Macrophage-activating Lipopeptide-2 Research Articles

Toll-Like Receptor Ligands Cause Proinflammatory and Prodiabetic Activation of Adipocytes via Phosphorylation of Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase But Not Interferon Regulatory Factor-3

Here, we aim to investigate the mechanisms of Toll-like receptor (TLR)-induced prodiabetic and proinflammatory activation of adipocytes and to detect differences in the responsiveness of TLRs to their respective ligands between adipocytes isolated from inflamed vs. noninflamed adipose tissue. Experiments using specific ligands for all known TLRs were performed in murine 3T3-L1 adipocytes and in human adipocytes isolated from noninflamed and inflamed adipose tissue. IL-6 and monocyte chemoattractant protein-1 (MCP-1) release were measured by ELISA. The expression of the signal transduction proteins phospho-extracellular signal-regulated kinase (P-Erk), P-c-Jun N-terminal kinase (JNK), and P-interferon regulatory factor-3 was investigated by Western blot analysis. Additionally, functional inhibitors of MAPK kinase-1/-2 and JNK-1/-2 were used in the stimulation experiments. Activation of TRL4 by lipopolysaccharide (LPS) and TLR1/2 by Pam(3)Cys up-regulates IL-6 and MCP-1 release in adipocytes via specific activation of Erk. Stimulation of adipocytes by macrophage activating lipopeptide-2 (MALP-2) induces MCP-1 but has no effect on IL-6 release. This stimulatory effect on MCP-1 release is antagonized by inhibition of both mitogen-activated protein kinase-1/-2 and JNK-1/-2. Phosphorylation of Erk and JNK is up-regulated after stimulation by MALP-2. In human adipocytes isolated from noninflamed adipose tissue, LPS and Pam(3)Cys, but not MALP-2, are potent inducers of IL-6 and MCP-1. MALP-2 is able to induce IL-6 and MCP-1 release in adipocytes isolated from inflamed adipose tissue, whereas these adipocytes lost their ability to respond to LPS. The present results point to a role of the adipose tissue in innate immunity. TLR-ligand-induced proinflammatory and prodiabetic activation of adipocytes might couple visceral adipose tissue dysfunction with insulin resistance and type 2 diabetes mellitus.

Beneficial Effects of TLR-2/6 Ligation in Pulmonary Bacterial Infection and Immunization with Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is the major pathogen in nosocomial and life-threatening infections of immunocompromised or critically ill patients. The macrophage-activating lipopeptide-2 (MALP-2) activates the immune system via Toll-like receptors (TLR) 2 and 6 and leads to an accumulation of immune cells in lungs of young adult (8-10 week old) rats after intratracheal application. This is characterized by a high increase of granulocyte numbers in the BAL 24 h after MALP-2 treatment. It was hypothesized that MALP-2 may have a positive effect on the clinical course of an experimental infection. Therefore, rats were treated with MALP-2 at different time points following an infection with P. aeruginosa. The effect of MALP-2 in combination with immunization with inactivated P. aeruginosa was also investigated. Rats (n = 10) were infected intratracheally (i.t.) with 1 x 10(8) CFU P. aeruginosa on day 0. They were treated on day -3, -1, 0 and +1 with 2.5 microg MALP-2 or the vehicle i.t. In additional experiments, rats were immunized on day -21 and -14 with 1 x 10(8) CFU of inactivated P. aeruginosa bacteria and 2.5 microg MALP-2 or vehicle with 1 x 10(8) CFU of inactivated bacteria and isopropanol. The clinical score, rectal temperature and weight of the rats were checked in both treatment and immunization experiments twice a day. On day 2 they were sacrificed, CFU were determined in the left lung, the right lung being used for histology. In the group treated with MALP-2 1 day prior to infection significant effects were seen: The rectal temperature was about 2 degrees C higher in comparison to the controls at 6 h and also 1 day after infection. Both the symptoms of the infection and the weight loss were significantly reduced. In addition, the CFU and the inflammation in the lung tissue were significantly lower. These effects were not observed after treatment on day -3, 0 or +1. The MALP-2 enhanced immunization only resulted in a tendency to clinical improvement. In conclusion, local immunostimulation at the appropriate time can enhance the host defense against bacteria in the lung.

