A role of N. aromaticivorans
or related alphaproteobacteria in human PBC was recently suggested based on their phylogenetic relationship with mitochondria, the target organ of PBC patients, particularly the conserved antibody epitope expressed by the enzyme PDC E2 (Kaplan, 2004; Padgett et al., 2005; Selmi et al., 2003
). In the current study, we hypothesized that NKT cells, a specialized lymphocyte population that is enriched in the liver might contribute to the pathogenesis of PBC. NKT cells were shown to specifically recognize the cell wall glycosphingolipids of various bacterial strains from the genus Sphingomonas
, which includes N. aromaticivorans
, through their semi-invariant CD1d-restricted TCR. By inoculating N. aromaticivorans
to mice, we established a model of liver disease resembling PBC and we further demonstrated the role of NKT cells in initiating a liver-specific autoimmune process mediated by T cells as well as in helping the production of signature anti-PDC E2 antibodies.
Inoculation of N. aromaticivorans
, whether intravenously or orally, consistently resulted in the development of chronic liver disease with liver hypertrophy and massive lymphocytic infitration in common strains of mice. Congestive splenomegaly also developed, possibly as an indirect consequence of liver hypertrophy. Key diagnostic elements of PBC were recorded, particularly in the NOD 1101 strain, including the striking infiltration of small bile ducts, their expression of MHC class II molecules, the formation of lympho-epithelioid granulomas, and the presence of eosinophils. Other lesions such as venulitis, hepatocyte lesions, and plasma cell expansion have been more often reported in autoimmune hepatitis, a poorly understood disease entity, or in graft versus host reactions (Czaja, 1994; Scheuer, 1998; Sherlock, 1998; Mieli-Vergani and Vergani, 2004
). A notable difference with human PBC, however, was the absence of marked fibrosis in the mouse system. Fibrosis, which is thought to be a tissue reaction to the bile extravasation associated with bile duct epithelium destruction, may involve Th2 cytokines that are suppressed in the mouse system, particularly in the NOD background (Delovitch and Singh, 1997
). Indeed, exposure to N. aromaticivorans
induced relatively little Th2 cytokines from mouse NKT cells in vitro compared with purified glycosphingolipid antigens (data not shown). Collectively, therefore, our findings support the notion that diverse susceptibility or resistance genes influence the final expression of the N. aromaticivorans
-induced liver disease. Future studies will explore the genetics of N. aromaticivorans-
induced liver disease in greater detail.
Importantly, liver disease was transferred upon injection of conventional T cells collected from the liver or the spleen of chronically diseased mice, into naive irradiated or SCID syngeneic recipients. While T cell responses to PDC-E2 have been previously demonstrated in PBC patients, consistent with a breakdown of tolerance to this antigen, their pathogenic nature remains uncertain. In contrast, the transfer experiment provided definitive proof of T cell-mediated autoimmunity as a cause of disease, although the autoantigens recognized by disease-causing T cells in this model remain to be elucidated. It is noteworthy that another bile duct disease reminiscent of biliary atresia, which develops in some pediatric patients, was recently observed in mice after rotavirus infection and could be transferred with T cells (Mack et al., 2006
Completing the cellular response against liver antigens, IgG autoantibodies against PDC-E2, the serological signature of PBC, were observed in all mouse strains examined. We detected anti-dsDNA as well, although at relatively low titer and frequency compared with mouse models of lupus such as NZBxNZW or MRL (data not shown). Nevertheless, it is worth noting that anti-DNA antibodies have also been reported in a fraction of PBC patients and that multiple forms of autoimmunity, including lupus, type I diabetes, Sjogren syndrome, or celiac disease can be found among relatives of some PBC patients (Kaplan and Gershwin, 2005
NKT cells specifically recognize N. aromaticivorans cell wall glycosphingolipids and, in the absence of LPS to activate TLRs, dominate the innate immune response. While they significantly accelerate the clearance of N. aromaticivorans, they are not required for recovery. Therefore, their activation upon microbial encounter may be deleterious for the host, providing innate signals that contribute to the breakdown of tolerance and unleash autoimmune effector cells. Indeed, CD1d-deficient mice were largely protected against manifestations of autoimmunity including humoral and cellular responses. Mixed chimera experiments further suggested that NKT cells could directly help CD1d-expressing but not CD1d-deficient anti-PDC-E2 B cells to switch to IgG2a. This finding would imply that B cells recognizing PDC-E2 can capture some glycosphingolipid ligand, presumably attached to the same microbial cell wall fragment to which the enzyme is appended by a lipoyl moiety, for loading onto CD1d in the lysosomal compartment and presentation at the cell surface. Such B cells would then receive NKT cell help, including CD40L and cytokines to induce isotype switch and perhaps somatic hypermutation as well. Consistent with this scenario, CD1d-deficient B cells mainly produced the T helper-independent IgM and IgG3 isotypes.
While NKT cells were clearly required to initiate the cellular and humoral components of disease, they were not necessary at the late chronic phase of the immunopathological process, as evidenced by disease transfer in the absence of NKT cells in the cellular inoculum or in the absence of CD1d in the recipients. This finding supports the notion that NKT cells mainly contribute to the innate phase of the response by provoking the breakdown of tolerance. The expansion of PDC-E2-specific B cells might in turn contribute to the expansion of PDC-E2-specific T cells through efficient surface antibody-mediated uptake of PDC-E2 and subsequent presentation of peptide/MHC complexes (Lin et al., 1991
). Other autoreactive B and T cells might be subsequently expanded through a similar process spreading to physically associated or proximal autoantigens (Lehmann et al., 1992; Vanderlugt et al., 1998
). In that respect, it is notable that very low levels of N. aromaticivorans
, as detected by PCR, persisted for several weeks after the bulk of the original microbial inoculum was cleared, perhaps providing continuous fuel for NKT cell activation, and that antibiotic treatment during that period prevented the evolution of chronic cellular and humoral autoimmunity. Consistent with a requirement for prolonged exposure to microbial elements, injection of heat-killed N. aromaticivorans
induced only transient anti-PDC-E2 response and liver lesions (data not shown). In that context, the preferential activation of NKT cells in the liver, where NKT cells are abundant, crawling along sinusoid endothelial cells and where N. aromaticivorans
also accumulates, may explain the biased autoreactivity toward autoantigens exposed in the liver environment and, ultimately, the severe organ-specific manifestations of N. aromaticivorans
infection. The nature of these pathogenic autoantigens, however, remains to be elucidated.
, like many other strains of the genus Sphingomonas
in the Sphingomonadaceae
family, is an ubiquitous organism found in various marine, terrestrial, and aerosol environments (Barbeau et al., 1996; Cavicchioli et al., 1999; Shi et al., 2001; Brodie et al., 2007
). It is also detected by PCR in human feces (Selmi et al., 2003
). While this genus only occasionally causes severe infection in immunodeficient individuals (Al-Anazi et al., 2008; Ensminger et al., 2006; Charity and Foukas, 2005
), it is likely that most individuals are chronically exposed to these microbial organisms. Our findings raise the question of whether and how aberrant activation of NKT cells might occur in response to this or related organism in the liver of some genetically predisposed patients, ultimately causing or promoting primary biliary cirrhosis. The ability of some strains such as N. aromaticivorans
to degrade xenobiotic and steroid aromatic compounds may be an important factor (Pinyakong et al., 2003; Shi et al., 2001
). Chemical xenobiotics might modify PDC-E2 or other self antigens and lead to loss of tolerance in individuals predisposed to autoimmunity (Gluud, 2002; Amano et al., 2005; Ju, 2005
). For example, guinea pigs injected with 6-bromohexanoate developed cholangitis and antimitochondrial antibodies (Leung et al., 2007
). Environmental factors have been suggested to induce or exacerbate PBC (Triger, 1980; Uibo and Salupere, 1999; Watson et al., 1995
), including urinary tract infections, tobacco, reproductive hormones, and exposure to nail polish. Other studies found increased prevalence of PBC near toxic waste sites (Ala et al., 2006
). Based on the mouse infection studies presented here and the previous studies showing crossreactivity of anti-PDC-E2 autoantibodies to N. aromaticivorans
in humans with PBC, we propose the following conclusions. First, a breakdown of tolerance is a critical component of PBC, as shown by the appearance of autoantibodies against PDC-E2 and the ability of T cells to transfer liver disease. These features also characterize other mouse models of PBC, including the NODc3c4 strain, IL2Rα-deficient mice, and mice transgenically expressing a dominant-negative form of the TGFβ receptor II (Irie et al., 2006; Koarada et al., 2004; Aoki et al., 2006; Wakabayashi et al., 2006; Oertelt et al., 2006
). Second, by virtue of coexpressing potent NKT ligands and 2-oxo-dehydrogenase enzymes with high degree of homology to their mitochondrial counterparts, N. aromaticivorans
has a unique ability to induce PBC. Thus, the present study provides an experimental demonstration and a mechanistic dissection of the role of NKT cells in the transition from early microbially driven immune responses to a stage of autonomous, microbial-independent, T and B cell autoimmunity. These findings warrant further studies on the role of the commensal flora, environmental microbes, and CD1d-restricted NKT cells in human PBC and its different experimental models. We do not suggest that N. aromaticivorans
is the only possible cause of PBC, but rather that it presents a model that has notable implications for immunological tolerance and autoimmunity.