Undenatured type II collagen protects against ...

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 experimental groups: (i) non-treated animals (Naïve), (ii) mice undergoing experimental arthritis (CIA) and (iii) mice undergoing arthritis with prophylactic 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 predominantly on disease incidence, or it was influencing both incidence and severity of disease, we divided OIT mice into symptomatic and asymptomatic 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.

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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. Disease incidence was calculated by dividing the number of cases by the total number of mice in the group at the indicated times. b Paw 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. c Hind 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. d Cell infiltration, pannus formation, cartilage and bone damage were quantified for individual mice in hematoxilin/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.

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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 undenatured 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.

a Anti type II collagen antibodies, IgG1 and IgG2 isotypes, were evaluated 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. b Total cell numbers isolated from draining lymph nodes (DLNs) collected from all naïve, CIA, asymptomatic and symptomatic OIT mice. c Total 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&#;c column 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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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 a significantly 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 regarded as immunological switches that 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, and IL-22, a cytokine we reported to promote the development of joint disease30,31,32,33. To confirm 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 correlate with disease severity, regardless of treatment. The impact of B cell-derived IL-17 and IL-22 production on pathology was diminished compared 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).

Naïve, CIA and CIA-OIT mice were culled at day 33 when tissue was collected for further analysis. a, b Correlation 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. c Expression 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 indicated by white arrows. Scale bars: 500&#;μm. Graphs show the quantification of the mean intensity of individual mice. d IL-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. e Relative 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. f Cell 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 asymptomatic vs OIT symptomatic].

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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). 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 asymptomatic groups as before. Mice in the OIT group that remained asymptomatic 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 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 consequence 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 epithelial 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 symptoms (Fig. 4c).

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. a Isolated gastrointestinal tract from a naïve mouse showing the four anatomical areas used for further study. b Duodenum, jejunum, ileum, and colon were fixed, and tissue sections 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. c Colon 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. d MLNs 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. Statistical significance was determined using Ordinary one-way ANOVA. Significance is indicated by asterisks, *p&#;<&#;0.05, **p&#;<&#;0.01.

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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+ T cells in MLNs 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 asymptomatic 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.

Naïve, CIA and OIT asymptomatic mice were culled at day 33, when mesenteric lymph nodes (MLNs), ileum and colon samples were collected. Single-cell suspensions 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&#;c Percentage 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&#;f Relative cell frequency and mean fluorescence 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 asymptomatic].

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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 peptides34,35,36. We did not 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 production in MLNs during CIA, whereas this was reduced both in the ileum (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 IL-22 as a protective or pathogenic factor, our results highlight that differential 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 in the tissue epithelium in the colon of 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).

Naïve, CIA and OIT asymptomatic mice were culled at day 33, when mesenteric lymph nodes (MLNs), ileum and colon samples were collected. Single-cell suspensions 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&#;c Percentage 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&#;f Relative 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. g Ileum 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. h Detailed images of colon sections stained for IL-22, scale bars&#;=&#;100&#;μm and 20&#;μm for zoomed areas.

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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 triggered by this cytokine in asymptomatic OIT mice. In health, IL-22 not only supports homeostatic expression of mucins in epithelial cells37, but also their 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 preserved 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 inflammation. 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 significantly 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 in asymptomatic 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 dependent 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 morbidity 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 composition 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) and colon (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). Firmicutes 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 unexpectedly, 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 symptomatic 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, and Anaerotruncus showed a consistent reduction in the ileum of asymptomatic OIT mice, Muribaculaceae species were increased (Fig. 8e).

a, b Ileum 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, d Relative 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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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 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. a Alpha Diversity analysis, including observed_ otus, shannon, simpson, chao1, dominance and pielou_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. b Beta 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. c Relative 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. d Clustering 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 Z value 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. e Relative abundance of the indicated genera. Data show mean&#;±&#;SEM and dots represent individual mice.

