Biosensors for inflammation as a strategy to engineer regulatory T cells for cell therapy.

Engineered regulatory T cell (Treg cell) therapy is a promising strategy to treat patients suffering from inflammatory diseases, autoimmunity, and transplant rejection. However, in many cases, disease-related antigens that can be targeted by Treg cells are not available. In this study, we introduce a class of synthetic biosensors, named artificial immune receptors (AIRs), for murine and human Treg cells. AIRs consist of three domains: (a) extracellular binding domain of a tumor necrosis factor (TNF)-receptor superfamily member, (b) intracellular costimulatory signaling domain of CD28, and (c) T cell receptor signaling domain of CD3-ζ chain. These AIR receptors equip Treg cells with an inflammation-sensing machinery and translate this environmental information into a CD3-ζ chain-dependent TCR-activation program. Different AIRs were generated, recognizing the inflammatory ligands of the TNF-receptor superfamily, including LIGHT, TNFα, and TNF-like ligand 1A (TL1A), leading to activation, differentiation, and proliferation of AIR-Treg cells. In a graft-versus-host disease model, Treg cells expressing lymphotoxin ß receptor-AIR, which can be activated by the ligand LIGHT, protect significantly better than control Treg cells. Expression and signaling of the corresponding human AIR in human Treg cells prove that this concept can be translated. Engineering Treg cells that target inflammatory ligands leading to TCR signaling and activation might be used as a Treg cell-based therapy approach for a broad range of inflammation-driven diseases.

Woronin bodies, their impact on stress resistance and virulence of the pathogenic mould Aspergillus fumigatus and their anchoring at the septal pore of filamentous Ascomycota.

Hyphae of filamentous Ascomycota consist of compartments that are connected via septal pores. To avoid a dramatic loss of cellular content after wounding, fungi developed mechanisms to occlude their septal pores. In most Pezizomycotina, so-called Woronin bodies are anchored in proximity to the pore. This is a prominent example for precise spatial positioning of organelles, but so far the underlying molecular organization has remained largely unknown. Using the pathogenic mould Aspergillus fumigatus, we provide evidence that Woronin bodies are important for stress resistance and virulence. Furthermore the molecular machinery anchoring them at the septum is described. Namely, we have identified Lah as the tethering protein and provide evidence that the Woronin body protein HexA binds to the septal pore in a Lah-dependent manner. Moreover, we demonstrate that a striking poly-histidine motif targets HexA to the septal cell wall. Thus, the axis HexA-Lah is an excellent candidate for the tether linking Woronin bodies to the septum. This model applies to A.fumigatus, but most likely also to the vast majority of the Pezizomycotina. Our findings shed light on the evolution of Woronin body anchoring and provide a basis for the development of novel strategies to combat fungal pathogens like A.fumigatus.

TLR5 stop codon polymorphism is associated with invasive aspergillosis after allogeneic stem cell transplantation.

Single nucleotide polymorphisms (SNPs) have been associated with an increased incidence of invasive aspergillosis (IA) after allogeneic stem cell transplantation (allo-SCT). We analyzed 41 patients with proven/probable IA after allo-SCT for an association of SNPs, within the TLR2, TLR4, TLR5, TLR9, and NOD2/CARD15 genes, with susceptibility to IA. The control group consisted of 130 patients who had allo-SCT but did not develop IA. While no association was found for donor SNPs and the recipients' risk of IA, analysis of recipient SNPs showed a significant association between the presence of recipient TLR5-Stop SNP (1174C> T) and the incidence of IA (P = 0.004). Multivariate analysis demonstrated that the recipient TLR5-Stop SNP appeared as an independent risk factor for IA after allo-SCT. Our study suggests that TLR5 is involved in host defense against Aspergillus fumigatus, and that the recipient TLR5-Stop SNP represents a risk factor for the development of IA after allo-SCT.

Genetic variation determines mast cell functions in experimental asthma.

Mast cell-deficient mice are a key for investigating the function of mast cells in health and disease. Allergic airway disease induced as a Th2-type immune response in mice is employed as a model to unravel the mechanisms underlying inception and progression of human allergic asthma. Previous work done in mast cell-deficient mouse strains that otherwise typically mount Th1-dominated immune responses revealed contradictory results as to whether mast cells contribute to the development of airway hyperresponsiveness and airway inflammation. However, a major contribution of mast cells was shown using adjuvant-free protocols to achieve sensitization. The identification of a traceable genetic polymorphism closely linked to the Kit(W-sh) allele allowed us to generate congenic mast cell-deficient mice on a Th2-prone BALB/c background, termed C.B6-Kit(W-sh). In accordance with the expectations, C.B6-Kit(W-sh) mice do not develop IgE- and mast cell-dependent passive cutaneous anaphylaxis. Yet, unexpectedly, C.B6-Kit(W-sh) mice develop full-blown airway inflammation, airway hyperresponsiveness, and mucus production despite the absence of mast cells. Thus, our findings demonstrate a major influence of genetic background on the contribution of mast cells in an important disease model and introduce a novel strain of mast cell-deficient mice.

Development of a Neurotensin-Derived 68Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS1 Receptor-Expressing Tumors.

Overexpression of the neurotensin receptor type 1 (NTS1R), a peptide receptor located at the plasma membrane, has been reported for a variety of malignant tumors. Thus, targeting the NTS1R with 18F- or 68Ga-labeled ligands is considered a straightforward approach towards in vivo imaging of NTS1R-expressing tumors via positron emission tomography (PET). The development of suitable peptidic NTS1R PET ligands derived from neurotensin is challenging due to proteolytic degradation. In this study, we prepared a series of NTS1R PET ligands based on the C-terminal fragment of neurotensin (NT(8-13), Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) by attachment of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an Nω-carbamoylated arginine side chain. Insertion of Ga3+ in the DOTA chelator gave potential PET ligands that were evaluated concerning NTS1R affinity (range of Ki values: 1.2-21 nM) and plasma stability. Four candidates were labeled with 68Ga3+ and used for biodistribution studies in HT-29 tumor-bearing mice. [68Ga]UR-LS130 ([68Ga]56), containing an N-terminal methyl group and a ß,ß-dimethylated tyrosine instead of Tyr11, showed the highest in vivo stability and afforded a tumor-to-muscle ratio of 16 at 45 min p.i. Likewise, dynamic PET scans enabled a clear tumor visualization. The accumulation of [68Ga]56 in the tumor was NTS1R-mediated, as proven by blocking studies.

Studies on galactofuranose-containing glycostructures of the pathogenic mold Aspergillus fumigatus.

Galactofuranose is a hexose that is exclusively found in microbes and in particular in certain pathogenic species. In the mold Aspergillus fumigatus, it is the characteristic constituent of the cell wall component galactomannan. Detection of this carbohydrate is currently a widespread method used for diagnosis of systemic A. fumigatus infections. In this study, we raised and characterized 2 monoclonal antibodies that specifically react with galactofuranose-containing glycostructures. We investigated the distribution of surface-accessible galactomannan on different A. fumigatus morphotypes. We provide evidence that the antibodies recognize distinct antigens and are suitable to detect A. fumigatus hyphae in immunohistology. A mutant that is impaired in synthesis of galactofuranose stimulated a normal cytokine response in murine macrophages, which argues against galactomannan being a relevant PAMP, at least in mice. Purified galactomannan-specific monoclonal IgM L10-1 failed to inhibit the hyphal growth under in vitro conditions, but L10-1 binding to hyphae led to an enhanced deposition of the complement protein C1q. However, administration of purified L10-1 antibodies prior to infection was not able to protect mice. In conclusion, we have found no evidence for galactomannan being a relevant A. fumigatus PAMP and describe 2 novel galactomannan antibodies that might be valuable tools for the diagnosis of A. fumigatus infections and further analysis of the biological significance of galactomannan.

The mitA gene of Aspergillus fumigatus is required for mannosylation of inositol-phosphorylceramide, but is dispensable for pathogenicity.

GDP-mannose:inositol-phosphorylceramide (MIPC)-derived glycosphingolipids are important pathogen-associated molecular patterns (PAMP) of Candida albicans and according to recently published data also of Aspergillus fumigatus. MIPC transferases are essential for the synthesis of MIPC, but have so far been studied only in Saccharomyces cerevisiae and C. albicans. Here, we have identified MitA as the only MIPC transferase in A. fumigatus. The DeltamitA mutant lacks MIPC and MIPC-derived glycosphingolipids and accumulates the precursor IPC. The mutant grows normally, shows no defects in cell wall or membrane organization and a normal resistance to different stressors. It is, however, sensitive to high Ca(2+) concentrations, especially during germination. Germination of DeltamitA mutant conidia is also decelerated under normal growth conditions, but neither the virulence of this mutant in a systemic model of infection nor its ability to trigger a cytokine response in macrophages is impaired, arguing against a role of MIPC-derived glycosphingolipids as important A. fumigatus PAMPs.

AfMkk2 is required for cell wall integrity signaling, adhesion, and full virulence of the human pathogen Aspergillus fumigatus.

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.

The putative alpha-1,2-mannosyltransferase AfMnt1 of the opportunistic fungal pathogen Aspergillus fumigatus is required for cell wall stability and full virulence.

Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative alpha-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A Deltaafmnt1 mutant is viable and grows normally at 37 degrees C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48 degrees C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48 degrees C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the Deltaafmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that alpha-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.

BMP6-induced modulation of the tumor micro-milieu.

