The Kallikrein-Kinin System: A Novel Mediator of IL-17-Driven Anti-Candida Immunity in the Kidney

The incidence of life-threatening disseminated Candida albicans infections is increasing in hospitalized patients, with fatalities as high as 60%. Death from disseminated candidiasis in a significant percentage of cases is due to fungal invasion of the kidney, leading to renal failure. Treatment of candidiasis is hampered by drug toxicity, the emergence of antifungal drug resistance and lack of vaccines against fungal pathogens. IL-17 is a key mediator of defense against candidiasis. The underlying mechanisms of IL-17-mediated renal immunity have so far been assumed to occur solely through the regulation of antimicrobial mechanisms, particularly activation of neutrophils. Here, we identify an unexpected role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in C. albicans-infected kidney, and we show that the KKS provides significant renal protection in candidiasis. Microarray data indicated that Klk1 was upregulated in infected kidney in an IL-17-dependent manner. Overexpression of Klk1 or treatment with bradykinin rescued IL-17RA-/- mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following C. albicans infection. Furthermore, combining a minimally effective dose of fluconazole with bradykinin markedly improved survival compared to either drug alone. These results indicate that IL-17 not only limits fungal growth in the kidney, but also prevents renal tissue damage and preserves kidney function during disseminated candidiasis through the KKS. Since drugs targeting the KKS are approved clinically, these findings offer potential avenues for the treatment of this fatal nosocomial infection.


Introduction
The commensal fungus Candida albicans causes several clinical conditions in immunocompromised individuals, including oropharyngeal candidiasis (OPC, thrush) and vaginal candidiasis [1]. However, the most severe Candida-induced disease is a systemic form of bloodstream candidiasis. Disseminated candidiasis is the fourth most common hospital acquired infection and is associated with a 40-60% mortality rate [2,3]. Intravascular catheters, abdominal surgery, prolonged use of antibiotics and immunosuppressive therapy are risk factors for this disease, and contribute to the concerning rise in the incidence of candidiasis [1]. Available antifungal medications are limited by drug-drug interactions, drug resistance, toxicity and high treatment costs. To date, there are no effective vaccines to fungal pathogens [1]. Thus, there is an unmet clinical need to develop alternative, safe and ideally inexpensive approaches to treat this fatal infection.
Candidiasis is often treated effectively with azoles, amphotericin B and echinocandins [4,5]. The extensive and prolonged use of antifungal medications to treat systemic fungal infections, however, has led to drug resistant fungal strains and host toxicity [5,6]. Thus, novel antifungals or improved therapeutic strategies are still needed. Indeed, in vitro studies combining azoles with other drugs such as tacrolimus, cyclosporine A, amiodarone or retigeric acid B yielded encouraging results [7][8][9][10][11]. These data justify the concept of novel combination therapies to treat candidiasis at lower dosage in preclinical animal models.
Death due to sepsis is a frequent outcome of disseminated candidiasis [1]. However, in 30-40% adults and 50% neonates, Candida hyphae invade and injure the kidney, leading to irreversible damage and fatal renal failure [12]. Once Candida invades the kidney, a robust innate response dominated by neutrophils and monocytes/macrophages contributes to pathogen clearance and sets the stage for the adaptive immune response [13]. During the course of fungal clearance, both innate effector cells and kidney-resident cells release tissue repair enzymes and anti-inflammatory proteins. While necessary to repair injured tissue, these factors also limit bystander damage caused by innate immune cells.
Considerable data implicate IL-17 (IL-17A) in immunity to C. albicans. For example, IL-17RA -/-, IL-17RC -/-, RORγt -/and IL-17A -/mice are all susceptible to systemic C. albicans infection [14][15][16][17]. At mucosal surfaces, IL-17 mediates antifungal activity by driving the expression of antimicrobial peptides and chemokines that facilitate neutrophil influx [18,19]. Unlike mucocutaneous candidiasis, which affects individuals with compromised IL-17 signaling, systemic C. albicans infection normally impacts individuals with no known underlying genetic defects in IL-17 signaling pathways [1]. One recent study reported that IL-17 also acts on NK cells to drive the production of GM-CSF, with protective activities in disseminated candidiasis [15]. However, the mechanisms of local IL-17-mediated antifungal activities within the kidney still remain unclear.
The Kallikreins (Klk) are a family of fifteen related serine proteases. Klk1 in particular plays a critical role in renal function and pathology [20]. Klk cleave kininogens to generate kinin peptides, known as bradykinin and kallidin. Collectively, this system is termed the Kallikreinkinin system (KKS) (Fig 1C). Bradykinin signals through two receptors; bradykinin receptor β2 (Bdkrb2) is constitutively expressed, whereas Bdkrb1 is inducible upon inflammatory signals. The primary known function of the KKS is to regulate blood pressure by promoting vasodilation [20]. In addition, studies in animal models implicate the KKS in regulating inflammation, tissue repair and homeostasis during kidney injury [21]. The renal protective function of the KKS is mediated through upregulation of tissue repair proteins, inhibition of profibrotic factors, and control of apoptosis [21]. Consistently, polymorphisms in KKS-related genes (ACE, BDKRB2, NOS3, KLK1) are associated with an increased risk of acute and chronic renal injury in humans [22][23][24][25]. While it is clear that the KKS protects the kidney in disorders associated with sterile inflammation, its role in renal immunity in infectious settings is less well defined.
In some bacterial and viral infections, bradykinin enhances vascular permeability to facilitate pathogen spread [26,27]. However, there are very few studies linking the KKS and C. albicans pathogenesis. Kininogens have been shown to bind the C. albicans cell wall, causing the fungal SAP2 protease to induce release of biologically active kinins [28,29]. Additionally, the KKS was implicated in IL-17-mediated skin inflammation and an IL-17-dependent model of autoimmunity (experimental autoimmune encephaloymyelitis, EAE) [30,31].
Here, we identified kallikrein genes as novel IL-17 targets in disseminated candidiasis, revealing an unanticipated link between IL-17 and KKS-mediated renal protection. Mice lacking the IL-17 receptor A subunit (IL-17RA) exhibited diminished Klk expression in the kidney. Moreover, overexpression of Klk1 restored protective renal immunity against systemic C. albicans infection in IL-17RA -/mice. The IL-17-KKS-axis activated bradykinin receptors, which served to enhance renal anti-C. albicans immunity. Addition of exogenous bradykinin in immunocompetent mice prevented renal damage by inhibiting apoptosis of kidney-resident cells, and prolonged animal survival during candidiasis. Finally, addition of bradykinin to a minimally effective dose of fluconazole significantly improved survival. These data identify a previously unrecognized link between IL-17 and KKS-mediated renal protection against disseminated candidiasis, which may provide the basis for clinical intervention in this disease.

