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Floxed anaphylatoxin receptor reporter mice as tools to study anaphylatoxin receptor expression and function

The anaphylatoxins receptors, i.e. C3a receptor (C3aR), C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2), contribute not only to tissue homeostasis but drive, perpetuate and resolve immune responses in many inflammatory diseases including infections, malignancies, autoimmune as well as allergic diseases. During the past few years, transcriptome expression data provided detailed insights into AT receptor tissue mRNA expression. In contrast, our understanding of cellular AT receptor expression in human and mouse tissues under steady and inflammatory conditions is still sketchy. Ligand binding studies, flow cytometric and immunohistochemical analyses convincingly demonstrated tissue-specific C5aR1 expression in various cells of myeloid origin. However, a detailed map for C3aR or C5aR2 expression in human or mouse tissue cells is still lacking. Also, reports about AT expression in lymphoid cells is still controversial. To understand the multiple roles of the ATs in the innate and adaptive immune networks, a detailed understanding of their receptor expression in health and disease is required. We have developed novel GFP-C5aR1 (Karsten et al. J. Immunol. 2015), C3aR- (Quell et al. J. Immunol. 2017) and C5aR2-tdTomato (Karsten et al. J. Immunol. 2017) knock-in mice that provided detailed insights into their expression pattern in tissue immune and stroma cells.

This project is designed to monitor intra- and extracellular AT receptor expression in models of allergy, autoimmunity, cancer and infection and to define the contribution of cell- and tissue-specific activation of individual AT receptors in such diseases.  

Figure 1 Generation of the floxed GFP-C5aR1 knock-in transgenic mice. (A) Schematic of the gene targeting strategy.An AcGFP1-IRES gene cassette flanked by two lox p sites was inserted directly upstream of exon 2 of the C5aR1gene by homologous recombination. This strategy was designed to produce a floxed GFP-C5aR1 knock-in mouse that allows for conditional knock-out studies. (B) Confocal microscopy of BM cells from C5aR1–/– (left), GFP-C5aR1flox/flox (middle) and C57BL/6 wt (right) mice, which were analyzed for GFP expression (shown in green) and stained with C5aR1-specific mAb (clone 20/70, shown in red) without permeabilization. Pictures are representative of at least three experiments.


References

  1. Karsten C.M., Laumonnier Y., Eurich B., Ender F., Bröker K., Roy S., Czabanska A., Vollbrandt T., Figge J., Köhl J. Monitoring and cell-specific deletion of C5aR1 using a novel floxed GFP-C5aR1 reporter knock-in mouse. 2015 J. Immunol. 194:1841-1855.
  2. Quell KM, Karsten CM, Kordowski A, Almeida L, Briukhovetska D, Wiese AV, Sun J, Ender F, Antoniou K, Schröder T, Schmudde I, Berger JL, König P, Vollbrandt T, Laumonnier Y, Köhl J. Monitoring C3aR expression using a novel floxed tdTomato-C3aR knock-in mouse. J. Immunol. 2017 199:688-706.
  3. Karsten CM, Wiese AV, Mey F, Figge J, Woodruff T, Reuter T, Scurtu O, Kordowski  A, Almeida L, Briukhovetska D, Quell KM, Sun J, Ender F, Schmudde I, Vollbrandt T, Laumonnier Y, Köhl J. Monitoring C5aR2 expression using a floxed tdTomato-C5aR2 knock-in mouse. 2017 J. Immunol. 199:3234-3248.
  4. Wiese AV, Ender F, Quell K, Antoninou K, Vollbrandt T, König P, Köhl J*, Laumonnier Y*. The C5a/C5aR1 axis controls the development of experimental allergic asthma independent of LysM-expressing pulmonary immune cells. PLoS One 2017 12(9).
  5. Ender F, Wiese AV, Schmudde I, Sun J, Vollbrandt T, König P, Laumonnier Y, Köhl J. Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase. PLoSOne. 2017 12:e0172446. 
  6. Laumonnier Y, Karsten CMK, Köhl J. Novel insights into the expression pattern of anaphylatoxin receptors in mice and men. Mol. Immunol. 2017 89:44-58.

Funding

EXC 306/ 2 Inflammation at Interfaces, CL XII Mouse models of inflammation