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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. a Alpha 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. b Beta 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. c Relative 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. d Clustering 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 Z value 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. e Relative abundance of the indicated genera. Data show mean&#;±&#;SEM and dots represent individual mice.

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A similar analysis was carried out on the colon faecal material, specifically 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 Bacteroidetes 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, and Ruminococcaceae 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 preventing 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 (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.

Daily supplementation with 40 mg of UC-II was well tolerated and led to improved knee joint extension in healthy subjects. UC-II also demonstrated the potential to lengthen the period of pain free strenuous exertion and alleviate the joint pain that occasionally arises from such activities.

After 120 days of supplementation, subjects in the UC-II group exhibited a statistically significant improvement in average knee extension compared to placebo (81.0 ± 1.3º vs 74.0 ± 2.2º; p = 0.011) and to baseline (81.0 ± 1.3º vs 73.2 ± 1.9º; p = 0.002). The UC-II cohort also demonstrated a statistically significant change in average knee extension at day 90 (78.8 ± 1.9º vs 73.2 ± 1.9º; p = 0.045) versus baseline. No significant change in knee extension was observed in the placebo group at any time. It was also noted that the UC-II group exercised longer before experiencing any initial joint discomfort at day 120 (2.8 ± 0.5 min, p = 0.019), compared to baseline (1.4 ± 0.2 min). By contrast, no significant changes were seen in the placebo group. No product related adverse events were observed during the study. At study conclusion, five individuals in the UC-II cohort reported no pain during or after the stepmill protocol (p = 0.031, within visit) as compared to one subject in the placebo group.

This randomized, double-blind, placebo-controlled study was conducted in healthy subjects who had no prior history of arthritic disease or joint pain at rest but experienced joint discomfort with physical activity. Fifty-five subjects who reported knee pain after participating in a standardized stepmill performance test were randomized to receive placebo (n = 28) or the UC-II (40 mg daily, n = 27) product for 120 days. Joint function was assessed by changes in degree of knee flexion and knee extension as well as measuring the time to experiencing and recovering from joint pain following strenuous stepmill exertion.

UC-II contains a patented form of undenatured type II collagen derived from chicken sternum. Previous preclinical and clinical studies support the safety and efficacy of UC-II in modulating joint discomfort in osteoarthritis and rheumatoid arthritis. The purpose of this study was to assess the efficacy and tolerability of UC-II in moderating joint function and joint pain due to strenuous exercise in healthy subjects.

The aim of this randomized, double blind, placebo-controlled study was to assess the impact of UC-II on knee function in otherwise healthy subjects with no prior history of arthritic disease who experienced knee pain upon strenuous physical exertion. The primary efficacy variable for assessing knee function included measurements of flexibility using range of motion (ROM) goniometry.

UC-II is a natural ingredient which contains a glycosylated, undenatured type-II collagen [ 24 ]. Previous studies have shown that small doses of UC-II modulate joint health in both OA and RA [ 24 - 26 ]. Tong et al. [ 27 ], using an in vivo model of collagen induced arthritis (CIA), demonstrated that ingesting microgram quantities of undenatured type II collagen significantly reduces circulating levels of inflammatory cytokines, potentially serving to decrease both the incidence and the severity of arthritis [ 28 ]. The ability to alter immunity via the ingestion of a food, or an antigen, is called oral tolerance. This is an ongoing normal physiological process that protects the alimentary tract against untoward immunological damage [ 29 , 30 ]. Research into its mechanism of action has revealed that several distinct types of T regulator cells mediate this phenomenon by releasing IL-10 and TGF-β [ 30 ]. It has also been shown that this effect is transitory in nature requiring that the food, or antigen, be consumed continuously in order to maintain the tolerogenic state [ 30 ]. Given these findings, plus our current understanding of the role of various cytokines in normal joint physiology, it was hypothesized that supplementation with UC-II might relieve joint discomfort and restore joint function in healthy subjects.

The overall findings discussed above point to a new, unifying view of joint physiology. It suggests that many of the biological processes occurring in knee joints affected by RA and OA also participate in the maintenance of healthy knees [ 1 , 4 , 5 ]. It therefore seems appropriate to test the efficacy of natural supplements or ingredients, which have been shown to moderate joint pain in RA and OA, as possible candidates for treating the joint discomfort that occasionally results from strenuous exercise in healthy individuals.

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Another protein released by dynamically compressed chondrocytes is transforming growth factor (TGF)-β [ 16 - 18 ]. This factor is secreted by many cell types and is known to interfere with the cell cycle and arrest differentiation [ 19 ]. With regard to chondrocytes, TGF-β induces cell proliferation in vitro and slows terminal differentiation into hypertrophic cells [ 20 ]. Numerous studies have shown that TGF-β reverses the in vitro catabolic effect of various proinflammatory cytokines on normal chondrocytes as well as chondrocytes harvested from RA and OA donors [ 21 - 23 ].

Mechanically stressed chondrocytes also produce a number of other molecules known to participate in inflammatory responses, including prostoglandin E2, NO, and vascular endothelial growth factor [ 14 ]. These are proinflammatory molecules that, in conjunction with TNF-α, IL-6 and IL-1β, result in a localized, and transitory inflammatory-like response that is part of the normal repair process occurring in knee joints, serves to moderate remodeling events [ 3 ]. Ostrowski et al. [ 15 ] showed that healthy individuals express up to 27-fold greater concentrations of the anti-inflammatory cytokine IL-10 in blood following a marathon run when compared to IL-10 blood levels at rest. This finding is not surprising given that these same individuals also show marked increases in the proinflammatory cytokines TNF-α, IL-1β, and IL-6. It therefore appears that in healthy subjects undergoing strenuous exertion, the induction of proinflammatory cytokines is offset by the synthesis of anti-inflammatory agents as part of the recovery process. This view is supported by the observation that IL-10 reduces the catabolic impact of IL-1β and TNF-α on cartilage explants from healthy volunteers, and this effect is enhanced by combining IL-10 with IL-4 [ 13 ].

When normal chondrocytes undergo strenuous mechanical stimulation under static conditions, their physiology shifts towards ECM breakdown, as indicated by the upregulation of several metalloproteinases (MMPs), such as MMP-13 as well as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and various aggrecanases [ 5 , 6 ]. This in vitro catabolic response is mediated by changes in the phosphorylation, expression, or translocation of several transcription factors to the cell nucleus such as NF-κB, p38 MAPK, Akt, and ERK [ 7 , 8 ]. By contrast, normal chondrocytes produce the anti-inflammatory cytokine IL-4 when mechanically stimulated under moderate and dynamic conditions [ 9 ]. The secretion of this autocrine molecule not only helps in shifting chondrocyte metabolism towards the synthesis of aggrecan and type II collagen, it also downregulates production of nitric oxide (NO) and various MMPs and aggrecanases [ 10 - 12 ]. This conclusion is corroborated by the finding that pretreatment of strenuously compressed normal chondrocytes with IL-4 attenuates their catabolic response [ 11 ]. This suggests that IL-4 plays a key role in downregulating remodeling functions, restoring articular cartilage homeostasis, as well as decreasing chondrocyte apoptosis following strenuous mechanical loading [ 12 , 13 ].

The impact of strenuous exercise on knee joints may cause localized pain and stiffness, which are hallmark features of pathologic inflammatory disease [ 1 ]. It has been shown that when dogs undergo a strenuous running regimen, significant losses in articular cartilage and glycosaminoglycans occur [ 2 ]. Such studies suggest that strenuous exercise may activate some of the same physiological processes that occur in arthritic disease [ 2 - 4 ]. In fact, in vitro studies have shown that many of the cytokines implicated in the onset and progression of both rheumatoid arthritis (RA) and osteoarthritis (OA) also appear to regulate the remodeling of the normal knee extracellular matrix (ECM) following strenuous exertion [ 5 ].

