Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host–microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel disease (IBD), however factors that mediate the host–microbiota interactions remain largely unknown. Here we collected mucosal-luminal interface samples from a pediatric IBD inception cohort and characterized both the human and microbiota proteins using metaproteomics. We show that microbial proteins related to oxidative stress responses are upregulated in IBD cases compared to controls. In particular, we demonstrate that the expression of human proteins related to oxidative antimicrobial activities is increased in IBD cases and correlates with the alteration of microbial functions. Additionally, we reveal that many of these human proteins are present and show altered abundance in isolated free extracellular vesicles (EVs). Therefore, our study suggests that the alteration of intestinal EV proteomes is associated with the aberrant host–microbiota interactions in IBD.

We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser4∼Val5 and Lys79∼Arg80. LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4∼5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg5-Ser6 and at Val7-Leu8 in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.

IntroductionAcute inflammation is the host response to tissue injury or infection that is characterized by the production of inflammatory mediators, culminating in the initial but transient recruitment of polymorphonuclear leukocytes (PMNs) that is followed by a prolonged macrophage accumulation. 1 However, the underlying, multifactorial mechanisms that shape the extent and kinetics of PMN and macrophage recruitment, apoptosis, and clearance remain unclear. Chemokines are an important class of chemoattractant cytokines produced locally in tissues that provide the directional cues for the movement of blood-derived leukocytes in development, homeostasis, and inflammation. 2 These potent chemoattractants are classified according to the position and spacing of their N-terminal cysteine residues, with 46 human chemokines currently divided into 4 families: C, CC, CXC, and CX 3 C. 3 The initial phase of inflammation involves a subset of CXC chemokines, which rapidly attract PMNs. 4 These PMN chemoattractants contain a conserved Glu-Leu-Arg (ELR) motif proximal to the CXC sequence, which is critical in cognate receptor binding and activation, 5 as well as PMN chemotaxis. 6 In humans, there are 7 ELR ϩ CXC chemokines: CXCL1, -2, and -3, also known as growth-related oncogenes ␣, ␤, and ␥, respectively; CXCL5/epithelial cell-derived neutrophil activating peptide-78 (ENA-78); CXCL6/granulocyte chemotactic protein-2 (GCP-2); CXCL7/neutrophil-activating peptide-2 (NAP-2); and CXCL8/interleukin-8 (IL-8).All bind the CXC-receptor (CXCR) 1; CXCL6 and -8 also signal through CXCR2. 7 Mice lack complete homologs of the 7 human ELR ϩ chemokines, having only 4: mCXCL1/ keratinocyte-derived chemokine (KC); mCXCL2/macrophage-inflammatory protein-2 (MIP-2); the more recently described mCXCL3/ dendritic cell inflammatory protein-1 (DCIP-1) 8 ; and mCXCL5/ lipopolysaccharide (LPS)-induced CXC chemokine (LIX). 9 All bind a single receptor that is homologous to human CXCR2. 10 After the initial PMN influx, the next stage of inflammation is directed in part by CC chemokines consisting of CCL2/monocyte chemoattractant protein (MCP)-1, CCL7/MCP-3, CCL8/MCP-2, and CCL13/MCP-4, which target multiple leukocyte subsets (monocytes, T lymphocytes, basophils, and eosinophils). 11 Monocytes can differentiate into macrophages whose role is to phagocytose and degrade microorganisms and foreign material and to present these antigens to initiate specific immune responses. In addition, macrophages ingest apoptotic PMNs from the inflamed site as a prelude to tissue resolution. 12 However, apoptosis alone cannot account for the entirety of the reduction in PMN numbers, because the potential for continued recruitment and replacement.Resident mast cells, macrophages, and epithelial cells have been proposed to produce the initial signals responsible for the accumulation of PMNs, eosinophils, and mononuclear cells in experimental models of inflammation by secretion of chemokines such as mCXCL1 and mCCL3. 13 Modulation of specific ELR ϩ chemokines by proteolytic ...