Øyvind Halaas

Endocytic pathways regulate Toll‐like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll‐like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor‐mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin‐binding endosomal sorting protein hepatocyte growth factor‐regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS‐associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Human Toll-Like Receptor 2 Mediates Monocyte Activation by Listeria monocytogenes, But Not by Group B Streptococci or Lipopolysaccharide

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-κB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.

Differential expression of Toll-like receptor 2 in human cells.

Human Toll‐like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti‐TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B‐cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B‐cells, CD3+ T‐cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after 20 h up‐regulated by LPS, GM‐CSF, IL‐1, and IL‐10 and down‐regulated by IL‐4, IFN‐γ, and TNF. On purified granulocytes, LPS, GM‐CSF, and TNF down‐regulated, and IL‐10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers.

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.

The Rab11a GTPase Controls Toll-like Receptor 4-Induced Activation of Interferon Regulatory Factor-3 on Phagosomes

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.

Intracellular Mycobacterium avium Intersect Transferrin in the Rab11+ Recycling Endocytic Pathway and Avoid Lipocalin 2 Trafficking to the Lysosomal Pathway

Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) binds the siderophore carboxymycobactin, an essential component of the iron acquisition apparatus of mycobacteria. Here we show that Lcn2 suppressed growth of Mycobacterium avium in culture, and M. avium induced Lcn2 production from mouse macrophages. Lcn2 also had elevated levels and initially limited the growth of M. avium in the blood of infected mice but did not impede growth in tissues and during long-term infections. M. avium is an intracellular pathogen. Subcellular imaging of infected macrophages revealed that Lcn2 trafficked to lysosomes separate from M. avium, whereas transferrin was efficiently transported to the mycobacteria. Thus, mycobacteria seem to reside in the Rab11(+) endocytic recycling pathway, thereby retaining access to nutrition and avoiding endocytosed immunoproteins like Lcn2.

IL-10 Enhances MD-2 and CD14 Expression in Monocytes and the Proteins Are Increased and Correlated in HIV-Infected Patients

Soluble proteins that bind LPS, like myeloid differentiation-2 (MD-2) and CD14, have essential roles in regulating LPS signaling through TLR4. During a Gram-negative bacterial infection, the host may control the response by adjusting the levels of soluble MD-2 and CD14. To address the surface expression of MD-2 on human leukocytes, we developed a mAb, IIC1, that recognized MD-2 both free and when bound to TLR4. MD-2 was found on the surface of freshly isolated monocytes, on a subpopulation of CD19+ B-cells and on CD15+ neutrophils. LPS transiently reduced the MD-2 levels on monocytes, which is most likely due to endocytosis of the LPS receptor complex since MD-2 colocalized with TLR4 in early endosomes after LPS stimulation. In the absence of LPS, MD-2 partly colocalized with TLR4 in Golgi trans and medial compartments. Cultivating monocytes for 18–20 h resulted in loss of MD-2 expression on the surface, which was reversed either by LPS or IL-10. Furthermore, addition of IL-10, but not LPS, resulted in a considerable increase in mRNA for both MD-2 and CD14. Using ELISA, we demonstrated that IL-10 had a profound dose- and time-related effect on the release of soluble MD-2 and soluble CD14 from monocytes. In HIV-infected patients, the amounts of MD-2, CD14, and IL-10 increased significantly in the patient group with AIDS. Of interest, we found that IL-10, CD14, and MD-2 levels were positively correlated, suggesting that IL-10 may be a driving force for increased release of MD-2 and CD14 during systemic inflammation.

Monocytes stimulated with group B streptococci or interferons release tumour necrosis factor-related apoptosis-inducing ligand.

Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) is a cytotoxic member of the TNF family. Some reports have shown that TRAIL is released from cells in a soluble form. In this work, we have investigated the mechanism of release of TRAIL from monocytes. First, we show that whole gram‐positive, gram‐negative and mycoplasmal bacteria as well as lipopolysaccharide (LPS), interferon‐α (IFN‐α), ‐β and ‐γ all induced upregulation of TRAIL on the surface of human monocytes. Next, we show that IFN‐α, ‐β and ‐γ all induced a dose‐dependent release of TRAIL, giving significant amounts of soluble TRAIL after 2 days. Of the bacteria, only the Group B streptococcus COH‐1 (GBS) induced release of TRAIL and concomittantly induced IFN‐α. Monocytes stimulated with GBS or IFN‐α also showed extensive cell death. When monocyte apoptosis was prevented by interleukin‐1, GM‐CSF, LPS or the caspase inhibitor zVADfmk, the IFN‐α‐induced release of TRAIL was reduced, whereas agents inducing necrosis caused increased release of TRAIL. LPS also prevented release of TRAIL from GBS‐stimulated monocytes. The release of TRAIL from IFN‐α‐stimulated monocytes was reduced by inhibitors of both cysteine and metalloproteases. We conclude that bacteria and IFN induce upregulation of membrane TRAIL and that release of TRAIL is associated with cell death.

The Journey of Toll-like Receptors in the Cell

Multicellular organisms are constantly challenged by microbes that are threatening to invade the host and causing genome or tissue destruction and pathology. In order to fight back attacks it is essential for the host to detect pathogens early before any tissue damage has occurred. For this purpose the Toll-like receptors (TLR) emerged as conserved microbial recognition proteins in species as different as worms (Caenorhabditis elegans) and humans. To date, 12 different TLRs have been found in mammals. TLR1, 2, 4, and 6 are found on the plasma membrane of immune cells and recognize lipoproteins and lipoglycans found on the surface of microbes. TLR5 is also on the plasma membrane and recognizes the motility apparatus protein flagellin. TLR3, 7, 8 and 9 are found intracellularly in immune cells and recognize a variety of nucleotides and nucleoside analogues found more frequently in microbes than in vertebrates. Lack of TLR signaling may results in severe loss of anti-microbial defense, and erroneous TLR signaling may result in allergies, auto-immunity or cancer (Bohnhorst, Rasmussen, Moen, Flottum, Knudsen, Borset, Espevik and Sundan 2006). This chapter will focus on localization and trafficking of TLRs and the reasons there may be for this compartmentalization.

Dynamics of immune effector mechanisms during infection with Mycobacterium avium in C57BL/6 mice

Opportunistic infections with non‐tuberculous mycobacteria such as Mycobacterium avium are receiving renewed attention because of increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanisms contributing to containment in a mouse infection model using virulent M. avium strain 104 in C57BL/6 mice. M. avium caused chronic infection in mice, as shown by sustained organ bacterial load. In the liver, bacteria were contained in granuloma‐like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in the adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti‐mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon‐γ were found in infected liver, spleen and serum of mice approximately 2 weeks post infection and coincided with a halt in organ bacterial growth. In contrast, expression of tumour necrosis factor was surprisingly low in spleen compared with liver. We did not detect interleukin‐17 in infected organs or M. avium‐specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens. This detailed characterization of M. avium infection in C57BL/6 mice may provide a basis for future studies aimed at gaining better insight into mechanisms leading to containment of infections with non‐tuberculous mycobacteria.