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Severe COVID-19 is accompanied by rampant immune dysregulation in the lung and periphery, with immune cells of both compartments contributing to systemic distress. The extent to which immune cells of the lung and blood enter similar or distinct pathological states during severe disease remains unknown. Here, we leveraged 96 publicly available single-cell RNA sequencing datasets to elucidate common and compartment-specific features of severe to critical COVID-19 at the levels of transcript expression, biological pathways, and ligand-receptor signaling networks. Comparing severe patients to milder and healthy donors, we identified distinct differential gene expression signatures between compartments and a core set of co-directionally regulated surface markers. A majority of severity-enriched pathways were shared, whereas TNF and interferon responses were polarized. Severity-specific ligand-receptor networks appeared to be differentially active in both compartments. Overall, our results describe a nuanced response during severe COVID-19 where compartment plays a role in dictating the pathological state of immune cells., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Inflammaging drives age-related pathologies. In this issue of Immunity, Barkaway et al. illustrate how aged endothelial cells and mast cells promote reverse migration of neutrophils from inflamed tissue back into circulation, causing tissue damage at distal sites., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections., Competing Interests: Declaration of interests T.A.C., H.H., M.B., P.S.L., P.C., and R.D.P. are inventors on provisional patent application US 63/038,186 titled “Methods and Compositions for treating coronavirus infectious disease.” H.H. and T.A.C. are co-founders of and hold equity in Alcea Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Several viruses target the human respiratory tract, causing different clinical manifestations spanning from mild upper airway involvement to life-threatening acute respiratory distress syndrome (ARDS). As dramatically evident in the ongoing SARS-CoV-2 pandemic, the clinical picture is not always easily predictable due to the combined effect of direct viral and indirect patient-specific immune-mediated damage. In this review, we discuss the main RNA (orthomyxoviruses, paramyxoviruses, and coronaviruses) and DNA (adenoviruses, herpesviruses, and bocaviruses) viruses with respiratory tropism and their mechanisms of direct and indirect cell damage. We analyze the thin line existing between a protective immune response, capable of limiting viral replication, and an unbalanced, dysregulated immune activation often leading to the most severe complication. Our comprehension of the molecular mechanisms involved is increasing and this should pave the way for the development and clinical use of new tailored immune-based antiviral strategies., (Copyright © 2021 American Society for Microbiology.)

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca 2+ mobilization. In DCs, Ca 2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP 3 ). Here, we showed that the IP 3 receptor 3 (IP 3 R3) and ITPKB, a kinase that converts IP 3 to inositol 1,3,4,5-tetrakisphosphate (IP 4 ), were both necessary for Ca 2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP 3 R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca 2+ mobilization through plasma membrane-localized IP 3 R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Endogenous oxidized phospholipids are produced during tissue stress and are responsible for sustaining inflammatory responses in immune as well as non-immune cells. Their local and systemic production and accumulation is associated with the etiology and progression of several inflammatory diseases, but the molecular mechanisms that underlie the biological activities of these oxidized phospholipids remain elusive. Increasing evidence highlights the ability of these stress mediators to modulate cellular metabolism and pro-inflammatory signaling in phagocytes, such as macrophages and dendritic cells, and to alter the activation and polarization of these cells. Because these immune cells serve a key role in maintaining tissue homeostasis and organ function, understanding how endogenous oxidized lipids reshape phagocyte biology and function is vital for designing clinical tools and interventions for preventing, slowing down, or resolving chronic inflammatory disorders that are driven by phagocyte dysfunction. Here, we discuss the metabolic and signaling processes elicited by endogenous oxidized lipids and outline new hypotheses and models to elucidate the impact of these lipids on phagocytes and inflammation., Competing Interests: IZ reports compensation for consulting services with Implicit Biosciences. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Di Gioia and Zanoni.)