IL-17 triggers Klk expression in the kidney during disseminated candidiasis
In C. albicans intravenous challenge in mice, the kidney is the most heavily colonized organ [32]. With a higher inoculum (>10 6 cfu), mice succumb to infection within 48-72 h due to sepsis. However, mice infected with a low dose of C. albicans (10 5 cfu) exhibit progressive loss of renal function over a period of~2 weeks, which more accurately reflects disease progression in humans [32]. During candidiasis, IL-17 is rapidly upregulated in the kidney, but its function in that organ is unknown [14]. To understand how IL-17 mediates kidney-specific immunity, we performed Illumina microarray analyses comparing WT and IL-17RA -/renal gene expression at 48 h p.i. Confirming previous reports, IL-17RA -/mice demonstrated significantly increased kidney fungal burden in comparison to WT following infection with C. albicans (CAF2-1 or SC5314) ( Fig 1A) [14,16,33].
The classic IL-17 gene signature includes neutrophil-related genes and antimicrobial peptides (AMPs), such as CXC chemokines (Cxcl1,2,5), defensins (Defb3), calprotectin (S100a8) and lipocalin 2 (Lcn2) [18]. Although a few genes previously shown to be controlled by IL-17 were differentially expressed in the kidney during candidiasis, overall we saw a surprisingly distinct gene profile compared to analyses of IL-17-dependent genes in other settings (Fig 1B and  S1A Fig) [18]. The expression of classical IL-17-responsive genes such as Cxcl1, Cxcl2 and Lcn2 were unaffected in IL-17RA -/mice (S1A Fig). Using the DAVID Gene Functional Classification algorithm (which uses a gene-to-gene similarity matrix based shared functional annotation), we identified several functional groups with enrichment scores over 1.0. Most striking to us based on their known role in kidney physiology was the enrichment of genes encoding the KKS (Fig 1B and 1C). Multiple Klk genes were suppressed in the kidney of IL-17RA -/mice compared to WT following C. albicans infection. These results were verified by measuring the renal expression of Klk1 and Klk1b27 by qPCR (Fig 1D).
We next analyzed the impact of differential fungal load on Klk gene expression in the kidney by inoculating WT mice with either a high (10 6 cfu) or low (10 5 cfu) dose of C. albicans and measuring expression at 48 h p.i. Either dose caused comparable Klk1 expression (S1B Fig), indicating that the differences in Klk expression are not due to differential fungal loads. We then verified protein expression of Klk1 by immunoblotting. Klk1 was constitutively expressed at comparable, albeit low, levels in the sham-infected WT and IL-17RA -/mice. However, Klk1 was upregulated in WT kidney following C. albicans infection. This observation was true upon infection with either the CAF2-1 or SC5314 strains of C. albicans. Confirming the gene expression data, we observed lower expression of renal Klk1 in the absence of IL-17RA during systemic infection (Fig 1E and 1F). Collectively, these data indicate that IL-17 signaling in the kidney regulates Klk1 expression during disseminated candidiasis. To our knowledge, this is the first demonstration that renal Klk1 is upregulated in an infectious setting, and certainly first demonstration that the KKS is controlled by IL-17.

Klk1 is required for IL-17-mediated renal protection against disseminated candidiasis
To understand the role of Kallikreins in candidiasis, we focused on Klk1 based on its connection to IL-17-driven diseases including EAE and systemic lupus erythematosus [30,31]. Kidney sections from C. albicans-infected WT mice were stained for Klk1 at 72 h p.i. Only kidney-resident cells, particularly renal tubular epithelial cells (RTEC), expressed Klk1 during infection (Fig 2A). These results agree with previous reports indicating that RTEC are the major producers of Klk1 in chronic kidney diseases [34,35]. Although Klk1 controls vital kidney functions, its regulation and function under inflammatory conditions are not well defined. We therefore asked whether changes in Klk1 expression were a direct result of IL-17 signaling in RTEC, or a by-product of generalized renal inflammation due to increased fungal burden. We treated primary RTEC in vitro with IL-17 together with TNFα, a cytokine with which IL-17 exhibits from WT and IL-17RA -/kidneys (n = 5-6) infected with C. albicans (E) CAF2-1 or (F) SC5314 were evaluated for Klk1 protein by western blotting. Sham infected mice received PBS. Images were quantified using ImageJ. Representative image of 1 of 2 independent experiments (E and F). For the bar diagram, data are combined from 2 independent experiments. Bars indicate mean ± S.D. P<0.05 (*), <0.01 (**), <0.001 (***). ns, not significant.  To verify the finding that IL-17 induces Klk1 in the kidney, we overexpressed IL-17 in WT mice using adenovirus (Ad-IL-17) [14]. Mice infected with Ad-IL-17 exhibited 400-fold more serum IL-17 than with a control vector (Ad-ctrl) (S2A Fig). This increased level of IL-17 was not associated with systemic inflammation, as serum TNFα and IL-1β levels were undetectable. By IHC, RTEC within kidney stained positively for Klk1 following overexpression of IL-17. The expression of Klk1 was restricted to RTEC, as no staining could be detected in the glomerular and vascular compartments of the kidney ( Fig 2C). Additionally, Ad-IL-17 administration upregulated multiple IL-17 target genes in the kidney (Il6, Cxcl5 and Lcn2) (Suppl. Fig 2B). Nonetheless, kidneys of Ad-IL-17-treated mice exhibited no overt inflammatory changes (S2C Fig). Thus, IL-17 induces the expression of Klk1 in RTEC following disseminated candidiasis.
Klk1 protects the kidney against acute and chronic disorders in sterile inflammation, but has not been linked to candidiasis or IL-17 signaling. To test the hypothesis that Klk1 plays a critical role in IL-17-driven renal protection against disseminated candidiasis, we overexpressed Klk1 with the adenoviral system (Ad-Klk1) and assessed disease susceptibility. Remarkably, overexpression of Klk1 significantly improved the survival of C. albicans-infected IL-17RA -/mice and in WT mice ( Fig 2D). A previous study suggested that Klk1 may induce inflammatory cytokines in human RTECs, at least in vitro [37]. However, we found that overexpression of Klk1 had very little impact on renal inflammatory gene expression or fungal load ( Fig 2E and S2D Fig). Overall, these data indicate that Klk1 enhances anti-C. albicans immunity in the kidney in an IL-17-dependent manner, but is not responsible for inducing inflammatory gene expression.

Activation of the bradykinin receptors is required for IL-17-Klk1 axisdriven protection against disseminated candidiasis
Klk1 mediates cleavage of kininogens to generate bradykinin, which signals through Bdkrb1 and Bdkrb2 (Fig 1C) [21]. Additionally, Klk1 activates protease-activated receptors (PAR) such as PAR4 to trigger the release of inflammatory mediators from RTEC [37]. To define the role of Bdkrb activation in renal immunity, IL-17RA -/mice were treated with bradykinin (300 nmol/kg) and survival evaluated following infection. As shown, 90% of the untreated IL-17RA -/mice succumbed to infection by day 5 p.i., while mortality in IL-17RA -/mice was delayed with bradykinin treatment (Fig 3A). Additionally, untreated WT mice had a modestly increased survival benefit compared to bradykinin treated IL-17RA -/mice, suggesting that there may also be a Bdkrb-independent pathway occurring in candidiasis.
Disseminated candidiasis typically impacts individuals with no known underlying immune defects [1]. Therefore, we assessed impact of Bdkrb signaling in mice with intact IL-17 independent experiments. (C) WT mice (n = 5) were injected with Ad-IL-17 or Ad-ctrl (1x10 9 pfu). Six days post-injection, kidney sections were stained with anti-Klk1 or isotype control Abs. Inset: Klk1 staining in RTEC and negative staining in glomerulus (GM) and blood vessels ( signaling capacity and normal levels of Klk1. Accordingly, WT mice were treated with Bdkrb1 and Bdkrb2 antagonists and evaluated for survival following systemic C. albicans infection. Mice given Bdkrb1 and Bdkrb2 antagonists had significantly reduced survival compared to untreated controls ( Fig 3B). Collectively, these results indicate participation of Bdkrb signaling in the renal host defense during disseminated candidiasis. Bradykinin prevents renal damage and preserves kidney function in immunocompetent mice following disseminated candidiasis Despite advances in antifungal therapy against disseminated candidiasis, mortality in patients with systemic C. albicans infection remains unacceptably high [1]. Since IL-17 is implicated in controlling candidiasis in experimental mouse models, targeting downstream mediators of IL-17 signaling pathway is an attractive approach to treat disseminated candidiasis [1]. In this regard, we hypothesized that manipulation of the IL-17-KKS pathway with bradykinin would ameliorate candidiasis in a host with intact IL-17 signaling. To provide proof-of-principle, WT mice were treated with bradykinin and survival assessed with two strains of C. albicans (CAF2-1 or SC5314). Indeed, the bradykinin-treated cohort exhibited significantly delayed mortality compared to an untreated control group (Fig 4A and 4B). Although bradykinin has been implicated in the development of angioedema and hypotesion [38], mice treated with bradykinin did not show an increased incidence of angioedema at day 7 p.i. (Fig 4C). Moreover, to investigate the hypotensive effect of bradykinin, we performed a study where blood pressure (BP) alterations were measured in real time in uninfected WT mice following bradykinin treatment. We find that i.p. injection of bradykinin exerted only a very brief hypotensive response that peaked within~1 minute with full recovery by 6 minutes (maximum reduction of Mean Arterial BP was 36%) (S3A Fig). This result indicates that bradykinin is not likely to have any long term consequences on renal function during candidiasis. Taken together, these results demonstrate a beneficial effect of exploiting the IL-17-KKS axis in treating disseminated candidiasis even in a host capable of mounting normal IL-17 response.
Following hyphal invasion, renal injury is mediated by unchecked fungal replication and bystander tissue damage caused by the local inflammatory response. Therefore, clearance of C. albicans and timely repair of damaged tissues is crucial to preserve renal function. To understand the mechanisms by which bradykinin mediates renal protection during disseminated candidiasis, WT mice were treated with bradykinin starting on day -1 and then daily for 7 days. Mice treated with bradykinin demonstrated significantly diminished serum blood urea nitrogen and creatinine levels compared to untreated animals (Fig 4D), indicating that bradykinin preserves normal renal function in systemic C. albicans infection.
We then asked whether improved renal function in bradykinin treated mice was due to reduced damage of kidney parenchyma following fungal invasion based on histological analyses. While sham-infected mice treated with bradykinin showed normal kidney histology, the renal parenchyma of infected mice showed overt pathological changes characterized by loss of brush border epithelium and tubular atrophy at day 7 p.i. Moreover, the damage was primarily restricted to the renal cortex and outer medullary region. In line with the kidney function results, renal damage was ameliorated upon bradykinin treatment (Fig 4E). Interestingly, the influx of inflammatory cells was comparable between the treated and untreated groups. Furthermore, we observed significantly diminished expression of neutrophil gelatinase-associated lipocalin (NGAL), a prototypical kidney injury marker, in bradykinin treated animals (Fig 4F and 4G). Collectively, these results indicate that bradykinin prevents C. albicans-mediated renal damage and preserves renal function during disseminated candidiasis.

Bradykinin-mediated renal protection is independent of fungal clearance or inflammatory cell influx
To identify the effector mechanisms by which bradykinin prevents renal insufficiency, we evaluated fungal load and inflammatory cell influx at days 3 and 7 p.i. Although differences in  7) and (D) serum BUN and creatinine levels (n = 6-10). Data pooled from 2-3 independent experiments. Each kidney function were already evident early as day 7 p.i. (Fig 4D), fungal loads were comparable between the bradykinin treated and untreated groups at these time points (Fig 5A). Previous studies have shown that neutrophils and monocytes/macrophages mediate fungal clearance in candidiasis [39,40]. Thus, we examined the frequency of infiltrating myeloid cells in kidney upon bradykinin treatment. In agreement with the fungal clearance rates, the percentages of kidney infiltrating inflammatory cells (CD45 + ), neutrophils (Gr1 + ) and macrophages (F4/80 + ) were similar between groups (Fig 5B and S3B Fig). We next assessed whether bradykinin impacted the candidacidal activity of innate cells. Bradykinin treatment of BM-derived neutrophils and macrophages (BMDM) did not alter their ability to kill unopsonized C. albicans yeasts, as determined by an in vitro fungal killing assay ( Fig 5C). Additionally, RTEC and BMDMs were stimulated in vitro with bradykinin and culture supernatants evaluated for cytokines and nitrite production. Bradykinin induced IL-6 in RTECs (Fig 5D), but did not induce IL-6, TNFα or nitrite in BMDMs (Fig 5E and 5F). Overall, these results suggest that renal protection observed in bradykinin treated mice is not due to diminished fungal load, inflammatory cell infiltration or proinflammatory function of innate cells in the kidney.