Outcome variables were assessed for conformance to the normal distribution and transformed as required. Within group significance was analyzed by non-parametric Sign test or by non-parametric Wilcoxon Signed Rank test, while Wilcoxon Mann&#;Whitney test was used to analyze between groups significance. The Fisher Exact test was used to evaluate the complete loss of pain between study cohorts whereas the binomial test was used to assess the likelihood of complete loss of pain at each visit. P-values equal to or less than 0.05 were considered statistically significant. All analyses were done on a per protocol basis using SPSS, v19 (IBM, Armonk, NY). Results were presented as mean ± SEM.

No rescue medications were allowed during the course of the study. At all study visits, subjects were given a list of the 43 prohibited medications and supplements (Table 3 ). Changes in overall medication history, or the use of these substances, were then recorded by the study coordinator. Subjects found to have used any of these prohibited substances were excluded from further participation in the study as per protocol.

The KOOS survey is a validated instrument consisting of 42 questions that are classified into sub-scales such as symptoms, stiffness, pain, daily activities, recreational activities and quality of life [ 34 ]. It measures the subjects&#; opinion about their knees and their ability to perform daily activities during the past week. The Stanford exercise behavior scale comprises 6 questions designed to assess exercise behaviors during the previous week [ 35 ].

Briefly, a stopwatch was started when subjects began climbing the stepmill. Time to onset of pain was recorded at the first sign of pain in the target knee. The baselines at each time point were normalized to account for dropouts. Percent change in time to complete recovery from pain was measured as follows: a new stopwatch was started when the subjects disembarked from the stepmill and the time to complete recovery from pain was recorded. The baselines at each time point were normalized to account for dropouts then compared against the reference interval which was defined as the percentage change between the study baseline and day 7.

Knee extension was measured by goniometry. Briefly, subjects were instructed to sit in an upright position on a table edge with their backs straight (knee position defined as 90°). The axis of a goniometer was placed at the intersection of the thigh and shank at the knee joint. Subjects were asked to bring their knees to full extension without changing the position of the pelvis and lumbar spine. The extended knee joint angle was measured and recorded. For knee flexion measurement, subjects were asked to actively flex their knees while lying in a prone position with their shins off the end of the table. The range of knee flexion motion was then measured and documented.

The study duration was 17 weeks with a total of 7 visits that included screening, baseline, days 7, 30, 60, 90 and 120 (final visit). Table 2 summarizes the study visits and activities. Figure 1 depicts the sequence of study procedures that subjects underwent during each visit. All subjects completed a medical history questionnaire at baseline and compliance reports during follow-up evaluations at 7, 30, 60, 90 and 120 days. Subjects were assessed for anthropometric measures, vital signs, knee range of motion (flexion and extension), six-minute timed walk, as well as the onset and recovery from pain using the Udani Stepmill Procedure. A Fitbit (San Francisco, CA) device was used to measure daily distance walked, steps taken and an average step length for study participants. Subjects were also asked to complete the KOOS survey as well as the Stanford exercise scales.

Simple randomization was employed using a software algorithm based on the atmospheric noise method ( http://www.random.org ). Sequential assignment was used to determine group allocation. Once allocated, the assignment was documented and placed in individually numbered envelopes to maintain blinding. Subjects, clinical staff, plus data analysis and management staff remained blinded throughout the study.

This randomized, double blind, placebo-controlled study was conducted at the Staywell Research clinical site located in Northridge, CA. Medicus Research (Agoura Hills, CA) was the contract research organization (CRO) of record. The study protocol was approved by Copernicus Group IRB (Cary, NC) on April 25th . The study followed the principles outlined in the Declaration of Helsinki (version ).

