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Recognition of infection and inflammation in the kidney

Heutinck, K.M.

Publication date 2011

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Citation for published version (APA): Heutinck, K. M. (2011). Recognition of infection and inflammation in the kidney.

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Download date:25 Sep 2021 4

SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I

Kirstin M. Heutinck1,2, Jorien Kassies1,2, Sandrine Florquin3, Ineke J.M. ten Berge2, Jörg Hamann1, Ajda T. Rowshani2

1 Dept of Experimental Immunology, 2 Dept of Internal Medicine, Renal Transplant Unit, 3 Dept of Pathology, Academic Medical Center, Amsterdam, The Netherlands

Submitted for publication

dsRNA induces serpinB9 expression in TECs

Abstract

Serine protease inhibitor B9 (serpinB9) protects cytotoxic lymphocytes and non- immune cells against granzyme B mediated apoptosis. In the kidney, lymphocytes contribute to tubular damage in interstitial nephritis of various etiology, like viral infection and transplant rejection. Under these inflammatory conditions, expression of serpinB9 could help to preserve the integrity and function of tubular epithelial cells (TECs). Indeed, we previously found that tubular serpinB9 expression was increased during subclinical rejection. Here, we studied the influence of inflammatory mediators on serpinB9 expression in primary human TECs. Of the various cytokines and Toll-like receptor (TLR) ligands tested, only the dsRNA analog poly(I:C) promoted serpinB9 expression. We found that TECs express the viral dsRNA receptors Toll-like receptor 3 (TLR3), melanoma differentiation associated 5 (MDA5) and retinoic acid inducible gene-I (RIG-I). The dsRNA receptor ligands poly(I:C) and 5’-triphosphate RNA enhanced serpinB9 expression independent of de novo synthesis, type I interferons and NFκB activity. To address the in vivo significance of these findings, 4 we analyzed the expression of serpinB9 in kidney transplant biopsies during infection with cytomegalovirus, Epstein-Barr virus and BK virus. We found that serpinB9 transcription was increased during each viral infection. Immunohistochemistry demonstrated that serpinB9 was expressed in the tubuli and in graft-infiltrating lymphocytes. In conclusion, dsRNA receptor activation may increase the threshold for granzyme B mediated apoptosis in TECs via upregulation of serpinB9 and thus help to protect the kidney against cytotoxic insults in case of viral infection.

Introduction

Serine protease inhibitors () are evolutionary conserved that restrict protease activity by a unique mechanism of ‘suicide inhibition’ resulting in a covalently bound protease- complex in which both protease and serpin are inactive 1. Serpins of the B clade, also known as serpins, lack an N-terminal secretion signal leading to an intracellular localization. Human clade B serpins regulate cellular processes like apoptosis, inflammation and cell migration 2;3. The best studied intracellular serpin, serpinB9 (PI-9), inhibits the cytotoxic enzyme granzyme B 4. Granzyme B producing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells express serpinB9 to protect themselves against self-inflicted injury 5-7. Furthermore, non-cytotoxic immune cells, epithelial and endothelial cells can also express serpinB9 8-12. Spi-6, the murine homolog of serpinB9, is known to protect dendritic cells and hepatocytes against granzyme B mediated apoptosis 13;14. Additionally, in human cancer cells serpinB9 expression is associated with an unfavorable clinical outcome and reduced susceptibility to NK cell-induced apoptosis

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15;16. These findings indicate that serpinB9 can protect various cell types against granzyme B released by cytotoxic lymphocytes. Tubulointerstitial nephritis is an inflammatory disease of the renal tubuli and interstitial tissue that can occur in several clinical conditions, like viral infection, allograft rejection, ischemia reperfusion injury and autoimmune disorders. Hallmark of the pathology is infiltration of immune cells, including CTLs and NK-cells, which can induce tubular damage leading to renal dysfunction 17-19. Renal cells and infiltrating immune cells are activated by pro-inflammatory cytokines and Toll-like receptor (TLR) ligands, the latter being derived from pathogens or damaged cells 20-24. A clinical example of interstitial nephritis is T cell-mediated transplant rejection, in which infiltrating alloreactive lymphocytes cause granzyme-mediated cell death in the tubuli 25. Interestingly, not all types of tubulointerstitial infiltrates immediately result in impaired renal function. A silent form of inflammation, named subclinical rejection may occur after kidney transplantation. We previously showed that tubular serpinB9 expression is increased during subclinical rejection suggesting that serpinB9 4 protects tubular epithelial cells (TECs) against the attack of CTLs9 . The question raised how serpinB9 expression is regulated in TECs. The few studies that have addressed the regulation of serpinB9 expression revealed induction by pro-inflammatory cytokines like LPS and TNFα in several human cell-lines 26-28 and by PMA and IL-2 in CD8+ T cells 4;29. Furthermore, estrogens enhanced serpinB9 expression in breast cancer cells 16. The regulation of serpinB9 expression in epithelial cells has not been studied so far. Here, we investigated the influence of inflammatory mediators on the expression of serpinB9 in human renal TECs. We analyzed serpinB9 expression in primary TECs after stimulation with pro-inflammatory cytokines and TLR- ligands 30. Only the dsRNA analog poly(I:C) was able to induce serpinB9 expression in TECs. We found that activation of the dsRNA sensors Toll-like receptor 3 (TLR3), melanoma differentiation associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) did directly induce serpinB9 expression. Since these receptors are known to recognize dsRNA of viral origin, we also analyzed the expression of serpinB9 in kidney transplant biopsies during infection with cytomegalovirus (CMV), Epstein-Barr virus (EBV) and BK virus (BKV). We observed that serpinB9 expression was upregulated during viral infection in vivo. Together our data suggest that serpinB9 induction is part of the cellular program engaged by dsRNA receptors in TECs.

