Journal of the Neurological Sciences 349 (2015) 249–250
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Short communication
Role of interferon-beta in Mycobacterium avium subspecies paratuberculosis antibody response in Sardinian MS patients Jessica Frau a,⁎, Davide Cossu b, Giancarlo Coghe a, Lorena Lorefice a, Giuseppe Fenu a, Gianluca Porcu a, Claudia Sardu c, Maria Rita Murru a, Stefania Tranquilli a, Maria Giovanna Marrosu a, Leonardo Antonio Sechi b, Eleonora Cocco a a b c
Multiple Sclerosis Centre, Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Italy Department of Biomedical Sciences, Section of Experimental and Clinical Microbiology, University of Sassari, Italy Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Italy
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 12 December 2014 Accepted 2 January 2015 Available online 9 January 2015 Keywords: Mycobacterium avium subspecies paratuberculosis Multiple sclerosis Interferon-beta Molecular mimicry Endocellular pathogen Immunomodulation
a b s t r a c t Background: Mycobacterium avium subspecies paratuberculosis (MAP) is associated with MS in Sardinia. Because anti-MAP antibodies (Abs) were more frequent in interferon-beta treated patients, we hypothesize that interferon-beta could interact with the immune system. Methods: Anti-MAP Abs were searched in the blood of 89 patients before commencing interferon-beta and after at least six months. Results: Anti-MAP Abs were detected before and during treatment in 18.7% and 34.7% of patients, respectively. Twenty-three (20.5%) patients became positive during therapy, and 5 (4.4%) patients became negative (p = 0.001). Conclusions: The study supports the hypothesis that interferon-beta could interact with the immune system, enhancing the immunological response against MAP.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction The pathogenesis of multiple sclerosis (MS) is multifactorial, involving both infectious and environmental factors [1]. Infectious agents could act as a “trigger” in autoimmunity within the “molecular mimicry” model. Currently, the most implicated infectious agent is the Epstein– Barr virus (EBV). [1] In recent years, in Sardinia, an island with a very high prevalence of MS [2], Mycobacterium avium subspecies paratuberculosis (MAP) was identified as being associated with the disease [3–5]. In a study involving 436 patients and 264 healthy controls, antibodies versus the antigen MAP2694 were found in 33.7% and 3.8%, respectively (p = 2.59 × 10− 23) [5]. MAP is an endocellular pathogen, which may interact with the immune system through cross-reactivity and molecular mimicry. This positivity was more frequent in patients who had received disease modifying therapy for MS at the time of sample collection [5]. Interferon-beta, a cytokine involved in viral and endocellular infections [6], was the drug most represented within the group of patients. Therefore, we hypothesized that this therapy could interact with the host immune system and enhance the immunological response to MAP. ⁎ Corresponding author. Tel.: +39 0706092930; fax: +39 0706092929. E-mail address: [email protected] (J. Frau).
http://dx.doi.org/10.1016/j.jns.2015.01.004 0022-510X/© 2015 Elsevier B.V. All rights reserved.
The aim of this work was to understand the role of interferon-beta in the immunological response to MAP by studying the presence of antiMAP2694 antibodies (Abs) before and during interferon-beta therapy in a subset of MS patients. 2. Materials and methods Patients with MS according to the McDonald 2010 criteria [7] were enrolled before commencing interferon-beta treatment and after obtaining informed written consent. The local ethics committee approved the study. Each patient was free of therapy for at least three months, and blood samples were obtained at the time of enrolment and after at least six month of treatment. All the samples were analyzed for the presence of anti-MAP2694 Abs with indirect ELISA, as described previously [3]. 2.1. Statistical analysis The McNemar test was used to evaluate the difference between two groups of patients. The first group showed Abs variation from positivity in the first sample to negativity in the second sample. The second group showed Abs variation that was initially negative and then became positive.
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J. Frau et al. / Journal of the Neurological Sciences 349 (2015) 249–250
Eighty-nine patients were enrolled in the study, 60 females and 29 males (F:M = 2.0). The mean duration of therapy between the first and the second sample collection was 20 months (minimum: 7, maximum: 62). At the beginning of the study Abs were found in 21 subjects (18.69%), while the second sample was positive in 39 subjects (34.71%). In particular, 23 patients (20.47%) became positive after interferon-beta therapy and only 5 (4.45%) patients who were positive at the first sample collection time became negative (p value = 0.001). The majority (45 patients, 40.05%) was always negative, while a group of positive patients did not change status (16 subjects, 14.24%) (see Table 1).
mechanisms could be the responsible for anti-MAP Abs production and for their higher frequency in subjects under interferon-beta treatment. Our study confirms previous data showing a higher frequency of anti-MAP Abs in patients with interferon-beta therapy. In particular, the negative/positive status could change over an individual's lifetime. After commencing interferon-beta treatment, I found that a change from negative to positive is much more frequent than from positive to negative. It is worth noting that more than half of patients who become positive had a higher optic density in ELISA. This supports the hypothesis that this drug could interact with the host immune system enhancing the immunological response to an endocellular pathogen such as MAP. The anti-MAP Abs negativity observed in a small number of persons could be explained as a physiological fluctuation in those with low Abs titre. This phenomenon has previously been observed in clinical practice for other common viruses. Our results are of particular importance for the interpretation of anti-MAP Abs test results. The timing of the sample collection and the possible presence of concomitant therapy should be taken into account in the interpretation of these results.
