Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus (SLE) (1) and may influence morbidity and mortality. In SLE, abnormalities of immune regulation lead to loss of self tolerance, which in turn triggers autoimmune responses. Those include T and B cell dysfunction and activation of autoreactive B cells that produce antibodies against nuclear antigens. Immune complexes are detected in the circulation, and their deposition, together with complement activation in glomeruli and along the tubular basement membrane, plays a major role in the pleomorphic histopathology of lupus nephropathy. The initiating event may be local binding of nuclear or other antigens to glomerular and tubular sites followed by in situ immune complex formation. Antigens contain nucleic acids, and antinuclear antibody (ANA) formation occurs as a consequence of stimulation of invariant receptors that recognize nucleic acid determinants (2).
Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved molecular patterns shared by a wide variety of microorganisms (3, 4). Among TLRs, TLR-9 specifically binds CpG DNA, a hypomethylated form of DNA typical of bacteria and virus (5). CpG DNA activates potentially autoreactive B cells and plasmacytoid dendritic cells to secrete Th1-like cytokines (6). Aberrant TLR-9 activation and release of cytokines and chemokines occurs in plasmacytoid dendritic cells after exposure to immune complexes containing DNA, rich in CpG motifs, isolated from the serum of patients with active lupus nephritis (7).
Lesions of lupus nephritis involve the renal glomerulus and tubulointerstitium. Whereas the glomerular lesions have been studied extensively, the pathophysiology of tubulointerstitial inflammation and damage remains ill-defined. Yet tubulointerstitial changes are prominent and contribute prominently to the unfavorable long-term prognosis (8). Proximal tubular epithelial cells play an active role in tubulointerstitial inflammation and fibrotic lesions, to the extent that when these cells are exposed to anti–double-stranded DNA (anti-dsDNA) antibodies from patients with active lupus, they produce cytokines that promote local recruitment of inflammatory cells (9). DNA-containing autoantibody complexes found in the serum of patients with lupus activate dendritic cells to generate cytokines and chemokines via TLR-9 (7).
Considering that proximal tubular cells have antigen-presenting capacity as dendritic cells under certain circumstances (10), we wondered whether TLR-9 could be involved in the development of tubular damage and interstitial inflammation in lupus nephritis. To this end, we investigated renal TLR-9 expression in (NZB × NZW)F1 lupus-prone mice at different stages of the disease, both when animals had elevated serum levels of anti-dsDNA antibodies and later on, when proteinuria and tubulointerstitial damage ensued. TLR-9 expression in resident renal cells was assessed by laser capture microdissection combined with TaqMan real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). The finding that increased tubular TLR-9 generation was associated with proteinuria provided the rationale for using seliciclib, a cyclin-dependent kinase (CDK) 2, 7, and 9 inhibitor, which effectively reduced proteinuria and ameliorated renal injury in this model (11). To assess the relevance of animal data to the pathophysiology of interstitial lesions in human lupus, we evaluated the expression of TLR-9 in renal biopsy specimens from patients with lupus nephritis. Based on the previously reported finding that endogenous DNA-containing autoantibody complexes in the serum of SLE patients activated dendritic cells through TLR-9 (7), we finally assessed whether DNA-containing immune complexes purified from SLE serum stimulated cultured human proximal tubular cells to express TLR-9.
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Previous studies in the MRL-lpr/lpr mouse model of lupus nephritis demonstrated that injection of synthetic or bacterial DNA rich in CpG motifs activated TLR-9 in inflammatory cells infiltrating the kidney, leading to aggravation of renal inflammation, proteinuria, and tissue damage (24, 25). The present study demonstrated that proximal tubular epithelial cells were sites of robust expression of TLR-9 mRNA and protein in NZB × NZW mice with overt nephropathy. Up-regulation of tubular TLR-9 expression was concomitant with the development of proteinuria and correlated with tubulointerstitial damage.
To our knowledge, this is the first study to show TLR-9 expression in proximal tubuli in experimental lupus. Laser capture microdissection combined with real-time quantitative RT-PCR allowed us to selectively retrieve proximal tubuli and inflammatory cell infiltrates and quantify the expression of TLR-9, which was remarkably increased in proximal tubuli of NZB × NZW mice compared with that in normal mice. Despite a number of reports (including the present one) which suggested a pathogenetic role of TLR-9 in lupus nephritis (24–26), studies in TLR-9–deficient mice have shown that lack of TLR-9 worsened the course of lupus disease (27, 28). Data obtained in studies of sepsis syndrome are relevant for reconciling the contrasting observations on TLR-9 in lupus (29). A direct link between lipopolysaccharide (LPS) poisoning and sepsis acting on TLR-4 was derived from the observation that TLR-4 activation by LPS caused fever, shock, and death, whereas Sultzer's mutant mice, bearing nonfunctional TLR-4, were protected against shock when given LPS (30). However, when these mice were infected with gram-negative bacteria, which release LPS and pathogen-associated molecules, the manifestations of sepsis worsened and the rate of death increased. These paradoxical results can be explained if TLR-4 both protects the host by sequestering infectious organisms and causes manifestation of sepsis when the system is maximally activated and protection is overwhelmed.
