Ex vivo Demonstration of Functional Deficiencies in Popliteal Lymphatic Vessels From TNF-Transgenic Mice With Inflammatory Arthritis.

Joshua P Scallan,Echoe M Bouta,Christopher T Ritchlin,Michael J Davis,Edward M Schwarz,Homaira Rahimi,H Mark Kenney

doi:10.3389/fphys.2021.745096

Joshua P Scallan, Echoe M Bouta + Show 5 more

Open Access

https://doi.org/10.3389/fphys.2021.745096

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Abstract

Background: Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. As the intrinsic differences between lymphatic vessels afferent to healthy, expanding, and collapsed draining lymph nodes are unknown, we characterized the ex vivo behavior of popliteal lymphatic vessels (PLVs) from WT and TNF-Tg mice. We also interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS).Methods: Popliteal lymph nodes (PLNs) in TNF-Tg mice were phenotyped as Expanding or Collapsed by in vivo ultrasound and age-matched to WT littermate controls. The PLVs were harvested and cannulated for ex vivo functional analysis over a relatively wide range of hydrostatic pressures (0.5–10 cmH2O) to quantify the end diastolic diameter (EDD), tone, amplitude (AMP), ejection fraction (EF), contraction frequency (FREQ), and fractional pump flow (FPF) with or without NOS inhibitors Data were analyzed using repeated measures two-way ANOVA with Bonferroni’s post hoc test.Results: Real time videos of the cannulated PLVs demonstrated the predicted phenotypes of robust vs. weak contractions of the WT vs. TNF-Tg PLV, respectively. Quantitative analyses confirmed that TNF-Tg PLVs had significantly decreased AMP, EF, and FPF vs. WT (p < 0.05). EF and FPF were recovered by NOS inhibition, while the reduction in AMP was NOS independent. No differences in EDD, tone, or FREQ were observed between WT and TNF-Tg PLVs, nor between Expanding vs. Collapsed PLVs.Conclusion: These findings support the concept that chronic inflammatory arthritis leads to NOS dependent and independent draining lymphatic vessel dysfunction that exacerbates disease, and may trigger arthritic flare due to decreased egress of inflammatory cells and soluble factors from affected joints.

Highlights

Rheumatoid arthritis (RA) is an inflammatory joint disease that affects 0.5–1% of the population (Firestein, 2003; Rudan et al, 2015)
Collapsed popliteal lymphatic vessels (PLVs) demonstrated significant increases in fractional pump flow (FPF) at all pressures following L-NAME administration (Figure 3I). These results indicate a substantial role for nitric oxide signaling in the reduction of PLV contractility during the Collapsed phase of TNF-mediated inflammatory arthritis
The retained deficiency in AMP during the Expanded phase following nitric oxide synthase (NOS) inhibition ex vivo (Figure 4C), and the incomplete resolution of lymphatic function following global inducible nitric oxide synthase (iNOS) ablation in vivo (Bell et al, 2019a), suggests additional mechanisms are likely associated with the deficiencies in PLV contractility during the progression of inflammatory arthritis, which is an active area of investigation

Summary

Introduction

Rheumatoid arthritis (RA) is an inflammatory joint disease that affects 0.5–1% of the population (Firestein, 2003; Rudan et al, 2015). Research is needed to elucidate nonautoimmune etiologies of RA Another major challenge for RA patients and caregivers is the tendency of the disease to flare, and the relative refractory nature of persistent disease despite aggressive therapy (Firestein, 2014). Recent gene expression studies have identified potential roles of immature IgD+ B-cells, circulating CD45−/CD31−/PDPN+ pre-inflammatory mesenchymal (PRIME) cells, and synovial macrophages in RA flare (Alivernini et al, 2020; Orange et al, 2020), it is possible that exacerbation of disease is due to the loss of protective mechanisms in the setting of chronic joint inflammation. Recent studies demonstrated lymphangiogenesis and expansion of draining lymph nodes during chronic inflammatory arthritis, and lymphatic dysfunction associated with collapse of draining lymph nodes in rheumatoid arthritis (RA) patients and TNF-transgenic (TNF-Tg) mice experiencing arthritic flare. We interrogated the mechanisms of lymphatic dysfunction through inhibition of nitric oxide synthase (NOS)

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