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Susann Wolf-Grosse,1 Anne Mari Rokstad,1–3 Syed Ali,4 John D Lambris,5 Tom E Mollnes,2,6–9 Asbjørn M Nilsen,1,* Jørgen Stenvik1,2,* 1Department of Cancer Research and Molecular Medicine, 2Centre of Molecular Inflammation Research (CEMIR), Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, 3Central Norway Regional Health Authority, Stjørdal, Norway; 4Division of Neurotoxicology, US FDA/National Center for Toxicological Research, Jefferson, AR, 5Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA; 6Department of Immunology, Oslo University Hospital, Rikshospitalet, Oslo, 7Research Laboratory, Nordland Hospital, Bodø, 8K.G. Jebsen Inflammatory Research Center, University of Oslo, Oslo, 9Faculty of Health Sciences, K.G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway *These authors contributed equally to this work Abstract: Iron oxide nanoparticles (IONPs) are promising nanomaterials for biomedical applications. However, their inflammatory potential has not been fully established. Here, we used a lepirudin anti-coagulated human whole blood model to evaluate the potential of 10 nm IONPs to activate the complement system and induce cytokine production. Reactive oxygen species and cell death were also assessed. The IONPs activated complement, as measured by C3a, C5a and sC5b-9, and induced the production of pro-inflammatory cytokines in a particle-dose dependent manner, with the strongest response at 10 µg/mL IONPs. Complement inhibitors at C3 (compstatin analog Cp40) and C5 (eculizumab) levels completely inhibited complement activation and secretion of inflammatory mediators induced by the IONPs. Additionally, blockade of complement receptors C3aR and C5aR1 significantly reduced the levels of various cytokines, indicating that the particle-induced secretion of inflammatory mediators is mainly C5a and C3a mediated. The IONPs did not induce cell death or reactive oxygen species, which further suggests that complement activation alone was responsible for most of the particle-induced cytokines. These data suggest that the lepirudin anti-coagulated human whole blood model is a valuable ex vivo system to study the inflammatory potential of IONPs. We conclude that IONPs induce complement-mediated cytokine secretion in human whole blood. Keywords: iron oxide nanoparticles, human whole blood, complement activation, cytokines, reactive oxygen species, complement inhibitors

Susann Grosse,1 Jørgen Stenvik,1,2 Asbjørn M Nilsen1 1Department of Cancer Research and Molecular Medicine, 2Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway Abstract: Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response. Keywords: iron oxide nanoparticles, primary human monocytes, PBMCs, cytokines, LPS adsorption, Toll-like receptors

Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNβ and pro-inflammatory cytokines (TNF, IL-1β, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo., (© 2023 Nilsen et al.)

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50-310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30-300 particles cell -1 . Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Dual stimuli-responsive nanogels (NGs) have gained popularity in the field of bio medicine due to their versatile nature of applicability. Poly(N-isopropylacrylamide)-co-poly(acrylic acid) (pNIPAm-pAAc)-based NGs provide such dual stimuli-response with pNIPAm and pAAc providing thermal and pH-based responses, respectively. Studying the growth of these NGs, as well as, understanding the effect of the incorporation of pAAc in the NG matrix, is important in determining the physico-chemical properties of the NG. Studies have been conducted investigating the effect of increasing pAAc content in the NGs, however, these are not detailed in understanding its effects on the physico-chemical properties of the pNIPAm-pAAc-based NGs. Also, the biocompatibility of the NGs have not been previously reported using human whole blood model. Herein, we report the effect of different reaction parameters, such as surfactant amount and reaction atmosphere, on the growth of pNIPAm-pAAc-based NGs. It is shown that the size of the NGs can be precisely controlled from ~130 nm to ~400 nm, by varying the amount of surfactant and the reaction atmosphere. The effect of increasing incorporation of pAAc in the NG matrix on its physico-chemical properties has been investigated. The potential of these NGs as drug delivery vehicles is investigated by conducting loading and release studies of a model protein drug, cytochrome C (Cyt C) from the NGs at temperature above the volume phase transition temperature (VPTT) and acidic pH. An ex vivo human whole blood model was used to investigate biocompatibility of the NGs by quantifying inflammatory responses during NG exposure. The NGs did not induce any significant production of chemokine IL-8 or pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), and the cell viability in human whole blood was maintained during 4 h exposure. The NGs did neither activate the complement system, as determined by low Terminal Complement Complex (TCC) activation and Complement Receptor 3 (CR3) activation assays, thereby overall suggesting that the NGs could be potential candidates for biomedical applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1β, TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1β, and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1β, and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus , GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus , GBS, Streptococcus pneumonia, Pseudomonas aeruginosa , and E. coli . The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus , GBS, and S. pneumonia , the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.

TLR8 is the major endosomal sensor of degraded RNA in human monocytes and macrophages. It has been implicated in the sensing of viruses and more recently also bacteria. We previously identified a TLR8-IFN regulatory factor 5 (IRF5) signaling pathway that mediates IFNβ and interleukin-12 (IL-12) induction by Staphylococcus aureus and is antagonized by TLR2. The relative importance of TLR8 for the sensing of various bacterial species is however still unclear. We here compared the role of TLR8 and IRF5 for the sensing of Group B Streptococcus (GBS), S. aureus , and Escherichia coli in human primary monocytes and monocyte-derived macrophages (MDM). GBS induced stronger IFNβ and TNF production as well as IRF5 nuclear translocation compared to S. aureus grown to the stationary phase, while S. aureus in exponential growth appeared similarly potent to GBS. Cytokine induction in primary human monocytes by GBS was not dependent on hemolysins, and induction of IFNβ and IL-12 as well as IRF5 activation were reduced with TLR2 ligand costimulation. Heat inactivation of GBS reduced IRF5 and NF-kB translocation, while only the viable E. coli activated IRF5. The attenuated stimulation correlated with loss of bacterial RNA integrity. The E. coli -induced IRF5 translocation was not inhibited by TLR2 costimulation, suggesting that IRF5 was activated via a TLR8-independent mechanism. Gene silencing of MDM using siRNA revealed that GBS-induced IFNβ, IL-12-p35, and TNF production was dependent on TLR8 and IRF5. In contrast, cytokine induction by E. coli was TLR8 independent but still partly dependent on IRF5. We conclude that TLR8-IRF5 signaling is more important for the sensing of GBS than for stationary grown S. aureus in human primary monocytes and MDM, likely due to reduced resistance of GBS to phagosomal degradation and to a lower production of TLR2 activating lipoproteins. TLR8 does not sense viable E. coli , while IRF5 still contributes to E. coli -induced cytokine production, possibly via a cytosolic nucleic acid sensing mechanism.

Toll-like receptors (TLRs) are innate immune receptors for sensing microbial molecules and damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (poly(I:C)) bind and activate TLR3. This stimulation leads to recruitment of the adaptor molecule TRIF (Toll/IL-1 resistance (TIR) domain-containing adapter-inducing interferon β) and activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNβ production. Here we report that, unlike non-metastatic intestinal epithelial cells (IECs), metastatic IECs express TLR3 and that TLR3 promotes invasiveness of these cells. In response to poly(I:C) addition, the metastatic IECs also induced the chemokine CXCL10 in a TLR3-, TRIF-, and IRF3-dependent manner but failed to produce IFNβ. This was in contrast to healthy and non-metastatic IECs, which did not respond to poly(I:C) stimulation. Endolysosomal acidification and the endosomal transporter protein UNC93B1 was required for poly(I:C)-induced CXCL10 production. However, TLR3-induced CXCL10 was triggered by immobilized poly(I:C), was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 can still signal from the cell surface of these cells. Furthermore, plasma membrane fractions from metastatic IECs contained both full-length and cleaved TLR3, demonstrating surface expression of both forms of TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli that trigger chemokine responses and promote invasiveness in these cells. We conclude that altered TLR3 expression and localization may have implications for cancer progression., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinflammatory cytokines via MyD88 -: NF-κB signaling. S. aureus can also induce the production of IFN-β, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-β induction by S. aureus are unclear. Surprisingly, we demonstrate that activation of TLR2 by lipoproteins does not contribute to IFN-β production but instead can suppress the induction of IFN-β in human primary monocytes and monocyte-derived macrophages. The production of IFN-β was induced by TLR8-mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IκB kinase (IKK)β, which thus reveals a physiological role of the recently described IRF5-activating function of IKKβ. TLR8 -: IRF5 signaling was necessary for induction of IFN-β and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-β and IL-12 production via a TAK1 -: IKKβ -: IRF5 pathway that can be inhibited by TLR2 signaling., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-β and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alginates from seaweed are used in chronic wound management, though the molecular and cellular effects of various alginate dressings are not well documented. We have developed ultrapure sodium-alginates from Pseudomonas fluorescens with different content and distribution of single guluronic acid (G) residues (0-45% G), and tested their biological activities on human primary keratinocytes (KCs). The alginates inhibited KC migration and induced expression of differentiation markers. The potency of the alginates correlated with the increasing percentage of single G residues. These findings were explained by different binding and release of ionic calcium (Ca++) from the alginates which subsequently triggered differentiation. Ca-free alginates had no effect on KC migration and differentiation, but the chemokine receptor CXCR7 was upregulated. Q-PCR revealed that also CXCL12/SDF-1, one of two known CXCR7-ligands, was induced by the alginates. Both CXCR7 and CXCL12-induction was dependent on the alginate G-content, and highest upregulation was induced by an alginate with 19% single G residues. In the epidermis, CXCR7 expression was restricted to the basal layer. This study defines two biological effects of ultrapure alginates on KCs, both being dependent on the alginate structure, and being either dependent or independent of Ca., (Copyright © 2012 Wiley Periodicals, Inc.)