Methods Isolation and culture of primary tubular epithelial cells Human kidney tissue was derived from non-tumor parts of kidneys with a renal cell carcinoma (Grawitz tumor) or donor kidneys intended for transplantation that were declined for technical reasons. TECs were isolated and cultured according to the

84 dsRNA induces serpinB9 expression in TECs method described previously 31. Experiments were performed in FCS coated culture plates with cryopreserved or fresh TECs of passages 3 till 6.

Stimuli Cells were stimulated with the following recombinant human cytokines and TLR- ligands: tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) (Invitrogen, Breda, The Netherlands), interferon γ (IFNγ), lipopolysaccharides (LPS) from Escherichia coli K-235, imiquimod (ImiQ), polyinosinic-polycytidylic acid (poly(I:C)), lipoteichoic acid (LTA) from Staphylococcus aureus (Sigma-Alderich, Zwijndrecht, The Netherlands), CpG oligonucleotide ODN2006 (CpG) (Invivogen, San Diego, CA, USA), interferon α2a (IFNα) and interferon β1a (IFNβ) (PBL InterferonSource, Piscataway, NJ, USA). The RIG-I ligand 5’-triphosphate RNA (3pRNA) was a kind gift of Prof. G. Hartmann and Dr. M. Schlee (Universitätsklinikum Bonn, Germany). Fugene HD transfection reagent (Roche, Mannheim, Germany) was used to deliver dsRNA mimics into the cytoplasm according to the manufacturer’s manual. Inhibitors used in this study were chloroquine, cycloheximide (Sigma-Alderich) and Bay 11-7082 (Merck 4 Chemicals, Nottingham, UK).

Messenger RNA measurements Total RNA was isolated from primary TEC cultures with the Invisorb Spin Cell RNA kit (Westburg, Leusden, The Netherlands) according to the instructions of the manufacturer. The isolated RNA was quantified and reverse transcribed to complementary DNA using oligo dT primer and SuperScript II RNase H-reverse transcriptase kit (Invitrogen). Serpin mRNA levels were measured by quantitative PCR in a LightCycler® PCR machine using the LightCycler® FastStart DNA Master SYBR Green I kit (Roche). Transcript levels were normalized to 18S rRNA. mRNA levels of dsRNA sensors and cytokines were analyzed by semi-quantitative PCR (35 cycles) using SALSA polymerase and enzyme dilution buffer (MRC Holland, Amsterdam, The Netherlands) and the Bio-Rad C1000 Thermal cycler (Bio-Rad laboratories, Veenendaal, The Netherlands). Primer sequences and annealing temperatures for all PCRs are depicted in supplementary Table 1.

Western blot analysis Cells were washed in cold PBS and resolved in 1% Triton-X in PBS supplemented with protease inhibitor cocktail (Roche) in PBS. Protein concentrations were determined with the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Proteins (50 μg per lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane and probed with primary antibodies against serpinB9 (clone PI9-17, kindly provided by Dr. S.M. van Ham, Sanquin, Amsterdam, The Netherlands 11) or serpinB8 (ab49634, Abcam, Cambridge, UK) and actin (I-19, Santa Cruz Biotechnology, CA, USA). Immunoreactive proteins were visualized with fluorescent-labeled secondary

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antibodies (IRDye680 donkey anti-mouse or IRDye800 donkey anti-goat, Westburg, Leusden, The Netherlands) and the Odyssey® Infrared Imaging System (LI-COR Biotechnology, Bad Homburg, Germany). Bands were quantified with the Odyssey software version 3.0 and normalized for the amount of actin.

Kidney transplant recipients We included 23 kidney transplant biopsies performed by protocol or upon clinical indication. We obtained material from patients with a stable graft function and normal histology (stable, n=5) or during infection with cytomegalovirus (CMV, n=7), Epstein- Barr virus (EBV, n=4) and BK virus (BKV, n=7). Viral infection was diagnosed by positive peripheral blood PCR, specific antibody conversion and/or immunohistochemistry. None of the patients received pre-emptive viral treatment or prophylaxis. Standard immunosuppression consisted of anti-CD25 monoclonal antibodies (Basiliximab), prednisone, calcineurin inhibitor (Tacrolimus or Cyclosporin) and mycophenolate mofetil. Of the seven patients with a CMV infection four received antiviral treatment 4 consisting of valganciclovir. Remaining patient characteristics are depicted in supplementary Table 2. Written informed consent was obtained from all patients, and the study was approved by the institutional ethical committee of the Academic Medical Center.

SerpinB9 expression in kidney transplant biopsies RNA was isolated from 50 μm sections of snap frozen renal biopsy specimens isolated with TRIzol reagent (Invitrogen, Breda, The Netherlands) according to the manufactures instructions. The isolated RNA was quantified and reverse transcribed to complementary DNA using a combination of oligo dT and random hexamer primers and M-MLV H-reverse transcriptase (Promega, Madison, WI, USA). SerpinB9 transcript levels were measured by quantitative PCR in the StepOnePlus Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the EXPRESS SYBR® GreenER™ reagent (Invitrogen). Transcript levels were normalized to a reference sample (primary TECs stimulated with poly(I:C) (50 μg/ml)) and GAPDH. Immunohistochemical staining for serpinB9 was performed on formalin-fixed paraffin-embedded tissue slides as described before 9. Scoring of the percentage serpinB9 positive tubuli was performed independently by two observers, using a semi-quantitative scoring system; 0, negative scoring (<5%); 1, weak expression (5- 25%); 2, mild expression (25-50%); 3, moderate expression (50-75%); and 4, strong expression (75-100%).

Statistical Analysis Statistical analyses were performed in SPSS (version 16.0, SPSS, Chicago, IL, USA) and Graph Pad Prism (version 5, GraphPad software, La Jolla, CA, USA). Individual values were compared with paired student T-tests (primary TECs) or Mann-Whitney U tests (biopsies)

86 dsRNA induces serpinB9 expression in TECs

Results TLR3 triggering induces serpinB9 expression in human TECs The expression of serpinB9 was analyzed in primary human TECs after stimulation with inflammatory cytokines or TLR ligands by quantitative RT-PCR and Western blot analysis. Unstimulated TECs contained little serpinB9 both on transcript (Fig. 1A) and protein (Fig. 1B) level. Expression of serpinB9 in primary TECs was not induced by TNFα, IFNγ, IL-1β or IL-6. Also agonists of TLR2, TLR4, TLR7 and TLR9 did not upregulate serpinB9. However, the TLR3 ligand poly(I:C), a synthetic mimic of dsRNA, strongly stimulated serpinB9 transcription and translation. Next to serpinB9, other clade B serpins have been reported to be protective against apoptotic stimuli32 . Therefore, we determined the expression levels of these serpins. SerpinB3, serpinB6 and serpinB8 transcripts were present (Suppl. Fig. 1A-C), while serpinB2 mRNA was constitutively very low and serpinB10 was absent (data not shown). IL-1β and poly(I:C) Figureinduced 1 serpinB3 expression, although mRNA levels remained close to the detection limit of 4 A SerpinB9 mRNASerpinB9 - primary mRNA TECs 10 16h 8 40h

6

4 Fold change Fold 2

0 TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG no stim

B SerpinB9 protein SerpinB9 - primary protein TECs 10 16h ** 8 40h ** 6

4 Foldchange 2

0 TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG no stim

serpinB9

actin

Figure 1: Poly(I:C) induces serpinB9 expression in primary tubular epithelial cells. Primary TECs were stimulated for 16 h (light grey bars) or 40 h (dark grey bars) with 100 ng/ml TNFα, 100 ng/ml IFNγ, 100 ng/ml IL-1β, 1000 pg/ml IL-6, 100 μg/ml LTA (TLR2 ligand), 100 μg/ml poly(I:C) (pIC, TLR3 ligand), 10 μg/ml LPS (TLR4 ligand), 100 μg/ml imiquimod (ImiQ, TLR7 ligand) or 10 μg/ml CpG oligonucleotides (CpG, TLR9 ligand). For each stimulus three concentrations with a one-log difference were tested, the highest concentration is depicted in the figure. A SerpinB9 expression was analyzed by quantitative PCR. An increase of mRNA above 2-fold (dotted line) was considered significant. B Protein levels of serpinB9 were determined by Western blot analysis. A representative blot of serpinB9 expression in TECs stimulated for 16 hours is depicted below the graph. The bars represent the fold change in serpinB9 expression compared to unstimulated TECs (mean ± SEM, n≥3).

