902
`
`Downloaded from https:u/aeademicoupcom/ndt/article/27/3/902/1896811 by guest on 31 July 2023
`
`EXHIBIT
`
`La
`
`Nephrol Dial Transplant (2012) 27: 902-905
`doi: 10.1093/ndt/gfr695
`Advance Access publication 13 December 2011
`
`Apoptotic cell-free DNA promotes inflammation in haemodialysis
`patients
`
`Johairna Atarnaniuk t, Chantal Kopeck Y2, Sonja Skoupy', Marcus D. Säemann2 and Thomas Weiclihart2
`
`'Institute of Laboratory Diagnostics, Kaiser Franz Josef Hospital, Vienna, Austria and 2Division of Nephrology and Dialysis,
`Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
`
`Correspondence and offprint requests to: Thomas Weichhart; E-mail: thomas.weichhart@meduniwien.ac.at
`
`Abstract
`Background. A proinflarnmatory environment character-
`ized by the continuous activation of the innate immune
`system is thought to contribute to the markedly elevated
`mortality in haemodialysis (HD) patients with end-stage
`renal disease (ESRD). The presence of circulating cell-free
`DNA (cfDNA) has been demonstrated as biomarker in
`many pathologies.
`Methods. We evaluated the occurrence of cfDNA in RD
`patients and its functional relevance for innate immunity
`and inflammation.
`
`Results. Here, we found that cfDNA was enhanced in the
`plasma of ESRD patients after RD compared to healthy
`controls. Functionally, cfDNA selectively stimulated the
`production of the proinflammatory cytokine interleukin
`(IL)-6 by human monocytes, whereas tumour necrosis
`factor-ct or IL-10 was not induced. Conversely, plasma
`from HD patients, but not from healthy controls or DNase
`1-treated RD plasma, induced IL-6 production from mono-
`cytes.
`Conclusion. We provide the first evidence that cfDNA has
`selective immunostimulatory effects on human monocytes.
`
`© The Author 201. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
`For Permissions, please e-mail: journals.permissious@oup.coin
`
`0001
`
`TwinStrand EX1094
`TwinStrand Biosciences v. Guardant Health
`IPR2022-01400
`
`

`

`Downloaded from https://acadomic.oupcorn/ndtfarticle/27/3/902/1896811 by guest on 31 July 202 3
`
`cfDNA promotes inflammation in HD
`
`This process may contribute to the proinflammatory milieu
`observed in HD patients.
`
`Keywords: apoptosis; cell-free DNA; haemodialysis; interleukin 6;
`monocytes
`
`Introduction
`
`End-stage renal disease (ESRD) patients under haemodial-
`ysis (HD) are characterized by a chronic inflammatory state
`that includes aberrant and chronic production of inflamma-
`tory cytokines such as interleukin (IL)-6 [1-3]. This prom-
`flammatory environment occurring in ESRD patients has
`been implicated in HD cornorbidity [4]. However, the ba-
`sis for the constitutive activation of IL-6 production is un-
`clear. It has been suggested that the systemic presence of
`uraemic proteins and the dialysis procedure itself may pro-
`mote immune cell activation and hence inflammation [5]. It
`has long been recognized that innate immune cells are
`dysregulated during the course of ESRD [6, 7].
`Over the years, it has been increasingly documented
`that cell-free DNA (cfDNA) is present in blood under
`various healthy and pathological conditions [8]. Hence,
`cfDNA is regularly detected in plasma or serum samples
`of patients suffering from cancer, lupus erythematosus or
`in individuals after exhaustive exercise [8-10]. cfDNA
`has been found in many cases, in which apoptosis or
`necrosis was involved suggesting that such events are
`the main source for its presence [8, 10]. Recently, it was
`described that cfDNA is released during the HD procedure
`[10-12]. However, the underlying reasons for the release
`of cfDNA and the functional effects of uraemic cfDNA
`have not been investigated.
`
`Materials and methods
`
`Studs' subjects
`
`903
`
`Agarose gel electrophoresis
`
`To separate plasma DNA, agsrose gel electrophoresis was performed. Gels
`were stained with Vistra Green (Atnersham Biosciences). As positive
`control for apnptotic DNA, we loaded genomic DNA that was isolated
`from peripheral blood mononuclear cells (PBMCs) treated with 5 mM
`staurosporine (STS).
`
`Monoct'te stimulation
`
`Monocytes were isolated as described [13]. Freshly isolated monocytes
`were seeded in 48-well plates adjusted to a final volume of 2.5 >< 105 cells,'
`0.5 mL. M000cytes were stimulated with 250 ng/mL cfDNA or with 10,
`100 and 200 pL of plasma from HD patients or healthy controls. In some
`experiments, we treated 200 p1.. HD plasma with 8 U DNase I (New
`England Biolabs) for 24 h. Cell-free supernatants were collected after
`24 h. IL-b, tumour necrosis factor (TNF)-ci and IL- 10 in the supernatants
`were measured by Lutninex (R&D Systems).
`
`Statistics
`
`Results are expressed as means di SEM. Student's t-test was used to detect
`statistical significance. For all statistical analyses, significant differences
`are marked in the graphics with * <0.05 or ***P < 0.001.
`
`Results
`
`cJDNA is enhanced in plasma of HD patients
`
`We found that plasma of ESRD patients, after the dialysis
`session (HD), contained a significantly elevated amount of
`cfDNA compared to healthy individuals (mean of 0.98 ng/
`pL plasma versus 0.43 nglpt, respectively) (Figure IA).
`To further characterize the plasma, we performed agarose
`gel electrophoresis. Interestingly, we observed that the
`cfDNA of HD patients had a pattern-like appearance that
`is typical for apoptosis (Figure IB). In contrast, the plasma
`cfDNA of healthy controls did not show this apoptotic
`pattern (Figure 113). These results indicate that the amount
`of cfDNA is augmented in plasma of HD patients poten-
`tially because of apoptosis of PBMCs induced by the dial-
`ysis procedure.
`
`Patients with ESRD undergoing maintenance MD for a minimum of 3 months
`were recruited for the study. Patients were in a stable condition and free from
`intercurrent illness and infection for at least 3 months. All patients were
`dialysed on standard bicarbonate basis for 4-5 h three times weekly using
`biocompatible polysulphone HD membranes (Fresenius). A group of healthy
`subjects with nomiat kidney function were used as control group. All indi-
`viduals gave written informed consent prior to their inclusion in the study,
`which was petfonned in accordance with the ethical standards laid down
`Declaration of Helsinki. Ethical approval has been provided by the locally
`ethics committee of the General Hospital of Vienna (EK 407/2007).
`
`Plasma isolation from blood
`
`Blood samples were drawn its EDTA tubes from a total of 23 HD patients
`and 14 healthy controls. The blood samples of the ED patients were drawn
`at the end of the dialysis session. Blood was immediately placed on ice and
`centrifuged at 1000 RCF for 15 ruin at 4°C to obtain plasma.
`
`Isolation aiul quuntilalion of cJDNA front blood
`
`Within the first 2 h of obtaining plasma samples, DNA from individual
`samples was either isolated from 800 pL of plasma using a DNA isolation
`mini kit (Qiamp Blood Mint Kit; Qiagen) for quantitation of cfDNA
`or from 10 mL plasma samples using the Qiamp Blood Maxi Kit (Qiagen)
`for functional assays according to the manufacturer's protocol. The DNA
`was eluted in 50 tiL (Mini Kit) or 500 ttL (Maxi Kit) of elution buffer.
`cfDNA concentration was measured using a Nanodrop measurement
`device (Themio Scientific).
