Abstract
Expressed strongly by myeloid cells, damage-associated molecular pattern (DAMP) proteins S100A8 and S100A9 are found in the serum of patients with infectious and autoimmune diseases. Compared to S100A9, the role of S100A8 is controversial. We investigated its biological activity in collagen-induced arthritis using the first known viable and fertile S100a8-deficient (S100a8-/-) mouse. Although comparable to the wild type (WT) in terms of lymphocyte distribution in blood and in the primary and secondary lymphoid organs, S100a8-/- mice had increased numbers of neutrophils, monocytes and dendritic cells in the blood and bone marrow, and these all expressed myeloid markers such as CD11b, Ly6G and CD86 more strongly. Granulocyte-macrophage common precursors were increased in S100a8-/- bone marrow and yielded greater numbers of macrophages and dendritic cells in culture. The animals also developed more severe arthritic disease leading to aggravated osteoclast activity and bone destruction. These findings were correlated with increased inflammatory cell infiltration and cytokine secretion in the paws. This study suggests that S100A8 is an anti-inflammatory DAMP that regulates myeloid cell differentiation, thereby mitigating the development of experimental arthritis.
Highlights
MethodsGeneration of S100a8-deficient miceThe animal protection committee at Universite Laval (Quebec City, QC, Canada) approved the experimental protocols (approval numbers 2012103 and 2013006)
No S100A8 mRNA was detected in bone marrow cells (S1C Fig) and S100A9 mRNA was present at reduced levels compared to wild type (WT)
Since myeloid cells express S100A8 strongly, we first performed a 13-parameter single-cell flow cytometry analysis combined with visualization using t-distributed stochastic neighbour embedding to study the leukocyte phenotypic diversity that develops in S100a8-/- mice
Summary
Generation of S100a8-deficient miceThe animal protection committee at Universite Laval (Quebec City, QC, Canada) approved the experimental protocols (approval numbers 2012103 and 2013006). The Lambda KOS phage library was screened by PCR using primers specific for exons 2 and 3 (Table 1). The yeast cassette was subsequently replaced with a LacZ/Neo selection cassette to complete the S100a8 targeting vector. Three targeted ES cell clones were identified and microinjected into C57BL/6 (albino) blastocysts to generate chimeric animals that were bred to C57BL/6 (albino) females, and the resulting heterozygous offspring were interbred to produce homozygous S100a8-deficient mice. The genotype at the S100a8 locus was determined by screening DNA from tail biopsies using quantitative PCR for the Neo cassette. This strategy allowed discrimination of 0, 1, or 2 gene disruptions representing respectively S100a8+/+, S100a8+/- and S100a8-/- mice
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