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IMMUNOLOGY AT STANFORD UNIVERSITY
Tissue integrity signals communicated by high-
molecular weight hyaluronan and the resolution
of inflammation
S. M. Ruppert •T. R. Hawn •A. Arrigoni •
T. N. Wight •P. L. Bollyky
ÓSpringer Science+Business Media New York 2014
Abstract The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior.
Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-
like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory.
We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a
novel form of pattern recognition that recognizes intact tissues and communicates ‘‘tissue integrity signals’’ that promote
resolution of local immune responses.
Keywords Hyaluronan Danger signals DAMP Integrity signal CD44 ECM
Abbreviations
HMW-HA High-molecular weight hyaluronan
LMW-HA Low-molecular weight hyaluronan
HA Hyaluronan
PAMPs Pathogen-associated molecular patterns
DAMPs Damage-associated molecular patterns
TLR Toll-like receptor
APC Antigen-presenting cell
DC Dendritic cell
Hyaluronan and the tissue response to injury
Hyaluronan (HA) is an extracellular matrix (ECM) glycos-
aminoglycan that is abundant in the ECM of inflamed tissues.
HA is a long, non-branching disaccharide made of glucu-
ronic acid and N-acetyl-glucosamine with diverse effects on
tissue structure and function (reviewed in [1–3]).
Both the size and the amount of HA are tightly regulated
during progression through the stages of an injury
response. Immediately upon injury, local HA production
increases substantially [2,3]. The three HA synthases
responsible for this production generate predominantly
high-molecular weight HA (HMW-HA) (defined here as
[5910
5
Da) [1,4–6]. During inflammation, this HA is
rapidly catabolized by a diverse group of host and (if
infection is present) microbial hyaluronidases (HA’ases),
mechanical forces, and oxidation [7,8], resulting in frag-
mentary, low-molecular weight HA (defined here as
\200 kDa) that are cleared via CD44-mediated
endocytosis. Upon the resolution of inflammation,
both the amount and size of HA return to basal levels.
However, in chronically inflamed tissues, shorter HA
polymers predominate. In light of these associations,
HA size has been termed a natural biosensor for the
state of tissue integrity [9,10].
S. M. Ruppert A. Arrigoni P. L. Bollyky (&)
Division of Infectious Diseases, Stanford University School
of Medicine, 300 Pasteur Drive, Rm. L-133, Stanford,
CA 94305-5107, USA
e-mail: pbollyky@stanford.edu
T. R. Hawn
Division of Allergy and Infectious Diseases, University of
Washington Medical Center, 1959 NE Pacific Ave, Seattle,
WA 98195, USA
T. N. Wight
Matrix Biology Division, Benaroya Research Institute, 1201 9th
Ave, Seattle, WA 98101, USA
P. L. Bollyky
123
Immunol Res
DOI 10.1007/s12026-014-8495-2
Here, we propose that the receptors that discriminate
between HMW-HA and low-molecular weight hyaluronan
(LMW-HA) together constitute an integrated system of
pattern recognition capable of communicating the presence
of either intact or fragmented ECM and, furthermore, that
the resulting contextual cues are relevant for integrating
wound healing with the local immune response to injury.
LMW-HA-mediated danger signals
Pattern recognition allows for efficient, choreographed
responses to environmental stimuli. During infection,
pathogen-associated molecular patterns (PAMPs) such as
lipopolysaccharide (LPS), instigate rapid, programmatic
responses that engender appropriately polarized immuno-
logic responses. Endogenous markers of inflammation,
termed danger-associated molecular patterns (DAMPs),
function in an analogous manner to microbial PAMPs and
trigger many of the same receptors [11,12]. DAMPs share
with PAMPs the properties of being small, structurally
repetitive molecules. However, unlike PAMPs, DAMPs are
also present in sterile inflammation. Examples of DAMPs
include heat-shock proteins [13,14], urate crystals [4,15],
and fragmentary components of the ECM [16,17].