Toll-like receptor 3 (TLR3) is an important sensor of viral infections and injury of self in keratinocytes. In this study, we stimulated primary keratinocytes with the TLR3-ligand polyI:C. This induced a toxic effect shown by up-regulation of the alarmin high-mobility group protein B1 and reduced responses in a MTT-assay. PolyI:C was a potent inducer of proinflammatory cytokines, and both these responses and the cytotoxic effects were found to be TLR3 dependent, as demonstrated by the use of siRNA for TLR3. Interestingly, co-stimulation with oligodeoxynucleotides (ODNs) inhibited all polyI:C induced effects. This inhibition was found to be mediated by the competition of endocytic uptake of polyI:C and ODNs. We have found polyI:C induced cytotoxicity and proinflammatory responses to be dependent of TLR3 and that this may be inhibited by ODNs. With these findings, we see a promising potential for ODNs in inhibiting TLR3-induced responses in inflammatory skin disorders., (© 2011 John Wiley & Sons A/S.)

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: The molecular pathogenesis of chronic skin wounds is complex and not fully understood. Although these wounds are often characterized as being in a state of persistent inflammation, the impact and participation of the innate immune responses in sustaining this inflammation needs further investigation., Objective: We investigated the cytokine profiles, Toll-like receptor (TLR)-stimulating activities and the levels of the antibacterial peptide Lipocalin-2 (Lcn-2) in a series of healing and non-healing chronic venous leg ulcers (CVLUs) through a study time of 8 weeks., Methods: Wound fluids from healing and non-healing CVLUs were run on a Human Cytokine Antibody Array, and Lcn-2 levels measured with ELISA. HEK 293 cells transfected with TLR2 or TLR4 and their respective co-receptors, and human peripheral blood monocytes were then stimulated with the wound fluids from healing and non-healing venous leg ulcers., Results: Healing wounds were associated with decreasing levels of IL-1alpha, IL-1beta and MIP-1delta, whereas in non-healing wounds decreasing levels of IL-8 and MIP-1alpha were found. Accordingly, wound fluid from non-healing CVLUs contained persistent Lcn-2 levels and TLR2- and TLR4-stimulating activities, while, in healing wounds, the TLR-stimulating activities decreased over time with significantly diminished levels of Lcn-2 (p<0.005)., Conclusions: Innate immune responses contribute to the chronic inflammation in non-healing CVLUs through participation of Toll-like receptors. The levels of the antimicrobial peptide Lcn-2 in wound fluids from these ulcers are elevated as a reflection of this contribution., (Copyright 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Objective: Meconium, the first intestinal discharge of the newborn, contains material accumulated during fetal life. Meconium activates complement and CD14 and may induce a systemic inflammatory response. Toll-like receptors are classical pattern-recognition receptors recognizing both exogenous and host-derived ligands. The cyanobacterial product CyP is a potent LPS antagonist binding to the TLR4/MD-2 complex. The aim of the present study was to investigate the role of the CD14/TLR4/MD-2 complex in meconium-induced inflammation., Methods: Whole blood from six donors was preincubated with anti-CD14 or CyP. Meconium was added and the samples were incubated for 4h. Twenty-seven inflammatory mediators were measured in a Bioplex Array Reader. Human embryonic kidney cells transfected with plasmids containing NF-kappaB dependent luciferase reporter, human MD-2, TLR4, TLR2 and/or CD14, were incubated with meconium or LPS for 18 h. Luciferase activity in cytoplasmic extracts was measured using a Luciferase Assay System kit., Results: Meconium induced formation of a broad panel of inflammatory mediators. CyP and anti-CD14 significantly (p<0.001) inhibited meconium-induced formation of (a) proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IFN-gamma) by 60-80% and 72-94%, respectively, (b) anti-inflammatory cytokines (IL-10, IL-1Ra) by 58-59% and 50-65%, respectively, (c) chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin, IP-10) by 43-77% and 57-87%, respectively, and (d) growth factors (G-CSF, GM-CSF, basic FGF, PDGFbb) by 53-71% and 40-78%, respectively, with no statistical significant difference between Cyp and anti-CD14. The inflammatory response could only partly be explained by LPS, suggesting that endogenous components of meconium contribute to the inflammatory response. Meconium activated NF-kappaB dose-dependently in cells expressing TLR4/MD-2 together with CD14, while no effect was seen in cells expressing TLR4/MD-2 alone or in TLR2/CD14 transfected cells., Conclusions: The results indicate that the CD14-dependent meconium-induced inflammatory reaction is mediated through the TLR4/MD2 complex. These data may have implications for future therapeutic strategies for meconium aspiration syndrome., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The clinical presentation of meningococcal disease is closely related to the number of meningococci in the circulation. This study aimed to examine the activation of the innate immune system after being exposed to increasing and clinically relevant concentrations of meningococci. We incubated representative Neisseria meningitidis serogroup B (ST-32) and serogroup C (ST-11) strains and a lipopolysaccharide (LPS)-deficient mutant (the 44/76 lpxA mutant) in human serum and whole blood and measured complement activation and cytokine secretion and the effect of blocking these systems. HEK293 cells transfected with Toll-like receptors (TLRs) were examined for activation of NF-kappaB. The threshold for cytokine secretion and activation of NF-kappaB was 10(3) to 10(4) meningococci/ml. LPS was the sole inflammation-inducing molecule at concentrations up to 10(5) to 10(6) meningococci/ml. The activation was dependent on TLR4-MD2-CD14. Complement contributed to the inflammatory response at >or=10(5) to 10(6) meningococci/ml, and complement activation increased exponentially at >or=10(7) bacteria/ml. Non-LPS components initiated TLR2-mediated activation at >or=10(7) bacteria/ml. As the bacterial concentration exceeded 10(7)/ml, TLR4 and TLR2 were increasingly activated, independent of CD14. In this model mimicking human disease, the inflammatory response to N. meningitidis was closely associated with the bacterial concentration. Therapeutically, CD14 inhibition alone was most efficient at a low bacterial concentration, whereas addition of a complement inhibitor may be beneficial when the bacterial load increases.