87 24 Figure 2 Chapter 4

A B 12 12 without HCQ 10 M HCQ 100 M HCQ * 10 10

8 8

6 6 Fold change Fold Fold change Fold 4 4

2 2

0 0 0 0.8 4 20 100 500 0 0.8 4 20 100 500 0 0,8 4 20 100 500 pIC 100 100 1000 1000 10pIC

No stim No IFN IFN serpinB9 serpinB9

actin actin

Figure 2: TLR3 triggering induces serpinB9 expression in primary tubular epithelial cells independent of type I IFNs. SerpinB9 expression was studied by Western blot analysis in stimulated primary TECs. The bars represent the fold change in serpinB9 expression compared to unstimulated cells (mean ± SEM, n=3-5).A The TLR3 inhibitor chloroquine (HCQ, 10-100 μM) was administered 30 minutes prior to stimulation with poly(I:C) (pIC, 0.1-500 μg/ml). B TECs were stimulated o/n with IFNα (100-1000 U/ml) and IFNβ (100-1000 U/ml). * p<0.5, paired 4 student T-test

the PCR. Transcription of serpinB8 was elevated by poly(I:C) as well and this could be confirmed at the protein level (Suppl. Fig. 1D). We continued our study by exploring the kinetics and mechanism of poly(I:C) induced serpinB9 expression in TECs. SerpinB9 was induced dose-dependently (Fig. 2A), occurred within 8 hours after stimulation and remained elevated up to 40 hours (Fig. 1B and data not shown). Chloroquine, a chemical that prevents endosomal acidification and hence impairs TLR3 signaling, completely inhibited poly(I:C) induced serpinB9 upregulation (Fig. 2A) strongly suggesting that TLR3 was indeed activated by endosomal uptake of the extracellularly delivered dsRNA mimic. TLR3 is known to mediate the production of inflammatory cytokines and type I IFNs via respectively the transcription factors NFκB and interferon regulating factors (IRFs) 3 and 7 33. Type I IFNs can subsequently turn on the transcription of a large set of IFN-stimulated that inhibit viral replication and promote antiviral immune responses. To test whether 25 type I IFNs regulate serpinB9 expression, we stimulated TECs with IFNα and IFNβ (Fig 2B). SerpinB9 levels were hardly affected by these cytokines indicating that poly(I:C) induced serpinB9 upregulation is not primarily the result of a type I IFN feed forward loop. Moreover, cycloheximide, an inhibitor of protein translation, did not prevent poly(I:C) induced serpinB9 mRNA (see below) confirming that serpinB9 is a direct product of TLR3 induced gene transcription.

88 Figure 3

dsRNA induces serpinB9 expression in TECs

A Fugene HD pIC pIC 3pRNA FG + pIC stim pIC 0.1 0.1 0 0.01 0.1 1 10 100 0 0.001 0.01 1 10 0.01 1 0.1 3pRNA + FG + 3pRNA 0.1 0.001 No 10 0.1

TLR3

MDA5

RIG-I

18S-rRNA

5 µM Bay B CC a ß β α + FG ß + FG + a α β pIC stim pIC stim stim pIC pIC stim pIC pIC No No No 10 No 10 0.1 100 IFN 100 IFN 1000 IFN 100 IFN 1000 10 0.1 0.1 No No 100 IFN 100 100 IFN 100 IFN 1000 1000 IFN 1000 10 TLR3 TLR3TLR3

MDA5 MDA5MDA5

RIG-I RIGRIG-I -I

18S-rRNA 18S18S-rRNA-rRNA 4

5 µg/ml CHX

Figure 3: Tubular epithelial cells express cytoplasmic and endo- somal dsRNA sensors. The ex- pression of dsRNA sensors was determined by PCR in stimulated primary TECs. The PCR bands shown are representative for at least three experiments. A TECs were stimulated for 16 h with 0.01-100 μg/ml poly(I:C) (pIC) or 0.001-1 μg/ml 3pRNA. The transfection rea-gent Fugene HD (FG) was used to accomplish cytoplasmic delivery of MDA5 ligand poly(I:C) and RIG-I ligand 3pRNA. The NFκB inhibitor Bay 11-7082 (Bay, 5 μM) was administered 30 minutes prior to stimulation with the dsRNA receptor ligands. B Protein translation was blocked with cyclohexi- mide (CHX, 5 μg/ml); at the same time dsRNA receptors were activated as described above. C Primary TECs were stimulated for 16 h with 100-1000 U/ml IFNα or IFNβ.