Propionibacterium acnes and Sebaceous Lipogenesis: A Love–Hate Relationship?

In this issue, Iinuma et al. show that Propionibacterium acnes (P. acnes)-conditioned medium and formalin-killed P. acnes augment intracellular lipid formation in hamster sebocytes by increasing the de novo synthesis of triacylglycerols. This commentary summarizes the current knowledge of the association of P. acnes with sebaceous lipogenesis, inflammation, and innate immunity, and points out the concurrent evidence that P. acnes-induced lipids may represent a recruitment of allies and/or enemies of the human skin.

MyD88 Adaptor-Like Is Not Essential for TLR2 Signaling and Inhibits Signaling by TLR3

Although a clear role for the adaptor protein myeloid differentiation factor-88 (MyD88) adaptor-like (Mal, or TIRAP) in TLR4 signaling has been demonstrated, there is limited information on its role in TLR2 signaling. Here we have systematically analyzed the role of Mal in signaling by TLR2, TLR4, and as a control TLR3 in murine macrophages and dendritic cells. Mal was not required for the induction of IL-6 or NFkappaB activation at high concentrations of the TLR1/2 ligand Pam(3)Cys-Ser-(Lys)(4) or the TLR2/6 ligand macrophage-activating lipopeptide-2 and was required for these responses only at low ligand concentrations. Similarly, induction of IL-6 by Salmonella typhimurium, which is sensed by TLR2, required Mal only at low levels of bacteria. Mal was required for IL-6 induction at all concentrations of the TLR4 ligand LPS. Mal deficiency boosted IL-6 induction by the TLR3 ligand polyinosinic-polycytidylic acid. Activation of JNK, but not p38 or IkappaB degradation, was similarly potentiated in response to polyinosinic-polycytidylic acid in Mal-deficient macrophages. MyD88 was vital for all TLR2 and TLR4 responses and, similar to Mal, was also inhibitory for TLR3-dependent IL-6 and JNK induction. MyD88 interacted with the Toll/IL-1R domains of TLR1, TLR2, TLR4, and TLR6. Mal interacted with the Toll/Il-1R domains of TLR1, TLR2, and TLR4 but not with TLR6. Our study, therefore, reveals that Mal is dispensable in TLR2 signaling at high ligand concentrations in macrophages and dendritic cells, with MyD88 probably coupling to the TLR2 receptor complex at sufficient levels to allow activation. An inhibitory role for Mal in TLR3 signaling to JNK was also demonstrated.

Vaccination approaches against Trypanosoma cruzi infection

In natural infection, the survival of Trypanosoma cruzi, despite the complex immune response elicited including several humoral and cellular components of innate and acquired immunity, suggests that the immune system’s natural responses are inherently inadequate. Consequently, it is of paramount importance to find alternatives to direct the immune system before infection, and redirect it after infection, to obtain a prophylactic and therapeutic vaccine. Herein, we review the recent advances in vaccine research and the development of the major antigen candidates, including cruzipain, trans-sialidase, amastigote surface protein, paraflagellar rod protein, among others. In the last 5 years, experimental works have been conducted to analyze DNA delivery systems, including viruses and bacteria, as well as immunomodulators such as CpG–oligodeoxynucleotide, macrophage-activating lipopeptide from Mycoplasma fermentans, glycolipid α-galactosylceramide, granulocyte-macrophage colony-stimulating factor, IL-12 and other cytokines and chemokines. The review also covers articles that shed light on some mechanisms of innate and adaptive immunity against T. cruzi, which improved our knowledge and provided potentially useful tools to fight infection. A better understanding of the protective immune responses that can effectively arrest T. cruzi survival in the mammalian host is critical for the development of vaccines against Chagas disease.