LMW-HA is an ECM molecule that functions as a pro-
inflammatory DAMP [3,18–24]. LMW-HA promotes the
activation and maturation of dendritic cells (DC) [1,25],
drives the release of pro-inflammatory cytokines such as
IL-1ß, TNF-a, IL-6, and IL-12 by multiple cell types [6,
26–28], drives chemokine expression and cell trafficking
[29–31], and promotes proliferation [32–34] (Fig. 1).
These signals may be particularly relevant in settings of
sterile inflammation.
Many of the pro-inflammatory effects of LMW-HA are
attributed to interactions with the pattern recognition
receptors Toll-like receptor 2 (TLR2) or Toll-like receptor
4 (TLR4). LMW-HA promotes TLR-mediated phosphor-
ylation of MAPK, nuclear translocation of NF-jB, and
TNF-aproduction (reviewed in [3,35]). While HA mole-
cules of all sizes share the same repeating disaccharide
structure, only LMW-HA can signal through TLR2 or
TLR4 [1,4,6,36]. Therefore, only products of HA
catabolism, indicative of active inflammation, promote
TLR signaling.
HMW-HA-mediated tissue integrity signals
HMW-HA predominates in healthy tissues and typically
inhibits inflammation. Specifically, HMW-HA prevents
cell growth and differentiation [7,37], diminishes the
production of inflammatory cytokines by multiple cell
types [9,38], and impairs phagocytosis by macrophages
[11,39]. Recently, HMW-HA has been implicated in the
inhibition of tumor progression [13,25]. Administration of
HMW-HA is anti-inflammatory in lung injury models [4,
40], collagen-induced arthritis [16,41], and a variety of
other in vivo model systems [18,20,22–24,42].
Most of these anti-inflammatory properties are attribut-
able to interactions of HMW-HA and CD44, the major cell-
surface HA-binding transmembrane glycoprotein. CD44 is
thought to translate cues from the ECM, including HA, into
signals that may influence growth, survival, activation, and
differentiation [25,43,44]. Consistent with this, CD44
-/-
mice are unable to efficiently resolve inflammation. Ini-
tially, this was demonstrated in a bleomycin-induced lung
injury model where CD44
-/-
mice have impaired clear-
ance of apoptotic neutrophils, persistent accumulation of
LMW-HA, and impaired activation of TGF-b1. Upon
reconstitution with CD44
?
leukocytes, the inflammation is
resolved [6,40,43]. Similar defects were subsequently
demonstrated in other injury models including bacterial
pneumonia [29,39]. Myocardial infarcts in CD44
-/-
mice
are associated with reduced collagen deposition and fewer
fibroblasts [32,45], suggesting that the loss of CD44
impairs the effectiveness and efficiency of wound repair.
In addition to these anti-inflammatory effects, CD44 is
reported to contribute to immune homeostasis via the
maintenance of type 1 helper T (Th1) memory cells.
Baaten and colleagues find that CD44 can counteract Fas-
mediated apoptosis of these cells [35,46]. These data
Fig. 1 Pro-inflammatory actions of LMW-HA and TLR signaling.
LMW-HA characterizes inflamed tissues with active matrix catabo-
lism. LMW-HA is an agonist of TLR signaling through interactions
with TLR2 and/or TLR4 and communicates ‘‘danger signals’’ to
infiltrating leukocytes. LMW-HA promotes leukocyte homing, pro-
liferation and production of pro-inflammatory cytokine production as
well as DC maturation and antigen presentation. LMW-HA also
induces turnover of CD44, potentially limiting the likelihood of CD44
cross-linking by intact ECM
Stanford Immunology
123
suggest that despite the multiple roles for CD44 in leuko-
cyte activation, survival, and costimulation, CD44 pri-
marily promotes homeostasis and the resolution, rather
than the propagation, of inflammation.
However, CD44 is not exclusively anti-inflammatory.
CD44 interacts with a large number of ECM components,
growth factors, and cytokines, and many of these factors,
including osteopontin, are pro-inflammatory [36,47].
