Undenatured type II collagen protects against collagen-induced arthritis by restoring gut-joint homeostasis and immunity

Abstract

Oral administration of harmless antigens can induce suppression of reactive immune responses, a process that capitalises on the ability of the gastrointestinal tract to tolerate exposure to food and commensal microbiome without triggering inflammatory responses. Repeating exposure to type II collagen induces oral tolerance and inhibits induction of arthritis, a chronic inflammatory joint condition. Although some mechanisms underlying oral tolerance are described, how dysregulation of gut immune networks impacts on inflammation of distant tissues like the joints is unclear. We used undenatured type II collagen in a prophylactic regime -7.33 mg/kg three times/week- to describe the mechanisms associated with protective oral immune-therapy (OIT) in gut and joint during experimental Collagen-Induced Arthritis (CIA). OIT reduced disease incidence to 50%, with reduced expression of IL-17 and IL-22 in the joints of asymptomatic mice. Moreover, whilst the gut tissue of arthritic mice shows substantial damage and activation of tissue-specific immune networks, oral administration of undenatured type II collagen protects against gut pathology in all mice, symptomatic and asymptomatic, rewiring IL-17/IL-22 networks. Furthermore, gut fucosylation and microbiome composition were also modulated. These results corroborate the relevance of the gut-joint axis in arthritis, showing novel regulatory mechanisms linked to therapeutic OIT in joint disease.

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communications biology Article
https://doi.org/10.1038/s42003-024-06476-z
Undenatured type II collagen protects
against collagen-induced arthritis by
restoring gut-joint homeostasis and
immunity
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Piaopiao Pan1,YilinWang
2, Mukanthu H. Nyirenda3,5, Zainulabedin Saiyed4, Elnaz Karimian Azari4,
Amy Sunderman4, Simon Milling3,MargaretM.Harnett
3& Miguel Pineda 1
Oral administration of harmless antigens can induce suppression of reactive immune responses, a
process that capitalises on the ability of the gastrointestinal tract to tolerate exposure to food and
commensal microbiome without triggering inflammatory responses. Repeating exposure to type II
collagen induces oral tolerance and inhibits induction of arthritis, a chronic inflammatory joint
condition. Although some mechanisms underlying oral tolerance are described, how dysregulation of
gut immune networks impacts on inflammation of distant tissues like the joints is unclear. We used
undenatured type II collagen in a prophylactic regime -7.33 mg/kg three times/week- to describe the
mechanisms associated with protective oral immune-therapy (OIT) in gut and joint during experimental
Collagen-Induced Arthritis (CIA). OIT reduced disease incidence to 50%, with reduced expression of
IL-17 and IL-22 in the joints of asymptomatic mice. Moreover, whilst the gut tissue of arthritic mice
shows substantial damage and activation of tissue-specific immune networks, oral administration of
undenatured type II collagen protects against gut pathology in all mice, symptomatic and
asymptomatic, rewiring IL-17/IL-22 networks. Furthermore, gut fucosylation and microbiome
composition were also modulated. These results corroborate the relevance of the gut-joint axis in
arthritis, showing novel regulatory mechanisms linked to therapeutic OIT in joint disease.
Rheumatoid arthritis (RA) is a chronic, autoimmune inflammatory con-
dition affecting primarily the synovial joints, although inflammation can
affect other tissues like the skin, eye, heart, lung, kidney and gastrointestinal
systems1,2. Despite advancements in immunosuppressive treatments,
approximately 20–30% of patients still do not respond to them, and a clear
increase of incidence rates in the global ageing population is described3.
Thus, new therapeutic interventions are needed, but the aetiology of RA is
not yet completely understood, and most disease triggers are still uni-
dentified. Although genetic factors are involved, it is known that environ-
mental effects can play a key role in disease initiation and progression. For
example, smoking can induce post-translational modifications leading to
major histocompatibility complex (MHC) presentation of modified self-
proteins to T cells. As a result, autoimmune responses are initiated,
including the production of self-reactive antibodies against immunoglo-
bulin G (Rheumatoid Factor), antibodies to citrullinated protein antigens
(ACPAs)4,5or the cartilage component collagen type II (C-II)6. Interestingly,
and contrary to the heterogenous pool of antigens recognised by ACPAs or
Rheumatoid Factor, type II collagen, being a single molecule, constitutes a
good target for clinical interventions based on oral tolerance mechanisms.
Experimentally, responses against collagen can be studied in vivo using the
collagen-induced arthritis (CIA) model7, in which intradermal injections of
type II collagen and Freud’s adjuvant break tolerance and initiate inflam-
matory responses in the joint. Data generated with the CIA model have
shown that repeated oral collagen administration stimulates systemic
1Centre for the Cellular Microenvironment, School of Molecular Biology, University of Glasgow, Glasgow, UK. 2Department of Bacteriology and Immunology,
Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, China. 3Institute of Infection and Immunity,
University of Glasgow, Glasgow, UK. 4Research and Development, Lonza Greenwood LLC, North Emerald Road, Greenwood, SC, USA.
5
Present address: Institute
of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
e-mail: miguel.pineda@glasgow.ac.uk
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tolerance8–10. Various underpinning mechanisms have been described
including expansion of tolerogenic dendritic cells in the gut-associated
lymphoid tissue (GALT), with subsequent induction of antigen-specific
Tregs, IL-10 and TGFβresponses9,11,12 and suppression of inflammatory IL-
17 pathways13. These positive results in pre-clinical studies encouraged
further development of collagen-based formulations. In this study, we used
undenatured type II collagen, a form of collagen isolated from the cartilage
of chicken sternum that preserves the physiological structure of collagen
fibres14. Unlike the production of denatured collagen molecules, the unde-
natured type II collagen manufacturing process maintains the intact gly-
cosylation and protein tertiary structure15 that is required to shape the
protein’s characteristic triple helix16. The preservation of this structure is key
because it determines the three-dimensional epitopes presented to immune
cells in the Peyer’s Patches of the gut17. Furthermore, by resisting the
hydrolytic action of human gastric fluid15, the preservation of triple helix
domains allows a more efficient presentation of epitopes and subsequent
regulatory responses. Supporting these findings, some studies indicated that
undenatured type II collagen reduces pain and joint stiffness in osteoar-
thritis, both in experimental models and patients18–20. Mechanistically, oral
collagen administration promotes regulatory T cell function in the gut tis-
sue, increasing IL-4, IL-10 and TGFβlevels20, whilst down-regulating
inflammatory cytokines (IL-2, TNF, IL-6 and IL-1) and metabolites, like β-
hydroxybutyrate21–23. Thus, undenatured type II collagen has also shown
efficacy in ameliorating pain in joint disease15,althoughtheprecise
immunological basis of protection remains unclear. Perhaps surprisingly,
less attention has been given to the structural aspects of the gastrointestinal
regions where the described effector mechanisms take place. Recent
research indicates that maintenance of the local gut tissue architecture and
microenvironmental cues are critical for correct immunological dis-
crimination and homoeostatic resolution of inflammation. Loss of mucosal
barrier function in the gut has been implicated in the aetiology of arthritis24,25
suggesting that restoration of the gut barrier integrity could be exploited
therapeutically. Moreover, it has also been postulated that RA could initiate
at mucosal sites before transitioning to more distant synovial joints26.Thus,
a better understanding of the microenvironmental changes in gut mucosal
sites in health and disease will allow us to understand and ultimately utilise,
induction of oral tolerance.
In this study, we use the murine model of CIA to further characterise (i)
immune mechanisms activated upon administration of prophylactic doses
ofundenaturedtypeIIcollageninmice,and(ii)functionallyrelevant
structural changes of the gut mucosal sites associated with undenatured type
II collagen protection against CIA. Using this system as a model of oral
immune therapy (OIT), we show that mice treated with undenatured type II
collagen present a significant reduction in disease incidence, which is
associated with the regulation of IL-17 and IL-22 cytokines in the joint and
rewiring of cellular and cytokine networks in the gut. Specifically, OIT was
associated with the protection of gut villi and crypt structure and the
maintenance of local levels of fucosylation. In describing the local gut
responses by which OIT acts to suppress inflammatory responses, these
findings increase our fundamental understanding of the role(s) of the gut in
autoimmunity and will support the development of alternative areas for
clinical intervention and prevention in RA.
