The Complex Role of Interleukin 6 in Regulating T-cell Responses during Acute Glomerulonephritis

Most forms of acute GN involve systemic or kidney-restricted autoimmunity meaning that the humoral (B cell/antibody) and/or cellular (T cell) compartments of the adaptive immune system have broken free of regulatory mechanisms that normally ensure tolerance to self-antigens and have directed effector functions that are usually reserved for eliminating harmful pathogens against molecular targets within the glomerulus.¹ In the case of rapidly progressive forms of acute GN, such as ANCA-associated vasculitis (AAV) and anti-glomerular basement disease, this loss of tolerance to glomerular self-antigens may be triggered or exacerbated by the proinflammatory activities of innate immune effectors including neutrophils, monocyte/macrophages and the complement system.¹ In this setting of coevolving inflammation and autoreactive antibodies and T cells, one important determinant of outcome is the nature of the CD4⁺ (“helper”) T cell repertoire that emerges—a process referred to as T-helper (Th) cell differentiation.² The recognition that distinctive combinations of cytokines polarize CD4⁺ helper T cells toward specific effector or regulatory phenotypes and that these different phenotypes subsequently mediate many of their immunologic effects via secretion of signature cytokines, has led to cytokine-blocking therapies for autoimmunity.³ In the case of acute GN, manipulating cytokine signals that determine the ratio between effector and regulatory T cells (Treg) represents a compelling new treatment strategy.³

Among the recently-described CD4⁺ T cell subtypes is the T helper 17 (Th17) cell—a proinflammatory immune effector characterized by expression of the transcription factor RAR-related orphan receptor gamma (RORγ)t and secretion of the signature cytokine IL-17A.² During bacterial and fungal infections, Th17 cells provide immune protection by stimulating a neutrophil-dominated form of inflammation. However, Th17-type immune responses are also important pathogenic mediators in acute GN.⁴ For example, adoptive transfer of Th17 cells to a T cell and B cell–deficient (RAG^−/−) mouse strain initiates acute GN in the nephrotoxic serum nephritis (NTN) model. Conversely, mice that are incapable of generating Th17 cells as a result of RORγt deficiency have reduced glomerular inflammation in the same model.⁴ Consistent with these findings, human subjects with acute GN due to AAV have increased numbers of Th17 cells in kidney biopsy samples and higher circulating Th17 cells and IL-17A compared with controls.^3–5

Intriguingly, a special relationship exists between Th17 cells and Tregs (which preferentially express the transcription factor forkhead box [FOX] P3). This is based, in part, on their responses to the cytokines TGF beta (TGF-β) and interleukin-6 (IL-6) whereby differentiation of Th17 cells was demonstrated to require TGF-β and IL-6, whereas induction of Treg occurred in the setting of TGF-β alone.⁶ One often cited implication of these observations is that the Th17/Treg ratio may be critically regulated by the presence of IL-6, further implying that blockade of IL-6 signaling to CD4⁺ T cells could restore immunologic balance in the setting of Th17-driven autoimmune disease. Perhaps unsurprisingly, recent basic immunology studies have shown the in vivo relationship between Th17 cells and Treg to be more complex. On the one hand, an IL-17A–producing RORγt⁺ Th17 phenotype may convert to an IL-10–producing Treg phenotype or vice versa. Alternatively, STAT-3–dependent FOXP3⁺ Treg (termed T-reg17 cells) have been found to preferentially suppress the activity of Th17 cells. Finally, a “bifunctional” population of CD4⁺ T cells that expresses both RORγt and FOXP3 has also been described.⁷

It is the latter, RORγt⁺/FOXP3⁺ T cell type that is the subject of a novel study conducted by Hagenstein et al.⁸ using the mouse NTN model and reported in the current issue of JASN. Of note, the authors previously demonstrated that RORγt⁺/FOXP3⁺ CD4⁺ T cells (which they refer to as “biTreg”) are a stable lineage in mice that is distinct from STAT3-dependent RORγt⁻/FOXP3⁺ Treg17 cells, is expanded in NTN and has the potential to mediate both suppressive and pro-inflammatory effects.⁷ They have now investigated the regulation of RORγt⁺/FOXP3⁺ CD4⁺ T cells by IL-6 during NTN.⁸ By generating a novel mouse strain in which the gene encoding the IL-6 receptor alpha chain (IL-6Ra) is selectively deleted in cells expressing CD4, the authors abolished direct IL-6–mediated signaling to all CD4⁺ cells. To eliminate potential confounding effects of the genetic manipulation on other immune cells, IL-6Ra–deficient or wild type (WT) CD4⁺ T cells were then adoptively transferred to RAG^−/− mice, in which NTN was induced. As expected, Th17 responses were impaired in recipients of IL-6Ra–deficient CD4⁺ T cells. Nonetheless, the severity of renal injury and functional impairment were not reduced suggesting the possibility that IL-6Ra signaling induces both damaging Th17 activity and a protective counter-regulatory mechanism. In support of this hypothesis, when adoptively transferred cells were first depleted of Treg, the authors observed both diminished Th17 response and a clear reduction in NTN severity in the recipients of IL6-Ra–deficient CD4⁺ T cells.⁷ In a meticulous series of additional NTN experiments, it was shown that a specific deficiency of RORγt⁺/FOXP3⁺ “biTreg” was present in the spleen and blood of mice with IL-6Ra–deficient CD4⁺ T cells (or in those with global deficiency of IL-6). In contrast, a reduction of both RORγt⁺ and RORγt⁻ Treg was observed in the kidneys. Taken together, the findings suggest that systemic expansion of RORγt⁺/FOXP3⁺ Treg is directly dependent on classic IL-6 signaling and is responsible for modulation of the early immunologic events in acute GN via suppression of Th17 responses as well as regulation of total Treg migration to the affected kidneys (Figure 1). This conclusion was further supported by well designed in vitro suppression assays and by in vivo experiments comparing the RORγt expression and disease-modulating effects of purified, adoptively transferred WT and IL-6Ra–deficient Treg.⁸

Figure 1.

