Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin.

Hemoglobin (Hb) released during red blood cell lysis can initiate TLR4-dependent signaling and trigger NF-κB activation in surrounding cells. Observations of chronic bleeding in various cancers leads us to hypothesize that Hb and Hb degradation products released from lysed RBC near cancer nests might modulate local TLR4-positive cells. We addressed the hypothesis in vitro by measuring Hb- and biliverdin (Bv)-induced NF-κB signaling in an engineered human TLR4 reporter cell model (HEK-BlueTM hTLR4). Therein, TLR4 stimulation was assessed by measuring NF-κB-dependent secreted alkaline phosphatase (SEAP). hTLR4 reporter cells incubated with 8 ηM lipopolysaccharide (LPS) or 20-40 µM fungal mannoprotein (FM) produced significant amounts of SEAP. hTLR4 reporter cells also produced SEAP in response to human, but not porcine or bovine, Hb. HEK-Blue Null2TM reporter cells lacking TLR4 did not respond to LPS, FM, or Hb. Bv was non-stimulatory in reporter cells. When Bv was added to Hb-stimulated reporter cells, SEAP production was reduced by 95%, but when Bv was applied during LPS and FM stimulation, SEAP production was reduced by 33% and 27%, respectively. In conclusion, Hb initiated NF-κB signaling that was dependent upon TLR4 expression and that Bv can act as a TLR4 antagonist. Moreover, this study suggests that hemorrhage and extravascular hemolysis could provide competitive Hb and Bv signaling to nearby cells expressing TLR4, and that this process could modulate NF-κB signaling in TLR4-positive cancer cells and cancer-infiltrating leukocytes.

Chimeric approach for narrowing a membrane-inserting region within human perforin.

Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved ß-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane ß-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40-50 µg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP's ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.

Therapeutic targeting of CCR1 attenuates established chronic fungal asthma in mice.

CC chemokine receptor 1 (CCR1) represents a promising target in chronic airway inflammation and remodeling due to fungus-associated allergic asthma. The present study addressed the therapeutic effect of a nonpeptide CCR1 antagonist, BX-471, in a model of chronic fungal asthma induced by Aspergillus fumigatus conidia. BX-471 treatment of isolated macrophages inhibited CCL22 and TNF-alpha and promoted IL-10 release. BX-471 also increased toll like receptor-9 (TLR9) and decreased TLR2 and TLR6 expression in these cells. When administered daily by intraperitoneal injection, from days 15 to 30 after the initiation of chronic fungal asthma, BX-471 (3, 10, or 30 mg kg(-1)) dose-dependently reduced airway inflammation, hyper-responsiveness, and remodeling at day 30 after conidia challenge. The maximal therapeutic effect was observed at the 10 mg kg(-1) dose. In summary, the therapeutic administration of BX-471 significantly attenuated experimental fungal asthma via its effects on both innate and adaptive immune processes.

A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment.

CCR5 antagonists: the answer to inflammatory disease?

Chemokines and their receptors mediate the inflammatory response during infectious and non-infectious disease. However, their continued activation and disregulation are commonly associated with chronic inflammation. Frequently, affected sites are characterised by inflammatory cell infiltrates expressing CC chemokine receptor 5 (CCR5) and high levels of CCR5 ligands. Neutralisation of CCR5 decreases the incidence and pathology of these diseases in murine models, and epidemiological studies in human patients corroborate these data. CCR5-deficiency has been associated with increased risk of hepatic disease and infection, but considering the pathological effects of chronic inflammation, pharmacologically targeting CCR5 is still a desirable and feasible goal. The discovery that CCR5 is a major HIV coreceptor initiated the race to produce effective CCR5 antagonists. This review summarises the progress made in CCR5 antagonist development and assesses their potential in the treatment of inflammatory disease.

CCR4 is a key modulator of innate immune responses.

CCR4 is recognized as a key receptor in Th2-associated immune processes, although very little is known about its role in innate immunity. Previous studies reported increased resistance to LPS-induced lethality in CCR4(-/-) mice compared with wild-type mice. This study demonstrates that CCR4(-/-) mice are similarly resistant to challenge with other TLR agonists, as well as bacterial peritonitis. Resistance was associated with enhanced early leukocyte recruitment, increased TLR expression, a skewed type 2 cytokine/chemokine profile, and improved bacterial clearance. Macrophages from CCR4(-/-) mice exhibited many features consistent with alternative activation, including elevated secretion of type 2 cytokines/chemokines and the found in inflammatory zone 1 (FIZZ1) protein. MyD88-dependent NF-kappaB signaling was significantly down-regulated in CCR4(-/-) macrophages, whereas p38 MAPK and JNK activation were conversely increased. These data stress the importance of CCR4 in macrophage differentiation and innate immune responses to pathogens, as well as the involvement of chemokine receptor expression in TLR signaling regulation.

Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1beta.

Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both immune response and inflammation-related diseases. The poxvirus-encoded viral CC chemokine inhibitor (vCCI) binds to many CC chemokines with high affinity, acting as a potent inhibitor of chemokine action. We have used heteronuclear multidimensional NMR to determine the structure of an orthopoxvirus vCCI in complex with a human CC chemokine, MIP-1beta (macrophage inflammatory protein 1beta). vCCI binds to the chemokine with 1:1 stoichiometry, forming a complex of 311 aa. vCCI uses residues from its beta-sheet II to interact with a surface of MIP-1beta that includes residues adjacent to its N terminus, as well as residues in the 20's region and the 40's loop. This structure reveals the strategy used by vCCI to tightly bind numerous chemokines while retaining selectivity for the CC chemokine subfamily.

Immunomodulatory role of CXCR2 during experimental septic peritonitis.

The loss of CXCR2 expression by neutrophils is a well-described, but poorly understood, consequence of clinical sepsis. To address the potential impact of this CXCR2 deficit during the septic response, we examined the role of CXCR2 in a murine model of septic peritonitis provoked by cecal ligation and puncture (CLP). CLP-induced mouse mortality was significantly attenuated with i.v. or i.p. administration of an affinity-purified murine CXCR2-specific polyclonal Ab. Mouse survival required Ab administration before and every 2 days following CLP. Furthermore, mice deficient in CXCR2 (CXCR2(-/-)) were significantly protected against CLP-induced mortality compared with control (CXCR2(+/+)) mice. The anti-CXCR2 Ab treatment delayed, but did not completely inhibit, the recruitment of leukocytes, specifically neutrophils, into the peritoneal cavity. Peritoneal macrophages from anti-CXCR2 Ab-treated mice exhibited markedly increased RNA and protein levels of several key proinflammatory cytokines and chemokines. Specifically, isolated preparations of these cells released approximately 11-fold more CXCL10 protein compared with peritoneal macrophages from control-treated or naive mice. CXCR2(-/-) mice had higher resting and CLP-induced levels of peritoneal CXCL10 compared with CXCR2(+/+) mice. Administration of a neutralizing, affinity-purified, murine CXCL10-specific polyclonal Ab before CLP in wild-type mice and every 2 days after surgery significantly increased mortality compared with control Ab-treated mice. Anti-CXCL10 treatment in CXCR2(-/-) mice negated the protective effect associated with the absence of CXCR2. In summary, these data demonstrate that the absence of CXCR2 protects mice from septic injury potentially by delaying inflammatory cell recruitment and enhancing CXCL10 expression in the peritoneum.

CCR1 and CC chemokine ligand 5 interactions exacerbate innate immune responses during sepsis.

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.