Subjects were required to undergo a 10 minute period of performance testing using a standardized stepmill test developed and validated by Medicus Research (Udani JK, unpublished observation). It involved exercising at level 4 on a StepMill® model PT (StairMaster® Health & Fitness Products, Inc., Kirkland, WA) until one or both knees achieved a discomfort level of 5 on an 11 point (0&#;10) Likert scale [ 33 ]. This pain threshold had to be achieved within a 10 minute period otherwise the subject was excluded. Once the requisite pain level was achieved the subject was asked to continue stepping for an additional two minutes in order to record the maximum pain level achieved before disembarking from the stepmill. The following knee discomfort measures were recorded from the start of the stepmill test: (1) time to onset of initial joint pain; (2) time to onset of maximum joint pain; (3) time to initial improvement in knee joint pain; (4) time to complete recovery from knee joint pain. Subjects who experienced a pain score of 5 (or greater) within one minute of starting the stress test were excluded. Out of 106 screened candidates, 55 subjects were enrolled in the study. Each subject voluntarily signed the IRB-approved informed consent form. After enrollment, the subjects were randomly assigned to either the placebo or the UC-II group.

One hundred and six subjects were screened for eligibility using the inclusion&#;exclusion criteria defined in Table 1 . Only healthy adults who presented with no knee joint pain at rest and no diagnosable markers indicative of active arthritic disease, as outlined by the American College of Rheumatology (ACR) guidelines [ 31 , 32 ], were admitted into the study. To accomplish this, all potential subjects were screened by a board certified clinician. Subjects presenting with any knee pain at rest and at least 3 of 6 clinical classification criteria, which included age greater than 50 years, morning stiffness in the joint lasting 30 minutes or less, crepitus on knee joint manipulation, body tenderness, bony enlargements, knee swelling or presence of excess fluid, and palpable warmth, were excluded. Potential subjects reporting the occasional use of NSAIDs, other pain relief medication, or anti-inflammatory supplements underwent a 2-week washout period before randomization.

The investigational study product UC-II is derived from chicken sternum. It is manufactured using a patented, low-temperature process to preserve its native structure. For the clinical study, 40 mg of UC-II material (Lot ), which provides 10.4 ± 1.3 mg of native type-II collagen, was encapsulated in an opaque capsule with excipients. Placebo was dispensed in an identical capsule containing only excipients (microcrystalline cellulose, magnesium stearate and silicon dioxide). Both study materials were prepared in a good manufacturing practice (GMP)-certified facility and provided by InterHealth Nutraceuticals, Inc. (Benicia, CA). Subjects were instructed to take one capsule daily with water before bedtime.

A total of eight adverse events, equally dispersed between both groups, were noted (Table 6 ). None of the adverse events was considered to be associated with UC-II supplementation. All events resolved spontaneously without the need for further intervention. No subject withdrew from the study due to an adverse event. Finally, no differences were observed in vital signs after seventeen weeks of supplementation, and no serious adverse events were reported in this study.

No significant differences were observed between the study groups for the six-minute time walk or the daily number of steps taken (p > 0.05). The distance walked in six-minutes by the UC-II (range = 505 to 522 meters) and the placebo (range = 461 to 502 meters) groups were within the reference range previously reported [ 39 ] for healthy adults (399 to 778 meters, males; 310 to 664 meters, females). Similarly, the average step length calculated from Fitbit data for both study groups (0.69 to 0.71 meters) also agreed with previously published results for normal adults [ 40 ].

During the course of this study it was noted that a number of subjects in both the placebo and the supplemented cohorts no longer reported any pain during the stepmill protocol. For the UC-II group, 5 subjects (21%) no longer reported pain by day 120, whereas only 1 subject (5%) in placebo group reported complete loss of pain (Table 5 ). This effect did not reach statistical significance between groups but there was an evident trend in the data towards a greater number of subjects losing pain in the UC-II cohort (p = 0.126). A binomial analysis for complete loss of pain at each visit demonstrated a statistical significance for the UC-II group by day 120 (p = 0.031). It is important to note that the complete loss of knee pain was not a random event. The pattern among the subjects indicates that loss of knee pain appeared to be a persistent phenomenon that spanned multiple visits (Table 5 ). A detailed review of the clinical report forms showed that none of these individuals consumed pain relief medication prior to their visits.

