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International audience; The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Infection of specific pathogen-free mice with lymphocytic choriomeningitis virus (LCMV) is a widely used model to study antiviral T-cell immunity. Infections in the real world, however, are often accompanied by coinfections with unrelated pathogens. Here we show that in mice, systemic coinfection with E. coli suppresses the LCMV-specific cytotoxic T-lymphocyte (CTL) response and virus elimination in a NK cell- and TLR2/4-dependent manner. Soluble TLR4 ligand LPS also induces NK cell-mediated negative CTL regulation during LCMV infection. NK cells in LPS-treated mice suppress clonal expansion of LCMV-specific CTLs by a NKG2D- or NCR1-independent but perforin-dependent mechanism. These results suggest a TLR4-mediated immunoregulatory role of NK cells during viral-bacterial coinfections., Exposure to multiple pathogens is common in nature, yet interactions between the immune components targeting bacterial and viral pathogens during co-infection are poorly understood. Here the authors show that bacteria-derived LPS induces cytotoxic NK cells that suppress antiviral CD8 T cell response.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses., Author summary Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. They phenotypically and functionally adapt to their local environment, for example, peritoneal macrophages are distinct from brain-resident microglia, from liver-resident Kupffer cells or lung macrophages in the lung. Airway macrophages are among the first cells to encounter human respiratory viruses, such as adenoviruses. They release pro-inflammatory cytokines, kill pathogens, present antigens, and restore tissues. Yet, interactions of viruses with lung macrophages are poorly understood, and it is unclear, how they lead to infection or virus clearance. Here we identified the murine scavenger receptor SR-A6 as a receptor for a subset of human adenoviruses on alveolar macrophage-like cells, so-called MPI cells. Scavenger receptors comprise a large family of trans-membrane proteins, and contribute to the clearance of endogenous proteins, lipoproteins and pathogens. In a series of robust experimentation, we show that adenoviruses use SR-A6 as an entry receptor for infection of MPI cells, and production of type I interferon. MPI cells are non-transformed, self-renewing macrophages derived from fetal murine liver, and closely resemble adult alveolar macrophages. The results demonstrate that SR-A6 binds virions on the surface of alveolar macrophage-like cells, and leads to infection.

Sarcoidosis is a complex disease with variable phenotypes that will require a multisystem approach to understand pathophysiology. One of the most challenging problems in sarcoidosis research is the absence of valid and widely accepted experimental models that accurately simulate human disease. The Foundation of Sarcoidosis Research (FSR) has funded five projects for the development of novel experimental models for sarcoidosis, presented and discussed in a workshop organized during the European Respiratory Society Congress held in Milan from September 9th to 13th. The experimental, in vivo or in sillico models presented may be quite helpful for investigating specific pathogenic and therapeutic questions, addressing especially severe forms of sarcoidosis. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 2-4)

mBio 8(5), e01445-17 (2017). doi:10.1128/mBio.01445-17, Published by American Society for Microbiology, Washington, DC

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo. Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors., IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo. The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

Toll-like receptors (TLRs) recognise pathogen-derived molecules and play a critical role during the host innate and adaptive immune response. Brucella spp. are intracellular gram-negative bacteria including several virulent species, which cause a chronic zoonotic infection in a wide range of mammalian hosts known as brucellosis. Recently it was isolated from wild rodents a new Brucella species, Brucella microti, that was found to be pathogenic in mice. Using this species-specific model, it was previously found that CD8+ T cells are required to control this infection. In order to find out the role of TLR-mediated responses in the control of this pathogen, the course of infection of B. microti was analyzed over 3 weeks in wild-type (WT) and TLR knock out (KO) mice including TLR2-/-, TLR4-/-, TLR9-/-, TLR2x4-/- and TLR 2x4x9-/-. WT and single TLR2, TLR4 and TLR9 KO mice similarly control infection in liver and spleen. In contrast, bacterial clearance was delayed in TLR2x4-/- and TLR2x4x9-/- mice at 7 and 14 days post-infection. This defect correlated with impaired maturation and pro-inflammatory cytokine production in B. microti-infected dendritic cells from TLR2x4-/- and TLR2x4x9-/- mice. Finally, it was found that Tc cells from TLR2x4-/- and TLR2x4x9-/- mice showed reduced ability to inhibit growth of B. microti in macrophages, suggesting the involvement of TLR2 and 4 in the generation of specific Tc cells. Our findings indicate that TLR2 and TLR4 are required to control B. microti infection in mice and that this effect could be related to its participation in the maturation of dendritic cells and the generation of specific CD8+ Tc cells. □ Published

Environmental signals can be translated into chromatin changes, which alter gene expression. Here we report a novel concept that cells can signal chromatin damage from the nucleus back to the surrounding tissue through the cytokine interleukin-1alpha (IL-1α). Thus, in addition to its role as a danger signal, which occurs when the cytokine is passively released by cell necrosis, IL-1α could directly sense DNA damage and act as signal for genotoxic stress without loss of cell integrity. Here we demonstrate localization of the cytokine to DNA-damage sites and its subsequent secretion. Interestingly, its nucleo-cytosolic shuttling after DNA damage sensing is regulated by histone deacetylases (HDAC) and IL-1α acetylation. To demonstrate the physiological significance of this newly discovered mechanism, we used IL-1α knockout mice and show that IL-1α signaling after UV skin irradiation and DNA damage is important for triggering a sterile inflammatory cascade in vivo that contributes to efficient tissue repair and wound healing.

Adenoviruses are important infectious agents and also emerging vectors in different biomedical applications. These viruses elicit a strong innate and adaptive immune response, which influences both the course of disease and the success of the applied vectors. Several Toll-like Receptor (TLR)-dependent and -independent mechanisms contribute to these responses. Understanding of the involved viral and cellular factors is crucial for the treatment of various adenovirus diseases and the optimal design of adenovirus vector applications. Here we summarize our current understanding of the complex nature of adenovirus-induced innate immune mechanisms.

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

Group B streptococcus (GBS) is a leading cause of both neonatal sepsis and meningitis, two diseases that are characterized by inflammation. However, the manner in which GBS organisms are recognized by monocytes and macrophages is poorly understood. In this study, we report that the recognition of GBS and other Gram-positive bacteria by macrophages and monocytes relies on bacterial single-stranded RNA (ssRNA). ssRNA interacts with a signalling complex, which comprises the Toll-like receptor adaptors MyD88 and UNC-93B, but not the established MyD88-dependent ssRNA sensors. The role of ssRNA in the recognition of Gram-positive bacteria--leading to the induction of inflammatory cytokines--has potential implications for sepsis pathogenesis, diagnosis and treatment.

In humans and mice, CD8α+ conventional dendritic cells are the primary source of interferon-λ released in response to the adjuvant and Toll-like receptor 3 agonist poly IC., Polyinosinic:polycytidylic acid (poly IC), a double-stranded RNA, is an effective adjuvant in vivo. IFN-λs (also termed IL-28/29) are potent immunomodulatory and antiviral cytokines. We demonstrate that poly IC injection in vivo induces large amounts of IFN-λ, which depended on hematopoietic cells and the presence of TLR3 (Toll-like receptor 3), IRF3 (IFN regulatory factor 3), IRF7, IFN-I receptor, Fms-related tyrosine kinase 3 ligand (FL), and IRF8 but not on MyD88 (myeloid differentiation factor 88), Rig-like helicases, or lymphocytes. Upon poly IC injection in vivo, the IFN-λ production by splenocytes segregated with cells phenotypically resembling CD8α+ conventional dendritic cells (DCs [cDCs]). In vitro experiments revealed that CD8α+ cDCs were the major producers of IFN-λ in response to poly IC, whereas both CD8α+ cDCs and plasmacytoid DCs produced large amounts of IFN-λ in response to HSV-1 or parapoxvirus. The nature of the stimulus and the cytokine milieu determined whether CD8α+ cDCs produced IFN-λ or IL-12p70. Human DCs expressing BDCA3 (CD141), which is considered to be the human counterpart of murine CD8α+ DCs, also produced large amounts of IFN-λ upon poly IC stimulation. Thus, IFN-λ production in response to poly IC is a novel function of mouse CD8α+ cDCs and their human equivalents.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Background The bacterial microflora aggravates graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation, but the underlying mechanisms of manifestations of intestinal GvHD (iGvHD) in the gut remain poorly understood. Aim To analyse the gut flora composition and the impact of bacterial sensing via Toll-like receptors (TLRs) in iGvHD. Methods By mimicking clinical low-intensity conditioning regimens used in humans, a novel irradiation independent, treosulfan and cyclophosphamide-based murine allogeneic transplantation model was established. A global survey of the intestinal microflora by cultural and molecular methods was performed, the intestinal immunopathology in TLR-deficient recipient mice with iGvHD investigated and finally, the impact of anti-TLR9 treatment on iGvHD development assessed. Results The inflammatory responses in iGvHD were accompanied by gut flora shifts towards enterobacteria, enterococci and Bacteroides/Prevotella spp. Analysis of iGvHD in MyD88 -/- , TRIF -/- , TLR2/4 -/- , and TLR9 -/- recipient mice showed that bacterial sensing via TLRs was essential for iGvHD development. Acute iGvHD was characterised by increasing numbers of apoptotic cells, proliferating cells, T cells and neutrophils within the colon. These responses were significantly reduced in MyD88 -/- , TLR2/4 -/- , TRIF -/- and TLR9 -/- mice, as compared with wild-type controls. However, TRIF -/- and TLR2/4 -/- mice were not protected from mortality, whereas TLR9 -/- mice displayed increased survival rates. The important role of TLR9-mediated immunopathology was independently confirmed by significantly reduced macroscopic disease symptoms and colonic apoptosis as well as by reduced T-cell and neutrophil numbers within the colon after treatment with a synthetic inhibitory oligonucleotide. Conclusions These results emphasise the critical role of gut microbiota, innate immunity and TLR9 in iGvHD and highlight anti-TLR9 strategies as novel therapeutic options.

