Machine-learning algorithms define pathogen-specific local immune fingerprints in peritoneal dialysis patients with bacterial infections.

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.

Toll-Like Receptors 2 and 4 Are Potential Therapeutic Targets in Peritoneal Dialysis-Associated Fibrosis.

Peritoneal dialysis (PD) remains limited by dialysis failure due to peritoneal membrane fibrosis driven by inflammation caused by infections or sterile cellular stress. Given the fundamental role of Toll-like receptors (TLRs) and complement in inflammation, we assessed the potential of peritoneal TLR2, TLR4 and C5a receptors, C5aR and C5L2, as therapeutic targets in PD-associated fibrosis. We detected TLR2-, TLR4-, and C5aR-mediated proinflammatory and fibrotic responses to bacteria that were consistent with the expression of these receptors in peritoneal macrophages (TLR2/4, C5aR) and mesothelial cells (TLR2, C5aR). Experiments in knockout mice revealed a major role for TLR2, a lesser role for TLR4, a supplementary role for C5aR, and no apparent activity of C5L2 in infection-induced peritoneal fibrosis. Similarly, antibody blockade of TLR2, TLR4, or C5aR differentially inhibited bacteria-induced profibrotic and inflammatory mediator production by peritoneal leukocytes isolated from the peritoneal dialysis effluent (PDE) of noninfected uremic patients. Additionally, antibodies against TLR2, TLR4, or the coreceptor CD14 reduced the profibrotic responses of uremic leukocytes to endogenous components present in the PDE of noninfected patients. Enhancing TLR2-mediated inflammation increased fibrosis in vivo Furthermore, soluble TLR2 (sTLR2), a negative modulator of TLRs that we detected in PDE, inhibited PDE-induced, TLR2- or TLR4-mediated profibrotic responses. Notably, sTLR2 treatment markedly reduced Gram-positive and -negative bacteria-induced fibrosis in vivo, inhibiting proinflammatory and fibrotic genes without affecting infection clearance. These findings reveal the influence of peritoneal TLR2 and TLR4 on PD-associated fibrosis and describe a therapeutic strategy against fibrosis.

Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling.

The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2(+) γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response.

Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis 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.

Targeting the TLR co-receptor CD14 with TLR2-derived peptides modulates immune responses to pathogens.

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.

CD200-expressing human basal cell carcinoma cells initiate tumor growth.

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Human skin cancer stem cells: a tale of mice and men.

Carcinomas, cancers of epithelial tissues, are the commonest malignancies and cause the greatest cancer mortality worldwide. Among these, the incidence of keratinocyte-derived non-melanoma skin cancers (NMSC), by far the greatest, is increasing rapidly. Yet despite access to tumor tissue, acceptance of human NMSC as a model carcinoma has been hindered by the lack of a reliable xenograft model. Instead, we have relied on the murine two-step carcinogenesis protocol as a reproducible squamous cell carcinoma (SCC) model, but this differs from their human counterpart in cause, site, genetic basis and biological behaviour. By xeno-engraftment of primary human SCC, we were recently successful in demonstrating the presence of primary human SCC cancer stem cells or tumor-initiating cells. These findings once more align the study human SCC as the archetypal carcinoma model. In this review, we describe the evidence for the existence of tumor-initiating cells, with emphasis on skin cancer, limiting our discussions to primary human cancer studies where possible.

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors.

BACKGROUND: Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC). METHODS: Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)-PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies. RESULTS: Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines. CONCLUSIONS: These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2.

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 Ca(2+) 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.

Soluble TLR2 reduces inflammation without compromising bacterial clearance by disrupting TLR2 triggering.

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-kappaB 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.

12/15-Lipoxygenase regulates the inflammatory response to bacterial products in vivo.

The peritoneal macrophage (Mphi) 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 CD11b(high) 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 Mphi numbers were significantly increased in 12/15-LOX(-/-) mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mphi 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-beta1, along with increased immigration of monocytes/Mphi, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-alpha. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MPhi population and regulates both the recruitment of monocytes/Mphi and cytokine response to bacterial products in vivo.

Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation.

Although the IL-6-related cytokine oncostatin M (OSM) affects processes associated with disease progression, the specific function of OSM in the face of an inflammatory challenge remains unclear. In this report, a peritoneal model of acute inflammation was used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared with wild-type and IL-6-deficient mice, peritoneal inflammation in oncostatin M receptor-beta-deficient (OSMR-KO) mice resulted in enhanced monocytic cell trafficking. In contrast to IL-6-deficient mice, OSMR-KO mice displayed no difference in neutrophil and lymphocyte migration. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression after peritoneal inflammation identified OSM as a prominent regulator of CCL5 expression. Specifically, OSM inhibited IL-1beta-mediated NF-kappaB activity and CCL5 expression in human mesothelial cells. This was substantiated in vivo where peritoneal inflammation in OSMR-KO mice resulted in a temporal increase in both CCL5 secretion and NF-kappaB activation. These findings suggest that IL-6 and OSM individually affect the profile of leukocyte trafficking, and they point to a hitherto unidentified interplay between OSM signaling and the inflammatory activation of NF-kappaB.

IL-6 regulates neutrophil trafficking during acute inflammation via STAT3.

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 gp130(Y757F/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 gp130(Y757F/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 gp130(Y757F/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 gp130(Y757F/Y757F) mice failed to rescue the altered responses observed in gp130(Y757F/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.

Receptor-interacting protein-2 deficiency delays macrophage migration and increases intracellular infection during peritoneal dialysis-associated peritonitis.

BACKGROUND: Early upregulation of receptor-interacting protein-2 (RIP2) expression during peritoneal dialysis (PD)-associated peritonitis correlates with a favorable clinical outcome, while failure to upregulate RIP2 correlates with a protracted course. We noticed that patients who do not upregulate RIP2 during PD-associated peritonitis have more peritoneal macrophages during the early phase of infection. METHODS: To study the mechanism behind this observation, we examined the role of RIP2 in the immune response to bacterial challenge in a mouse model of acute peritonitis. We injected RIP2(+/+) and RIP2(-/-) mice intraperitoneally with a Staphylococcus epidermidis cell free-preparation, and peritoneal cells were isolated 3, 6 and 24 h after challenge. RESULTS: Surprisingly, RIP2(-/-) mice had a comparable influx of inflammatory leukocytes, but had a significantly higher number of peritoneal macrophages at 3 h, indicating delayed emigration of these cells. No significant differences were seen at later times suggesting that migration was delayed but not inhibited. In addition, RIP2(-/-) macrophages were more permissive to intracellular infection by Staphylococcus aureus, indicating that, in the absence of RIP2, resident peritoneal macrophages could become reservoirs of bacteria. CONCLUSION: These findings provide a mechanism for the observation that upregulation of RIP2 expression is required for rapid resolution of peritonitis, by decreasing intracellular infection and by regulating the migration of antigen-presenting cells in the early stages of an inflammatory response.

Viral IL-6 blocks neutrophil infiltration during acute inflammation.

Pathologies arising as a consequence of human herpesvirus-8 (HHV8) infections are closely associated with the autocrine activity of a HHV8 encoded IL-6 (vIL-6), which promotes proliferation of infected cells and their resistance to apoptosis. In this present report, studies show that vIL-6 may also be important in influencing the host's immunological response to secondary infections. Using peritoneal inflammation as a model of acute bacterial infection, vIL-6 was found to specifically block neutrophil recruitment in vivo through regulation of inflammatory chemokine expression. This response was substantiated in vitro where activation of STAT3 in human peritoneal mesothelial cells by vIL-6 was associated with enhanced CCL2 release. Although vIL-6 did not effect CXCL8 production, IL-1beta-induced secretion of this neutrophil-activating chemokine was significantly suppressed by vIL-6. These data suggest that vIL-6 has the capacity to suppress innate immune responses and thereby influence the outcome of opportunistic infections in HHV8-associated disease.

Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P1 in murine embryonic fibroblasts and vascular smooth muscle cells.

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P1 (also known as EDG-1), a Gi-linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P1 null mice. Our data suggest that the S1P1 receptor is critical for S1P-induced, Gi-dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P1 GPCR is a critical downstream component of PDGF-induced chemotaxis.

Extracellular export of sphingosine kinase-1 enzyme. Sphingosine 1-phosphate generation and the induction of angiogenic vascular maturation.

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 rate-limiting. 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 degrees 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.