DE GRUYTER
`
`C. Garbers and J. Scheller: Properties of interleukin-6 and interleukin-41 === T1147
`
`lead to up-regulation of IL-6R in T celis are not known,
`and as hepatocytes do not express the receptors for IL-2
`(Tato and Cua, 2008), it is unlikely that IL-? is a general or
`the sole regulator of IL-6Rexpression. In at least one case
`it was reported that IL-6 can induce the expression ofits
`own LL-68 (Thabard et al., 2001). Whether and howIL-8
`expression is regulated has not been studied thus far.
`
`IL-6 and IL-i1 signaling: similarities
`and differences
`
`iL-6 signaling via gp130 and the membrane-bound IL-6R
`is called classic signaling (Garbers et al., 2012) (Figure 1}.
`In addition to the membrane-boundIL-6R, up te 50 ng/ml!
`solubleforms of the [L-6R (s0L-6R) were foundin the serum
`of man, which can rise up to 170 ng/ml! under pathophysi-
`ological conditions (Gaillardet al., 1993; Mitsuyamaeial.,
`1995; Montero-hulian, 2001). More than 90%of the sIL-6R
`is generated by limited proteclysis of the membrane-
`bound IL-6R by the proteases ADAMIO or ADAMI7 and
`up to 10%of the sIL-6R is gencrated by translation from
`adifferentially spliced mRNA. In differential splicing, the
`exon coding for the trans-membranc region of the IL-6R is
`skipped, resulting in premature tcomination of the IL-6R
`open reading frame anc a new short C-terminus (Chalaris
`etal., 2013). Importanily, both forms of the sIL-6Rcan still
`bind to IL-6. In contrast te most other soluble receptors,
`s{L-6R does not act as an antagonist but as an agonist.
`Consequently, the interaction of the IL-6/sTL-6R complex
`with 2p130 can activate cells, which do not necessarily
`express the membrane-hound IL-6R and are therefore
`unresponsive to IL-6 classic signaling. IL-6 signaling via
`the sIL-6R was called IL-6 trans-signaling (Figure 1) (ose-
`John and Heinrich, 1994; Chalaris et al., 2011).
`The complex of [1-6 and sfL-6R can stimulate all
`cells of the body and thereby mimic signals from other
`cytokines of the IL-6 family, especially [1-11 signaling
`tones et al., 2011). Interestingly, IL-6 trans-signaling is
`mimicked by the viral orthologue of IL-6 (viL-6) encoded
`by the humanherpes virus 8 (HHV8), which directly binds
`to and stimulates gp130 and does not depend on {1-6R
`tAdam et al., 2009; Suthaus et al., 2022).
`IL-1i signaling is mediated by #p130 and the speci-
`ficity factor IL-IR (Figure 1) (Pflanz et al., 1999}, which
`means that both IL-6 ancl {L-11 signal via the same 9p130
`homodimeric signal-transducing receptor complex.
`In
`principle, a soluble IL-IR can also form biologically
`active soluble complexes with IL-tt (Pflanz et al., 1999), A
`naturally occurring soluble LL-T1R has, however, not been
`
`described thus far, leaving open the question of if an IL-11
`trans-signaling pathwayexists in vivo (Figure 1).
`To date, no functional role in sional transduction was
`assigned to the o-recepiors IL-6R and 1L-118. The early
`signaling pathways of IL-6 and IL-li via homodimeric
`#pi30 appear similar or identical and inchide phospho-
`rylation of the JAKs, STAT1 and 3, and activation of the
`mitogen-activated protein (MAP) kinase pathway. Only
`the local cytokine expression and the a-receptor expres-
`sion pattern decide whether a cell reacts towards IL-6 or
`iL-i1, Detailed comparative analysis of dynamic signal
`transduction of 11-6 and IL-li have, however, not been
`performed to date, Future experiments have to function-
`ally compare IL-6 and IL-1 signal transductionin vitro and
`in vive, This is of particular importance because IL-6 and
`IL-1 have in part opposingroles in vivo and at least some
`cells are not able to conduct the fidl spectrumof signal
`transduction pathways after
`IL-6/IL41 activation (see
`section ‘Comparison of IL-6 and IL-11 in vivo’).
`The single nucleotide polymorphism (SNP) 1s2228145,
`located within exon 9 of the IL-6R gene Jocus, is charac-
`terized by a coding mutation resulting in an amino acid
`exchange from Asp(G58)Ala. This SNP is quitc common,
`as up to 20% of people genotyped were homozygous
`(Galicia ct al. 2004), These individuals are character-
`ized by reduced cellular 1L-6R level and increased sIL-6R
`level
`resulting in redueed responsiveness to classic
`signaling IL-6 (Ferreira et al., 2013). Furthermore,
`the
`increased level of sIL-6R might lead to increased IL-6
`trans-signaling (Scheller and Rose-John, 2012).