Results
Undenatured type II collagen protects against experimental
arthritis
We chose the CIA model to study the effects of oral administration of
undenatured collagen in arthritis progression. We defined three experi-
mental groups: (i) non-treated animals (Naïve), (ii) mice undergoing
experimental arthritis (CIA) and (iii) mice undergoing arthritis with pro-
phylactic undenatured type II collagen oral immunotherapy (OIT). OIT
started 2 weeks prior to CIA induction, and oral gavage of undenatured type
II collagen was applied 3 times per week. All mice in the CIA group
developed symptoms. Monitoring of disease progression showed that OIT
suppressed joint inflammation as evidenced by the significant reduction in
disease scores and incidence (Fig. 1a), showing that ~50% of the mice did not
show any clinical symptoms. To evaluate whether OIT impacted pre-
dominantly on disease incidence, or it was influencing both incidence and
severity of disease, we divided OIT mice into symptomatic and asympto-
matic groups to measure paw swelling and relative weight change (Fig. 1b).
OIT symptomatic mice were not significantly different to CIA controls, in
terms of paw swelling and weight loss, whilst OIT asymptomatic mice
showed similar values to healthy naïve controls. Values for individual mice
are represented in Supplementary Fig. 1.
Corroborating the clinical results, histopathological analysis of CIA
joints showed high numbers of infiltrating immune cells, causing bone
damage and severe loss of cartilage that was absent in those OIT mice that
were asymptomatic (Fig. 1c). Quantification of these disease indicators
showed that asymptomatic OIT mice were completely protected against
cartilage and bone damage in all cases, although they presented low levels of
cell infiltration and pannus formation in some cases (Fig. 1d). Symptomatic
OIT mice showed higher percentages of mice with no pathological signs for
cell infiltration, cartilage damage and bone damage, although the results
were not statistically significant and overall, scores were similar to those
observed in the CIA control group (Fig. 1d).
OIT inhibits systemic inflammatory and cellular pathways
Disease scores and histology demonstrated the therapeutic effect of unde-
natured type II collagen, protecting around 50% of the CIA mice from
developing symptomatic arthritis. To gain insight into the protective
mechanisms, we first evaluated the effect on pathogenic humoral responses
as determined by the levels of IgG1 and IgG2a anti-CII antibodies27.As
expected, all mice undergoing CIA had increased levels of anti-collagen
antibodies in serum, with OIT mice exhibiting slightly lower anti-CII IgG1
but similar IgG2a levels compared to CIA (Fig. 2a). Nevertheless, both
symptomatic and asymptomatic mice generated high levels of anti-CII
antibodies, suggesting that the inhibition of humoral responses was not a
pivotal factor in preventing arthritis development in our model.
Hence, we next assessed cellular immunity, working under the
hypothesis that cellular immune responses were rewired in protected OIT
mice. First, we counted the total number of cells in joint-draining lymph
nodes (DLN), i.e., axillary, brachial and popliteal. As expected, CIA mice had
asignificantly higher number of cells compared to naïve, whereas the
numbers in naïve and asymptomatic OIT were not significantly different
and symptomatic OIT mice resembled CIA controls (Fig. 2b). Further
analysis of distinct immune cell populations by flow cytometry showed that
this trend was conserved in B and T cells, including CD4+and CD8+subsets,
as all these lymphocyte groups in asymptomatic OIT were not significantly
different from the levels found in naïve mice (Fig. 2c).
Since regulatory T cells (Tregs) have been extensively implicated in the
establishment of tolerance, we evaluated the levels of CD3+CD25+FoxP3+
Tregs in the DLNs of asymptomatic OIT mice compared to those of the
naïve and CIA groups (Supplementary Fig. 2a). We also evaluated CD39
and CD73 expression as functional indicators of their effector suppressive
capacity, since these markers are regardedasimmunologicalswitchesthat
shift ATP-driven pro-inflammatory immune cell activity towards an anti-
inflammatory state mediated by adenosine28 (Supplementary Fig. 2b, c). We
did not observe any expansion of Tregs in asymptomatic OIT mice at the full
day (day 33), suggesting that (systemic) Treg-mediated mechanisms were
not responsible for the observed undenatured type II collage n protection
against CIA during this late stage. Although we cannot rule out that Tregs
play a role in protection during pre-clinical disease stages, we hypothesised
that protection in CIA-OIT mice was associated with a reduction in pro-
inflammatory cytokines. Specifically, we investigated the expression of IL-
17, a highly pathogenic factor in CIA29,andIL-22,acytokinewereportedto
promote the development of joint disease30–33.Toconfirm the pathogenic
role of IL-17 and IL-22 in the CIA joint, we first collated mice from all groups
to directly compare their clinical scores with IL-17 and IL-22 expression in
CD4+T cells (Fig. 3a) and B cells (Fig. 3b), analysed by flow cytometry.
Results indicate that levels of IL-17+and IL-22+CD4 T cells significantly
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correlate with disease severity, regardless of treatment. The impact of B cell-
derived IL-17 and IL-22 production on pathology was diminished com-
pared to that of CD4+T cells, corroborating the leading role of Th17 cells in
progressing joint inflammation. To corroborate these findings, we next
analysed IL-17 and IL-22 expression in the joint tissue of CIA and
asymptomatic OIT mice by immunofluorescence (Fig. 3c). In line with our
previous results, non-inflamed tissue from naive mice exhibited minimal
cytokine expression, whereas effective OIT mitigated the heightened
expression observed in CIA, with a significant reduction in IL-22 (Fig. 3c).
OIT also reduced production of IL-17 by DLN cells from OIT mice,
including both symptomatic and asymptomatic animals, compared with
their CIA counterparts upon TPA/Ionomycin stimulation in vitro (Fig. 3d).
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Fig. 1 | Disease scores and histological analysis in response to oral immu-
notherapy (OIT) in CIA mice. a Disease scores (left panel) and incidence (right
panel) were evaluated at the indicated time points for naïve (grey), CIA (red) and
CIA-OIT (blue). OIT consisted of administration of undenatured type II collagen by
oral gavage 3 times a week, starting 2 weeks before induction of arthritis. Cumulative
disease scores were given for each limb, 0 =no disease and 4 = highest score. Each
dot represents the mean of individual mice and error bars show SEM (n= 15 for CIA,
n= 16 for OIT groups, n= 9 for naïve mice, data from 2 independent experiments).
Statistical significance was determined by one-way ANOVA; *p< 0.05, ***p< 0.001
between CIA and naïve, #p< 0.05, ##p< 0.01 comparing CIA and OIT groups. Dis-
ease incidence was calculated by dividing the number of cases by the total number of
mice in the group at the indicated times. bPaw width (left panel) and weight change
(percentage of initial weight, right panel) were evaluated at the indicated time points.
Each dot represents the mean of individual mice, error bars show SEM (n= 15 for
CIA, n= 7 for OIT asymptomatic, n= 9 for OIT symptomatic, n= 9 for naïve mice,
data from 2 independent experiments). Statistical significance was determined by
one-way ANOVA; *p< 0.05, ***p< 0.01,***p< 0.001, ns = non-significant. cHind
paws were collected at the end of the experiment (day 34) for each mouse, sectioned,
and stained with haematoxylin and eosin for histological analysis. Images show one
representative mouse (Disease score = median for each group). Superimposed
dotted lines show bone limits; scale bar = 500 μm. dCell infiltration, pannus for-
mation, cartilage and bone damage were quantified for individual mice in hema-
toxilin/eosin-stained sections. Dot plots show histological scores (from 0 to 4,
0 = healthy tissue, 4 = highest possible score). Individual dots represent individual
paws (n= 32 for CIA, n= 8 for OIT asymptomatic, and n= 24 for OIT symptomatic,
data collected from 2 independent experiments). Bars show the mean value for each
group. Statistical significance was determined by the Kruskal–Wallis test; **p< 0.01,
***p< 0.001. The percentage of mice with no sign of pathology (green) for the
indicated parameter is shown in vertical column graphs.
Fig. 2 | Cellular and humoral responses in response to oral immunotherapy (OIT)
in the joint. a Anti type II collagen antibodies, IgG1 and IgG2 isotypes, were eval-
uated by ELISA in serum from naïve control mice (n= 8), CIA (n= 7, mean of
disease score 4.86) and OIT mice (n= 7 for OIT symptomatic, mean of disease
scores = 7; n= 4 for OIT asymptomatic) at day 33 after induction of arthritis. Values
represent Optical Density (OD) at 450 nm. Each dot represents the mean values of
individual mice analysed in technical triplicates. bTotal cell numbers isolated from
draining lymph nodes (DLNs) collected from all naïve, CIA, asymptomatic and
symptomatic OIT mice. cTotal number of B cells, total T cells, CD4 and CD8 T cells
in DLNs from (b) were evaluated by Flow Cytometry. Each dot in a–ccolumn bars
represents values of individual mice collated from 2 independent experiments. Error
bars show mean ± SEM. Statistical significance was determined using ordinary one-
way ANOVA. Significance is indicated by asterisks, *p< 0.05, **p< 0.01
and ***p< 0.001.