The cytoplasmic dsRNA receptors MDA5 and RIG-I upregulate serpinB9 expression in TECs MDA5 and RIG-I are dsRNA sensors like TLR3 that recognize viral replication in the cytoplasm. Activation of these cytoplasmic receptors can be accomplished in vitro by transfection of respectively poly(I:C) and 5’-triphosphate RNA (3pRNA). The expression of TLR3, MDA5 and RIG-I was analyzed in TECs stimulated with these dsRNA mimics since it is known that receptor expression can be induced by their26 ligands 34;35. Figure 3A (left panel) demonstrates that primary TECs constitutively express small amounts of TLR3, MDA5 and RIG-I. Triggering of the dsRNA receptors dose-dependently induced the transcription of TLR3, MDA5 and to a lesser extent RIG-I. This induction was apparent at very low amounts of dsRNA suggesting that activation of receptors renders TECs more equipped to sense dsRNA. Induction of dsRNA receptor expression occurred in the absence of de novo protein expression

89 Figure 4

Chapter 4

A 8 Figure 4: The dsRNA receptors TLR3, MDA5 and RIG-I directly 6 promote serpinB9 expression in 4 primary TECs. SerpinB9 expression was analyzed by Western blot Fold change Fold 2 analysis in primary TECs. The depicted blots are representative 0 0 0.01 0.1 1 10 0 0.01 0.1 1 10 pIC for at least three experiments + FG with actin as loading control. serpinB9 A SerpinB9 expression in TECs actin stimulated 16 h with 0.01-10 μg/ ml poly(I:C) (pIC). The transfection reagent Fugene HD (FG) was used B C to accomplish delivery to the +F G cytoplasm where MDA5 is located. pIC stim Fugene HD pIC The bars represent the fold change 0.1 No 10 in serpinB9 expression compared serpinB9 to unstimulated cells (mean ± pIC stim stim pIC actin SEM, n=3). B TECs stimulated as NoBay No 10 0.1 0.13pRNA No described above or with 0.1 μg/ 4 serpinB9 serpinB9 ml 3pRNA and Fugene HD to M Bay

actin actin µ activate RIG-I. C NFκB signaling 5 was blocked by adding 5 μM Bay 11-7082 30 minutes prior to D activation of TLR3 (pIC) or MDA5 + + FG +FG (pIC + FG) as described above. pIC pIC stim stim pIC pIC D Primary TECs were stimulated as 10 10 0.1 No 0.1 No No described above in the presence serpinB9 of cycloheximide (CHX, 5 μg/ml). 18S-rRNA SerpinB9 expression was analyzed by semi-quantitative PCR after 16 5 µg/ml CHX hours.

(Fig. 3B) and in response to stimulation with via type I IFNs (Fig. 3C). To discriminate between the transcription factors that are activated by dsRNA receptors, NFκB and IRFs, we tested the effect of the NFκB inhibitor Bay 11-7082. The inhibitor blocked poly(I:C) induced TNFα production completely (data not shown), but did not abrogate ligand induced upregulation of dsRNA receptor mRNA (Fig. 3A, right panel). Thus, TECs are equipped with functional endosomal and cytoplasmic dsRNA sensors, which in response to activation are collectively upregulated by direct and indirect type I IFN mediated gene activation. Next, we wondered whether triggering of MDA5 and RIG-I would promote serpinB9 expression like TLR3 signaling does. Protein levels of serpinB9 were indeed elevated in TECs transfected with either poly(I:C) (Fig. 4A) or 3pRNA (Fig. 4B) and27 this induction was not blocked by Bay 11-7082 (Fig. 4C) or cycloheximide (Fig. 4D). In conclusion, we showed that activation of endosomal and cytoplasmic dsRNA receptors promote the expression of serpinB9 in TECs independent of new protein synthesis, type I IFNs and NFκB activity.

90 dsRNA induces serpinB9 expression in TECs

SerpinB9 expression in the kidney is enhanced during viral infection Since TLR3, MDA5 and RIG-I sense viral dsRNA, we wondered whether serpinB9 was expressed in the kidney during viral infection. For that reason, transcript levels of the granzyme B inhibitor were analyzed by quantitative PCR in snap-frozen biopsy material obtained from kidney transplant recipients during infection with CMV, EBV and BKV. In comparison with stable biopsies, viral infection was associated with significantly increased levels of serpinB9 mRNA (Fig. 5A). To address which cell types expressed the granzyme B inhibitor, immunohistochemical staining for serpinB9 was performedFigure 5 on formalin-fixed and paraffin-embedded biopsy material, collected simultaneously

A SerpinB9 mRNA B SerpinB9 protein ** 3 * 4 ** 3 2 2 1 4 1 Relative expression Relative SerpinB9 expression SerpinB9 0 0 Stable CMV EBV BKV Stable CMV EBV BKV ATN C Stable CMV

