The Th17/IL-17 Axis and Kidney Diseases, With Focus on Lupus Nephritis

Systemic lupus erythematosus (SLE) is a disease characterized by dysregulation and hyperreactivity of the immune response at various levels, including hyperactivation of effector cell subtypes, autoantibodies production, immune complex formation, and deposition in tissues. The consequences of hyperreactivity to the self are systemic and local inflammation and tissue damage in multiple organs. Lupus nephritis (LN) is one of the most worrying manifestations of SLE, and most patients have this involvement at some point in the course of the disease. Among the effector cells involved, the Th17, a subtype of T helper cells (CD4+), has shown significant hyperactivation and participates in kidney damage and many other organs. Th17 cells have IL-17A and IL-17F as main cytokines with receptors expressed in most renal cells, being involved in the activation of many proinflammatory and profibrotic pathways. The Th17/IL-17 axis promotes and maintains repetitive tissue damage and maladaptive repair; leading to fibrosis, loss of organ architecture and function. In the podocytes, the Th17/IL-17 axis effects include changes of the cytoskeleton with increased motility, decreased expression of health proteins, increased oxidative stress, and activation of the inflammasome and caspases resulting in podocytes apoptosis. In renal tubular epithelial cells, the Th17/IL-17 axis promotes the activation of profibrotic pathways such as increased TGF-β expression and epithelial-mesenchymal transition (EMT) with consequent increase of extracellular matrix proteins. In addition, the IL-17 promotes a proinflammatory environment by stimulating the synthesis of inflammatory cytokines by intrinsic renal cells and immune cells, and the synthesis of growth factors and chemokines, which together result in granulopoiesis/myelopoiesis, and further recruitment of immune cells to the kidney. The purpose of this work is to present the prognostic and immunopathologic role of the Th17/IL-17 axis in Kidney diseases, with a special focus on LN, including its exploration as a potential immunotherapeutic target in this complication.


INTRODUCTION
Systemic lupus erythematosus (SLE) is a disease characterized by hyperreactivity to the self, with the polarization of the immune response to a proinflammatory profile (1,2), autoantibodies production (3), immune complex formation (4) and deposition in tissues (5). It also occurs with local production of inflammatory mediators, and additional recruitment of inflammatory cells, resulting in tissue damage in various organs (6). These events together express the dysregulation of the local and systemic immune response, which characterize the disease (1,7,8). Lupus nephritis is one of the most worrying organic affectations of lupus, one of the strongest predictors of a poor outcome in SLE, being responsible for the greater burden attributable to the disease, mainly in the low-income populations (9,10).
Several immunological pathways are involved in the pathogenesis of SLE (11), which calls us to go deeper in the knowledge about the immunopathologic complexity of this disease, aiming to explore new opportunities for targeted therapies (12). Among the effector cells, the Th17, a subtype of T helper cells (CD4+), is one of those that has shown significant hyperactivation (1,(13)(14)(15); correlates with disease activity (1,16,17), being involved in many manifestations of SLE as neuropsychiatric (18,19), cutaneous (20,21), and lupus nephritis (1,22,23); and correlates with the fatality of the disease (24).
Other studies highlight the predominant role of the Th17/IL-17 axis in LN that even in models deficient in TNF receptors (another potent proinflammatory cytokine), Th17-associated pathways were sufficient to cause the clinical and pathological changes of lupus nephritis (41). In addition, other Th17-related elements of the immune response participate in this process (16,42,43). Thus, IL-23 (involved in the differentiation and maintenance of Th17 by an autocrine mechanism) is also increased in lupus and correlates with disease activity (44,45). In experimental models, the RORγt (the Th17-defining transcription factor) promoted itself glomerulonephritis; and RORγt ablation or deficiency (RORγt-/-) conferred protection to experimentally induced glomerulonephritis (46,47). This review focuses on the role of the Th17/IL-17 axis in the immunopathology and prognosis of lupus nephritis and its exploration as a potential immunotherapeutic target in this complication.

Proteinuria, Hematuria, and Anemia
Serum IL-17 levels are significantly associated with proteinuria (48, 49); and its concentration at the baseline keeps a positive correlation with the severity of proteinuria (50). In a study involving 15 patients (who underwent kidney biopsy), using the laser microdissection technique, the percentage of IL-17+ TCR+ among kidney-infiltrating cells correlated positively with hematuria in LN (51). In another study, elevated serum levels of IL-17 and IL-6 were associated with anemia (52).

Severity Scores and Histological Activity
Th17 cell frequencies significantly correlated with SLEDAI and inversely with C3 (53) and the concentration of IL-17 at the baseline kept a positive correlation with other parameters of severity (ESR, SLEDAI scores, and ANA titers) (50). In relation to histological activity, Th17 cell frequencies in peripheral blood and IL-17 levels in serum correlated significantly with renal biopsy classification for LN (43,49,53). A significant positive association has been found between serum IL-17 (and TWEAK) levels and nephritis activity index (54). In another study, the frequencies of circulating Th17-cells correlated positively with histological activity index, cellular crescent, and endocapillary proliferation. Additionally, intraglomerular levels of IL-17 and IL-23 were significantly higher in class IV LN than in MCN patients or HC (43). In another study, with measurement of urinary , the levels of uIL-17 were significantly higher in the severe LN than in the control group (P < 0.05); and increased with disease severity seen in biopsy (mean ± SD: 43.96 ± 24.04, 55.69 ± 33.21, and 124.02 ± 256.74 pg/ml; for HC, class I-II, and class III-IV LN, respectively) (55). Another study observed that serum levels of IL-17A were significantly elevated in proliferative forms compared to non-proliferative LN (56).

Requirement for Pulse Steroids and Response to Treatment
A study found that the presence of IL-17 in renal tissue correlated with the requirement for pulse steroids (p < 0.05) (49). In relation to response to treatment, in a study involving 52 patients with active LN (who underwent kidney biopsy at baseline and after immunosuppressive therapy), higher IL-17 levels at baseline were associated with persisting active nephritis after treatment (WHO III, IV, V) (42). At follow-up, non-responders had higher IL-17 (and IL-23) expression by inflammatory cells infiltrating renal tissue than responders (42). On the other hand, IL-17 and IL-23 decreased significantly in patients with active LN after 6 months of therapy (P < 0.001) (45). Another study showed that despite a progressive decrease in serum concentrations of IL-17A and IL-21 during induction therapy, the concentration of these cytokines remained higher in the non-remission than in the remission group (50).

Renal Function, ESRD, and Mortality
Th17 cell frequencies significantly correlated with serum creatinine (53) and IL-17 was an independent risk factor for poor prognosis of LN (48). In another study, IL-17 immunostaining in biopsy correlated negatively with GFR (49). Table 1 summarizes the clinical studies that have assessed the role of the Th17/IL-17 axis in lupus nephritis.  There was a progressive decrease in serum concentrations of IL-17A and IL-21 (P < 0.01, P = 0.001, respectively) during induction therapy. The concentration of these cytokines remained higher in the non-remission than in the remission group. Additionally, the concentration of these cytokines at the baseline kept a positive correlation with the severity of proteinuria, ESR, SLEDAI scores, and ANA titers.

THE TRACK AND FOOTPRINTS OF TH17/IL-17 AXIS HYPERACTIVITY IN LUPUS From Extracellular Chromatin to APCs Maturation
An early event in the classical immunopathogenesis of SLE is the easy release of intracellular content into the extracellular space, the breakdown of immune tolerance to self, and autoantibodies production (4,67). Components released into extracellular space function as danger-associated molecular patterns (DAMPs) (4,68) and are recognized by dendritic cells, and other antigenpresenting cells (APCs), through toll-like receptors (TLR4) present in their plasma membrane (69-72). On the other hand, autoreactive B cells respond to immunogenic DNA with autoantibodies production (67,71,73), which APCs also internalizes (through FCγRII) as DNA-containing immune complexes (68) and then recognized by TLR7 and TLR9 present in endosome (69, 74,75). The binding of DAMPs to TLRs in APCs induces their maturation (76,77). Mature APCs, in turn, drive lymphocyte activation (78).

Activation and Differentiation of Th17 Cells
Th17 cells differentiate from naive T auxiliary cells, according to microenvironmental factors, in the presence of IL-1β, IL-6, IL-23, and TGF-β, which are the key cytokines for its differentiation (78)(79)(80) and requires the lineage-specific transcription factor retinoid-related orphan receptor-gamma (RORγt) (80, 81). As described above, Mature APCs (after the binding of DAMPs to TLRs) trigger lymphocyte activation by the interaction of MHC II with TCR and several co-stimulatory molecules (15,78,80,82).
In the context of this interaction, mature APCs produce the key cytokines for Th17 differentiation (78,80), using nuclear factor kappa B (NF-κB) and/or mitogen-activated protein kinase (MAPK) as signaling pathways (72,77,83). These cytokines bind to their respective receptors in naive CD4+ T Cells and trigger a chain of events downstream involving the Signal transducer and activator of transcription 3 (STAT3), which stimulates the synthesis of IL-17 and IL-21 either by binding directly to their genes or by activating RORγt (80,84). Interestingly, T cells from SLE patients presented enhanced Stat3 activity added to higher RORγt expression (85). Once differentiated, Th17 cells secrete their cytokines (IL-17A, IL-17F, IL-17C, IL-21, and IL-22), most of them with a pathogenic role in the kidney (32,(86)(87)(88), in addition to its systemic effects (1,15). Regarding Th17 cells differentiation, it is also worth mentioning that podocytes, mesangial cells, and renal tubular epithelial cells can behave as antigen-presenting cells (89)(90)(91)(92). So, these intrinsic renal cells can alone trigger the local activation of Th17 cells after recognizing, processing, presenting eventual DAMPs that cross the glomerular filtration barrier, as seen in other kidney disease models (89,93,94). Interestingly, a study showed that IL-17 (and IFNγ) upregulated the expression of MHC-I, MHC-II, and co-stimulatory molecules (CD80 and CD86) on the podocyte surface. Moreover, under IL-17 stimulation, podocytes increased the uptake and processing of antigen, resulting in the presentation of its peptide on the cell surface (93). This fact brings robustness to the idea that, in part, naïve T cells can enter the kidney and continue, under local factors, in the path of differentiation and activation to Th17 (95,96). A recent study reinforces this thesis by demonstrating that, in kidneys of patients with ANCA-associated glomerulonephritis, Th17 cells develop from CD4+ tissue-resident memory T cells and exacerbate renal pathology by secreting IL-17A (97,98).

Th17 Polarization in SLE and Related Immunes Imbalances
Although the same general mechanisms regulate the activation of all T-cell subtypes (effector and regulatory) it is worth emphasizing that in the lupus autoimmunity environment, there is favoritism of self-reactive effector (4,73,99); with the detriment of regulatory cells (17,97,100). One of the bases of this polarization lies in the fact that there are plasticity and reciprocity between Th17 and Treg (100-102), a balance influenced by various factors (103), and the inflammatory and autoimmunity environment of lupus favors to the side of the Th17 cells (2,79,101). Added to Th17/IL-17 axis overactivity, LN is characterized by decrease, suppression, or dysfunction of Treg cells (57,104) and impairment of other protective factors like IL-2 (97) and IL-10 (105,106).
Several aspects present in SLE favor the polarization of CD4+ cells to a proinflammatory profile (Th1 and Th17) (2,107). This range from phenotypic and functional aberrations of APCs (2,108,109) to T cells specific aspects, like changes in immunometabolism (marked glycolysis, lipid synthesis, glutaminolysis, and hyperactivation of the mTOR pathway) (110,111), and abnormalities in signaling pathways (112,113). In addition, epigenetic changes, such as histone hypomethylation at naive CD4+ T Cells level, have also been described to favor Th17 polarization (114,115) and were early events before lupus flares (114). Additionally, dysbiosis, a characteristic also present in lupus (116,117), is a potentiating factor for Th17/IL-17 polarization (118); and a study has even shown that autoimmune kidney disease is exacerbated by the migration of pathogenic Th17 cells from the intestine to the kidney (119).
In the lupus autoimmunity environment, other elements of the immune response participate in Th17 polarization, as shown in an experimental study with lupus-prone mice, in which dendritic and B cells increased Th17 expansion, associated with limited Treg expansion, and increased renal infiltration by Th1 and Th17 cells (2). In another study, basophils obtained from patients with SLE promoted Th17 differentiation from SLE naïve CD4+ T cells in vitro coculture (120). Even cells with a dominant protective role, like Treg cells, in the lupus nephritis background, have been shown to facilitate the proliferation of Th17 lymphocytes and are less suppressive (47, 63). Several other elements of the immune response favor polarization to Th17 in the context of lupus (121).

Recruitment of Th17 Cells to the Kidney
Th17 cells are attracted to the kidney by chemokines CCL20, CXCL9, and CXCL10 (33, 122, 123) through binding to their receptors (CXCR3 and CCR6) expressed on the surface of these cells (124)(125)(126). Recruitment is facilitated by the enhanced migratory capacity of Th17 cells from SLE patients, through high Akt activity (60) and involvement of calcium/calmodulindependent kinase IV (CaMK4) (96,127). Most intrinsic kidney cells (podocytes, mesangial, and kidney tubular cells) secrete CXCL9, CXCL10, and CCL20 in response to injury (23,123,128). In an experimental study, stimulation of mesangial cells with nucleosome-containing immune complexes resulted in their activation and expression of CCL20 (129). More recently, it has been shown that components of the extracellular matrix, produced by injured cells, stimulate resident macrophages to produce CCL20, CXCL9, and CXCL10, cooperating in this way in the recruitment of Th17 cells (130).
Once in the kidney, Th17 cells maintain the phenotypic and functional features through several other factors, as demonstrated in models where local T cells had elevated expression of inducible T cell costimulator (ICOS) coreceptor and were protected from apoptosis by elevating the activity of the PI3K-Akt signaling pathway. These features together result in facilitating the accumulation of active T cells in the kidney (131,132). Additionally, a clinical study revealed that LES patients had elevated serum autoantibodies against co-inhibitory PD-1, facilitating T cell proliferation and maintaining the hyperactive phenotype; and this kept a close association with disease activity, particularly renal involvement (133).

Effects of Th17/IL-17 Axis in the Kidney
In the kidney, the Th17/IL-17 axis participates in several points of the damage chain. In summary, this involves changes in the structure and functioning of intrinsic specialized renal cells, promoting and maintaining an inflammatory environment, participating in repetitive tissue damage and maladaptive repair, leading to renal fibrosis and loss of function (23,25). Thus, the Th17/IL-17 axis behaves as a true chief orchestrator of immunity (134,135). The available evidence on the Th17/IL-17 axis effects on specific renal cells or compartments is described below in this review.

Glomerular Compartment
Regarding the effect of the Th17/IL-17 axis on the filtration barrier elements, there is growing evidence about the harmful effect of IL-17 on cellular elements of this barrier; however, it remains a field in need of an extensive investigation.

Podocytes
On podocytes, an experimental study raised the possibility that Th17 cells would produce a soluble mediator that enhances podocyte motility, causing rearrangement of the actin cytoskeleton and increased permeability (136). This finding may be the basis of the correlation found between IL-17 levels and proteinuria and its severity (50, 53). According to the study, this soluble factor mimics the protease-activated receptors-1 (PAR-1) activation signaling pathways (136).
Still focusing on the potential impact on the cytoskeleton of podocytes, the exposure of mice podocytes to recombinant IL-17 induced overproduction of Cmaf-inducing protein(c-mip), with consequent induction of cytoskeletal disorganization and apoptosis in adriamycin-induced nephropathy model (137). Interestingly, silencing c-mip prevented IL-17 related podocyte apoptosis by promoting persistent activation of NF-κB and upregulation of anti-apoptotic protein Bcl-2 (137). C-mip is a protein whose expression is suppressed in healthy glomeruli (138) and increased in pathological conditions and has been associated with cytoskeletal disorganization in podocytes and proteinuria (139,140).
In another experimental study with mouse podocytes, IL-17A stimulation disrupted the podocyte morphology by decreasing podocin expression and increasing desmin expression. In this study, podocytes expressed IL-17RA, and stimulation with IL-17A induced changes associated with activation of the NLRP3 inflammasome-caspase-1 pathway, production of intracellular reactive oxygen species (ROS), and increased IL-1β secretion. Interestingly, the blockade of these downstream signaling pathways restored the podocyte morphology (141).
Additional evidence about the harmful effect of IL-17 on podocytes comes from studies involving patients with primary nephrotic syndrome (PNS) (142). In these patients, IL-17 was highly expressed in renal tissue, being higher in patients with focal segmental glomerulosclerosis (FSGS), the glomerular disease with greater fibrosing behavior. As increased the expression of IL-17 Messenger RNA (mRNA) in the tissue, decreased the expression of podocalyxin (PCX) mRNA; and the IL-17 mRNA correlated directly with the number of podocytes lost in the urine. In the complement, with in-vitro experiment, IL-17 induced podocytes apoptosis and reduced podocyte health proteins such as nephrin, synaptopodin, and PCX. At the same time, IL-17 induced the expression of proteins like Fas, Fas ligand (FasL), active-caspase-3, active-caspase-8, and phosphorylated-p65. These effects occurred with the involvement of NF-κB pathways, and its inhibition attenuated the IL-17-induced podocyte apoptosis, decreasing or suppressing the molecular pathways described above (142). In another study, exposure of murine podocytes to recombinant IL-17 also induced apoptosis, increased the expression of caspase-3, caspase-8, and Fas; associated with decreased PCX expression, in a dose-and time-dependent manner (143).

Mesangial Cells
In mesangial cells, stimulation with IL-17A or IL-17F induces the production and release of chemokines CCL2 and CXCL2 in a MAPK-dependent manner. Both IL-17RA and IL-17RC are expressed in these cells, and the production of the chemokines was in a dose-and time-dependent manner (32).

Glomerular Endothelial Cells
Concerning glomerular endothelial cells (GEC), there are no specific studies in these cells. What is known are the effects of the axis on the endothelium from other vascular beds (see description in the section hypertension and thrombosis). However, these effects we believe to be applicable (in whole or part) to GEC. Specific studies are needed to assess potential local-specific effects.