A major finding of the present study is the positive correlation between tubular TLR-9 expression and the development of proteinuria and tubulointerstitial damage, which was further suggested by experiments using the CDK 2, 7, and 9 inhibitor, seliciclib, known to ameliorate autoimmune disease in NZB × NZW mice (11). Targeting aberrant renal cell proliferation by CDK inhibitors was found to be an effective therapeutic strategy in animal models of proliferative glomerulonephritis (31, 32). Here we report that the renoprotective effect of seliciclib in NZB × NZW mice was associated with inhibition of TLR-9 expression. To explain such an effect, one should consider the intracellular pathways leading to TLR-9 activation.
TLR-9 is localized in the endoplasmic reticulum and traffics to the endosomal–lysosomal compartment after cellular activation, becoming accessible to endocytosed foreign CpG DNA (33–35). It has been shown that CDK-2–cyclin E complexes control the vesicular fusion reaction of the endosomal apparatus in liver parenchyma cells (36). It is possible that under proteinuric conditions, seliciclib could have influenced endocytosis in proximal tubular cells, preventing TLR-9 activation at the endosomal sites as well as the downstream pathologic events. Seliciclib prevented tubular injury and exerted a remarkable antiinflammatory effect by limiting the interstitial accumulation of monocyte/macrophages, a major source of TLR-9. Taken together, these observations implicate TLR-9 as a trigger of tubulointerstitial damage in lupus nephritis.
We then sought to determine whether animal data were relevant to the pathophysiology of interstitial lesions in human lupus nephritis. We found intense staining for TLR-9 localized at proximal tubular cells in biopsy specimens from patients with active lupus nephritis, which would extend the significance of our observation to humans. In immune-mediated glomerular diseases, which include lupus nephritis, alteration of size-selective properties of the glomerular capillary wall is associated with abnormal filtration of plasma proteins, which conceivably include immune complexes (37) containing DNA enriched in hypomethylated CG motifs (38, 39). Ultrafiltered macromolecules and proteins are then actively reabsorbed by receptor-mediated endocytosis in proximal tubular cells, giving rise to endosomes that, upon acidification, progress to lysosomes (40). In vitro studies have shown that internalization of SLE immune complexes into subcellular lysosomes containing TLR-9 induces a signaling cascade leading to activation of plasmacytoid dendritic cells to produce cytokines and chemokines (7).
It is conceivable that a mechanism of TLR-9 activation by DNA-containing immune complexes similar to that described in dendritic cells occurs in proximal tubular cells. Proximal tubular cells possess Fc neonatal receptors on the plasma membrane, which could favor the uptake of immune complexes containing DNA (41), followed by DNA-induced activation of TLR-9. Our in vitro experiments showing that sera from patients with lupus nephritis, but not from patients with another autoimmune disease, stimulated cultured proximal tubular epithelial cells to produce TLR-9 would confirm such a possibility.
We found that immune complexes present in sera from patients with lupus induced up-regulation of TLR-9 in proximal tubular cells. Short ODNs rich in CCGG motifs, specific antagonists of CpG DNA, prevented the TLR-9 mRNA increase induced by immune complexes from lupus patient sera, suggesting that the DNA component of immune complexes is required for the stimulatory activity. IgG-depleted sera from patients with lupus still retained the ability to stimulate TLR-9 in proximal tubular cells, but this was prevented by the inhibitory short ODNs. This indicates that additional molecules in lupus sera participate in the engagement of the receptor. In this context, nucleosomes that bear hypomethylated DNA might act as endogenous ligands responsible for TLR-9 up-regulation (23).
In conclusion, we provide evidence that resident proximal tubular epithelial cells are activated to express TLR-9 in experimental and human lupus nephritis. Tubular TLR-9 expression correlates with tubulointerstitial damage. Cultured proximal tubular cells produce TLR-9 after exposure to immune complexes from lupus patient sera. DNA complexed with IgG is required for TLR-9 stimulatory activity. These findings suggest that tubular TLR-9 activation has a pathogenetic role in tubulointerstitial inflammation and damage in lupus nephritis, and they indicate a novel target for future therapies.
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Dr. Benigni had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Benigni, Zoja, Remuzzi.
Acquisition of data. Caroli, Longaretti, Gagliardini, Galbusera, Moioli, Romagnani.
Analysis and interpretation of data. Benigni, Caroli, Longaretti, Gagliardini, Zoja, Galbusera, Moioli, Remuzzi.
Manuscript preparation. Benigni, Caroli, Zoja, Remuzzi.
Statistical analysis. Caroli, Longaretti, Gagliardini, Galbusera.
Provision of human sera and purified IgG. Tincani, Andreoli.