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus Moritella was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We developed an orally administered, engineered, bacterium-based, RNA interference-mediated therapeutic method to significantly reduce the symptoms in the most frequently used animal model of inflammatory bowel disease. This bacterium-mediated RNA interference strategy was based on the genomically stable, non-pathogenic E. coli MDS42 strain, which was engineered to constitutively produce invasin and the listeriolysin O cytolysin. These proteins enabled the bacteria first to invade the colon epithelium and then degrade in the phagosome. This allowed the delivery of a plasmid encoding small hairpin RNA (shRNA) targeting tumor necrosis factor (TNF) into the cytoplasm of the target cells. The expression levels of TNF and other cytokines significantly decreased upon this treatment in dextran sulfate sodium (DSS)-induced colitis, and the degree of inflammation was significantly reduced. With further safety modifications this method could serve as a safe and side effect-free alternative to biologicals targeting TNF or other inflammatory mediators., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1β, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy., Competing Interests: Declaration of Interests J.C.K. holds equity and consults for IFM Therapeutics, Quench Bio, and Corner Therapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Orche Bio, and Dahlia Biosciences. D.Z. holds equity and consults for Corner Therapeutics. Boston Children’s Hospital has filed patents related to the use of hyperactivating stimuli and hyperactive DCs in the treatment of disease. J.C.K. serves on the advisory board of Cell Reports., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests.

Humans are constantly exposed to fungi, either in the form of commensals at epithelial barriers or as inhaled spores. Innate immune cells play a pivotal role in maintaining commensal relationships and preventing skin, mucosal, or systemic fungal infections due to the expression of pattern recognition receptors that recognize fungal cell wall components and modulate both their activation status and the ensuing adaptive immune response. Commensal fungi also play a critical role in the modulation of homeostasis and disease susceptibility at epithelial barriers. This review will outline cellular and molecular mechanisms of anti-fungal innate immunity focusing on C-type lectin receptors and their relevance in the context of host-fungi interactions at skin and mucosal surfaces in murine experimental models as well as patients susceptible to fungal infections., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here, we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide. While cells activated by lipopolysaccharide rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine, and favor the accumulation of oxaloacetate in the cytoplasm. This metabolite potentiates interleukin-1β production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

TLRs are a class of pattern recognition receptors (PRRs) that detect invading microbes by recognizing pathogen-associated molecular patterns (PAMPs). Upon PAMP engagement, TLRs activate a signaling cascade that leads to the production of inflammatory mediators. The localization of TLRs, either on the plasma membrane or in the endolysosomal compartment, has been considered to be a fundamental aspect to determine to which ligands the receptors bind, and which transduction pathways are induced. However, new observations have challenged this view by identifying complex trafficking events that occur upon TLR-ligand binding. These findings have highlighted the central role that endocytosis and receptor trafficking play in the regulation of the innate immune response. Here, we review the TLR4 and TLR9 transduction pathways and the importance of their different subcellular localization during the inflammatory response. Finally, we discuss the implications of TLR9 subcellular localization in autoimmunity., (©2019 The Authors. Society for Leukocyte Biology Published by Wiley Periodicals, Inc.)

Lambda interferons (IFNλs, type III IFNs or interleukins-28/29) were described fifteen years ago as novel cytokines sharing structural and functional homology with IL-10 and type I IFNs, respectively. IFNλs engage a unique receptor complex comprising IFNLR1 and IL10R2, nevertheless they share signaling cascade and many functions with type I IFNs, questioning their possible non-redundant roles and overall biological importance. Here, we review the latest evidence establishing the primacy of IFNλs in front line protection at anatomical barriers, mediating antiviral immunity before type I IFNs. We also discuss their emerging role in regulating inflammation and limiting host damage, a major difference to type I IFNs. IFNλs come thus to light as dual function cytokines mediating antiviral immunity and damage control., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress., Competing Interests: The authors have declared that no competing interests exist.

Vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. Despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. To address this issue, one possibility is to exploit the high efficiency of dendritic cells (DCs) as antigen-presenting cells for T cell priming. DCs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. Therefore, the choice of adjuvants to guide and sustain DCs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target DCs represent the key issues currently explored in both preclinical and clinical settings. Here, we review advances in DCs-based vaccination approaches, which exploit direct in vivo DCs targeting and activation options. We also discuss the recent findings for efficient antitumor DCs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Type III interferons (IFNs) (or IFN-λ) are the latest addition to the IFN family. Even though they share little protein homology with type I IFN, both exhibit remarkable functional similarities: each can be induced in response to viral infections, and both lead to Janus kinases (JAK) and signal transducer and activator of transcription (STAT) activation. The JAK/STAT pathway induces antiviral responses and IFN-stimulated gene transcription. However, despite the similarities in their effector functions with type I IFNs, IFN-λ also has a non-redundant role in protecting barrier organs: epithelial cells preferentially produce IFN-λ rather than type I IFNs; and interferon lambda receptor 1 (IFNLR1), the specific receptor for IFN-λ, is highly expressed on cells of epithelial lineage. Thus far, IFN-λ has been considered mainly as an epithelial cytokine, which restricts viral replication in epithelial cells and constitutes an added layer of protection at mucosal sites. However, it is now increasingly recognized that IFNLR1 is expressed broadly, and that immune cells such as neutrophils and dendritic cells also respond to IFN-λ. Moreover, in many in vivo models, IFN-λ modulates immune cell functions and thereby configures itself less as a cytokine that is only specific to the epithelium, and more as a cytokine that directly controls the inflammatory response at mucosal sites. Here, we critically review the recent literature on immune modulatory roles for IFN-λ, and distinguish between the direct and indirect effects of this IFN on immune cell functions in different inflammatory settings.

Interferon-λ (IFN-λ) is a central regulator of mucosal immunity; however, its signaling specificity relative to that of type I interferons is poorly defined. IFN-λ can induce antiviral interferon-stimulated genes (ISGs) in epithelia, while the effect of IFN-λ in non-epithelial cells remains unclear. Here we report that neutrophils responded to IFN-λ. We found that in addition to inducing ISG transcription, IFN-λ (but not IFN-β) specifically activated a translation-independent signaling pathway that diminished the production of reactive oxygen species and degranulation in neutrophils. In mice, IFN-λ was elicited by enteric viruses and acted on neutrophils to decrease oxidative stress and intestinal damage. Thus, IFN-λ acted as a unique immunomodulatory agent by modifying transcriptional and non-translational neutrophil responses, which might permit a controlled development of the inflammatory process.

Nuclear factor of activated T cells (NFAT) is activated in innate immune cells downstream of pattern recognition receptors, but little is known about NFAT's functions in innate immunity compared with adaptive immunity. We show that early activation of NFAT balances the two major phases of the innate response to Candida albicans skin infections: the protective containment (abscess) and the elimination (expulsion) phases. During the early containment phase, transforming growth factor- β (TGF- β ) induces the deposit of collagen around newly recruited polymorphonuclear cells to prevent microbial spreading. During the elimination phase, interferon- γ (IFN- γ ) blocks differentiation of fibroblasts into myofibroblasts by antagonizing TGF- β signaling. IFN- γ also induces the formation of plasmin that, in turn, promotes abscess capsule digestion and skin ulceration for microbial discharge. NFAT controls innate IFN-γ production and microbial expulsion. This cross-talk between the innate immune and the fibrinolytic systems also occurs during infection with Staphylococcus aureus and is a protective response to minimize tissue damage and optimize pathogen elimination., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chronic inflammation is one of the causes of neurodegeneration in Amyotrophic lateral sclerosis (ALS). Here we examined whether circulating dendritic cells (DCs) can contribute to disease progression. We found ALS patients show a significant reduction in the number of circulating DCs. Also, patients' DCs present an increased expression of CD62L and a tendency to overexpress CCR2 compared with healthy donors. Moreover, DCs derived from a subpopulation of ALS patients produced higher levels of IL-8 and CCL-2 upon lipopolysaccharide (LPS)-stimulation. Finally, we found a significant inverse correlation between the time from onset of the pathology to its diagnosis and the levels of IL-6 secretion induced by LPS. Our data support the hypothesis, in a subpopulation of patients, DCs recruited at the diseased tissue produce high levels of CCL-2 and IL-8 and contribute to the inflammatory process promoting the recruitment of other inflammatory cells. An increased efficiency of IL-6 production may accelerate only the initial phases of disease progression. Blood DC analysis can be used to identify ALS patients with an altered course of inflammatory cell recruitment at the diseased central nervous system (CNS). The high levels of CD62L expression suggests this molecule could be a target for treatment of CNS inflammation.