`It is cur-
`rently unknown whether the increased sIL-6R levels are
`caused by increased shedding of mernbrane-bound[L-6R
`or other mechanisms. For the IL-lIR, no such dominant
`SNP is known. Nevertheless, single case reports of indi-
`viduals with missense mutations within the IL-IIR have
`
`been described (Nieminenet al., 2011). These amino acid
`alterations lead to IL-IIR variants that are not capable to
`fulfill IL-P-mediated signal transduction, and the patients
`suffered from craniosynostosis, delayed tooth eruption,
`and supernumerary teeth (Nieminen et al., 2011).
`
`Therapeutic intervention of IL-6 and
`iL-11 signaling
`
`The signaling pathway of IL-6 and iL-11 offers multiple
`ways of intervention, either through inhibition or enhance-
`meni, and nearly all of them have been used cither experi-
`mentally or therapeutically in the clinic. In this section,
`we will discuss the possible steps of Intervention and
`
`
`
`Ex. 2001 - Page1332
`Ex. 2001 - Page1332
`
`

`

`148 ———
`
`C. Garbers and }. Scheller Properties of Interleukin-6 and interleukin-11
`
`DE GRUYTER
`
`
`
`
`8go130Fc
`
`rep
`gp agptsa
`
`
`
`iL TH antagonists
`
` BTS}
`
`digs’ 301
`
`ARIML-GR mAb
`
`ANTHL-G HAR
`
`
`
` Opta@on730
`
`
`Figure 3 Strategies of therapeutic (L-6/iL-11 blockade.
`Antibodies to IL-6 or IL-6 neutralize IL-6/IL-6R complexes and block both, IL-6 classic and trans-signaling and interfere with binding of IL-6
`to IL-6R. Anti-tl-6 mAbs might lead to high- evel accumulation of IL-6 because of decreased clearance (Lu et al., 1992). IL-6R neutralizing
`antibodies only lead to moderate elevation ofIL-6 levels because of impaired internalization and subsequent degradation ofIL-6 (Nishimoto
`etal., 2608). The sep130Fc protein specifically blocks IL-6 trans-signaling without affecting classical signaling via the membrane-bound
`ILG6R, as 1-6 alone has no measurableaffinity to s2p130Fc. IL-11 antagonists bind to IL-Z1R. Typically, these antagonists have not binding
`site(s} for 26136 and inhibit complex formation of IL-11/iL-1iR/gp139.
`
`highlight which compounds actually made their way from
`the bench io the bedside (Figure 3).
`AS mentioned previously, sohible forms of the IL-6R
`can be found in human body fluids, which Icd to the dis-
`crimination of IL-6 classic and tvans-signaling. In addition
`io sIL-6R, soluble forms of ep130 (sep130) were also found
`in human serum at 100-200 ne/ml.
`it was shownthat
`sepi30 acts as natural inhibitor of IL-6 trans-signaling
`to inactivate circulating IL-6 in IL-6/sIL-6R/sgpi30 cor-
`plexes (Miiller-Newen et al., 1998; Jostock et al., 2001).
`Despite a molar excess of sIL-6R over IL-6, free IL-G and
`IL-6 in IL-6/s1L-6R complexes are present, which allows
`both classic ancl trans-signaline. Under these conditions,
`sep130 was, however, able to trap all free IL-6 molecules
`in IL-6/sIL-6R/sgp130 complexes, resulting in inhibition
`of classic signaling by sep130 (Garbers et al., 2611). IL-6
`is known to exhibit pro-inflammatory and regenerative
`activities (Scheller et al., 2011b}. For example, IL-6 defi-
`cient mice are more susceptible in an inflammatory bowel
`disease model as compared to wild-type mice (Griven-
`nikov et al., 2009} but protected in mouse models of rheuw-
`matoid arthritis (Nowell et al, 2003, 2009). As szpi30
`is not able to directly inhibit IL-6 classic signaling via
`the membrane-bound IL-6R, recombinant sepi30G was
`used to distinguish between classic- and trans-signaling
`in vivo, It turned out that IL-6 trans-signaling has mostiy
`pro-inflammatory properties and is the main diiving
`force of [L-6-signaling during chronic inflammation. IL-6
`classic signaling has, however,
`regenerative activities
`and is needed, for example for induction of the hepatic
`
`acute-phase response (Scheiler ct al, 2011b). This has
`led to the cvaluation of an Fc-fusion protein of sgpi30
`(sgp130Fc) as a therapeutic principle to neutralize the
`pro-inflammatory activitics of 1L-6, which would not com-
`promise other bencficial activities of IL-6 classic signal-
`ing (Rose-Jchn ct al., 2007; Wactzig and Rose-John, 2012).
