REVIEW
`published: 15 May 2019
`doi: 10.3389/fimmu.2019.01093
`
`Coordination of Immune-Stroma
`Crosstalk by IL-6 Family Cytokines
`
`Nathaniel R. West*
`
`Department of Cancer Immunology, Genentech, South San Francisco, CA, United States
`
`Stromal cells are a subject of rapidly growing immunological interest based on their ability
`to influence virtually all aspects of innate and adaptive immunity. Present in every bodily
`tissue, stromal cells complement the functions of classical
`immune cells by sensing
`pathogens and tissue damage, coordinating leukocyte recruitment and function, and
`promoting immune response resolution and tissue repair. These diverse roles come
`with a price: like classical immune cells, inappropriate stromal cell behavior can lead to
`various forms of pathology, including inflammatory disease, tissue fibrosis, and cancer.
`An important immunological function of stromal cells is to act as information relays,
`responding to leukocyte-derived signals and instructing leukocyte behavior in kind. In
`this regard, several members of the interleukin-6 (IL-6) cytokine family, including IL-6,
`IL-11, oncostatin M (OSM), and leukemia inhibitory factor (LIF), have gained recognition
`as factors that mediate crosstalk between stromal and immune cells, with diverse roles in
`numerous inflammatory and homeostatic processes. This review summarizes our current
`understanding of how IL-6 family cytokines control stromal-immune crosstalk in health
`and disease, and how these interactions can be leveraged for clinical benefit.
`
`Keywords: stromal cells, cytokines, inflammation, fibrosis, immune
`
`THE DIVERSE ROLES OF STROMAL CELLS IN IMMUNITY AND
`INFLAMMATION
`
`The term “stroma” refers to the non-parenchymal components of tissues that form a supportive
`matrix in which parenchymal cells reside (1). While a confusingly broad array of cell types
`have been described as “stromal cells,” in this review they are defined as non-hematopoietic,
`non-epithelial mesenchymal cells, including fibroblasts, myofibroblasts, bone marrow stromal cells,
`and the specialized fibroblast-like stromal cells of secondary lymphoid organs. Other mesenchymal
`populations such as endothelial cells, adipocytes, and muscle cells, while of great interest, are
`largely omitted from this discussion for the sake of brevity and clarity. Long considered to
`be mere structural entities without specialized functions, an explosion of data in the last two
`decades has established stromal cells as key regulators of both protective and pathological immune
`responses (2).
`Regulation of immune function by stromal cells has been most extensively studied in the context
`of secondary lymphoid organs. First identified in 1992, podoplanin (PDPN)+ fibroblastic reticular
`cells (FRC) form a dense reticular network in lymph nodes that facilitates leukocyte migration and
`antigen presentation (2–5). By producing soluble chemokines, cytokines, and other factors—such
`as CCL19 (C-C motif chemokine ligand 19), CCL21, and IL-7 (interleukin 7)—FRC are crucial for
`controlling leukocyte recruitment, survival, and proliferation. FRC-like stromal cells play similar
`roles in other lymphatic tissues, such as in tertiary lymphoid organs of the intestinal mucosa (6, 7).
`
`Edited by:
`
`Teruki Dainichi,
`
`Kyoto University, Japan
`
`Reviewed by:
`
`Carl D. Richards,
`
`McMaster University, Canada
`
`Nathan Karin,
`
`Technion Israel Institute of
`
`Technology, Israel
`
`*Correspondence:
`
`Nathaniel R. West
`
`west.nathaniel@gene.com
`
`Specialty section:
`
`This article was submitted to
`
`Immunological Tolerance and
`
`Regulation,
`
`a section of the journal
`
`Frontiers in Immunology
`
`Received: 28 February 2019
`
`Accepted: 29 April 2019
`
`Published: 15 May 2019
`
`Citation:
`
`West NR (2019) Coordination of
`
`Immune-Stroma Crosstalk by IL-6
`
`Family Cytokines.
`
`Front. Immunol. 10:1093.
`
`doi: 10.3389/fimmu.2019.01093
`
`Frontiers in Immunology | www.frontiersin.org
`
`1
`
`May 2019 | Volume 10 | Article 1093
`
`Lassen - Exhibit 1030, p. 1
`
`

`

`West
`
`IL-6 Family Cytokines and Stromal Cells
`
`In non-lymphoid tissues, stromal cells can exert similar effects
`to those of the secondary lymphoid organs by acting as scaffolds
`for leukocyte migration and by producing a diverse array of
`cytokines and chemokines (2).
