A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells

BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

Leishmaniasis is a neglected disease that affects more than 12 million people worldwide. In Brazil, the cutaneous disease is more prevalent with about 28,000 new cases reported each year, and L. braziliensis is the main causative agent. The interesting data about the infection with this parasite is the wide variety of clinical manifestations that ranges from single ulcerated lesions to mucocutaneous and disseminated disease. However, experimental models to study the infection with this parasite are difficult to develop due to high resistance of most mouse strains to the infection, and the mechanisms underlying the distinct manifestations remain poorly understood. Here, the authors use a mouse experimental model of infection with different L. braziliensis isolates, known to induce diseases with distinct severity in the human hosts, to elucidate immune mechanisms that may be involved in the different manifestations. They showed that distinct parasite isolates may modulate host response, and increased IL-4 production and Arg I expression was related to more severe disease, resulting in longer length of disease with larger lesions and reduced parasite clearance. These findings may be useful in the identification of immunological targets to control L. braziliensis infection and potential clinical markers of disease progression

Bone marrow-derived macrophage obtained from fresh or cryopreserved bone marrow cells similarly express CD80 and CD86 in response to LPS and <i>Legionella pneumophila</i>.

<p>BMDMs obtained from fresh or cryopreserved (frozen) bone marrow cells were left untreated (NS), stimulated with 1 µg/ml LPS or infected with <i>L. pneumophila</i> at an MOI of 0.5, 1 and 2. Cultures were cultivated in non-tissue culture-treated 6 well-plates at a density of 1×10⁶ BMDM per well and stimulated/infected for 18 hours. Cells were stained for CD11b, F4/80, CD80 and CD86 and evaluated by flow cytometry. Plots were gated on CD11b⁺ cells. Shown are the percentage of cells F4/80⁺CD80⁺ (A) and F4/80⁺CD86⁺ (C). The mean fluorescence intensities are shown for CD80 (B) and CD86 (D). Data are representative of those found in three independent experiments. Asterisks indicate statistically significant differences in relation to NS (<i>P</i><0.05). No statistically significant differences were detected between BMDM obtained from fresh or frozen BM cells (<i>P</i>>0.05).</p

Fresh or cryopreserved bone marrow cells show similar expression of cell markers.

<p>Cells from fresh or cryopreserved (frozen) bone marrow were harvested and labeled with phycoerythrin (PE)-conjugated Abs against CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, Sca-1 and PDGF or control IgGs and analyzed by FACS. (A) Dot plot showing the two gates analyzed (G1 and G2). (B and C) Averages and standard deviation of the triplicates show the percentages of the cells positive for each marker within the gated population: G1 (B) or G2 (C). Data are one representative experiment out of two independent experiments performed. No statistically significant differences were detected between fresh or frozen BM cells (<i>P</i>>0.05).</p