Indeed, CD44 contributes to leukocyte trafficking [37,48]
and antibodies against CD44 that inhibit this trafficking
have been proposed as a therapy for inflammatory and
autoimmune diseases [38,49]. CD44 is also implicated in
TLR signaling in certain contexts [39,50,51]. We propose
that CD44 is anti-inflammatory in those contexts in which
it is cross-linked by HMW-HA and potentially other ECM
ligands.
How HMW-HA and CD44 inhibit inflammation
Most downstream effects of CD44-binding HA and other
ligands are mediated either via cellular re-organization (i.e.,
cytoskeleton or lipid raft architecture) or through receptor
complexes that CD44 partners with (reviewed in [25,52]).
Several such mechanisms are highlighted in Fig. 2.
HMW-HA negatively regulates pro-inflammatory TLR
signaling. Mice treated with HMW-HA prior to LPS
exposure have greatly decreased serum IL-6 and TNFa
levels and are protected from symptoms of sepsis [36,40]. In
other studies, oral administration of HMW-HA likewise
suppresses inflammation in a TLR4-dependent manner [41,
53] and actively downregulates LPS-induced NF-jB trans-
location [42,54]. Consistent with these findings, both
MyD88 expression levels and nuclear translocation of NF-
jB are increased in CD44
-/-
mice [43,44,55,56]. These
mice also have diminished levels of several negative regu-
lators of TLR signaling, including A20 and IRAK-M [35,40,
43], suggesting that CD44 could govern expression of these
molecules. In addition to inducing effects on TLR signaling
downstream, CD44 physically associates with TLR4 and co-
expressed MD-2 and facilitates the binding of LMW-HA to
this complex [39,57], presumably in the absence of HMW-
HA. Together, these data suggest that HMW-HA and CD44
modulate TLR signaling at multiple points.
CD44 cross-linking may disproportionately impact cell
types that are important for immune suppression, such as
regulatory T cells (Tregs). The suppressive capacity of
Tregs correlates with expression levels of the transcription
factor, Foxp3 [45]. Foxp3
?
Tregs are a specialized sub-
population of CD4
?
T cells that maintain immune
homeostasis via production of IL-10, an anti-inflammatory
cytokine critical for both immune regulation and for wound
healing [58]. Depletion of Tregs leads to multi-systemic
Fig. 2 Anti-inflammatory actions of HMW-HA and CD44 cross-
linking. Several mechanisms of anti-inflammatory activities propa-
gated via HA and CD44 have been reported. 1. HMW-HA and CD44
negatively regulate pro-inflammatory TLR signaling at multiple
levels. 2. CD44 is thought to play an important role in clearance of
pro-inflammatory LMW-HA. 3. HMW-HA promotes the function and
phenotypic stability of regulatory T-cell populations, including
Foxp3
?
Treg, TR1, and NKT cells. 4. CD44 cross-linking promotes
production of anti-inflammatory cytokines. 5. HA is a potent
antioxidant that limits the damage caused by free radicals generated
at sites of inflammation. HA length probably does not impact this
anti-oxidant property but is likely to contribute to its longevity. In
light of these anti-inflammatory roles, we propose that HMW-HA
functions as a tissue integrity signal that dampens inflammation at
sites of intact tissues
Stanford Immunology
123
autoimmunity in both mice and humans [46], but can be
prevented or ablated upon Treg adoptive transfer [47,48].
HMW-HA promotes the function and phenotypic sta-
bility of Tregs. Firan and colleagues were the first to
demonstrate an association between HMW-HA binding
and Foxp3 expression [49,59]. We subsequently reported
that HMW-HA enhanced the function and viability of
Tregs, particularly in low IL-2 environments [50,51,60].
Moreover, HMW-HA promotes Treg function via
increased Foxp3 expression and production of IL-10 [36,
51,52,61]. Along similar lines, we find that HMW-HA, in
the context of a TCR signal, induces conventional T cells
to produce IL-10 and behave like type 1 regulatory cells
(TR1), a subset of Tregs [36,52]. Similarly, stimulation of
CD44 through Ab cross-linking or HMW-HA causes
immunoregulatory natural killer T cells (NKT) to secrete
cytokines, up-regulate activation markers, and resist acti-
vation-induced cell death [50,51,53].