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Following this, we conducted flow cytometry (Supplementary Fig. 3 for
gating strategy) to investigate intracellular IL-17 and IL-22 expression in all
DLN cells (Fig. 3e), categorising OIT mice into symptomatic and asymp-
tomatic groups as before. Mice in theOITgroupthatremainedasympto-
matic exhibited markedly reduced expression levels of both IL-17 and IL-22
producers. Symptomatic mice displayed immune profiles that resembled
those of arthritic mice, although they still showed a reduction, albeit not
significant, in IL-17 producers. We further analysed which IL-17- and IL-
22-producing DLNs were modulated in response to OIT. CD4+and CD8+
T cells, and CD19+B cells data (cell frequency and numbers) were collated
and normalised in radar charts for visualisation (Fig. 3f, raw data shown in
Supplementary Fig. 4). This revealed that whilst IL-17+and IL-22+CD4 and
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CD8 T cells were significantly reduced, B cell-dependent production of IL-
17 and, to a lesser extent, IL-22 were less affected in asymptomatic OIT
relative to CIA mice. Moreover, an intriguing response was observed in
asymptomatic OIT mice, which exhibited a significantly higher frequency of
IL-17+B cells (Fig. 3f, Supplementary Fig. 4).
Oral administration of undenatured type II collagen protects
against damage to gut tissue in CIA
The establishment of oral tolerance by feeding of antigens is a direct con-
sequence of specific immune responses triggered in the gastrointestinal tract
and gut-associated lymphoid tissue (GALT), which can lead to loss of
mucosal barrier function in RA, suggesting that gut tissue architecture can
modulate gut immunity and host-microbiome interactions25. Our previous
work showed that gut pathology precedes and perpetuates chronic systemic
inflammation driving autoimmunity and joint damage in CIA24. Therefore,
we evaluated the integrity of the gastrointestinal tract (duodenum, jejunum,
ileum and colon) in our experimental OIT model (Fig. 4a). CIA mice
showed substantial damage in all gut areas, such as disruption of the epi-
thelial layer, cell infiltration and thickening of the muscular layer (Fig. 4b).
We quantified the ratio of villi to crypt length in the gut to compare the
health and function of the intestinal mucosa. CIA mice exhibited a
diminished villi-to-crypt ratio, reminiscent of findings in inflammatory gut
conditions. This alteration was rectified in the duodenum of asymptomatic
OIT mice, with a similar trend in the jejunum and ileum. This effect was not
observed in the colon. Interestingly, symptomatic OIT mice, characterised
by joint pathology, also exhibited an absence of gut damage or pathology.
Indeed, symptomatic OIT tended to exhibit even higher villi-to-crypt ratios
than healthy mice, particularly in the distal areas of the small intestine such
as the ileum but not in the colon (Fig. 4b). Given the persistently reduced
villi-to-crypt ratio observed in the colon of all OIT mice, we conducted
Periodic Acid-Schiff (PAS) staining to visualise further tissue damage
associated to redistribution or changes in glycogens and mucins. PAS
staining revealed attachment/effacement lesions in the colon of arthritic
mice (Fig. 4c), in line with previous reports24,25. However, such lesions were
absent in asymptomatic OIT mice but not in those showing joint symp-
toms (Fig. 4c).
Finally, to assess how the safeguarding of gut tissue in asymptomatic
OIT mice translated into gut immune responses, we examined the total
number of cells in mesenteric lymph nodes (MLNs), as well as those of B
cells and CD4+and CD8+T cells. We did not observe any significant
difference in the number of DLN cells amongst these experimental groups.
Nonetheless, both asymptomatic and symptomatic OIT mice exhibited an
expansion of B cells in mesenteric lymph nodes (MLNs), not only in
comparison to the CIA group but also in relation to the naive controls, albeit
statistical significance was attained only for the symptomatic group
(Fig. 4d). In contrast, the numbers of CD4+and CD8+TcellsinMLNs
remained unaltered in both cases.
Overall, our results indicate that OIT modulates gut immunity and
triggers protective mechanisms to preserve gut integrity, even in those mice
who ultimately develop joint pathology. Hence, our next objective was to
conduct a comprehensive characterisation of the local immunological
pathways responsible for protection. We, therefore, worked with asymp-
tomatic OIT mice, a cohort demonstrating neither joint nor gut pathology.
We used cells from MLNs and cells isolated directly from the gut, following
enzymatic tissue digestion (Gating strategy shown in Supplementary Fig. 5).
In line with our experiments in the joint tissue (Fig. 3), we investigated IL-17
expression by flow cytometry in the mesenteric lymph nodes (MLNs) and
ileum and colon tissue (Fig. 5), to assess correlations between gut cellular
networks with distant responses within the joint. Contrary to the DLN data
(Fig. 3), the levels of IL-17 producers in total MLN cells (Fig. 5a), ileum
(Fig. 5b) or colon (Fig. 5c) tissue were not significantly elevated in CIA mice.
Perhaps surprisingly, there was a trend towards reduced levels of IL-17+cells
in the ileum, although there tended to be higher levels of these cells in the
colon of CIA mice, inverse patterns that were reversed in asymptomatic OIT
mice (Fig. 5b, c). However, analysis of specific cell types revealed a distinct
rewiring of the IL-17-producing networks associated with healthy animals,
in both the CIA and asymptomatic OIT groups, in each of the MLNs
(Fig. 5d), ileum (Fig. 5e) and colon (Fig. 5f) tissue. Overall, a broad analysis
indicates that in healthy conditions, IL-17 is generally produced by innate
cells (NKT, ILC3 and γδ T cells), whilst in CIA this predominantly switches
to adaptive CD4/CD8 cell responses in MLNs, and expansion of more
specialised cell types in the gut tissue, with OIT showing a mixed pattern
distinct to both naive and CIA networks (Raw data from radar charts are
shown in Supplementary Fig. 6). Intriguingly, OIT protection tended to
correlate with increased levels of IL-17+producers in the ileum, although the
results did not reach statistical significance. Moreover, their production of
IL-17 showed a general increase in the OIT group when assessing the mean
fluorescence intensity in all cell types.
We conducted a similar approach to evaluate IL-22 expression in
MLNs and gut tissue (Fig. 6), bearing in mind that IL-22 exerts key mucosal
healing mechanisms, promoting epithelial regeneration and integrity,
mucus production and synthesis of antimicrobial peptides34–36.Wedidnot
observe any significant differences among groups in total IL-22+MLNs
(Fig. 6a), or in the cells isolated from the ileum (Fig. 6b) or colon (Fig. 6c).
Nevertheless, and in line with the previous results shown for IL-17, flow
cytometric analysis of the distinct populations of producers suggests that IL-
22 networks are actively rewired in asymptomatic OIT mice compared to
CIA and naïve controls. Thus, production of IL-22 in naïve MLNs comes
mostly from B cells and innate ILC3 and NKT cells whilst in CIA mice it
extends to CD4+T cells, NKTs and γδ T cells, the latter being the only
enhanced populations in asymptomatic OIT (Fig. 6d). Analysis of IL-22
mean fluorescence intensity revealed a generalised increase in IL-22 pro-
ductioninMLNsduringCIA,whereasthiswasreducedbothintheileum
(Fig. 6e) and colon (Fig. 6f), thereby corroborating the protective role of
local IL-22 in the gut tissue. In fact, and in contrast to the IL-22 results in
MLNs, CIA mice did not increase IL-22 production, whilst all innate
populations located in the gastrointestinal tract increased the expression
of IL-22 in asymptomatic OIT mice, as measured by mean fluorescence
intensity (MFI) (Fig. 6e, f) (Raw data from radar charts are shown in
Supplementary Fig. 7). Although we cannot unequivocally define intestinal
Fig. 3 | OIT protection is associated with reduced upregulation of inflammatory
IL-17 and IL-22 in the joint and draining lymph nodes. Naïve, CIA and CIA-OIT
mice were culled at day 33 when tissue was collected for further analysis.
a,bCorrelation between numbers of IL-17 and IL-22 positive lymph node cells and
clinical scores in T cells (a) and B cells (b) isolated from draining lymph nodes
(DLNs). Every dot represents values for individual mice from all groups. Data are
presented as mean ± SEM, r: Pearson’s coefficient. cExpression of IL-17 and IL-22
(red) was evaluated in the joint tissue by immunofluorescence in naïve, CIA and OIT
asymptomatic mice. DAPI (Blue) was used to stain nuclei as counterstaining.
