The etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.
Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.
The 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.
Citation: Paccagnini D, Sieswerda L, Rosu V, Masala S, Pacifico A, Gazouli M, et al. (2009) Linking Chronic Infection and Autoimmune Diseases: Mycobacterium avium Subspecies paratuberculosis, SLC11A1 Polymorphisms and Type-1 Diabetes Mellitus. PLoS ONE 4(9): e7109. doi:10.1371/journal.pone.0007109
Editor: Stefan Bereswill, Charité-Universitätsmedizin Berlin, Germany
Received: August 13, 2009; Accepted: August 25, 2009; Published: September 21, 2009
Copyright: © 2009 Paccagnini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was made possible due to support under the FP6 European project n. 036812 acronym: Nanomyc, and the Italian PRIN 2007W52X9M_005. Niyaz Ahmed would like to thank the University of Hyderabad for startup grants (OBC/XI plan). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Type 1 diabetes mellitus (T1DM) is a multifactorial autoimmune disease in which T-lymphocytes infiltrate the islets of the pancreas and destroy the insulin-producing beta cell populations . The exact cause of T1DM is not clearly known. However T1DM constitutes interactions of polygenic traits with environmental factors that are not clearly defined in the available literature and it is not known what triggers autoimmunity to self-antigens such as those expressed in the pancreatic islets of Langerhans cells , .
Accumulating line of evidence points to role for Mycobacterium avium subsp. paratuberculosis (MAP) in the development of T1DM as an environmental trigger , , . MAP bacteria have been generally known to harness molecular mimicry as a strategy to avoid clearance . Recently our group observed immune responses to MAP in T1DM patients, thus supporting an infectious cause for T1DM . Moreover, the presence of MAP was confirmed in T1DM patients by culture and was isolated from blood of T1DM patients . It has long been held that genetic susceptibilities, epitope homologies, and endemic bacterial load in the environment might support the case for an infectious trigger, such as MAP, to be the probable agent of T1DM in genetically susceptibility individuals , , , .
Regarding genetic susceptibility, the SLC11A1 gene (previously known as NRAMP1) is a functional and a positional candidate gene that associates with T1DM as well as susceptibility to mycobacterial infections , , , . Additionally, Kissler et al.  demonstrated that SLC11A1 gene silencing using RNAi approach in mice reduced the frequency of T1DM and protected against experimental autoimmune encephalomyelitis, advocating thereby for a role for SLC11A1 in autoimmunity. Moreover, it was recently demonstrated that association of variants of the gene encoding SLC11A1 with T1DM may reflect its function in processing and presentation of islet cell self-antigens to dendritic cells (DCs) .
Thus, non-MHC genes could affect the MHC-restricted T-cell response through altered antigen processing and presentation.
To date, a number of polymorphisms at the SLC11A1 locus have been associated with susceptibility to infectious agents and to autoimmune disorders . Specifically, a 5′(GT)n repeat polymorphism in the promoter region of the SLC11A1 gene seems to be of particular interest, since it has been shown to affect the levels of gene expression . In vitro studies of this polymorphism suggested direct contribution of particular alleles either to autoimmune (allele 3) or to infectious (allele 2) disease susceptibility , . Nevertheless, variants of the SLC11A1 located within the coding region, the introns, and the 3′-UTR have been shown to influence susceptibility to autoimmune disorders and T1DM , .
Our study aimed at examining the association of the SLC11A1 polymorphisms in relation to the presence of MAP infection, with T1DM in patients from Sardinia.
Patients and controls
A total of 131 participants comprising of 59 T1DM patients (28 females and 31 males with age ranging between 18–94 years) and 76 healthy controls (Table 1) were tested for the detection of SLC11A1 polymorphisms and the presence of MAP specific IS900 signature using total DNA extracted from peripheral blood mononuclear cells collected at the Institute of Diabetology, medical clinic of Sassari University, Italy. Informed written consents from patients including other necessary clearances were obtained before blood samples were drawn. Institutional review board of the University of Sassari approved the study.
Briefly, 5 ml blood from patients was centrifuged and plasma supernatant used in ELISA. Remaining plasma samples were aliquoted and stored frozen at −20°C for short-term storage (<6 months) and −80°C for long term storage (>6 months).
To extract MAP DNA, samples were processed by using the lysor instrument - 500 µl of glass beads (Sigma) were added, followed by standard phenol-chloroform extraction and DNA precipitation with absolute ethanol and 10M ammonium acetate; the pellet was washed with 70% ethanol and resuspended in 70 µl of TE buffer.
