Peter F. Mühlradt

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.

Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.

Synergy and Cross-Tolerance Between Toll-Like Receptor (TLR) 2- and TLR4-Mediated Signaling Pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.

The Mycoplasma‐derived lipopeptide MALP‐2 is a potent mucosal adjuvant

The adjuvanticity of MALP‐2, a 2‐kDa synthetic lipopeptide with macrophage‐stimulatory activity, was evaluated in BALB/c mice using β‐galactosidase (β‐gal) as model antigen. When co‐administered with β‐gal by either the intranasal (i.n.) or i.p. route, MALP‐2 (0.5 μg) was capable of increasing β‐gal‐specific serum IgG titers by 675–3,560‐fold (i.n.) and 64–128‐fold (i.p.), respectively, as compared to immunization with β‐gal alone. Using MALP‐2, almost maximal IgG responses were already stimulated following the first immunization, and the IgG titers were similar to those observed using 10 μg of cholera toxin B subunit (CTB) as adjuvant. The mucosal immune system was also effectively stimulated (p<0.05) when MALP‐2 was administered by the i.n. route (36% and 23% of β‐gal‐specific IgA in lung and vaginal lavages, respectively). The i.n. co‐administration of MALP‐2 stimulated a stronger cellular immune response than CTB, both in submandibular lymph nodes and spleen (p<0.05). The analysis of β‐gal‐specific IgG isotypes and the profiles of cytokines secreted by in vitro re‐stimulated cells showed that co‐administration of MALP‐2 triggered a dominant Th2‐response pattern. A recruitment of B220+ and MAC‐1+ cells with an up‐regulated expression of MHC class I, CD80 (B7.1) and CD54 (ICAM‐1) was observed in nasal associated lymphoid tissues from MALP‐2 treated mice. Taken together, our results demonstrated that the synthetic lipopeptide MALP‐2 represents a very promising adjuvant for the mucosal delivery of vaccine antigens.

The Mucosal Adjuvant Macrophage-Activating Lipopeptide-2 Directly Stimulates B Lymphocytes via the TLR2 without the Need of Accessory Cells

The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity as mucosal adjuvant, promoting strong humoral and cellular responses. Although B cells expressing TLR2/6 are potential targets, very little is known about the effect of MALP-2 on B cells. Studies were performed using total spleen cells or purified B cells from WT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2 promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) via TLR2. MALP-2 also increased the frequency of IgM- and IgG-secreting cells, but bystander cells were required for IgA secretion. Activated B cells exhibited increased expression of activation markers and ligands that are critical for cross-talk with T cells (CD19, CD25, CD80, CD86, MHC I, MHC II, and CD40). Immunization of mice lacking T cells showed that MALP-2-mediated stimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunization of mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve the immunogenicity of vaccine formulations.

Efficient mucosal delivery of the HIV‐1 Tat protein using the synthetic lipopeptide MALP‐2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell‐mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV‐1 Tat protein as antigen and the synthetic lipopeptide, macrophage‐activating lipopeptide‐2 (MALP‐2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti‐Tat antibody responses, and Tat‐specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freunds adjuvant. Major linear B cell epitopes mapped within aa 1–20 and 46–60, whereas T cell epitopes were identified within aa 36–50 and 56–70. These epitopes have also been described in vaccinated primates and in HIV‐1‐infected individuals with better prognosis. Analysis of the anti‐Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP‐2, as opposed to the Th2 response observed with Tat plus incomplete Freunds adjuvant. Tat‐specific IFN‐γ‐producing cells were significantly increased only in response to Tat plus MALP‐2. These data suggest that Malp‐2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

Toll-like receptor 2/6 stimulation promotes angiogenesis via GM-CSF as a potential strategy for immune defense and tissue regeneration