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Allergic contact hypersensitivity (CHS) is a T cell-mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rbeta2-deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12-independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) beta2, or both, we show that the concomitant absence of TLR4 and IL-12Rbeta2, but not the absence of TLR4 or IL-12Rbeta2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rbeta2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12-independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12-competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rbeta2-deficient mice, but not in germ-free TLR4/IL-12Rbeta2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8 + T cells. This coincides with up-regulation of p53 and dysregulation of NF-κB. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors.

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.

Background: In the course of inflammatory bowel diseases (IBD) and acute murine ileitis following peroral Toxoplasma gondii infection, commensal Escherichia coli accumulate at inflamed mucosal sites and aggravate small intestinal immunopathology. Aim: To unravel the molecular mechanisms by which commensal E coli exacerbate ileitis. Methods: Ileitis was investigated in mice that lack Toll-like receptors (TLR) 2 or 4, specific for bacterial lipoproteins (LP) or lipopolysaccharide (LPS), respectively. Gnotobiotic mice, in which any cultivable gut bacteria were eradicated by antibiotic treatment, were used to study the role of LPS in ileitis. Results: Microbiological analyses revealed that E coli increase in the inflamed ileum. TLR4 −/− , but not TLR2 −/− , mice displayed reduced mortality and small intestinal immunopathology. Decreased interferon (IFN)-γ and nitric oxide (NO) levels in the inflamed terminal ileum of TLR4 −/− mice indicated that TLR4 signalling aggravates ileitis via local mediator release from immune cells. E coli strains isolated from the inflamed ileum activated cultured mouse macrophages and induced TLR4-dependent nuclear factor κB activation and NO production in human embryonic kidney 293 cells and in peritoneal macrophages, respectively. Most strikingly, in contrast with wild-type mice, gnotobiotic TLR4 −/− mice were protected from induction of ileitis by treatment with purified E coli lipid A or colonisation with live E coli . Finally, prophylactic treatment with the LPS scavenger polymyxin B ameliorated T gondii -induced ileitis. Conclusion: These findings highlight the innate immune system as a key player in T gondii -induced ileal immunopathology. Treatment with LPS or TLR4 antagonists may represent a novel strategy for prophylaxis and/or therapy of small intestinal inflammation in IBD.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam 3 CSK 4 and MALP-2, on Ag (DNP–HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam 3 CSK 4 , but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam 3 CSK 4 , most probably by electrostatic forces, reduced the level of active DNP–HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE + Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam 3 CSK 4 indicate that one must be cautious when interpretating results obtained with “model” substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcɛR1) and TLR2 are examined.

