Skip to main content
. 2021 Apr 8;10:e66190. doi: 10.7554/eLife.66190

Figure 6. In vivo analysis of Ccl19/Ccr7 axis during inflammation.

Pharmacological regulation of lipopolysaccharide (LPS)/Toll-like receptor (TLR) signaling pathway. (A) Annexin-V binding to membrane phosphatidylserine (PS) on lymph node (LN) myeloid populations from wild-type (WT) (left mosaic bars) and Traf6∆∕∆ (right mosaic bars) (n = 4 mice per group) after PBS or LPS administration. LN suspension cells were stained for myeloid surface markers including annexin-V and analyzed by flow cytometry. (B) Transwell migration of low-density bone marrow (LDBM)-derived LK cells toward gradient generated by pre-treated LN cells with dimethylsulfoxide (DMSO) (solid bars) as vehicle control and inhibitors (mosaic bars) against Irak1/4 (right lined), Ubc13 (left lined), and IKK (white squares), and following TLR signaling pathway activation by PBS (black) or LPS (blue). (C) Analysis of SNAP23 phosphorylation (Ser95) in LN myeloid cells previously treated with DMSO (solid bar) as vehicle control, Irak1/4 (right lined mosaic bar), Ubc13 (left lined mosaic bar), or IKK (white squares mosaic bar) inhibitors. Values represent two independent experiments as mean ± SD of two independent experiments performed in triplicate. (D) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 into LN-residing CD11blow/CD11c+ cDC from non-manipulated mice by flow cytometry. Values represent mean ± SD of two or three independent experiments. p<0.05, **p<0.01, ***p<0.001.

Figure 6.

Figure 6—figure supplement 1. Traf6 does not associate with changes in survival of hematopoietic cells, and progenitors and Ccl19 can be detected in a specific subpopulation of lymph node (LN) conventional dendritic cells (cDCs).

Figure 6—figure supplement 1.

(A) Apoptosis (TUNELassay) of LK progenitors, Lin- and Lin + bone marrow (BM) cells. (B) Representative overlap histograms of phospho-SNAP23 (pSNAP23) into LN myeloid cells treated with dimethylsulfoxide (DMSO) (upper left), Irak1/4-inh (upper right), Ubc13-inh (lower left), and IKK-inh (lower right) and stimulated with PBS (red lines) or lipopolysaccharide (LPS) (blue lines). (C) Gating strategy to determine Ccl19 pre-stored into LN-residing myeloid cells basally. (D) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 in pDC populations in basal conditions.