Cellular and immunological consequences arising from impaired L-plastin function

Abstract

Inborn errors of immunity (IEI) are genetic disorders affecting the immune system. Their disease burden is complex, with increased susceptibility to infections, autoimmunity, allergies, and malignancies.

In this project, we studied a family presenting with an IEI resulting from a novel LCP1 variant (LCP1c.740-1G>A). Lymphopenia, neutropenia, and thrombocytopenia were reported in the affected individuals, spanning at least two generations. Whole-exome analysis revealed a mutation in the LCP1 gene affecting the splicing site upstream of exon eight. LCP1 encodes for L-plastin, a cytoskeleton protein predominantly expressed in leukocytes. Its actin-bundling activity is central to the immune response, particularly during integrin-mediated events, such as migration and the establishment of the immune synapse. The LCP1c.740-1G>A variant leads to two aberrant transcripts, both predicted to affect protein integrity: an in-frame deletion of 24 nucleotides; and a whole exon eight deletion resulting in a frameshift and the introduction of an early stop codon. We conducted further protein and cellular analysis using a mouse model expressing the orthologous variant. Expression of the small in-frame deletion transcript, and the reduction of protein synthesis were confirmed using mouse splenocytes. The mouse model exhibited a similar haematopoietic phenotype to patients. Circulating lymphocytes were proportionally decreased in Lcp1c.740-1G>A mice. T cells exhibited aberrant cytoskeleton organisation and impaired chemotaxis after CXCL12 stimulation. Similarly, the spreading capacity of platelets was reduced upon activation with collagen. By immunisation of the mouse model with sheep red blood cells, we confirmed impairments in the germinal centre reaction.

We also generated a mouse model expressing the Lcp1c.694A>T variant, previously reported to cause human neutropenia, and observed a similar but more severe defect in response to immunisation. Significantly, we did not observe an immunisation defect in Lcp1-/- mice. In vitro T cell proliferation confirmed a decrease in cell proliferation for the three mouse models. However, the Lcp1c.740-1G>A and Lcp1c.694A>T variants selectively resulted in an accumulation of large cells with abnormally increased DNA content, as identified by flow cytometry analysis. We confirmed a gene-dose dependent accumulation of polyploid cells by microscopy, indicating a cytoskeleton defect in completing cytokinesis in the presence of abnormal forms of L-plastin, but not by the absence of it (Lcp1-/-). Finally, we performed transcriptome analysis of proliferating cells expressing Lcp1c.694A>T/c.694A>T and identified a signature of aberrant cell cycle progression and upregulation of inflammatory signals.

In summary, LCP1 deficiency leads to a novel IEI arising from cytoskeleton defects, including a previously uncharacterized mechanism during cytokinesis. Show more