`importantly, sgpi30Fc dees not inhibit the signaling of
`the other [L-6-type cytokines (Scheller et al., 2005), but
`vould interfere with a hypothetical IL-11 trans-signaling
`pathway. Whether the recently uncovered 1L-30/sIL-6R
`complex, which also can be blocked by sgpl3GFc, plays a
`role in vivo is currently unknown (Garbers et al, 2013). A
`phase I clinical tial with the sgp130Fe protein is planned
`for 2013.
`
`In addition to sgp130, monoclonal antibodies are
`used to specifically inhibit IL-6 signaling. The most prom-
`inent example is the humanized antibody tocilizumab
`marketed as Actemra (RoActemra in the EU), which is
`approved for the treatment of rheumatoid arthritis and
`systemic juvenile idiopathic arthritis in Europe and the US
`(Nishimoto et al., 2005; Tanaka et al., 2012}. Tocilizumab
`specifically binds to site 1 of the 1L-6R, thereby blocking
`binding of IL-6 to IL-6R. Besides blocking IL-6-induced
`signal transduction,
`tocilizumab also prevented cellu-
`lar internalization and degradation of IL-6. As a conse-
`guence, IL-6R neutralizing antibodies lead to moderate
`elevation of overall IL-6 levels (Nishimoto et al., 20068},
`Other IL-6-directecl neutralizing antibodies are in clini-
`cal development as antiinflammatory and anticancer
`therapeutics. lt remains to be seen whether these novel
`
`
`
`Ex. 2001 - Page1333
`Ex. 2001 - Page1333
`
`

`

`DE GRUYTER
`
`C. Garbers and j. Scheller: Properties of interleukin-6 and interleukin-11 === L149
`
`anti-IL-6 mAbs also result in high-level accumulation of
`IL-6 because of decreased clearance (Lu et al., 1992}. In
`contrast to sepi30, the anti-IL-6R antibody tocilizumab
`and all other agents block IL-6 classic and trans-sisnaling
`(jones et al., 2019}.
`Because of the broad pro-inflammatory properties of
`IL-6, the therapeutic use of the cytokine itself does not
`seem to be a good choice. However, in a recent paper it
`was shown that treatment with recombinant iL-6 was pro-
`iective via classic signaling in a mouse moctel of epider-
`molysis bullosa acquisita (EBA), an autoimmune disease
`induced by autoantibodies against
`type VII collagen,
`whereas inhibition of IL-6 trans-signaling by sgp130Fc
`was not beneficial (Samaveram et al, 2013), This example
`Ulustrates that also recombinant {1-6 might be an option
`to treat certain diseases,
`In contrast, recombinant IL-4 is used to accelerate
`recovery of the hematopoietic system after cancer therapy,
`especially to treat severe throrsbocytopenta associated
`with chemotherapy, for which it is approved in the USA
`by the US Food and Drug Administration (FDA) (Du and
`Williams, 1997; Putoczki and Emst, 2010). Inhibition
`of IL-l1 signaling is an attractive therapcutic option (or
`details in IL-lt-dependent malignancies sec the following
`section ‘Comparison of IL-6 and iL-11 in vive’), but to date
`no antl-IL-ll or anti-TL-1R inhibitory monoclonal antibod-
`ios wore described. However, two 0L-11 antagonists have
`been described (Underhill-Day et al., 2003; Lee et al,
`2008). In the W147A antagonist, the amino acid 147 is
`mutated from a tryptophan to an alanine, which destroys
`the so-called ‘site TT’ of ILAL This mutant can therefore
`
`bind to the IL-l1R, but engagement of the zp130 homedi-
`mer fails, resulting in efficient blockade of IL-11 signaling
`(UnderhillDay et al., 2003). However, itis unclear if these
`two proteins are beneficial in inflammatorydiseases that
`depend upon IL-11, as systematic explorations in animal
`models are still missing.
