YAP and TAZ in Fibroblastic Reticular Cells Support Hematopoiesis and Retention of Lymphocytes in Lymph Nodes

INTRODUCTION: Fibroblastic reticular cells (FRCs) are essential for adaptive immune response and maintaining lymph node (LN) homeostasis. FRCs regulate immune cell entry into the LN and confer compartmentalization of lymphocytes by secretion of essential chemokines. Indeed, FRCs are activated and display an undifferentiated phenotype in LN of patients with follicular lymphoma, but their meaning and mode action are unclear. The mammalian Hippo signaling pathway is an essential regulator of cell fate, differentiation, and homeostasis. Its final downstream effectors YAP and TAZ (YAP/TAZ) are inhibited by LATS1 and LATS2 (LATS1/2) in a phosphorylation-dependent manner. The roles of YAP/TAZ in hematopoiesis are considered to be dispensable under healthy physiological condition, but their significance appears to be context dependent in pathologies including hematologic malignancies.

OBJECTIVES: We hypothesized that the Hippo pathway plays important roles in regulating FRCs by interacting with lymphocytes, with relevance to structural and functional properties of LNs. Using FRC-specific YAP/TAZ depletion or hyperactivation mouse models, we sought to investigate the roles of YAP/TAZ in orchestrating lymphocyte homeostasis by FRCs.

METHODS: Animal care and experimental procedures were performed under the approval of the Institutional Animal Care and Use Committee (KA2016-12) of KAIST. Yap/Taz^∆FRC or Lats1/2^∆FRC mouse were generated by crossing FRC-specific Ccl19-Cre or inducible Pdgfrb-Cre-ER^T2 mouse with Yap^fl/fl/Taz^fl/fl or Lats1^fl/fl/Lats2^fl/fl mouse, respectively. Cre-ER^T2-negative but flox/flox-positive littermates were defined as wild-type mice (WT). In order to induce Cre activity, 2 mg of tamoxifen (Sigma-Aldrich) was dissolved in corn oil and intra-peritoneally injected for four consecutive days. Skin-draining LNs were analyzed with immunofluorescence staining, flow cytometry, or fluorescence activated cell sorting. Chemokine expression was measured from sorted FRCs with qRT-PCR, immunoblotting, or ELISA. APC BrdU flow kit (BD Biosciences) was used for cell cycle analysis. Blood count in mice was measured with hemocytometer (pocH-100iV DiFF, Sysmex).

RESULTS: Both YAP/TAZ were highly and equally distributed in nucleus and cytoplasm of mouse LN FRCs, in addition to blood and lymphatic endothelial cells. LNs of Yap/Taz^∆FRC mice compared with WT revealed a slight reduction in LN weight and reduced total cell number (~64.9%), decreased proportion (~42.9%), and number (~61.1 %) of FRCs among CD45^- stromal cells, which were less proliferative (~65.2%) but had no difference in apoptosis. However, neither proliferation nor apoptosis of immune cells was altered. Analysis of isolated FRCs revealed that mRNA levels of lymphoid chemokines for immune cell trafficking and function were significantly attenuated in Yap/Taz^ΔFRC mice compared with WT. Intriguingly, Lats1/2^∆FRC mice looked pale from 1 week after birth. We found lower red blood cell (RBC) count (~16.1%) with decreased concentration of hemoglobin (~25.8%) in Lats1/2^∆FRC mice compared with WT, suggesting hypochromic anemia. Moreover, although whole body length and overall organ size were small in Lats1/2^∆FRC mice compared with WT at 2 weeks after birth, we could not observe any gross or microscopic abnormalities in major organs including brain, heart, lung, liver, and kidney in Lats1/2^∆FRC mice. We further analyzed the primary lymphoid organ thymus and the secondary lymphoid organ spleen, as they are both supported by CCL19⁺ FRCs. Similar to major organs, the overall size of thymus and spleen was smaller in Lats1/2^∆FRC mice compared with WT at 2 weeks after birth. However, microscopic analysis of thymus revealed that the border between the cortex and medulla was disrupted by the expanded medulla at the periphery that blends into the surrounding cortex in Lats1/2^∆FRC mice, and analysis of spleen also revealed unclear lymphoid follicles with significantly reduced number of immune cells within the expanded white pulp in Lats1/2^∆FRC mice compared with WT.

CONCLUSIONS: Our findings present YAP/TAZ as essential governors of FRCs by supporting hematopoiesis and lymphocyte homeostasis. Considering that YAP/TAZ are enriched in bone marrow stromal cells, additional work to elucidate their role in promoting bone marrow microenvironment will improve our understanding on hematopoietic stem cell niche.