Lena Alexopoulou

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis.

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3−/−) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3−/− mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-α receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.

Toll-like receptor 3 promotes cross-priming to virus-infected cells.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8α+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus

Systemic autoimmune disease in humans and mice is characterized by loss of immunologic tolerance to a restricted set of self-nuclear antigens. Autoantigens, such as double-stranded (ds) DNA and the RNA-containing Smith antigen (Sm), may be selectively targeted in systemic lupus erythematosus because of their ability to activate a putative common receptor. Toll-like receptor 9 (TLR9), a receptor for CpG DNA, has been implicated in the activation of autoreactive B cells in vitro, but its role in promoting autoantibody production and disease in vivo has not been determined. We show that in TLR9-deficient lupus-prone mice, the generation of anti-dsDNA and antichromatin autoantibodies is specifically inhibited. Other autoantibodies, such as anti-Sm, are maintained and even increased in TLR9-deficient mice. In contrast, ablation of TLR3, a receptor for dsRNA, did not inhibit the formation of autoantibodies to either RNA- or DNA-containing antigens. Surprisingly, we found that despite the lack of anti-dsDNA autoantibodies in TLR9-deficient mice, there was no effect on the development of clinical autoimmune disease or nephritis. These results demonstrate a specific requirement for TLR9 in autoantibody formation in vivo and indicate a critical role for innate immune activation in autoimmunity.

Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways

Both lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) are adjuvants for the adaptive immune response, inducing upregulation of costimulatory molecules (UCM) on antigen-presenting cells. Trif, an adapter protein that transduces signals from Toll-like receptor 4 (TLR4) and TLR3, permits the induction of many cytokines, including interferon-β, which signals through the type I interferon receptor. We show here that LPS-induced UCM was strictly dependent on the TLR4→Trif axis, whereas dsRNA-induced UCM was only partly dependent on the TLR3→Trif axis. But both LPS- and dsRNA-induced UCM were entirely dependent on type I interferon receptor signaling. These findings show that UCM involves an autocrine or paracrine loop, and indicate that an alternative TLR3-independent, Trif-independent pathway contributes to dsRNA-induced UCM.

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice.

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-α and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2−/−) or TLR1 (TLR1−/−) produced low titers of antibodies against OspA. Notably, macrophages from TLR2−/− mice were unresponsive to OspA and PGN, whereas those from TLR1−/− mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia.

Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3 −/− mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3 −/− animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology.

Interleukin‐10 (IL‐10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL‐10‐deficient mice requires the function of TNF, indicating that the IL‐10/TNF axis regulates mucosal immunity. We further show that IL‐10 targets the 3′ AU‐rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL‐10 does not alter TNF mRNA stability, and its action does not require the presence of the stability‐regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL‐10 is exerted mainly by inhibition of the activating p38/MAPK‐activated protein kinase‐2 pathway. These results demonstrate a physiologically significant cross‐talk between the IL‐10 receptor and the stress‐activated protein kinase modules targeting TNF mRNA translation. This cross‐talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut.

Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria

Toll-like receptors (TLRs) are key components of the immune system that detect microbial infection and trigger antimicrobial host defense responses. TLR5 is a sensor for monomeric flagellin, which is a component of bacterial flagella known to be a virulence factor. In this study we generated TLR5-deficient mice and investigated the role of TLR5 signaling in the detection of flagellin and antibacterial immune responses to Salmonella typhimurium and Pseudomonas aeruginosa. We found that TLR5 is essential for the recognition of bacterial flagellin both in vivo and ex vivo. TLR5 contribution to antibacterial host response to i.p. infection with S. typhimurium or intranasal administration of P. aeruginosa may be masked by TLR4 or other sensing mechanisms. By using radiation bone marrow chimera, we showed that upon i.p. injection of flagellin immune responses are mediated by lymphoid cells, whereas resident cells are required for the initiation of response upon intranasal flagellin administration. These results suggest that flagellin recognition in different organs is mediated by distinct TLR5-expressing cells and provide insights into the cooperation of the TLR5 and TLR4 signaling pathways used by the innate immune system in the recognition of bacterial pathogens.

Brucella Control of Dendritic Cell Maturation Is Dependent on the TIR-Containing Protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyers patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.