`
`cJDNA selectively stimulates IL-6 production in
`human monocytes
`
`Next, we wanted to characterize the functional properties of
`cfDNA. To this end, we purified cfDNA from the plasma of
`RD patients and healthy controls and tested if cfDNA is able
`to activate primary human monocytes, one of the cardinal
`innate immune cells in the blood. We observed that cIDNA
`isolated from RD patients induced the production of the pro-
`inflammatory cytokine IL-6 in monocytes (Figure 2A). Nei-
`ther TNF-cr nor IL- 10 was induced by cfDNA indicating that
`cfDNA selectively induces IL-6 expression in monocytes
`(Figure 2A). Interestingly, cfDNA from healthy controls like-
`wise stimulated IL-6 production in a comparable fashion to
`cfDNA from HD patients (Figure 2A). These results provide
`the first evidence that cfDNA, irrespective of the origin, pro-
`motes the production of IL-6 in human monocytes.
`
`Plasma front RD patients promotes IL-6 production in
`human monocytes
`
`Although cfDNA from HD patients and healthy controls
`was similarly potent to induce IL-6, we speculated that RD
`
`0002
`
`

`

`M STS
`
`HD
`
`clii
`
`J. Atamasiiuk C/ (if
`
`904
`A
`1.5
`
`S
`
`U,
`
`0i
`
`0
`
`ctrl
`
`RD
`
`cfDNA
`
`B
`
`2072
`1050
`
`600
`500
`400
`300
`200
`100
`
`Fig. 1. cNA in plasma of HD patients and healthy controls. Plasma was obtained from 13 HD patients and 9 healthy controls. (A) DNA was isolated
`and concentrations were measured as described in the Materials and methods section. cfDNA concentrations in plasma of HD patients and controls (ctrI).
`5P < 0.05. (B) Agarose gel electrophoresis was performed to characterize DNA samples. Lane 1, molecular size marker (the size of the bands is shown in
`bp); Lane 2: apoptotic DNA; Lanes 3-6: cfDNA samples of HD patients; Lanes 7-10: cfDNA samples of healthy controls.
`
`A
`80
`
`65 -
`-J
`0
`a 40 -
`
`-J
`
`20 -
`
`B
`40
`
`30
`
`10
`
`80-
`
`80
`
`.j 60-
`S
`
`40-
`
`20
`
`med
`
`I
`tri
`
`-
`
`HO
`
`cfDNA
`
`med
`
`ctrl
`
`HO
`
`med
`
`ctrl
`
`HD
`
`cfDNA
`
`C
`
`cfDNA
`
`0
`
`25-
`
`a
`1 0
`DO
`
`med 100 100.>l 200pl l5,j
`
`lOOp] 200j>I
`
`ctrl plasma
`
`HO plasma
`
`med
`
`HO Plasma HO Plasma +
`DNase I
`
`Fig. 2. cfDNA in plasma of 1-ID patients promote expression of IL-6 in human monocytes. (A) Human monocytes were incubated with individual
`samples of cfDNA (125 ng) from HD patients (n = 10) or healthy controls for 24 ii. The amount of IL-6, TNF-n and IL-10 in the supernatants was
`measured by Luminex. Data are representative three experiments. u.s., not significant. (B) Human monocytes from three different donors were incubated
`with 10. 100 and 200 p1. plasma from HD patients (ii = 9) or healthy controls (si = 5) overnight. IL-ti concentrations in the supernatant ofmonocytes were
`measured by Luminex. *** P <0.001. (C) Monocytes from two different donors were incubated for 24 It with 200 pL HD plasma (ii = 3) that was either
`treated with DNase I or left untreated. IL-6 concentration in the supernatants were measured by Luminex. UP < 0.05.
`
`plasma might be able to more strongly activate monocytes
`as cfDNA is present in higher amounts in the plasma of HD
`patients. As shown in (Figure 213), pure plasma of HD
`patients dose dependently induced the production of IL-6
`in human monocytes, whereas plasma from healthy con-
`trols did not stimulate IL-6. To more directly address
`whether the presence of cfDNA in the plasma of HD
`patients induces IL-6 production, we treated RD plasma
`with DNase I to degrade DNA. We observed that the
`DNase I-treated HD plasma showed a reduced capacity to
`induce IL-6 in human monocytes. These results suggest
`that the cfDNA present in plasma of RD patients is a driver
`of IL-6 production.