Percent change in time to complete recovery from pain. Values are presented as Mean ± SEM. *p &#; 0.05 indicates a statistically significant difference from baseline. Number of completers: n = 18 in UC-II group (n = 3 dropouts; n = 5 did not have pain; n = 1 time to complete recovery from pain was not achieved); n = 20 in placebo group (n = 6 dropouts; n = 1 did not have pain; n = 1 did not use stepmill).

The time to complete recovery from joint pain showed significant reductions at days 60, 90 and 120 compared to baseline for both the UC-II group as well as the placebo group (Figure 4 ). Percent changes in times were calculated after normalizing the baselines against the reference range of baseline to day 7. The UC-II group exhibited average reductions of 31.9 ± 11.7% (p = 0.041), 51.1 ± 6.1% (p = 0.004) and 51.9 ± 6.0% (p = 0.011) at days 60, 90 and 120, respectively. By contrast, the reductions for the same time points for the placebo cohort, 21.9 ± 10.2% (p = 0.017), 22.2 ± 15.5% (p = 0.007) and 30.0 ± 11.8% (p = 0.012), were of lower magnitude but nonetheless statistically significant versus baseline. None of these between group differences achieved statistical significance.

The time to offset of joint pain was recorded immediately upon the subject stepping off the stepmill. Both groups began to recover from pain with the same rate resulting in no significant differences between groups in the time to initial offset of joint pain (p > 0.05).

Five individuals in the UC-II group and one in the placebo group reported no onset of pain by the end of study (see below and Table 5 ). Given this unexpected finding, an additional analysis was undertaken which included these individuals in the time to onset of initial pain analysis. The 10 minute limit of the stepmill procedure was used as the lower limit to pain onset. Under these conservative assumptions, supplementation with UC-II yielded statistically significant increases in time to onset of pain at day 90 (3.65 ± 0.7 min, p = 0.011) and day 120 (4.31 ± 0.7 min, p = 0.002) versus a baseline of 1.4 min for each visit. The between-group comparison at day 120 approached the statistical level of significance favoring the UC-II cohort (p = 0.051).

Impact of stepmill procedure on the onset of pain. Values are presented as Mean ± SEM. *p &#; 0.05 indicates a statistically significant difference from baseline. Number of completers: n = 19 in UC-II group (n = 3 dropouts; n = 5 did not have pain); n = 20 in placebo group (n = 6 dropouts; n = 1 did not have pain; n = 1 did not use stepmill).

Supplementation with UC-II resulted in statistically significant increases in the time to onset of initial joint pain at day 90 (2.75 ± 0.5 min, p = 0.041) and at day 120 (2.8 ± 0.5 min, p = 0.019) versus a baseline of 1.4 min for each visit. No statistically significant differences were noted for either the placebo group or between groups (Figure 3 ).

Figure 2 summarizes the average knee extension changes over time for subjects supplemented with either UC-II or placebo. The UC-II supplemented cohort presented with a statistically significant greater increase in the ability to extend the knee at day 120 as compared to the placebo group (81.0 ± 1.3º vs 74.0 ± 2.2º, p = 0.011) and to baseline (81.0 ± 1.3º vs 73.2 ± 1.9º, p = 0.002). The UC-II group also demonstrated a significant increase in knee extension at day 90 (78.8 ± 1.9º vs 73.2 ± 1.9º, p = 0.045) compared to baseline only. An intent to treat (ITT) analysis of these data also demonstrated a statistically significant net increase in knee extension at day 120 versus placebo (80.0 ± 1.3º vs 73.7 ± 1.8º, p = 0.006). No statistically significant changes were observed in the placebo group at any time during this study. With respect to knee flexion, no significant changes were noted in either study group (p > 0.05). The power associated with the former per protocol statistical analyses was 80%.