Human genomic DNA extraction for SCL11A1 gene amplification was performed as previously published .
MAP DNA detection
MAP DNA detection was performed by PCR as previously reported , . Sequence analysis of the amplicons confirmed IS900 identity. For SCL11A1 polymorphisms, 100 ng of template (genomic) DNA, obtained from blood cells, was amplified. The primer sequences and PCR cycling parameters that we used were previously reported .
Nine polymorphisms (-274C/T, D543N G/A, 823C/T, -237C/T, INT4G/C, 577-18G/A, A318V C/T, 1465-85G/A, and 1729+55del4) were genotyped across NRAMP1 . Candidates were genotyped across these polymorphisms by PCR restriction fragment length polymorphism (PCR-RFLP), in which primers and restriction enzymes were used as previously described . Restriction enzyme digestion products were resolved by electrophoresis on 3% agarose gels stained with ethidium bromide.
Statistical analysis was performed by using the Chi square test with Yate's correction. Inference was aided by GraphPad InStat (version 3.00, GraphPad Software, Inc., San Diego, CA, USA)
MAP DNA was amplified from the blood of 33 out of 59 T1DM patients (55.9%) whereas MAP was detected only in 18 out of 79 healthy controls (22.7%) indicating a statistically highly significant difference (Table 1).
SLC11A1 allele frequencies
SLC11A1 allele frequencies for 274 C/T polymorphism differed significantly between T1DM patients and control group, this polymorphism was significantly associated with diabetes type 1, in particular allele 1 and 2 generated a P value of <0.0005 (Table 1).
There was no significant difference in the allele frequencies of the other 8 polymorphisms for 823 G/T, 577-18G/A, A318V and 1465-85G between T1DM patients and control group (Table 1).
Bivariate and multivariate logistic regression analysis was very significant both for the presence of MAP and SLC11A1 274C/T genotype to predict diabetes status among patients with T1DM and non-diabetic controls as shown in Table 2. In particular, allele 2 alone showed an odds ratio of 2.7 (1.2, 6.3) in a bivariate model and 2.7 (1.2, 6.3) in a multivariate model whereas allele 1 showed an odds ratio of 7.9 (2.7, 23.5) in a bivariate model and 9.4 (2.9, 30.3) in a multivariate model.
SLC11A1 gene has a vital function in the pathway of macrophage activation . It is involved in the expression of chemokines, interleukin-1β, inducible nitric oxide synthase, MCH II molecules, and TNF-α , . The localization of SLC11A1 is within the late endosomal/lysosomal compartments in phagocytes and it has been hypothesised to perform a transporter function as a divalent cation , . Recent reports show a role of the SLC11A1 gene in different autoimmune diseases such as Crohn's disease , rheumatoid arthritis and juvenile rheumatoid arthritis ,  multiple sclerosis , diabetes type 1  and infectious diseases including tuberculosis , ,  and leprosy , . There is evidence that SLC11A1 gene is involved in modifying susceptibility to T1DM as shown by RNA silencing that inversely correlated with increased susceptibility to infection .
Antigen presenting cells have long been considered associated to diabetes susceptibility ,  due to their exclusive expression of MHC class II molecules, the major genetic factors underlying T1DM. Different workers have identified diabetes-associated genes that are also involved in antigen-processing pathways , . SLC11A1 functions as a membrane transporter of divalent cations but its mechanism of mediating natural resistance to bacterial and parasitic infections remains unclear , . Previous studies on the innate response of macrophages during intracellular bacterial infection suggested that SLC11A1-mediated deprivation of divalent cations might change the phagosomal microenvironment that impairs the pathogenesis of intracellular pathogens . Dai et al. found that SLC11A1 contributes to enhanced phagosomal acidification in macrophages and dendritic cells . Based on these data, Dai et al.  suggest that SLC11A1-mediated changes in antigen processing within the vesicular system may have general effects in autoimmune as well as in infectious diseases.
MAP is the causative agent of Johne's disease in ruminants  and it has been associated with Crohn's disease , . We have previously reported the presence of MAP DNA in the PBMC of T1DM patients . These finding were confirmed by detecting MAP specific antibodies in the blood of T1DM patients but not in type 2 diabetic patients and controls . More recently two MAP strains were isolated from the blood of T1DM patients by our group .
