difference was observed in the anti-CII IgM

levels between groups (Fig. 3A). Conversely, none of Natura-treated

mice had detectable amounts of circulating anti-CII IgG, whereas, in

the other groups, (either those receiving vehicle or untreated), mice

secreted variable and significant levels of anti-CII IgG (Fig. 3B). The

CII specific humoral response in mice given an early treatment with

Natura-a was seen to be impaired on day 40 when only three mice

had mild inflammation of digits, but tended to normalize at the end

Table 1. Effect of treatment with Natura-a following different regimens on various arthritis parametersa)

Treatment groups Incidence of CIA n (%) Onset (days) Maximal severityb)

CII1vehicle 9/10 (90%) 36.272.5 13.871.6

CII1Natura-a (D1-56) 5/10 (50%) 38.273.2 8.873.2

a) Data are mean7SEM of n diseased mice/group.b) The maximal severity is the higher clinical score reached during CIA course by each affected mouse.

Eur. J. Immunol. 2010. 40: 460–469Simon Glatigny et al.462


of the experiment (Fig. 3C). These findings indicate that, when it

was administered at the moment of CIA induction, Natura-aquantitatively reduced the secretion of anti-CII Ab.

To study whether the drug also qualitatively altered the humoral

response, levels of CII-specific Ab expressing IgG1 and IgG2a

isotypes were determined in samples collected on day 40 post

priming. In line with the data described above, levels of anti-CII

total IgG were seen to be impaired in the group that underwent

early treatment with Natura-a. Interestingly, the drop in Ab

response significantly reduced the IgG2a isotype (p 5 0.01) whereas

it weakly reduced the IgG1 resulting in an IgG1/IgG2a ratio

increase in both treated groups (1.7070.31 for vehicle treated

animals at day 40 compared with 5.8271.19 (po0.01) in the early

treatment group from day 1 and 2.8270.61 (po0.05) in the late

treatment group) (Fig. 3D). Regarding the late-treatment schedule,

CII-specific IgG2a levels were identical to those in the control group

and IgG1 was somewhat increased. Since Ab production of IgG2a

and IgG1 isotypes are known to be associated with Th-1 and Th-2

responses, respectively, the present findings suggest that Natura-amay induce a shift from a Th-1 dominated type of response to Th-2

response. Thus, Natura-a induced a marked and isotype-specific

modulation of the anti-CII Ab response.

Effect of Natura-a on in vivo cytokine production

Immunization with CII elicits a cellular response in all mice that is

characterized by the synthesis of various proinflammatory cyto-

kines such as IFN-g and IL-17 produced by T cells. To investigate

the effects of Natura-a on production of different cytokines, we

quantified IL-4, IL-6, IL-12, IL-17, IL-23 and IFN-g cytokines in sera

of treated or vehicle mice, at different times after treatment. On day

20, only IL-17A was significantly decreased in the treated group

compared with vehicle (Fig. 4A). On the other hand, neither IL-23

nor IL-6 was decreased in this group. Moreover, Natura- treatment

did not affect the Th-1 response because the IFN-g level remained

elevated and constant in all groups. At all time points, IL-4 and

IL-12 were either undetectable (IL-4) or barely detectable in sera

(IL-12) (data not shown). At day 40, IL-17A was the only

significantly decreased proinflammatory cytokine (data not

shown). Thus, Natura-a treatment seems to act in vivo on IL-17A

synthesis or induction.

Natura-a alters in vivo Treg development

To explore whether the beneficial effect of Natura-a on arthritis

could be associated with late changes in T cells, the proliferative

response to CII and the percentage of Treg (defined as CD41

CD251Foxp31) were evaluated at time of sacrifice. Treg in the

spleen and lymph nodes of the mice treated with Natura-a were

compared with those of the control mice, from day of

immunization to time of sacrifice. In all the mice tested from

the two groups, specific proliferative responses to varying

concentrations of CII were very low (if any); i.e., having

proliferation indexes below 2. This was in both the spleen and

lymph nodes (Table 2) indicating that CII-specific T cells could

not be detected in the lymphoid organs 2 months after

immunization. Regarding the detection of Treg, a 31.2% increase

of spleen CD41CD251Foxp31 cells was observed in mice treated

with Natura-a (14.170.6 versus 9.771.9) but was not observed

in lymph node cells.

