Matthew J. Robinson

Myeloid C-type lectins in innate immunity

C-type lectins expressed on myeloid cells comprise a family of proteins that share a common structural motif, and some act as receptors in pathogen recognition. But just as the presence of leucine-rich repeats alone is not sufficient to define a Toll-like receptor, the characterization of C-type lectin receptors in innate immunity requires the identification of accompanying signaling motifs. Here we focus on the known signaling pathways of myeloid C-type lectins and on their possible functions as autonomous activating or inhibitory receptors involved in innate responses to pathogens or self.

Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan

Zymosan is a particulate yeast preparation that elicits high levels of IL‐2 and IL‐10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk‐coupled receptor dectin‐1 and the MyD88‐coupled receptor TLR2. Here, we show that induction of IL‐2 and IL‐10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk‐deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL‐2 and IL‐10 response in DC and emphasises the importance of Syk‐coupled pattern recognition receptors such as dectin‐1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.

CLEC-2 signaling via Syk in myeloid cells can regulate inflammatory responses.

Myeloid cells express a plethora of C‐type lectin receptors (CLRs) that can regulate immune responses. CLEC‐2 belongs to the Dectin‐1 sub‐family of CLRs that possess an extracellular C‐type lectin‐like domain and a single intracellular hemITAM motif. CLEC‐2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC‐2 and found that CLEC‐2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb‐mediated crosslinking of CLEC‐2 leads to hemITAM‐dependent signaling via Syk, Ca2+ and NFAT and, in myeloid cells, modulates the effect of toll‐like receptor (TLR) agonists to selectively potentiate production of IL‐10. A macrophage/dendritic cell‐dependent increase in IL‐10 is also observed in mice given anti‐CLEC‐2 mAb together with LPS. Collectively, these data indicate that CLEC‐2 is expressed in myeloid cells and acts as a Syk‐coupled CLR able to modulate TLR signaling and inflammatory responses.

Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells

We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M‐CSF or GM‐CSF. When differentiated in GM‐CSF (GM‐MØP) the resultant cells resemble GM‐CSF bone marrow‐derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM‐MØP to effectively present antigen to a T‐cell hybridoma, these cells are comparatively poor at priming the expansion of IFN‐γ responses from naïve CD4+ T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM‐MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM‐CSF represents a unique in vitro model of inflammatory monocyte‐like cells, with important differences from bone marrow‐derived dendritic cells, which will facilitate functional studies relating to the many ‘sub‐phenotypes’ of inflammatory monocytes.

Granulocyte macrophage colony-stimulating factor receptor α expression and its targeting in antigen-induced arthritis and inflammation

BackgroundBlockade of granulocyte macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFRα) is being successfully tested in trials in rheumatoid arthritis (RA) with clinical results equivalent to those found with neutralization of the current therapeutic targets, TNF and IL-6. To explore further the role of GM-CSF as a pro-inflammatory cytokine, we examined the effect of anti-GM-CSFRα neutralization on myeloid cell populations in antigen-driven arthritis and inflammation models and also compared its effect with that of anti-TNF and anti-IL-6.MethodsCell population changes upon neutralization by monoclonal antibodies (mAbs) in the antigen-induced arthritis (AIA) and antigen-induced peritonitis (AIP) models were monitored by flow cytometry and microarray. Adoptive transfer of monocytes into the AIP cavity was used to assess the GM-CSF dependence of the development of macrophages and monocyte-derived dendritic cells (Mo-DCs) at a site of inflammation.ResultsTherapeutic administration of a neutralizing anti-GM-CSF mAb, but not of an anti-colony-stimulating factor (anti-CSF)-1 or an anti-CSF-1R mAb, ameliorated AIA disease. Using the anti-GM-CSFRα mAb, the relative surface expression of different inflammatory myeloid populations was found to be similar in the inflamed tissues in both the AIA and AIP models; however, the GM-CSFRα mAb, but not neutralizing anti-TNF and anti-IL-6 mAbs, preferentially depleted Mo-DCs from these sites. In addition, we were able to show that locally acting GM-CSF upregulated macrophage/Mo-DC numbers via GM-CSFR signalling in donor monocytes.ConclusionsOur findings suggest that GM-CSF blockade modulates inflammatory responses differently to TNF and IL-6 blockade and may provide additional insight into how targeting the GM-CSF/GM-CSFRα system is providing efficacy in RA.

Pulmonary pharmacodynamics of an anti-GM-CSFRα antibody enables therapeutic dosing that limits exposure in the lung

ABSTRACT Pulmonary alveolar proteinosis is associated with impaired alveolar macrophage differentiation due to genetic defects in the granulocyte macrophage colony-stimulating factor (GM-CSF) axis or autoantibody blockade of GM-CSF. The anti-GM-CSFRα antibody mavrilimumab has shown clinical benefit in patients with rheumatoid arthritis, but with no accompanying pulmonary pathology observed to date. We aimed to model systemic versus pulmonary pharmacodynamics of an anti-GM-CSFRα antibody to understand the pharmacology that contributes to this therapeutic margin. Mice were dosed intraperitoneal with anti-GM-CSFRα antibody, and pharmacodynamics bioassays for GM-CSFRα inhibition performed on blood and bronchoalveolar lavage (BAL) cells to quantify coverage in the circulation and lung, respectively. A single dose of 3 mg/kg of the anti-GM-CSFRα antibody saturated the systemic cellular pool, but dosing up to 10 times higher had no effect on the responsiveness of BAL cells to GM-CSF. Continued administration of this dose of anti-GM-CSFRα antibody for 7 consecutive days also had no inhibitory effect on these cells. Partial inhibition of GM-CSFRα function on cells from the BAL was only observed after dosing for 5 or 7 consecutive days at 30 mg/kg, 10-fold higher than the proposed therapeutic dose. In conclusion, dosing with anti-GM-CSFRα antibody using regimes that saturate circulating cells, and have been shown to be efficacious in inflammatory arthritis models, did not lead to complete blockade of the alveolar macrophages response to GM-CSF. This suggests a significant therapeutic window is possible with GM-CSF axis inhibition.

Haplosaurus computes protein haplotypes for use in precision drug design

Selecting the most appropriate protein sequences is critical for precision drug design. Here we describe Haplosaurus, a bioinformatic tool for computation of protein haplotypes. Haplosaurus computes protein haplotypes from pre-existing chromosomally-phased genomic variation data. Integration into the Ensembl resource provides rapid and detailed protein haplotypes retrieval. Using Haplosaurus, we build a database of unique protein haplotypes from the 1000 Genomes dataset reflecting real-world protein sequence variability and their prevalence. For one in seven genes, their most common protein haplotype differs from the reference sequence and a similar number differs on their most common haplotype between human populations. Three case studies show how knowledge of the range of commonly encountered protein forms predicted in populations leads to insights into therapeutic efficacy. Haplosaurus and its associated database is expected to find broad applications in many disciplines using protein sequences and particularly impactful for therapeutics design.Proteoforms arise as protein isoforms or as protein haplotypes, which are the result of genetic variation. Here, the authors develop Haplosaurus, a database that computes protein haplotypes genome-wide from existing genotype data and analyse protein haplotype variability in the 1000 Genomes dataset.

Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection

1. 1. Robinson, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20082818 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20082818%26rft_id%253Dinfo%253Apmid%252F19703985%26rft.genre%253Darticle%26rft_val_fmt%