Superimposed dotted lines show bone tissue and areas of cell infiltration are indi-
cated by white arrows. Scale bars: 500 μm. Graphs show the quantification of the
mean intensity of individual mice. dIL-17 concentration was evaluated by ELISA in
the supernatants of draining lymph node cells upon PMA (50 ng/ml)/Ionomycin
(500 ng/ml) stimulation for 12 h. Data show naïve, CIA and OIT (symptomatic and
asymptomatic). Each dot represents cells from one individual mouse. Error bars
show mean ± SEM; *p< 0.05, **p< 0.01 analysed by one-way ANOVA from one
experimental model. eRelative frequency and total cell number of IL-17+and IL-
22+DLN cells were evaluated by flow cytometry. Data show mean ± SEM; each dot
represents individual mice from two independent experimental models; *p< 0.05,
analysed by one-way ANOVA. fCell frequency and total cell numbers of IL-17+and
IL-22+CD4 T cells, CD8 T cells and B cells in DLNs, represented at the corners of
radar charts: Naïve (grey), CIA (red) and OIT asymptomatic (orange) and OIT
symptomatic (blue); data were normalised to maximum expression in each group.
Significance on the raw data among groups was evaluated by ordinary one-way
ANOVA, where *p< 0.05, **p< 0.01 [CIA vs naïve]; $p< 0.05 [naïve vs OIT
symptomatic]; ▲p< 0.05 [naïve vs OIT asymptomatic]; +p< 0.05 [CIA vs OIT
asymptomatic]; §p< 0.05 [CIA vs OIT asymptomatic]; #p< 0.05 [OIT asymptoma tic
vs OIT symptomatic].
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IL-22 as a protective or pathogenic factor, our results highlight that differ-
ential rewiring of the IL-22+cell network in the gut is associated with
inflammatory or homoeostatic conditions, with a general expansion of
IL-22+innate populations and significant increase in IL-22 production by
NKT cells in asymptomatic OIT mice. Finally, IL-22 staining in gut tissue
provided further support for the protective role of local gut IL-22 expression
in OIT mice, as there was a significant increase in IL-22 secretion/deposition
inthetissueepitheliuminthecolonof asymptomatic OIT mice compared to
that CIA mice (Fig. 6g, h). Interestingly, increased staining of IL-22 was also
observed in symptomatic OIT mice (Fig. 6g, h), perhaps explaining why the
gut of symptomatic OIT mice also exhibited protected tissue integrity
(Fig. 4b, c).
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Gut fucosylation and microbiome composition are reshaped in
OIT and arthritic mice
Since our results suggest that intestinal IL-22 networks correlate with
effective OIT, we next investigated potential protective mechanisms trig-
gered by this cytokine in asymptomatic OIT mice. In health, IL-22 not only
supports homeostatic expression of mucins in epithelial cells37,butalsotheir
fucosylation stage38. Fucosylation is a specific type of post-translational
glycosylation, that prevents infections and enhances the integrity of the
epithelial gut layer39,40. Therefore, because asymptomatic OIT mice pre-
served gut tissue integrity and had enhanced gut IL-22 expression, we first
hypothesised that such protection was associated with increased epithelial
fucosylation, which would, in turn, prevent gut damage during inflamma-
tion. To investigate this, we stained the tissue with two lectins that recognise
fucosylated glycans, Ulex european Agglutinin (UEA) and Auleria aureate
lectin (AAL), that bind to terminal and core fucosylation respectively.
Consistent with the protective role proposed for terminal fucosylation, UEA
binding (recognising terminal alpha[1,2] linked fucose residues) was sig-
nificantly reduced in the ileum of CIA mice, while asymptomatic OIT mice
more resembled the profile of healthy tissue, as they were not significantly
different to the Naïve group (Fig. 7a). By contrast, no significant difference
was seen in the colon across the groups in this model (Fig. 7a). Moreover,
neither the ileum nor the colon, showed significant differences in core
fucosylation as detected by AAL binding (Fig. 7b). Next, we evaluated
mRNA expression of fucosyltransferases (FUTs) in whole gut tissue,
enzymes responsible for fucosylated glycan biosynthesis. In the ileum, only
expression of FUT8 mRNA was significantly different inasymptomatic OIT
mice (Fig. 7c), whereas no significant changes were seen in the colon
(Fig. 7d). These results in FUT expression do not explain the observed
maintenance of fucosylation in OIT mice compared to the reduced levels in
CIA mice (Fig. 7a), suggesting that other factors may be involved. This is not
completely unexpected since gut epithelial fucosylation is strongly depen-
dent on environmental factors, such as microbiome composition, which is
also regulated by cytokine-dependent mechanisms41. Dysregulation of some
microorganisms in the gut has been linked to RA pathogenesis and mor-
bidity in multiple studies, perhaps through subsequent variation of intestinal
metabolites that promote inflammation in the target tissue42,43. Therefore, an
alternative hypothesis is that the rewiring of IL-17/IL22 immune networks
upon undenatured type II collagen administration modifies the composi-
tion of the gut microbiome, or vice versa, which in turn, can affect mucin
fucosylation consolidating dysregulation of the microbiome to perpetuate
systemic inflammatory response. To provide support for this hypothesis, we
conducted 16 S amplicon sequencing to investigate the microbial diversity
in the ileum (Fig. 8)andcolon(Fig.9), using faecal samples from naïve, CIA
and OIT mice. As before, OIT mice were separated into symptomatic
(disease scores 5 ± 0.6, n= 3) and asymptomatic mice. We also separated
CIA mice into established disease with high scores (9.3 ± 2.8, n=3), and
mice with more recently initiated joint inflammation and lower disease
scores (3.6 ± 0.22, n= 3), to identify changes in microbial content led by OIT
and not a reduced inflammatory environment. To understand the microbial
community diversity in the samples (Within-community) we looked at α-
diversity indexes Dominance, Simpson, Observed_otus, Shannon and
Chao1 (Fig. 8a). The ileum microbiome of OIT asymptomatic group
showed significant differences in the Dominance (higher) and Simpson
index (lower), suggesting a microbial community that is characterised by
lower diversity, skewed abundance distribution, and less evenness across
different taxa. Differences in β-diversity visualised by the UniFrac distance
suggest that the microbial communities in the OIT asymptomatic mice have
distinct compositions (Fig. 8b). The composition and relative abundance of
the ileum microbiota at the phylum level were examined (Fig. 8c). Firmi-
cutes was the dominant phylum in all groups, followed by Bacteroidetes
(Bacteroidota). CIA mice at earlier joint disease stages (CIA low) exhibited a
fivefold reduction in the proportion of the Bacteroidota, a loss that was
absent in both symptomatic and asymptomatic OIT mice. Perhaps unex-
pectedly, CIA mice exhibiting more established, high-score arthritis, rather
than the more recently developed low-score arthritic mice, displayed
microbiota profiles and indexes closer to those of naïve mice. This is likely
related to the appearance of self-resolving mechanisms described in the CIA
model that can occur around two weeks after joint disease onset such as TGF
production44, which can affect the microbiome composition, particularly the
Bacteroidetes45, thereby suggesting that CIA mice with lower scores due to
them being at earlier stages of disease may provide a better reference for
identification of pathogenic microbiome changes. Compared to sympto-
matic mice, the asymptomatic OIT group increased the diversity of bacteria
in the colon, expanding the Campylobacteria and Proteobacteria phyla.
Moreover, the analysis of the top 35 genera in abundance (Fig. 8d) revealed
that whilst the Lachnospiraceae,Mucispirillium,andAnaerotruncus showed
a consistent reduction in the ileum of asymptomatic OIT mice, Mur-
ibaculaceae species were increased (Fig. 8e).
A similar analysis was carried out on the colon faecal material, speci-
fically showing alpha diversity indexes (Fig. 9a), beta diversity by UniFrac
distance (Fig. 9b), and relative abundances at the phyla (Fig. 9c) and genera
(Fig. 9d, e) levels. Contrary to the ileum, the colon microbiome of OIT
asymptomatic mice presents a lower dominance index and a higher
Simpson index compared to symptomatic OIT and arthritic mice with lower
disease scores (Fig. 9a), suggesting a more diverse and even population that
is not strongly dominated by a few specific taxa in asymptomatic OIT.
