Nanoparticles reveal reversible and permanent changes in lymph node tissue structure after a single inflammatory insult

Introduction: The lymph node (LN) is a critical immune tissue whose main function is to coordinate adaptive immune responses. During inflammatory challenges like infections, LNs rapidly expand to withstand the influx of migrating and proliferating B and T cells. During the resolution of the inflammation, the LNs then slowly contract again. The extracellular spaces of the LN support its overall shape and structural integrity. Collagen fibers and their associated fibroblastic reticular cells work together to bear the extra tension during the expansion phase and produce extra collagen fibers during the contraction phase. Research has shown that the extracellular microenvironment plays a large role in the functions of immune cells including cell migration, proliferation, and differentiation. For example, aging and chronic inflammation cause fibrosis in LNs, which has been correlated with reduced T cell migration speeds and directionality, specifically in proximity to extracellular matrix-rich regions. Naïve T cell migration is critical for immune activation and reduced motility may contribute to reduced immune function. However, we have limited understanding of LN extracellular microenvironments in part due to lack of techniques that can assess biomechanical properties with enough detail to determine changes in the intricately structured LNs. We have previously developed nanoparticles that can be used to perform multiple particle tracking of the lymph nodes to assess changes in lymph node biomechanics including viscoelastic properties and pore size. Here we used this technology to assess how LN extracellular spaces during an inflammatory response.

Materials and

Methods: We used lipopolysaccharide (LPS), to model bacterial-induced inflammation of the skin draining LNs of mice and assessed the viscoelastic properties (including viscosity and elastic and viscous moduli) of the LN extracellular tissue using our previously developed nanoparticle systems. We then determined how the LN changes throughout expansion and contraction phases and if there are permanent changes associated with a single inflammatory event. We assessed biomechanical properties during the course of the inflammatory process on days 1, 3 (peak inflammation), 5, 7, and 14 (resolution). We then used multiple particle tracking (MPT) analysis which determines the mean squared displacement (MSD) of nanoparticles undergoing Brownian motion within live LN slices. This MSD value is then used to extrapolate pore size, viscosity, elastic modulus, and viscous modulus. Pore size is calculated using the equation PS=MSD^(1/2)+a, where a is the particle radius. Viscosity is calculated using the equation D = kBT/6πas⟨∆r^2(s)⟩, where kB is the Boltzmann constant, T is the temperature, and η is the material viscosity. Elastic modulus and viscous modulus is calculated by using a generalized Stokes-Einstein relationship and the Laplace transform of MSD and the relaxation modulus: G ̃(s) = kBT/πas⟨∆r^2(s)⟩. Using the frequency-dependent, complex modulus equation and substituting s with iω, G*(ω) = Gi(ω) +iG”, we can solve for the elastic and loss modulus. In addition, we use flow cytometry to assess changes in immune cell infiltration during inflammation.

Results, Conclusions, and Discussions: We found that by day 3 (peak inflammation) nodes have approximately doubled in size and gradually return to their original size by day 14 (full resolution). LNs had an elastic moduli of 0.48 ± 0.22 Pa in untreated mice and decreased on day 1 to 0.04 ± 0.04 Pa, showing that tissues relax during initial inflammation. As inflammation continued to day 3, tissues reached a max elastic modulus of 1.6 ± 0.57 Pa (Figure 1). Interestingly, the viscosity of LNs increased 29-fold from untreated mice to day 3. The increased stiffness and high viscosity also correlated with a 1.1-fold decrease in pore size averages on day 3. Our data suggests that B and T cell zones restructure differently, despite the dense extracellular network found in the T cell zone. T cell zones are stiffer with an elastic moduli of 3.7 ± 1.2 Pa on day 3 compared to 2.0± 0.69 Pa in B cell zones (Figure 2A). We saw that both B and T cell zones had a 20+ fold decrease in elastic moduli on day 3 versus day 14 (Figure 2A). B cell zones had, on average, a 95-fold increase in viscosity from day 1 to day 14, while T cell zones had only a 4-fold increase (Figure 2B). Interestingly, pore sizes in the T cell zone first increased on day 1 and then decreased on day 14, while B cell zones followed a seesaw trend (Figure 2C). Overall, we discovered that tissue is permanently restructured, regardless of zone. Even after full resolution at day 14, LNs are stiffer and have smaller pores and interstitial fluid is more viscous compared to untreated LNs (Figure 1). Our data corroborates previous claims that study LN mechanics and propose tissue relaxation at the onset of inflammation for the influx of proliferating and migrating cells. Our data suggests that B and T cell zones restructure their microenvironment independently, which may be caused by ECM restructuring in the T cell zone alongside germinal center formation in the B cell zone. Finally, we found that LNs are permanently restructured after only one inflammatory challenge.

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