Franziska Ampenberger

The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily.

Loxoribine (7‐allyl‐7,8‐dihydro‐8‐oxo‐guanosine) acts as synthetic adjuvant in anti‐tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll‐like receptor (TLR) 7/MyD88‐dependent signaling pathway. TLR7‐ and MyD88‐deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7‐deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R‐848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG‐DNA‐driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen‐associated molecular patterns in endosomal/lysosomal compartments.

Fatty acid–induced mitochondrial uncoupling elicits inflammasome-independent IL-1α and sterile vascular inflammation in atherosclerosis

Chronic inflammation is a fundamental aspect of metabolic disorders such as obesity, diabetes and cardiovascular disease. Cholesterol crystals are metabolic signals that trigger sterile inflammation in atherosclerosis, presumably by activating inflammasomes for IL-1β production. We found here that atherogenesis was mediated by IL-1α and we identified fatty acids as potent inducers of IL-1α-driven vascular inflammation. Fatty acids selectively stimulated the release of IL-1α but not of IL-1β by uncoupling mitochondrial respiration. Fatty acid–induced mitochondrial uncoupling abrogated IL-1β secretion, which deviated the cholesterol crystal–elicited response toward selective production of IL-1α. Our findings delineate a previously unknown pathway for vascular immunopathology that links the cellular response to metabolic stress with innate inflammation, and suggest that IL-1α, not IL-1β, should be targeted in patients with cardiovascular disease.

Nrf2 is essential for cholesterol crystal-induced inflammasome activation and exacerbation of atherosclerosis.

Oxidative stress and inflammation — two components of the natural host response to injury — constitute important etiologic factors in atherogenesis. The pro‐inflammatory cytokine interleukin (IL)‐1 significantly enhances atherosclerosis, however, the molecular mechanisms of IL‐1 induction within the artery wall remain poorly understood. Here we have identified the oxidative stress‐responsive transcription factor NF‐E2‐related 2 (Nrf2) as an essential positive regulator of inflammasome activation and IL‐1‐mediated vascular inflammation. We show that cholesterol crystals, which accumulate in atherosclerotic plaques, represent an endogenous danger signal that activates Nrf2 and the NLRP3 inflammasome. The resulting vigorous IL‐1 response critically depended on expression of Nrf2, and Nrf2‐deficient apolipoprotein E (Apoe)−/− mice were highly protected against diet‐induced atherogenesis. Importantly, therapeutic neutralization of IL‐1α and IL‐1β reduced atherosclerosis in Nrf2+/−Apoe−/− but not in Nrf2−/−Apoe−/− mice, suggesting that the pro‐atherogenic effect of Nrf2‐signaling was primarily mediated by its permissive role in IL‐1 production. Our studies demonstrate a role for Nrf2 in inflammasome activation, and identify cholesterol crystals as disease‐relevant triggers of the NLRP3 inflammasome and potent pro‐atherogenic cytokine responses. These findings suggest a common pathway through which oxidative stress and metabolic danger signals converge and mutually perpetuate the chronic vascular inflammation that drives atherosclerosis.

CD40–CD40L cross-talk integrates strong antigenic signals and microbial stimuli to induce development of IL-17-producing CD4+ T cells

IL-17-producing CD4+ T cells have been recognized as key players in organ-related autoimmune disease; however, the parameters that govern their development are yet to be elucidated fully. By using both in vivo and in vitro systems, we have investigated the role of antigen dose, pathogen-associated molecular patterns, and CD40–CD40 ligand (CD40L) cross-talk in Th17 differentiation. We found that the strength of antigenic stimulation critically influenced the extent of Th17 differentiation, because high, but not low or intermediate, antigen concentrations led to IL-17 production. Strong antigenic stimulation of T cells up-regulated CD40L expression, which in concert with certain microbial stimuli (i.e., cytosine phosphate guanine, curdlan, and zymosan) synergistically increased dendritic cell (DC) IL-6 production and Th17 polarization. CD40-deficient DCs exhibited reduced cytokine release and failed to drive Th17 development in vitro. These results were confirmed in vivo where the absence of CD40–CD40L cross-talk was found to prevent the expansion of IL-17-producing cells and accordingly the development of experimental autoimmune encephalitis. Our data demonstrate that CD40–CD40L cross-talk is important for Th17 development by translating strong T cell receptor and microbial stimuli into IL-6 production.

Lymph Node Resident Rather Than Skin-Derived Dendritic Cells Initiate Specific T Cell Responses after Leishmania major Infection

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11chighCD11bhighGr-1−CD8−langerin− DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11chigh CD11bhigh DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.

Dyslipidemia inhibits Toll-like receptor–induced activation of CD8α-negative dendritic cells and protective Th1 type immunity

Environmental factors, including diet, play a central role in influencing the balance of normal immune homeostasis; however, many of the cellular mechanisms maintaining this balance remain to be elucidated. Using mouse models of genetic and high-fat/cholesterol diet–induced dyslipidemia, we examined the influence of dyslipidemia on T cell and dendritic cell (DC) responses in vivo and in vitro. We show that dyslipidemia inhibited Toll-like receptor (TLR)–induced production of proinflammatory cytokines, including interleukin (IL)-12, IL-6, and tumor necrosis factor-α, as well as up-regulation of costimulatory molecules by CD8α− DCs, but not by CD8α+ DCs, in vivo. Decreased DC activation profoundly influenced T helper (Th) cell responses, leading to impaired Th1 and enhanced Th2 responses. As a consequence of this immune modulation, host resistance to Leishmania major was compromised. We found that oxidized low-density lipoprotein (oxLDL) was the key active component responsible for this effect, as it could directly uncouple TLR-mediated signaling on CD8α− myeloid DCs and inhibit NF-κB nuclear translocation. These results show that a dyslipidemic microenvironment can directly interfere with DC responses to pathogen-derived signals and skew the development of T cell–mediated immunity.

Tick Saliva Inhibits Dendritic Cell Migration, Maturation, and Function while Promoting Development of Th2 Responses

Similarly to other blood-feeding arthropods, ticks have evolved immunosuppressive mechanisms enabling them to overcome the host immune system. Although the immunomodulatory effect of tick saliva on several cell populations of the immune system has been extensively studied, little is known about its impact on dendritic cells (DCs). We have examined the effect of Ixodes ricinus tick saliva on DC function in vitro and in vivo. Exposure of DCs to tick saliva in vitro resulted in impaired maturation, upon CD40 or TLR9, TLR3 and TLR7 ligation, as well as reduced Ag presentation capacity. Administration of tick saliva in vivo significantly inhibited maturation and early migration of DCs from inflamed skin to draining lymph nodes, and decreased the capacity of lymph node DCs to present soluble Ag to specific T cells. Moreover, saliva-exposed DCs failed to induce efficient Th1 and Th17 polarization and promoted development of Th2 responses. Our data reveal a complex inhibitory effect exerted by tick saliva on DC function. Given the role of DCs as the key instigators of adaptive immune responses, alteration of their function might represent a major mechanism of tick-mediated immune evasion.