Another way CD44 may impact regulatory cell popu-
lations is through effects on antigenic responses. CD44
cross-linking is known to impact formation of TCR com-
plexes [53,54] and immune synapse function [55,56,62].
Therefore, CD44 may magnify low-level antigenic
responses and provide tonic signals to these cells. As Treg
and NKT cells both constitutively express high levels of
CD44 receptor, they are poised to respond to such signals.
Memory T cells are another group that is constitutively
CD44
high
and have impaired homeostasis in the absence of
CD44 [35,63].
CD44 cross-linking by HMW-HA as a novel form
of pattern recognition
The ability of HA to bind to CD44 is dependent upon
interactions with multiple CD44 molecules. This is because
the interaction between HA and CD44 is mediated by low-
affinity hydrogen bonds [25,57] such that interactions with
multiple receptors or increasing amounts of HA are
required for efficient binding. In a study in which HA
molecules bound to lipid nanoparticles were used to probe
a CD44-coated surface, it was demonstrated that only HA
molecules of [700 kDa could bind CD44 reliably [6,59].
HA binding is further influenced by the density of CD44
receptors on the cell surface and their relative affinity for
HA [60,64]. While the precise size cutoffs vary between
model systems, these data suggest that efficient HA binding
to CD44 is predicated upon CD44 cross-linking.
The ability of HMW-HA to cross-link spatially isolated
CD44 molecules and the inability of LMW-HA to do so
may explain how leukocytes discriminate between HMW-
HA and shorter HA polymers. Consistent with this, the
anti-inflammatory effects attributed to HMW-HA in mul-
tiple systems can be recapitulated using antibody-mediated
CD44 cross-linking [36,51,52,61,65]. We propose that
CD44 cross-linking functions as a form of pattern recog-
nition that distinguishes between tissue microenvironments
that are actively inflamed, characterized by LMW-HA, and
those that are healing or uninjured, characterized by HMW-
HA. This model does not exclude other roles for CD44 but
attributes the homeostatic and anti-inflammatory properties
of HMW-HA to CD44 cross-linking. Consistent with this
view, cross-linking is implicated in CD44-mediated effects
on IL-10 production [36,52,66], Treg homeostasis [50,51,
67], NKT cell function [53], neutrophil inhibition [62], and
cell survival [63]. CD44 interacts with multiple other cell-
surface receptors whose signaling influences leukocyte
proliferation, maturation, activation, and trafficking [25],
and we speculate that the contribution of HMW-HA and
CD44 cross-linking to these pathways will prove to pro-
mote homeostasis and the resolution of inflammation.
Of note, HMW-HA and CD44 also reduce inflammation
in ways that do not necessitate cross-linking. CD44 medi-
ates clearance of HA fragments such that, in the absence of
CD44, these fragments accumulate and drive inflammation
through interactions with TLR [6]. Further, HA acts as an
antioxidant and the greater the longevity of HMW-HA
could translate into a prolonged ability to mitigate oxida-
tive damage.
The integration of HA contextual cues
We propose a model whereby HA size-specific interactions
with TLR2/TLR4 and CD44 together constitute an inte-
grated system of pattern recognition that discriminates
between actively inflamed and healing tissues based on the
predominant size of HA. In this model, LMW-HA, typical
of actively inflamed tissues, communicates pro-inflamma-
tory ‘‘danger’’ signals via TLR signaling while HMW-HA,
typical of post-inflammatory or uninflamed tissues, com-
municates ‘‘tissue integrity’’ signals via CD44 cross-link-
ing. Together, these HA-mediated contextual signals
constitute an integrated system for sensing changes in the
inflammatory milieu and coordinating appropriate respon-
ses (Fig. 3). By ‘‘contextual signals,’’ we mean the infor-
mation gained from the immune environment, specifically
in the form of factors that engage co-stimulatory receptors.
