Phagosomal signalling of the C-type lectin receptor Dectin-1 is terminated by intramembrane proteolysis

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal β-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.


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No sample size calulation was performed. Instead, sample sizes were chosen based on established and accepted standards of the field. In general, experiments were repeated at least 3 times using independent samples. When differences were small, experiments were repeated with up to 15 mice to be sure to give a good estimation of normal variation within the experiment.
Individual values were excluded from the following Figures due to high deviation from other data and are highlighted in the accompanying Source data file: Fig. 6d: one sample from SPPL2b KO MOI 10 (reason: deviation more than 5-fold standard deviation) Fig. 6n: data originating from a single knockout animal due to an in general impaired response of cells derived from this mouse to the applied ligands (more than 2-3 fold of standard deviation) Fig. 6o: data from the same Knockout animal as in Firgure 6n were excluded for the same reasons mentioned above. Suppl. Fig. 7h: One sample from Knockout BMDC treated with HKCA was excluded due to high deviation (approx. 5-fold standard deviation) Suppl. Fig. 7e: Two values from wild type BMDC treated with Curdlan were excluded due to high deviation (more than 5-fold standard deviation) All experiments were replicated as indicated in the respective figure legends.
For mouse experiments, animals were grouped to the experimental sets to obtain age-and sex-matched cohorts based on the assigned genotypes without further selection. For PBMC experiments, blood samples were provided by the German Red Cross Blood Donation Service North-East, Dresden or the Transfusion Medicine of the University Hospital Schleswig-Holstein (UKSH) in Kiel, which randomly selected healthy volunteers.
Blinding was not employed since most of the readouts were based on machine readouts (ROS, ELISA, FACS) and thereby could be only minimally influenced by the experimentator. Western Blot results were also quantified to minimise subjective judgements. Based on limited staff and special restrictions in the context of working with viable C. albicans yeasts, the person performing the experiment usually had to be the same also evaluating it.  Fig. 3g) anti-mCD74: Validated for detection of endogenous levels of CD74 and its corresponding NTF in Schneppenheim et al., 2013. Knockout validated in Bikoff et al., 1993: validated by company for Western Blotting using human N87 cell lysates. Additionally validated in Roche et al., 1991 andKong et al., 2018. anti-Syk: validated by company for IP (human Syk) and Western blotting (murine) by appearance of a band at the corresponding size anti-GFP: validated by company for detection of GFP in transiently GFP-transfected cells vs untransfected cells anti-Actin: validated for Western Blotting of human and murine cells by company based on a band at corresponding size anti-Cofilin: validated for Western Blot analysis of Cofilin protein levels in human and murine cell lines based on a band at the corresponding size anti-EEF2: validated by company for Western Blotting of lysates of human and murine cell lines as well as murine tissue samples anti-alphaTubulin: validated by company for Western Blotting using whole cell lysates of murine and human origin. Also validated using recombinant protein.

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anti-phospho-ERK1/2: validated for use in Western Blot applications by company for human and murine cell lines based on a specific double band only occuring after stimulation of cells with TPA or other activators. Band can be blocked by application of an ERK1/2 inhibitor. anti-ERK1/2: validated by company for Western Blotting in human and murine samples. Detecting a double band at the expected size. Specificity of the antibody has been validated by siRNA in HEK293T cells. anti-phospho-Tyrosine: Validated by company for Western Blot application using lysates of unstimulated or EGF-treated A-431 cells (human). Specifically precipitates overexpressed Dectin-1 only upon stimulation with Zymosan known to induce tyrosinephosphorylation of the receptor. anti-HA: validated for Western Blot and IF detection of HA-tagged proteins in several publications from our laboratory, e.g. Mentrup et al., 2019. anti-LAMP2: Specifically detects human LAMP2 in immunofluorescence analysis as indicated by clear vesicular/lysosomal staining. Was employed before several times in studies from our laboratory (e.g. Mentrup et al., 2019).