Natura-a action on T-cell differentiation

We observed that Natura-a treatment at the time of immuniza-

tion decreased the IL-17A sera level in all mice. To better

understand the Natura-a effects on T-cell differentiation, we

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Figure 2. Therapeutic effect of Natura-a on CIA development. (A and B)On day 35 after CIA immunization, a timepoint when 40% of the micehave already developed arthritis, mice were orally treated with Natura-a or vehicle three times per week and evolution of disease severity wasmonitored at the indicated timepoints after initial Natura-a treatment(day 0 after initial treatment 5 day 35 after collagen immunization.Evolution of arthritic score 5 score at day after initial treatment–scoreat day 35. Data are mean1SEM of ten mice/group. ypo0.05; 8po0.01versus vehicle-treated group (Student’s t test). (B) Time course of CIAincidence. Data are percent of affected mice in each group.



studied the effect of Natura-a on the polarization of naive CD4

T cells. We cultured in vitro CD41 CD25- T cells from naive mice

under conditions favoring Th-1, Th-2, Treg and Th-17 with or

without Natura-a throughout the time of culture. After 4 days of

culture IL-4, IL-17A and IFN-g levels were quantified in the

supernatants of all conditions. As expected, the different

polarizing conditions led to selective secretion of the signature

cytokines. As shown in Fig. 4B, one of the main actions of

Natura-a was to decrease the IL-17A synthesis in all conditions

tested, except that of the strongest Th-17 condition. Of note, IL-4

production was not modified in presence of Natura-a, showing

that Natura-a seems to have no specific action on this T-cell

differentiation pathway. Moreover, the addition of Natura-a to

the medium abrogated or reduced IFN-g production. These

results were evidenced by intracellular cytokine staining, which

demonstrated that the presence strongly inhibited IL-17 and

IFN-g production at cellular level (Fig. 4C). The Natura-a effect

was dose dependent with a strongest and marked effect at 2mM

dose. In the various conditions tested, we did not observe

difference in CFSE labeling in untreated compared with Natura-

treated cells. On the other hand, we did observe a non-significant

difference in cell counting when using Natura-a at 2mM. Thus,

although we presently cannot categorically exclude that the use

of Natura at high doses (2 mM) could have some cell toxicity, the

effect on reduction of IL-17 and IFN-g producing cells

in the presence of Natura is still clear (Supporting Information

Fig. S1). These results showed that Natura-a can strongly act on

in vitro T-cell differentiation.

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Figure 3. Kinetics of sera anti-CII Ab levels in CII-immunized mice treated with Natura-a or vehicle. Treatment was either maintained from day 1(i.e. day after collagen immunization) up to day 56 (Natura 1–56) or started on day 35 after collagen immunization and continued thereafter untilday 56 (Natura 35–56). (A and B) Levels of anti-CII Ab were detected in sera from individual mouse collected on day 20 post-priming using ELISA.(A) Total IgG. (B) Total IgM. (C) Sera from individual mice were collected at days 20, 40 and 60 after collagen immunization and CII-specific IgG weremeasured by ELISA. (D) IgG1/IgG2a ratio was calculated following anti-CII IgG1 and IgG2a determination by ELISA in samples collected on day 40post-immunization. Data are mean7SEM of ten mice/group.

Table 2. Determination of Treg in the spleen and lymph node cells of mice at the end of the in vivo experiment

Percentages of Treg (CD41CD251Foxp31)

Vehicle-treated mice Natura-a-treated (D1-56) mice

n Arthritis Spleen Lymph nodes n Arthritis Spleen Lymph nodes

1 Yes 11.3 9.7 4 No 13.1 9.8

2 Yes 10.2 9.1 5 No 14.4 9.2

3 No 7.6 9.9 6 Yes 14.9 11.1

Mean7SEM 9.771.9 9.670.3 14.170.6 10.370.9



Discussion

The findings reported herein provide strong evidence that a

preventive therapeutic protocol using oral administration of

Natura-a ameliorates CIA in DBA/mice. Early and continuous

administration of the drug seems to be required for this protection.

Indeed, treatment beginning at immunization but discontinued on

day 25 post-priming induces transient inhibitory effects. Treat-

ments starting when arthritis is already well established (day 35)

showed that Natura-a can limit the course of CIA.

Beneficial effect elicited by early administration of Natura-awas manifested by several criteria. First, the drug strongly

reduced the disease incidence, since half of the mice from this

group never exhibited clinical symptoms of arthritis. Second, in

the other mice that were considered as ‘‘arthritic’’, inflammation

developed slowly, affecting only their digits. Thus, compared

with controls, fewer mice exhibited late symptoms of inflamma-

tion. However, once the first signs of arthritis occurred, severe

inflammation progressed in the Natura-a-treated mice as rapidly

as it increased in vehicle-treated mice.