`All the above-mentioned interventions take place
`outside of the cell, thereby preventing the binding of
`the cytokine or cytokine/receptor complex to the signal-
`transducing S-receptor gp130. Another way to inhibit
`IL-6/IL-11 signaling is the use of small cell-permeable
`compounds that block proteins further downstream of
`the signaling cascade. The most promising candidates
`to date ave inhibitors of the Janus kinases, which are
`responsible for phosphorylation of gpt30 as well as
`STATI and STAT3. The importance of these proteins is
`further underlined by the fact that numerous patients
`suffering from myeloproliferative disorders show gain-
`of-function mutations in the JAK2 gene, Ruxolitinib, a
`chemical that blocks both Jaki and Jak2, is approved for
`
`the use against myelofibrosis. Furthermore, it was tested
`in a phase I] study in patients with rheumatoid arthritis
`(RA) (Williams et al., 2008), showing promising results.
`Another Jak1/2 inhibitor, baracitinib, is also currently
`evaluated for the clinical use against RA (O’Shea et al.,
`2013). Tofacitinib is already approved by the FDA and
`can be therapeutically used in RA patients, who cannot
`be properly treated with methotrexate anymore. Tofaci-
`tinib blocks Jaki, Jak2 and Jak3 (Jiang et al., 2008; Haan
`et al, 2011). Efficacy of tofacitinib has been shown in
`clinical studies against, for example, RA (Fleischmann
`et al., 2012; van Vollenhoven et al., 2012) or ulcerative
`colitis (Sandborn et al., 2012}.
`However, it has to be noted that the blockade of Jak
`family members by small chemical compoundsis rather
`unselective compared to the specific inhibition of IL-6 or
`IL-lt mentioned before. Both cytokines (and murnerous
`other cytokines and growth factors} trigger activation of
`JAKs, and all these beneficial and/or destructive effects
`are simultaneously gone when the mentioned inhibi-
`tors are applied. Therefore, we think that blocking the
`binding of a single cytokine to its receptor is an altemma-
`tive therapeutic strategy, which might lead to reduced
`side effects,
`
`Comparison of IL-6 and IL-11
`in vivo
`
`In this section, we will directly compare and discuss the
`current viewof the biclogical function of 1-6 and [L-11
`in vive (Figure 4}. We will, however, not list all functions
`of IL-6 but concentrate on main functions of IL-l and
`
`discuss the role of IL-6 in these settings.
`
`THE calls’
`
`Figure & Overviewof IL-6 and H-11 in (patho-physiology.
`The table summarizes the main finding of the opposite functions of
`IL-6 and {L-11 fn vive described in this review.
`
`
`
`Ex. 2001 - Page1334
`Ex. 2001 - Page1334
`
`

`

`2150 = C. Garbers andj. Scheller: Properties of interleukin-6 and interleukin-14
`
`DE GRUYTER
`
`IL-6 and IL-11 promote opposite
`functions in heart failure
`
`Heart fatluye is the leading causeof mortality. The onset of
`heart failure is caused by cardiovasculardiseases, includ-
`ing myocardial infarction, hypertension, viral infections
`and others. [L-6 type cytokines, in particular CT-1 and LIF,
`have a wide range of biological functions in the heart, e.g.,
`in cardiomyocytes and in controlling immune reactions of
`cardiovascular diseases (Fujio et al., 201).
`TL-lIR has been shown to be expressed by cardiac
`myocytes and cardiac fibroblasts (Kimura et al., 2007),
`thereby making this organ a potential target of [L-11
`in vive. Indeed, several studies have underlined a func-
`tional role of IL-11 within the heart. Intravenous admin-
`
`istration of recombinant JL-11 before coronary arteryliga-
`tion greatly reduced the infarct size in a murine model of
`ischemia/reperfusion (1/R) (Kimura et al., 2007). Injection
`of IL-1t has been shown to reduce cardiac fibrosis, thereby
`attenuating cardiac dysfunction, in a myocardial infarc-
`tion model by coronary ligation in mice (Obana et al.,
`2010). The authors showed that a cardiac-specific knock-
`ut of STATS resulted in a cornplete loss of IL-lbenefi-
`cial effects, and conversely that this could be mimicked
`througha cardiac-specific transgenic expression of a con-
`stitutively active STATS mutant (Obanaet al., 2010). They
`further showthat IL-11 mRNAis 60-fold up-regulated one
`day after myocardial infarction, suggesting an endoge-
`nous role of T1-11 in the heart besices its beneficial effects
`
`when administered intravenously (Obana et al., 2010).
`Within the ischemia/reperfusion model, IL-11 prevented
`not only cardiac damage when administered in advance
`of the TR, but was still effective when injected at the start
`of the reperfusion, thereby reducing cardiac infury and
`preserving cardiac function (Obana et al., 2012). This post-
`conditioning effect was mediated by activation of STATS,
`as mice with a cardiac-specific STAT3 knock-out were not
`protected in an IL-ii- dependent manner against 1/R injury
`(Obana et al., 2012}. Also in hindlimk ischemia, a mouse
`model for peripheral vascular disease, therapeutic admin-
`istration of recombinant [L-11 has heen shownto he ben-
`
`eficial, as it leads to an increase in recoveryafter femoral
`axtery ligation and enhanced collateral vessel growth
`(Aitsebaomo et al., 2011).
`In humans, increased [1-6 and sIL-6Rlevel were found
`in acute myocardial infarction (Kandaet al., 2000), Inter-
`leukin-6 is produced by hypoxic myocytes and plays an
`iraportant role in neutrophil-mediated reperfusion injury
`in the myocardium (Yarnauchi-Takihara et al., 1995; Sawa
`et al, 1998). Transgenic mice overexpressing IL-6 and
`
`membrane-bound IL-6R (actin promoter) develop cardiac
`hypertrophy, whereas either transgenic mice for IL-6 or
`IL-6R alone did not show detectable myocardial abnor-
`malities (Hirota et al., 1995). In rats, 1L-6 trans-signaling
`leads to myocardial fibrosis, hypertension, and diastolic
`dysfunction (Meléndez et al., 2010}. Moreover, 1L-6 neu-
`tralization partially reduced cardiac hypertrophy as well
`as collagen synthesis in rat heart in a STAT3-dependent
`manner (Mir et al., 2012}, In ischemia/reperfusion, infarct
`size in IL-6 deficient mice was similar to wild-type mice.
`However, preconditioning reduced infarct size in wild-
`ype but not in IL-6 deficient mice (Dawnet al., 2004).
`Treatment with a neutralizing anti-IL-6R antibody,
`tested in a murine model of myocardial infarction, also
`did not reduce initial Infarct size, but reduced neutrophil
`and macrophage infiltration, matrix-metalloproteinase 2
`activity, and led to an improvement of contractile func-
`tion, which resulted in improved survival 28 days after
`surgery (Kobara et al., 2010).
`These results suggested that production of IL-6 in
`the heart and in particular IL-6 wans-signaling may he a
`pathophysiological response that contributes to hypertro-
`phy and progresses into heart failure. Therefore, inhibi-
`tion of IL-6 trans-signaling was suggested as a potential
`thorapy for hypertension and cardiac hypertrophy (Colcs
`et al., 2007). The importance of IL-6 in cardiovascular dis-
`eases was further supported by a recent study, showing
`that
`in murine experimental autoimmune myocarditis
`IL-6 and IL-6R expression was up-regulated and disease
`development was prevented by neutralizing anti-IL-GR
`antibodies and pharmacological
`inhibition of STAT3
`(Camporeale et al, 2013). Specific inhibition of IL-6 trans-
`signaling by sep120 was, however, sufficient to effectively
`block the development of atherosclerosis in mice under-
`lining the pro-inflammatory role of IL-6 trans-signaling
`(Schuett et al., 2012). The recently described conditional
`IL-6R deficient mice have not heen studied so far in car-
`
`diovascular disease models (McFarland-Mancini et al.,
`2010). As IL-6R can also transmit signals of p26 (cytokine
`subunit of 1L-27) and CNTF Gchuster et al., 2003; Garbers
`et al., 2613), it would be interesting to see if LL-6 and 1L-6R
`deficient mice have the same phenotype in cardiovascular
`disease models.
`
`Furthermore, the coding 1L-6R rs2228145 SNP leads
`to reduced production of acute-phase proteins such as
`C-reactive protein and fibrinogen and a lower risk of
`coronary heart disease (Collaboration, 2012; Consortium,
`2012), This is caused by a reduced level of membrane-
`bound IL-6R and/or an increased soluble IL-6R level.
`Mechanistically,
`the decreased risk of coronary heart
`disease for homozygous carriers of the 1522728145 SNP
`
`
`
`Ex. 2001 - Page1335
`Ex. 2001 - Page1335
`
`

`

`DE GRUYTER
`
`C, Garbers and |. Scheller: Properties of interleukin-6 and interleukin-11 === Ti54
`
`might be caused by reduced IL-6 classic signaling on
`target cells, such as hepatocytes, monocytes and mac-
`rophages and/or an increased buffering of secreted IL-6
`by the sIL-G6R and sep130 proteins (Ferreira et al, 2013;
`Scheller & Rose-John, 2612}. Both modes would reduce the
`overall IL-6 activity.