Consistent with previous reports, analysis at the phyla level reveals higher
diversity in the colon than in the ileum in naïve mice. Firmicutes remain the
most abundant phylum, but there is an increased proportion of Bacter-
oidetes and Deferribacteriota. OIT asymptomatic mice showed a more
diverse profile overall but exhibited a reduction in the Bacteoridetes and
Patesciabacteria phyla relative to the CIA low-score arthritic mice (Fig. 9c).
At the genus level, the abundance of the top 35 genera present changed
between asymptomatic OIT mice and the rest of the groups (Fig. 9d).
Clostridia_vadinBB60 were reduced, whereas others like Colidextribacter,
Roseburia, Rikenella,andRuminococcaceae were increased, relative to the
CIA-low score group (Fig. 9e). Critically, the distinct profiles of healthy
(Naïve) and OIT asymptomatic mice suggest that rather than simply pre-
venting CIA-induced changes in the microbial species, OIT rewires the
interactive network between the gut immunoregulatory pathways and
commensal bacteria to a phenotype that protects against arthritis. Such a
network is likely to be bidirectional, with bacterial metabolites further
adjusting host immunity. To gain some insight into the potential biological
activities of microbial communities without directly measuring gene
expression or protein function, we conducted some metagenomic function
prediction based on the 16 S sequencing using the bioinformatic tool
PICRUSt246. This analysis indicated differences among naïve, CIA and OIT
groups, particularly in the ileum of OIT asymptomatic mice
Fig. 4 | OIT protects against gastrointestinal damage associated with inflam-
matory arthritis in both symptomatic and asymptomatic cases. Naïve, CIA and
OIT asymptomatic mice and OIT symptomatic mice were culled at day 33 when gut
tissue and total mesenteric lymph nodes (MLNs) were collected. aIsolated gastro-
intestinal tract from a naïve mouse showing the four anatomical areas used for
further study. bDuodenum, jejunum, ileum, and colon were fixed, and tissue sec-
tions were subjected to hematoxylin and eosin staining. The length of villi and crypts
was measured using Image J software, and the ratio of villi/crypt was quantified. Each
dot represents the villi/crypt ratio for individual mice, and data were collated from
two independent experiments. Statistical significance was evaluated by ordinary
one-way ANOVA, where *p< 0.05, **p< 0.01 and ***p< 0.001. cColon tissue
sections were stained with PAS to detect changes in the mucus layer and associated
pathology. Scale bars = 500 μm. The depicted mice are representative of individual
mice whose disease scores fall within the median value for each group. dMLNs were
collected to generate single-cell suspensions, and a total number of cells was counted.
Cell number and percentage of B cells, total T cells, CD4 and CD8 T cells were
evaluated by Flow Cytometry. Each dot represents one individual mouse and error
bars show mean ± SEM. Mice were pooled from 2 independent experiments. Sta-
tistical significance was determined using Ordinary one-way ANOVA. Significance
is indicated by asterisks, *p< 0.05, **p< 0.01.
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(Supplementary Fig. 8a), but also in the colon (Supplementary Fig. 8b).
Although further experimental work is required to demonstrate this, this
data suggest that effective OIT could impact of the functional host-
microbiome crosstalk during chronic arthritis.
Discussion
Oral tolerance mechanisms have been the subject of investigation since the
beginning of the 20th century, but translation of these findings into a clinical
setting has not always been successful, indicating that key pathways are still
Fig. 5 | Expression of IL-17 in mesenteric lymph nodes gastrointestinal tract.
Naïve, CIA and OIT asymptomatic mice were culled at day 33, when mesenteric
lymph nodes (MLNs), ileum and colon samples were collected. Single-cell suspen-
sions were obtained from MLNs and digested gut tissue, and IL-17 expression was
subsequently evaluated by flow cytometry in total isolated cells (a–c) and specific cell
populations, including CD4 T cells, CD8 T cells, group 3 innate lymphoid cells
(ILC3), γδ T cells and NK cells (d–f). a–cPercentage and number of total IL-17+
cells in MLNs (a), ileum samples (b) and colon (c). Each dot represents values of
individual mice; bars show mean values for each group ±SEM; Naïve n= 10. CIA
n= 15, asymptomatic OIT n= 7. Statistical significance was determined using
ordinary one-way ANOVA, *p< 0.05. d–fRelative cell frequency and mean fluor-
escence intensity (MFI) of IL-17 in the indicated cell populations in MLNs (d), single
cells isolated from the ileum (e) and colon (f). Each corner of the radar charts
represents the indicated normalised parameter for naïve (grey), CIA (red) and
asymptomatic OIT (orange) mice. Data were normalised to maximum expression in
each group; naïve n= 5, CIA n= 5, asymptomatic OIT n= 4. Statistical significance
was determined using raw data and ordinary one-way ANOVA. *p< 0.05,
**p< 0.01 in CIA versus Naïve; ▲p< 0.05 [naïve vs OIT asymptom atic].
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unknown. Our results show that a high percentage of animals (~55%) that
received OIT were fully protected against experimental arthritis, with no
symptoms of disease, supporting the previously described protective role of
undenatured type II collagen in inflammatory joint disease15,22,47–49.Our
analysis of asymptomatic and symptomatic OIT mice, in comparison with
the CIA group, therefore, contributes to our understanding of the
mechanisms triggered by OIT to achieve a therapeutic effect. Asymptomatic
OIT mice are strongly protected against cartilage and bone damage, which
may be a direct consequence of the lower IL-17 and IL-22 levels in the joint.
We still observed some cell infiltration in these mice, however, suggesting
that this was not sufficient to initiate any inflammatory response or reflected
recruitment of inflammation resolving/suppressing cells. By contrast,
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symptomatic OIT animals exhibited immune cell infiltration and clinical
joint scores similar to those of CIA mice, suggesting that OIT impacted
disease incidence rather than disease severity. Nevertheless, both sympto-
matic and asymptomatic mice showed protection against gut damage,
indicating that all OIT mice had undergone some local immunoregulation
regardless of whether they developed clinical joint symptoms or not. Such
gut local protection could be a consequence of the elevated IL-22 gut levels
observed in both groups. IL-22 promotes the protection of the gut tissue,
primarily through the maintenance of mucosal homoeostasis and activation
of anti-microbial responses50. Thus collectively, the high IL-22 in the joint
and gut of symptomatic OIT mice may reflect pro-inflammatory effects in
the joint, but homeostatic mechanisms in the gut. IL-22, therefore, could
play a central role in OIT, a factor that has been involved not only in joint
inflammation and epithelial repair, as discussed, but also in the regulation of
fucosylation of mucins and modulation of the gut microbiome, factors that
are also modulated in our experimental model. However, we still do not
know the factors that ultimately drove protection in the asymptomatic OIT
mice. Environmental factors could be affecting the functional outcomes of
IL-22 in some mice, and further studies should address these to harness all
the therapeutic potential of undenatured type II collagen.
Following the comparative analysis of symptomatic and asymptomatic
OIT mice, we next focused on the investigation of the protective mechan-
isms activated in mice with no clinical symptoms. This demonstrated that
the protective effect of undenatured type II collagen occurs, at least in part,
through the protection of targeted organs distal to the joint such as the gut,
where it acts broadly to rewire cytokine networks and regulate the gut
dysbiosis observed in arthritic mice. Interestingly, asymptomatic OIT mice
displayed a distinct ileum and colon microbiome rather than a preservation
of the bacteria found in healthy animals, indicating an active modulation of
the bacterial community and potentially, the subsequent metabolite pro-
duction to effect protection. Interestingly, therefore, Roseburia species were
expanded in asymptomatic OIT, a genus which produces immunor-
egulatory Short Chain Fatty Acids (SFCA) and inhibits Th17 but increases
IL-22 responses in the gut51–54.Theseactionsoverlapwithourinvivo
findings in the intestine of asymptomatic OIT mice, providing support to
the hypothesis that rewiring of functional microbiome-cytokine networks is
boosted in protected OIT. Such networks will likely include more players,
increasing the level of complexity and bidirectional regulation. For example,
Ruminococcaceae, another genus increased in the colon of asymptomatic
OIT mice, produces SFCA which has been directly implicated in activating
the Wnt/β-catenin pathway responsible for gut epithelium regeneration55.
Likewise, modulation of local gut immune system populations in OIT may
exert some selective pressure on the microbiome composition, to facilitate
the dominance of protective genera. For example, the expansion of ILC3
cells observed in asymptomatic OIT mice could contribute to this by
modulating the microbiota composition via epithelial fucosylation40,41.