4. Discussion
Conflict of interest statement
Table 1 Research of anti-MAP Abs before (1° sample) and during (2° sample) treatment with interferon-beta. 1° sample
2° sample
Positive Negative
Positive
Negative
16 (14.24%) 23 (20.47%)
5 (4.45%) 45 (40.05%)
3. Results
MAP is an endocellular pathogen responsible for Johne's disease in ruminants. It is not directly responsible for human diseases, although in Sardinia it is associated with autoimmune diseases such as MS [3–5]. The route of infection is fecal–oral. MAP colonizes the mucosa associated with lymphoid tissue of the small intestine, from where it could interact with the immune system. It is likely that MAP has a role in autoimmunity via molecular mimicry whereby its persistence presence triggers autoantibodies [4]. In our previous work, no associations were found between the presence of MAP Abs and age, age at onset, duration of disease, EDSS, previous immunomodulant and immunosuppressive therapy and clinical course. However, the frequency of MAP Abs was higher in patients exposed to immunomodulatory treatment at the sample collection time, and the most represented therapy was interferon-beta [5]. It is noteworthy that interferon-beta is a cytokine involved in infections, especially due to virus and endocellular agents [6]. It should also be noted that the first clinical trial of interferon-beta was based on the hypothesis of a viral role in the pathogenesis of MS and on the antiviral properties of the molecule [8]. The mechanisms of action for interferon-beta are not fully understood. However, this treatment could regulate many genes, some of which have anti-viral action and could modify the level and the function of some cell populations [8]. In particular, an increase of T-helper 2 cells, T regulatory cells, NK regulatory cells, dendritic cells and monocytes and a decrease of CD4 and CD8 T cells, B cells and NK cells were observed [9]. Moreover, this therapy could enhance both blood leukocytes and serum levels of B-cellactivating factor (BAFF), which promote the inflammation supporting B-cell survival [10]. It has been demonstrated that the induction of BAFF could lead to the production of autoantibodies and interferonbeta neutralizing antibodies [10]. Moreover, the upregulation of BAFF levels inducted by interferon-beta and, consequently, of autoreactive B-cells could lead to worsening in Devic's disease [10]. Similar
There is no conflict of interest. Acknowledgments This investigation was supported by grants from the following: the Sardinian Region (protocol number 2009ZYECWZ) L.R.7 2009, 2010 Progetti di ricerca di base, Prin Miur Programmi Di Ricerca Scientifica Di Rilevante Interesse Nazionale b Bando Prin 2009N protocollo: 2009ZYECWZ and Fondazione Banco di Sardegna (grant number: 723/ 2012-0206). References [1] Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann Neurol 2007;61(4):288–99. [2] Cocco E, Sardu C, Massa R, et al. Mult Scler 2011;17(11):1282–9. [3] Cossu D, Cocco E, Paccagnini D, et al. Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients. PLoS One 2011; 6(4):e18482. [4] Cossu D, Masala S, Cocco E, et al. Are Mycobacterium avium subsp. paratuberculosis and Epstein–Barr virus triggers of multiple sclerosis in Sardinia? Mult Scler 2012; 18(8):1181–4. [5] Frau J, Cossu D, Coghe G, et al. Mycobacterium avium subsp. paratuberculosis and multiple sclerosis in Sardinian patients: epidemiology and clinical features. Mult Scler 2013;19(11):1437–42. [6] Nagarajan U. Induction and function of IFNβ during viral and bacterial infection. Crit Rev Immunol 2011;31(6):459–74. [7] Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69(2):292–302. [8] Rudick RA, Goelz SE. Beta-interferon for multiple sclerosis. Exp Cell Res 2011; 317(9):1301–11. [9] Kantor AB, Deng J, Waubant E, et al. Identification of short-term pharmacodynamic effects of interferon-beta 1a in multiple sclerosis subjects with broad-based phenotypic profiing. J Neuroimmunol 2007;188(1–2):103–16. [10] Krumbholz M, Faber H, Steinmeyer F, et al. Interferon-beta increases BAFF levels in multiple sclerosis: implication for B cell autoimmunity. Brain 2008;131(Pt 6): 1455–63.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Short communication
Role of interferon-beta in Mycobacterium avium subspecies paratuberculosis antibody response in Sardinian MS patients Jessica Frau a,⁎, Davide Cossu b, Giancarlo Coghe a, Lorena Lorefice a, Giuseppe Fenu a, Gianluca Porcu a, Claudia Sardu c, Maria Rita Murru a, Stefania Tranquilli a, Maria Giovanna Marrosu a, Leonardo Antonio Sechi b, Eleonora Cocco a a b c
Multiple Sclerosis Centre, Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Italy Department of Biomedical Sciences, Section of Experimental and Clinical Microbiology, University of Sassari, Italy Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Italy
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 12 December 2014 Accepted 2 January 2015 Available online 9 January 2015 Keywords: Mycobacterium avium subspecies paratuberculosis Multiple sclerosis Interferon-beta Molecular mimicry Endocellular pathogen Immunomodulation