A total of 55 individuals met the eligibility criteria and were randomized to the placebo (n = 28) or to the UC-II (n = 27) group. Baseline demographic characteristics for subjects in both groups were similar with respect to age, gender, height, weight and BMI (Table 4 ). A total of nine subjects, three in UC-II group and six in placebo group, were lost to follow-up. The results presented herein encompass 46 total subjects, 22 subjects in the placebo group plus 24 subjects in the UC-II group. It should be noted that the average age of the study participants was approximately 46 years which is about 16 years younger than the average age observed in many OA studies [ 36 - 38 ].

Discussion

In this study, the UC-II supplement, consisting of undenatured type II collagen, was investigated for its ability to improve joint function in healthy subjects who develop joint pain while undergoing strenuous exercise. The rationale behind this approach centered on the hypothesis that strenuous exercise might uncover transient joint changes due to daily physical activities that are not attributable to a diagnosable disease. In the same way that nominally elevated blood levels of lipids, glucose plus high blood pressure and obesity can be predictive of future progression to diabetes and heart disease [41], the development of joint pain upon strenuous exercise may be indicative of possible future joint problems.

At study conclusion, we found that subjects ingesting the UC-II supplement experienced a significantly greater forward ROM in their knees versus baseline and placebo as measured by knee extension goniometry. Knee extension is necessary for daily function and sport activities. Loss of knee extension has been shown to negatively impact the function of the lower extremity [42,43]. For example, loss of knee extension can cause altered gait patterns affecting ankles and the hip which could result in difficulty with running and jumping [42,43]. Studies have further shown that a permanent loss of 3-5º of extension can significantly impact patient satisfaction and the development of early arthritis [44].

By contrast, when knee flexion, another measure of knee function, was assessed via goniometry, no differences in clinical outcomes were observed between the two study cohorts. From a structure-function perspective this outcome is not surprising. During the earliest characterized phases of OA there is an apparent preferential loss of knee extension over knee flexion, and this loss has been shown to correlate with WOMAC pain scores [45,46]. In addition, MRI imaging of the early osteoarthritic knee has shown that initial changes in knee structure appear to center on articular cartilage erosions (fibrillations) about the patella and other weight bearing regions of the knee [47]. Such changes might favor a loss in knee ROM that preferentially affects extension over flexion. The pathophysiology of the early osteoarthritic knee, we believe, provides insight regarding the effect of daily physical activities on the healthy knee insofar as it helps explain the discordance in clinical outcomes between knee extension and flexion.

Both the time to onset of initial joint pain as well as time to full recovery were measured in this study. For each of these measures the clinical outcomes favored the UC-II supplemented cohort versus their baseline status. The ability of UC-II to modulate knee extension may relate to its ability to moderate knee joint pain. Crowley et al. [26] and Trentham et al. [25] demonstrated that UC-II effectively enhances joint comfort and flexibility thereby improving the quality of life (QoL) in both OA and RA subjects, respectively. This effect may be attributable to the finding that microgram quantities of undenatured type II collagen moderate CIA in both the rat and the mouse via the induction of T regulator cells [27,28,48]. The induction of these T regulators takes place within gut associated lymphatic tissues (GALT), including mesenteric lymph nodes, in response to the consumption of undenatured type II collagen [27]. Studies have shown that these regulatory cells produce IL-10 and TGF-β [30,49]. A special class of CD103+ dendritic cells, found almost exclusively in the GALT, facilitates this process [48,50]. Once activated, T regulator cells appear to downregulate a wide range of immunologic and proinflammatory activities resulting in the moderation of the arthritic response initiated by undenatured type II collagen [27]. The phenomenon of oral tolerance has also been demonstrated in humans, and appears to involve a similar set of T regulators [30,51-53].