Glomerular Basement Membrane
Although little is known about the potential effect of IL-17 on glomerular basement membrane (GBM), the IL 17 presence was associated with GBM thickening in a model of accelerated diabetic nephropathy; while IL-17A blockade with antibody reduced this effect (144). Additionally, in a model of antiglomerular basement membrane glomerulonephritis (anti-GBM GN), the Th17/IL-17 pathways were drivers of inflammation and autoantibody-induced renal injury; and the knockout or inhibition of IL-17 ameliorated these effects associated with decreased proinflammatory cytokines (145,146).

Tubulointerstitial Compartment: Tubular Epithelial Cells, Fibrosis, and Epithelial-Mesenchymal Transition Renal Tubular Epithelial Cells and Inflammation
The Exposure of RTEC to IL-17 induces the production of various mediators, from cytokines, chemokines, and growth factors as shown in several studies and experimental models, and both receptors (IL-17RA and IL-17RC) are expressed in these cells. Stimulation of RTEC with IL-17 increases the expression of various cytokines like IL-6, IL-1β, TNF-α (31,147).
In a model of crescent glomerulonephritis by lupus, RTEC stimulated with IL-17 and IFN-α significantly increased the expression of CCL2, which is chemotactic for dendritic cells and macrophages. In mice lacking IL-17RA, renal infiltration by macrophages was severely impaired, despite unchanged systemic response (147). In addition, stimulation of tubular epithelial cells with IL-17 increased mRNA expression of other chemokines like Cxcl1, Cxcl2, and Cxcl8, which are chemotactic for monocytes and neutrophils, as found in models of autoimmune glomerulonephritis. These effects were synergically potentiated by TNF-α (148). Thus, under IL-17 stimulation, RTEC produces mediators that recruit dendritic cells and macrophages that are important sources of TGF-β to promote renal fibrosis (149) putting IL-17 as an important driver of RTEC-mediated immunopathogenesis in LN.
The stimulation of RTEC with IL-17A impacts neutrophil kinetics, leading to the synthesis of the granulocyte colonystimulating factor (G-CSF) in a dose-and time-dependent manner and this effect occurred in synergy with TNF-αor IL-1β. The downstream signaling pathways of this effect involved MAPK activation (31). Added to G-CSF secretion, the stimulation of RTEC with IL-17A induced the expression of chemokines CXCL1 and CXCL5 that are responsive for massive neutrophil recruitment and consequent renal tissue injury (86,150). Taken together, IL-17 is a potent orchestrator of neutrophil-mediated damage, promoting both differentiation and the recruitment of neutrophils to the kidney (31,150).

IL-17, Renal Tubular Epithelial Cells, and Fibrosis
The Th17/IL-17 axis is a potent promoter of renal fibrosis (39, 151) as found in an experimental model of unilateral ureter obstruction (UUO), where TGF-β1 expression (mRNA and protein) were increased in the obstructed kidney (39). In the complement of the study, the addition of IL-17A to cultured renal proximal tubular epithelial cells or renal fibroblasts increased the production of fibronectin using the TGF-β/Smad signaling pathway; associated with increased expression of TGF-β1 mRNA and protein (39). Interestingly, the IL-17A-mediated fibronectin production was abrogated neutralizing TGF-β1 pathways, either by administering an anti-TGF-β1 antibody or TGF-β1 receptor I inhibitor (39).
In another study, IL-17A promoted myofibroblast activation and extracellular matrix deposition, and IL-17 deficient mice were protected from fibrosis secondary to obstruction (36). In an experimental model of hypertension and angiotensin II-induced fibrosis, the IL-17A or IL-17RA blockade with specific antibodies significantly reduced the fibrosis marker TGF-β1 (152). On the other hand, the antifibrotic effect of many agents in the kidney has been associated with reducing IL-17 (153)(154)(155)(156).

IL-17 and Epithelial-Mesenchymal Transition
Some scant literature shows that the Th17/IL-17 axis induces Epithelial-Mesenchymal Transition (EMT) on tubular epithelial cells (157,158). In one of these studies, with cultured cells, IL-17A promoted the cellular proliferation and secretion of extracellular matrix and induced inversion from epithelial to mesenchymal phenotype in a TGF-β1-dependent pathway (157).
Despite few studies in the kidney, the effect of the axis on EMT is well-known in many organs such as bronchoalveolar epithelium (159,160); epithelial cells of the salivary glands in Sjögren's syndrome (161,162); biliary epithelial cells (163,164); and peritoneal mesothelial cells (165). Concerning the promotion of the same effect on other intrinsic cells (mesangial and glomerular endothelial cells), it is an open gap to be elucidated in future investigations.

Vascular Compartment: Thrombotic Microangiopathy and Hypertension
One of the factors of poor prognosis in renal biopsy in lupus is thrombotic microangiopathy (which is the combination of endothelial injury and thrombosis). No primary studies evaluated the role of the Th17/IL-17 axis in thrombotic events in patients with lupus. However, IL-17A, IL-17RA, or IL-23 probably participate in this process because they are described as mediators of endothelial dysfunction (166,167); and have been associated with the occurrence of arterial thrombosis (167,168). In experimental studies, with psoriasis models, IL-17A shown to be a mediator of thrombotic events and vascular dysfunction (169,170). In another study with endothelial cells from patients with rheumatoid arthritis (RA), IL-17 (in combination with TNF-α) induced a procoagulant and prothrombotic phenotype (beyond the inflammatory state). Mechanistically, this occurred due to the strong inhibition of the expression of CD39/ATPDase (an inhibitor of platelet activation), enhancement of tissue factor (the cellular receptor for FVII and FVII), combined to decreased thrombomodulin (167). Additionally, studies have found an increase in Th17/IL-17 axis activity in primary antiphospholipid syndrome (171,172).
Hypertension is one of the manifestations of kidney involvement in lupus, and its presence is one of the factors of poor prognosis in LN (173,174). There is a lack of primary studies evaluating the direct effect of the Th17/IL-17 axis in this event in LN. However, there is evidence associating the Th17/IL-17 axis with primary hypertension and renal inflammation in both experimental and human studies (175,176). Additionally, basic research with angiotensin II-induced hypertension models shows that IL-17A deficiency or the blockade of IL-17A or IL-17RA with specific antibodies significantly reduces the pressure and inflammation in target organs (152,177). In another experimental study, IL-17A appeared to be a key mediator of vascular remodeling of the small arteries. Increased IL-17A levels increased blood pressure by induction of arterial remodeling and stiffness. In addition, treatment with antihypertensive drugs lowered blood pressure without modifying structural changes. Conversely, blocking the IL-17A with antibodies decreased blood pressure and vascular remodeling, suggesting that it has a sustained effect on vascular structure, more than merely hemodynamic (178). So, despite the lack of primary studies focusing IL-17 and hypertension on NL, it is believed that there is a participation of the axis in this outcome since lupus is a disease that occurs with significant hyperactivity of the Th17/IL-17 axis.

The Th17/ IL-17 Axis and Local Immune Response Orchestration Chemokines Production, Recruitment of More Immune Cells, Tertiary Lymphoid Structures Formation
As already described, the Th17/IL-17 axis induces the expression of chemokines like CXCL5, CXCL2, and CXCL8 for the recruitment of neutrophils (by binding to the receptors CXCR1 and CXCR2) (23,32,150), CCL2, CCL5 to attract monocytes and macrophages (by binding to receptors CCR1, CCR2, CCR5) (32,36). In addition to IL-17-induced chemokine production, several other chemokines like CXCL13 (179,180), CCL2, CCL7, CXCL1, CXCL2, and CXCL5 are produced by injured cells and resident macrophages, promoting the infiltration of B cells, dendritic cells, NK, Th1 cells (181)(182)(183) increasing the recruitment of more immune cells to the organ (184). The Sustained recruitment of immune cells can lead to the formation of kidney tertiary lymphoid structures (TLS), with some autonomy, in local activation of effector T cells, and in situ production of autoantibodies and components of the complement system (185,186), sustaining by itself the inflammatory flame at the local level (95,187,188). A recent experimental study showed that IL-17A is an orchestrator of TLS formation in the kidney, and this formation is associated with intrarenal inflammation, fibrosis, and progression of kidney damage (189). Interestingly, genetic depletion of IL-17A or blockade with anti-IL-17A antibody significantly reduced TLS formation, associated with attenuation of renal inflammation and fibrosis (189).

Production of Autoantibodies in situ, Complement Activation, Immunocomplex Formation, and Tissue Deposition
The IL-17 seems to participate in the production of autoantibodies, in situ, probably involving tertiary lymphoid structures, as found the correlation between IL-17 and increased anti-double-stranded DNA (dsDNA) production in an experimental study with kidney biopsy (185). IL-17 also seems to participate in other in situ events, including complement activation, immunocomplex formation, and tissue deposition, as found in the association of its expression level with these critical events in LN (185). In another study, it was evidenced that in IL-17A-/-mice, there was a decreased glomerular IgG and complement deposition and decreased intrarenal expression of Th1-associated proinflammatory mediators (190).

Crosstalk Between Th17; Intrinsic Renal Cells and Resident Immune Cells in Kidney Diseases
There is a Crosstalk between the kidney cells and Th17/IL-17 axis since the intrinsic cells of the kidney can induce the polarization of the lymphocytic response to the Th17 profile; as shown in an experimental study, in which stimulation of podocytes with bacterial products Polarized Naive CD4+ T Cells into Th17 cells (94). In addition, there is a crosstalk between Th17/IL-17 and intrarenal immune cells as shown that resident dendritic cells and infiltrating monocytes secret IL-1β that activated intrarenal Th17 cells and enhanced the IL-17 secretion (95). Elevated levels of IL-17, in turn, stimulate intrinsic kidney cells to produce chemokines and G-CSF/GM-CSF, inducing the differentiation of neutrophils and macrophages from bone marrow and recruitment to the kidney (31,191,192). Together, these aspects show that IL-17 participates in the crosstalk between Th17, neutrophils, monocytes, and intrinsic kidney cells (32).
In this orchestrator role of local immune response, it was demonstrated, in a model of obstructive nephropathy, that monocytes and macrophages express the IL-17RA receptor, and the absence of this receptor in all myeloid cells resulted in a reduction in macrophage accumulation in the kidney and significant attenuation of fibrosis (192). IL-17 participates as a mediator or potentiator of renal damage caused by several other cells and molecules of the immune response. For example, in a model of obstructive nephropathy, the C3 component produced locally by macrophages promoted renal fibrosis through increasing T-cell proliferation and IL-17A expression. Furthermore, the blockade of C3a reduced IL-17A expression and tubulointerstitial fibrosis (38). The Th17/IL-17 axis even seems to be able to initiate the chain of kidney damage by itself as it possesses the property of activating the inflammasomes and the toll-like receptors (193). Figure 1 is a schematic representation of the chain of events from hyperreactivity to self-DNA, activation and polarization of the Th17/IL-17 axis, to kidney damage and ESRD.
Th17/IL-17 axis seems to increase the risk of CKD itself, as seen in a genetic study with 650 elderly, where single nucleotide polymorphism (SNP) of IL17RA (rs4819554 AA homozygotes) was significantly more frequent among individuals with eGFR < 60 ml/min/1.73 m 2 ; and was associated to the risk of developing ESRD (40). Another study including 290 non-diabetic ESRD patients and 289 normal controls found that patients had a significantly higher frequency of IL17E rs10137082 * C and IL17RA rs4819554 * A alleles compared to control. At the same time, the genotyping analysis found that SNPs for IL17E (rs10137082) and IL17RA (rs4819554) were significantly more frequent among patients than in controls, after adjusting for confounders (208). It is important to highlight that this is an association and not necessarily a causal relationship because SNPs are hardly a causal factor alone. This is even less likely in a disease like lupus, with heterogeneous and multifactorial etiology (5,209). However, associated SNPs may be players with additive  74,75). The binding of DAMPs to TLRs in APCs induces their maturation (76,77). Part of dendritic cells migrates to draining lymph nodes to present the processed antigen to T cells and induce their activation and differentiation. The differentiation of Th17 cells is promoted by proinflammatories cytokines (IL-1β, IL-6, IL-21, IL-23) that dendritic cells secrete using the NF-κB and MAPK signaling pathways (77,83). Dendritic cells that remained in the tissue secrete various chemokines like CXCL9, CXCL10, and CCL20 that drive the recruitment of Th17 cells to the kidney through binding to receptors (CXCR3 and CCR6) (96,125,126). (B) In the Kidney, Th17 releases its cytokines (IL-17A, IL-17F, IL-17C, IL-21, and IL-22) that act directly on intrinsic kidney cells (mesangial cells, podocytes; glomerular endothelial cells, renal tubular epithelial cells). IL-17 family cytokines are responsible for changes in the cytoskeleton of the podocytes, activation of inflammasome and caspases, and induction of oxidative stress and podocytes apoptosis. In addition, in tubular epithelial cells, IL-17 promotes the activation of the profibrotic pathways with the increase of the expression of TGF-β (36, 87), promotion of EMT (158) with consequent increase of extracellular matrix proteins and fibrosis (87). (C) Besides local effects, IL-17 amplifies the systemic inflammatory response by stimulating the synthesis of inflammatory cytokines, growth factors, and chemokines, resulting in granulopoiesis/myelopoiesis and recruitment of more immune cells to the kidney (31,32,194). In addition, it promotes autoantibody production by its effects on Tfh and GC (195,196), and plasma cells (197). DC, dendritic cells; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; GBM, glomerular basement membrane; GEC, glomerular endothelial cells; IC, immune complex; IL-1, Interleukin-1; IL-17, Interleukin-17; IL-21, Interleukin-21; IL-23, Interleukin-23; IL-6, Interleukin-6; MAPK, Mitogen-activated protein kinase; MC, mesangial cell; Mo/mΦ, Monocytes/macrophages; NF-κB, Nuclear factor-κ B; PO, podocyte; RTEC, renal tubular epithelial cells; Tfh, follicular helper T cells; TGF-β, transforming growth factor-beta; Th17, T helper lymphocytes, subtype 17; TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4. or synergistic effects at the confluence of the multi-players that characterize the disease (11,111).
Additional evidence about the role of the Th17/IL-17 axis on Kidney diseases comes from the observation of the increased risk of CKD associated with renal inflammation in human diseases that occur with the hyperactivation of the Th17/IL-17 axis, like psoriasis (210)(211)(212), rheumatoid arthritis (213,214), and ankylosing spondylitis (215)(216)(217). In two studies (in the United Kingdom and Taiwan), psoriasis was associated with an increased risk of chronic kidney disease independent of traditional risk factors (211,212). The myriad of exposed situations suggests that Th17/IL-17 is a permanent participant, or at least as a pivotal element, in the pathogenesis of many kidney diseases independent of the initial insult (218); and its role extends from initial mechanisms, ESRD, to consequences of CKD and dialysis (88,205,207,219).
The role of the Th17/IL-17 axis has been found in other organic diseases that combine both inflammatory and fibrosing courses (219)(220)(221). Thus, the axis is involved in intestinal fibrosis in inflammatory bowel disease (222,223), in pulmonary fibrosis in systemic sclerosis and cystic fibrosis (220,224), liver cirrhosis (221,225) and peritoneal fibrosis (219,226). Its blockage and/or suppression has emerged as promising to prevent/mitigate the inflammatory and/or fibrosing behaviors in such conditions (226)(227)(228). Figure 2 summarizes the Th17/IL-17 axis effects on intrinsic renal cells and immune cells with potential implications in kidney damage, particularly in lupus nephritis.

Production of Autoantibodies
The Th17/IL-17 axis participates in the production of autoantibodies by B cells, as demonstrated in studies with autoimmune models in which the IL-17 drives the development of autoreactive germinal center (GC); and mice lacking the IL-17 receptor have reduced B cell development and humoral responses (229,230). In another study with an autoimmune disease model, the blockade of IL-17 signaling was associated with a significant reduction in both the number and size of germinal centers (231).
The IL-17RA receptor is essential for the optimal location of follicular helper T cells (Tfh) in the light zone (LZ) of the GC to promote the production of autoantibodies by B cells (195). Additionally, the production of IL-17 initially correlates with a reduced migratory response of B cells to chemotactic like CXCL12, suggesting that IL-17 not only facilitates the interaction between Tfh and responder B cells but also prolongs this interaction by increasing the time of permanence of B cells in GC (229).
In relation to the structure and functioning of the germinal center, a lymph node study showed that IL-17 is a critical requirement for the proliferation of lymph node and splenic stromal cells, particularly fibroblastic reticular cells (FRCs), during experimental autoimmunity. Without IL-17 signaling, there was a failure in FRC proliferation (nutrient stress, arrested cell cycle, and apoptosis), resulting in the impaired germinal center formation and antigen-specific antibody production (196).
The IL-17 importance in the production of autoantibodies was also evidenced in another study in which the PBMC supernatants from LN patients expressed higher levels of IgG, anti-dsDNA under IL-17 stimulation than in a normal culture medium. This effect occurred in a dose-dependent manner, and could be blocked completely by IL-17 monoclonal antibodies or partially by dexamethasone (58). Another experimental study showed that IL-17 increased anti-double-stranded DNA antibody production, and this was the link in the correlation between cytokine levels and disease severity (185). A recent study has ratified the crucial role of IL-17 by demonstrating that IL-17 promotes autoantibody production and increases plasma cell survival. In this study, the subset of plasmocytes expressing the IL-17RC receptor had an exponential increase in the production of anti-dsDNA IgG upon IL-17A stimulation in both patients and mice. Additionally, the transfer of Th17 depleted PBMC resulted in a significant reduction of autoantibody production and attenuation of renal damage. This attenuating effect was also observed in IL-17 or IL-17RC deficient mice, while the adoptive transfer of Th17 to IL-17deficient mice restored the plasma cell response and renal lupus damage. The most important is that IL-17 significantly promoted plasma cell survival, through phosphorylation of p38, stabilizing Bcl2l1 mRNA, which encodes the anti-apoptotic protein Bcl-xL (197).