a b s t r a c t Background: Mycobacterium avium subspecies paratuberculosis (MAP) is associated with MS in Sardinia. Because anti-MAP antibodies (Abs) were more frequent in interferon-beta treated patients, we hypothesize that interferon-beta could interact with the immune system. Methods: Anti-MAP Abs were searched in the blood of 89 patients before commencing interferon-beta and after at least six months. Results: Anti-MAP Abs were detected before and during treatment in 18.7% and 34.7% of patients, respectively. Twenty-three (20.5%) patients became positive during therapy, and 5 (4.4%) patients became negative (p = 0.001). Conclusions: The study supports the hypothesis that interferon-beta could interact with the immune system, enhancing the immunological response against MAP.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction The pathogenesis of multiple sclerosis (MS) is multifactorial, involving both infectious and environmental factors [1]. Infectious agents could act as a “trigger” in autoimmunity within the “molecular mimicry” model. Currently, the most implicated infectious agent is the Epstein– Barr virus (EBV). [1] In recent years, in Sardinia, an island with a very high prevalence of MS [2], Mycobacterium avium subspecies paratuberculosis (MAP) was identified as being associated with the disease [3–5]. In a study involving 436 patients and 264 healthy controls, antibodies versus the antigen MAP2694 were found in 33.7% and 3.8%, respectively (p = 2.59 × 10− 23) [5]. MAP is an endocellular pathogen, which may interact with the immune system through cross-reactivity and molecular mimicry. This positivity was more frequent in patients who had received disease modifying therapy for MS at the time of sample collection [5]. Interferon-beta, a cytokine involved in viral and endocellular infections [6], was the drug most represented within the group of patients. Therefore, we hypothesized that this therapy could interact with the host immune system and enhance the immunological response to MAP. ⁎ Corresponding author. Tel.: +39 0706092930; fax: +39 0706092929. E-mail address: [email protected] (J. Frau).
http://dx.doi.org/10.1016/j.jns.2015.01.004 0022-510X/© 2015 Elsevier B.V. All rights reserved.
The aim of this work was to understand the role of interferon-beta in the immunological response to MAP by studying the presence of antiMAP2694 antibodies (Abs) before and during interferon-beta therapy in a subset of MS patients. 2. Materials and methods Patients with MS according to the McDonald 2010 criteria [7] were enrolled before commencing interferon-beta treatment and after obtaining informed written consent. The local ethics committee approved the study. Each patient was free of therapy for at least three months, and blood samples were obtained at the time of enrolment and after at least six month of treatment. All the samples were analyzed for the presence of anti-MAP2694 Abs with indirect ELISA, as described previously [3]. 2.1. Statistical analysis The McNemar test was used to evaluate the difference between two groups of patients. The first group showed Abs variation from positivity in the first sample to negativity in the second sample. The second group showed Abs variation that was initially negative and then became positive.
250
J. Frau et al. / Journal of the Neurological Sciences 349 (2015) 249–250
Eighty-nine patients were enrolled in the study, 60 females and 29 males (F:M = 2.0). The mean duration of therapy between the first and the second sample collection was 20 months (minimum: 7, maximum: 62). At the beginning of the study Abs were found in 21 subjects (18.69%), while the second sample was positive in 39 subjects (34.71%). In particular, 23 patients (20.47%) became positive after interferon-beta therapy and only 5 (4.45%) patients who were positive at the first sample collection time became negative (p value = 0.001). The majority (45 patients, 40.05%) was always negative, while a group of positive patients did not change status (16 subjects, 14.24%) (see Table 1).
mechanisms could be the responsible for anti-MAP Abs production and for their higher frequency in subjects under interferon-beta treatment. Our study confirms previous data showing a higher frequency of anti-MAP Abs in patients with interferon-beta therapy. In particular, the negative/positive status could change over an individual's lifetime. After commencing interferon-beta treatment, I found that a change from negative to positive is much more frequent than from positive to negative. It is worth noting that more than half of patients who become positive had a higher optic density in ELISA. This supports the hypothesis that this drug could interact with the host immune system enhancing the immunological response to an endocellular pathogen such as MAP. The anti-MAP Abs negativity observed in a small number of persons could be explained as a physiological fluctuation in those with low Abs titre. This phenomenon has previously been observed in clinical practice for other common viruses. Our results are of particular importance for the interpretation of anti-MAP Abs test results. The timing of the sample collection and the possible presence of concomitant therapy should be taken into account in the interpretation of these results.
4. Discussion
Conflict of interest statement
Table 1 Research of anti-MAP Abs before (1° sample) and during (2° sample) treatment with interferon-beta. 1° sample
2° sample
Positive Negative
Positive
Negative
16 (14.24%) 23 (20.47%)
5 (4.45%) 45 (40.05%)
3. Results
MAP is an endocellular pathogen responsible for Johne's disease in ruminants. It is not directly responsible for human diseases, although in Sardinia it is associated with autoimmune diseases such as MS [3–5]. The route of infection is fecal–oral. MAP colonizes the mucosa associated with lymphoid tissue of the small intestine, from where it could interact with the immune system. It is likely that MAP has a role in autoimmunity via molecular mimicry whereby its persistence presence triggers autoantibodies [4]. In our previous work, no associations were found between the presence of MAP Abs and age, age at onset, duration of disease, EDSS, previous immunomodulant and immunosuppressive therapy and clinical course. However, the frequency of MAP Abs was higher in patients exposed to immunomodulatory treatment at the sample collection time, and the most represented therapy was interferon-beta [5]. It is noteworthy that interferon-beta is a cytokine involved in infections, especially due to virus and endocellular agents [6]. It should also be noted that the first clinical trial of interferon-beta was based on the hypothesis of a viral role in the pathogenesis of MS and on the antiviral properties of the molecule [8]. The mechanisms of action for interferon-beta are not fully understood. However, this treatment could regulate many genes, some of which have anti-viral action and could modify the level and the function of some cell populations [8]. In particular, an increase of T-helper 2 cells, T regulatory cells, NK regulatory cells, dendritic cells and monocytes and a decrease of CD4 and CD8 T cells, B cells and NK cells were observed [9]. Moreover, this therapy could enhance both blood leukocytes and serum levels of B-cellactivating factor (BAFF), which promote the inflammation supporting B-cell survival [10]. It has been demonstrated that the induction of BAFF could lead to the production of autoantibodies and interferonbeta neutralizing antibodies [10]. Moreover, the upregulation of BAFF levels inducted by interferon-beta and, consequently, of autoreactive B-cells could lead to worsening in Devic's disease [10]. Similar
There is no conflict of interest. Acknowledgments This investigation was supported by grants from the following: the Sardinian Region (protocol number 2009ZYECWZ) L.R.7 2009, 2010 Progetti di ricerca di base, Prin Miur Programmi Di Ricerca Scientifica Di Rilevante Interesse Nazionale b Bando Prin 2009N protocollo: 2009ZYECWZ and Fondazione Banco di Sardegna (grant number: 723/ 2012-0206). References [1] Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann Neurol 2007;61(4):288–99. [2] Cocco E, Sardu C, Massa R, et al. Mult Scler 2011;17(11):1282–9. [3] Cossu D, Cocco E, Paccagnini D, et al. Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients. PLoS One 2011; 6(4):e18482. [4] Cossu D, Masala S, Cocco E, et al. Are Mycobacterium avium subsp. paratuberculosis and Epstein–Barr virus triggers of multiple sclerosis in Sardinia? Mult Scler 2012; 18(8):1181–4. [5] Frau J, Cossu D, Coghe G, et al. Mycobacterium avium subsp. paratuberculosis and multiple sclerosis in Sardinian patients: epidemiology and clinical features. Mult Scler 2013;19(11):1437–42. [6] Nagarajan U. Induction and function of IFNβ during viral and bacterial infection. Crit Rev Immunol 2011;31(6):459–74. [7] Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69(2):292–302. [8] Rudick RA, Goelz SE. Beta-interferon for multiple sclerosis. Exp Cell Res 2011; 317(9):1301–11. [9] Kantor AB, Deng J, Waubant E, et al. Identification of short-term pharmacodynamic effects of interferon-beta 1a in multiple sclerosis subjects with broad-based phenotypic profiing. J Neuroimmunol 2007;188(1–2):103–16. [10] Krumbholz M, Faber H, Steinmeyer F, et al. Interferon-beta increases BAFF levels in multiple sclerosis: implication for B cell autoimmunity. Brain 2008;131(Pt 6): 1455–63.