In this study we report the novel association of T1DM with the 274 C/T polymorphism within the SLC11A1 gene and the presence of MAP DNA in Sardinian T1DM patients. This polymorphism may be the link of a permissive infection by MAP in these people. Recent studies have reported that the 4-bp TGTG deletion located 55 nucleotides downstream of the last codon in exon 15 (1729 +del55del4) is associated with tuberculosis susceptibility in Chinese children . We did not find any statistical difference at this locus between T1DM and healthy controls. SCLA11 polymorphisms were analyzed also by stratification by sex and no differences were observed (data not shown). It may be possible that polymorphisms in different loci of the SCL11A1 gene may confer susceptibility to different intracellular pathogens although genetically very close. Further studies are necessary in order to elucidate the role of SCL11A1 polymorphisms and how it may influence MAP infection in humans.
We would like to thank prof Giovanni Fadda and prof Seyed Hasnain for discussion and encouragement.
Conceived and designed the experiments: LAS. Performed the experiments: DP VR SM. Analyzed the data: LS Ji NA SZ LAS. Contributed reagents/materials/analysis tools: LS AP MG. Wrote the paper: MG NA LAS.
- 1. Akerblom HK, Vaarala O, Hyöty H, Ilonen J, Knip M (2002) Environmental factors in the etiology of type 1 diabetes. Am J Med Genet 115: 18–29.
- 2. Dow CT (2006) Paratuberculosis and Type I diabetes: is this the trigger? Med Hypotheses 67: 782–5.
- 3. Knip M, Veijola R, Virtanen SM, Hyoty H, Vaarala O, et al. (2005) Environmental triggers and determinants of type 1 diabetes. Diabetes 54: 125–136.
- 4. Rosu V, Ahmed N, Paccagnini D, Gerlach G, Fadda G, et al. (2009) Specific immunoassays confirm association of Mycobacterium avium subsp. paratuberculosis with Type-1 but not Type 2 diabetes mellitus. PLoS ONE 4(2): e4386. Epub 2009 Feb 10.
- 5. Sechi LA, Paccagnini D, Salza S, Pacifico A, Ahmed N, et al. (2008) Mycobacterium avium subsp. paratuberculosis bacteraemia in Type-1 diabetes cases: An infectious trigger? Clin Inf Dis 46: 148–149.
- 6. Polymeros D, Bogdanos DP, Day R, Arioli D, Vergani D, et al. (2006) Does cross-reactivity between Mycobacterium avium paratuberculosis and human intestinal antigens characterize Crohn's disease? Gastroenterology 131: 85–96.
- 7. Sechi LA, Rosu V, Pacifico A, Fadd GA, Ahmed N, et al. (2008) Humoral immune responses of Type-1 Diabetes patients to M. avium subspecies paratuberculosis lend support to the infectious trigger hypothesis. Clin Vaccine Immunol 15: 315–26.
- 8. Daneman D (2006) Type 1 diabetes. Lancet 367: 847–58.
- 9. Guarner F, Bourdet-Sicard R, Brandtzaeg P, Gill HS, McGuirk P, et al. (2006) Mechanisms of disease: the hygiene hypothesis revisited. Nat Clin Pract Gastroenterol Hepatol 3: 275–84.
- 10. Rook GA, Adams V, Hunt J, Palmer R, Martinelli R, Brunet LR (2004) Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders. Springer Semin Immunopathol 25: 237–255.
- 11. Blackwell JM, Barton CH, White JK, Searle S, Baker AM, et al. (1995) Genomic organization and sequence of the human NRAMP gene: identification and mapping of a promoter region polymorphism. Mol Med 1: 194–205.
- 12. Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, et al. (1998) Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 338: 640–644.
- 13. Nishino M, Ikegami H, Fujisawa T, Kawaguchi Y, Kawabata Y, et al. (2005) Functional polymorphism in Z-DNA-forming motif of promoter of SLC11A1 gene and type 1 diabetes in Japanese subjects: association study and meta-analysis. Metabolism 54: 628–33.
- 14. Sechi LA, Gazouli M, Sieswerda L, Molicotti P, Ahmed N, et al. (2006) Relationship between Crohn's disease, infection with Mycobacterium avium sub. paratuberculosis and Slc11a1 gene polymorphisms in Sardinian patients. World Journal of Gastroenterology 12: 7161–7164.
- 15. Kissler S, Stern P, Takahashi K, Hunter K, Peterson LB, et al. (2006) In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nat Genet 38: 479–83.
- 16. Dai YD, Marrero IG, Gros P, Zaghouani H, Wicker LS, et al. (2009) Slc11a1 enhances the autoimmune diabetogenic T-cell response by altering processing and presentation of pancreatic islet antigens. Diabetes 58: 156–6.