Additional experiments are needed to provide further support to these data,
but our findings provide additional evidence for the role of a pathogenic
gut–joint axis in arthritis and describe new networks that may underpin the
induction of anti-inflammatory responses upon exposure to fed antigens
such as undenatured type II collagen.
Amelioration of joint inflammation and bone damage during protec-
tive OIT was also associated with the downregulation of pro-inflammatory
IL-17 in the joint and draining lymph nodes. Inflammatory factors pro-
duced systemically and in the joint, such as IL-17, could induce damage in
the gut tissue to exacerbate disease pathology, but a breach of tolerance at
mucosal surfaces is thought to be an initial event in RA that can occur many
yearsbeforediseaseonset.Whichfactoristhecauseoreffectisnotknown,
although both scenarios appear to happen in specific clinical cases. More-
over, we and others have shown that microbiome dysbiosis and gut
pathology precede systemic inflammation, autoimmunity and joint disease
in the CIA model24,56. In any case, gut dysbiosis and metabolite production
seem to be a driving factor to ultimately lead to disease progression in mouse
models and human studies57,58.
Some well-defined tolerogenic mechanisms did not appear to be cri-
tical to protection in OIT mice, including the reduced production of anti-
collagen antibodies or induction of regulatory T cells (Treg) in the gut and
draining lymph nodes, although it has previously been reported that type II
collagen increased levels of Treg and TGFβ, in addition to the reduction of
IL-1759. This lack of effect on Tregs can be a consequence of the antigen dose
used in our study since Treg activity is stimulated by lower doses60.Thedose
of 7.33 mg/kg was chosen because our preliminary studies suggested this to
be more efficient under our experimental conditions, perhaps because of the
strong inhibitory effect of IL-17-dependent pathways which are the main
drivers of CIA. A dose of 2 mg/kg was efficient in a recent study of
experimental osteoarthritis, both in young and older animals49. Our study
focused on the late stages of the disease, and we cannot rule out that Tregs
are involved prior to the induction of IL-17-mediated pathways. Likewise,
we did not evaluate the numbers of regulatory B cells (Bregs), but there is
experimental evidence showing that aberrant regulation of this cell type
modulates arthritis progression via gut-dependent mechanisms24,25.Infact,
we observed a significant increase in the number of B cells in the gut in
response to undenatured collagen treatment but no effects in the production
of anti-collagen antibodies, perhaps indicating a role for Bregs, an area we
plan to address in follow-up studies. For example, since Tregs are generated
in the gut during the induction phase of disease61, additional studies,
including pre-clinical disease stages are required to provide a full under-
standing of undenatured type II collagen-dependent regulatory responses.
The impact of oral administration of undenatured type II collagen on B cells
in MLNs prior to the initiation of disease might also be needed to fully
understand its protective mechanisms, although due to the lack of lineage-
specificmarkers,theseexperimentswillbemorechallengingtoconduct.
Reduction of IL-17 can be directly related to bone protection, as it
increases RANKL and osteoclastogenesis62. Interestingly, IL-22 expression
in draining lymph nodes and joints was increased in CIA mice and
decreased in the asymptomatic OIT group (both IL-17+/IL-22+CD4 T cell
levels show positive correlations with increasing articular score). This is
contrary to the observations in the gut, where IL-22 was generally up-
regulated in OIT mice and provides further support for the dual role
described for IL-22 during arthritis31,63, perhaps related to the ability of this
cytokine to promote gut epithelial cell regeneration50.Thus,IL-22canbea
critical contributor to both disease pathology and protective responses, as
the newly defined gut–joint axis theory64 proposes that the gut mucosa is the
first site of disease initiation. Consistent with this, CIA mice displayed
significant gut pathology and structural damage that was absent in OIT
mice. IL-17 acts to regulate mucosal host defence against many invading
Fig. 6 | Expression of IL-22 in mesenteric lymph nodes and gastrointestinal tract.
Naïve, CIA and OIT asymptomatic mice were culled at day 33, when mesenteric
lymph nodes (MLNs), ileum and colon samples were collected. Single-cell suspen-
sions were obtained from MLNs and digested gut tissue, and IL-22 expression was
subsequently evaluated by flow cytometry in total isolated cells (a–c) and specific cell
populations, including CD4 T cells, CD8 T cells, B cells, group 3 innate lymphoid
cells (ILC3), γδ T cells, NK cells and NKT cells (d–f). a–cPercentage and number of
total IL-22+cells in MLNs (a), ileum samples (b) and colon (c). Naïve n= 10. CIA
n= 15, asymptomatic OIT n= 7. Each dot represents values for individual mice; bars
show mean values for each group ± SEM. d–fRelative cell frequency and mean
fluorescence intensity (MFI) of IL-22 in the indicated cell populations in MLNs (d),
single cells isolated from the ileum (e) and colon (f). Each corner of the radar charts
represents the indicated normalised parameter for naïve (grey), CIA (red) and
asymptomatic OIT (orange) mice. Data were normalised to maximum expression in
each group; naïve n= 5, CIA n= 5, asymptomatic OIT n=4.gIleum and colon
sections were stained with anti-IL-22 antibodies and specific secondary antibodies
(Red) and DAPI (Blue) as counterstaining . Scale bars = 500 μm. Pixel intensity for
IL-22 staining was quantified using ImageJ, each dot shows the mean value of 10
different areas for each individual mouse. Error bars show standard error (SEM).
Statistical significance was determined using raw data and ordinary one-way
ANOVA, where *p< 0.05, **p< 0.01. hDetailed images of colon sections stained for
IL-22, scale bars = 100 μm and 20 μm for zoomed areas.
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Fig. 7 | Effect of protective OIT on fucosylation and fucosyltransferase expression
in the gut tissue during arthritis. a,bIleum and colon sections from naïve, CIA and
asymptomatic OIT mice were stained with UEA (a) and AAL (b) biotinylated lectins
and fluorescence streptavidin (yellow) to detect terminal and core fucosylation
respectively. DAPI (Blue) was used as counterstaining. Scale bars: 500 μm. Images
show one representative example of each group. Graphs show the mean pixel
intensity for lectin staining in individual mice; each dot shows the mean value from
10 different analysed areas, quantified using Image J software. c,dRelative
expression of fucosyltransferases mRNA was evaluated by RT-PCR in the ileum (c)
and colon (d) samples, including FUT1, FUT2, FUT4, FUT7, FUT8 and FUT9.
Expression is shown as relative to actin expression. Statistical significance was
evaluated by one-way ANOVA where *p< 0.05. Each dot represents values of
individual mice where data are collected from two independent experiments. Error
bars represent standard error (SEM).
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pathogensinthegut,butunresolved IL-17-associated inflammation may
cause gut pathology65. In addition, IL-22 has been proposed to play both
pathologic and protective roles in gut integrity and homeostasis. Thus, to
identify pathological mechanisms, we examined the cells producing IL-17
and IL-22 in MLNs, ileum and colon as sites of Th17/22 induction and
tolerance. Interestingly, whilst our results show upregulation of IL-17 in the
gut tissue from CIA but not asymptomatic OIT mice, the most striking
changes appear to reflect OIT-induced switches in the cellular networks of
IL-17+and IL-22+cells in the gut, with these cytokines being predominantly
produced by innate effector cells like NKT and ILCs, rather than the CD4+
helper and γδ T cells driving systemic autoimmunity in CIA29.IL-22pro-
duction was increased in all cell types isolated from the ileum in response to
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undenatured type II collagen administration, and this reflected an expan-
sion of IL-22+γδT cells, NKT cells and CD4 T cells. Overall, our data shows
that the protective role of gut IL-17/IL-22 likely depends on the relative
contribution coming from innate and adaptive immune cells, although we
have not investigated all the molecular mechanisms underlying these pro-
tective cytokine and cellular networks.
Future studies plan to focus on the ability of undenatured type II
collagen to harness protective IL-22 actions in the mucosal epithelium50.IL-
22 promotes epithelial fucosylation, which is crucial to maintaining barrier
integrity and immunity66. Indeed, perhaps linking the gut-joint axis, fucose
can play a protective role in both local gut and systemic inflammation67.
