Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation

Abstract

While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

Fig. 1. Strategy for generation of CCL2 floxed mice and Cre-mediated excision

Schematic shows the wild-type CCL2 gene targeting construct, and the homologous recombinant allele in ES cells. A loxP site was inserted upstream of exon 1 and an FRT-PGK-neo-FRT-loxP cassette from PL452 plasmid was inserted downstream of exon 3. The positions of PCR primers used for each screening step are displayed at the approximate locations. P1, P2, P3 and P4 show the location of the different primers used in the PCR genotyping analysis. Scr1, Scr2, Scr3 and Scr4 show the location of nested PCR primers for 3′ arm region confirmation, while Scr5, Scr6, Scr7 and Scr8 show the same for 5′ arm region confirmation.

Fig. 2. Molecular characterization of cell-targeted CCL2 deletions

(A) Identification of homologous recombination in ES cells. Correctly targeted ES cells were identified by nested PCR. The primers Scr1, Scr2, Scr3 and Scr4 for nested PCR of 3′ region confirmation amplify a 3013 bp fragment; the primers Scr5, Scr6, Scr7 and Scr8 for nested PCR for 5′ region pair amplify a 2992 bp fragment. The lane designations (+) and (−) on the far right indicate positive and negative controls, respectively. (B) Genotyping of CCL2^loxP/+ and CCL2^loxP/loxP mice Mouse DNA was analyzed by PCR: primers P1/P2 result in a 300 bp fragment for the CCL2 wild type allele: primers P3/P4 result in a 390 bp fragment for the CCL2 mutant allele; primers P5/P6 result in a 256 bp fragment for the CCL2 wild type allele; and primers P7/P8 result in a 356 bp fragment for the CCL2 mutant allele. (C and D) Genotyping of CCL2^loxP/loxP/GFAP-Cre and CCL2^loxP/loxP/Tie2-Cre mice. Mouse DNA was analyzed by PCR: primers P5/P6 result in a 300 bp fragment for the Tie2-Cre gene; primers P7/P8 result in a 190 bp fragment for the GFAP-Cre gene. The lane designations (+) and (−) reflect presence or absence, respectively, of the Cre gene.

Fig. 3. Targeted CCL2 gene deletion in astrocyte and BMEC cultures from conditional CCL2 KO mice

Separate astrocyte and BMEC cultures were derived from opposite cerebral hemispheres of the following groups of mice at postnatal day 5–6: CCL2^loxP/loxP/GFAP-Cre (astrocyte KO); CCL2^loxP/loxP/Tie2-Cre (endothelial KO); and CCL2^loxP/loxP (W.T. littermates of the respective KOs). (A, B) PCR analysis of genomic DNA (100 ng) from astrocytes and BMEC, indicating (A) the deletion band (256 bp) representing the excised CCL2 gene, and (B) Presence of the Cre gene. (C, D) RT-PCR analysis (28 cycles) of RNA (200 ng) from astrocytes (+LPS) and BMEC, showing results for (C) CCL2 mRNA and (D) β-actin mRNA (indicating loading efficiency).

Fig. 4. Quantification of CCL2 protein and RNA expression in astrocyte and BMEC cultures from conditional CCL2 KO mice

Cultures were established as in Fig. 4 from CCL2^loxP/loxP/GFAP-Cre (astrocyte KO), CCL2^loxP/loxP/Tie2-Cre (endothelial KO) and CCL2^loxP/loxP (W.T. littermate)mice. Astrocytes were stimulated with LPS (10 μg/ml, 6 hr), while BMEC were unchallenged. (A) Supernatants were collected from cultures and analyzed for CCL2 protein by ELISA. (B) Following removal of supernatants, cells were processed for RNA, and analysis performed by qRT-PCR (40 cycles). The relative difference in CCL2 RNA levels between KOs and controls was > 32-fold in all cases (some CCL2 RNA coming from minor contaminating cells).

Fig. 5. In situ retrieval of astrocytes and BMEC by immuno-LCM

Arrowheads point to CD31+ BMEC in microvessels, and arrows demarcate GFAP+ astrocytic processes. Top figure shows selective capture of astrocyte, while bottom figure shows retrieval of a single microvessel. before, shows tissue section before LCM; after, shows same section following cellular retrieval. The small images to the side show the astrocyte and microvessel, respectively, each deposited onto a cap, from which the tissue is extracted and RNA isolated. Notably, the LCM process is highly selective in retrieving the targeted cell types; even the closely apposed astrocyte foot processes are not disturbed upon BMEC/microvessel retrieval (bottom figure).

Fig. 6. In vivo confirmation of cell conditional CCL2 KOs

CCL2^loxP/loxP/GFAP-Cre (astrocyte KO) and CCL2^loxP/loxP/Tie2-Cre (endothelial KO) mice, along with their corresponding CCL2^loxP/loxP (W.T.) littermates, were injected with LPS (i.p., 4 mg/kg). After 4 hr, brain tissue was subject to immuno-LCM to selectively retrieve BMEC (top) or astrocytes (bottom). RNA was isolated from the respective astrocyte and BMEC samples and subject to qRT-PCR to quantify relative CCL2 levels. For BMEC, CCL2 mRNA level was expressed relative to either the housekeeping gene RPL19 (left) or the endothelial marker CD31 (right). For astrocytes, CCL2 mRNA levels were normalized to either RPL19 (left) or the astrocyte marker GFAP (right).

Fig. 7. E- and P-selectin expression in response to LPS

Following LPS injection of CCL2^loxP/loxP/GFAP-Cre (astrocyte KO), CCL2^loxP/loxP/Tie2-Cre (endothelial KO), and CCL2^loxP/loxP (W.T.) littermate mice (as described in Fig. 6), brain tissue was frozen and cut at 7 μm. Individual brain sections (‘tissue scrapes’) representing each of these groups was then subject to TRIzol^®-mediated RNA extraction/isolation and qRT-PCR analysis for E- and P-selectin mRNA.