Background Propionibacterium acnes was found in lungs and lymph nodes of patients with sarcoidosis and may induce hypersensitivity type granuloma formation. Data regarding the immune response to P. acnes of European sarcoid patients are scarce. Methods We assessed the total IgG and IgA amount and specific antibodies to P. acnes and to Staphylococcus aureus, serving as a control, in BAL fluid of 64 patients with sarcoidosis and of 21 healthy volunteers. In a subcohort of sarcoid patients and controls, TNF-α and GM-CSF production of BAL cells stimulated with heat-killed P. acnes were measured. Results In sarcoid patients, the total IgG and IgA levels in BAL fluid were significantly elevated compared to healthy volunteers. IgG and IgA titres against P. acnes and S. aureus were increased in sarcoid patients, yet based on the total amount of antibodies, only antibodies directed against P. acnes were relatively and significantly increased. Furthermore, BAL cells of sarcoid patients produced significantly more TNF-α and GM-CSF upon stimulation with heat-killed P. acnes compared to controls. Conclusions Patients with sarcoidosis had elevated levels of specific antibodies against P. acnes which suggest contact with this bacterium in the past. Furthermore, BAL cells of sarcoid patients produced inflammatory cytokines (TNF-α and GM-CSF) upon stimulation with P. acnes indicating potential involvement of this pathogen in the pathogenesis of sarcoidosis in some patients.

We investigated the effect of a primary non-lethal infection with lymphocytic choriomeningitis virus (LCMV) on the course and outcome of a secondary infection with the Gram-negative Salmonella enterica serovar Typhimurium or the Gram-positive Listeria monocytogenes in mice. We found that at each stage of the viral infection the susceptibility of mice to bacterial super-infections changes dramatically and depends also on whether the secondary infection is a Gram-positive or Gram-negative one. The study shows that the outcome of the secondary infection is determined by a delicate balance between the overproduction of and the hypersensitivity to inflammatory cytokines (TNF-alpha and IFN-gamma), as well as by the changes in blood leukocytes occurring in mice in the course of viral infection.

Toll-like receptors (TLR) and IL-12 represent key elements of innate immunity. Using C57BL/10 ScCr mice it was shown that TLR4 is important for control of infection with respiratory syncytial virus (RSV). Since these mice have an additional defect in the IL-12R, we reinvestigated immunity to RSV in several C57BL/10 and BALB/c mouse strains lacking a functional TLR4, a functional IL-12-IL-12R interaction or both. In the absence of a functional IL-12 axis, early virus control was impaired in C57BL/10 mice, but not in BALB/c mice. By contrast, TLR4 had no impact on RSV elimination. Pulmonary NK cell recruitment was impaired in IL-12 deficient BALB/c mice and NK cytotoxicity was reduced in IL-12/IL-12R-deficient mice of both genetic backgrounds. Absence of TLR4 had no impact on NK cell recruitment or NK activity nor on recruitment of other pulmonary inflammatory cells. Activation of RSV-specific T cell immunity, including T cell mediated immunopathology, was normal in all mutant strains. These findings clearly argue against a significant role for TLR4 and define a limited role for IL-12 in primary murine RSV infection.

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR40/0) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rβ20/0] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

The contribution of murine Toll-like receptors 2 and 4 (TLR2 and -4, respectively) to cytokine induction by heat-killed bacteria was analyzed in vitro and in vivo. Gram-negative bacteria induced cytokines primarily via TLR4; the contribution of TLR2 was only minor. Neither TLR4 nor, surprisingly, TLR2 was required in the MyD88-dependent response toStaphylococcus aureus.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.

We used biotinylated LPS (LPSb) and flow cytometry to study LPS-monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-alpha on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-alpha after LPS stimulus was higher in HV than SP (mean +/- SD = 25.2 +/- 14.2% and 2.2 +/- 2.6%, respectively, P0.001). LPS-induced CD69 on T CD8+ and CD8- lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean +/- SD = 22.3 +/- 14.6 and 8.6 +/- 5.0, respectively, P = 0.01) and CD8- cells (GMFI, mean +/- SD = 28.3 +/- 7.7 and 14 +/- 4.3 respectively, P0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.

Background: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. Methods: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-α). Results: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-α and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-α in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. Conclusion: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood. Cytometry (Clin. Cytometry) 50:14–18, 2002. © 2002 Wiley-Liss, Inc.

Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow–derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2–deficient mice were still susceptible to sHA. In accordance, addition of an anti–TLR-4 mAb to human monocyte–derived DCs blocked sHA-induced tumor necrosis factor α production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-κB, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