The question of involvement of T cells in the protection

conferred by administration of Natura-a was addressed. As far as

anti-CII T cells were concerned, no functional deficiency (in

terms of proliferation) could be detected upon oral treatment

with Natura-a (data not shown). These observations suggest that

the pathogenic T cells specific for the arthritogenic epitope of CII

may not be the major target of the drug. Our results suggest

that Natura-a’s mechanism of action may be dependent on

polarization of CD4 T-cell subsets and/or the activation of

suppressor T cells. This hypothesis is supported by several results.

First, the measure of cytokine levels in the sera of treated and

untreated mice revealed a drop of IL-17 in the group of mice fed

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Figure 4. Effect on Natura-a on cytokine production. (A) In vivo cytokine production. Sera IL-4, IL-6, IL-12, IL-17, IL-23 and IFN-g cytokine levels inNatura-a- or vehicle-treated mice were assessed on day 20 post collagen immunization by ELISA. Data are mean1SEM of ten mice/group. po0.01versus vehicle-treated group (Student’s t-test). (B) Natura-a inhibits Th-1 and Th-17 induction. CD41CD25� T cells isolated by Dynabeads fromDBA/1. mice were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of Natura-a for 2 days underTh-0 (control), Th-1, Th-2 Th-17 or iTreg conditions. The cells were then let to proliferate for 2 days in IL-2 and IL-4, IFN-g and IL-17 levels in thesupernatants were measured by ELISA. These experiments are mean of three independent experiments. ypo0.05; �po0.02; 8po0.01 versus untreatedcells.(C) Purified CD41CD25� T cells were cultured in Th-0, Th-1 and Th-17 polarized conditions with or without Natura-a at the indicatedconcentration. On day 4, CD4 T cells were harvested, activated 4 h with PMA and ionomycin in the presence of Brefeldin A, stained intracellularlywith anti-IFN-g, and anti-IL-17A, and analyzed by FACS. Data are representative of two independent experiments.



Natura-a. Second, the presence of Natura-a during in vitro T-cell

differentiation resulted in a profound alteration of the ratio

Th-1/Th-2/Th-17 subsets. Third, quantification of CD41CD251

Foxp31 T-cells (referred to as Treg) by FACS analysis in the

spleen of immunized mice showed an increase of this population

in mice administered Natura-a compared with the control mice.

Our results showed that Natura-a has a strong effect on Th-1

and Th-17 differentiation. Recent papers show that CIA is

governed by two proinflammatory CD4 T-cell types called Th-1

and Th-17 cells. These cells are mainly producers of IFN-g and

IL-17A and these syntheses are induced by distinct cytokines in the

medium; IL-12 for Th-1 and IL6, TGF-b for Th-17 cells. Moreover,

IL-23 is involved in the amplification and survival of Th-17 cells. In

our experiments, neither IL-23 nor IL-6 was decreased in the

groups of treated mice suggesting that Natura-a action is down-

stream of IL-6 and IL-23. Furthermore, in vitro results from Th cell

differentiation experiments also showed that Natura had a limited

effect on TGF-b action. Because a strong in vitro system was used,

it is highly possible that Natura-a acts by limiting both Th-1 and

Th-17 differentiation, and that the presence of TGF-b could lead to

increase ratio of Treg over other T-cell types as suggested by the

increase of CD41CD251Foxp31 in the spleen of Natura-a treated

animals. In future, it will be interesting to elucidate precisely the

molecular mechanism of action of Natura-a on T-cell differentia-

tion and how TGF-b could interfere.

Altogether, our results showed that Natura-a’s treatment

seems to act on IL-17A synthesis, or induction, and this decrease

results in low incidence and severity of the disease in the group

treated from day 0. Although numerous small-molecules have

been tested for the treatment of CIA, few have been reported to

have a modifying beneficial effect on the disease. Among them,

imatinib mesylate, a selective tyrosine kinase inhibitor has been

shown to prevent and treat CIA [26]. Interestingly, imatinib has

been shown to inhibit IFN-g production [26, 27]. It would be

interesting to know whether it also acts on Th-17 differentiation

as does Natura-a. Our results demonstrated that Natura-a could

be considered as a direct regulator of CD41 T-cell differentiation.