Bradykinin prevents apoptosis of kidney-resident cells during disseminated candidiasis
Activation of bradykinin receptors protects the kidney from end-stage renal damage by inducing tissue-protective growth factors and matrix-degrading enzymes [21]. Therefore, we examined the effect of bradykinin treatment on profibrotic changes and tissue-protective growth factors expression in candidiasis. We observed minimal extracellular matrix protein deposition in bradykinin treated mice (S4A Fig). Additionally, there were no significant differences in expression of genes encoding tissue-protective growth factors (Hgf and Ctgf) or matrix-metalloproteinases at day 7 p.i. (Mmp2 and Mmp9) (S4B Fig). Previous studies have described an anti-apoptotic function of bradykinin in kidney injury [41,42]. Therefore, we assessed apoptosis in kidney-resident cells following C. albicans infection. At day 7 p.i., flow cytometry analysis revealed a significantly reduced frequency of both early (AnnexinV + PI -) and late (AnnexinV + PI + ) apoptotic kidney-resident cells (CD45 -) in bradykinin treated compared to untreated mice (Fig 6A). Concurrently, there was a significant reduction in the number of TUNEL positive cells in the renal cortex following bradykinin treatment (Fig 6B). C. albicans can regulate survival of kidney-resident macrophages via Caspase-3 [39]. In agreement with the reduced apoptosis (Fig 6A), bradykinin treatment resulted in a significantly diminished number of cleaved Caspase-3 + kidney-resident cells (CD45 -) ( Fig  6C). Moreover, IHC indicated that cleaved Caspase-3 is localized in the renal cortex and outer medullary region of untreated mice (Fig 6D). The extent of cleaved Caspase-3 was markedly reduced following bradykinin treatment (Fig 6D). Although expression of Bax (a pro-apoptotic gene) in kidney-resident cells was comparable between the groups, there was an increase in expression of Bcl-xL (an anti-apoptotic gene) after bradykinin treatment (Fig 6E). Thus, bradykinin preserves renal function during systemic fungal infection by limiting apoptosis of kidney-resident cells.  Fungal clearance correlated well with survival, as mice treated with FLC (5 mg/kg) showed the same susceptibility as untreated animals (S5B Fig). Therefore, the 5 mg/kg dose of FLC was chosen to evaluate the impact of the combination therapy. Next, C. albicans infected WT mice were treated with FLC and/or bradykinin and evaluated for survival over 14 d (Fig 7A). Strikingly, mice receiving the combination of bradykinin and FLC showed a significant increase in survival compared to untreated mice or mice given FLC or bradykinin alone ( Fig 7B). As expected, mice treated with bradykinin alone but not FLC demonstrated increased survival compared to untreated mice ( Fig 7B). These data show that a combination of bradykinin and FLC confers better protection against disseminated candidiasis than either drug individually. Consequently, use of bradykinin could potentially permit reducing the dose of antifungal drugs without compromising efficacy against fungal infection.

Discussion
Kidneys in a healthy state are sterile. However, renal infections occur via hematogenous routes or from ascending spread from the bladder or urethra [43]. In recent years, considerable data have implicated IL-17 in immunity against disseminated candidiasis [14][15][16]. Nevertheless, it is unclear how IL-17 regulates immunity within the kidney, the most heavily colonized organ during blood-borne C. albicans infection. In the present report, we have identified renal-protective kallikreins as novel IL-17 target genes in systemic candidiasis, thereby revealing a new connection between IL-17 and KKS-mediated renal defense. IL-17 not only limits fungal growth in the kidney, but also prevents renal tissue damage and preserves kidney function during C. albicans invasion (S6 Fig). Consequently, therapeutic manipulation of the IL-17-KKS pathways protected mice from early mortality in disseminated candidiasis. Our data provide important and potentially translatable insights into the renal functions of IL-17 in the context of this fatal hospital-acquired infection.
Kidney-specific immune responses are mediated by both kidney-infiltrating immune effectors and kidney resident cells. Although IL-17RA is ubiquitously expressed, most documented IL-17R signaling occurs in non-hematopoietic cells, particularly cells of epithelial and mesenchymal origin [44,45]. A recent study also suggested a role for IL-17RA signaling in NK cell development in the context of disseminated candidiasis [15], although this report conflicts with a study showing that NK cells are redundant for antifungal defense in immunocompetent hosts [46]. Consistent with the latter finding, we and others have observed upregulation of transcripts encoding IL-17A and IL-17-responsive genes in the kidney [14]. We also showed that kidney resident cells express the IL-17R and are responsive to IL-17 [47]. Taken together,  IL-17-Kallikrein-Axis and Disseminated Candidiasis these results argue that there is a bona fide, kidney-specific role of IL-17 in immunity to candidiasis.
Unlike mucocutaneous candidiasis, disseminated infection typically occurs in individuals with no known defects in IL-17 signaling pathways. In line with these observations, we were intrigued by the surprisingly different gene profile seen in C. albicans-infected kidney compared to prior studies involving mucosal Candida infection such as the tongue [18]. These results highlight the fact that different cell types "interpret" IL-17 signals differently, with nonidentical patterns of gene expression depending on setting. Therefore, lessons derived from studies of anti-C. albicans immunity at mucosal sites cannot always be applied to local kidney immune responses. Although we show that IL-17 (in conjunction with TNFα) induces Klk1 gene in primary RTEC, there is little known about Klk1 gene regulation at the transcriptional level. We have identified conserved CCAAT Enhancer Binding Protein (C/EBP)-β binding sites within the putative proximal promoters of the Klk1 gene, hinting that IL-17 may regulate Klk1 expression in a C/EBPβ-dependent manner. Indeed, C/EBPβ -/mice are susceptible to systemic candidiasis [48,49]. Since TNFα is required for protection against disseminated candidiasis [50], further studies should focus on Klk gene expression in the kidney of TNFα-deficient mice.
Klk1 cleaves kininogens to form bradykinin, a process known as the bradykinin-dependent pathway [20]. In addition, Klk1-mediated activation of PAR4 induces cytokine production and prevents apoptosis in RTEC, known as the bradykinin-independent pathway [37]. Mice deficient in specific PARs or treated with PAR antagonists exhibited compromised renal inflammatory changes [51,52]. Nevertheless, the contribution of PARs in IL-17-Klk1-mediated renal protection is unknown. Moreover, the relative contributions of Bdkrb1 and Bdkrb2 in renal defense against candidiasis need to be determined, which is possible with specific knockout mouse strains.
Our data show that bradykinin treatment improved renal function as early as 7 days p.i. Interestingly, serum creatinine and BUN levels did not correlate with the survival benefit at late time points, the basis for which is unclear. Late mortality despite improved renal function likely indicates side effects of bradykinin in mice. Although bradykinin has been implicated in causing angioedema [37], mice treated with bradykinin did not show any signs of angioedema following fungal infection (Fig 4C). Additionally, bradykinin at the dose levels used in this study did not seem to exert any long term consequences on blood pressure. Therefore, these studies argue against the likelihood of bradykinin toxicity and point towards alternative possibilities to explain the lack of correlation between kidney function and survival benefit. First, extremely short half-life of bradykinin (approx. 30 sec) and internalization of bradykinin receptors may lead to bradykinin unresponsiveness at the late time points. Second, daily administration of bradykinin may lead to the activation of a negative feedback regulatory mechanisms leading to increased degradation of bradykinin by Angiotensin converting enzymes. Finally, bradykinin has been shown to modulate glomerular function by regulating podocyte permeability [53], an effect independent of its renal tissue protective function. Given the potential side effects in bradykinin therapy, future studies investigating these questions could help better understand the means to harness the beneficial impact of IL-17-KKS axis without compromising the safety in treatment against disseminated candidiasis.
An Ab targeting IL-17 (secukinumab) was approved in 2016 to treat moderate-severe plaque psoriasis [54]. Abs against IL-17/IL-17RA are in clinical trials for other autoimmune conditions [55]. One obvious concern with anti-IL-17 therapy is compromising IL-17-driven antifungal immunity. Although systemic C. albicans infection has not been reported thus far with secukinumab, patients on this medication may have a higher risk of developing disseminated candidiasis in the face of predisposing factors, such as an indwelling catheter, abdominal surgery or long-term antibiotic use. Our data show that activation of the KKS pathway restored protection in IL-17RA -/mice. Thus, drugs targeting the IL-17-KKS axis may be considered to treat or prevent disseminated C. albicans infection in patients receiving anti-IL-17 therapy.
Amphotericin B, azoles and echinocandins are used to treat systemic C. albicans infections, but there are concerns due to drug-resistance and toxicity. Here, we show proof-of-concept that combination therapy with bradykinin and low-dose FLC is effective in treating candidiasis in mice. Notably, the survival rate of mice treated with the combination therapy was similar to the survival rate in mice given four times the FLC dose. This approach may be especially valuable for anti-fungal drugs such as amphotericin B due its intrinsic renal toxicity. Future studies will test the efficacy of combination therapy with amphotericin B and bradykinin, once we have access to oral or injectable form of amphotericin B suitable for administration in mice. In addition, the efficacy of ACE inhibitors, known to increase the levels of bradykinin and are routinely used to treat patients with obstructive nephropathy, need further evaluation in pre-clinical animal models of disseminated candidiasis. Overall, our data show that defining the IL-17 anti-fungal pathway has highlighted a potentially translatable and testable approach to treating systemic candidiasis. Additionally, the novel convergence between IL-17 and the KKS pathways in renal defense against fungal infection represents a major advance in our understanding of IL-17 signaling in the kidney inflammation.

Ethics Statement
Wild type (WT) C57BL/6J mice were purchased from The Jackson Laboratory (Bar Habor, ME). IL-17RA -/mice were kindly provided by Amgen (San Francisco, CA) and bred in-house. All mice were housed under specific pathogen-free conditions, and age-matched male mice were used for all experiments. Animal protocols were approved by the University of Pittsburgh IACUC (Protocol # 14094427), and adhered to the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health.

