A pathogenic role for Th17 cells in inflammatory renal disease is well established. The mechanisms underlying their counter-regulation are, however, largely unknown. Recently, Th17 lineage-specific regulatory T cells (Treg17) that depend on activation of the transcription factor Stat3 were identified. We studied the function of Treg17 in the nephrotoxic nephritis (NTN) model of crescentic GN. The absence of Treg17 cells in Foxp3Cre 3Stat3

Cells expressing both the regulatory T cell (Treg)-inducing transcription factor Foxp3 and the Th17 transcription factor RORgt have been identified (biTregs). It is unclear whether RORgt + Foxp3 + biTregs belong to the Th17-specific Treg17 cells, represent intermediates during Treg/Th17 transdifferentiation, or constitute a distinct cell lineage. Because the role of biTregs in inflammatory renal disease is also unknown, we studied these cells in the nephrotoxic nephritis (NTN) model of acute crescentic GN. Induction of NTN resulted in rapid renal and systemic expansion of biTregs. Notably, analyses of the biTreg expression profile revealed production of both anti-inflammatory (IL-10, IL-35) and proinflammatory (IL-17) cytokines. Additionally, biTregs expressed a signature of surface molecules and transcription factors distinct from those of Th17 cells and conventional Tregs (cTregs), and biTregs were identified in Treg17-deficient mice. Finally, fate reporter and cell transfer studies confirmed that biTregs are not Treg/Th17 transdifferentiating cells. Therapeutic transfer of biTregs suppressed the development of nephritis to an extent similar to that observed with transferred cTregs, but in vitro studies indicated different mechanisms of immunosuppression for biTregs and cTregs. Intriguingely, as predicted from their cytokine profile, endogenous biTregs displayed additional proinflammatory functions in NTN that were abrogated by cellspecific deletion of RORgt. In summary, we provide evidence that RORgt + Foxp3 + biTregs are a novel and independent bifunctional regulatory T cell lineage distinct from cTregs, Treg17 cells, and Th17 cells. Furthermore, biTregs appear to contribute to crescentic GN and hence may be novel therapeutic targets.

Systemic lupus erythematosus (SLE) is a complex and potentially fatal autoimmune disorder. Although Th17 cells are thought to be central mediators of SLE, mechanisms underlying their counter regulation remain largely unknown. To help define this, we studied the function of the newly defined Stat3-dependent Th17-specific regulatory T cells (Treg17). Treg-specific deletion of Stat3 was achieved by generating Foxp3(Cre) × Stat3(fl/fl) mice and SLE was induced by intraperitoneal injection of pristane. Lack of Treg17 cells in these mice caused selectively enhanced peritoneal Th17 inflammation. Importantly, Treg17 deficiency also resulted in aggravated pulmonary vasculitis with increased percentages of Th17 cells and significantly higher mortality. Similarly, 4 and 9 months after pristane injection, analysis of renal and systemic immunity showed overshooting Th17 responses in the absence of Treg17 cells, associated with the aggravation of lupus nephritis. Expression of the Th17 characteristic trafficking receptor CCR6 was strikingly reduced on Tregs of Foxp3(Cre) × Stat3(fl/fl) mice, resulting in impaired renal Treg infiltration. Thus, Stat3-induced Treg17 cells are novel antiinflammatory mediators of SLE. One mechanism enabling Treg17 cells to target pathogenic Th17 responses is shared expression of the chemokine receptor CCR6.

IL-6 can mediate proinflammatory effects, and IL-6 receptor (IL-6R) blockade as a treatment for inflammatory diseases has entered clinical practice. However, opposing effects of IL-6 have been observed in models of GN. Although IL-6 is proinflammatory in murine lupus nephritis, protective effects have been observed for IL-6 in the nephrotoxic nephritis (NTN) model of acute crescentic GN. In light of the potential dangers of IL-6-directed treatment, we studied the mechanisms underlying the contradictory findings in GN. IL-6 can signal through the membrane-bound IL-6R, which is expressed only on hepatocytes and certain leukocytes (classic), or through the soluble IL-6R, which binds the ubiquitously expressed gp130 (alternative). Preemptive treatment of mice with anti-IL-6R or anti-IL-6 worsened NTN, whereas selective blockade of alternative IL-6 signaling by the fusion protein sgp130Fc did not. FACS analysis of mouse spleen cells revealed proinflammatory macrophages express the highest levels of IL-6Ra, and in vitro treatment with IL-6 blocked macrophage proliferation. Furthermore, proinflammatory macrophages were expanded during inflammation in IL-6 2/2 mice. Late application of anti-IL-6 after establishment of adaptive nephritogenic immunity was sufficient to aggravate NTN within 2.5 days, a period when macrophages are active. Finally, NTN was aggravated in mice with macrophage-specific impairment of IL-6 classic signaling, coincident with enhanced macrophage proliferation and accumulation in the kidney. Our data thus reveal a novel mechanism in which IL-6-mediated dampening of macrophage activation protects tissues from overshooting immune responses. This finding has important implications for potential IL-6-directed therapies and supports the careful choice of recipient patients and timing.

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immunopathogenesis. However, a pathogenic role for the T helper type 17 (Th17) axis was demonstrated by many studies, while regulatory T cells (T ) were shown to mediate protection. Recently, we and others characterized a novel and independent T cell population expressing both the T characteristic transcription factor forkhead box protein 3 (FoxP3) and the Th17-defining retinoic acid receptor-related orphan nuclear receptor γt (RORγt). Studies in a model of acute glomerulonephritis unveiled potent regulatory, but also proinflammatory, functions of RORγt FoxP3 T . This bi-functional nature prompted us to suggest the name 'biT '. Importantly, the pathogenic biT effects were dependent upon expression of RORγt. We thus aimed to evaluate the contribution of RORγt FoxP3 biT to pristane-induced SLE and explored the therapeutic potential of interference with RORγt activation. Our analyses revealed expansion of IL-17 producing biT in a distinctive time-course and organ-specific pattern, coincident with the development of autoimmunity and tissue injury. Importantly, specific ablation of RORγt activation in endogenous biT resulted in significant amelioration of pristane-induced pulmonary vasculitis and lupus nephritis. As potential mechanisms underlying the observed protection, we found that secretion of IL-17 by biT was abrogated completely in FoxP3 × RORC mice. Furthermore, T showed a more activated phenotype after cell-specific inactivation of RORγt signalling. Finally, and remarkably, biT were found to potently suppress anti-inflammatory Th2 immunity in a RORγt-dependent manner. Our study thus identifies biT as novel players in SLE and advocates RORγt-directed interventions as promising therapeutic strategies.