`To date it is not completely understood, why IL-l1
`and 1L-6 have opposing functions in cardiovascular dis-
`eases, IL-6 but not IL-l] contributes to the activation and
`
`attraction of immune cells, e.g., neutrophils’ attraction
`to the damaged heart. Inhibition of neutrophil invasion
`after heart damage has been shown to be beneficial (Litt
`et al, 1989). Unlike IL-lIR, IL-6R is not expressed on car-
`diomyocytes (Meléndez et al, 2010), which might explain
`different biological outcomes. However, stimulation of
`cardiomyocytes with IL-6 trans-signaling via the sIL-6R
`or IL-11 induces STAT3 phosphorylation, but detrimental
`effects are only observed for IL-6 trans-signaling. There
`are, however, at least two possible explanations for these
`phenomenon: firstly, signal transduction might be differ-
`ent in terms of signal kinetics (duration and intensity),
`even though 0-6 and TL-11 use the same signal transduc-
`ing 2pi30 receptors. Secondly, IL-6 trans-signaling unse-
`lectively activates all cells within the heaxt, which may
`cause the overall negative outcome in comparison to IL-11,
`which specifically activates only cardiomyocytes and car-
`diofibrobiasts. These issues might be addressed by activa-
`tion of IL-1] trans-signaling in all heart cells via an TL-11/
`sIL-L1R fusion protein, as IL-6 and IL11 trans-signaling
`would not be restricted to IL-6R and IL-1iR expressing
`cells, respectively. We would expect that the net result of
`IL-6/sIL-6R and IL-11/sIL-11R during ischemia/reperfusion
`injury would he the same. However, this has to be shown
`in side-by-side experiments. Alternatively, [L-6 might be
`modified in a way, that it selectively targets only cardio-
`myocytes/fibroblasts in vivo. This might be achieved by an
`IL-6 variant that uses IL-17R but not 1L-6R as an a-receptor,
`Here, we would expect that this IL-I1R-specific IL-6 variant
`would act like IL-l1. However, if these predictions are not
`true, than the signal kinetics of IL-6 and IL-11 must be
`different. lt is not clear how different signal kinetics are
`regulated on the molecular level.
`
`Similarities and dissimilarities of
`
`IL-6 and IL-i1 in asthma
`
`IL-l] mRNA and proteinis largely absent from normal lung
`tissue and primary lung cells and lung cell ines. However,
`several stimuli have been shownto induce LL-H expression
`
`in these cells (lias ct al., 1994a,h, 1997). Furthermore,
`IL-1] is present in secretions from patients with viral res-
`piratory infections (Einarsson et al., 1996). Specific over-
`expression of [L-11 in the lung using the CC10 promoter
`(Clara cell 10 kDa protein), resulted in airway obstruction,
`remodeling of the bronchial space through filbrosis of the
`subephithelium, and lymphocytic inflammation through
`accumulation of peribronchiolar mononuclear cells (Tang
`et al, 1996), Interestingly, the phenotype of transgenic
`mice for IL-6 under the control of the CC10 promoter was
`verysimilar to [L-1i transgenic mice (DiCosmoet al, 1994;
`Doganci et al., 2005b). in mice, 50-60%of the airwaycells
`are Clara cells (Pack et al., 1981), ensuring alocal, lung-
`restricted distribution of IL-6 and IL-tt.
`
`This phenotype observed in mice mimicked in several
`aspects the pathophysiological situation in human asth-
`matic patients. Furthermore, (L-l1 is highly expressed in
`patients with moderate and severe forms of asthma, but
`not in ritd forms of asthmatic or healthy humans, [L-11
`was expressed in eosinophils as well as epithelial cells,
`and the amount of TL-11 expression correlated with the
`severity of the discasc (Minshall ct al., 2000). However,
`IL-11 was also shownto selectively block pulmonary cosin-
`ophilia caused by acroalicrgens and the accompanying
`TH2-type inflammation (Wang et al., 2000).
`Increased levels of IL-6 are found in blood (Yokoyama
`et al., 1995}, bronchoalveclar lavage fluid (BALF) (Broide
`et al., 1997), and hing tissues (Marini et al., 1992) of asth-
`matic patients, Moreover, increased levels of sTL-GR have
`been observed in the airways of patients with allergic
`asthma (Doganci et al., 2?005a,b). TH2 cells are critical
`allergic driver celis. IL-6 tans-signaling via siL-GR sup-
`ports expansion of TH2 effector cells and cytokine produc-
`tion in the lung. At the same time, [L-6 classic signaling
`suppresses the activity of reculatoryT cells (Treg) leading
`to reduced peripheral tolerance (Pasare and Medzhitov,
`2003; Doganci et al., 2005a).