`Importantly, the immunological functions of stromal cells
`can vary substantially depending on their host organ and
`physiological context. For example, lymph node FRC recruit
`CCR7 (C-C chemokine receptor type 7)+ T cells (naïve
`and central memory) and CCR7+ dendritic cells (DC) to
`lymph nodes by producing the chemokines CCL19 and
`CCL21, as well as the pro-survival cytokines IL-7 and IL-
`15, thereby coordinating T cell activation and maintenance
`(4). In contrast, stromal cells in peripheral tissues generally
`lack expression of CCL19 and CCL21; accordingly, naïve
`and central memory T cells are infrequent in the periphery.
`However, expression of various pattern recognition and cytokine
`receptors by non-lymphoid tissue stromal cells allows them
`to sense microbial molecules and endogenous danger signals
`(1, 8, 9). In response, they produce chemokines [including
`CCL20 and CXCL10 (C-X-C motif chemokine ligand 10)] that
`attract effector T cells to sites of inflammation. Furthermore,
`inducible expression of leukocyte adhesion molecules including
`ICAM-1 (intercellular adhesion molecule 1) and VCAM-1
`(vascular cell adhesion molecule 1) allows
`tissue-resident
`stromal cells to further influence the balance between leukocyte
`recruitment, retention, and recirculation (1, 2, 9). Finally,
`stromal cells contribute directly to immune response resolution
`and tissue repair, the latter being one of their best studied
`functions. Examples of “pro-resolution” factors produced by
`stromal cells include NOS2 (nitric oxide synthase 2) and NO
`(nitric oxide), which are released by lymph node FRC to
`constrain T cell proliferation (10–12), and IDO1 (indoleamine
`2,3-dioxygenase 1) produced by peripheral
`stromal cells,
`which similarly limits T cell proliferation by depleting the
`critical T cell metabolite tryptophan (13, 14). Thus, stromal
`cells in different
`tissues collectively regulate the strength,
`quality, and duration of immune responses via diverse and
`complementary mechanisms.
`As with most
`immunological processes, communication
`between stromal and immune cells is highly dependent on
`cytokines. Stromal cells bear receptors to a variety of biologically
`diverse cytokines
`that
`represent virtually all branches of
`innate and adaptive immunity, including innate inflammatory
`cytokines [e.g., TNF (tumor necrosis factor) and IL-1β], Th1
`cytokines [e.g., IFN-γ (interferon gamma)], Th2 cytokines
`(e.g., IL-13), Th17 cytokines (e.g., IL-17A), and tolerogenic
`cytokines [e.g., TGF-β (transforming growth factor beta)]
`(7, 9, 15, 16).
`In turn, stromal cells can be prodigious
`producers of other cytokines and chemokines, such as IL-6
`(1, 2, 7, 9). In recent years, cytokines of the IL-6 family
`have gained increasing attention for their roles in various
`homeostatic and pathological processes, which in many cases
`can be attributed to their ability to co-ordinate immune-
`stroma crosstalk. This review aims to provide a focused update
`on the contributions of IL-6 family members to immune-
`stromal interactions.
`
`AN OVERVIEW OF THE IL-6 CYTOKINE
`FAMILY
`
`The IL-6 family includes IL-6, IL-11, IL-27, IL-31, oncostatin
`M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic
`factor (CNTF), cardiotrophin 1 (CT-1), and cardiotrophin-
`like cytokine factor 1 (CLCF1) (17, 18). With the exception
`of IL-27, which is a heterodimeric protein comprised of IL-
`27p28 and EBI3 (Epstein-Barr virus-induced gene 3) (19), IL-6
`family members are compact 4-helix bundle cytokines made
`from a single polypeptide. Glycoprotein 130 (gp130, encoded
`by the IL6ST gene) is a crucial receptor subunit utilized by
`all members of the IL-6 family except IL-31. While gp130
`expression is relatively ubiquitous in a wide variety of tissues and
`organs, cell-type specificity for different IL-6 family members is
`bestowed by the more restricted expression patterns of ligand-
`specific co-receptors, including IL-6R (IL-6 receptor), IL-11R
`(IL-11 receptor), IL-27Rα (IL-27 receptor alpha), OSMR (OSM
`receptor), LIFR (LIF receptor), and CNTFRα (CNTF receptor
`alpha). Three distinct forms of receptor-ligand complexes have
`been described (Figure 1). First characterized was that of
`IL-6, which engages IL-6R along with two subunits of gp130.
`Intriguingly, although this implies the formation of a trimeric
`complex, a series of cooperative interactions can ultimately
`produce an interlocked hexamer comprised of two subunits
`each of IL-6, IL-6R, and gp130 (20). A similar structure is
`likely formed in response to IL-11/IL-11R interaction (21, 22).
`In this arrangement, only gp130 drives signal transduction,
`due to an absence of intracellular signaling motifs in IL-6R
`and IL-11R. In contrast, OSMR, LIFR, and IL-27Rα form
`heterodimers with gp130 in the presence of
`their cognate
`ligands (23–28). Unlike IL-6R and IL-11R, OSMR, LIFR, and
`IL-27Rα are capable of driving signal transduction via their
`own suite of signaling motifs. Finally, CNTF and CLCF1
`drive formation of a trimeric complex that includes gp130,
`LIFR, and CNTFRα (29–31). The gp130-independent outlier
`of the family, IL-31, engages a heterodimeric complex of IL-
`31Rα (previously known as gp130-like receptor) and OSMR
`(18). Notably, while mouse OSM binds with high affinity only
`to the gp130/OSMR heterodimer, human and rat OSM can
`bind with high affinity to either gp130/OSMR or gp130/LIFR
`heterodimers (32–34). Thus, in rats and humans, manipulation
`of LIFR would be expected to affect both OSM and LIF signaling
`(as well as CLCF1, CT-1, and CNTF), while manipulation
`of OSMR would influence OSM and IL-31 signaling. As a
`corollary, changes in human or rat OSM bioavailability would
`influence cells that express OSMR and/or LIFR, while changes
`in LIF or IL-31 would affect only LIFR- or IL-31Rα-expressing
`cells, respectively.
`All members of the IL-6 family drive signal transduction via
`receptor-associated Janus kinases (primarily JAK1 and JAK2),
`which phosphorylate a variety of conserved tyrosine residues in
`the cytoplasmic domains of signaling receptor subunits (gp130,
`OSMR, LIFR, IL-27Rα, and IL-31Rα) (17, 18, 35). Several
`downstream signaling pathways are activated in response,
`including signal
`transducer and activator of
`transcription
`
`Frontiers in Immunology | www.frontiersin.org
`
`2
`
`May 2019 | Volume 10 | Article 1093
`
`Lassen - Exhibit 1030, p. 2
`
`

`

`West
`
`IL-6 Family Cytokines and Stromal Cells
`
`FIGURE 1 | Receptor usage of IL-6 family cytokines. With the exception of IL-31, IL-6 family cytokines transduce signals via receptor complexes that include gp130
`and one or more additional ligand-specific subunits. IL-6 and IL-11 signaling requires IL-6R and IL-11R, respectively. The cytoplasmic domains of these receptor are
`short and lack signaling motifs, making gp130 the sole source of signal transduction downstream of IL-6 and IL-11. The heterodimeric cytokine IL-27 (comprised of
`IL-27p28 and EBI3) requires a complex of gp130 and IL-27RA. LIF and CT-1 use a heterodimeric complex of gp130 and LIFR, while CNTF and CLCF1 signal via a
`trimeric complex of gp130, LIFR, and CNTFRα, a GPI-anchored protein that does not directly contribute to signaling beyond facilitation of ligand binding. OSM
`displays species-specific receptor usage. In humans and rats, OSM signals via either gp130/OSMR or gp130/LIFR complexes, while in mice OSM is primarily
`recognized by OSMR. IL-31 does not require gp130, and instead uses a complex of OSMR and IL-31R. Aside from IL-6R, IL-11R, and CNTFRα, all receptors in the
`IL-6 family are capable of directly activating signal transduction in response to ligand binding. IL-6 family cytokines employ classical JAK-mediated signaling. Major
`downstream mediators include STAT3 (the main STAT for all except IL-27), STAT1 (activated preferentially by IL-27 and to a lesser extent by other IL-6 family
`members), additional STATs that depend on cell type and physiological context (including STATs 4, 5, and 6), the MAPK cascade, PI3K/Akt/mTOR signaling, and
`SRC/YAP/NOTCH signaling. Akt, protein kinase B; CLCF1, cardiotrophin-like cytokine factor 1; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin 1; EBI3,
`Epstein-Barr virus induced 3; ERK, extracellular signal-regulated kinase; gp130, glycoprotein 130, also known as IL-6 signal transducer; IL, interleukin; IL-6R, IL-6
`receptor; IL-11R, IL-11 receptor; IL-27RA, IL-27 receptor; CNTFRα, CNTF receptor; LIF, leukemia inhibitory factor; LIFR, LIF receptor; MAPK, mitogen activated
`protein kinase; JAK, janus kinase; JNK, c-jun n-terminal kinase; mTOR, mammalian target of rapamycin; OSM, oncostatin M; OSMR, OSM receptor; PI3K,
`phosphatidylinositol-3-kinase; STAT, signal transducer and activator of transcription; SRC, Proto-oncogene tyrosine-protein kinase Src; YAP, yes-associated protein.
`
`(STAT) proteins (including STAT1, STAT3, STAT4, STAT5,
`and STAT6), the mitogen-activated protein kinase (MAPK)
`cascade, the phosphatidylinositol-3-kinase (PI3K)/Akt pathway,
`and the SRC/YAP/NOTCH pathway (Figure 1). While signal
`transduction by individual IL-6 family members is broadly
`similar, the relative strength of activation of specific pathways
`can differ depending on the cytokine, cell type, and physiological
`context. For example, unlike gp130, OSMR efficiently recruits
`the adapter protein SHC, allowing OSM to drive more potent
`activation of the MAPK pathway than IL-6, which signals via
`SHP-2 bound to gp130 (35, 36). Similarly, although STAT3
`is generally considered to be the dominant STAT protein
`activated by the IL-6 family, IL-27 preferentially activates
`STAT1 (37). Further complexity is provided by the capacity
`of IL-6, IL-11, and CNTF to signal via soluble receptor forms
`in a process known as trans signaling. In this process, soluble
`versions of IL-6R, IL-11R, or CNTFRα are produced either
`through proteolytic cleavage of membrane-bound receptors,
`
`in either
`or via expression of alternatively spliced mRNA;
`case, the soluble receptor form can dimerize with its cognate
`ligand in solution, and subsequently produce a functional
`signaling complex in association with membrane-bound gp130
`(18, 38–40). Cells thus require only gp130 to be sensitive
`to trans signaling, which allows many cell types that lack
`IL-6R, IL-11R, or CNTFRα to respond to these cytokines.
`In the case of IL-6, trans signaling is thought to be a critical
`mechanism by which IL-6 promotes disease pathogenesis,
`particularly arthritis and colorectal cancer (18, 41, 42). Thus,
`while many similarities exist between IL-6 family cytokines,
`differences
`in their
`receptor usage,
`signal
`transduction
`profiles, and patterns of
`receptor expression collectively
`foster a substantial degree of functional pleiotropy. Indeed,
`IL-6 family members influence cell survival, proliferation,
`differentiation, metabolism, and migration, thus contributing to
`a plethora of physiological processes that are critical for both
`homeostasis and pathology.
`
`Frontiers in Immunology | www.frontiersin.org
`
`3
`
`May 2019 | Volume 10 | Article 1093
`
`Lassen - Exhibit 1030, p. 3
`
`

`

`West
`
`IL-6 Family Cytokines and Stromal Cells
`
`EXPRESSION OF IL-6 FAMILY CYTOKINES
`BY STROMAL CELLS
`
`Although some members of the IL-6 family are produced
`primarily by hematopoietic cells (notably OSM and IL-27),
`stromal cells can be important sources of several others,
`including IL-6, IL-11, and LIF. Diverse factors appear to regulate
`the expression of these cytokines by stromal cells, including
`microbial sensing, detection of endogenous alarmins, stimulation
`by other cytokines (including those within the IL-6 family
`itself), and cell stress (Figure 2). Although these inputs are
`known drivers of cytokine production, the critical drivers in
`vivo, particularly under physiological conditions, are rarely
`well defined.