The clearance and activity of different types of lipopolysaccharide (LPS) released during infection with Gram-negative bacteria were investigated. When highly purified preparations differing in their specific endotoxin activity were administered intravenously to mice, the clearance of rough (R)-form LPS preparations from Salmonella minnesota and Escherichia coli was much faster than that of a smooth (S)-form LPS preparation from Salmonella abortus equi, but slower than that of lipooligosaccharides (LOS) preparations from Bordetella pertussis and Helicobacter pylori. After intraperitoneal infection with 107and 108CFU E. coli O111:B4, relatively high levels of LPS were detected dose-dependently in the plasma of infected mice and persisted for a long time. In addition, plasma sCD14 levels in infected mice were higher than in LPS-administered mice. These results indicate that continuously higher levels of plasma LPS followed by stronger host responses occur during infection and suggest that these differences between LPS-administered and infected mice should be taken into consideration when analyzing host responses induced by LPS.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

IFN-γ-dependent hypersensitivity to LPS is inducible in mice by infection or pre-treatment with killed bacteria. Hypersensitive mice exhibit enhanced inflammatory responses to LPS, including the overproduction of TNF-α. Using Lpsn BALB/c and Lpsd BALB/c/l mice, primed with Propionibacterium acnes or infected with Salmonella typhimurium, we show that concurrently to hypersensitivity to LPS, a hypersensitivity to other constituents of killed Gram-negative or Gram-positive bacteria and to staphylococcal enterotoxin B (SEB) develops. The TNF-α hyper-responses in sensitized mice induced by different Gram-positive bacteria, are generally weaker than those by Gram-negative bacteria and vary significantly, due to the absence of a common, LPS-equivalent component. Using IFN-γR—/— and the respective wild-type mice, we demonstrate that although sensitization to LPS and killed Listeria monocytogenes is exclusively IFN-γ-dependent, an IFN-γ-independent, moderate sensitization to certain TNF-α-inducing constituents in bacteria may develop in parallel.

Using a combined pretreatment methodology employing an alkaline reagent and a kinetic chromogenic Limulus test using Endospecy®, different types of endotoxins in murine plasma were able to be measured quantitatively. The detection of smooth (S)-form Salmonella abortus equi LPS (S-LPS) was approximately twice as sensitive as Helicobacter pylori LPS in aqueous solution. S-LPS added to whole mouse blood was recovered quantitatively from plasma, but the rough (R)-form S. minnesota LPS was not. The rates of clearance of S-LPS from the circulation following intravenous injection were shown to be different among BALB/c, C3H/HeN and B10Sn mice. Infection of mice with Salmonella typhimurium 3 days earlier resulted in accelerated clearance of subsequently injected LPS from the circulation. Significantly higher amounts of free non-microbe-associated LPS were detected in the blood of mice 1 h after infection with Escherichia coli O111:B4 followed by immediate treatment with ceftazidime, in comparison with non-antibiotic-treated but infected mice.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.

One important feature of the Propionibacterium acnes-induced hypersensitivity to LPS is the enhanced production of TNFα induced in the sensitized mice upon LPS challenge [Katschinski T., Galanos G., Coumbos A., Freudenberg M.A. Gamma interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect Immun 1992; 60: 1994-2001]. We investigated the role of LPS binding protein (LBP) in the TNFα response of normal and P. acnes-sensitized mice to LPS in LBP+/+ and LBP-/- mice. Treatment of LBP+/+ (BalbC and 129 Sv) mice with P. acnes enhanced their TNFα response to LPS by 75-200-fold compared to the non-treated controls. Unsensitized LBP-/- (129 Sv x BalbC) mice were also stimulated by LPS to produce low amounts of TNFα. These were enhanced by prior P. acnes treatment, however, only by a factor of 4. The characteristic TNFα hyper-response was absent suggesting that the enhanced activity of LPS in sensitized mice is expressed only in the presence of LBP. Evidence for this was obtained by showing that administration of exogenous LBP restored fully the inducibility of the TNFα hyper-response in P. acnes-sensitized LBP-/- mice. Their response to LPS was 1000-fold higher then that of sensitized controls without LBP. A similar LBP treatment of unsensitized LBP-/- mice increased the TNFα response by a factor of only 5. In an analogous experiment, also IFN-γ-sensitized LBP-/- mice exhibited a TNFα overproduction in response to LPS only in the presence of exogenous LBP.