Synthetic Lipopeptide MALP-2 Inhibits Intracellular Growth of Mycobacterium bovis BCG in Alveolar Macrophages—Preliminary Data

Alveolar macrophages (AM) are the primary target cell of the lung for inhaled mycobacterial pathogens. We investigated the effect of the synthetic lipopeptide MALP-2 on the interaction between AM from rats and Mycobacterium bovis BCG. AM were infected with M. bovis BCG at a multiplicity of infection (MOI) of 10 and then cultured in medium alone or medium supplemented with either synthetic macrophage activating lipopeptide-2 (MALP-2), or IFN-gamma, or both. Mycobacterial CFU were counted on days 3 and 7 and cell-free supernatants were collected for cytokine measurements. Treatment of macrophages with MALP-2 led to reduced bacterial loads by day 3 and 7 post-infection and at the same time enhanced the release of TNF-alpha, IL-6 und IL-10 compared to non-stimulated, M. bovis BCG-infected AM. Macrophages co-treated with MALP-2 and IFN-gamma or IFN-gamma in the absence of MALP-2 limited the growth of M. bovis BCG only by day 3, but not day 7 post-stimulation. Our data show that MALP-2 is effective in decreasing bacterial loads in lung sentinel cells.

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL-6, IL-1beta, TGF-beta, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor gammabeta was detected. To clarify if IL-17(+)cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c(+)/langerin(+) monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1beta, and IL-23. TGF-beta, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1beta, and IL-23 by MoLC, while TGF-beta was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-gamma expression was higher in MoLC- than in monocyte-derived DC-cocultured Th cells. Anti-IL-1beta, -TGF-beta and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

Murine mast cells secrete a unique profile of cytokines and prostaglandins in response to distinct TLR2 ligands

Mast cells (MCs) are important effector cells in host defense against bacteria. In the course of a bacterial infection, MCs can be activated by various mechanisms, i.e. bacterial toxins, endogenously produced infection-associated peptides or via complement receptors, fimbrial adhesion molecules and toll-like receptors (TLRs). While some of these mechanisms are well established, the effects of TLR2 ligand-driven MC activation are far less understood. Here, we show that murine mature connective tissue-type MCs, but not immature bone marrow-derived cultured mast cells, express significant amounts of full length TLR2 on their surface. Activation by various TLR2 ligands only induces the selective release of cytokines in peritoneum-derived cultured mast cells (PCMCs) with preferential secretion of pro-inflammatory cytokines (IL-6 > IL-17 > IFN-gamma TNF > IL-1 > GM-CSF) upon stimulation with lipoteichoic acid (LTA). This response is much lower in PCMCs stimulated with the TLR2/6 agonist macrophage-activating lipopeptide-2 (MALP-2), which most prominently triggers the release of the immunomodulatory cytokine IL-10. Furthermore, only LTA but not MALP-2 induces prostaglandin D2 secretion which is again restricted to the mature MC phenotype. These findings suggest that TLR2 ligand-mediated activation of mature MCs, i.e. tissue-residing cells, which most likely occurs during infection, can selectively raise a potent inflammatory or anti-inflammatory response, depending on TLRs which are engaged.

Monocyte antimicrobial activities are differentially triggered by TLR2/1 and TLR2/6 activation (134.91)

Abstract Human innate immune responses against microbial pathogens are mediated in part by Toll-like receptors (TLR). We have previously demonstrated that the 19kD lipoprotein, a TLR2/1 ligand, induces a vitamin D-dependent killing of Mycobacterium tuberculosis in infected monocytes; however the ability of the other TLR family members to induce this pathway is unclear. Given that TLRs instruct the innate immune response to different types of pathogens, we hypothesize the TLR family members will differentially regulate antimicrobial activity. While 19kD is able to induce killing in infected macrophages, macrophage activating lipopeptide 2 (MALP-2), a TLR2/6 ligand, did not. We investigated the differences in gene expression profiles between 19kD and MALP-2 treated monocytes, which yielded differentially expressed gene programs. Interleukin-10 family members were found to be upregulated in 19kD treated monocytes as compared to monocytes treated with MALP-2. In MALP-2 treated monocytes, leukocyte immunoglobulin-like receptors (LIR) were found to be upregulated. Investigating these differentially regulated genes can yield information that could results therapeutic targets against intracellular pathogens.