Amplification of Systemic Inflammatory Response
Amplification of the inflammatory response is among the systemic effects of IL-17. This includes granulopoiesis and myelopoiesis by stimulating the synthesis of GM-CSF and G-CSF, increased production of chemokines, and inflammatory cytokines (31,192); in addition to associated paralysis or impairment of anti-inflammatory pathways (105). In these effects, IL-17 makes synergy with several other inflammatory mediators such as IFN-γ, TNF-α, and IL- 23 (42, 49, 232, 233). In a PBMC culture medium, stimulation with IL-17 induced significant IL-6 mRNA transcription in PBMC from LN patients than from HC (58). This study also brings the notion that, compared to controls, cells from lupus patients are hyperresponsive with higher production of inflammatory mediators under the same stimulus conditions (58).
In neutrophil kinetics, especially, IL-17 participates in various points of the chain, from differentiation through the synthesis of colony-stimulating factors (31); recruitment of neutrophils to the target organs through endothelial cell activation in a STAT3 and/or MAPK-dependent manner (34, 234) and by the synthesis of attracting chemokines like CXCL5, CXCL1; and CXCL8/IL-8 (86,235). In relation to CXCL1, a potent chemoattractant for neutrophils, it is worth mentioning that IL-17 participates in regulating its production and in the stability of its mRNA (232,235) and increases its biological half-life (236).
The Th17/IL-17 axis seems to have a cooperative relationship with other pathways whose importance is highlighted in the pathogenesis of SLE, like Type I interferons (6,11,237). This was evidenced by studies that found increased IL-17A and IL-17A-producing cells in IFN+ than in IFN-patients and HCs (238,239). In one of these studies involving 31 patients with SLE, patients displaying high IFN-α bioactivity (58.1% of them) had a higher frequency of Th17 cells in peripheral blood than those with low IFN-α bioactivity (mean ± SD 1.9 ± 1.0 vs. 1.2 ± 0.9). Additionally, subjects with high IFN-α bioactivity and elevated Th17 cells had significantly higher disease activity and serum IL-6 levels than those with low IFN-α and Th17 cells. Suggesting that IFN-α and Th17 cell pathways co-exist and co-regulate the disease pathogenesis (238). Other studies found a significant correlation between the Th17/IL-17 axis and Blymphocyte stimulator (BLyS/BAFF), a factor strongly correlated with IFN type I (239,240). In another study involving 33 patients with cutaneous lupus erythematosus who underwent biopsy, the level of IL-17A in tissue correlated positively with the IFN-α expression (Spearman's ρ = 0.56) (20).
The repercussion of this relationship between Type I IFN and the Th17/IL-17 axis in kidney damage was evident in an experimental model in which mice deficient in IL-17RA were protected from Type I Interferondependent crescentic glomerulonephritis. This effect was associated with impaired renal infiltration by activated macrophages, despite unaffected systemic response (147). As the underlying mechanism, the authors have shown that IL-17 in association with IFN-I differentially regulates the expression of macrophage chemoattractants genes, including Ccl2 (encoding CCL2) in RTEC (147). However, no primary study evaluated this relationship in LN in humans. A gap in knowledge to be filled in next studies, and combining genetic studies with integrative Bayesian network approaches may bring additional information to current knowledge in this disease characterized by heterogeneity (209).
The Th17/IL-17 axis may be the bridge (or part of it) between LES and other organs comorbidities and outcomes (241,242), such as cardiovascular disease because it is known to promote endothelial activation (233,234), prothrombotic states (167,168), hypertension (166,177), and atherogenesis (207,243,244); and osteoporosis because it is known to increase bone catabolic activity (245).

THE POTENTIAL OF TARGETING THE TH17/IL-17 AXIS AND RELATED PATHWAYS ON NEPHROPROTECTION
As described above, the Th17/IL-17 axis is involved in several points in the renal damage chain. Its effects include the induction of changes in the cytoskeleton of the podocytes with increased motility, decreased expression of the podocyte health proteins, increased oxidative stress, activation of inflammasome and caspases, and induction of podocytes apoptosis. The axis also promotes the activation of the profibrotic pathways, such as increasing the expression of TGF-β and the promotion of EMT with consequent increase of extracellular matrix proteins. In addition, it stimulates the synthesis of inflammatory cytokines by intrinsic and immune cells, synthesis of growth factors and chemokines which together result in granulopoiesis/myelopoiesis, and recruitment of more inflammatory cells. Therefore, inhibition of the Th17/IL-17 axis (and its signaling pathways) represents a promising strategy in treating lupus nephritis in an early view.

Agents Targeting Directly the Th17/IL-17 Axis
Several agents directly interfere with the axis and are approved to treat diseases in which the Th17/IL-17 axis clearly drives the inflammation. So, secukinumab and ixekizumab are agents targeting IL17A, both approved for ankylosing spondylitis, plaque psoriasis, and psoriatic arthritis (246); Bimekizumab that neutralizes both IL-17A and IL-17F is in preclinical phases for psoriatic arthritis and ankylosing spondylitis (247) and brodalumab an anti-IL17R, is approved for plaque psoriasis (248). Regarding the use of these agents in lupus, there are no studies completed so far; however, there are two ongoing trials to assess the safety, efficacy, and tolerability of secukinumab in patients with active lupus nephritis (NCT04181762); and the safety and efficacy of secukinumab in cutaneous manifestation of lupus (NCT03866317). In a case report involving a patient with lupus nephritis complicated by psoriasis vulgaris, the use of secukinumab was reported to be effective for both conditions with improvement in clinical and laboratory parameters (249).

