Olivier Demaria

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.

TLR8 deficiency leads to autoimmunity in mice

TLRs play an essential role in the induction of immune responses by detecting conserved molecular products of microorganisms. However, the function of TLR8 is largely unknown. In the current study, we investigated the role of TLR8 signaling in immunity in mice. We found that Tlr8(-/-) DCs overexpressed TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster NF-κB activation upon stimulation with the TLR7 ligand R848. Tlr8(-/-) mice showed splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and increased serum levels of IgM and IgG2a. Furthermore, Tlr8(-/-) mice exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither Tlr7(-/-) nor Tlr8(-/-)Tlr7(-/-) mice showed any of the phenotypes observed in Tlr8(-/-) mice. These data provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-γ. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-γ production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-γ production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-γ production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I–specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity

Significance Tumor recognition by the immune system can occur spontaneously but has usually little impact on tumor growth. However, the cellular and molecular mechanisms that drive these responses could be exploited therapeutically to generate efficacious antitumor immunity. Here, we show that stimulator of IFN genes (STING), a molecule involved in cytosolic DNA sensing and required for the generation of spontaneous antitumor immune responses, can be targeted by intratumoral injection of cGAMP to boost antitumor immunity and to control tumor growth. The immune response induced by therapeutic but also spontaneous STING activation was dependent on type I IFN produced by endothelial cells in the tumor microenvironment, unraveling an unexpected role of the tumor vasculature in the initiation of spontaneous and therapeutic antitumor immunity via STING. Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice.

Significance Toll-like receptors (TLRs) that sense microbial or endogenous DNA and RNA have been well implicated in systemic lupus erythematosus (SLE), a multisystem disease characterized by an autoimmune response to nuclear antigens. In mice, both TLR8 and TLR9 control TLR7-mediated lupus, but it is unknown if they have an additive effect in controlling TLR7. We demonstrate that double TLR8/9-deficient mice have increased abnormalities characteristic of SLE and that both TLR8 and TLR9 keep under control TLR7-mediated lupus, but they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells. These TLR interactions have to be taken into account when novel therapeutic approaches are developed that target the blocking of TLRs. Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9−/−) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8−/− or TLR9−/− mice. On the cellular level, TLR8−/− and TLR8/9−/− dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9−/− cells responded normally. Moreover, B cells from TLR9−/− and TLR8/9−/− mice were hyperesponsive to R848, but TLR8−/− B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.

Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self-DNA

Psoriasis is a T‐cell‐mediated skin autoimmune disease characterized by the aberrant activation of dermal dendritic cells (DCs) and the sustained epidermal expression of antimicrobial peptides. We have previously identified a link between these two events by showing that the cathelicidin antimicrobial peptide LL37 has the ability to trigger self‐nucleic acid mediated activation of plasmacytoid DCs (pDCs) in psoriatic skin. Whether other cationic antimicrobial peptides exert similar activities is unknown. By analyzing heparin‐binding HPLC fractions of psoriatic scales, we found that human beta‐defensin (hBD)2, hBD3, and lysozyme are additional triggers of pDC activation in psoriatic skin lesions. Like LL37, hBD2, hBD3, and lysozyme are able to condense self‐DNA into particles that are endocytosed by pDCs, leading to activation of TLR9. In contrast, other antimicrobial peptides expressed in psoriatic skin including elafin, hBD1, and psoriasin (S100A7) did not show similar activities. hBD2, hBD3, and lysozyme were detected in psoriatic skin lesions in the vicinity of pDCs and found to cooperate with LL37 to induce high levels of IFN production by pDCs, suggesting their concerted role in the pathogenesis of psoriasis.

Homeostatic NF-κB Signaling in Steady-State Migratory Dendritic Cells Regulates Immune Homeostasis and Tolerance

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKβ in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

[Toll-like receptor 8: the awkward TLR].

Toll-like receptors (TLR) sense a variety of microbial products and play an important role in the mounting of innate and adaptive immune responses. TLR1 to TLR9 are common in mice and humans and recognize similar ligands in both species, with the exception of TLR8. Human TLR7 and TLR8 and mouse TLR7 detect viral single-stranded RNA and imidazoquinoline compounds, while mouse TLR8 not. Based on this discrepancy, for long time it was believed that mouse TLR8 is not functional and as a consequence the contribution of TLR8 to innate immunity remained poorly understood. Our recent studies revealed an important role for TLR8 in the regulation of TLR7-mediated autoimmunity in the mouse. This review illustrates our current understanding regarding the function of TLR8 and its potential for future clinical use for the treatment and/or prevention of various pathological conditions.

Interleukin 23–Helper T Cell 17 Axis as a Treatment Target for Pityriasis Rubra Pilaris

Importance Treatment of pityriasis rubra pilaris (PRP) is solely based on its resemblance to psoriasis rather than any knowledge of its pathomechanism. Insight into pathogenic mediators of inflammation is essential for targeted and valid treatment options that could replace previous serendipitous therapeutic approaches in refractory PRP. Objective To determine whether blockade of the interleukin 23–helper T cell 17 (IL-23–TH17) pathway with ustekinumab represents an efficacious and, based on its proinflammatory cytokine profile, targeted treatment option in PRP. Design, Setting, and Participants In this case report, a patient with PRP received outpatient treatment at a university hospital department of dermatology with ustekinumab according to the dosing regimen approved for psoriasis. Lesional skin biopsy samples were taken from this patient and 2 others with refractory PRP. Messenger RNA (mRNA) expression of proinflammatory innate and T-cell–derived cytokines were measured and compared with skin samples from patients with psoriasis and healthy donors. From 1 patient, lesional skin samples were taken before ustekinumab treatment and 4 and 28 weeks after treatment initiation. Follow-up was completed after 6 months. Intervention Subcutaneous ustekinumab, 45 mg, at weeks 0 and 4 and quarterly thereafter. Main Outcomes and Measures The primary outcome was to determine the changes in expression of proinflammatory innate and T-cell–derived cytokines during ustekinumab therapy. The secondary objective was to evaluate the clinical and histopathologic phenotype in relation to the mRNA expression profile of proinflammatory cytokines. Results In lesional PRP skin samples from a single patient, upregulated expression levels were found for most proinflammatory innate cytokines, including tumor necrosis factor (TNF), IL-6, IL-12, IL-23, and IL-1&bgr;. Among adaptive T-cell cytokines, an increase of TH1 cytokines and, in particular, TH17 cytokines IL-17A, IL-17F, and IL-22 was seen in PRP. The patient with PRP who received ustekinumab showed regression of skin lesions after 2 weeks and almost complete resolution after 1 month. Clinical and histopathologic improvement paralleled the expression levels of TH17 cytokines but not of interferon-&ggr; and TNF, which lagged behind the amelioration. Conclusions and Relevance In this case report, a role of the IL-23–TH17-axis in PRP was identified, suggesting a shared pathogenic inflammatory pathway with psoriasis, despite evident clinical and histopathologic differences. In addition, this report provides a rationale for targeting the IL-23–TH17-pathway as a treatment option for refractory PRP.