Innate Immunity and Adipocyte Function: Ligand‐specific Activation of Multiple Toll‐like Receptors Modulates Cytokine, Adipokine, and Chemokine Secretion in Adipocytes

The aim of this study was to analyze Toll-like receptor (TLR) expression in preadipocytes and mature adipocytes and to investigate whether TLR ligands influence the release of cytokines, chemokines, and adipokines. Murine 3T3-L1 preadipocytes and mature adipocytes were used for stimulation experiments. The effects of lipopolysaccharide (LPS), flagellin, Poly (U), Poly (I:C), macrophage-activating lipopeptide-2 (MALP2), Pam3Cys, and CpG on the release of interleukin-6 (IL-6), resistin, and monocyte chemoattractant protein-1 (MCP-1) were determined by enzyme-linked immunosorbent assay (ELISA). Nuclear translocation and promoter binding of NFkappaB were analyzed by electrophoretic mobility shift assays. TLR expression was investigated by reverse-transcriptase (RT-PCR). All TLRs except TLR5 and TRL7 are expressed in the stromal vascular cell (SVC) fraction and in mature adipocytes of different fat stores. Whereas basal and LPS-induced IL-6 release is higher in preadipocytes, basal and LPS-induced MCP-1 release is higher in mature adipocytes. Mature adipocytes respond to corticosterone regarding MCP-1 and resistin release. The ligands for TLRs influence IL-6, MCP-1, and resistin release differentially. Some of these ligands induce nuclear translocation and promoter binding of NFkappaB. Besides TLR5, that is not expressed in mature adipocytes, all TLR family members are involved. There exists a functional TRL pathway in adipocytes that connects innate immunity with adipocyte function. As a consequence, the role of the adipose tissue in both immunity and metabolism has to be investigated in future studies. The results of this approach will help to explain the metabolic changes such as insulin resistance observed during infection and the immunological phenomena such as macrophage infiltration of adipose tissue seen in obesity.

Toll‐like receptor ligand activation of murine bone marrow‐derived dendritic cells

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses.

Polyanionic Microbicides Modify Toll-Like Receptor-Mediated Cervicovaginal Immune Responses

Topical microbicides are being developed as a preventative approach to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1) and other infections. For them to be efficacious, it is believed that they should avoid inducing inflammation while allowing the vaginal epithelium to initiate protective Toll-like receptor (TLR)-mediated innate responses against pathogens. In this study, human cervical and vaginal epithelial cells were exposed to polyanionic HIV entry inhibitors and the following synthetic TLR ligands: (i) the bacterial lipoprotein Pam(3)CSK(4), binding cell surface TLR1/TLR2; (ii) macrophage activating lipopeptide 2 (MALP-2), binding cell surface TLR2/TLR6; and (iii) the viral double-stranded RNA analog poly(I:C), recognized by intracellular TLR3. Cell activation was assessed by nuclear factor kappaB (NF-kappaB) reporter gene transactivation and cytokine production. In spite of enhancing TLR-triggered NF-kappaB activation, the polyanionic microbicide compounds dextran sulfate and polystyrene sulfonate significantly inhibited TLR-mediated cytokine production. They decreased cytokine mRNA and protein levels of proinflammatory (interleukin-8 [IL-8] and IL-1beta) and antiviral (beta interferon) cytokines following epithelial cell stimulation with Pam(3)CSK(4), MALP-2, or poly(I:C). These activities were associated with the sulfate/sulfonate moieties of the polyanionic compounds, since the unsulfated dextran control did not show any effects. Our data demonstrate that these microbicide compounds are capable of selectively interfering with TLR-mediated epithelial responses at different points in their signaling pathways and underscore the importance of expanding the assessment of microbicide compatibility with vaginal innate immune function. Further studies are warranted to determine the impact of this interference on HIV-1 transmission risk.