Interestingly, OIT restored the reduced gut fucosylation observed in CIA
mice. We did not identify the mechanism(s) controlling such tissue fuco-
sylation, but since this was not associated with changes in the expression of
fucosyltransferases, environmental factors could be controlling local fucose
content. For example, our results indicate that OIT induces microbiome
changes that could be responsible for the modulation of fucosylation status
as well as some of the therapeutic anti-inflammatory effects. In line with this,
rewiring of immune-glycan-microbiome networks could affect the pro-
duction of protective metabolites such as short-chain fatty acids with known
anti-inflammatory effects in models of autoimmunity68,69.Additionally,
specific microbial species may play an integral role in the induction of the
protective glycan-dependent mechanisms associated with OIT. For exam-
ple, segmentedfilamentous bacteria (SFB) is a c ommongut resident that can
induce differentiation of Th17+CD4 T cells in arthritis70,71, but also induce
IL-22 production in the small intestine lamina propria to modulate fuco-
sylation of the ileum72,73. Thus, understanding the gut microenvironment,
including cytokine networks, regulation of microbiome composition, and
mechanisms linking both sides, such as epithelial glycosylation, may fill
some of the gaps in the field, opening new avenues for translational
opportunities, which consider human disease heterogeneity. For example,
several clinical subgroups have been described in RA based on the presence
of specific autoantibodies and/or specific immune responses; where for
instance, the myeloid RA phenotype is driven by TNF, contrary to lymphoid
RA type that is IL-17/IL-1-mediated, and these RA phenotypes correlate
with response to treatments74. Thus, it is likely that specificgroupsof
patients (with distinct pathogenic signatures) respond differently to unde-
natured type II collagen therapies, and the search for predictive biomarkers
should be considered in future clinical trials. Additionally, patients’
responses may differ in early or late disease stages, since anti-collagen
antibodies show a higher concentration during early disease stages, and it
has been associated with specific disease phenotypes75,76.
Collectively, our study demonstrates the importance of differential IL-
17- and IL-22-producing cell networks in the joint-gut inflammatory axis
and the therapeutic potential of undenatured type II collagen. Regulation of
cytokine networks correlates with protection against arthritis, indicating
that factors affecting them may determine effective OIT. For translational
purposes, understanding the impact of exogenous factors in regulating these
immune networks can offer a way to enhance the clinical value of oral
immunomodulation, and to identify patient groups which may benefitfrom
these interventions. In this regard, we have identified molecular and cellular
parameters in the gastrointestinal tract correlating with low inflammatory
conditions, including regulation of cytokines, cellular networks, tissue gly-
cosylation and microbial profiles. These findings could offer new oppor-
tunities to treat arthritis and other chronic inflammatory disorders affected
by changes in mucosal tissues.
Methods
Collagen-induced arthritis and undenatured type II collagen
treatment
Male DBA/1 mice were purchased at 8–10 weeks of age (Envigo; Bicester,
UK) and housed and maintained in the Central Research Facility of the
University of Glasgow. We have complied with all relevant ethical reg-
ulations for animal use. All experiments were approved by and conducted
in accordance with the Animal Welfare and Ethical Review Board of the
University of Glasgow, UK Home Office Regulations and Licences PPL
P8C60C865, PIL I62988261, PIL I675F0C46. To induce arthritis, mice
were immunised with type II chicken collagen emulsion (1 mg/mL; in
complete Freund’s adjuvant [CFA] i.d.) and then administered type II
chicken collagen in PBS i.p. at 21 days after the first injection. Mice were
divided into three experimental groups, naïve, CIA, and mice receiving
undenatured collagen. Undenatured type II collagen (7.33 mg/kg) was
given to the animals by oral gavage three times a week, starting two weeks
before induction of arthritis. The dose was selected based on pilot studies,
suggesting that 7.33 mg/kg was the most protective under our lab
experimental conditions. A concentrate of undenatured type II collagen
was obtained from Lonza Greenwood LLC (UC-II®undenatured type II
collagen). Monitoring of mouse health, clinical scores and weight were
evaluated every 2 days. Clinical scoring of each limb was used to assess
disease progression, where each paw received a score ranging from 0 (no
inflammation) to 4 (highly inflamed/loss of functionality) as described
previously29. An aggregated Clinical score of 10 for an individual mouse
was considered an experimental endpoint, with any such mice imme-
diately euthanized.
Hematoxylin and eosin (H&E) staining
Gut tissue and paws from individual mice were collected for histology. Gut
tissue was processed according to the Swiss roll technique, including duo-
denum, jejunum, ileum and colon. Gut tissue and paws were fixed in 4%
paraformaldehyde for up to 24 h, then transferred into 70% ethanol for
storage. Following dehydration, tissue was embedded in paraffinblocksand
sectioned on a standard microtome at 7 μm thickness using a microtome
(Leica RM2125). For joint blocks, bone decalcification, paraffin sectioning
and H&E staining were completed at Histology Research Service, School of
Veterinary Medicine, University of Glasgow. For gut tissue paraffinblocks,
tissue processor Lecia Asap300 was used. Sections were heated for 35 min at
60 °C, then followed by H&E staining, which was performed on all tissues
for identification of pathological changes. For analysis of gut sections, the
length of gut villi and crypt depth were determined using Image J software to
calculate villi/crypt length ratios. Pathology of the joint tissue (cell infiltra-
tion, pannus formation, bone damage and cartilage damage) was assessed by
visual evaluation according to a score system ranging from 0 (no effect) to 4
(high pathology). The final score for each mouse paw is representative of two
hind paws. No cut off was used to exclude any joints in the analysis.
Fig. 8 | Analysis of the microbial composition in the ileum. Faecal matter was
taken from the ileum of naïve mice (n= 4), severe arthritis (CIA high, n= 3, disease
scores 11, 10 and 7), mild arthritis (CIA low, n= 3; disease scores 3, 4 and 4),
symptomatic OIT mice (OIT high, n= 3; disease scores 5, 6 and 4) and asympto-
matic OIT mice (OIT low, n= 3) at day 33 when DNA was isolated and subjected to
16 S ribosomal RNA (rRNA) amplification and sequencing. aAlpha Diversity
analysis, including observed_ otus, shannon, simpson, chao1, dominance and pie-
lou_e indices. Each dot represents individual mice; graphs show the mean ± SEM.
Kruskal–Wallis test was used to analyse whether the differences in species diversity
between groups were significant, *p< 0.05. bBeta diversity indices heatmap of
unweighted unifrac distance Matrix. The size and colour of the circle in the square
represent the differences in coefficient between the two samples. The larger the circle
is, the darker the corresponding colour is, indicating that the differences between the
two samples are greater. cRelative abundance of the indicated phyla. Each column
shows data from individual mice. Data are also presented as pie charts, presenting
proportion values for each group as means. dClustering of Species Abundance. The
top 35 genera in abundance were clustered from the species and sample levels
according to their abundance information in each sample. Heatmap in grey scale
shows the mean value of all mice in the group for Zvalue of taxonomic relative
abundance after standardisation. The coloured heatmap represents the values for
individual mice: the x-axis represents the sample name, and the y-axis represents the
function annotation. The cluster tree on the left side is the species cluster tree.
eRelativeabundance of the indicated genera. Data show mean ± SEM and dots
represent individual mice.
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Assessment was conducted blindly, by two independent researchers. Images
were acquired using an EVOS microscope.
Periodic acid-Schiff (PAS) staining
Paraffin sections were dewaxed using the same method used for H&E
staining and rehydration. The slides were stained with Periodic acid for
5 min and rinsed with water before staining with Schiff’s reagent for
15 min. After washing with tap water for 5 min, the sections were
counterstained with haematoxylin for 1 min. Sections were then rinsed
with water and dehydrated using a series of ethanol and xylene, mounted
with DPX and coverslip. Images were acquired on an EVOS brightfield
microscope.
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Immunofluorescence
Tissue sections (7 μm thickness) were dewaxed in xylene followed by
100% EtOH, 90% EtOH, 70% EtOH, 50% EtOH, and 30% EtOH for
3 min x2, respectively. Sections were then put in antigen retrieval buffer
(Citrate buffer, Ph: 5.0) for 20 min at 95 °C31. To block non-specific
antibody binding, sections were incubated with Carbo-Free Blocking
Solution (Vector labs) for 30 min. The slides were washed in PBS-T (PBS
0.05% Tween20), and Streptavidin/Biotin blocking was performed
following kit instructions (Vector laboratories, SP-2002). Sections
were incubated with biotinylated lectins (Vector laboratories, 1:200) or
primary antibodies in PBS and incubated overnight at 4 °C. Sections
were washed with PBS-T at least 3 times. Streptavidin-Alexa 647 or
secondary antibodies (1:400) were applied in PBS at room temperature
for 60 min. Slides were rinsed with PBS and mounted with mounting
media containing DAPI. Images were acquired with an EVOS micro-
scope and confocal microscopy and were later analysed in Image J
software.