C. albicans culture and disseminated infection
C. albicans strains CAF2-1 or SC5314 were used as indicated. C. albicans was grown in YPD at 30°C for 18-24 h. Mice were injected via the tail vein with PBS (sham-infected) or 1x10 5 cfu (unless otherwise indicated) C. albicans yeast cells resuspended in PBS. Mice were weighed and monitored daily. Mice were sacrificed if they showed >20% weight loss or signs of severe pain or distress. Mice were evaluated for survival over a period of 14 days. At sacrifice on days 2, 3 and 7 p.i., as indicated, kidneys were weighed and homogenized in sterile PBS using a Gentle-MACS (Miltenyi Biotec, Cambridge MA). Serial dilutions of organ homogenates were plated on YPD agar with antibiotics, and fungal burden represented as colony forming units (cfu) per gram of tissue.

Measurement of serum creatinine and blood urea nitrogen
Serum was collected by retro-orbital bleeding at day 7 p.i. Creatinine and blood urea nitrogen levels were assessed using the QuantiChrom Creatinine Assay kit (BioAssay Systems, Hayward CA) and MaxDiscovery Blood Urea Nitrogen Enzymatic kit (Bioo Scientific Corp., Austin TX), respectively.

Measurement of vascular permeability
Vascular permeability in mice was assessed as described before [56]. Briefly, 100 μl sterile solution of Evans Blue dye (30mg/kg) (Sigma Aldrich., St Louis, MO) in PBS was injected intravenously. The stain was allowed to circulate for 30 min. After 30 min, mice were sacrificed and the hind feet were removed, blotted dry and weighed. The Evans blue was extracted from the feet with 1 ml of formamide overnight at 55°C and measured spectrophotometrically at 600 nm. Evans Blue stain was quantified according to a standard curve. The results are presented as ng of Evans Blue stain/mg of tissue. Bone marrow derived macrophages (BMDM) from C57BL/6J mice were cultured for 7 days in the presence of L929 supernatants. BMDM and RTEC (1x10 6 cells/well) were treated with bradykinin (R&D Biosystems, Minneapolis MN) or left untreated for 24 h. LPS (Sigma Aldrich, St Louis, MO) was used as positive control. Supernatants were subjected to analyses using commercially available IL-6, IL-1β and TNFα ELISA kits (Ebiosciences, Dallas TX. Nitrite concentrations were measured by tri-iodide based reductive chemiluminescence as previously described [57]. Briefly, samples were injected into tri-iodine to reduce nitrite to NO gas that was detected by a Nitric Oxide Analyzer (Sievers, GE).

In vitro fungal killing assay
Neutrophils isolated from bone marrow using Neutrophil Isolation Kit (Miltenyi Biotech, San Diego, CA) were plated at 1×10 5 cells/well. Non-opsonized C. albicans was added to neutrophils at 0.5 × 10 5 yeast cells/well (ratio of 2:1). If indicated, bradykinin was added to the wells. Cultures were incubated with unopsonized C. albicans for 3 h and lysed in cold double-distilled H 2 O. Killing was assessed by cfu counts in triplicate. The results are reported as percentage killing of C. albicans which is calculated as 1-[cfu of treatment group/cfu of control group] X 100. The same protocol was followed for bone marrow derived macrophages which were cultured with L929 supernatants supplemented media for 7 days prior to carrying out the killing assay.

Gene expression profiling and quantitative real-time PCR analysis
At sacrifice, kidneys were stored at -80°C. Total RNA was extracted from the homogenized kidney tissue with the RNeasy Micro Kit (Qiagen, Valencia CA) and submitted to Genomics Research Core at University of Pittsburgh. Gene expression analysis was performed using Mouse WG6 Gene Expression Bead Chip (Illumina). All test, normalization and transformation analyses were performed using caGEDA, a feely available informatics tool. The data sets were analyzed for differentially expressed genes. Efficiency analysis was performed by Random Resampling Validation using a Naïve Bayes Classifier and PACE analysis. The cluster analysis was performed by Unweighted Pair Group Method with Arithmetic Mean and similarity measure was determined by Euclidean distance. The raw and normalized microarray data have been submitted to the Gene Expression Omnibus (GEO), and study ID is GSE88800 at: https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE88800.For real time PCR analysis, complementary DNA was synthesized with SuperScript III First-Strand (Invitrogen, Carlsbad CA). Gene expression was determined by qPCR with PerfeCTa SYBR Green FastMix ROX (Quanta BioSciences, Gaithersburgh MD) on a 7300 Real-Time PCR System (Applied Biosystems, Carlsbad CA). Primers were obtained from Quantitect (Qiagen, Valencia CA). The expression of each gene was normalized to Gapdh.