TLR2/6 stimulation of the rat lung: effects on lymphocyte subsets, natural killer cells and dendritic cells in different parts of the air‐conducting compartments and at different ages

The composition of lymphocyte subsets in the lung has been found to be compartment-specific. To characterize the effect of age, weanling, young adult and adult rats were studied in control conditions and after a single intratracheal dose of the Toll-like receptor 2/6 (TLR2/6) agonist macrophage activating lipopeptide-2 (MALP-2). In all age groups, T, B and natural killer (NK) cells increased dramatically in the epithelium and lamina propria of the bronchi. Male adult rats were found to have responded to MALP-2 to a much greater extent than females when lymphocyte subsets were counted in the epithelium and the lamina propria. In a second series of experiments the time kinetics of regulatory T-cell (Treg) subsets and dendritic cells (DCs) in the lung was studied after local stimulation with MALP-2. Different time-dependent patterns were found in the Treg subsets CD4(+) CD25(+), CD4(+) CD25(+) neuropilin 1(+) and CD4(+) CD25(+) Foxp3(+) cells. Neutrophils and DCs also showed different patterns. Thus, the local application of a TLR agonist increased the number of lymphocyte subsets in a compartment-specific pattern. However, data should not be generalized or extrapolated from one age group, sex or lymphocyte subpopulation to another.

Intracutaneous injection of the macrophage‐activating lipopeptide‐2 (MALP‐2) which accelerates wound healing in mice – a phase I trial in 12 patients

Chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, are a challenge to physicians and medical personnel and a cause of tremendous discomfort and ensuing loss of quality of life to the patients. Wound healing involves production and action of various growth factors. A novel approach, distinct from the application of single growth factors, is the administration of the macrophage stimulator macrophage-activating lipopeptide-2 (MALP-2). The rationale is based on the finding that macrophages are the main source of several growth factors required for wound healing, which are sequentially released during this process. MALP-2 has previously been shown to be effective in an established animal model with diabetic mice. The purpose of the present phase I study was to establish tolerability of MALP-2 when applied into small cutaneous wounds in human beings. Twelve patients (six females and six males; mean age 66.8 years; range 52-87 years) with different diagnoses were enrolled into the study. An artificial wound was created with a 2-mm diameter skin biopsy punch and a volume of 30 microl MALP-2 (0.125-1 microg) or vehicle control, respectively, was injected intracutaneously into the wound and closed with a water-resistant transparent adhesive. Photos were taken daily from every patient up to 6 days, and skin biopsies were performed after 1 week from six patients. We could show in the present study for the first time that MALP-2 caused a transient erythema and was tolerated without any systemic side effects up to a dose of 1 microg per wound in human beings. In healthy as well as in diabetic patients, MALP-2 induced local inflammation that faded after 48 h. The effectiveness of MALP-2 in the healing of chronic wounds in humans, e.g. in chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, could now be addressed in further studies.

Immunostimulation with Macrophage-Activating Lipopeptide-2 Increased Survival in Murine Pneumonia

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

Nickel and the Microbial Toxin, MALP-2, Stimulate Proangiogenic Mediators from Human Lung Fibroblasts via a HIF-1α and COX-2–Mediated Pathway

Hypoxia-inducible factor (HIF-1alpha) and cyclooxygenase-2 (COX-2) have been implicated in the regulation of inflammatory-like processes that lead to angiogenesis and fibrotic disorders. Here we demonstrate that in human lung fibroblasts (HLFs) treated with mixed exposures to chemical and microbial stimuli, HIF-1alpha stabilization plays a pivotal role in the induction of COX-2 mRNA and protein, driving the release of vascular endothelial growth factor (VEGF) and proangiogenic and profibrotic chemokines. Upon costimulation with Ni and the mycoplasma-derived lipopeptide macrophage-activating lipopeptide-2 (MALP-2), there was a synergistic induction of CXCL1 and CXCL5 mRNA and protein release from HLF, as well as an enhanced response in VEGF compared to either stimulus alone. Consistent with our previous findings that Ni and MALP-2 stimulates the induction of CXCL8 via a COX-2-mediated pathway, CXCL1, CXCL5, and VEGF release were also regulated by COX-2. Ni induced the stabilization of HIF-1alpha protein in HLF, which was further enhanced in the presence of MALP-2. Depletion of HIF-1alpha using siRNA blocked COX-2 induction by Ni and MALP-2 along with the release of VEGF, CXCL1, CXCL5, and CXCL8. Our results indicate that Ni and MALP-2 interact to promote an angiogenic profibrotic phenotype in HLF. Moreover, these findings reveal a potential role for HIF-1alpha in mediating chemical-induced alterations in cellular response to microbial stimuli, modulating pulmonary inflammation and its consequences such as fibrosis and angiogenesis.

