Multiple particle tracking demonstrates sex differences in the lymph node microenvironment

Introduction: The lymph node (LN) is a highly specialized organ within the immune system, crucial in orchestrating the adaptive immune response. LNs are critical hubs where naïve B and T lymphocytes encounter cognate antigens, sparking the activation and response of the immune system to foreign substances. LNs uniquely modify their size in response to an immune challenge, to accommodate increased numbers of infiltrating and proliferating lymphocytes. This behavior is supported by a robust three-dimensional network of extracellular matrix (ECM) fibers and fibroblastic reticular cells (FRCs). ECM fibers and FRCs work synergistically to alternate load bearing between them, allowing LNs to withstand forces exerted by lymphocytes during different stages of expansion. Mechanical cues from the extracellular microenvironment have been shown to influence immune cell functionality, however the effect of sex-based differences on these microenvironments is less explored. Males and females exhibit significant disparities in their response to self and foreign antigens, with females generally displaying higher lymphocyte counts, greater T cell proliferation and stronger antibody responses. While this allows for higher vaccine efficacy and improved recovery times, it also increases susceptibility to autoimmune disorders and other pathologies. Therefore, exploring how sexual dimorphism influences the LN microenvironment will clarify structure-function relationships within the LN and enhance our understanding of sex-based variations in immune response. This study utilizes multiple particle tracking microrheology to evaluate and compare mechanical properties (loss/elastic moduli) on LN extracellular spaces in male and female mice and highlight the role of mechanical cues in their differential immune response.

Materials and

Methods: 6–12-week-old male and female C57BL/6 mice are euthanized via CO2 inhalation, and their inguinal skin draining lymph nodes collected. The lymph nodes are sliced using a vibratome and their mechanical properties are assessed using multiple-particle tracking (MPT). MPT involves tracking nanoparticles, moving via Brownian motion within live LN slices and calculating their mean-square displacement (MSD). The MSD values can then be used to extrapolate the microrheological properties of the tissue, including the pore-size, elastic and loss modulus and the microviscosity. Additionally, using lipopolysaccharide (LPS) to inflame the LNs, we perform MPT on collected tissue on day 1, 3 (peak inflammation) and day 14 (resolution) to understand mechanical differences between male and female mice over the duration of an inflammatory response.

Results, Conclusions, and Discussions: We found that sex affects the diffusive behavior of nanoparticles in LN extracellular tissues. When comparing nanoparticle MSDs in the B cell (outer cortex) and T cell (inner paracortex) zones between male and female mice, female mice exhibit higher MSD values both overall and within the B-Cell zone (figures 1a/1b). Interestingly, the diffusive behavior in the T-cell zones is similar between males and females, but a striking difference is observed in the B cell zone. There were significant differences in nanoparticle MSDs between the B-cell and T-cell zones in both male and female mice. In male mice, we observed higher MSD values in the T-cell zones (figure 1c) than the B-cell zone. Notably, these trends are reversed in female mice (figure 1d), with higher MSDs in the B-cell zones. We posit that this could a result of varied pore sizes caused by differential cell counts, with females known to have higher B-cell counts than their male counterparts. For uninflamed lymph nodes in female mice, we observe an average pore size of 690 ± 10 (SD) nm. Additionally, the pore size increases 1 day after LPS stimulation (1030 ± 10 (SD) nm) but decreases at day 3 (880 ± 10 (SD) nm), likely due to expansion and the increased cell density over the course of inflammation. LNs also exhibit viscoelastic behavior, with female LNs 3 days post LPS injection exhibiting the highest elastic (G’) and loss (G’’) moduli (1.33 and 0.94Pa respectively), with a G’/G’’>>1 indicating solid or elastic tissue response. For male LNs we expect similar trends in pore size and moduli, but smaller magnitudes than female mice due to lower cell numbers.

Understanding the role of sex-based differences in immunological studies not only improves their rigor and reproducibility but will also help guide the development of more personalized interventions to various pathologies. Our work underscores differences in lymph node biomechanics between males and females and provides perspective on how the biomechanics change after inflammatory cues. Further studies are needed to investigate the underlying cause of these mechanical differences and if they affect cellular behavior within the lymph node.

Acknowledgements (Optional):