Importancia de la comunicación en la resiliencia

Nuclear lupus autoantigens trigger autoantibody production and the release of proinflammatory mediators by activating Tlr7 and Tlr9 in B cells and dendritic cells in vitro (Leadbetter et al, 2002, Lau et al, 2005; Means et al, 2005; Barrat et al, 2005; Lovgren et al, 2004; Savarese et al, 2006; Vollmer et al, 2005). Blocking Tlr9 signaling with injections of specific oligodeoxynucleotides could ameliorate lupus nephritis in MRLlpr/lpr mice (Patole et al, 2005; Dong et al, 2005), hence we hypothesized that antagonism of Tlr7 or Tlr7 plus Tlr9 may be effective in vivo.

Injections with TLR7 antagonist IRS661 were started from week 11 of age till 24 weeks. This could substantially reduced autoimmune tissue lung and kidney injury at week 24 of age which is also consistent with the phenotype of Tlr7-deficient MRLlpr/lpr mice (Christensen et al, 2006). These data support a role of Tlr7 for the mechanisms that foster the progression of autoimmune tissue injury in MRLlpr/lpr mice, e.g. the expansion of the autoreactive CD4/CD8 double negative autoreactive T cell population, immune complex-mediated local complement activation, and the local inflammatory response involving macrophage and T cell recruitment (Kotzin et al, 1996; Lipsky et al, 2001;

Singh et al, 2006). TLR7 antagonist, IRS661 significantly reduced the number of CD4/CD8 double negative T cells in spleen, a population which continuously expands in MRLlpr/lpr, because of the inability to delete autoreactive and activated T cells in mice via the interaction of Fas with the Fas-ligand in these mice (Cohen et al, 1991). Autoantibody production is another characteristic feature of MRLlpr/lpr mice and contributes to autoimmune tissue injury via immune complex formation and local complement activation. Interestingly, Tlr7 is particularly required to generate anti-Sm RNP IgG (Christensen et al, 2006) and serum levels of anti-Sm RNP IgG were also reduced with injection of IRS661. Furthermore, we found that IRS661 reduced the serum levels of anti-dsDNA IgG2a and IgG2b. We also observed that, the glomerular deposits of

IgG2a and complement factor C3c were both reduced in IRS661-treated mice, indicating a

role for Tlr7 in the production of selected nephritogenic autoantibodies and immune complex-glomerulonephritis in MRLlpr/lpr mice. In this study we used identical assay systems as reported by Christensen et al, and we found that the data from both the labs consistently show that blockade or lack of Tlr7 does not substantially reduce homogenous nuclear and mitotic ANA staining of Hep2 cells (Christensen et al, 2006). In reports from same lab (Christensen et al, 2005 and 2006), 20-30 % of lpr mice showed speckled nuclear pattern indicating the presence of anti-SmRNP, autoantibodies which was confirmed by western blots for Sm binding activity, which supports our results that inhibition of Tlr7 lupus mice leads to decrease in levels of anti sm-RNP or anti-Sm antibodies. Our data is consistent and supported by results from other groups recently (Christensen et al, 2006) which showed that Tlr7 deficient lpr mice produced anti- DNA/anti-chromatin autoantibodies but not anti Sm-RNP antibodies. The Tlr7 deficient

lpr mice also have lower IgG2a and IgG titres than their Tlr-sufficient wild-type littermates as well as reduced numbers of activated T cells and PDCs (nevertheless, renal disease was decreased only moderately) (Christensen et al, 2006). Another study also showed similar results with lpr mice deficient in MyD88 failed to produce both DNA and Sm-reactive antibodies (Lau et al, 2005). Tlr7 also appears to play a role in activation of B cells that express transgene encoded 564 Igi B cells spontaneously produce antibodies that give cytoplasmic Hep-2 staining pattern and form ICs that deposit in kidney. Tlr7 deficient 564Igi B cells no longer spontaneously produce 564 Igi antibodies (Berland et al, 2006).

Together, the beneficial effects of Tlr7 blockade with IRS661 on autoimmune tissue injury of MRLlpr/lpr mice were associated with a reduction of CD4/CD8 double negative T cells, reduction in levels of pathogenic autoantibodies, and reduction in levels of proinflammatory chemokines in kidney, known to mediate immune cell recruitment into the tissue.