- 17. Blackwell JM, Searle S (1999) Genetic regulation of macrophage activation: understanding the function of Nramp1 ( = Ity/Lsh/Bcg). Immunol Lett 65: 73–80.
- 18. Takahashi K, Satoh J, Kojima Y, Negoro K, Hirai M, et al. (2004) Promoter polymorphism of SLC11A1 (formerly NRAMP1) confers susceptibility to autoimmune type 1 diabetes mellitus in Japanese. Tissue Antigens 63: 231–236.
- 19. Rosu V, Ahmed N, Paccagnini D, Pacifico A, Zanetti S, et al. (2008) Mycobacterium avium subspecies paratuberculosis is not associated with type-2 diabetes mellitus. Ann Clin Microbiol Antimicrob Apr 22 ;7: 9.
- 20. Canonne-Hergaux F, Gruenheid S, Govoni G, Gros P (1999) The Nramp1 protein and its role in resistance to infection and macrophage function. Proc Assoc Am Physicians 111: 283–289.
- 21. Alter-Koltunoff M, Ehrlich S, Dror N, Azriel A, Eilers M, et al. (2003) Nramp1-mediated innate resistance to intraphagosomal pathogens is regulated by IRF-8, PU.1, and Miz-1. J Biol Chem 278: 44025–32.
- 22. Blackwell JM, Searle S, Mohamed H, White JK (2003) Divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the Ity/Lsh/Bcg/Nramp1/Slc11a1 gene story. Immunol Lett 85: 197–203.
- 23. Hofmeister A, Neibergs HL, Pokorny RM, Galandiuk S (1997) The natural resistance-associated macrophage protein gene is associated with Crohn's disease. Surgery 122: 173–8.
- 24. Yang YS, Kim SJ, Kim JW, Koh EM (2000) NRAMP1 gene polymorphisms in patients with rheumatoid arthritis in Koreans. J Korean Med Sci 15: 83–87.
- 25. Yen JH, Lin CH, Tsai WC, Ou TT, Wu CC, et al. (2006) Natural resistance-associated macrophage protein 1 gene polymorphisms in rheumatoid arthritis. Immunol Lett 102: 91–97.
- 26. Comabella M, Altet L, Peris F, Villoslada P, Sanchez A, et al. (2004) Genetic analysis of SLC11A1 polymorphisms in multiple sclerosis patients. Mult Scler 10: 618–20.
- 27. Bellamy R (2003) Susceptibility to mycobacterial infections: the importance of host genetics. Genes Immun 4: 4–11.
- 28. Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, et al. (1998) Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 338: 640–644.
- 29. Jin J, Sun L, Jiao W, Zhao S, Li H, et al. (2009) SLC11A1 (Formerly NRAMP1) gene polymorphisms associated with pediatric tuberculosis in China. Clin Infect Dis 48: 733–8.
- 30. Abel L, Sanchez FO, Oberti J, Thuc NV, Hoa LV, et al. (1998) Susceptibility to leprosy is linked to the human NRAMP1 gene. J Infect Dis 177: 133–145.
- 31. Ferreira FR, Goulart LR, Silva HD, Goulart IM (2004) Susceptibility to leprosy may be conditioned by an interaction between the NRAMP1 promoter polymorphisms and the lepromin response. Int J Lepr Other Mycobact Dis 72: 457–467.
- 32. O'Keeffe M, Brodnicki TC, Fancke B, Vremec D, Morahan G, et al. (2005) Fms-like tyrosine kinase 3 ligandadministration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development. Int Immunol 17: 307–314.
- 33. Piganelli JD, Martin T, Haskins K (1998) Splenic macrophages from the NOD mouse are defective in the ability to present antigen. Diabetes 47: 1212–1218.
- 34. Litherland SA, Grebe KM, Belkin NS, Paek E, Elf J, et al. (2005) Nonobese diabetic mouse congenic analysis reveals chromosome 11 locus contributing to diabetes susceptibility, macrophage STAT5 dysfunction, and granulocyte-macrophage colonystimulating factor overproduction. J Immunol 175: 4561–4565.
- 35. Chacon O, Bermudez LE, Barletta RG (2004) Johne's disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Annu Rev Microbiol 58: 329–363.
- 36. Sechi LA, Scanu AM, Molicotti P, Cannas S, Mura M, et al. (2005) Detection and Isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia. Am J Gastroenterol 100: 1529–1536.