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Introduction: Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Methods and Results: Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2ß1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2ß1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2ß1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2ß1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2ß1 in TNTs and filopodia of integrin α2ß1-expressing OC precursors. Discussion: Our findings define integrin α2ß1 as an early marker of OC differentiation.

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The S100A8/A9 heterocomplex is an abundant damage-associated molecular pattern and mainly expressed by monocytes, inflammatory activated keratinocytes and neutrophilic granulocytes. The heterocomplex as well as the heterotetramer are involved in a variety of diseases and tumorous processes. However, their detailed mode of action and especially which receptors are involved hereby remains to be fully revealed. Several cell surface receptors are reported to interact with S100A8 and/or S100A9, the best studied being the pattern recognition receptor TLR4. RAGE, CD33, CD68, CD69, and CD147, all of them are involved as receptors in various inflammatory processes, are also among these putative binding partners for S100A8 and S100A9. Interactions between S100 proteins and these receptors described so far come from a wide variety of cell culture systems but their biological relevance in vivo for the inflammatory response of myeloid immune cells is not yet clear. In this study, we compared the effect of CRISPR/Cas9 mediated targeted deletion of CD33, CD68, CD69, and CD147 in ER-Hoxb8 monocytes on S100A8 or S100A9 induced cytokine release with TLR4 knockout monocytes. Whereas deletion of TLR4 abolished the S100-induced inflammatory response in monocyte stimulation experiments with both S100A8 and S100A9, knockouts of CD33, CD68, CD69, or CD147 revealed no effect on the cytokine response in monocytes. Thus, TLR4 is the dominant receptor for S100-triggered inflammatory activation of monocytes.

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Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

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The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but are completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of S100a8 and S100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout (KO)-based screening approach in immortalized murine monocytes, we identified the transcription factor C/EBPδ as a central regulator of S100a8 and S100a9 expression. We showed that S100A8/A9 expression and thereby neutrophil recruitment and cytokine release were decreased in C/EBPδ KO mice in a mouse model of acute lung inflammation. S100a8 and S100a9 expression was further controlled by the C/EBPδ antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within S100a8 and S100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100a8 and S100a9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterized by S100a8 and S100a9 overexpression.

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