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are important components of the mammalian innate defence system against Gram-negative infections. cDNA encoding a protein related to mammalian BPI and LBP have been cloned from several teleosts including the Atlantic cod (Gadus morhua L.). Using real-time PCR an increase in cod BPI/LBP expression in whole blood and peritoneal cells was demonstrated one, two and four days after intraperitoneal injection of inactivated Vibrio anguillarum. Although constitutively produced in the head kidney, a moderate rise of BPI/LBP mRNA production was seen on day two in this organ. After seven days the BPI/LBP mRNA levels at the three locations examined were almost back to normal. In situ hybridisation demonstrated a leucocytic localisation and morphology of the BPI/LBP expressing cells in various tissues. A combination of in situ hybridisation and peroxidase staining of head kidney leucocytes showed that the BPI/LBP producing cells are peroxidase positive and possible neutrophil like cells. The results suggest that the cod BPI/LBP is important in the first-line defence against bacterial infections and has a function more related to the mammalian BPI molecule than the LBP counterpart.

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.

Suppression subtractive hybridization was used to examine gene expression in Atlantic cod head kidney cells treated with polyinosinic polycytidylic acid (poly I:C). One of the most abundant genes was ISG15, showing 24-53% amino acid similarity to ISG15 from both mammals and teleosts. The promoter was cloned by genome walking and three potential interferon-stimulated response elements (ISREs) were identified. Analysis of the gene structure revealed a single intron in the 5' untranslated region (UTR) of cod ISG15, which also seems to be present in zebrafish and pufferfish ISG15. A quantitative real time PCR assay was established to monitor the gene expression of cod ISG15. Injection of cod with poly I:C strongly induced the expression of ISG15 in all organs investigated. Stimulation was most pronounced the first day with a gradual decline the following days. The expression of ISG15 in head kidney cells was also induced in vitro by treatment with poly I:C, but not significantly with LPS. However, injection of formalin killed Vibrio anguillarum-induced ISG15 expression in head kidney.