Natural killer (NK) cells are critical players against tumors. The outcome of anti-tumor vaccination protocols depends on the efficiency of NK-cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK-cell activation dynamics is needed. NK-cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti-tumor functions. NK-cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two-photon microscopy. Close DC and NK-cell contacts are essential for the localized delivery of DC-derived IL-18 to NK cells, a strict requirement in NK-cell activation., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Dendritic cells (DCs) use pattern recognition receptors to detect microorganisms and activate protective immunity. These cells and receptors are thought to operate in an all-or-nothing manner, existing in an immunologically active or inactive state. Here, we report that encounters with microbial products and self-encoded oxidized phospholipids (oxPAPC) induce an enhanced DC activation state, which we call "hyperactive." Hyperactive DCs induce potent adaptive immune responses and are elicited by caspase-11, an enzyme that binds oxPAPC and bacterial lipopolysaccharide (LPS). oxPAPC and LPS bind caspase-11 via distinct domains and elicit different inflammasome-dependent activities. Both lipids induce caspase-11-dependent interleukin-1 release, but only LPS induces pyroptosis. The cells and receptors of the innate immune system can therefore achieve different activation states, which may permit context-dependent responses to infection., (Copyright © 2016, American Association for the Advancement of Science.)

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

Pattern recognition receptors (PRRs) are generally recognized as the initiators of all immune responses. PRRs bind molecular patterns associated with microorganisms or endogenous mediators released by stressed tissues. Upon ligand binding, PRRs induce the activation of an inflammatory process that ultimately leads to pathogen clearance or restoration of tissue homeostasis. PRRs govern these processes, regulating the activation of a complex network of transcription factors able to induce the appropriate immune response to a specific ligand. Toll-like-receptors (TLRs) are the first and best characterized PRR family, and for a long period of time they were believed to be autonomous proteins able to recognize and initiate all the immune response to a given stimulus. Recently this view was challenged by the discovery that so-called TLR co-receptors, such as CD14 and CD36, not only favor TLR-dependent signaling but can also transduce their own signal in a TLR-independent manner. Here we will discuss the capacity of TLR co-receptors to bind different microbial and endogenous ligands and to integrate TLR functions inducing specific signaling modules., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lactobacilli are able to induce upregulation of co-stimulatory molecules in DCs with Th1 cytokines production and increase in Treg activity. This could explain the observed effectiveness of the prolonged administration of lactobacilli in the prevention of allergic disorders in infants and envisage the possible use of bacteria expressing the allergen for the specific immunotherapy of allergic diseases. Hence, we evaluated Streptococcus thermophilus (ST) expressing rBet v 1 as allergen delivery tool and adjuvant factor for immunotherapy in Betv1-sensitized mice. rBet v 1 gene was introduced and expressed in ST (ST[rBet v 1]). BALB/c mice were sensitized with rBet v 1 and then treated with either ST alone, ST[rBet v 1], or the combination of ST and rBet v 1, for 20 days. After 2 aerosol challenges, Treg frequency, in vitro allergen-induced cytokines, rBet v 1-specific IgE and IgG2a, and bronchial histology were made in harvested spleen, sera, and lung. Results were compared with those obtained from not-treated/sensitized mice. ST[rBet v 1] induced immunological and histological changes typical of successful SIT: increased frequency of Tregs and expression of Foxp3; decreased allergen-specific IgE/IgG2a ratio; decrease of in vitro rBet v 1-induced IL-4 from spleen cells; increased allergen-induced IL-10 and IFN-γ; drop of bronchial eosinophilia. ST and ST+rBet v 1 combination, even though induced a slight increase in the frequency of Tregs and moderate allergen-induced IL-10, were ineffective in reducing bronchial eosinophilia, allergen induced IL-4 and rBet v 1-specific IgE/IgG2a ratio. ST[rBet v 1] has tolerogenic and Th-1 skewing properties and efficiently delivers the allergen to the gut immune-system restraining and readdressing the established specific Th2 response toward the allergen in mice.

Toll-like receptors (TLRs) are essential players in the innate immune response to invading pathogens. Although extensive research efforts have provided a considerable wealth of information on how TLRs function, substantial gaps in our knowledge still prevent the definition of a complete picture of TLR signaling. However, several recent studies describe additional layers of complexity in the regulation of TLR ligand recognition, adaptor recruitment, posttranslational modifications of signaling proteins, and the newly described, autonomous role of the TLR4 co-receptor CD14. In this review, by using it as model system for the whole TLR family, we attempt to provide a complete description of the signal transduction pathways triggered by TLR4, with a particular emphasis on the molecular and cell biological aspects regulating its function. Finally, we discuss a recently reported model of CD14-dependent signaling and highlight its biological implications.

Dendritic cells (DCs) and macrophages contribute to the activation of immune responses against infectious agents. They sense the presence of microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs). Among the different PAMPs, the response to lipopolysaccharide (LPS) is one of the best characterized. Upon LPS encounter DCs undergo an activation process and acquire the ability to prime both natural killer and T-cell responses after migration to lymph nodes. Once they completed the effector phase, DCs reach a terminal differentiation stage and eventually die by apoptosis. By contrast, macrophages do not leave the tissue upon LPS recognition. They first initiate inflammatory processes and then switch to an anti-inflammatory phenotype to restore tissue homeostasis. In this review we will focus on the molecular bases of the divergent responses of DCs and macrophages to LPS., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Dendritic cells (DCs) sense the presence of pathogens through germline-encoded pattern recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms and endogenous stimuli. Toll-like receptors (TLRs) are the best characterized PRRs. TLR activation has a profound effect on a number of DC activities, including endocytosis, cytoskeleton rearrangement, migration, antigen processing and presentation, survival, and death. The goal of TLR-induced DC reprogramming is to promote the appropriate activation and differentiation of lymphocytes bearing clonally distributed antigen-specific receptors. In this review, we will focus on the functional consequences of TLR engagement for conventional DCs.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

The recognition of microbial stimuli by Toll-like receptors (TLRs) expressed on dendritic cells (DCs) is essential for the regulation of immune responses. DC activation via TLRs leads to the production of proinflammatory cytokines, chemokines and surface molecules that play a key role in the regulation and control of inflammatory reactions and adaptive immunity. Minor imbalances in the feedback control of TLR-activated innate immune cells have been associated with autoimmunity in genetically prone individuals. We review here recent studies indicating how TLR-mediated activation of innate immune cells, including DCs, may be involved in the development and/or maintenance of autoimmune responses in the presence of both endogenous and exogenous ligands.

Dendritic cells are professional antigen-presenting cells able to initiate innate and adaptive immune responses against invading pathogens. In response to external stimuli dendritic cells undergo a complete genetic reprogramming that allows them to become, soon after activation, natural killer cell activators and subsequently T cell stimulators. The recent observation that dendritic cells produce interleukin 2 following microbial stimulation opens new possibilities for understanding the efficiency of dendritic cells in regulating immune system functions. This review discusses how dendritic cells control natural killer, T- and B-cell responses and the relevance of interleukin 2 in these processes.