Neutrophils and macrophages recognize pathogens by means of a variety of surface receptors. These include non-opsonic pattern recognition receptors (PRR) as well as opsonic Fc-receptors and complement receptors. Among the various classes of PRR are the leucin-rich repeat-containing families of Toll-like receptors (TLR) and NOD-like receptors (NLR). While the pathogen-associated molecular patterns that act as ligands for the membrane-expressed TLR are relatively well defined, the activation mode of the intracellular NLR has remained enigmatic.
The NLR member NLRP3 is part of a protein complex known as the inflammasome that mediates caspase-dependent activation of the inflammatory cytokine IL1b and related cytokines. While previous studies suggested a role for reactive oxygen species (ROS) generated by a NADPH oxidase upstream of this inflammasome, our results show that in human phagocytes NLRP3-inflammasome function is independent of NADPH oxidases when using cells from patients with chronic granulatomous disease (CGD). Similarly, other functions like the formation of neutrophil extracellular traps, that is believed to be relevant in the context of bacterial clearance, also appeared NADPH oxidase independent, in contrast to earlier claims.
We have also continued our efforts to identify and characterize novel mutations in the various subunits of the phagocyte NADPH oxidase that cause CGD. Among others we identified a patient with the insertion of a partially exonized retrotransposon of the TMF-1 gene into intron 1of the CYBB gene encoding the gp91phox subunit of the oxidase. This represents a unique example of mRNA splicing ‘caught in action’ and causing a genetic disorder. The mutation analysis in CGD is performed by us and other laboratories and is also incorporated into the European CGD registry. This is a genetic database of mutations that includes information from about 500 patients. The database is hosted by our laboratory.
We also identified a patient with a CARD9 deficiency allowing us to define the role of this protein in human phagocytes for the first time. CARD9 is expressed in myeloid cells and is known to function downstream of the β-glucan receptors dectin-1 and dectin-2, which play a critical role in the host defense against fungi. Strikingly, the patient suffers from a rare invasive Candida infection in the brain. Our further analyses indicate a prominent role of CARD9 in Candida-induced cytokine production by monocytes and in the induction of Th17 responses.
Role of immunoreceptors
In addition to the pathways relevant for pathogen recognition and immune cell activation we are studying the function and genetics of various families of immunoreceptors expressed on phagocytes and other innate immune cells. These include Fc-receptors (FcR), signal regulatory proteins (SIRP), and killerlike immunoreceptors (KIR).
SIRPa, the prototypic member of the SIRP family, is a typical inhibitory immunoreceptor expressed primarily on myeloid and neuronal cells. It acts as a receptor for the broadly expressed surface molecule CD47, and the ligation of SIRPa by CD47 results in the recruitment and activation of tyrosine phosphatases, such as SHP-1 and SHP-2, to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic tail of SIRPa. We have proposed that CD47 acts as a ‘self’ molecule to control phagocyte functions.
Indeed, there is good evidence now that CD47-SIRPa interactions negatively regulate the clearance of host cells by macrophages in vivo. We are investigating whether other phagocyte activities are also controlled via SIRPa. We have demonstrated, for instance, that SIRPa is a negative regulator of the phagocyte NADPH oxidase both in vitro and in vivo. In fact, it appears that CD47-SIRPa interactions do this by modulating the developmentally controlled expression of gp91phox in phagocytes and we are now characterizing the signaling pathway involved (see figure). This mechanism is anticipated to control the magnitude of the respiratory burst and thereby to reduce ‘collateral’ tissue damage of this toxic mechanism during infection. In addition, we have obtained evidence that interactions between CD47 on tumor cells and SIRPa on neutrophils form a critical limitation for antibody-dependent cellular cytotoxicity (ADCC) of phagocytes towards tumor cells. This is based on both in vivo experiments using SIRPa-mutant mice, as well as on in vitro evidence from ADCC experiments using a variety of human tumors and therapeutically relevant antibodies (e.g. trastuzumab and rituximab). We have also generated novel antibodies against SIRPa that have the capacity to improve ADCC and these may be instrumental for enhancing the efficacy of antibody therapy in cancer patients.
Of interest, the various immunoreceptor families, like FcR, SIRP and KIR, are subject to an extraordinary genetic variation within the population. But which of these individual differences, that include polymorphisms and copy number variation (CNV), are the critical determinants of individual immunogenicity and disease susceptibility is unknown. We are investigating this by multi-ligation probe amplification (MLPA) and large scale sequencing technology.
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