EBV BKV

Figure 5: SerpinB9 expression in the kidney is enhanced during viral infection. A SerpinB9 transcription in frozen biopsy specimens obtained from stable patients (n=5) and patients infected with cytomegalovirus (CMV, n=6), Epstein-Barr virus (EBV, n=4) or BK virus (BKV, n=7) depicted as relative expression (median). B+C Immunohostochemical stainings for serpinB9 on formalin-fixed paraffin-embedded tissue slides. B Semi-quantitative scoring of the tubular SerpinB9 expression, scored as described in the methods section (median). C Representative pictures of the serpinB9 staining pattern observed in stable, CMV, EBV and BKV biopsies, with serpinB9 expression found in the tubuli (black arrows) and infiltrating lymphocytes (red arrow) (magnification 200x). For color figure, see appendix. 28

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with the frozen material used for PCR. An increased percentage of serpinB9 positive tubuli was found in CMV and BKV positive biopsies (Fig. 5B+C). During EBV infection, the number of serpinB9 positive tubuli was on average comparable to stable biopsies (Fig. 5B+C). Positive serpinB9 staining was also found in graft infiltrating lymphocytes, which were focally present in the majority of biopsies taken during viral infection but absent in stable biopsies (Fig. 5C). Thus, during viral infection serpinB9 expression in the kidney is increased, which reflects both an enhanced tubular expression and an increase in the number of graft infiltrating serpinB9 positive lymphocytes.

Discussion

We here demonstrate that the granzyme B inhibitor serpinB9 is constitutively expressed by primary human renal TECs and that its transcription and translation is enhanced by activation of the dsRNA sensors TLR3, MDA5 and RIG-I. Remarkably, 4 pro-inflammatory cytokines and other TLR agonists, which are known to be elevated under inflammatory conditions like in interstitial nephritis, did not modulate serpinB9 levels in TECs. dsRNA sensors recognize dsRNA of viral origin, leading to activation of pro-inflammatory, anti-viral and pro-apoptotic pathways 36;37. Viral infection of TECs, for example by BKV, is rare in healthy individuals but may occur in immunocompromised patients 38. CMV, EBV and BKV infection are a well known complication in kidney transplant recipients. These DNA viruses are latently present in the majority of adults and have the capacity to infect TECs 39-41. We found that transcript levels of serpinB9 were significantly increased in biopsies obtained during viral infection compared to biopsies from stable controls. SerpinB9 was expressed in the tubuli and by graft infiltrating lymphocytes. In agreement with our findings, elevated serpinB9 expression has been shown in lymphocytes of EBV infected patients, but not in patients with a bacterial infection 27. Increased levels of serpinB9 are also found in EBV-transformed lymphoblastoid cell-lines, in serum during symptomatic CMV infection and in urine during BKV nephropathy, providing further evidence that serpinB9 expression is associated with viral infection 42-44. dsRNA formed during replication of CMV, EBV and BKV might serve as ligand for TLR3, MDA5 and/or RIG-I. Literature about the role of these dsRNA sensors in the recognition of CMV and BKV is limited, yet it is known that EBV infected cells produce small EBV encoded RNAs (EBERs) that can activate both TLR3 and RIG-I 45-47. ssRNA and DNA of viral origin, which are recognized by respectively TLR7/8, TLR9 and DNA- dependent activator of IFN-regulatory factors (DAI) 48;49, can also elicit an anti-viral immune response like dsRNA. Ligands of TLR7 and TLR9 did not promote serpinB9 expression, yet we cannot exclude involvement of DAI or other cytoplasmic DNA sensors in the recognition of CMV, EBV and BKV.

92 dsRNA induces serpinB9 expression in TECs

Key to the interpretation of our data is the question why dsRNA receptor activation should render TECs less sensitive to granzyme B mediated apoptosis. It seems paradoxical to protect virus infected cells against the attack of cytotoxic lymphocytes. TECs are known to support innate and adaptive immune responses by monitoring their environment for danger signals and producing cytokines and other inflammatory mediators in response to these signals 17. The expression of TLRs like TLR3 provides TECs with versatile sensors for pathogens mainly utilized by specialized cells of the immune system. The downside of the ability to detect virus with high affinity is that invading cytotoxic lymphocytes may damage the epithelial barrier between urine and blood. SerpinB9 may protect infected and non-infected TECs against cell death provoked by exaggerated or misdirected granzyme B. Of note, TLR3, MDA5 and RIG-I can be activated not only by viral dsRNA, but also by endogenous ligands. For example, several studies have shown that RNA released from necrotic cells can activate TLR335;50 . Thus, another function of dsRNA receptors in TECs might be to sense tissue injury, as may occur during acute tubular necrosis induced by toxic compounds or by ischemia. In that condition, activation of repair mechanisms and protection of viable cells are 4 essential to maintain the integrity and function of the kidney. Our observation that expression of dsRNA receptors in TECs is directly induced by low concentrations of poly(I:C) as well as by type I IFN signaling is in line with other publications 34;35;51. TLR3, MDA5 and RIG-I have overlapping signaling pathways in which the transcription factors IRF3/7 and NFκB are activated 36;37. IRFs are known to induce the production of type I IFNs, which in turn can trigger IRF9 regulated genes after binding to the IFNα/β receptor 33. IFNα/β did not significantly induce serpinB9 expression and dsRNA receptor induced serpinB9 transcription was independent ofde novo protein synthesis, indicating that the inhibitor is a direct target of dsRNA receptor activated transcription factors. Most of the cytokines and TLR ligands that we have tested are capable to activate NFκB, demonstrating that this pathway by itself does not promote serpinB9 expression in primary TECs. Moreover, NFκB was not required since blockade with a specific inhibitor did not abrogate serpinB9 induction by dsRNA. Our findings contrast to studies that have shown that pro-inflammatory cytokines and NFκB activation promote serpinB9 expression in monocytes, hepatocytes and hepatocarcinoma cell-lines 26-28. In favor of a cell-type specific regulation, we found that serpinB9 in TECs is directly regulated by IRF3 and/or IRF7, as illustrated in Figure 6. In conclusion, we here show that the expression serpinB9 in TECs is regulated by dsRNA receptors. In the kidney, dsRNA receptor ligands can originate from viral infection as well as from necrotic cells. We indeed found that serpinB9 expression in the kidney was increased during infection with CMV, EBV and BKV and that both renal tubuli and infiltrating lymphocytes expressed the granzyme B inhibitor. Expression of serpinB9 may render TECs less sensitive to the attack from allo-, auto- or virus- specific cytotoxic lymphocytes and thereby help to preserve tubular integrity and

93 Figure 6

Chapter 4

MDA5

TEC RIG-I

dsRNA

TNFα IL-6 NFκB

serpinB9 IRF-3/7 IFNα/β IFNβ dsRNA receptors IRF-9 IFNR 4 IFNβ

Figure 6: Schematic overview of dsRNA receptor signaling in tubular epithelial cells. Human TECs express the dsRNA sensors TLR3, MDA5 and RIG-I. TLR3 is located in the membrane of endo- lysosomes and becomes activated after endocytosis of viral particles or dsRNA. The cytoplasmic receptors MDA5 and RIG-I recognize dsRNA generated during viral replication in infected cells. The three dsRNA receptors activate the same transcription factors; namely NFκB and IRF-3/7. Triggering of NFκB induces transcription of pro-inflammatory cytokines like TNFα while IRF-3/7 initiate gene-expression of type I IFNs, serpinB9 and the dsRNA receptors themselves. Type I IFNs sequentially trigger IFN receptors leading to IRF-9 mediated transcription of the dsRNA receptor genes. This feed forward loop renders TECs more sensitive to dsRNA.

function. Future research is needed to better understand the role of serpinB9 in renal TECs and elucidate in which clinical settings the inhibitor plays a significant role.

Acknowledgements

This work is supported by a Clinical Fellowship award from The Netherlands Organization for Health Research and Development to AT Rowshani (90700157) and research grants from the Academic Medical Center of the University of Amsterdam and the Roche Organ Transplantation Research Foundation (ROTRF, 445257295). We kindly thank Prof. C. van Kooten for his advice about the culture of primary TECs, colleagues of the Departments of Pathology and Urology of the AMC for providing human kidney samples, N. Claessen for her help with the immunohistochemical stainings and Prof. R.A.W. van Lier and Dr. E. Eldering for their critical views on the manuscript.

94 29 dsRNA induces serpinB9 expression in TECs

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97 Chapter 4 Supplementary figure 1

Supplementary material

A SerpinB3 SerpinB3 mRNA - primarymRNA TECs 12 16h 10 24h 8 6 4 Foldchange 2 0 TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG

B SerpinB6 SerpinB6 mRNA - primarymRNA TECs 10 16h 8 24h

6 4 4 Foldchange 2

0 TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG

C SerpinB8 SerpinB8 mRNA - primarymRNA TECs 10 16h 8 24h

6

4 Foldchange 2

0 TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG

D TNFa IFNg IL-1b IL-6 LTA pIC LPS ImiQ CpG IFNa no stim

serpinB8

actin

Supplementary Figure 1: Human tubular epithelial cells express serpinB3, serpinB6 and serpinB8. Primary TECs were stimulated with cytokines or TLR-ligands for 16 (light grey bars) or 40 (dark grey bars) hours with 100 ng/ml TNFα, 100 ng/ml IFNγ, 100 ng/ml IL-1β, 1000 pg/ ml IL-6, 100 μg/ml LTA (TLR2 ligand), 100 μg/ml poly(I:C) (pIC, TLR3 ligand), 10 μg/ml LPS (TLR4 ligand), 100 μg/ml imiquimod (ImiQ, TLR7 ligand) or 10 μg/ml CpG oligonucleotides (CpG, TLR9 ligand). Expression of serpinB3 (A), B6 (B) and B8 (C) was analyzed by quantitative PCR. The bars represent the fold change in serpin expression compared to unstimulated cells (mean +/- SEM, n=3). An increase of mRNA above 2-fold (dotted block line), was considered significant. D SerpinB8 protein levels determined by Western blot analysis.

