Investigation of the role of toll-like and interleukin-6 receptors in peritoneal inflammatory responses to infection

Bacterial infection is a feature of long-term peritoneal dialysis (PD) and a cause of loss of peritoneal function and treatment failure. Understanding how local inflammation is initiated and peritoneal host defence mechanisms are activated in PD patients is key to reducing the detrimental consequences of peritonitis. The capacity of human peritoneal mesothelial cells (HPMC) to ingest bacteria has been described, and the ability of the Toll-like family of innate immune receptors (TLR) to trigger inflammatory responses to pathogens has been demonstrated. However, the pathogen recognition ability, the potential role of TLRs, the specific role in the early inflammatory response, and the regulation of HPMC’ putative ability for pathogen recognition have not been fully investigated. To address these issues, the present study aimed at characterising TLR expression and responses in HPMC and evaluating the capacity of modulators of pro-inflammatory responses, namely soluble TLR2 (sTLR2) and the IL-6/sIL-6R complex, to regulate TLR-mediated HPMC and peritoneal responses in vitro and in vivo. HPMC were found to respond to an array of bacterial pathogens via expression and function of a specific set of TLRs. HPMC responses were susceptible to modulation by sTLR2 and sIL-6R, resulting in inhibition of TLR2-driven HPMC responses. In vivo, sTLR2 and IL-6/sIL- 6R reduced neutrophil influx partly by inhibiting NF-κB activation in stromal cells of the peritoneum. IL-6 signalling counteracted TLR2-mediated responses by reducing peritoneal leukocyte recruitment and chemokine production. Notably, following the establishment of a mouse model of peritoneal bacterial infection, IL-6 signalling was confirmed to be beneficial to bacterial clearance. The results of this thesis confirm and extend the knowledge of the pivotal role that HPMC play in peritoneal responses to infection. The capacity of sTLR2 and IL-6/sIL-6R to modulate peritoneal responses demonstrated in this study may inform the design of new therapeutic strategies to reduce PD-associated peritonitis and thus improve treatment outcomes

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 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

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

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-1-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

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

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

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 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

Oncostatin M receptor signalling regulates monocytic cell trafficking during acute inflammation [Abstract]

Although the interleukin (IL)-6 related cytokine oncostatin-M (OSM) affects a variety of inflammatory events associated with disease progression, the function of OSM in the face of an inflammatory challenge remains unclear. In this current report a peritoneal model of acute inflammation has been used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared to wild type mice (WT) the induction of peritoneal inflammation in oncostatin-M receptor-β deficient mice (OSMR-KO) resulted in enhanced monocytic cell trafficking. No difference in neutrophil and lymphocyte migration was however noted suggesting that OSM control of leukocyte recruitment is functionally distinct from that of IL-6. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression following peritoneal inflammation inferred that OSM regulation of CCL5 might account for the observed difference in monocytic cell trafficking. Indeed through comparative analysis of inflammatory events triggered in OSMR-KO and IL-6KO mice it is evident that certain chemokines commonly regulated in vitro by both IL-6 and OSM are preferentially regulated in vivo by one prevailing factor. In this respect, it is evident that IL-6 acts as a more prominent in vivo regulator of CCL2 expression than OSM. In contrast, OSM was found to uniquely inhibit the IL-1β driven expression of CCL5. This was substantiated in vivo where induction of peritoneal inflammation in OSMR-KO mice resulted in significantly raised CCL5 levels, as compared to WT and IL-6KO mice. No difference in CCL3 and CCL4 was however noted. Mechanistically, these studies inferred a hitherto unidentified interplay between OSM-mediated STAT signaling and NF-κB activation. In this respect, EMSA analysis of nuclear extracts from peritoneal membranes isolated during course of the inflammatory response showed that OSMR-KO mice display an enhanced profile of NF-κB activation as compared to WT mice. These findings suggest that activation of gp130 by IL-6 and OSM trigger distinct inflammatory responses to affect individual aspects of leukocyte trafficking. The outcome of such a response may ultimately rely on a defined ability to modulate NF-κB signaling