Ingested (oral) thyrotropin releasing factor (TRH) inhibits EAE

Cytokine 61 (2013) 323–328

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Ingested (oral) thyrotropin releasing factor (TRH) inhibits EAE q Staley A. Brod ⇑, Victoria Bauer Department of Neurology, University of Texas–Houston, Health Science Center, 6431 Fannin St., Houston, TX 77030, United States

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Article history: Received 27 July 2012 Received in revised form 26 September 2012 Accepted 19 October 2012 Available online 11 November 2012 Keywords: Oral proteins Thyrotropin releasing factor TRH EAE Adoptive transfer

a b s t r a c t Background: Ingested immunoactive proteins type I IFN, SIRS peptide 1–21, a-MSH, ACTH, SST inhibit clinical attacks and inflammation in acute EAE by decreasing Th1-like cytokines, increasing Th2-like cytokines or increasing Treg cell frequencies. Objective: We examined whether another protein, thyrotropin releasing factor (TRH), would have similar anti-inflammatory effects in EAE after oral administration. Design/methods: B6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or TRH during ongoing disease. Splenocytes from mock fed or TRH fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. Results: Ingested (oral) TRH inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from TRH fed donors protected against actively induced disease and decreased inflammation. In actively fed mice, oral TRH decreased IL-17 and TNF-a cytokines in both the spleen and the CNS. In recipients of donor cells from TRH fed mice there was a reduction of Th1 and Th17 and induction of Th2-like IL-13 cytokines in both the spleen and CNS. Oral TRH decreased clinical score and decreased inflammatory foci in both actively fed and recipients of actively fed mice. There was no significant increase in Treg cell frequencies in actively fed or recipients of TRH fed donor cells. Conclusions: Ingested (orally administered) TRH can inhibit clinical disease, inhibit CNS inflammation by decreasing Th1-like, Th17 and TNF-a cytokines and increasing Th2-like cytokines (IL-13) in the CNS. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction EAE is a T cell mediated inflammatory autoimmune process of the CNS that resembles in some aspects the human demyelinating disease multiple sclerosis (MS) [1] and provides a useful animal model for the evaluation of potential therapies for T cell mediated autoimmune diseases [15,31,39]. Ingested proteins such as type I IFN [5], SIRS peptide 1–21 [6], a-MSH [7], ACTH [8] and SST [9] inhibit clinical attacks and inflammation in acute EAE [5,11]. Ingested proteins act by reduction in Th1-like encephalitogenic activity (ingested IFN-a) [6,7,10,11], induction of Th2-like counterAbbreviations: ACTH, adrenocorticotropin hormone; a-MSH, alpha-melanocyte stimulating hormone; PBMC, peripheral blood mononuclear cell; EAE, chronic relapsing experimental autoimmune encephalomyelitis; DTH, delayed type hypersensitivity; DPBS, Dulbecco’s phosphate buffered saline; SIRS, soluble immune response suppressor; SPF, specific pathogen free; SST, somatostatin; Treg, T regulatory cell. q Supported in part by a grant from the Clayton Foundation for Research. The authors have nothing to disclose. ⇑ Corresponding author. Address: Multiple Sclerosis Research Group, University of Texas–Houston, 6431 Fannin St., 7.044, Houston, TX 77030, United States. Tel.: +1 713 500 7046, mobile: +1 713 213 1105; fax: +1 713 500 7040. E-mail addresses: [email protected] (S.A. Brod), Victoria.L.Bauer@ uth.tmc.edu (V. Bauer).

1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.10.015

regulatory cytokines (oral SIRS peptide) [6], reduction in CNS Th1-like encephalitogenic cytokines (a-MSH) [7], reduction in Th1-like encephalitogenic cytokines IL-2, IFN-c and IL-17 along with CD4+CD25+FoxP3+ frequency induction (Treg) (ACTH) [8] and reduction of Th1 and Th17 with induction of Th2-like IL-4 cytokines and Treg cells (SST) [9]. Considerable evidence supports a pivotal role for TRH in the pathophysiology of the inflammatory process [21]. TRH is found outside the brain in intrinsic nerve fibers of the GI tract and modulates PBMC polyclonal IgG providing evidence for a functional link between the immune system and the endocrine system [19]. TRH receptors are found in mesenteric lymph nodes [25] and small intestine epithelial cells [38]. In veterinarian science oral TRH supplementation increases relative bursa weights in chicks and significantly increased lymphocyte cell population without affecting serum concentrations of either T4 or T3 cells [17]. TRH can significantly influence the development of lymphoid cells associated with intestinal intraepithelial lymphocytes (IELs) [37]. These data suggest that TRH may not necessarily be absorbed but nonetheless involved in the growth of lymphoid organs and the production of lymphocytes in the gut. We therefore examined whether oral TRH would have a similar anti-inflammatory effect in EAE in vivo by decreasing Th17,