`In response to infection or an inflammatory challenge, IL-6
`production is rapidly increased by stromal cells. Depending
`on their location and the nature of the challenge, this could
`be due to direct sensing of danger signals, responses to other
`inflammatory cytokines, or both. As an NF-κB (nuclear factor
`kappa B) response gene (43), IL-6 is induced by stromal cells
`downstream of several pattern recognition receptors including,
`but probably not limited to, toll-like receptor (TLR)2, TLR4, and
`NOD2 (nucleotide binding oligomerization domain 2) (44–46).
`
`The NF-κB activating cytokines IL-1β, IL-17A, and TNF (tumor
`necrosis factor alpha) are also potent inducers of stromal IL-6
`production, and can do so in synergy with one another (43, 47–
`54). Although NF-κB is thought to be the dominant driver of
`IL-6 production downstream of these cytokines, contributions
`by MAPK signaling have also been observed. Indeed, signaling
`by alternative pathways such as the MAPK and PI3K cascades
`may underlie the ability of cytokines like OSM (55, 56), IL-4 (49),
`and TGF-β (54, 57) to promote stromal IL-6 expression, since
`these are not classical activators of NF-κB. Beyond cytokines and
`danger signals, cadherin-11 (CDH11), a mesenchymal cadherin
`that engages in homophilic interactions between adjacent cells,
`has also been shown to drive IL-6 production via NF-κB and
`MAPK signaling (53). Indeed, blockade of CDH11 attenuates
`inflammation in mouse models of arthritis, an effect that may
`be due in part to reduced IL-6 production by CDH11+ synovial
`fibroblasts (53). Finally, IL-6 is a well-known product of the
`senescence-associated secretory phenotype (SASP) in fibroblasts,
`a feature associated with aging and cancer (58). Indeed, IL-6
`produced by prostate tumor fibroblasts in response to metabolic
`stress has been proposed to mediate malignant progression (59).
`Less is known about the regulation of LIF and IL-11 expression
`by stromal cells, but the mechanisms involved may be similar
`
`FIGURE 2 | IL-6 family cytokine production by stromal cells and their biological effects. Stromal cells are important contributors to production of three members of the
`IL-6 family: IL-6, LIF, and IL-11. Expression of these cytokines is regulated by various stimuli including recognition of bacterial products via TLR2, TLR4, or NOD2, and
`diverse cytokines that drive activation of NF-κB, MAPK, PI3K, and STAT3. LIF has been shown to promote IL-6 expression via STAT4 signaling, while IL-4 and IL-13
`can suppress LIF and IL-11 expression through activation of STAT6. Following production by stromal cells, IL-6, LIF, and IL-11 can influence diverse biological
`processes including CD4+ T cell polarization, regulation of chemokine production, promotion of alternative macrophage differentiation, and tissue remodeling through
`effects on stromal and epithelial cells. In this figure, arrows indicate stimulatory effects, and capped lines indicate inhibitory effects. All processes illustrated are
`described further in the main text. CCL, C-C motif chemokine ligand; ECM, extracellular matrix; IL, interleukin; LIF, leukemia inhibitor factor; MAPK, mitogen activated
`protein kinase; NF-κB, nuclear factor kappa B; NOD2, nucleotide-binding oligomerization domain-containing protein 2; OSM, oncostatin M; PI3K,
`phosphatidylinositol-3-kinase; STAT, signal transducer and activator of transcription; TFH, T follicular helper cell; TGFβ, transforming growth factor beta; Th, T helper;
`TLR, toll-like receptor; Treg, regulatory T cell.
`
`Frontiers in Immunology | www.frontiersin.org
`
`4
`
`May 2019 | Volume 10 | Article 1093
`
`Lassen - Exhibit 1030, p. 4
`
`

`

`West
`
`IL-6 Family Cytokines and Stromal Cells
`
`to those of IL-6. Like IL-6, LIF and IL-11 expression by stromal
`cells can be induced by IL-1β, TNF, and TGF-β (60–64). Notably,
`induction of both IL-11 and LIF in response to TGF-β stimulation
`of cancer-associated fibroblasts is thought to promote tumor
`progression (61, 62). Intriguingly, IL-4 and IL-13 were shown
`to counteract TNF and IL-1β-induced expression of LIF and
`IL-11, but not IL-6, by gingival fibroblasts (64). This effect was
`dependent on STAT6, and provides a potential mechanism for
`selective modulation of individual IL-6 family members.
`
`RESPONSIVENESS OF STROMAL CELLS
`TO IL-6 FAMILY CYTOKINES
`
`Stromal cells express the necessary receptor subunits to respond
`to the majority of gp130-dependent IL-6 family cytokines. In
`general, gp130 and OSMR are ubiquitously expressed by stromal
`cells from essentially all anatomical locations studied thus far.
`OSM is therefore a major activating factor of stromal cells,
`as well as various other mesenchymal populations including
`endothelial cells, muscle cells, adipocytes, and osteoblasts (34,
`56, 65). Expression of other ligand-specific receptor subunits
`is more variable and depends on the cell type, anatomical
`location, and physiological context. IL-6R, for example, tends
`to be expressed at relatively low levels, and stromal cells are
`correspondingly less sensitive to classical IL-6 signaling than
`OSM. Indeed, expression of OSMR mRNA in human colon
`fibroblasts is roughly 10x higher than that of IL-6R (55).
`However, inflammatory conditions that yield soluble IL-6R can
`increase stromal cell sensitivity to IL-6 due to trans signaling.
`Responsiveness of stromal cells to LIF appears to vary widely
`depending on anatomical location. For example, LIF induces
`contractile and inflammatory phenotypes in dermal and synovial
`fibroblasts, respectively, but has little effect on colon fibroblasts
`(55, 62, 63, 66). Sensitivity of stromal cells to IL-11 and IL-27
`has also been documented (67–73). In contrast, IL-31Rα does
`not seem to be expressed by most stromal cells at physiologically
`relevant levels (74, 75).
`
`CONTROL OF INFLAMMATION AND
`ADAPTIVE IMMUNITY BY THE
`IL-6-STROMA AXIS
`
`Exposure of stromal cells to factors such as microbial ligands or
`inflammatory cytokines can drive IL-6 production during both
`acute and chronic inflammation. Following infection of mice
`by Toxoplasma gondii, for example, IL-6 expression was shown
`to be elevated in a population of bone marrow stromal cells
`characterized by high VCAM-1 and low CD146 expression, and
`stroma-derived IL-6 was required for the increased myelopoiesis
`that occurs as part of the host response to infection (76).
`Bone marrow stromal cells also induce IL-6 in response to
`viral infections such as CMV (cytomegalovirus) (77). During
`Helicobacter hepaticus-driven colitis in mice, non-hematopoietic
`stromal cells are the dominant intestinal producers of IL-6, with
`expression levels that substantially exceed those of MHC-II+
`myeloid cells (55). Interestingly, IL-6 expression may be a feature
`
`of specific intestinal stromal cell subsets with distinct ontogeny
`or activation states. For example, human OSMRhigh intestinal
`stromal cells were found to be enriched in IL-6 expression
`relative to their OSMRlow counterparts (55), consistent with
`the well-established ability of OSM to induce IL-6 expression
`in mesenchymal cells (78–86). Single-cell RNA-sequencing has
`similarly revealed substantial heterogeneity in the intestinal
`stromal cell compartment. High IL-6 expression is enriched
`in a stromal cell subset that is rare in healthy individuals,
`but dramatically expanded in patients with inflammatory bowel
`disease (IBD) (87). Notably, these cells were further characterized
`by expression of a variety of additional immunostimulatory
`molecules, including IL-33 and the FRC-associated chemokines
`CCL19 and CCL21,
`implying a specialized role in immune
`regulation (87). Notably, a disease-associated single nucleotide
`polymorphism (SNP) in the human IL6 promoter was shown
`to control production of IL-6 by fibroblasts, but had no effect
`on IL-6 expression by CD14+ monocytes, suggesting that host
`genetics can also play an important role in determining stromal
`IL-6 output (88).
`Following initiation of acute inflammation, IL-6 can act on
`several cell types to shape the quality of the ensuing immune
`response. For example, IL-6 controls the balance between
`inducible regulatory T cell (Treg) and Th17 differentiation
`following activation of naïve CD4+ T cells (41). Although stromal
`cells have not conclusively been demonstrated to contribute to
`this process, FRC-derived IL-6 has been suggested to support
`the development and maintenance of B cell responses. Medullary
`FRC were shown to be important regulators of plasma cell
`homeostasis, in part by producing the plasma cell survival factor
`IL-6 (89, 90). IL-6 is also necessary for the differentiation of
`follicular helper T cells (TFH), which drive the maturation of B
`cells and the generation of protective antibody responses (91, 92).