Melanoma is the deadliest form of skin cancer with rising incidence, creating a significant health problem. We discovered increased expression of bone morphogenetic protein 6 (BMP6) in melanoma cells and tissues, and observed that BMP6 deficiency caused significantly delayed tumor onset and decelerated tumor progression in a melanoma mouse model. Moreover, we determined that BMP6 inhibits dermal mast cell recruitment and found that mast cell-derived mediators significantly reduced melanoma growth in vitro. In line with this, mast cell deficiency accelerated tumor onset and progression in a melanoma mouse model. Analysis of human melanoma tissues revealed a strong negative correlation between melanoma proliferation and mast cell infiltration. This study elucidates a novel role of BMP6-induced modulation of the tumor microenvironment.

Localization of the mouse defense lectin ficolin B in lysosomes of activated macrophages.

Ficolins are pattern-recognition molecules of the innate immune system able to trigger the lectin pathway of the complement activation upon binding to microbial surfaces. In humans, two plasma ficolins have been identified and characterized, whereas a third cell-associated ficolin (M-ficolin) was found on monocyte surfaces. The mouse homologue of M-ficolin is called ficolin B. Although the spatial-temporal expression patterns of mouse ficolins have been described recently, the subcellular localization of ficolin B protein is so far unknown. By using ficolin B-specific antibodies and confocal microscopy, we show that ficolin B is expressed within mouse peritoneal exudate macrophages and is co-localized with Lamp-1, a marker for lysosomes and late endosomes. In addition, the data indicate that ficolin B expression is up-regulated upon macrophage activation.

Failure of interferon-gamma and tumor necrosis factor in mediating anemia of chronic disease in a mouse model of protracted septic peritonitis.

Interferon-gamma (IFN-gamma) is considered one of the main inflammatory cytokines contributing to the generation of anemia of chronic disease (ACD). In this study, we used a previously described murine model for ACD based on sublethal cecal ligation and puncture (CLP) with ensuing protracted peritonitis. Within 2 weeks after CLP, a moderate normochromic anemia with low serum iron concentration and preserved iron stores develops, which is consistent with ACD. In order to determine whether IFN-gamma contributes to the development of ACD in vivo, we neutralized IFN-gamma after CLP shortly before and during the phase of most severe bone marrow depression in order to prevent anemia. Additionally, we studied IFN-gamma receptor-deficient mice that underwent CLP. Two weeks after CLP, we determined the red blood cell count, hemoglobin concentration, hematocrit, serum iron concentration, and iron stores in spleens of wild-type mice, IFN-gamma receptor-deficient mice, and mice after neutralization of IFN-gamma. Neutralization of IFN-gamma after CLP could not prevent mice from becoming anemic. Accordingly, IFN-gamma receptor-deficient mice developed anemia to the same extent as wild-type mice. Serum iron concentration was lowered both in IFN-gamma receptor-deficient and wild-type mice. Iron stores in untreated IFN-gamma receptor-deficient mice were elevated compared to untreated wild-type mice. After CLP both IFN-gamma receptor-deficient and wild-type mice had equally overloaded iron stores. Additional neutralization of TNF in IFN-gamma receptor-deficient mice also did not attenuate CLP-induced anemia. Our results clearly demonstrate that neither IFN-gamma alone nor in combination with TNF is a mediator of ACD in our model with transient anemia induced by protracted septic peritonitis.

TNF and regulatory T cells are critical for sepsis-induced suppression of T cells.

The immune system in sepsis is impaired as seen by reduced numbers and function of immune cells and impaired antigen-specific antibody responses. We studied T cell function in septic mice using cecal ligation and puncture (CLP) as a clinically relevant mouse model for sepsis. The proliferative response of CD4(+) and CD8(+) T cells was suppressed in septic mice. Adoptive transfer experiments demonstrated that the T cells were not intrinsically altered by CLP. Instead, the septic host environment was responsible for this T cell suppression. While CLP-induced suppression was dependent on TNF activity, neither the activation of TNF receptors type 1 nor TNF receptor type 2 alone was sufficient to generate sepsis-induced suppression showing that the two TNF receptors can substitute each other. Specific depletion of regulatory T (Treg) cells improved the impaired T cell proliferation in septic recipients demonstrating participation of Treg in sepsis-induced suppression. In summary, sepsis leads to TNF-dependent suppression of T cell proliferation in vivo involving induction of Treg cells.

Requirement of TNF and TNF receptor type 2 for LPS-induced protection from lethal septic peritonitis.

Pretreatment of mice with low quantities of LPS induces endotoxin tolerance characterized by enhanced resistance to lethal doses of LPS and to a number of infectious challenges. Mice subjected to cecal ligation and puncture (CLP) survived the ensuing septic peritonitis significantly better when they had been pretreated with LPS. This LPS-induced protection was dependent on endogenous TNF production capacity since LPS pretreatment did not protect TNF-deficient mice from death after CLP. While mice deficient in the TNF receptor type 2 (p75TNFR) were as sensitive to CLP-induced mortality as control mice, LPS pretreatment could not reduce mortality in p75TNFR-deficient mice after CLP. Therefore, activation of the TNF receptor type 2 by endogenous TNF constitutes an important interaction for the development of LPS-induced resistance to bacterial infection.

Treatment of experimental sepsis-induced immunoparalysis with TNF.

Following a severe septic abdominal infection induced by sublethal cecal ligation and puncture (CLP) in mice, a phase of depressed immune reactivity occurred two days after CLP characterized by a reduced capacity to produce TNF. To determine whether this reduced TNF production causes immunoparalysis as determined by increased susceptibility to bacterial infection and whether therapeutic TNF substitution can be beneficial during this phase, a super-infection with Salmonella enterica Serovar typhimurium or Listeria monocytogenes was induced two days after sublethal CLP. After CLP a state of true immunoparalysis developed during which Salmonella or Listeria super-infection led to increased lethality paralleled by increased bacterial numbers in spleens and livers. Injection of recombinant human TNF before or at the time of super-infection conferred protection to Salmonella but not to Listeria. In the latter case, the infection mortality was even enhanced. Thus, super-infection during the state of sepsis-induced immunoparalysis leads to increased lethality. TNF substitution during this state of immunoparalysis can be beneficial or deleterious, depending on the location of TNF activity in the animal, timing of TNF administration, or the type of super-infection. These results demonstrate that impaired TNF production capacity can account for some aspects of immunoparalysis, however, diagnostic parameters are required for a safe TNF substitution therapy.

Hypoferraemia during the early inflammatory response is dependent on tumour necrosis factor activity in a murine model of protracted peritonitis.

On the grounds of clinical, in vitro and in vivo studies, tumour necrosis factor (TNF) is considered to be one of the inflammatory cytokines that contributes to to the generation of hypoferraemia and anaemia of inflammation (AI). We used a recently described murine model for AI and hypoferraemia, based on sublethal caecal ligation and puncture (CLP) with ensuing protracted peritonitis, to investigate the contribution of TNF to the generation of hypoferraemia. During the early inflammatory response to CLP, a marked decrease in serum iron concentration occurs within 8 h. To determine whether TNF contributes to the generation of hypoferraemia at this time point, we studied TNF-deficient mice and wild-type mice that underwent CLP. The serum iron concentration was decreased in wild-type mice whereas TNF-deficient mice maintained normal serum iron levels following CLP. Hypoferraemia in wild-type mice was accompanied by the downregulation of ferroportin 1 (Fp1) in macrophages. In the macrophages of TNF-deficient mice, Fp1 was not downregulated following CLP. The initial expression of hepcidin was detectable at the mRNA level but not at the protein level by immunohisto-chemistry in wild-type and TNF-deficient mice. Therefore, hepcidin does not appear to be involved in the regulation of early hypoferraemia. TNF appears to regulate the expression of Fp1 by transcriptional control. Our results demonstrate that TNF mediates hypoferraemia during the early inflammatory response by regulating the expression of Fp1 in macrophages.

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression.

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)Il6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

Approaching the secrets of N-glycosylation in Aspergillus fumigatus: characterization of the AfOch1 protein.

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.

Properdin plays a protective role in polymicrobial septic peritonitis.

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

Investigations on the involvement of the lectin pathway of complement activation in anaphylaxis.

BACKGROUND: Systemic anaphylaxis is the most severe form of immediate hypersensitivity reaction. The activation of the complement system occurs during anaphylactic shock. The purpose of this study was to determine in a mouse model whether the lectin pathway of complement activation is involved in anaphylaxis. METHODS: To see whether the lectin pathway is involved in anaphylactic shock, serum mannan-binding lectin (MBL) levels were measured after passive anaphylaxis. Also MBL expression and binding to potential ligands were investigated. To determine whether complement or mast cell activation is essential for hypothermia in anaphylactic shock, mouse strains deficient in MBL-A and MBL-C, C1q, factors B and C2, C5, C5aR, or mast cells were tested. RESULTS: After antigenic challenge a marked drop in body temperature as well as a rapid decrease in serum MBL levels were observed. The decrease of serum MBL levels in shock could not be attributed to MBL binding to immune complexes or tissues, but an interaction of MBL with mast cell-derived proteoglycans was seen. In contrast to mast cell-deficient mice, none of the complement-deficient mouse strains were protected from shock-associated hypothermia. CONCLUSIONS: These results indicate that neither MBL nor activation of the complement cascade is crucial for the induction of anaphylaxis. In contrast mast cell activation is associated with the development of hypothermia and possibly the observed decrease in serum MBL levels.