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decreasing Th1-like cytokines or increasing Th2-like cytokines with Treg induction in the CNS target organ in murine EAE. 2. Materials and methods 2.1. Induction of active EAE C57BL/6 6–8 week old females were actively immunized, maintained, handled and surveiled as outlined previously [6]. Briefly, C57BL/6 6–8 week old females (Jackson Labs, Bar Harbor, ME) were actively immunized by subcutaneous injection (s.c.) of 0.2 ml inoculum containing 200 lg MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) in IFA (DifcoLabs, Detroit, MI) with 800 lg Mycobacterium tuberculosus hominis H37Ra (MT) on day 0 and 7 following [36], with pertussis toxin (PTx) (List Biologicals) 200 ng i.p. on day 0 and day 2 and followed for evidence of disease. Clinical severity was graded daily as follows by a blinded observer: 0 = no disease; 1 = minimal or mild hind limb weakness (associated with limp tail); 2 = moderate hind limb weakness or mild ataxia (waddling gait and/or poor righting ability); 3 = moderate to severe hind limb weakness; 4 = severe hind limb weakness or moderate ataxia; 5 = paraplegia with no more than moderate four limb weakness; 6 = paraplegia with severe four limb weakness or severe ataxia. 2.2. Adoptive transfer Thirty days after inoculation and after peak score of clinical attack, all spleens from each treatment group were aseptically removed, single cell suspensions prepared, and red cell lysis performed by adding 2–3 ml sterile water to single cells for 5 s, and once the solution became transparent, adding AIM-V media to a 50 ml tube. Splenocytes from grouped saline (mock) fed or 100 lg TRH fed mice were re-stimulated with MOG peptide 35–55 at a final concentration of 10 lg/ml for 48 h in serum free medium (AIM-V medium, Gibco BRL, Grand Island, NY) with 2  105 cells/200 ll in triplicate in 96 well U-bottomed plates in a humidified 5% CO2/95% air incubator at 37 °C. Following incubation, cells were collected, washed twice in PBS, and viability determined by standard Trypan blue exclusion. Viable concentrations were adjusted to 107 cells/0.5 ml Dulbecco’s PBS immediately prior to i.p. injection into active MOG peptide 35–55 immunized recipient mice during ongoing disease (day 17 post immunization). Following administration of TRH or adoptive transfer, clinical outcome was measured by comparing the difference between group mean active treatment and placebo group scores from day 17–34 post immunization. 2.3. Immuno-active protein Protirelin – TRH (synthetic human – tripeptide) was purchased from Cell Sciences. 2.4. Dosing (feeding) regimen Once non-treated inoculated mice attained a clinical score 1.5– 2.0, B6 mice were randomized to one of three treatment groups, and gavaged (fed) with 0.1 ml of saline (mock), 10 lg, or 100 lg of TRH using a 2.5 cm syringe fitted with a 22–24 gauge ball point needle (Thomas Scientific, Swedesboro, NJ) as previously described [6]. 2.5. Histology Following sacrifice, spinal cords were removed and immersion fixed in 10% neutral buffered formalin for a minimum of 2 weeks.

