Double Deletion of C5aR1 and C5aR2 during Streptococcal Pneumonia

Abstract

Abstract The complement system is essential for the clearance of encapsulated bacteria such as Streptococcus pneumoniae (Spn), recognized as a major pathogen of pneumonia. Yet, it is not entirely clear whether C5 and its activation product C5a have beneficial or detrimental effects on the outcome of Spn infection. C5a binds to its two homologous C5a receptors (C5aR1 and C5aR2). The extent of functional overlap and role distribution between C5aR1 and C5aR2 remains enigmatic. A critical barrier to allow studies on the role distributions of C5aRs has been the absence of a mouse strain with dual deletion of both receptors - which are encoded as adjacent, linked genes on mouse chromosome 7. CRISPR/Cas9 guided gene editing was combined with zygote/pronucleus microinjections in C57BL/6J mice to generate a C5aR1/C5aR2 double-knockout strain (C5aR1/2−/−). PCR-based screening and Sanger sequencing confirmed a 12.6 kB deletion of the coding regions of C5aR1 and C5aR2 of the new mouse strain, C57BL/6J-Del( 7C5ar2-C5ar1)1Bosm. Phenotyping of C5aR1/2−/− mice was followed by functional studies including intra-tracheal injections of recombinant mouse C5a or intranasal injections of Spn TIGR4. The airway influx of Ly6G+SiglecF- neutrophils by C5a was abrogated in C5aR1/2−/− mice. Surprisingly, the dual genetic absence of C5a receptors was associated with a higher build-up of neutrophil numbers in alveolar spaces after Spn TIGR4 infection. In a transcriptome-wide screen, FACS-sorted myeloid cells from infected lungs of double and single knockout mice were subjected to RNA-sequencing. Identifying novel differentially expressed genes in the C5aR1/2−/− mice during Spn infection will help to uncover potential synergisms and redundancies of C5aR1 and C5aR2.