Toll-like receptors (TLRs) are known primarily as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiologic and pathologic processes has been reported. Because many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the TLR2/6 agonist macrophage-activating lipopeptide of 2 kDa (MALP-2), a common bacterial lipopeptide. In vivo and in vitro Matrigel assays demonstrated that MALP-2 promoted angiogenesis in a TLR2/6-dependent manner. Moreover, MALP-2 induced endothelial cell proliferation and migration and a strong secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF release in response to MALP-2 from isolated vascular segments was completely prevented when the endothelium was removed. MALP-2 containing Matrigel implants exhibited vascular structures as well as CD45(+) cells. MALP-2 induced migration of leukocytes and likewise GM-CSF release, particularly from the monocyte population. Inhibition of GM-CSF by siRNA or antibodies suppressed MALP-2-induced angiogenesis in vitro and in vivo. These results clearly identified a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2, which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.

Synthetic mycoplasma-derived lipopeptide MALP-2 induces maturation and function of dendritic cells

Dendritic cells (DC) modulate immune responses depending on the nature of the antigens. Receptors capable of discriminating these antigens on the basis of the pathogen-associated molecular patterns (PAMP) belong to the Toll-like receptor (TLR) family. The macrophage-activating lipopeptide 2 kDa (MALP-2), a synthetic lipopeptide derived from Mycoplasma fermentans, signals through TLR-2 and TLR-6. The aim of this study was to examine whether MALP-2 can modulate the functional properties of human monocyte-derived DC. The effects of this treatment were compared to those of the TLR-4 agonist lipopolysaccharide (LPS). To ensure clinical applicability, DC were generated under serum-free conditions. MALP-2 and LPS stimulation induced the expression of CD83 and increased the expressions of CD80, CD86, HLA-ABC and CD40. Furthermore, both substances decreased the endocytotic capacity of DC and induced the release of bioactive TNF-alpha and IL-10, whereas LPS additionally increased IL-12 release. Pretreatment with both substances boosted the allostimulatory capacity of DC. In a coculture with autologous lymphocytes, either MALP-2 or LPS pretreated DC induced a marked proliferation of lymphocytes, but only DC prestimulated with MALP-2 activated lymphocytes to produce the cytokines IL-4, IL-5 and IFN-gamma. No polarisation of lymphocytes into T-helper (Th)1 or Th2 was detected. These data indicate that MALP-2 is a potential candidate to modulate DC for clinical applications.

In Vivo Effects of a Synthetic 2-Kilodalton Macrophage-Activating Lipopeptide of Mycoplasma fermentans after Pulmonary Application

ABSTRACT Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals. Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes. The present study deals with the question of which mycoplasmal components cause this host reaction. The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection. To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2. Application of 2.5 μg induced a pronounced leukocyte accumulation in the bronchoalveolar space. At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3. Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation. The leukocyte count of the lung interstitium was increased on day 3 after treatment. After 10 days all investigated cell populations returned to control levels. Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates. MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2. Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction. The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.

The macrophage‐activating lipopeptide‐2 accelerates wound healing in diabetic mice

Abstract:  Wound healing in healthy individuals proceeds at an optimal rate. However, in patients, with – e.g.– locally impaired blood flow or diabetes, chronic wounds develop and often become infected. Chronic wounds mean a low quality of life for the afflicted patients, not to mention enormous costs. Rather than using recombinant growth factors to accelerate wound healing, we employed the toll‐like receptor agonist macrophage‐activating lipopeptide‐2 (MALP‐2) to improve the healing of full‐thickness excision skin wounds in an animal model with obese, diabetic mice. A gene array experiment suggested that MALP‐2 stimulates the release of various mediators involved in wound healing. Further data to be presented in this study will show (i) that MALP‐2 is capable of stimulating the appearance of the monocyte chemoattractant protein‐1 at the wound site, (ii) that this leads to increased leucocyte and, in particular, macrophage infiltration and (iii) that MALP‐2‐treated wounds closed 2 weeks earlier than vehicle‐treated controls. MALP‐2, thus, appears to stimulate the early inflammatory process needed to set in motion the ensuing consecutive natural steps of wound healing resulting in wound closure.