98 dsRNA induces serpinB9 expression in TECs

Supplementary Table 1: PCR primer specifications

Gene Sense Anti-sense Tannealing SerpinB9 I,II 5’-GCGTCGAGCTGCTTTGTAGTT-3’ 5’-CAGAATGCTGTTGGCTCTGTTGT-3’ 60 °C SerpinB2 I 5’-GAAGACCAGATGGCCAAGGT-3’ 5’-GAGAGCGGAAGGATGAATGGAT-3’ 60 °C SerpinB3 I 5’- GCGGTCTCGTGCTATCT-3’ 5’-GGCAGCTGCAGCTTCTG-3’ 60 °C SerpinB6 I 5’-CCCTCCCGCGGTTTAAACTA-3’ 5’-CCTCCGTGCCTTCCTCAT-3’ 52 °C SerpinB8 I 5’-CATTCTGCTTCCCGATGACAACA-3’ 5’-CGAAGGAAAGGCTCCAAGTCATAA-3’ 52 °C TLR3 II Commercial (Invivogen, San Diego, CA, USA) 55 °C MDA5 II 5’-GGCATGGAGAATAACTCATCAG-3’ 5’-CTCTTCATCTGAATCACTTCCC-3’ 56 °C RIG-I I,II 5’-ATCCCAGTGTATGAACAGCAG-3’ 5’-GCCTGTAACTCTATACCCATGTC-3’ 56 °C 18s-rRNA I,II 5’-GGACAACAAGCTCCGTGAAGA-3’ 5’-CAGAAGTGACGCAGCCCTCTA-3’ 60 °C TLR3 I 5’-TGGTTGGGCCACCTAGAAGTA-3’ 5’-TCTCCATTCCTGGCCTGTG-3’ 55 °C MDA5 I 5’-TGGTCTCGTCACCAATGAAA-3’ 5’-CTCCTGAACCACTGTGAGCA-3’ 57 °C GAPDH I 5’-TGCACCACCAACTGCTTAGC-3’ 5’-GGCATGGACTGTGGTCATGAG-3’ 60 °C I used for quantitative mRNA measurements,II used for semi-quantitative mRNA measurements 4

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Supplementary Table 2: Patient characteristics Stable CMV EBV BKV P- Characteristic (n=5) (n=7) (n=4) (n=7) value Recipient Age (median (year), quartiles) 55 (49-65) 48 (32-54) 54 (30-62) 53 (48-66) 0.59 Gender (% male) 80 86 75 14 0.18 CMV status (% positive) - Pre-transplant 100 29 25 86 - At time of biopsy 100 100 25 86 EBV status (% positive) - Pre-transplant 100 100 25 86 - At time of biopsy 100 100 100 100

Donor Age (median (year), quartiles) 35 (33-48) 53 (40-62) 51 (42-56) 52 (46-54) 0.10 Gender (% male) 40 14 25 57 0.57 CMV status (% positive) 80 71 25 43 0.31

Transplantation 4 Cold ischemia (median (h), quartiles) 2 17 8 16 0.20 (2-13) (7-22) (2-20) (3-24) Delayed graft function (% present) 0 14 0 0 0.54 Transplantation type (%) 0.64 - Deceased donor, heart beating 20 57 50 71 - Deceased donor, non-heart beating 0 29 0 0 - Living donor, related 80 0 25 0 - Living donor, unrelated 0 14 25 29

Characteristics at time of biopsy GFR (median (ml/min/1.73 m2), 64 (59-85) 19 (16-35) 55 (36-58) 31 (16-44) 0.01 quartiles) Time after transplantation (median 28 (24-82) 19 (6-32) 28 (23-52) 86 (18-110) 0.34 (weak), quartiles)

Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; BKV, BK-virus; GFR, glomerular filtration rate. The different patient groups were compared by either Kruskal-Wallis tests for continuous variables or χ2-tests for binary parameters. GFR (ml/min/1.73 m2) is calculated with the modification of diet in renal disease (MDRD) study equation: 186 x (serum creatinine (mg/dl))-1.154 x (age (year))-0.203 x (0.742, if female) x (1.1210, if African Americans) [Myers et al. (2006) Clin Chem 52:5-18]

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