Serum cytokine and antibody ELISAs
Single-cell suspensions of lymph nodes (LN) were obtained, and cells were
then cultured (106/ml) and stimulated with TPA (50 ng/ml)/Ionomycin
(500 ng/ml) for 12 h when supernatants were collected for detection of
secreted cytokines. Interleukin-17 (IL-17) was measured by ELISA
according to the manufacturer’s instructions (Invitrogen). For determina-
tion of anti-collagen type II-specific IgG1 and IgG2a antibodies in serum,
high-binding 96-well ELISA plates were coated with chicken collagen (5 μg/
ml) overnight at 4 °C before washing and blocking with PBS 5% BSA. Serum
from individual animals was diluted (1:100) and incubated with HRP-
conjugated goat anti-mouse IgG1 or IgG2a (1:10,000) in PBS 10% FBS prior
to developing with TMB. Samples were read in a microplate reader (Sunrise)
at an optical density of 450 nm.
Flow cytometry
LN cells were suspended in FACS buffer (1% FBS; 0.5 mM EDTA, in PBS).
Lymphocytes were identified by labelling with antibodies against CD3
(FITC) and CD4 (AF700). CD8+T cells were identified by labelling with
antibodies against CD3 (FITC) and CD8 (PE-Cy5). B cells were identified by
labelling with antibodies against CD19 (BV421). Data were acquired using
an LSR-II Flow Cytometer, and populations were gated based on isotype
controls using FlowJo software. For gut sample preparation, the ileum and
colon were collected into HBSS 10% FBS following removal of all excess fat
and Peyer’s patches and immediately digested following established pro-
tocol in our group77.Briefly, tissue was washed with PBS and cut into small
pieces (around 1 cm), rinsed with warm HBSS and transferred to wash
buffer (HBSS no Ca++,Mg
++ and containing 2 mM EDTA) for 15 min
(37 °C, 220 rpm). Ileum samples were digested with 0.5 mg/mL of Col-
lagenase IV (15 min, 37 °C) and the colon was digested with 0.5 mg/mL of
Collagenase IV containing 24 μg/mL DNase I (20 min, 37 °C) to generate
single cell suspensions prior to resuspending for antibody staining and flow
cytometric analysis.
RT-PCR
Gut tissues were dissolved in Trizol Reagent (1 ml/50–100 mg of tissue)
following tissue homogenisation, and the lysates were centrifuged for 5 min
at 12,000 rpm at 4 °C. The supernatants were transferred to a fresh tube, and
chloroform (0.2 ml/mL of Trizol) was added to the samples for 5 minutes of
incubation followed by centrifugation (15 minutes at 12,000g4°C).RNAin
the aqueous phase was precipitated by adding 100% EtOH. Isolated RNA
was cleaned using the RNeasy Plus Mini kit (Qiagen, Germany) according to
the manufacturer’s instructions. The High-Capacity cDNA Reverse Tran-
scriptase kit (Applied Biosystems, Life Technology, UK) was used to gen-
erate cDNA. qPCR reactions were conducted using StepOne PlusTM real-
time PCR system (Applied Biosystems, UK) and KiCqStart®qPCR Ready
Mix (Sigma-Aldrich). Taqman probes were used to evaluate gene expres-
sion, including fucosyltransferases (FUT1, FUT2, FUT4, FUT7, FUT8 and
FUT9). Data were normalised to the reference gene β-actin to obtain ΔCT
values.
Microbial diversity sequencing and analysis
Genomic DNA from the ileum and colon faecal matter was purified using
QIAamp DNA Stool Mini Kit (Qiagen, Germany) and stored at −20 °C. All
samples were subjected to paired-end sequencing by Novogene (UK)
Company Limited, according to established protocols. Briefly, specific16S
target hypervariable regions (amplicons) were amplified by PCR. Amplified
DNA fragments were end-repaired, and A-tailed. The sequencing adapters
were ligated to the ends of the DNA fragments using a DNA-binding
enzyme, and the DNA fragments were purified using AMpure PB magnetic
beads to construct an SMRTbell library. Finally, the sequencing primer was
annealed to the SMRTbell templates, followed by the binding of the
sequence polymerase to the annealed template. The library was checked
with Qubit for quantification. Quantified libraries were pooled and
sequenced on PacBio Sequel II/IIe systems. For sequencing data processing,
the PacBio BAM file was split according to barcode and filtered to get clean
data, used to generate Amplicon Sequence Variants (ASVs). Denoising,
species annotation and alpha diversity indices (including observed_ otus,
shannon, simpson, chao1, goods_ coverage, dominance and pielou_e
indices), were performed by the DADA2 and Qiime2 software. Sequences
with less than 5 abundance were filtered out to obtain the final ASVs and
feature table. Then, the Classify-sklearn moduler in QIIME2 software was
used to compare ASVs with the database and to obtain the species anno-
tation of each ASV.
Statistics
All data were analysed using one-way ANOVA with Fisher’sLSDpost-tests
for parametric data or Kruskal–Wallis test and Dunn’s post-test for non-
parametric data (GraphPad Prism software). Shapiro–Wilk test was used to
assess normality.
Reporting summary
Further information on research design is available in the Nature Portfolio
Reporting Summary linked to this article.
Fig. 9 | Analysis of the microbial composition in the colon. Faecal matter was taken
from the colon of naïve mice (n= 3), severe arthritis (CIA high, n= 3, disease scores
11, 10 and 7), mild arthritis (CIA low, n= 3; disease scores 3, 4 and 4), symptomatic
OIT mice (OIT high, n= 3; disease scores 5, 6 and 4) and asymptomatic OIT mice
(OIT low, n= 3) at day 33, when DNA was isolated and subjected to 16 S ribosomal
RNA (rRNA) amplification and sequencing. aAlpha Diversity analysis, including
observed_ otus, shannon, simpson, chao1, dominance and pielou_e indices. Each
dot represents one individual mouse; graphs show the mean ± SEM. Kruskal–Wallis
test was used to analyze whether the differences in species diversity between groups
were significant, *p< 0.05. bBeta diversity indices heatmap of unweighted unifrac
distance Matrix. The size and colour of the circle in the square represent the dif-
ferences in coefficient between the two samples. The larger the circle is, the darker the
corresponding colour is, indicating that the differences between the two samples are
greater. cRelative abundance of the indicated phyla. Each column shows data from
individual mice. Data are also presented as pie charts, presenting proportion values
for each group as means. dClustering of species abundance. The top 35 genera in
abundance were clustered from the species and sample levels according to their
abundance information in each sample. Heatmap in grey scale shows the mean value
of all mice in the group for Zvalue of taxonomic relative abundance after standar-
disation. The coloured heatmap represents the values for individual mice: the x-axis
represents the sample name, and the y-axis represents the function annotation. The
cluster tree on the left side is the species cluster tree. eRelative abundance of the
indicated genera. Data show mean ± SEM and dots represent individual mice.
https://doi.org/10.1038/s42003-024-06476-z Article
Communications Biology | (2024) 7:804 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Data availability
Metagenomic data have been deposited in NCBI’s Gene Expression
Omnibus SRA under the Bioproject accession code PRJNA1117859. The
source data behind the graphs in the paper can be found in Supplementary
Data 1. All other data are available in the article and Supplementary files or
from the corresponding authors upon reasonable request. Numerical source
data for all graphs in the manuscript can be found in Supplementary
Data 1 File.
Received: 9 June 2023; Accepted: 20 June 2024;
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Acknowledgements
This work was funded by an Industrial partnership PhD between the
Universityof Glasgow and Lonzaand a Versus Arthritis Career Development
Fellowship Award grant 21221.
Author contributions
P.P., M.M.H., S.M. and M.P. conceivedand designedthe study. P.P., Y.W.,
M.H.N. and M.P. performed the lab work. P.P., Z.S., E.K.A., A.S., S.M.,
M.M.H. andM.P. contributed to data interpretation and manuscript revision.
https://doi.org/10.1038/s42003-024-06476-z Article
Communications Biology | (2024) 7:804 18
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P.P., S.M., M.M.H and M.P. wrote the paper. All authors read and approved
the submitted version.
Competing interests
Z.S., E.K.A.and A.S. are employeesof Lonza GreenwoodLLC., Greenwood,
SC, USA. The remaining authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s42003-024-06476-z.
Correspondence and requests for materials should be addressed to
Miguel Pineda.
Peer review information Communications Biology thanksZhihua Liu, Julie
Gibbs and the other, anonymous, reviewer for their contribution to the peer
review of this work. Primary Handling Editors: Martina Rauner and Joao
Valente. A peer review file is available.
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