Synergistic effects of chemical insult and toll-like receptor ligands on dendritic cell activation

There is considerable interest in the development of in vitro methods for the identification of contact sensitizers, including those that use cultured dendritic cells (DC), key players in cutaneous immune responses. Chemical allergens, such as dinitrobenzene sulfonic acid (DNBS), or skin irritants, such as benzene sulfonic acid (BS), induce modest changes in DC phenotype. In an attempt to increase the sensitivity of DC responses, DC have been co-cultured with chemical and DC activators (toll-like receptor [TLR] ligands). Cells were cultured with DNBS or BS at doses of equivalent cytotoxicity, together with sub-optimal doses of selected TLR ligands (Pam(3)Cys-Ser-(Lys)(4) [PAM], TLR1-2; macrophage-activating lipopeptide-2 [MALP-2], TLR6-2; or flagellin; TLR5). Both chemicals caused a decline in cell viability. DNBS induced a higher proportion of late apoptotic/necrotic cells whereas BS was associated with early apoptotic cells, suggesting different mechanisms of cell death. Some synergy was observed for interleukin (IL)-6 and tumor necrosis factor alpha production for DC co-cultured with BS and MALP-2/PAM. In contrast, there were marked synergistic effects on IL-6 secretion when DC were cultured with DNBS and flagellin. It may be possible to exploit this enhanced sensitivity of flagellin-activated DC for chemical allergen for the development of in vitro skin sensitization assays.

Efficient immune responses against Intimin and EspB of enterohaemorragic Escherichia coli after intranasal vaccination using the TLR2/6 agonist MALP-2 as adjuvant

Mucosal vaccine formulations based on purified recombinant C 280 γ-Intimin and EspB ( Escherichia coli secreted protein B) from enterohaemorragic E. coli co-administered with a pegylated derivative of the TLR2/6 agonist MALP-2 (macrophage-activating lipopeptide) as adjuvant were evaluated in BALB/c mice. After intranasal vaccination, strong humoral and cellular immune responses were observed against C 280 γ-Intimin and EspB. Sera of immunized mice inhibit bacterial haemolytic activity in vitro. Antigen-specific T-cell proliferation, IL-4, IL-2 and IFN-γ producing cells, and secretory IgA were mostly detected in animals receiving MALP-2 as adjuvant. These results suggest that C280 γ-Intimin and EspB are good candidate antigens to be incorporated into mucosal vaccines against this important pathogen.

Intratumoural injection of the toll-like receptor-2/6 agonist ‘macrophage-activating lipopeptide-2’ in patients with pancreatic carcinoma: A phase I/II trial

15604 Background: This phase I/II trial examined safety and efficacy of the toll-like receptor 2/6 agonist MALP-2 in combination with gemcitabine in patients with incompletely resectable pancreas carcinomas. MALP-2 is a toll-like receptor-2/6 agonist, acts an immunological adjuvant and has been described recently to prolong survival in a mouse model of an orthotopic, syngeneic pancreas tumor. Methods: Men and women with incompletely resectable pancreas carcinomas were eligible while patients with R0 or R1 resections or with peritoneal carcinosis were excluded. Ten patients were injected intratumorally during surgery with 20–30μg MALP-2 followed by postoperative chemotherapy. Samples were taken from peripheral blood and wound secretion, and assayed for cell content, cytokine and CRP levels, and NK activity. Results: A MALP-2 dose of 20 μg was well tolerated. Clear signs of local MALP-2 effects were: influx of lymphocytes and monocytes in wound secretions, and abolishment of inhibition of NK activity. The actual mean survival is 17.9 ± 4.6 months (CI 8.9, 26.9); the median survival being 9.3 months. Two patients are still alive after 34 months. Conclusions: Up to 20μg MALP-2 were well tolerated, and no systemic effects were noted. The mean survival of 17.9 months is remarkably high. No significant financial relationships to disclose.

Ex vivo testing of immune responses in precision-cut lung slices

The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon γ (IFNγ), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1α, TNFα, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNγ resulting in increased levels of TNFα, IL-12(p40), RANTES, and IL-1α. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.