Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

Martina B Lorey,Katriina Rossi,Kari K Eklund,Tuula A Nyman,Sampsa Matikainen

doi:10.1074/mcp.m116.064840

Martina B Lorey, Katriina Rossi + Show 3 more

Open Access

https://doi.org/10.1074/mcp.m116.064840

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Abstract

Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.

Highlights

From the ‡Rheumatology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, Finland; §Department of Immunology, Institute of Clinical Medicine, University of Oslo and Rikshospitalet Oslo, Oslo 0424, Norway
Intracellular LPS that activates the non-canonical inflammasome in macrophages triggers pyroptosis, which is associated with LDH release from the cells [27]
To study the kinetics of IL-18 secretion, macrophages were transfected with LPS for different time periods after which cell culture supernatants were collected, and IL-18 secretion was studied by Luminex assay

Summary

Introduction

We studied protein secretion in human macrophages in response to non-canonical caspase4/5 inflammasome activation using label-free quantitative proteomics combined with bioinformatics. Intracellular LPS that activates the non-canonical inflammasome in macrophages triggers pyroptosis, which is associated with LDH release from the cells [27]. To further characterize activation of EV secretion in human macrophages in response to LPS transfection, we studied the secretion kinetics of known EV marker proteins Alix, annexin-1, galectin-3, and ITGAX.

Results

Conclusion