Regarding the humoral response to CII, we observed that

levels of anti-CII were strongly reduced in the blood of mice with

early treatment, compared with the other groups, and this

impairment persisted until the end of the experiment. Notably,

the drop in total IgG directed to the immunizing antigen essen-

tially concerned the IgG2a isotype, which is known to be that of

pathogenic Ab in CIA model and drive by IFN-g. In keeping with

this assumption, high titers of anti-CII IgG2a were secreted by

control mice and also by the mice with late treatments that failed

to fully down-regulate the pathological process and the ongoing

inflammation.

In mice, in vitro activation of naive T cells by DC and TGF-b,

together with proinflammatory cytokines particularly IL-6, leads

to the differentiation of Th-17 cells [28–30]. It was also shown

that IL-6 was an important cytokine in Th-17 differentiation

in vivo and that this effect of IL-6 is at least partially explained via

tyrosine residues of the signal transducer gp130 and depends on

STAT-3 activation [31–34]. Interestingly, the STAT-3 signaling is

involved in the host inflammatory response and in the innate and

adaptive immune functions [35, 36]. Hyperactivated STAT-3

signaling by cytokines/infectious agents has been demonstrated

to play a critical role in pathologenesis of a numerous of acute

and chronic inflammatory diseases [37–42]. Natura-a has been

shown to inhibit expression of various pro-inflammatory cyto-

kines, including IL-1b, IL-6 and TNF-a, and activation of STAT-3

in LPS-stimulated human monocytes (see Supporting Information

materials). Thus, Natura-a could act through down-regulation of

IL-17A production by inhibiting STAT-3 phosphorylation. Taken

together, Natura-a exerts potent anti-inflammatory effect via

regulation of autoimmune response and appears to be a potential

drug candidate to control inflammation.

Materials and methods

Induction and assessment of arthritides

Male DBA/1 (H-2q) mice were purchased from Harlan (Bicester,

UK) and were used at 7–10 wk of age. Immunization with bovine

native CII (100 mg emulsified in CFA, id), boosted with CII

(100 mg in IFA) on day 121 and a blind evaluation of arthritis

were performed as previously described in detail [14]. The

date of disease onset was recorded, and the clinical severity of

each joint or group of joints (toes, tarsus, ankle, wrist and knee)

was graded: 0 (normal), 1 (erythema), 2 (swelling), 3 (defor-

mity) or 4 (necrosis). Clinical scores of each joint (graded 0–4)

were summed to yield the arthritic score and the severity

of CIA was expressed both as the mean score observed on a

given day and as the mean of the maximal arthritic score

reached by each mouse. In curative treatment protocols, the

results were expressed as means of individual net scores

calculated by subtracting arthritic scores evaluated on the last

day of treatment. The studies were approved by the Cochin

institute committee on animal care; agreement number to

perform experiments on living animals: no. 75-777 and animal

facility agreement number no. 3991.

Natura-a

Natura-a is a cytokine regulator being developed by Natrogen

Therapeutics International (NY, USA). It is small synthetic

molecule, and chemically belongs to dihydroindole family with

a molecular weight 276 and a melting point of 235–237 C1. The

chemical name of Natura-a is N-methyl-D3, 30-dihydroindole-2,

20 diketone. Experiments demonstrate that Natura-a significantly

inhibits expression of various pro-inflammatory cytokines includ-

ing IL-1b, IL-6 and TNF-a while stimulates expression of the

regulatory cytokine IL-10 in LPS-stimulated THP-1 cells (Study

NTI-2001 Report, Supporting Information support S1). Natura-aalso shows efficacy against acute and chronic ulcerative colitis in

dextran sulfate sodium salt induced mouse models (Manuscript



in Preparation). Pharmacokinetic studies demonstrated that

the t1/2 of Natura-a was approximately 6 h with a relatively little

variability in the values for ke in rats, dogs as well as in humans.

No genotoxicity of Natura-a has been observed. Clinical trials are

currently underway under US FDA guidelines for the treatment of

inflammatory related diseases (protocol NTI-2007-1).

Treatment with Natura-a

Natura-a was provided by Natrogen Therapeutics International

with a purity Z98.5%. Immunized mice were divided into four

groups corresponding to different treatments regimens. All

groups were treated orally by gavage three times per week with

Natura-a at (50 or 100 mg/kg). Saline solution was used as

vehicle to prepare a suspension of Natura-a, which was

administered to mice by gavage using flexible probes. For

preventive effect, mice were treated throughout the duration of

the experiment. For therapeutic effect, mice were treated from

day 35 up to day 56. In each experiment, control group of mice

received only the vehicle.