The above description of how UC-II might modulate joint function is most easily understood in the context of RA given that the CIA animal model resembles this disease most closely [27,28,54]. However, the case for T regulators and immune cytokines having a moderating effect on healthy or OA knee joint function appears less apparent. This view has changed in recent years due to a growing body of evidence suggesting that both OA and normal chondrocyte biology appears to be regulated by some of the same cytokines and chemokines that regulate inflammation [5,6,55]. For example, Mannelli and coworkers [56] recently reported that feeding microgram amounts of native type II collagen (porcine) prevents monoiodoacetate-induced articular cartilage damage in this rat model of osteoarthritis, as measured by pain thresholds and by circulating levels of cross linked c-telopeptides derived from type II collagen. This finding corroborates the efficacy of undenatured type II collagen in improving joint comfort in osteoarthritic conditions [26].

In the present study, we show for the first time that UC-II can improve joint function in healthy subjects undergoing strenuous physical exercise. This observation, when considered in context with normal chondrocyte physiology, suggests that activated T regulator cells, specific for undenatured type II collagen, home to an overstressed knee joint where their release of the anti-inflammatory cytokines, IL-10 and TGF-β reverse the catabolic changes caused by strenuous exertion [13,21,57]. In addition, the IL-10 and TGF-β produced by these T regulators may tilt the TH balance in the knee joint towards TH2 [30,58] responses which preferentially result in IL-4 production further fostering a shift in chondrocyte metabolism towards ECM replenishment.

Several additional tests were used in this study to assess overall joint function, QoL, and physical activity. The additional parameters and tests measured included a six minute timed walk plus the Stanford exercise scale and KOOS survey. With respect to the KOOS survey, both cohorts were statistically significant versus baseline for symptoms, pain, daily function, recreational activities and QoL but were not significant from each other. This is not an unexpected finding given that this study was carried out with healthy subjects who do not present with any joint issues at rest. It is only when the knee is stressed via the stepmill do subjects report any joint discomfort. Under these conditions, and as indicated above, the UC-II group appears to experience less joint discomfort and greater joint flexibility. No difference in clinical outcomes between groups was seen in the six minute timed walk, the daily distance walked, or the Stanford exercise scale questionnaire. Once again we are not surprised by these results given that these tests and questionnaires are designed and clinically validated to assess the severity of arthritic disease in unhealthy populations.

No clinical biomarkers associated with arthritic diseases were assessed in this study. Healthy subjects would not be expected to present with significant alterations in their inflammatory biomarker profile as they lack clinical disease [59]. In addition, it should be noted that the joint discomfort measured in this study is acute pain induced by a stressor rather than due to an ongoing inflammatory event. Therefore, any elevation in inflammation markers that might occur in these healthy subjects may simply be due to the physiological impact of strenuous exercise.

There are two study limitations to consider when reviewing these results. The first, time to onset of initial pain, was limited to a 10-minute interval. The current study design did not address the possibility that subjects might cease to experience pain on the stepmill. Future studies should allow for an extension of the exertion interval in order to gauge how much longer a subject can exercise before reporting pain. In this way better defined parameters can be placed upon the degree to which UC-II supplementation results in the cessation of joint pain due to strenuous exercise in healthy subjects.

The second limitation that merits consideration is the possibility that study subjects may have early signs of arthritis that do not meet the ACR criteria. This possible limitation was addressed by performing an extensive medical examination for signs and symptoms of OA and by excluding volunteers who experienced pain levels of 5 or greater within one minute of using the stepmill.

UC-II is a unique ingredient that supports healthy joints. Previous studies have focused on the efficacy of this ingredient in OA subjects. By including healthy subjects in this study, and using non-disease endpoints as a measure of efficacy, it is believed that the benefits that derive from UC-II usage now extends to include healthy individuals. Further, this ingredient appears to be safe for human consumption based on an extensive series of in vivo and in vitro toxicological studies as well as the absence of any adverse events in this and in previous human studies [24,26,60]. In conclusion, daily supplementation with 40 mg of UC-II supports joint function and flexibility in healthy subjects as demonstrated by greater knee extension and has the potential both to alleviate the joint pain that occasionally arises from strenuous exercise as well as to lengthen periods of pain free exertion.

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