`
`Discussion
`
`Large clinical studies have shown that the mortality rate of
`individuals with ESRD undergoing HD is markedly ele-
`vated. This has been attributed among other things to the
`constitutive occurrence of inflammation in many patients,
`and hence, inflammatory markers such as C-reactive pro-
`tein or IL-6 are powerful predictors of mortality in this
`patient group [14-1 6]. The inflammatory environment in
`HD patients has been attributed Co the incomplete removal
`of uraernic toxins that promote inflammatory reactions but
`also to the RD procedure itself [5]. Dialysis membranes
`and uraemic plasma can promote apoptosis of human
`
`0003
`
`

`

`Downloaded from https:l1academcoup.com/ndVartideI27/3I9O2I189681 1 by guest an 31 July 202 3
`
`cfDNA promotes inflammation in HD
`
`polyrnorphonuclear neutrophils and mononuclear cells
`directly as well as through their interactions with mono-
`cytes [17-22]. It is thought that these apoptotic leucoctes
`then activate the innate immune system to contribute to
`the proinflamrnatory environment in ESRD patients under-
`going HD.
`cfDNA has been found in many pathologies, in which
`apoptosis or necrosis is involved suggesting that such
`events are a source for its presence [8]. cfDNA has been
`evaluated primarily as biomarker so far, whereas potential
`functional effects of cfDNA have not been investigated [8].
`Also in ESRD patients on HD, the presence of cfDNA has
`been described [10-12]. We speculated that cfDNA present
`in RD patients may activate the innate immune system to
`promote proinflamrnatoiy events. Indeed, we observed that
`cfDNA isolated from HD patients induced the production
`of IL-6 from human monocytes. This effect was selective as
`cfDNA did not induce TNF-u, or IL-10. Moreover, it is
`noteworthy that cfDNA from healthy individuals similarly
`induced IL-6 indicating that the main difference between
`HD patients and healthy controls regarding cfDNA is the
`absolute amount of cfDNA, which is elevated in patients
`with RD. Conversely, plasma from RD patients but not
`from healthy individuals was able to induce the expression
`of IL-6 from monocytes. Moreover, DNase I-treated RD
`plasma showed a severely reduced ability to induce IL-6.
`These results suggest that cfDNA in the RD plasma con-
`tributes to the constitutive proinflammatory events in these
`patients.
`In conclusion, we found in the current study that cfDNA
`released from blood cells by ongoing apoptosis is abun-
`dantly present in the plasma of HD patients. Importantly,
`we have shown for the first time that cfDNA is able to
`selectively induce the production of the proinflammatoiy
`cytokine IL-6 in human monocytes. We further established
`that the plasma of RD patients, but not of healthy controls,
`mimicked the capacity of cfDNA to induce IL-6 in human
`monocytes indicating that this process may contribute to
`the proinflammatory environment observed in HD patients.
`
`Acknowledgements. We thank M. Merio for excellent technical assistance.
`This work was supported by the Medical Scientific Fund of the Mayor of
`the City of Vienna (to LA.) and by the Else-Kr6ner Fresettius Stiflung
`(to T.W.).
`
`Conflict of interest statement. None declared.
`
`References
`
`1. Bologa RM, Levine DM, Parker TS et al. Interleukin-6 predicts hy-
`poalbuminemia, hypocholesterolemia, and mortality in hemodialysis
`patients. Am JKidney Di.r 1998; 32: 107-114
`
`905
`
`2. Ishizuka T, Nuts K, Yokoyama T ci at. Increased serum levels of
`inierleukin-12 may be associated with Thl differentiation in hemo-
`dialysis patients. Nephron 2002; 90: 503-504
`3. Rysz J, Majewslca E, Stolasek RA eta!, increased levels of soluble TNF-
`alpha receptors and cellular adhesion molecules in patients undergoing
`bioincompatible hemodialysis. Am JNephrol 2006; 26: 437-444
`4. Panichi V, Paoletti S. Consani C. Inflammatory pattern in hemodiafil-
`tration. Contrib Nephrol 2008; 161: 185-190
`5. Jofre R. Rodriguez-Benitez P, Lopez-Gomez Jivl et at. Inflammatory
`syndrome in patients on hemodialysis. JAm Soc Nephrol 2006; 17
`(12 Suppl 3): S274-S280
`6. Descamps-Latscha B. The immune system in end-stage renal disease.
