CD14+ CD15- HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure.

OBJECTIVE: Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR- myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. DESIGN: Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15-CD11b+HLA-DR- cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. RESULTS: Circulating CD14+CD15-CD11b+HLA-DR- M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. CONCLUSION: Immunosuppressive CD14+HLA-DR- M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent

Interleukin 1-Beta (IL-1) Production by Innate Cells Following TLR Stimulation Correlates With TB Recurrence in ART-Treated HIV-Infected Patients

BACKGROUND: Tuberculosis (TB) remains a major cause of global morbidity and mortality, especially in the context of HIV co-infection, since immunity is not completely restored following antiretroviral therapy (ART). The identification of immune correlates of risk for TB disease could help in the design of host-directed therapies and clinical management. This study aimed to identify innate immune correlates of TB recurrence in HIV+ ART-treated individuals with a history of previous successful TB treatment. METHODS: Twelve participants with a recurrent episode of TB (cases) were matched for age, sex, time on ART, pre-ART CD4 count with 12 participants who did not develop recurrent TB in 60 months of follow-up (controls). Cryopreserved peripheral blood mononuclear cells from time points prior to TB recurrence were stimulated with ligands for Toll like receptors (TLR) including TLR-2, TLR-4, and TLR-7/8. Multi-color flow cytometry and intracellular cytokine staining was used to detect IL-1β, TNF-α, IL-12 and IP10 responses from monocytes and myeloid dendritic cells (mDCs). RESULTS: Elevated production of IL-1β from monocytes following TLR-2, TLR-4 and TLR-7/8 stimulation was associated with reduced odds of TB recurrence. In contrast, production of IL-1β from both monocytes and mDCs following Bacillus Calmette-Guérin (BCG) stimulation was associated with increased odds of TB recurrence (risk of recurrence increased by 30% in monocytes and 42% in mDCs respectively). CONCLUSION: Production of IL-1β by innate immune cells following TLR and BCG stimulations correlated with differential TB recurrence outcomes in ART-treated patients and highlights differences in host response to TB

Cooperative Stimulation of Dendritic Cells by Cryptococcus neoformans Mannoproteins and CpG Oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.National Institutes of Health (RO1 AI25780, RO1 AI37532, K08 AI 53542

Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis

OBJECTIVE: To analyze the expression, regulation, and biologic relevance of Toll-like receptors (TLRs) 1-10 in synovial and skin fibroblasts and to determine the expression levels of TLRs 2, 3, and 4 in synovial tissues from patients with early rheumatoid arthritis (RA), longstanding RA, and osteoarthritis (OA). METHODS: Expression of TLRs 1-10 in RA synovial fibroblasts (RASFs), OASFs, and skin fibroblasts was analyzed by real-time polymerase chain reaction (PCR). Fibroblasts were stimulated with tumor necrosis factor alpha, interleukin-1beta (IL-1beta), bacterial lipopeptide, poly(I-C), lipopolysaccharide, and flagellin. Production of IL-6 was determined by enzyme-linked immunosorbent assay and induction of TLRs 2-5, matrix metalloproteinases (MMPs) 3 and 13 messenger RNA by real-time PCR. Expression of TLRs 2-4 in synovial tissues was analyzed by immunohistochemistry. RESULTS: Synovial fibroblasts expressed TLRs 1-6, but not TLRs 7-10. Among the expressed TLRs, TLR-3 and TLR-4 were the most abundant in synovial fibroblasts, and stimulation of synovial fibroblasts with the TLR-3 ligand poly(I-C) led to the most pronounced increase in IL-6, MMP-3, and MMP-13. In contrast, skin fibroblasts did not up-regulate MMP-3 or MMP-13 after stimulation with any of the tested stimuli. In synovial tissues from patients with early RA, TLR-3 and TLR-4 were highly expressed and were comparable to the levels of patients with longstanding RA. These expression levels were elevated as compared with those in OA. CONCLUSION: Our findings of high expression of TLRs, particularly TLRs 3 and 4, at an early stage of RA and the reactivity of synovial fibroblasts in vitro to TLR ligands suggest that TLR signaling pathways resulting in persistent inflammation and joint destruction are activated early in the disease process

Caspase-8 controls the gut response to microbial challenges by Tnf-alpha-dependent and independent pathways

Objectives: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear. Design: We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo. Results: TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α. Conclusions: Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies

TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence

Assessment of the immunomodulatory properties of the probiotic strain Lactobacillus paracasei K5 in vitro and in vivo

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1&alpha; (IL-1&alpha;), &Iota;L-1&beta;, IL-6, tumor necrosis factor-alpha (TNF-&alpha;), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes

BACKGROUND: Monocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc. METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells. RESULTS: Infectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes. CONCLUSION: This study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells