Allergy-induced systemic inflammation impairs tendon quality

Abstract

Background: Treatment of degenerating tendons still presents a major challenge, since the aetiology of tendinopathies remains poorly understood. Besides mechanical overuse, further known predisposing factors include rheumatoid arthritis, diabetes, obesity or smoking all of which combine with a systemic inflammation.

Methods: To determine whether the systemic inflammation accompanying these conditions contributes to the onset of tendinopathy, we studied the effect of a systemic inflammation induced by an allergic episode on tendon properties. To this end, we induced an allergic response in mice by exposing them to a timothy grass pollen allergen and subsequently analysed both their flexor and Achilles tendons. Additionally, we analysed data from a health survey comprising data from more than 10.000 persons for an association between the occurrence of an allergy and tendinopathy.

Findings: Biomechanical testing and histological analysis revealed that tendons from allergic mice not only showed a significant reduction of both elastic modulus and tensile stress, but also alterations of the tendon matrix. Moreover, treatment of 3D tendon-like constructs with sera from allergic mice resulted in a matrix-remodelling expression profile and the expression of macrophage-associated markers and matrix metalloproteinase 2 (MMP2) was increased in allergic Achilles tendons. Data from the human health study revealed that persons suffering from an allergy have an increased propensity to develop a tendinopathy.

Interpretation: Our study demonstrates that the presence of a systemic inflammation accompanying an allergic condition negatively impacts on tendon structure and function.

Funding: This study was financially supported by the Fund for the Advancement of Scientific Research at Paracelsus Medical University (PMU-FFF E-15/22/115-LEK), by the Land Salzburg, the Salzburger Landeskliniken (SALK, the Health Care Provider of the University Hospitals Landeskrankenhaus and Christian Doppler Klinik), the Paracelsus Medical University, Salzburg and by unrestricted grants from Bayer, AstraZeneca, Sanofi-Aventis, Boehringer-Ingelheim.

Keywords: Allergy; Systemic inflammation; Tendinopathy; Tendon.

Figure 1

Induction of an allergic response in mice. (A) Experimental setup. (B) Multiplex bead array analysis reveals significantly elevated serum levels of TH1/pro-inflammatory (IFNγ, TNFα, IL1β, IL2, IL6), TH2/Treg related cytokines (IL4, IL5, IL10, IL13), TH17 cytokines IL17A and IL17F and selected chemokines in the blood of allergic mice compared to control or Alum-treated animals; data are shown as Mean + SEM, *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA (Kruskal-Wallis and Dunn's Multiple Comparison Test), n (control/allergy) = 16, n (Alum) = 6. (C) Concentrations of selected cytokines used to formulate a cocktail simulating the cytokine concentrations measured in mouse sera.

Figure 2

Biomechanical testing parameters. (A) Flexor tendon diameters (red line) were measured at 0.1N preload using two cameras positioned at an angle of 90° to each other. (B) The cross sectional area of tendons from allergic mice was significantly larger than that from control animals; *p<0.05, Mann-Whitney U test, n≥22. (C) Tendons from allergic animals displayed a significantly reduced stiffness; *p<0.05, Unpaired 2-tailed t test, n≥21). (D) No difference in maximal tensile load could be detected; Unpaired 2-tailed t test, n≥21. (E) Young‘s modulus and (F) stress to failure were significantly lower in tendons from allergic mice; *p<0.05, Unpaired 2-tailed t test, n≥21. Scale bar: 1mm.

Figure 3

Structural and biochemical analysis of mouse Achilles tendons. (A) Examination of histological sections under polarized light revealed a significantly less dense collagen packing in tendons from allergic mice; *p<0.05, Mann-Whitney U test, n≥11. (B) Collagen fibers were less parallelly oriented in tendons from the allergy group; *p<0.05, **p<0.01, Mann-Whitney U test, n≥5. (C, D) No significant difference between groups could be seen in shape and orientation of cell nuclei; Mann-Whitney U test, n≥5. (E) Polarized light microscopic images. Scale bar: 100 µm. (F, G) Ultrastructural analysis of mouse Achilles tendons. Shown are frequencies of fibril diameters of control and allergic mice determined by TEM (n = 600 fibrils of ≥5 animals each). Mean ± SEM are displayed. Analysis of electron microscopic images of Achilles tendons did not show a significant difference in fibril size distribution (insert). The statistical model of Linear mixed model fit by maximum likelihood (‘lmerMod’) was used yielding a p-value of p = 0.55. Scale bar: 500 nm. (H) Glycosaminoglycan (GAG) analysis revealed a significantly increased amount of GAGs in tendons from allergic animals; *p<0.05, Unpaired 2-tailed t test, n = 3.

Figure 4

qPCR and western blot analysis on 3D tendon-like constructs. (A) Image of tendon-like constructs spread between silk sutures mounted on a collagen-coated petri dish. (B) Treatment of tendon-like constructs with an allergy stimulating cytokine cocktail (ASCC) results in upregulation of selected target genes; *p<0.05, **p < 0.01, ***p<0.001, ****p<0.0001, Mann-Whitney U test (◊) or Unpaired 2-tailed t test (○) depending on normal distribution, n = 9. (C) Western blot analysis of tendon-like constructs exposed to the ASCC shows a significant upregulation of Mmp1 and Mmp2; Bars represent mean ± SEM; *p<0.05, Mann-Whitney U test, n = 4. Scale bar: 1cm.

Figure 5

MMP2 expression in mouse Achilles tendons. (A) Quantification of immunofluorescence staining on cryosections reveals a significantly increased MMP2 expression in tendons from allergic mice; representative image and quantification; *p<0.05, Mann-Whitney U test, n ≥ 5. (B) Western blot analysis shows upregulation of MMP2; representative image and densitometric quantification, normalization to ß-actin; *p<0.05, Unpaired 2-tailed t test, n = 3. (C) MMP2 activity is upregulated in allergic animals as shown by gelatin zymography; representative image and densitometric quantification, normalization to ß-actin; **p<0.01, Unpaired 2-tailed t test, n = 3. Scale bar: 50 µm.

Figure 6

Macrophage marker expression in vivo: Achilles tendons from allergic mice show a clearly increased expression of (A) F4/80, (B) CD68, (C) CD163, and (D) IBA-1 (p=0.0519) compared to control animals as evidenced by fluorescent staining of cryosections. Representative images and densitometric quantification are shown; *p<0.05, Mann-Whitney U test, n ≥ 6. Scale bar: 50 µm.

Figure 7

Perilipin expression in vivo and in vitro. (A) Perilipin is significantly higher expressed in Achilles tendons from allergic mice; representative image and quantification of perilipin expression, normalized to control settings; *p<0.05, Unpaired 2-tailed t test, n ≥ 11. (B) In vitro the number of perilipin positive cells in 3D tendon-like constructs exposed to ASCC is increased compared to controls; **p<0.01, Mann-Whitney U test, n ≥ 8. Blue DAPI stain indicates cell nuclei. White arrows point toward perilipin positive cells. White asterisk indicates adipose tissue. The dotted blue line demarcates the tendon proper. Scale bar: 100 µm.

Figure 8

Graphical summary (Created with BioRender.com.)