`Consequently, aclministration of anti-IL-6R antibod-
`ies that block classic and trans-signaling in experimen-
`tal asthma leads to cell death of lung effector T cells via
`activation of regulatoryTcells (Finotto et al., 2607). More-
`over, a combination of extracellular matrix deposition,
`inflammation, angiogenesis, and airway smooth muscle
`(ASM) mass results in airway wall
`thickening during
`asthma. Because ASMcells dic not express membrane-
`bound IL-6R, IL-6 trans-signaling may contribute to vessel
`expansion in airway walls of asthmatic subjects (Ammit
`et al., 2007).
`The IL-6R SNP 1s2228145 is a potential modifier of hing
`function in subjects with asthma and might identify sub-
`jects at risk for more severe asthma Qlawkinset al., 2012),
`
`
`
`Ex. 2001 - Page1336
`Ex. 2001 - Page1336
`
`

`

`TS2Z mm C. Garbers andj. Scheller: Properties of interleukin-6 and interleukin-14
`
`DE GRUYTER
`
`Whereas IL-6 promotes expansion of TH2 cells, [L-11
`restricts TH2-driven inflammation in allergic lung dis-
`eases, A vole of IL-1 on the development of regulatory
`T cells was not investigated so fax. Again on the celhilar
`and molecular level, opposing functions of LL-6 and IL-11
`are not understood, A recent study showed that depending
`on the cell type, 1L-11] is able to activate STATI and STAT3
`or solely STATI (Onnis et al., 2613), and differential acti-
`vation of signal transduction pathways might contribute
`io divergent outcomes of IL-6 and iL-1] signaling, Again,
`a detailed analysis of cellular [L-6R and IL-W1R expression
`profiles and cell type specific signal ansduction analysis
`in allergic asthmaand other diseases is mandatory.
`
`Regenerative and inflammatory
`potential of IL-6 and IL-11 in the
`colon
`
`The TL4iR is expressed in human colonic epithcHal cclis
`(Kiessling et al., 2004), alsc making the colon a target for
`IL-l in vive. The pathogen Citrobacter rodentium induces
`barricr disruption of the colon and infiltration of immunc
`cclis such as macrophages and neutrophils, which makes
`ita well-established model for infectious colitis Eickmann,
`2006). Application of recombinant IL-1] to C. rodentium-
`caused colitis in TLR2deficient mice prevented lethal-
`ity. IL-l fed to activation of STAT3 in ard regeneration of
`intestinal epithelial cells. In Hne with this, signaling via
`TLR? induced [1-11 expression, which in turn helped to
`maintain the barrier fumetion of the intestinal epithelium
`(Gibson et al, 2010). The human leucocyte antigen A
`variant HLA-B27 is associated with a class of inflammatory
`diseases known as spondyloarthropathies (Brown et al.,
`1996). Transgenic rats, which overexpress the human §2-
`microglobulin together with HLA-B27, have been shown
`to be a good model of inflammatory diseases, including
`inflammatory bowel disease (Hammeret al., 1990). These
`rats showed down-regulationof IFNy, TNFaand 1L43 when
`treated with recombinant IL-i1 and an overall reduced
`
`clinical disease severity score compared to control animals
`(Peterson et al., 1998). First clinical trials with recombi-
`nant hIL-1i for the treatment of Crohn’s disease have also
`
`shownpromising results (Sandset al, 1999, 2002).
`Levels of IL-6 in sera correlate with clisease severity in
`inflammatory bowel disease (Hosokawaet al,, 1999). Mainly
`lamina propria mononuclear cells and Tcells secrete IL-6
`and sIL-6R in Crohn's disease patients. In a T cell ansfer
`eolids mouse model, anti-IL-6R monoclonal antibodies
`
`prevented the development of signs and symptoms of colitis
`(Yamamoto et al., 2000) and the humanized anti-IL-6R
`monoclonal antibodytocilizumab maybe a promising drug
`for Crohn’s disease (ito et al., 2004), LL-6 trans-signaling
`but not classic signaling caused detrimental anti-apoptosis
`of T cells ancl tissue damage. Consequently, blockageof IL-6
`trans-signaling with sep130Fc suppressed T cell responses
`in experimental colitis and improved the clinical severity
`score (Atreya et al., 2000}, Blockade of 1L-6 trans-signaling
`alsa prevented the development of spontaneous Heitis in
`SAMPI/Yit mice via reduction of STAT3 phosphorylation
`(Mitsuyama et al., 2006}. in addition, like [L-11, IL-6 sig-
`naling can stimulate survival and proliferation of intestinal
`epithelial cells, Abrogation of regeneralive pathways in the
`intestine may explain why IL-6 deficient mice displayed a
`widespread damage of the colonic mucosa in the non-T ceil-
`dependent DSS-colitis model (Grivennikovet al, 2009), tis
`not clear if epithelial regeneration is induced byclassic or
`trans-signaling, since intestinal epithelial cells largely lack
`membrane-bound IL-6R.