When we were performing these studies in our lab, some new results were published about Yaa mice, as reported by Subramanian et al (2006) and Pisitkun et al (2006), transgenic models with autoimmune accelerator on Y chromosome. The Yaa loci consisting of Tlr7 and 16 other genes from X chromosome which were duplicated on Y chromosome in these mice. Another study had reported Tlr7 transgene founder lines in transgenic BL6 mice, which were showed to have Tlr7-gene dosage dependent disease severity of lupus in such mice and Tlr7 gene dependent increase in RNA antibodies and increased nucleolar and speckled nucleolar patterns by such mice sera in Hep-2 slides (Deane et al, 2007). It also had severe effects on survival of these mice dependent on Tlr7

gene dosage compared to WT-BL6 mice. Altogether the results suggest that Tlr7 is responsible in progression and development of lupus.

Given the opposing effects of Tlr7 and Tlr9 deficiency in MRLlpr/lpr mice (Christensen

et al, 2006; Wu et al, 2006) the effects of Tlr7/Tlr9 dual antagonist IRS954 were quite different. Injections with IRS954 from week 11 to 24 of age improved kidney and lung disease in MRLlpr/lpr mice and no additive effects were observed as compared to IRS661. The effects of IRS954 on lupus nephritis and lung injury are comparable to what has been observed with oligonucleotide antagonists specific for Tlr9 only in the same lupus model (Patole et al, 2005) or in NZB/NZW mice (Dong et al, 2005). These findings support the concept that recognition of endogenous DNA and RNA molecules via Tlr7 and Tlr9 contribute to the progression of autoimmune tissue injury. This concept was developed from studies showing that lupus autoantigens in immune complexes with IgG prepared from autoimmune mice or lupus patients can activate B cells and dendritic cells

in vitro (Leadbetter et al, 2002; Lau et al, 2005; Means et al, 2005; Savarese, 2006; Vollmer et al, 2005). These in vitro experiments and in vivo studies with Tlr9 antagonists are inconsistent with the aggravated phenotype of Tlr9-deficient MRLlpr/lpr mice, and have questioned the specificity of the antagonists and the assay systems used for the analysis of serum autoantibodies (Christensen et al, 2006; Lartigue et al, 2006; Wu et al, 2006, Yu et al, 2006; Marshak Rothstein et al, 2006). Similar to our results, other groups have found out that Tlr9 deficient FcRIIB deficient 56R mice have decreased IgG2a and IgG2b titers (Ehlers et al, 2006).

The data reported by Barrat, et al, and our own study have demonstrated the specificity of IRS954 for Tlr7 and Tlr9 (Barrat et al, 2005). Furthermore, by applying identical assay

systems as in the study reported by Christensen et al, we show that IRS954 has distinct effects on serum autoantibody levels as compared to IRS661 (Christensen et al, 2006). For example, in contrast to IRS661, IRS954 did not affect serum dsDNA autoantibodies, and may be Tlr9 antagonism provided by IRS954 ‘neutralized’ the suppressive effect of on Tlr7-mediated anti-dsDNA IgG2a, IgG2b, anti-Sm and anti-Sm-RNP IgG production.

Furthermore, IRS954 injections were associated with fewer mice with homogenous nuclear staining on Hep2 cells which was consistent with what has been observed in Tlr9- deficient MRLlpr/lpr mice (Christensen et al, 2006). The finding that IRS954 did not reduce dsDNA autoantibodies is potentially interesting because we previously found that another oligo ODN2114, blocking Tlr9 only, reduced these antibodies in MRLlpr/lpr mice (Patole et al, 2005). These reports suggests that the roles of Tlr7 and Tlr9 for the evolution of specific autoantibodies may even be more complex and requires a detailed analysis of immune cell subsets from Tlr7 and Tlr9 double-knock out cells which are not yet available. Our results were also supported by the results from another group about TLR7 and TLR9 dual inhibition and amelioration of disease symptoms in lupus mice (Barrat et al, 2007). Recent studies published about 5 separate patient cohorts have identified atleast two SNPs associated with IRF as high risk factor for SLE (Sigurdsson et al, 2005 and 2007; Graham et al, 2007), and since activation of Tlr7 and Tlr9 mediates the Type 1 IFN production via IRF5 and IRF7, role of these 2 receptors in pathogenesis of lupus has become the major contributing factor.

In summary these data suggest that delayed onset of oligonucleotide-based inhibition of Tlr7 reduces autoantibody production and prevents autoimmune tissue injury in experimental lupus. Combined blockade of Tlr7 and Tlr9 has no additive effects. This

data support the concept that endogenous ligands of Tlr7 contribute to the pathogenesis of autoantibody production and autoimmune tissue injury in SLE and propose Tlr7 blockade as a novel therapeutic target for lupus.