Agents Targeting Indirectly the Th17/IL-17 Axis and Related Pathways
The Th17/IL-17 axis can be targeted indirectly in several ways, from interfering in the differentiating pathways, inhibition of migratory capacity, acting on mechanisms that favor polarization, including immunometabolism.
The differentiating pathways of Th17 cells are also a therapeutic target to be explored to prevent the prosperity of the axis. In fact, in a clinical trial, the addition of ustekinumab, a human monoclonal antibody against IL-12 and IL-23, to standard care resulted in better efficacy in clinical and laboratory parameters (250). It is noteworthy that il-23 not only drives the expansion, survival of pathogenic Th17 and other IL-17producing cells (84) but also decreases Treg by decreasing the production of IL-2 (the positive regulator of Treg) (251). Thus, the beneficial effect of its inhibition should involve as mechanisms the decrease of Th17 and the increase of Treg, the impairement of the IL-23/IL-17 synergisms, among other potential mechanisms. Still, on the path of differentiation, the inhibition of STAT3, the main Signal transducer in Th17 differentiation, delayed/limited the installation of lupus nephritis in experimental models (252)(253)(254). In another experimental model of LN, renal pathological damage was attenuated with the use of α-mangostin and 3β-acetyloxy-oleanolic, compounds with inhibitory activity on retinoic acid receptor-related orphan receptor gamma t (RORγt), the transcription factor for Th17 differentiation. These compounds significantly decreased serum anti-dsDNA antibody levels, IL-17A, and IFN-γ expression (255,256).
Since metabolic changes at the level of T cells are important in Th17 polarization and immune hyperreactivity, targeting the immunometabolism is another potentially promissory indirect strategy in SLE (110,111). In experimental studies, metformin, which inhibits oxygen consumption and glucose oxidation, inhibited the activation of T cells, with a consequent decrease in the production of IFN-γ and IL-17 (257,258). In another study with glucose transport inhibitors (CG-5), there was a decrease in Th1 and Th17 polarization by inhibiting their differentiation, accompanied by induction of regulatory T (Treg) (259). In addition to the effect on T cells, CG-5 treatment reduced the expansion of B cells in GC and autoantibodies' production (259). However, in a clinical trial, the addition of metformin to standard care could not demonstrate an additional benefit in reducing SLE recurrence (260). The hyperactivation of the mTOR pathway, a feature that favors Th17 polarization, is another potential target. In two studies with SLE patients, the use of rapamycin (an inhibitor of mTOR pathway) in combination with IL-2 or alltrans retinoic acid (ATRA) showed clinical efficacy decreasing the disease activity, associated with reduced Th17 cells, and restoration and long-term maintenance of Treg/Th17 ratio balance (261,262). Aligned with this data, in a 12-months prospective open-label study, rapamycin significantly reduced the disease activity scores (SLEDAI and BILAG), associated with a reduction in IL-17 production (either by Th17 cells or double-negative T cells) (263). A trial is registered to assess the efficacy and safety of rapamycin in patients with active SLE (NCT04582136).
Several other agents have shown their potential in improving SLE interfering with the axis. Thus, the immunomodulatory efficacy of stem cell therapies (either Umbilical cord, Bone Marrow or adipose-derived) involves the suppression of the axis or restoration of the Treg/Th17 balance (12,154); and defects in the functioning of stem cells trigger the disease (121). The beneficial effect of specific MicroRNA as miR-125a-3p and MicroRNA-10a-3p also involve interference on the axis (153,264). In an experimental study, punicalagin (a bioactive antagonist of PAR2) ameliorated lupus nephritis, in association with a significant reduction in splenic Th17 populations compared to the vehicle controls (265). Remembering that PARs are involved in Th17-induced rearrangement in the cytoskeleton and increased permeability (136). In an experimental study with MRL/lpr mice, a traditional Chinese medicinal formula suppressed the IL-17 production and Th17 activity by inhibiting the expression of CaMK4, which was associated with a decrease in renal hypercellularity and infiltration by neutrophils (266). It is worth remembering that CaMK4 is involved in Th17 activity and enhances its migratory capacity (96).

The Effect of Lupus Current Medicines on Th17/IL-17 Axis and Related Pathways
Many of the drugs with just known efficacy in the treatment of lupus, and which act on other biological targets, have a parallel effect on the Th17/IL-17 axis. Thus, for example, the effect of methylprednisolone on improving lupus nephritis was also associated with the rebalancing of Splenic CD4+ cells with a significant reduction in Th17 populations compared to controls in an experimental study (265). This corroborates the clinical observation that induction treatment was associated with the progressive reduction of IL-17A, IL-6, and IL-21 (50). Hydroxychloroquine, an immunomodulator in lupus, inhibited Th17 differentiation (267), and reduced Th17-related cytokines in patients (268). Mycophenolic acid, used in an experimental study, inhibited the production of IL-17A, which occurred with the reduction of granulopoiesis; and this effect was completely abolished in mice lacking the IL-17 receptor (269). The same drug showed an effect of reducing STAT3 phosphorylation in patients with SLE (270), which is crucial in synthesizing IL-17 and IL-21 (84). Even Belimumab, a recombinant human IgG-1λ monoclonal antibody that inhibits B-cell activating factor (BLyS/BAFF), effective in lupus nephritis (271), shown to occur, in its effectiveness, with the restoration of the Treg/Th17 balance (272).

CONCLUSIONS AND FUTURE DIRECTIONS
Dysregulated immunity at the Th17/IL-17 axis level plays a significant role in lupus nephritis pathogenesis and ongoing damage, following the initial activation of APC by immunogenic DNA or DNA-containing immune complexes.
The Th17/IL-17 axis orchestrates a chain of events that promote a proinflammatory and profibrotic environment stimulating intrinsic renal and resident immune cells to synthesize inflammatory cytokines and chemokines, promoting further recruitment of immune cells into the kidney. The Th17/IL-17 axis also exercises this driver and amplifier role systemically. All resident kidney cells express receptors for IL-17 and respond to IL-17 exposure in many ways, including changes on the cytoskeleton with increased motility, decreased expression of health proteins, increased oxidative stress, and activation of the inflammasome and caspases resulting in podocytes apoptosis. In renal tubular epithelial cells, IL-17 increases the expression of profibrotic and proinflammatory factors, such as TGF-β and fibronectin; and probably induces EMT of RTEC, promoting the further synthesis of the extracellular matrix, with all consequent changes in microstructure and renal functioning.
Despite considerable evidence on the contribution of the Th17/IL-17 axis in the pathogenesis of NL, studies directed to the Th17/IL-17 axis as a therapeutic target did not change the course of the disease as expected-a real gap in translation from bench to bedside. More works are needed to dissect the role of the Th17/IL-17 axis in the pathogenesis of the disease, and the underlying signaling pathways, to open the opportunity to target it effectively, preferably in a multitarget instead of single-cell based approach. In addition, clinical trials with the best designs are necessary, taking into account the clinical and immunological heterogeneity that characterize lupus.