After fixation, cords were sectioned in entirety in the horizontal plane at approximately 3 mm intervals and processed to paraffin. Paraffin blocks were sectioned at 6–8 lm, and step sections were stained with hematoxylin and eosin and examined by light microscopy. Cord sections were evaluated independently for foci of inflammation by a blinded observer (SAB), without knowledge of the treatment status of the mice prior to sacrifice. Spinal cord tissue was sampled in an identical fashion for each animal and numbers of inflammatory foci per high powered field (HPF) (>20 perivascular lymphocytes) in the parenchyma were counted. 2.6. Measurement of cytokine secretion Spleens and spinal cords (CNS) from each treatment group were aseptically removed and single cell suspensions prepared. In spinal cords, whole cords were passed through a cell strainer for CNS lymphocytes (B and D, Franklin Lakes, NJ) and spun at 600 rpm several times to separate lymphocytes from CNS tissue. Splenocytes and cord lymphocytes from grouped saline fed or 100 lg TRH fed mice were stimulated with 10 lg MOG peptide 35–55  48 h as previously described [5,6]. Murine cytokine responses were examined using a customized RayBio Mouse Cytokine Inflammatory Antibody Array including innate cytokine TNF-a, IL-17 (Teff), Th1-like (IL-2, IFN-c), Th2-like cytokines (IL-4, IL-10, IL-13) and IL-12p70 using the RayBioantibody array Analysis tool application (RayBiotech, Inc., Norcross, GA). Results were grouped from mice fed saline or mice fed with TRH from grouped samples of two separate experiments (each sample performed in duplicate) and expressed as pg/ml ± SEM (student t-test). 2.7. Phenotypic analysis CD25 and FOXP3 expression by CD3+CD4+ lymphocytes was analyzed using the Beckman Coulter 10-Color Gallios Flow Cytometer and mouse regulatory T Cell Staining Kit with PE Foxp3 FJK16s, FITC CD4, APC CD25 (eBioscience, San Diego, CA) following the manufacturer’s instructions. 2.8. Statistics Statistical analysis was performed using ANOVA and student t test. (Prism 4.0). 3. Results Preliminary experiments determined the immuno-modulatory capability of 10 and 100 lg ingested (orally administered) TRH compared to saline placebo in EAE. Mice were immunized and separated into three groups once each mouse attained a clinical score 1.5–2 (day 17 post immunization) at which time oral dosing was started. The placebo group showed a slight decreased group clinical score from day 17 and plateaued at clinical score = 2.0 after 34 days post immunization and 17 days after the initiation of feeding. Active treatment groups fed with 10 and 100 lg showed significant decreases in group clinical scores after initiation of therapy (day 17) in several experiments (p < 0.001, ANOVA) with 100 lg showing the most persistent clinical effect and reduction of disease severity compared to placebo (Fig. 1). Thirty-four days following immunization, there were significantly less inflammatory foci in the 10 lg fed group (mean group inflammatory score = 4.8 ± 0.38) and in the 100 lg fed group (mean group inflammatory score = 2.9 ± 0.25) compared to the control mock fed group (mean group inflammatory score = 9.6 ± 0.58) (Fig. 2).

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Fig. 1. Ingested TRH inhibits clinical EAE attacks. B6 mice (n = 8/group) were immunized with MOG peptide 35–55 and were gavaged with 0.1 ml of control saline or 10 lg or 100 lg TRH as described in methods. Both 10 and 100 lg ingested TRH significantly inhibits clinical EAE progression compared to control (p < 0.001, ANOVA, day 17–34, group clinical score ± SEM). The figure shows combined results from three separate experiments (total n = 24/group).

After adoptive transfer of MOG-restimulated splenocytes into actively immunized recipient mice with early clinical disease on day 17 (mean group clinical score 2.0, respectively), recipients of donor splenocytes from placebo fed mice slightly decreased their group clinical disease severity over 17 days to a plateau of 2.0. In contrast, recipients of donor splenocytes from 100 lg TRH fed mice decreased their group clinical score at day 34 to a score = 0.98 (Fig. 3) (p < 0.001). Seventeen days following adoptive transfer, the number of CNS inflammatory foci in the 100 lg fed group compared to the control mock placebo group was significantly different (mean group inflammatory score) for recipients of placebo fed donors (15.0 ± 3.2) vs recipients of 100 lg TRH fed donors (7.48 ± 0.28) (t test, p < 0.001) (Fig. 4).

Fig. 2. Spinal cords from TRH fed mice show significantly less inflammatory foci in the CNS. Spinal cords were harvested as outlined in methods. There were significantly fewer group mean inflammatory foci in 10 lg and 100 lg fed mice compared to placebo fed mice (p < 0.001, ANOVA) (n = 24/group).

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Fig. 3. Recipients of adoptively transferred TRH fed donor cells protect against active EAE. Thirty-four days after inoculation and after peak score of clinical attack, spleens from mock and 100 lg TRH fed mice were isolated and re-stimulated with MOG peptide 35–55 and adoptively transferred as described in methods. Recipients of saline fed donor cells slightly decreased their group clinical disease severity. In contrast, recipients of TRH fed donor cells decreased their group clinical score significantly compared to recipients of saline control cells (p < 0.005, days 17–34, group clinical score ± SEM). This experiment shows a combination of three separate experiments (total n = 24/group).

