Figure 6. Association of LVS with a specific cell population in infected lymph nodes.
Mice were infected intra-nasally with 105 CFU of LVS. Forty-eight hours later MdLNs were collected from 10 animals and single cell suspensions were prepared and examined by several assays. (A) Association between viable bacteria and MdLN cells was assayed by examining non-washed (total LN suspension) and washed cells for presence of viable LVS prior or post gentamicin treatment. Bacterial counts are presented as CFU per a single MdLN. (B) Association between LVS and RTDC cells was assayed by subjecting the washed cell fraction (∼108 cells) to sorting by magnetic microbeads coated with either anti-CD11b (B, top panel) or anti-CD11c (B, lower panel). Viable bacteria (no gentamicin treatment) were counted in the input cell suspension, in the bound cell fraction, as well as in cells that did not bind to the microbeads, and presented as CFU/106 cells. The amounts of cells in the input and in the bound fractions were ∼108/10 MdLNs and in the bound fractions only ∼106/10 MdLNs. CD11b and CD11c sorted cells were also examined by fluorescence microscopy for presence of intracellular bacteria. Typical cells carrying bacteria are presented in the insets to (B). (C) The cell/bacteria association in the CD11b-sorted fraction was characterized by determining bacterial count in non-treated cells, in cells treated with gentamicin (genta), in cells treated with gentamicin followed by washing and saponin lysis (genta+sap), as well as in cells where saponin treatment preceded gentamicin treatment (sap+genta). All bacterial counts were performed in triplicates, error bars represent variation of these counts. Data presented in this figure were collected from a single experiment. Two additional CD11b sorting experiments and one additional CD11c sorting experiment were conducted, exhibiting similar enrichment of intracellular bacteria.