Cytokine detection

Mice were anesthetized and bled by retro-orbital puncture at days

20 and 40 following immunization and sera kept at �801C until

use. IL-4, IL-6, IL-12, IL-17, IL-23 and IFN-g cytokines were

measured by ELISA (eBioscience San Diego, CA, USA) in serial

twofold dilution starting 1/4 to 1/16 in sera of treated or control

mice as indicated by manufacturer.

Levels and isotypes of Ab to CII

Mice were anesthetized and bled by retro-orbital puncture and by

cardiac puncture at the time of death. Sera were stored at �201C

until use. Ab to CII were detected by ELISA as previously

described [43, 44]. The plates were read at 405 nm with a

Titertek multiscan spectrophotometer (Dynatec MR 5000,

Guyancourt, France). Isotypes of Ab were determined in

individual sera by serial twofold dilutions using alkaline

phosphatase-conjugated goat anti-mouse IgG1 and IgG2a (South-

ern, Birmingham, USA). The standard serum was a pool of mouse

sera with high amounts of anti-CII Ab.

CD4 T-cell purification and in vitro differentiation

CD41CD25� T cells from the whole spleen of DBA/1 mice were

enriched. Briefly, splenocytes were incubated on ice for 20 min

with anti-CD8 (LYT2), anti-CD11b (Mac1), anti-GR1 (8C5), anti-

CD19 (1D3) and anti-CD25 (PC61), and then with magnetic

beads coupled to anti-rat Ig (Dynal Biotech, Compiegne, France).

Purified T-cell subsets were usually 90–95% pure. To generate

Th-1 cells, 5� 105 purified CD41CD25� T cells were stimulated

with plate-bound anti-CD3 (10 mg/mL) and soluble anti-CD28

(2 mg/mL) for 3 days in complete medium (RPMI 1640 glutamax

medium supplemented with 10% FBS, 2% sodium-pyruvate, 1%

penicillin-streptomycin, 1% Hepes, 0,1% b-mercaptoethanol)

supplemented with IFN-g (2 ng/mL) and anti-IL-4 (10 mg/mL).

To generate Th-2 cells, CD41CD25� T cells were stimulated in

complete medium, supplemented with anti-IFN-g (10 mg/mL)

and IL-4 (2 ng/mL). To generate Treg cells, CD41CD25� T cells

were stimulated in complete medium supplemented with

TGF-b1 (2 ng/mL). To generate Th-17 cells, CD41CD25�

T cells were stimulated in complete medium supplemented

with anti-IFN-g (10 mg/mL), anti-IL4 (10 mg/mL), IL-6 (2 ng/mL)

and TGF-b1(2 ng/mL). After 3 days of culture, cells were

supplemented with IL-2 (1 nM) and at day 4 supernatants

were collected, and cytokine content was detected by ELISA kits

for IFN-g, IL-4 and IL-17A (eBioscience). Cytokine producing

cells were determined by intracellular staining using PE-

conjugated anti-mouse IL-4, Alexa 488-conjugated anti-mouse

IL-17A (BD Pharmingen San Diego, CA, USA) and APC-

conjugated anti-mouse IFN-g. Cells were harvested, washed and

stimulated in medium containing phorbol myristate acetate,

ionomycin and brefeldin A for 4 h (Sigma, L’Isle d’Abeau Chesnes,

France). Cells were fixed in 2% paraformaldehyde, permeabilized

with 0.1% saponin and stained with fluorescent Ab. Then, the

cells were analyzed using an FC500 cytometer (Beckman Coulter,

Villepinte, France), and the CxP analysis software (Beckman

Coulter).

Acknowledgements: This work was supported by institutional

grants from Institut National de la Sante et de la Recherche

Medicale (INSERM), CNRS, and Natrogen Therapeutics

International, Inc, New York, USA

Conflict of interest: S.K.M. and L.G.W. are employees and share

holders of Natrogen Therapeutics International, Inc.

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Abbreviations: CIA: collagen-induced arthritis � CII: type II collagen �RA: rheumatoid arthritis

Full correspondence: Dr. Gilles Chiocchia, Institut Cochin Departement

d’Immunologie, Pavillon Hardy A, 27 rue du Faubourg Saint-Jacques,

75674 Paris, Cedex 14, France

Fax: 1331-40-51-66-41

e-mail: [email protected]

Received: 29/4/2009

Revised: 16/9/2009

Accepted: 12/11/2009



Treatment of collagen-induced arthritis by Natura-α via regulation of Th-1/Th-17 responses

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