We compared the cytokine profiles of MOG re-stimulated spleen and cord lymphocytes in mock dosed vs 100 lg TRH dosed mice (from Fig. 1). Splenic lymphocytes showed significant decreases in levels of Th17-like cytokine IL-17 and TNF-a in TRH fed groups compared to the mock fed group (Fig. 5). CNS lymphocytes showed significant decreases in levels of Th1-like cytokines IL-2 and IL-12, IL-17 (Teff) and TNF-a in the TRH fed group compared to the mock fed group (Fig. 6). There was increased CNS lymphocyte production of MOG induced IL-13 in TRH dosed vs mock dosed mice (Fig. 6). We also compared the cytokine profiles of MOG re-stimulated spleen and cord lymphocytes in recipients of mock fed or 100 lg TRH fed donor cells (from Fig. 3). Splenic lymphocytes showed significant decreases in levels of Th1-like cytokines IL-2 and IFN-c, IL-

Fig. 4. Recipients of adoptively transferred TRH fed donor cells show significantly less inflammatory foci compared to control. There were significantly fewer group mean inflammatory foci in recipients of 100 lg fed cells compared to recipients of mock fed cells (p < 0.008, ANOVA) (n = 24/group).

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Fig. 5. Ingested TRH decreases Th17 and decreases TNF-a in spleen cells of actively immunized mice. Lymphocytes isolated from spleens from mock fed mice or TRH dosed mice were stimulated with MOG peptide 35–55 and measured using an inflammatory cytokine antibody array as described in methods. Splenic lymphocytes showed decreased levels of Th17-like cytokines IL-17 (p < 0.01) and TNF-a (p < 0.01) in TRH dosed vs mock dosed mice. This experiment shows a combination of three separate experiments (total n = 24/group). ND = none detected.

Fig. 6. Ingested TRH decreases pro-inflammatory Th17 and Th1-like cytokines and increases of Th2-like IL-13 in the CNS of actively immunized mice. Lymphocytes isolated from spinal cords from mock fed mice or TRH fed mice were stimulated with MOG peptide 35–55 and measured using an inflammatory cytokine antibody array as described in methods. Splenic lymphocytes showed decreased levels of Th1-like cytokines IL-2 (p < 0.05) and IL-12 (p < 0.05), IL-17 (p < 0.001) and TNF-a (p < 0.01) and also showed increased Th2-like IL-13 (p < 0.001). This experiment shows a combination of three separate experiments (total n = 24/group).

17 (Teff), and IL-12 in TRH fed groups compared to the mock fed group (Fig. 7). There was increased peripheral splenic lymphocyte production of MOG induced IL-13 in TRH dosed vs mock dosed mice (Fig. 7). CNS lymphocytes showed significant decreases in levels of IL-17 (Teff) in TRH fed group compared to the mock fed group (Fig. 8). CNS lymphocytes showed significant decreases in levels of IL-2, IFN-c, IL-12, IL-17 in TRH fed group compared to the mock fed group (Fig. 8). There was increased CNS lymphocyte production of MOG induced IL-4 and IL-13 in both TRH dosed vs mock dosed mice (Fig. 8).

Fig. 7. Recipients of donor cells from TRH fed mice show decreases in splenic Th17, Th1-like cytokines and increased Th2-like IL-13. Whole splenocytes from recipients of mock fed or TRH fed donor cells were stimulated with MOG peptide 35–55 and measured using an inflammatory cytokine antibody array as described in methods. Splenic lymphocytes showed decreased levels of Th1-like cytokines IL-2 (p < 0.03), IL-12 (p < 0.03), IL-17 (p < 0.01), IFN-c (p < 0.05) and increased production of MOG induced IL-13 (p < 0.03) in TRH dosed vs mock dosed mice. This experiment shows a combination of three separate experiments (total n = 24/group).

Fig. 8. Recipients of donor cells from TRH fed mice show decreases in CNS Th17, Th1-like cytokines and increased Th2-like IL-4 and IL-13. Lymphocytes isolated from spinal cords from recipients of mock fed or TRH fed donor cells were stimulated with MOG peptide 35–55 and measured using an inflammatory cytokine antibody array as described in methods. CNS lymphocytes showed decreased levels of Th1-like cytokines IL-2 (p < 0.05), IL-12 (p < 0.03), IFN-c (p < 0.03), IL-17 (p < 0.03) and increased peripheral splenic lymphocyte production of MOG induced IL-4 (p < 0.03) and IL-13 (p < 0.03) in TRH dosed vs mock dosed mice. This experiment shows a combination of three separate experiments (total n = 24/ group). ND = none detected.

We next determined if CD4+CD25+FoxP3+ Treg might be induced by TRH feeding and explain protection in actively treated and recipients of adoptively transferred cells from TRH fed donors. FACS analysis shows no significant increase in CD4+CD25+FoxP3+ cell frequency in TRH fed compared to mock fed mice in actively fed or recipients of actively fed donor cells (data not shown).