`Importantly, IL-6 induces production of IL-21 by TFH cells,
`which is critical for both TFH maintenance and plasma cell
`differentiation in germinal centers (93, 94). Publicly available data
`provided by the ImmGen project suggest that FRC constitutively
`express IL-6, and do so at levels that far exceed those of other
`lymph node-resident cell types (95). Thus, FRC-derived IL-6 is
`likely to be a central linchpin in the regulation of both T cell and
`B cell responses in secondary lymphoid organs.
`In inflamed peripheral tissues, IL-6 controls the temporal
`switch from recruitment of granulocytes
`to preferential
`recruitment of mononuclear cells by modulating chemokine and
`cytokine production in local mesenchymal cells, including the
`suppression of TNF and IL-1β production, possibly via STAT3-
`mediated repression of NF-κB signaling (96, 97). IL-6 promotes
`the differentiation of monocytes into macrophages rather than
`dendritic cells in vitro, but genetic IL-6 deficiency does not
`affect dendritic cell frequencies in vivo (98–101). However,
`IL-6 appears to mediate alternative macrophage differentiation
`in vivo and inhibits inflammatory cytokine production and
`microbicidal activity by macrophages (102–105). IL-6 can also
`promote survival and regeneration of damaged epithelia during
`inflammatory challenges, a feature that can be subverted to
`promote cancer progression (106). Thus, while IL-6 is important
`for initiation of immune responses, it also promotes resolution
`
`Frontiers in Immunology | www.frontiersin.org
`
`5
`
`May 2019 | Volume 10 | Article 1093
`
`Lassen - Exhibit 1030, p. 5
`
`

`

`West
`
`IL-6 Family Cytokines and Stromal Cells
`
`of inflammation and tissue repair (54, 62). Notably, IL-6 protects
`mice from the lethal inflammatory effects of Staphylococcal
`enterotoxin B (SEB; a model of toxic shock), in direct contrast
`with TNF (107).
`Although IL-6 is an important regulator of physiological
`immune responses, excess or chronic IL-6 production can
`promote inflammatory or fibrotic pathology. IL-6 has been
`implicated in a variety of inflammatory diseases, but is perhaps
`best studied in the context of arthritis, a condition that can be
`effectively treated via blockade of IL-6 signaling (42). Synovial
`fibroblasts in inflamed joints are thought to be the major
`source of IL-6, which is likely produced in response to a
`variety of inflammatory factors including TNF, IL-1β, LIF, and
`CDH11 (47, 53, 63). IL-6 is necessary for pathology in pre-
`clinical models of antigen-induced and spontaneous arthritis,
`in which it orchestrates a variety of inflammatory processes
`including activation of CCL2 production by synovial fibroblasts,
`differentiation of autoinflammatory Th17 cells, and bone erosion
`via increased osteoclastogenesis (108–111). IL-6 has also been
`shown to promote CCL20 production by fibroblasts, which may
`further promote recruitment of inflammatory Th17 cells (112).
`IL-6 appears to promote arthritis primarily via trans signaling,
`likely because synovial fibroblasts and activated CD4+ T cells do
`not express sufficient IL-6R to respond to IL-6 alone (108, 109).
`Indeed, CCL2 production following IL-6 stimulation of synovial
`fibroblasts requires the presence of soluble IL-6R, or a chimeric
`IL-6/IL-6R protein known as “hyper-IL-6” (108). IL-6 has also
`been shown to mediate fibrosis in the skin, lung, and heart (113–
`116). Notably, in a phase 2 clinical trial of patients with systemic
`sclerosis, a disease characterized by skin fibrosis, treatment with
`actemra (tocilizumab; anti-IL6R) dramatically attenuated fibrotic
`behavior and transcriptional signatures in dermal fibroblasts,
`along with significant attenuation of disease severity (113).
`
`REGULATION OF INFLAMMATION AND
`HEMATOPOIESIS BY THE OSM-STROMAL
`CELL AXIS
`
`OSM is a pleiotropic cytokine with roles reported in a plethora
`of homeostatic and disease settings (34, 56, 65). Unlike IL-6,
`OSM is not generally produced at significant levels by stromal
`cells, but is instead a product of various hematopoietic cell types,
`including monocytes, macrophages, dendritic cells, neutrophils,
`eosinophils, mast cells, and T cells (34, 56, 65). OSM is further
`distinguished from IL-6 by the cellular distribution of its specific
`receptors (OSMR and LIFR in humans; OSMR in mice), which
`are largely restricted to non-hematopoietic cell types, notably
`epithelial cells, fibroblasts, endothelial cells, adipocytes, and
`neurons (34, 56, 65). OSM thus provides a means for leukocytes
`to deliver information to non-hematopoietic cells in inflamed or
`damaged tissues.
`While little is known about the role of OSM in infectious
`disease or other host-defense settings, OSM can clearly
`influence hematopoietic homeostasis. OSM is necessary
`for the maintenance of granulocyte-macrophage, erythroid,
`megakaryocyte, and multipotential hematopoietic progenitor
`
`populations in the bone marrow, an effect that likely involves
`stimulation of bone marrow stromal cells by OSM (117–120).
`The ability of OSM to drive expression of CXCL12 (SDF1) in
`stromal cells may partly explain its effects on hematopoiesis
`(119, 121–123). However, aging studies have shown that OSM-
`deficient mice develop progressive hematological defects that
`include reduced numbers of circulating leukocytes, erythrocytes,
`and platelets, along with pronounced bone marrow adiposity.
`OSM was shown to suppress adipose differentiation of murine
`PDGFRα+ Sca1+ mesenchymal stem cells, thereby preventing
`the development of “fatty” marrow and safeguarding the
`hematopoietic niche (120). Several additional studies have
`confirmed that OSM acts on stromal progenitors to suppress
`adipocyte differentiation in favor of osteoblast development
`(124–130), suggesting that OSM plays a fundamental role
`in regulating the bone marrow microenvironment. Notably,
`overexpression of OSM in bone marrow stromal cells promotes
`the development of lethal myeloproliferative neoplasms and
`bone marrow fibrosis in mice (131, 132).
`Numerous studies have implicated OSM in the pathogenesis
`of inflammatory conditions, including arthritis, inflammatory
`bowel disease, psoriasis, and allergic airway inflammation.
`Intra-articular adenoviral delivery of OSM causes arthritis-
`like pathology characterized by robust leukocyte infiltration,
`synovial hyperplasia, and erosion of bone and cartilage (133–
`135). Consistent with these findings, antibody blockade of OSM
`can reduce pathology in the collagen-induced and pristane-
`challenge pre-clinical models of inflammatory arthritis (136).
`Synovial fibroblasts respond to OSM by producing a wide
`variety of inflammatory factors including cytokines (e.g., IL-
`6), chemokines, (e.g., CCL2, CCL13, CXCL1), and leukocyte
`adhesion factors such as ICAM-1 (78, 82, 83, 134, 137–139).
`Furthermore, cytokine receptors such as IL1R1 (IL-1 receptor,
`type 1), gp130, and OSMR are induced by OSM, suggesting that
`OSM can sensitize synovial fibroblasts to additional cytokine
`stimulation. OSM can synergize with TNF and IL-1β to promote
`increased cytokine and chemokine expression, as well as high
`MMP (matrix metalloprotease) to TIMP1 (tissue inhibitor of
`metalloproteases) ratios to promote tissue damage. Remarkably,
`OSM alone drives high TIMP1 expression (134, 135, 138, 140,
`141), and OSM only promotes net tissue degradation when
`acting in synergy with TNF, IL-1β, or IL-17A, suggesting that its
`pathogenicity may depend on the presence of other inflammatory
`factors (135, 137, 138, 142).
`Emerging data suggest an important role for OSM-stromal cell
`interactions in barrier tissues, such as skin and intestinal mucosa.
`Dermal fibroblasts express extracellular matrix components such
`as collagens and glycosaminoglycans in response to OSM, and
`display an interferon-like response featuring upregulation of
`the viral RNA sensors RIG-I and MDA5 (143–146). While
`sufficient
`to induce skin inflammation, OSM may not be
`required for psoriasis-like pathology, as it is dispensable in
`the aldara (imiquimod) challenge model of psoriasis (55, 147,
`148). OSM and OSMR are also overexpressed in the lesional
`skin of patients with atopic dermatitis, but whether OSM
`signaling is required for pathogenesis of
`this condition is
`unclear (147). A potentially non-redundant inflammatory role
`
`Frontiers in I