`
`Increased expression of pro-inflammatory cytokines,
`inckuding IL-6, were found in colitis-associated cancer
`(CAC) paticnts (Mitsuyama ct al., 1991). In a murine model
`of CAC, IL-6 deficient mice had a decreased tumor load
`sugecsting that IL-6 was necessaryfor tumor development
`and growth (Grivennikov ct al., 2009). Among others,
`TGE-B signaling is considered to be a tumor-supprossive
`pathway, and therefore inactivating mutations within
`the TGF-6 signaling play an important role in CAC clevel-
`opment. TGE-B is secreted by tumor infiltrating T cells.
`Expression of a dominant-negative form of the TGE-QRIT
`{dnTGF-BRID chain in murine T celis resulted in signifi-
`cantly higher IL-6 levels in the colonic tissue, suggesting a
`negative regulatory circuit between TGF-B and IL-6 produc-
`tion (Becker et al., 2004, 2005). Consequently, TGF-B sup-
`pressed colon cancer tumor progression through the inhi-
`bition of IL-6 trans-signaling (Becker et al., 2004). When
`crossed on an IL-6 deficient background, dnTGF-BRU/IL-6
`deficient mice showed reduced signs of colitis but autoim-
`mune cholangitis was exaggerated. lt was suggested that
`therapeutic blockadeof LL-6 in autoimorunediseases such
`as colitis might be undertaken with caution in patients
`who have accompanying liver diseases (Zhang et al., 2010).
`Recently, an unexpected role of 1L-11 in CAC-associ-
`ated metastasis has been described (Calon et al., 2012).
`Secreted TGF-3 from the cancer cells stinvulates [1-11
`secretion from cancer-associaied Hbroblasts, which in
`
`turn activates STAT3 signaling via gp130 in tumor cells.
`This circuit increases the survival rate of metastatic cells,
`thereby enhancing metastasis formation (Calon et al,
`2012), In line withthis, expressionof the IL-11R was shown
`
`
`
`Ex. 2001 - Page1337
`Ex. 2001 - Page1337
`
`

`

`DE GRUYTER
`
`€. Garbers and j. Schelter: Properties of interleukin-6 and interleukin-11 ---—- 2353
`
`to correlate with tumor invasion and lymphatic infiltra-
`tion of human colorectal adenocarcinomas (Yoshizaki
`etal., 2006),
`Taken together, murine colitis models, which are
`based on the detrimental activity of the acquired immune
`response, appear to benefit from the blockade of IL-6 sig-
`naling, However, in DSS-induced colitis, which is character-
`ized by a massive destabilization of the intestinal barrier
`and mainly activates the innate immune system, iL-6 con-
`irfbutes to regeneration of the epithelial barrier and has
`protective functions. It remains to be seen how both func-
`tions of IL-6 contribute to human forms of inflammatory
`howel disease, e.g., Crohr’s disease and ulcerative colitis,
`The role of IL-1] maybe restricted to the maintenance of the
`intestinal barder. However, data from IL-1] deficient mice or
`blockade of IL-11 are still missing. TGF-B appears to block
`iL-6 butstiroulate IL-l production, suggesting that 1-6 and
`IL-1 have in part opposing roles in colitis and CAC.
`
`Requirement of IL-11 but not of IL-6
`in gastric tumors
`
`Cytokine signaling is tightly regulated by negative feed-
`back loops. Both the phosphatase SHP2 and SOCS3 bind
`to the phosphorylated tyrosine residuc pY759 (human) /
`pY757 (mouse) of the IL-6 cytokine familysignal transducer
`gpi30. Whereas SHP2 is constantly expressed in the cell
`and gets phosphorylated and therefore dissociates from the
`receptor after ligand binding (Lu et al., 2001, 2003}, SOCS3
`is usually absen