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4. Discussion We show here for the first time that TRH by the oral route can decrease clinical autoimmune disease. Our data shows an overall anti-inflammatory effect of ingested TRH in MOG immunized mice. Both 10 lg and 100 lg ingested (oral) TRH showed significant clinical effect with 100 lg demonstrating the most robust activity. Incidently, mice given oral 10 lg and 100 lg TRH daily for 7 days prior to immunization showed statistically attenuated EAE severity (data not shown). Adoptive transfer of TRH fed MOG-re-stimulated splenocytes into recipient mice with early clinical disease suppressed disease compared to splenocytes from mock fed donors. Both active treatment with oral TRH or adoptive transfer of splenocytes from TRH fed mice showed significantly less CNS inflammation in the TRH groups compared to control. We found that there was a decrease in innate inflammatory cytokines TNF-a, Th1-like cytokines IL-2 and IFN-c, IL-17 (Teff), IL-12p70 as well as increases in Th2-like counter-regulatory cytokines, in particular IL-13. However, we saw no increased frequency of Treg cell frequencies in spleen of TRH fed mice compared to controls. Inoculation of B6 mice with MOG peptides can activate pathogenic neuroantigen-specific Th1 T helper cells in vivo and produce inflammation in murine EAE [36]. IL-17 is produced by Th17 cells distinct from the traditional Th1- and Th2-cell subsets and is involved in generation of autoimmunity [13,16]. IL-12 induces Th1like cells [14]. Th2-like lymphocytes produce IL-4 [24], IL-10 and IL-13 [24] and inhibit EAE [26]. Splenic IL-13 reduces infiltrating mononuclear cells into CNS during EAE [28]. TNF-a is important in CNS pathology in EAE [32] and induces EAE [20]. Previous investigators have shown that TRH significantly inhibits monocyte activity [23] and TRH receptors are expressed in mesenteric lymph nodes [25]. Immunization of rats with sheep red blood cells (SRBC, a T cell-dependent antigen) increases TRH mRNA [29]. PBMC, rat splenocytes and transformed T cell lines produce TSH in response to thyrotropin-releasing hormone (TRH) [18,30] and can enhance or modulate the in vitro antibody response [19,22]. Although we did not measure TSH, oral TRH does not increase serum T4 or T3 [17] so it is unlikely that we stimulated a potential immuno-modulatory TSH. It is more likely that oral TRH interacted with immune cells in the gut directly. Oral TRH can inhibit clinical disease and inflammation by decreasing Th17, the generator of autoimmunity [27]. Th17 cells are constitutively present throughout the intestinal lamina propria (LP) and Peyer’s Patch (PP) [12]. Understanding the mechanism of action of oral TRH as either inhibiting T cell Th17 activity directly in LP or PP, or indirectly via defined subsets of immune-activating or immune-modulatory dendritic cells (DCs) in the gut associated lymph tissue (GALT) is important. Future in vitro and in vivo experiments will examine DC [34,35,2] that are uniquely positioned for interaction with peptides passing into the GALT and antigen-specific CD3+ and CD4+ T cells that accumulate in interfollicular T cell regions (IFRs). Activation of such cell populations may partly explain the decreases in inflammatory foci in treated or recipients of treated donor cells in addition to inhibition of Th17 effector cells. We have shown that oral immunoactive proteins such as SIRS peptide [6], a-MSH [7], (ACTH) [8] and (SST) [9] can all have immunomodulatory activity in EAE. However, we have not seen reduction of innate immune cytokines TNF-a in recent EAE experiments with oral SIRS peptide, a-MSH, ACTH or SST. Our study shows that a neuropeptide, TRH with intrinsic immune activity, can reduce Th1-like activity, induce Th2-like activity and decrease innate immune cytokines without induction of Treg cell. Thus, oral TRH shows a unique pattern of immunomodulation for an ingested neuropeptide.

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Therapeutically, administration of immuno-active proteins via the gut offers an alternative to systemic application with ease of administration in chronic clinical use [33], patient convenience [40], and would be a great step forward because gut delivery is easy, well tolerated, and inexpensive with a favorable therapeutic index [3,4]. Oral TRH may beneficially interact with the GALT generating immunomodulation and as an endogenous peptide found in humans may have a high therapeutic index. Because oral TRH shows activity in an animal model of multiple sclerosis, it would be a good candidate for the potential treatment of multiple sclerosis. First in human (FIH) trials using oral TRH will examine potential toxicity and immunological effects of this novel immunoactive neuroprotein for the potential treatment of autoimmune disease.

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