Interacting chemokine signals regulate dendritic cells in acute brain injury

Abstract

Brain trauma is known to activate inflammatory cells via various chemokine signals although their interactions remain to be characterized. Mice deficient in Ccl3, Ccr2 or Cxcl10 were compared with wildtype mice after controlled cortical impact injury. Expression of Ccl3 in wildtypes was rapidly upregulated in resident, regularly spaced reactive microglia. Ccl3-deficiency enhanced endothelial expression of platelet selectin and invasion of peripheral inflammatory cells. Appearance of Ccr2 transcripts, encoding the Ccl2 receptor, reflected invasion of lysozyme 2-expressing phagocytes and classical antigen-presenting dendritic cells expressing major histocompatibility complex class II. Ccr2 also directed clustered plasmacytoid dendritic cells positive for the T-cell attracting chemokine Cxcl10. A reduction in Ccr2 and dendritic cells was found in injured wildtype cortex after cyclophosphamide treatment resembling effects of Ccr2-deficiency. The findings demonstrate the feasibility to control inflammation in the injured brain by regulating chemokine-dependent pathways.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Traumatic brain injury (TBI) in wildtype (wt) and chemokine-deficient (Ccl3−/− and Ccr2−/−) mice.

(a) In situ hybridization revealed intense Ccl3 expression in the perilesional zone four hours after trauma, in a pattern resembling distribution of activated microglial cells. (b) Temporal patterns of Ccl3 and Ccr2 transcript levels in the injured wt neocortex revealed by qRT-PCR. (c) The Ccr2 transcript was further upregulated in Ccl3−/− mice as compared to wt mice three days postinjury. Gfap expression was also strongly upregulated but with no differences between the strains. (d) The Ccl2 transcript, encoding a Ccr2 ligand, was upregulated after injury with no differences between wt and Ccl3−/− mice as shown by microarray analysis. In contrast, Selp transcript expressed by endothelial cells was more upregulated in Ccl3−/− compared to wt brains. (e) Lyz2 expression and the microglial surface marker isolectin B4 (IB4) showed only partial overlap three days post-injury in wt brains (in situ hybridization and histochemistry). The Bst2, characterizing pDCs, exhibited a different expression pattern (insert). (f) At three days postinjury, the Lyz2 increase shifted in opposite directions in the Ccl3−/− and Ccr2−/− compared to wt brains. (g) Cavity volume was reduced in the Ccr2−/− compared to wt mice seven days after injury in accordance with downregulation of inflammatory Lyz2-response.

Figure 2. Interactions among chemokine transcripts analyzed with qRT-PCR and in situ hybridization in neocortex three days after TBI.

(a) The Ccl3 transcript expression did not differ between Ccr2−/− and wt brains. (b) Compared to wt mice, Cxcl10 transcript showed opposite changes in expression in Ccl3−/− and Ccr2−/− brains. (c) A clustered cell pattern was seen for the Cxcl10 transcript by in situ hybridization in all injured brains, although with different intensities depending on genotype. Expression was stronger in the injured Ccl3−/− mice and weaker in the Ccr2−/− mice compared to wt brains. (d) Ccl3 and Ccr2 upregulation showed no differences in wt compared to Cxcl10−/− injured brains by qRT-PCR. (e) Suggested model of interactions among chemokine ligands Ccl3 and Cxcl10 and the chemokine receptor Ccr2 three days after brain injury.

Figure 3. Inflammatory cells in the injured wt and Ccr2−/− cortex examined by flow cytometry and qRT-PCR.

(a) Cells detected with FITC-CD45 in combination with either APC-PDCA-1 (Bst2) or APC-Cd11c (Itgax) in neocortex of wt or Ccr2−/− mice three days after injury. Upper panel left shows gating for Cd11c. The middle and right panels show wt and Ccr2−/− results, respectively, demonstrating reduced number of Cd11c-positive cells in the Ccr2−/− mice. Lower panel shows that PDCA-1 positive cells were reduced in Ccr2−/− brains. (b) Quantitative flow cytometry data from wt and Ccr2−/− mice three days postinjury. Isotype controls showed only trace signals. (c) Temporal expression patterns of six inflammatory-related transcripts (Itgax, H2-Aa, Bst2, Cxcl10, Lyz2 and Hmox1) after injury in wt and Ccr2−/− brains.

Figure 4. Inflammatory-related transcripts measured in Ccl3−/− and Ccr2−/− mice with F2 hybrid background three days postinjury.

Lyz2 expression was upregulated in homozygous Ccl3 knockouts but downregulated in Ccr2−/− as in the parental strains, as was Bst2. Both Itgax and Cxcl10 levels were less upregulated in the injured F2 Ccr2−/− brains. Gfap expression increased to the same extent in injured wt and F2 hybrid brains.

Figure 5. Cell sorting and immunodepletion of dendritic cells in neocortex three days after TBI in wt mice analyzed by qRT-PCR.

(a) Cd11c-positive cells sorted on magnetic microbeads expressed enhanced H2-Aa and Itgax levels. In contrast, Bst2 was enriched in cells sorted on anti-PDCA-1 microbeads. (b) Cortical levels of inflammatory transcripts in mice injected with control immunoglobulin or with antibodies directed to PDCA-1. Depletion of pDCs was not accompanied by shifts in Itgax or H2-Aa transcript levels whereas a distinct reduction of Bst2 and Cxcl10 occurred.

Figure 6. Cyclophosphamide treatment in wt mice analyzed three days after TBI.

(a) Microarray showed markedly weakened injury-activated Ccr2 and H2-Aa levels while upregulation of the fractalkine receptor Cx3cr1 was not affected. (b) Itgax was also dampened in mice subjected to cyclophosphamide treatment as shown by qRT-PCR whereas Ccl3 upregulation was not impaired. (c) Flow cytometry demonstrated reduced number of brain cells gated for Cd11c in mice treated with cyclophosphamide. Isotype controls (upper middle panel) showed only trace labeling of inflammatory cells. Quantitative data from flow cytometry assays (right), show a substantial reduction of Cd11c-positive cDCs in the injured brains treated with cyclophosphamide compared to mice injected with saline.