Mechanisms must exist for the integration of these sig-
nals, given that HA molecules of disparate size are likely to
coexist in injured tissues. Since HA molecules of different
sizes may compete for binding to CD44, another variable
that might impact competitive binding interactions is the
molar predominance of LMW-HA generated from a single
HMW-HA molecule. The relative expression of HA
receptors may also be impacted by the HA molecules
themselves. For example, LMW-HA is known to induce
Stanford Immunology
123
CD44 cleavage [64], and this could make less CD44
available for cross-linking. Additionally, HA may regulate
its own catabolism; the enzymatic activity of the primary
extracellular hyaluronidase, HYAL-2, is dependent on
CD44 [65] along with other factors [66].
Other ECM components are likely to influence both HA
integrity as well as interactions with CD44. In vivo, a diverse
group of HA-binding molecules, called hyaladherins, con-
tribute to particular HA structural architectures and may also
contribute to CD44 cross-linking [67]. Hyaladherins and the
nature and extent of HA superstructures may be essential to
the anti-inflammatory properties of HMW-HA, an idea first
proposed by Day and De La Motte [68].
An example of a hyaladherin with established roles in
the cross-linking of CD44 is tumor necrosis a-stimulated
gene 6 (TSG-6). TSG-6 catalyzes the covalent transfer of
heavy chains of inter-a-inhibitor (IaI) present in serum to
HA polymers, forming complex, cross-linked HA net-
works, promoting further interactions of HA with CD44
[69]. Such structures form the basis of provisional wound
matrix, the scaffold that is a crucial early component of
healing tissue. Additionally, CD44 binds heavy chain
bound HA with greater avidity than unbound HA, pro-
moting more efficient wound healing [70]. TSG-6 prevents
HA degradation and inhibits enzymes involved in ECM
catabolism, including HA’ases [68][71]. TSG-6 levels are
diminished at sites of chronic inflammation [72,73], and,
indeed, treatment with TSG-6 has been shown to be of
benefit in some inflammatory disorders, particularly in the
context of autoimmunity [74,75]. Additionally, there are
other hyaladherins, such as pro-inflammatory versican [76],
that can inhibit HA–CD44 interactions.
Conclusions
The integration of wound healing and immune regulation is
critical for the resolution of inflammatory responses but is
poorly understood. Here, we have proposed that tissue
repair and the immune response to tissue damage are
synchronized by contextual cues that reflect the local
integrity of the ECM, in particular the size of HA. More-
over, we have proposed that the receptors that discriminate
between HA molecules of different sizes together consti-
tute an integrated system of pattern recognition. This sys-
tem of HA size-specific pattern recognition offers a
powerful model for how tissue repair and immune regu-
lation are integrated in injured and healing tissues.
This model also suggests that catabolic HA at sites of
sterile inflammation may drive autoimmunity and predicts
that it may be possible to impact HA integrity in vivo in ways
that promote wound healing and immune tolerance. Indeed,
delivery of exogenous HMW-HA is beneficial in both the
amelioration of airway inflammation [77] and in limiting
scar formation in dermatologic wounds [78]. Supplementa-
tion with TSG-6 to promote HA integrity is likewise a
strategy for immunotherapy with known benefit in autoim-
mune arthritis [74] and other inflammatory settings.
However, many questions remain. For example, are
there additional fragmentary ECM components, that are
also CD44 ligands, that function in an analogous manner to
HA? How are CD44 and TLR-mediated signals integrated?
How do other HA receptors (e.g., RHAMM, LYVE-1,
HARE) and the various sizes of HA polymers fit within this
model of pattern recognition? Do clinical preparations of
HMW-HA used to prevent abdominal adhesions (e.g., Se-
prafilm) or reduce joint inflammation (e.g., Synvisc) in fact
provide tissue integrity signals? These questions and others
warrant continued investigation into the role of HA in
immune regulation.
Acknowledgments This work was supported by National Institutes
of Health grants T32 AI07290 (to SMR); R01 DK096087-01, R01
HL113294-01A1, and U01 AI101984 (to PLB); and HL018645 and a
BIRT supplement AR037296 (to TNW). The authors declare that they
have no conflict of interest.
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