Flow cytometry
At sacrifice on day 7 p.i., kidneys were harvested following perfusion with PBS. Briefly, kidney homogenates were digested in PBS with 1 mg/ml collagenase type I (Worthington, Lakewood NJ) for 30 m at 37°C. Cells were stained with the following antibodies: CD45 (BioLegend; clone 30-F11), Ly6G (BD Biosciences; clone IA8), F/480 (eBiosciences; clone BM8). For detection of apoptotic cells and cleaved Caspase-3 positive cells, cells were stained with Annexin V (BD Pharmingen) and CaspGLOW Fluroscein active Caspase-3 staining kit (eBioSciences), respectively as per manufacturer's protocol. Samples were acquired and sorted on a Fortessa and FACS ARIA II, respectively (BD Biosciences, San Jose CA) and analyzed with FlowJo (Tree Star, Ashland OR).

Histology and Immunohistochemistry
Kidneys were fixed in formalin, dehydrated and paraffin embedded. Serial kidney sections were stained with H&E, Periodic-acid Schiff or Masson Trichrome stains for morphological analysis and determination of kidney injury.
Immunohistochemistry staining was done on formalin-fixed, paraffin embedded sections. Sections were rehydrated and antigen retrieval was performed with heated citrate. Primary antibodies against the following proteins were used: Klk1 (LifeSpan Biosciences, Seattle WA), NGAL (Santa Cruz Biotechnology, Dallas TX) and cleaved caspase-3 (Cell Signaling, Danvers MA). Secondary antibodies used were horseradish peroxidase coupled antibodies (Jackson ImmunoResearch, West Grove, PA). To detect apoptotic cells TUNEL staining was done on frozen kidney sections using the TUNEL apoptosis detection kit according to manufacturer's protocol (Millipore, Temecula CA). The number of TUNEL + cells was counted in 15 randomly selected high powered fields (400X) per slide. All images were obtained with EVOS FL Auto microscope (Life Technologies CA).

Western blots
Kidneys were homogenized in RIPA buffer. Concentration of protein was quantified by the BCA quantitation assay (Thermo Scientific, Pittsburgh PA). Equal amounts of sample were subjected to electrophoresis and transferred to PVDF membranes (Millipore, Billerica MA). After blocking with 5% milk in TBS, the blots were incubated with anti-mouse Klk1 (LifeSpan Biosciences, Seattle WA), anti-mouse NGAL (R&D Biosystems, Minneapolis MN), anti-mouse cleaved Caspase-3 (Cell Signaling, Danvers MA) or anti-mouse beta-actin (Abcam, Cambridge, MA) overnight in 4°C. The blots were then washed and incubated for 1 hour at room temperature with individual secondary antibodies. Bands were detected using an enhanced chemiluminescence detection system (ThermoScientific, Pittsburgh PA) and developed with a FluorChem E imager (ProteinSimple, San Jose CA). Band corresponding to proteins of interest were analyzed by ImageJ software.

Adenoviruses
Adenoviruses expressing IL-17A (Ad-IL-17) and control vector (Ad-ctrl) were kindly provided by Dr. J. Kolls (U. Pittsburgh). Ad-Klk1 and corresponding Ad-ctrl vector were from Applied Biological Materials Inc. (Richmond, British Columbia, Canada). Mice were injected via the tail vein with 1x10 9 pfu virus 72 h prior to induction of disseminated candidiasis.

Fluconazole treatment
C. albicans infected mice were treated with Fluconazole (FLC) (Diflucan: obtained from University of Pittsburgh Medical Center, Pittsburgh PA) as described before with minor modifications [58]. Briefly, mice were treated with 5, 10, 20 and 40 mg/kg body weight FLC by oral gavage at 2 h and 26 h post infection. Untreated mice received equal volume of PBS.

Statistics
Data were analyzed by Kaplan-Meier, ANOVA, Mann-Whitney or unpaired Student's t test using GraphPad Prism (La Jolla, CA). P values <0.05 were considered significant. All experiments were performed a minimum of twice to ensure reproducibility. direct arterial cannulation method in mice (n = 2) every 2 min (Baseline) and every 1 min after i.p. injection of bradykinin (300 nmol/kg/day). PBS was injected as control. (B) WT mice were treated daily with bradykinin (300 nmol/kg/day) starting day -1 (relative to infection). At day 0, mice were subjected to systemic candidiasis. As a negative control, sham-infected WT mice were treated with bradykinin only. At day 7 p.i. (n = 4-6), kidney infiltrating neutrophils (Gr1 + ) and macrophages (F4/80 + ) (gated on CD45 + ) cells were evaluated by flow cytometry. In response to disseminated candidiasis, kidney infiltrating innate and adaptive IL-17-producing cells is the major source of IL-17. IL-17 in turn binds its receptor (IL-17RA/RC) on kidney-resident target cells, activating downstream signaling events leading to expression of IL-17-responsive cytokines, chemokines and AMP genes. Innate effectors (neutrophils, macrophages) recruited in response to IL-17-induced signals facilitate fungal clearance. IL-17 also induces expression of kallikreins in target cells. Kallikreins cleave kininogens to form bradykinin. Activation of bradykinin receptors (Bdkrb) on renal cells prevents apoptosis and controls of tissue damage. (TIF)