`Qsrr Opin Nephrol H,iperten.s 1993; 2: 883-891
`7. Descamps-Latacha B, Herbelin A, Nguyen AT et al. Immune system
`dysregulation in uremia. Semin Nephrol 1994; 14: 253-260
`S. Jung K, Fleischhacker M, Rabien A. Cell-free DNA in the blood as a
`solid tumor biomarker—a critical appraisal of the literature. Cl/n c/tim
`Ac/u 2010; 411: 1611-1624
`9. Atamaniuk J, Hsiao YY, Mustak M ci al. Analysing cell-free plasma
`DNA and SLE disease activity. Earl ('fin Invest 2010; 41: 579-583
`10. Atamaniuk 3, Ruzicka K, Stuhhneier KM et al. Cell-free plasma DNA: a
`marker for apoptosis during hemodialysis. Cl/n Client 2006; 52: 523-526
`11. Garcia Moreira V, de la Cera Martinez T, Gago Gonzalez E et al.
`Increase in and clearance of cell-free plasma DNA in hemodialysis
`quantified by real-time PCR. Cl/n ('hem Lab Med 2006; 44: 1410-1415
`12. Korabccna M, Opatrna S. Wirth Jet at. Cell-free plasma DNA during
`peritoneal dialysis and hemodialysis and in patients with chronic kid-
`ney disease. Ann N YAcad Sci 2008; 1137: 296-301
`13. Weichhart T, Costantino G, Poglitsch Met al. The TSC-mTOR sig-
`naling pathway regulates the innate inflammatory response. Immunity
`2008:29:565-577
`14. Zimmermann J, Herrlinger S. Pruy A ci at. Inflammation enhances
`cardiovascular risk and mortality in hemodialysis patients. Kidne,v hit
`1999;55:648-658
`15. Haverkate F, Thompson SG, Pyke SD et al. Production of C-reactive
`protein and risk of coronary events in stable and unstable angina.
`European Concerted Action on Thrombosis and Disabilities Angina
`Pectoris Study Group. Lancet 1997; 349: 462-466
`16. Yeun JY, Levine RA, Mantadilok V et al. C-Reactive protein predicts
`all-cause and cardiovascular mortality in hemodialysis patients. Am J
`Kidney Dis 2000; 35: 469-476
`17. Nahar N, Shah H, Sin 3 at at. Dialysis membrane-induced neutrophil
`apoptosis is mediated through free radicals. Cl/n Nephrol 2001; 56: 52-59
`IS. Cendoroglo M. Jaber BL, Balakrishnan VS et at. Neutrophil apop-
`tosis and dysfunction in uremia. JAm Soc Nap/trot 1999; 10:
`93-100
`19. Saber BL, Balakrishnan VS, Cendoroglo MN et al. Modulation of
`iteutrophil apoptosis by uremic plasma during hemodialysis. Blood
`.Purif 1998; 16: 325-335
`20. Andrikos E, Buoricristiani E, D'Intini V et al. Effect of daily hemo-
`dialysis on monocytes apoptosis. Blood Purif 2005; 23: 79-82
`21. Koller H, Hochegger K, Zlabinger GJ et al. Apoptosis of human
`polymorphonuclear neutrophils accelerated by dialysis membranes
`via the activation of the complement system. NephrolDiat Transplant
`2004; 19: 3104-3111
`22. Carracedo J, Ramirez R, Soriano S et al. Caspase-3-dependent path-
`way mediates apoptosis of human mononuclear cells induced by cel-
`lulosic haemodialysis membranes. Nephrol Dial Tramplant 2002; 17:
`1971-1977
`
`Received for publication: 24.5.11; Accepted in revised form: 4.11. 11
`
`0004
`
`