The group of teleosts is highly diverse, comprising more than 23000 extant species. Studies of the teleost antibody repertoire have been conducted in many different species within different orders, though some species and families have been better characterised than others. The Atlantic cod (Gadus morhua L.) and several species within the Salmoninae (e.g. Salmo salar and Oncorynchus mykiss) are among the best-studied teleosts in terms of the antibody repertoire. The estimated size of the repertoire, the organisation of immunoglobulin (IG) gene segments, the expressed IG repertoire, the IgM serum concentration, and the serum antibody responses reveal some fundamental differences between these species. The serum IgM concentration of G. morhua is some ten times higher than that of S. salar, though G. morhua is characterised as a 'low' (or 'non') responder in terms of specific antibody production. In contrast, an antibody response is readily induced in S. salar, although the response is strongly regulated by antigen induced suppression. The IGHD gene of G. morhua has a unique structure, while the IGHM and IGHD genes of S. salar have a characteristic genomic organisation in two parallel loci. In addition, salmonids, express a broad repertoire of IGH and IGI V-region gene segments, while a single V gene family dominates the expressed heavy and light chain repertoire of G. morhua. Little is known about the developing antibody repertoire during ontogeny, in different stages of B-cell maturation, or in separate B-cell subsets. Information on the establishment of the preimmune repertoire, and the possible role of environmental antigens is also sparse.

Using the differential screening technique, a cDNA related to the mammalian family of lipid transfer/lipopolysaccharide-binding proteins was cloned from the Atlantic cod (Gadus morhua L.). The gene is an ortholog of a recently identified gene of rainbow trout (Oncorhynchus mykiss). Phylogenetic analyses suggest that teleost BPI/LBP are modern descendants of the ancestor of mammalian bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), and a gene of the urochordate Ciona intestinalis is related to this gene family. Molecular modeling suggests that the structure of cod BPI/LBP is similar to mammalian BPI and LBP, while its highly basic character is similar to BPI. Cod BPI/LBP is constitutively expressed in head-kidney (HK) leukocytes. After intraperitoneal injection of bacterin high levels of cod BPI/LBP mRNA were detected also in peripheral blood cells and spleen, while moderate to low levels of transcript were found in heart, liver, gills, skin, brain, and intestine. We conclude that the patterns of charge and expression of cod BPI/LBP are more similar to mammalian BPI than to mammalian LBP.

Cytokines are important mediators in inflammation, and play a key role in inflammation induced by Gram-negative bacteria. Cytokines are released into serum during different pathological conditions, such as meningococcal disease, and the cytokine levels in serum seem to correlate with fatal outcome of septic shock (1-3). Consequently, detection of cytokines in serum samples from patients with pathological diseases may be of prognostic and clinical value. Cytokines can be detected using bioassays and immunoassays, and this chapter focuses on description of bioassays for detection of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6 in serum samples.

This study was undertaken to determine whether a lack of VH domain diversity could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod VH regions was studied in 113 cDNA and 2 genomic clones. A fourth VH family and a second putative JH element were identified. The expressed VH repertoire showed a clear bias in the pattern of VH family utilization, with about 80% of the clones belonging to the VH-III family. Furthermore, the VH-III family was complex and could be subdivided into several subfamilies, while little variation was seen within the other families. The VH family bias gives a somewhat reduced variability of the VH gene region of cod, but not lower than that of the rabbit IgM repertoire. The H chain CDR3 region of cod was longer than that of trout, frog and mouse, and also highly variable in sequence, probably reflecting a relative importance of this region in cod. On the other hand, the CDR3 length variability was restricted, and this may reduce the diversity of the cod VH region.

A new immunoglobulin heavy-chain gene with some homology to mammalian IgD was recently cloned from the channel catfish and Atlantic salmon, two species of teleost fish. We have cloned and sequenced a new H-chain gene from Atlantic cod (Gadus morhua L.) which has clear similarities to these genes, but which also differs in several ways. The similarities of catfish, salmon, and cod delta to the mammalian delta genes are sequence homology, location immediately downstream of IgM (mu), and expression by alternative splicing rather than class switching. A unique feature of catfish, salmon, and cod delta is the chimeric nature of the gene product, as the mu1 exon is spliced to the delta1 exon. Several unique features of cod IgD were found: (1) a deletion of the delta3, delta4, delta5, and delta6 domains described in catfish and salmon IgD, (2) a tandem duplication of a part of the delta locus including the delta1 and delta2 domains, (3) the presence of a truncated delta7 domain downstream of the deltaTM exons, and (4) the separation of the duplicated domains by a short exon (deltay) which has homology to a conserved part of the transmembrane exon 1 (TM1) of some H-chain isotypes. This unique organization of the delta locus of cod probably developed after the evolutionary split from the catfish and salmon branches.