The use of immortalized mast cells (P815) and macrophages (J774) as surrogates to study mast cell-macrophage interactions during Francisella tularensis infection

Francisella tularensis is a Gram negative coccobacillus and the etiological agent of the zoonotic disease tularemia. Due to its low infectious dose, high mortality, and ability to be aerosolized it has been studied as a potential future bioweapon. If such an attack were to occur, pulmonary tularemia would be the most common manifestation of the disease, consequently, immune mechanisms that control and resolve Francisella infection are currently being studied. Most studies involving Francisella use an attenuated live vaccine strain (LVS) due to its low infectivity in humans but high virulence in mice. Previously, an in vitro bone marrow derived mast cell and macrophage system has been established to study interactions between these two cell types during infection. However, due to the length of time needed for generating bone marrow derived cells and the variability of these cells, a cell line model system was investigated in this study. It was found that the P815 mastocytoma cell line and J774 macrophage cell line serve as reliable surrogates for the bone marrow derived cells. These cells were infected with Francisella tularensis LVS for various periods and assayed for intracellular bacterial replication as well as cytokine production. P815 mast cells are not susceptible to high levels of intracellular LVS replication and also produce an inhibition of replication within the J774 macrophages, a highly permissive cell type. In addition, supernatants from these cultures show increased levels of IL-4. These results mimic those found in the bone marrow derived system, indicating that this cell line model is an appropriate substitute. To determine the mast cell to macrophage ratio required for optimal inhibition of intramacrophage LVS replication, various titrations of mast cells were co-cultured with J774 macrophages. As P815s were removed from the co-cultures, LVS replication within J774 macrophages increased. This increase also correlates with decreasing levels of IL-4 in the cultures. It was found in co-culture that a ratio of up to 5 macrophages to 1 mast cell resulted in optimal inhibition of intramacrophage LVS replication. Collectively, these results provide a cell line model to study mast cell and macrophage interactions during Francisella infection. Additionally, the P815 and J774 cell lines can be genetically manipulated to study various mechanisms involved in the innate immune response to tularemia.