The illustration depicts antigen-driven expansion of effector and regulatory CD4⁺ T cell subpopulations in the spleen along with their migration to and further expansion within the kidney during nephrotoxic serum nephritis in the settings of intact and deficient IL-6 direct signaling within CD4-expressing cells (as demonstrated experimentally by Hegenstein et al.⁸). Left panel: When IL-6 signaling is intact, antigens derived from nephrotoxic sheep IgG drive splenic proliferation of RORγt⁺ T helper (Th)17 and Th1 effectors in addition to FOXP3⁺ conventional regulatory T cells (cTreg) and RORγt⁺/FOXP3⁺ bifunctional Treg (“biTreg”). Migration to and further proliferation of these CD4⁺ T cell subpopulations within the kidneys results in neutrophil-associated Th17-dependent injury (counter-regulated by biTreg) as well as Th17-independent injury (counter-regulated by cTreg and possibly also by biTreg). Right panel: When IL-6 signaling is deficient in CD4⁺ T cells, Th17 expansion in the spleen and kidney is diminished along with associated renal neutrophil infiltration. However, splenic and renal expansion of biTreg is similarly prevented and migration of cTreg to the kidney is reduced. Thus, the loss of IL-6–driven biTreg expansion results in a deficiency of intra-renal regulatory T cell populations and more severe Th17-independent injury. Unchanged intra-renal Th1 cell numbers suggest the presence of additional Th17-independent mediators of glomerular injury.

Although not directly tested in this study, the findings imply that a mAb or other therapeutic intervention to block IL-6 signaling during the early course of Th17-mediated acute GN could prove ineffective or even harmful as a result of inhibiting the induction of RORγt⁺/FOXP3⁺ T-reg. It must be acknowledged that elucidating such refined immunologic detail in mice comes at the price of a limited direct applicability to human disease. Species-specific differences between mouse and human immune systems; the exclusive use of young, genetically homogenous animals maintained in a pathogen-free environment; and the highly-contrived experimental conditions involving genetic manipulations, adoptive transfer of selected cell types and precisely timed triggering of glomerular injury all create distance between this preclinical study and the medical conditions for which we seek improved therapies. Nonetheless, bifunctional FOXP3⁺ T-reg sharing the features of Th17 cells (IL-17A production and IL-6–inducible RORγt expression) have been shown to be expanded in patients with rheumatoid arthritis and to have potential suppressive effects.⁹ In a more general sense, all of the recent exciting clinical developments in immunotherapy for cancer, autoimmunity and transplantation (checkpoint inhibitors, CAR T cells, cytokine inhibition, costimulatory blockade) can be traced back to origins in such small animal-based fundamental immunology.

The potential clinical relevance of the work by Hagenstein et al. is enhanced by the fact that a range of therapeutic products targeting IL-6 signaling has been developed with some of these now approved for use in immune/inflammatory conditions– particularly rheumatoid arthritis, Castleman’s disease and inflammatory bowel disease.¹⁰ Significantly, however, the biology of IL-6 is remarkably complex, encompassing metabolic homeostasis, bone turnover, acute phase response to infection/inflammation, B cell activation/antibody generation and T cell effector function and regulation.¹⁰ Furthermore, IL-6–mediated signaling is delivered to various target cell types through three distinct molecular configurations involving the IL-6 cytokine itself, the IL-6Ra (which may be surface bound or soluble) and the ubiquitously expressed surface receptor protein gp130.¹⁰ Several reagents have been developed to specifically target one or more of these signaling pathways and there is evidence that these can be effectively matched to the mechanistic role of IL-6 in individual diseases. Based on their findings, Hagenstein et al. discuss the potential need for cell-type specific targeting of IL-6Ra in acute GN. However, additional preclinical experiments will be needed to determine whether the effects of IL-6 signaling on pathogenic Th17 cells and potentially protective “biTreg” can be separated from each other. Alternatively, the results of this study raise the possibility of ex vivo-engineered bifunctional T-reg (e.g., by RORγt transduction) as a personalized cell therapy to counteract pathogenic Th17 responses. In this case, however, the coexisting proinflammatory effects previously described by Kluger et al.⁷ could represent a limitation.

To conclude, the elegant experimental study of Hagenstein et al. provides a distinctive insight into the complex role played by IL-6 during the evolution of pathogenic and counter-regulatory T cell responses in acute GN. Their results raise an important note of caution in regard to the application of anti–IL-6 and anti-IL6Ra therapies to AAV, anti-glomerular basement disease and other forms of rapidly progressive GN. More importantly, perhaps, they illustrate the unique role of fundamental immunology research to generate new hypotheses about the inter-woven mechanisms by which the immune system mediates individual kidney diseases and how these may be more effectively targeted in the future.

Disclosures

None.

Funding

Prof. Griffin is supported by grants from the European Commission [Horizon 2020 Collaborative Health Project NEPHSTROM (grant number 634086) and FP7 Collaborative Health Project VISICORT (grant number 602470)] and from Science Foundation Ireland [CÚRAM Research Centre (grant number 13/RC/2073)] and by the European Regional Development Fund.