This study investigates the effect of orally administered testosterone on serum testosterone levels and immune responses including outcome of Plasmodium chabaudi malaria. Female C57BL/10 mice were fed on a diet impregnated with 17α-methyl-testosterone for 3 weeks. This raised the circulating testosterone levels from 0.28 ng ml to 2.69 ng ml on the average. In these mice, blood-stage infections of P. chabaudi resulted in a lethal outcome, whereas protective immunity developed in about 80% of mice fed on control diet without testosterone. Dietary 17α-methyl-testosterone reduced the capacity of peritoneal cells to generate reactive oxygen intermediates after stimulation with C3b-coated zymosan and phorbol-myristate-acetate. Also, mice fed on dietary 17α-methyl-testosterone responded to heat-killed Salmonella typhimurium with a higher increase in serum TNF, whereas the induced increase in the production of IL-10 by spleen cells was largely suppressed and no effect was found with respect to the production of IFN-γ and IL-4. Our data indicate that the method of oral administration of 17α-methyl-testosterone raises circulating testosterone to levels that impair protective immune responses to P. chabaudi malaria.

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF–induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Sepsis-induced immune reactions are reduced in TNF receptor 2 (TNFR2)-deficient mice as previously shown. In order to elucidate the underlying mechanisms, the functional integrity of myeloid cells of TNFR2-deficient mice was analyzed and compared to wild type (WT) mice. The capacity of dendritic cells to produce IL-12 was strongly impaired in TNF-deficient mice, mirroring impaired production of IL-12 by WT dendritic cells in sepsis or after LPS or TNF pre-treatment. In addition, TNFR2-deficient mice were refractory to LPS pre-treatment and also to hyper-sensitization by inactivated Propionibacterium acnes, indicating habituation to inflammatory stimuli by the immune response when TNFR2 is lacking. Constitutive expression of TNF mRNA in kidney, liver, spleen, colon and lung tissue, and the presence of soluble TNFR2 in urine of healthy WT mice supported the conclusion that TNF is continuously present in naïve mice and controlled by soluble TNFR2. In TNFR2-deficient mice endogenous TNF levels cannot be balanced and the continuous exposure to enhanced TNF levels impairs dendritic cell function. In conclusion, TNF pre-exposure suppresses secondary inflammatory reactions of myeloid cells; therefore, continuous control of endogenous TNF by soluble TNFR2 seems to be essential for the maintenance of adequate sensitivity to inflammatory stimuli.

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lpsd mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsn (LPS sensitive) and Lpsd mice, and its modulation by LPS, killed gram-negative and gram-positive bacteria and double-stranded (ds)RNA. Untreated Lpsn and Lpsd cultured macrophages (M phi), expressed similar amounts of CD14 mRNA and membrane-bound (m)CD14. LPS enhanced CD14 expression only in Lpsn M phi, while all bacteria, or dsRNA, enhanced CD14 in Lpsn and Lpsd M phi. Similarly, in vivo administration of LPS induced or enhanced CD14 mRNA in different organs of Lpsn mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD14 expression in Lpsn, Lpsd and also in TNF receptor 2-deficient (TNFR2-/-) mice, but failed to do so in TNFR1-/- mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2-/- and TNFR1-/- mice to a similar extent, revealing that this induction does not require TNF signaling.

Background/objectiveToll-like receptors (TLR) mediate the recognition of microbial constituents and stress-induced endogenous ligands by the immune system. They may also be involved in the maintenance or break down of tolerance against autologous antigens. The aim of our investigation was to study the consequence of TLR4 deficiency on the development of insulin-deficient diabetes in the NOD mouse.MethodsThe TLR4 defect of the C57BL/10ScN mouse was backcrossed onto the NOD background and the effect of TLR4 deficiency on diabetes development was analysed by in vivo and in vitro studies.ResultsCompared to animals with wildtype TLR4 expression (TLR4(+/+)), female NOD mice carrying a homozygous TLR4 defect (TLR4(-/-)), showed significant acceleration of diabetes development, with a younger age at diabetes onset (TLR4 (+/+) 177±22 d, TLR(-/-): 118±21 d; pConclusionsOur findings demonstrate that TLR4 deficiency results in an acceleration of diabetes development and immune cell infiltration of islets in NOD mice. We conclude that TLR4 is involved in the progression of the insulitis process thereby controlling the development of insulin-deficient diabetes in NOD mice.

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) of Rhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4'bisphosphorylated beta (1 --6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlcN --GlcN, 2,3-diamino-2,3-dideoxy-D-Glc-(DAG --DAG, and DAG --GlcN. Lipid A of R. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked and cis delta 11-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cis delta 11-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine, phosphorylethanolamine, nor by 4-amino-4-deoxy-L-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1 --4)HexA(1 --5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS of R. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10(-1)-10(-2) of that reported for Salmonella abortus equi LPS, and it was also capable of inducing TNF alpha and IL6 in macrophages of C57BL/10ScSN mice.