Human lymph node derived fibroblastic and double negative reticular cells alter their chemokines and cytokine expression profile following inflammatory stimuli

Lymph node is a secondary lymphoid organ that has structural organization related to their immune function, which is highly ordered and compartmentalized. Lymph nodes shelter immune cells and also fibroblastic-like cells such as fibroblastic reticular cells (FRCs). FRCs have mesodermal origin and are classically characterized by the expression of podoplanin (PDPN/gp38) and lack of CD31 expression. FRCs are implicated in several immune processes but the pathways subjacent their function are still a matter of research. Another cell population found in LNs, the double negative cells (DNCs), are even less known. They do not express PDPN or CD31 markers and a specific marker is absent, their localization within the LN is undefined and their phenotype or function, remain to be clarified. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. This study evaluates gene expression and secretion of cytokines and chemokines in human-derived FRCs and DNCs during homeostasis and following inflammatory stimuli. Our results demonstrate that cytokines and chemokines secreted by human FRCs and DNCs partially differ from those identified in murine models. In addition, our study demonstrates that DNCs expressed a more varied of cytokines when compared with FRCs, while FRCs expressed a wider chemokines pattern. Such differences maybe related with specific functional differences between those cell populations within LNs. Peripheral (PLN) and mesenteric human LN (MLN) were obtained from 04 individuals with different ages and clinical status (larynx cancer-LN03, diverticulitis-LN12, breast cancer-LN15 patients and a liver donor-LN16) submitted to surgical procedures.The FRCs and DNCs were cultured with or without inflammatory stimuli provided by TNFα (25ng/mL) in combination with IL-1β (5ng/mL) treatments (17, 18). The culture supernatant was removed 24 hours after treatment and frozen for ELISA assays. The cells that remained attached to the culture plate were harvested for RNA extraction. In total 4 LNs were used for gene expression analysis, 2 LNs isolated from cancer patients (LN04 and LN15), 1 from a diverticulitis patient (LN12) and 1 isolated from a healthy liver donor (LN16). We compared treated and control FRCs and DNCs: cells were treated or not with INF-γ (50ng/mL) or TNFα (25ng/mL) in combination with IL-1β (5ng/mL).