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Genome-wide analysis of the CARD9/Dectin-1 transcriptional program in neutrophils and macrophages by RNA-seq

ABSTRACT: Purpose: To characterize Card9-dependent and Card9-independent Dectin-1 mediated gene expression changes in neutrophils and to compare response to Dectin-1 stimulation in neutrophils and macrophages. Method: BM neutrophils from WT and Card9-/- mice were isolated by MACS column purification and treated with or without the Dectin-1 agonist, curdlan for 3 hrs. WT BM-derived macrophages were also treated with or without curdlan for 3hrs before sample collection. Results: We identified both Card9-dependent and Card9-independent gene expression changes with Dectin-1 stimulation in neutrophils. We also identified cell-type specific differences in the response to Dectin-1 stimulation in neutrophils and macrophages. Conclusions: Dectin-1 stimulation induces significant gene expression changes regulated by either canonical Card9-dependent or non-canonical Card9-independent mechanisms.

ORGANISM(S): Mus musculus

PROVIDER: GSE148850 | GEO | 2021/02/26

REPOSITORIES: GEO

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Project description:Acanthamoeba castellanii (Ac) and macrophages share structural, morphological, physiological and biochemical similarities, including the ability to phagocyte a myriad of microorganisms in the environments they live. Whereas there is voluminous information on phagocytic receptors of macrophages, for Ac, the understanding of how the recognition of extracellular microorganisms works still awaits elucidation. Recently, our group described mannose-binding proteins expressed on the surface of the trophozoites. However, as soluble mannose did not inhibit entirely the process, other interactions might be possible. In the present work, we aimed to characterize the potential Ac proteins able to recognize the polysaccharide β-1,3-glucan on fungal surfaces. Our data demonstrate that Ac could bind curdlan or laminarin on its surface as detected by Dectin-1-Fc and fluorescent conjugate, suggesting the presence of β-1,3-glucan binding molecules. Optical tweezers detected higher adhesion affinity of laminarin or curdlan coated beads to A. castellanii (characteristic time of 46.9 s and 43.9 s, respectively) in comparison control beads (BSA or dextran-coated). In agreement, a H. capsulatum (Hc) G217B having β-1,3-glucan as the most external layer strongly adhered to Ac (characteristic time of 5.3 s), whereas Hc G186A, an α-1,3-glucan expressing strain, displayed much lower adhesion forces (characteristic time of 83.6 s). The specificity of our system was confirmed with addition of soluble β-1,3-glucan, which inhibited dramatically the adhesion of Hc G217B to Ac (characteristic time of 38,5 s). By indirect ELISA, the biotinylated extract of Ac showed higher binding to Hc G217B surface than Hc G186A, as similar results were observed when using Dectin-1-Fc. By interaction assays, association rates to Ac and RAW macrophages were twice higher for Hc G217B when compared to Hc G186A. Inhibitions with mannose, or its combinations with curdlan or laminarin demonstrated inhibitions higher than 50% during Ac and Hc G217B interaction. For RAW macrophages, the combinations mannose + laminarin and mannose + curdlan had inhibition of 64.4% and 51.5%, respectively, in the interaction with Hc G217B. The killing assay show that for A. castellanii, there was a decrease in the number of viable fungi when either laminarin and curdlan were added, which is similar to results observed with macrophages, suggesting the participation of this receptor for fungal entrance and survival within phagocytes. Proteomics identified several proteins with the capacity to bind β-1,3-glucans, including a membrane integral component (L8HDD6) displaying a legume lectin domain and also belonging to the Concanavalin A-like lectin/glucanase domain superfamily. By the demonstrated binding specificity of this receptor, our data reinforce other pathways of fungal recognition and suggests to a possible parallel or even divergent evolution, between A. castellanii and macrophages.

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Genome-wide analysis of the CARD9/Dectin-1 transcriptional program in neutrophils and macrophages by RNA-seq

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