SummaryFibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

The successful resolution of inflammation is dependent upon the coordinated transition from the initial recruitment of neutrophils to a more sustained population of mononuclear cells. IL-6, which signals via the common receptor subunit gp130, represents a crucial checkpoint regulator of neutrophil trafficking during the inflammatory response by orchestrating chemokine production and leukocyte apoptosis. However, the relative contribution of specific IL-6-dependent signaling pathways to these processes remains unresolved. To define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we used a series of gp130 knockin mutant mice displaying altered IL-6-signaling capacities in an experimental model of acute peritoneal inflammation. Hyperactivation of STAT1 and STAT3 in gp130Y757F/Y757F mice led to a more rapid clearance of neutrophils, and this coincided with a pronounced down-modulation in production of the neutrophil-attracting chemokine CXCL1/KC. By contrast, the proportion of apoptotic neutrophils in the inflammatory infiltrate remained unaffected. In gp130Y757F/Y757F mice lacking IL-6, neutrophil trafficking and CXCL1/KC levels were normal, and this corresponded with a reduction in the level of STAT1/3 activity. Furthermore, monoallelic ablation of Stat3 in gp130Y757F/Y757F mice specifically reduced STAT3 activity and corrected both the rapid clearance of neutrophils and impaired CXCL1/KC production. Conversely, genetic deletion of Stat1 in gp130Y757F/Y757F mice failed to rescue the altered responses observed in gp130Y757F/Y757F mice. Collectively, these data genetically define that IL-6-driven signaling via STAT3, but not STAT1, limits the inflammatory recruitment of neutrophils, and therefore represents a critical event for the termination of the innate immune response.

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2) to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overexpressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-κB activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leukocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derived components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compromise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2 mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with its coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions.

The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P 1 /EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are ratelimiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4°C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.The membrane phospholipid sphingomyelin is metabolized by a complex series of enzymatic steps, resulting in the formation of polar sphingolipid metabolites such as sphingosine, ceramide, and sphingosine 1-phosphate (S1P) 1 (1). Such lipids are utilized as signaling molecules within or among cells (2).For example, ceramide and sphingosine were proposed to be intracellular mediators (2, 3). In contrast, S1P appears to act extracellularly in vertebrates by interacting with the G protein-coupled receptors (GPCRs) of the S1P 1 /EDG-1 subfamily (4 -6). However, in early studies, S1P was proposed to act as an intracellular second messenger (7). Although S1P (and/or sphingosine) may act as an intracellular messenger in lower eukaryotes such as yeast (8), an unequivocal demonstration of a second messenger/intracellular role for S1P is lacking at present, especially for higher eukaryotes. S1P interaction with the S1P 1 /EDG-1 family of GPCRs may regulate the formation and/or maintenance of the cardiovascular system. In zebrafish, the EDG-5-like receptor, termed Miles Apart, regulates the development of the heart in the embryo (9). Null mutation of the S1P 1 /EDG-1 gene in the mouse results in embryonic lethality because of a vascular maturation defect (10). S1P cooperates with polypeptide angiogenic factors such as vascular endothelial cell growth factor and fibroblast growth factor (FGF) to induce vascular maturation in mice (11). In vitro studies indicate that S1P induces vascular endothelial cell migration,...

The immune system has evolved to sense invading pathogens, control infection, and restore tissue integrity. Despite symptomatic variability in patients, unequivocal evidence that an individual's immune system distinguishes between different organisms and mounts an appropriate response is lacking. We here used a systematic approach to characterize responses to microbiologically well-defined infection in a total of 83 peritoneal dialysis patients on the day of presentation with acute peritonitis. A broad range of cellular and soluble parameters was determined in peritoneal effluents, covering the majority of local immune cells, inflammatory and regulatory cytokines and chemokines as well as tissue damage–related factors. Our analyses, utilizing machine-learning algorithms, demonstrate that different groups of bacteria induce qualitatively distinct local immune fingerprints, with specific biomarker signatures associated with Gram-negative and Gram-positive organisms, and with culture-negative episodes of unclear etiology. Even more, within the Gram-positive group, unique immune biomarker combinations identified streptococcal and non-streptococcal species including coagulase-negative Staphylococcus spp. These findings have diagnostic and prognostic implications by informing patient management and treatment choice at the point of care. Thus, our data establish the power of non-linear mathematical models to analyze complex biomedical datasets and highlight key pathways involved in pathogen-specific immune responses.

Dysregulation of Toll-like receptor (TLR) responses to pathogens can lead to pathological inflammation or to immune hyporesponsiveness and susceptibility to infections, and may affect adaptive immune responses. TLRs are therefore attractive therapeutic targets. We assessed the potential of the TLR co-receptor CD14 as a target for therapeutics by investigating the magnitude of its influence on TLR responses. We studied the interaction of CD14 with TLR2 by conducting peptide screening and site-directed mutagenesis analysis and found TLR2 leucine-rich repeats 5, 9, 15, and 20 involved in interaction with CD14. Peptides representing these regions interacted with CD14 and enhanced TLR2- and TLR4-mediated proinflammatory responses to bacterial pathogens in vitro. Notably, the peptides' immune boosting capacity helped to rescue proinflammatory responses of immunosuppressed sepsis patients ex vivo. In vivo, peptide treatment increased phagocyte recruitment and accelerated bacterial clearance in murine models of Gram-negative and Gram-positive bacterial peritonitis. Up-modulating CD14's co-receptor activity with TLR2-derived peptides also enhanced antigen-induced dendritic cell (DC) maturation and interleukin-2 production and, most notably, differentially affected DC cytokine profile upon antigen stimulation, promoting a T helper 1-skewed adaptive immune response. Biochemical, cell imaging, and molecular docking studies showed that peptide binding to CD14 accelerates microbial ligand transfer from CD14 to TLR2, resulting in increased and sustained ligand occupancy of TLR2 and receptor clustering for signaling. These findings reveal the influence that CD14 exerts on TLR activities and describe a potential therapeutic strategy to amplify responses to different pathogens mediated by different TLRs by targeting the common TLR co-receptor, CD14.

TLR and complement activation ensures efficient clearance of infection. Previous studies documented synergism between TLRs and the receptor for the pro-inflammatory complement peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. We demonstrate a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identify an underlying mechanistic target. Pre-exposure of PBMCs and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4 signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR upregulation or modulation of C5a-induced Ca2+ mobilization. Rather, TLRs targeted another C5a receptor, C5L2 (acting as a negative modulator of C5aR), by reducing C5L2 activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defense at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation.