Nucleic acids play a complex role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Nucleic acids are both the primary self-antigens and potent DAMPs, activating DCs through TLR7 and TLR9 and inducing the production of Type I IFNs (Barrat et al., 2005; Marshak-Rothstein and Rifkin, 2007). Nucleic acids often activate DCs either as part of an immune complex with autoantibodies (Leadbetter et al., 2002) or in complex with chaperones like heat shock proteins (HSP) and HMGB1. Oligonucleotides that inhibit the response to TLR7 and TLR9 ameliorate disease in lupus-prone mice (Barrat et al., 2007). Similarly the genetic deficiency of TLR7, or TLR7 and TLR9 combined prevent disease, while the single deficiency of TLR9 renders the disease worse, suggesting a double-edge effect of TLR9 (Santiago-Raber et al., 2009). In our hands, DCs from lupus-prone mice express constitutively high levels of Type I IFNs and IFN responsive genes and this IFN Signature was diminished by treatment with oligonucleotides inhibitory for TLR7 and TLR9, supporting a role for the response of DCs to nucleic acids in the excessive production of Type I IFNs in lupus (Sriram et al., 2012). The importance of TLR7 is further highlighted by the discovery that the Yaa translocation of a piece of chromosome X, containing 16 genes including TLR7, to the Y chromosome induces in male mice a severe lupus-like disease by creating a duplication of TLR7 (Pisitkun et al., 2006). Necrotic cells are a source of immunogenic nucleic acids (Eloranta et al., 2009) as well as neutrophils undergoing NETosis (Garcia-Romo et al., 2011).

A subset of PAMPs that can also be considered a form of special endogenous danger signals are endogenous viral elements (EVEs). EVEs are sections of viral DNA that have integrated into the mammalian genome during evolution (Stoye, 2012). The best characterized EVEs come from retroviruses, but may also derive from other viruses (Katzourakis and Gifford, 2010). Recent evidence demonstrates that defects in controlling retroviral elements are linked to autoimmunity. Indeed, mice deficient in the cytoplasmic three prime repair exonuclease 1 (Trex1) (formerly DNase III) express elevated IFN alpha (IFNα) levels in many organs and develop inflammatory myocarditis in the absence of viral infection (Morita et al., 2004). Trex1-deficient (Trex1−/−) murine embryonic fibroblasts accumulate endogenous retroviral DNA in the cytosol which induces excessive Type I IFN production via STING (Yang et al., 2007; Stetson et al., 2008; Gall et al., 2012). Loss-of-function mutations in Trex1 are present in patients with Aicardi–Goutières syndrome as well as other autoimmune diseases such as familial chilblain lupus (Lee-Kirsch et al., 2007a; Gunther et al., 2009) and SLE (Lee-Kirsch et al., 2007b; Hur et al., 2008). This evidence suggests that cytoplasmic DNA derived from endogenous retroviruses is an endogenous danger signal that, if not scavenged by Trex1, can trigger an intracellular nucleic acid-sensing machinery (Pichlmair and Reis e Sousa, 2007; Thompson et al., 2011), leading to excessive production of the pro-autoimmunogenic Type I IFNs (Elkon and Stone, 2011). Although DCs are activated by Type I IFNs (Gallucci et al., 1999), and might therefore become hyperactivated, and thus initiate the autoimmune process, phenotypic, or specific functional abnormalities of DCs in Trex1−/− mice or in patients with Aicardi–Goutières syndrome have not yet been reported.

Other forms of nucleic acids acting as danger signals are the intracellular single nucleotides ATP and UTP. Extracellular ATP concentration is typically kept very low (10 nM) by ectonucleotidases (Robson et al., 2006). Under pathologic conditions, however, local concentrations of ATP can be increased – when liberated from damaged tissue or when released by an active process such as platelet or phagocyte activation (Zeiser et al., 2011). These adenosine derivatives activate DCs (Marriott et al., 1999) by stimulating the high affinity purinergic receptors P2Z/P2X₇ (Mutini et al., 1999). ATP and UTP activate the inflammasome in collaboration with TLRs (Mariathasan et al., 2004) and mediate at least some of the sterile inflammation induced by necrotic cells (Iyer et al., 2009; Aymeric et al., 2012). P2X₇-activated DCs have been shown to be crucial for the induction of tumor immunity (Ghiringhelli et al., 2009), the lack of P2X₇ signaling decreases the severity of EAE (Sharp et al., 2008), and oxidized ATP (oxATP), an inhibitor of the ATP receptor P2rx7, ameliorated autoimmune type I diabetes and autoimmune encephalitis in mice (Lang et al., 2010). Apoptotic cells can also release small amounts of ATP through the pannexin family of plasma membrane channels. At this low concentration, ATP binds low affinity purinergic receptors, including P2Y₂ and P2X₇ on phagocytes and recruits them to accelerate non-inflammatory clearance of apoptotic cells (Elliott et al., 2009). In a model of allergic lung inflammation, P2Y₂ is required for myeloid DC and eosinophil migration, although this receptor does not seem to be involved in DC maturation (Muller et al., 2010). Therefore, the ATP molecule may represent an important DAMP, conserved from prokaryotes (Atarashi et al., 2008) to plants (Heil et al., 2012) to mammals (Aymeric et al., 2012), acting as an activation signal at high doses when released by damaged cells, while at lower doses it may act as a “find-me” signal that helps housekeeping clearance of apoptotic cells and prevents immune activation (Elliott et al., 2009).

Uric acid was one of the first DAMPs to be discovered and is partly responsible for the adjuvant properties of dying cells (Shi et al., 2003). Uric acid is a product of purine metabolism and is normally soluble inside cells. When released in the extracellular space uric acid precipitates and forms insoluble crystals of monosodium urate (MSU) capable of activating DCs in vitro and of acting as an adjuvant in vivo (Shi et al., 2003; Rock et al., 2010). Extracellular uric acid is highly inflammatory when it accumulates in joints to cause gout (Rock et al., 2013) or when it is released in large amounts during anti-cancer therapy causing tumor lysis syndrome (Muslimani et al., 2011). The inflammatory nature of uric acid, as well as of other crystals like silica and alum, is in part due to their ability to activate the inflammasome (Martinon et al., 2006) with the extracellular delivery of endogenous ATP, the stimulation of IL-1β, and other pro-inflammatory cytokines, and the induction of reactive oxygen species (ROS) (Schorn et al., 2010; Riteau et al., 2012). These crystals may activate the inflammasome by causing direct cell damage to phagocytes, including DCs, inducing phagolysosome rupture (Hornung et al., 2008). Indeed, membrane fragments of these organelles can activate several responses, including polyubiquitination, autophagy, and pyroptosis and may play a role in host defense against intracellular parasites (Hilbi, 2009). Excessive activation of the inflammasome has been strongly linked to a group of hereditary autoinflammatory diseases called cryopyrin associated periodic syndromes (Lamkanfi and Dixit, 2012) but it may also play a role in more classic autoimmune diseases such as multiple sclerosis (MS) as mice deficient for components of the inflammasome (Nlrp3 and ASC) are protected from the development of EAE (Gris et al., 2010; Shaw et al., 2010).

Heat shock proteins are the most abundant proteins in cells, responsible for maintaining the correct folding of nascent and misfolded proteins, and of proteins that must remain inactive for long periods. HSPs are up-regulated in response to tissue stressors such as elevated temperature, osmotic shock, and cytotoxic agents (Srivastava, 2002). During cellular necrosis, HSPs are released into the extracellular space where they activate the innate immune response by triggering TLRs (Vabulas et al., 2002). As HSPs are protein chaperones, they also actively participate in DC antigen processing and presentation to initiate adaptive immunity (Srivastava, 2002). Srivastava’s group showed that necrotic cells, but not apoptotic cells, release many HSPs such as HSP70, HSP90, calreticulin, and GP96 (Basu et al., 2000), which may activate immunity through the common receptor CD91 (Binder et al., 2000). In contrast, other HSPs, like the human heat shock protein 60, induce DC maturation and promote a Th1 phenotype via TLR4 (Flohe’ et al., 2003). Like HMGB1 and autoantibodies, HSPs may contribute to the development of autoimmunity not only as endogenous adjuvants but also as chaperones that physically deliver autoantigens to APCs (Biswas et al., 2006). This is the mechanism by which HSP70 promotes autoimmunity in a mouse model of diabetes (Liu et al., 2003; Millar et al., 2003). Furthermore, autoantibodies against HSPs are found in SLE patients and allelic variants of the Hsp70 genes are significantly associated with SLE (Furnrohr et al., 2010) and with several other autoimmune diseases such as MS (Ramachandran and Bell, 1995), Crohn’s disease (Debler et al., 2003), Grave’s disease (Ratanachaiyavong et al., 1991), and insulin-dependent diabetes mellitus (Pociot et al., 1993).

High mobility group box protein 1 (HMGB1) is a well-established endogenous danger signal (Lotze and Tracey, 2005), released by necrotic cells that have lost membrane integrity (Scaffidi et al., 2002) as well as secreted by phagocytic cells as a late mediator of inflammation (Gardella et al., 2002). HMGB1 normally has a nuclear localization and was originally described as a DNA binding protein (Javaherian et al., 1978; Bianchi et al., 1989), but a variety of other cellular functions have been attributed to HMGB1 largely depending on different post-translational modifications such as oxidation and hyperacetylation (Harris et al., 2012). Intracellular HMGB1 regulates gene transcription and autophagy (Tang et al., 2012). Extracellular HMGB1 acts as a classic DAMP when released by necrotic cells, or as a secondary danger signal when secreted by macrophages and DCs in response to LPS and TNF-α (Yang et al., 2005; Bianchi, 2007). Autocrine HMGB1 mediates DC activation and the ability to induce Th1 polarization (Dumitriu et al., 2005). The secretion of HMGB1 by monocytic cells is mediated by hyperacetylation of HMGB1, which localizes it to the cytoplasm where, upon a secondary signal, it is liberated into the extracellular compartment (Bonaldi et al., 2003).

HMGB1 is significantly increased in the serum of patients with brain and myocardial ischemia (Goldstein et al., 2006) and in septic patients in which it mediates the late phase of septic shock (Wang et al., 1999). HMGB1 is recognized by many receptors, like the receptor for advanced glycation end products (RAGE) (Kokkola et al., 2005) as well as by TLR2 and TLR4 (Park et al., 2004), CD24-Siglec10, and the recently reported TIM-3 (Chiba et al., 2012). When bound to the chemokine CXCL12, HMGB1 acts as a chemokine to recruit leukocytes to the site of inflammation by binding CXCR4 (Schiraldi et al., 2012). The shift from functioning as a chemokine to acting as a danger signal depends on its redox state: intracellular HMGB1 has three cysteines that are in a reduced state (all-thiol state); when it is secreted or released, the all-thiol HMGB1 can bind to CXCL12 and chemoattract unless two cysteines are oxidized, forming a disulfide bond and promoting HMGB1 danger signal functions (Yang et al., 2013). During apoptosis HMGB1 is kept intracellular, and uptake of apoptotic cells induces release of oxidized HMGB1 so that its pro-inflammatory activities are neutralized. Possible functions of this fully oxidized state are still under investigation (Yang et al., 2013).

Evidence suggests that HMGB1 participates in the pathogenesis of many autoimmune diseases like Rheumatoid Arthritis (RA), SLE, EAE, and diabetes (Zhang et al., 2009; Harris et al., 2012). For example, RA patients have increased levels of HMGB1 in the serum and synovial fluid that decreases upon therapy-induced amelioration of joint inflammation (Zetterstrom et al., 2008). Similarly, HMGB1 blockade ameliorates arthritis in animals, while its administration in the joints induces arthritis (Kokkola et al., 2003). In SLE patients, serum HMGB1 levels, as well as anti-HMGB1 autoantibody titers, positively correlate with disease activity (Jiang and Pisetsky, 2008; Abdulahad et al., 2011). HMGB1-containing nucleosomes from apoptotic cells have been shown to induce secretion of pro-inflammatory cytokines and expression of costimulatory molecules in macrophages and DCs and the administration of HMGB1-nucleosome complexes in mice induces lupus-like autoantibodies (Urbonaviciute et al., 2008; Urbonaviciute and Voll, 2011). In MS patients, extracellular HMGB1 is increased in the cerebrospinal fluid (CSF) and microglia and macrophages expressing cytosolic HMGB1 are increased in MS active lesions (Andersson et al., 2008). In mice, HMGB1 drives neuroinflammation in EAE and the inhibition of HMGB1 signaling with a neutralizing antibody ameliorates disease (Robinson et al., 2013). Clinical and experimental evidence also suggest a role for HMGB1 in the pathogenesis of type I diabetes. In diabetes-prone NOD mice, HMGB1 blockade significantly inhibited insulitis progression and delayed diabetes onset, decreased the number, and maturation of DCs in the pancreatic lymph nodes, and increased the number of regulatory T cells (Han et al., 2008). Once diabetes has developed, hyperglycemia can induce HMGB1 secretion (Yao and Brownlee, 2010; Dandona et al., 2013) and HMGB1 may contribute to the nephropathy and the vascular complications of diabetes by fueling inflammation and promoting tissue damage, especially in the kidney (Lin et al., 2012). Higher levels of serum HMGB1 were associated in type 1 diabetes patients with a higher risk of mortality, cardiovascular disease (Nin et al., 2012a), and kidney damage (Nin et al., 2012b).

In summary, HMGB1 is a powerful danger signal that plays a complex role in the pathogenesis of autoimmune diseases. Blockade of HMGB1 has thus far proven to be beneficial in ameliorating a number of types of experimental autoimmunity, making its therapeutic potential very promising. Further investigation is still needed to dissect the role of HMGB1 as an activator of DCs in terms of its multiple functions as a cytokine, chemokine, transcription regulator, and possible functions yet to be discovered.

Type I IFNs activate DCs in vitro and act as adjuvant in vivo (Gallucci et al., 1999). They are secreted by virally infected cells (Taniguchi and Takaoka, 2002) as a primary danger signal to alert both neighboring tissue cells and local immune cells to the presence of a viral infection. They are also secreted by immune cells as an amplifier of innate immunity (Liu, 2005). Type I IFNs (IFNs α and β) mediate the activation of DCs to promote adaptive immune responses such as cross-priming and isotype switching of responding murine B cells toward IgG2 (Theofilopoulos et al., 2005). Type I IFNs are particularly important in autoimmunity and especially in SLE in which an IFN Signature is present – an abnormally high expression of Type I IFN responsive genes in immune cells and tissues (Baechler et al., 2003; Bennett et al., 2003; Crow et al., 2003; Elkon and Stone, 2011). The hyper-activation of Type I IFNs may play a role in the early and most acute phases of disease because the IFN Signature is common in pediatric patients (Bennett et al., 2003) and in adults with central nervous system involvement and nephritis (Baechler et al., 2003). Supporting this notion, we have recently discovered that myeloid DCs and pDCs from lupus-prone Sle1,2,3 mice express an IFN Signature before the onset of autoimmunity. Even when generated in culture from bone marrow precursors, depleted of mature T and B cells, macrophages, and DCs, and thus, severed from the in vivo pro-autoimmune environment, DCs from Sle123 mice expressed an IFN Signature (Sriram et al., 2012). These results indicate that DCs are an independent cellular source of the pathogenic danger signals implicated in lupus (Elkon and Stone, 2011; Sriram et al., 2012). Since polymorphisms in genes that are part of the signaling pathway of Type I IFNs, such as IRF5, IRF7, and STAT4, are associated with a higher risk of developing SLE (Harley et al., 2009), the over-expression of Type I IFNs in these patients may be the result of the combination of genetic predisposition to produce high levels of Type I IFNs and exposure to danger signals that stimulate IFN production (Elkon and Stone, 2011; Niewold, 2011).

During tissue damage, not only are cells stressed and dying but components of the extracellular matrix (ECM) are disrupted leading to the production of low molecular weight degradation products such as hyaluronic acid (HA) and heparan sulfate, which have been shown to activate DCs and initiate inflammation (Termeer et al., 2002; Shirali and Goldstein, 2008; Brennan et al., 2012). Moreover, other components of the ECM such as fibrinogen, fibronectin extra-domain A (EDA), biglycan, and tenascin-C are up-regulated in response to tissue injury, and they all act as danger signals involved in the pathogenesis of RA (Goh and Midwood, 2011). Many of these molecules have been found increased in RA tissues; administration of danger signals, such as fibronectin EDA or tenascin-C, induces joint inflammation in vivo, while mice deficient in tenascin-C show a rapid resolution of inflammation (Goh and Midwood, 2011), indicating an important role for ECM molecules as initiator/amplifiers of the autoimmune process in RA.

The term nanoparticle may apply to almost any material with individual units at the nano scale. Nanomaterials can be generated from organic (e.g., liposomes, organic polymers) or inorganic materials (e.g., gold, silver) and engineered to display a molecule of interest or contain a therapeutic payload. Using nanoparticles and synthetic material systems, it may be possible to modulate the immune response through direct manipulation of DCs (Elamanchili et al., 2007).

The usefulness of pro-inflammatory nanoparticles has been demonstrated in a number of fields (Fadeel, 2012; Fadeel et al., 2012). For example, nanoparticle-based co-delivery of antigens and danger signals directly to DCs may be a promising strategy to direct anti-tumor immune responses (Kim and Mooney, 2011), as well as useful in next-generation vaccine adjuvants (Demento et al., 2009). However, because materials designed as next-generation therapies are sometimes capable of inducing DC-mediated inflammation similarly to PAMPs or DAMPs (Koike et al., 2008; Li et al., 2010), nanomaterials may raise concerns for safety (Dobrovolskaia and McNeil, 2007; Stern and McNeil, 2008). For example, nanoparticle debris from prosthetic alloys can activate the inflammasome (Caicedo et al., 2009), nanoparticles are known to modulate various forms of inflammatory cell death (Andón and Fadeel, 2013), and some nanomaterials are strong activators of complement (Salvador-Morales et al., 2006).

Additionally, it is now clear that nanoparticles do not stay “naked” when introduced in vivo – they become coated by biomolecules (e.g., proteins) to form what is termed a bio-corona (Fadeel, 2012). The composition and biophysical properties of the bio-corona is likely to have major implications for safety and effectiveness of future therapies (Aggarwal et al., 2009). The protein bio-corona first develops as a “soft” coat which eventually becomes a compact “hard” shell, becoming difficult to remove (Mohamed et al., 2012). Proteins within the hard coat are therefore aggregated and may become unfolded or modified (for example, citrullinated), possibly resulting in DCs recognizing altered self-antigen as danger signals, although evidence for this is still lacking (Fadeel, 2012).

The study of nanoparticle characteristics is also likely to uncover as yet unknown characteristics of danger signals. With the ability to control the characteristics and the delivery of the “danger signal,” we may better understand the immune response to those factors. For example, size of the nanoparticle seems to determine which DC populations interact with an injected particle: namely, larger particles require peripheral DCs for transport into the draining lymph nodes, whereas smaller particles act on lymph node resident cells directly (Manolova et al., 2008). Large particles also tend to be more inflammatory, with smaller particles often eliciting no reaction at all (Fadeel, 2012), possibly because larger sized particles more potently destabilize the phagolysosome.

Although most studies focus on the pro-inflammatory nature of particulate matter, some nanomaterials may suppress immune function (Mitchell et al., 2009; Zolnik et al., 2010) or can be engineered to evade the innate immune system (Moghimi, 2002). Indeed, gold has anti-inflammatory properties that have been exploited for many years in treatment for RA and gold nanoparticles are being engineered as novel treatments for a number of inflammatory diseases (Ulbrich and Lamprecht, 2010). Recently, Yeste et al. used gold nanoparticles to generate tolerogenic DCs in a murine model of EAE by delivering a tolerogenic compound in combination with oligodendrocyte antigen to DCs. These DCs in turn induced the generation of regulatory T cells which mitigated disease progression (Yeste et al., 2012).

Although gold may have some anti-inflammatory properties and may be useful as a delivery system, it also has many toxic side effects, earning itself a black box warning and precluding it as a practical therapy. Indeed, heavy metals can cause autoimmune-like syndromes (Schiraldi and Monestier, 2009) and therefore should be implemented cautiously as therapeutics. However, it is important to note that nanoparticles are being developed which are both biocompatible and biodegradable. For example, liposomes have been used as a delivery system to successfully treat experimental murine arthritis (Metselaar et al., 2003; Sethi et al., 2013), and biodegradable nanoparticles have been used to localize and target T cells (Fahmy et al., 2007). Nanoparticles coated with glycans have been used to target antigen to DCs (García-Vallejo et al., 2013), but the therapeutic use of DC-targeted nanoparticles in spontaneous autoimmunity remains largely unexplored (Ulbrich and Lamprecht, 2010). Nanomaterials therefore represent a promising strategy for next-generation therapy in the autoimmune diseases and an important area for future investigation to insure their safety and efficacy.

Physiologic pH is normally held within relatively narrow range (pH 7.35–7.45). During localized inflammation (e.g., infection, arthritic joint) or tissue ischemia (e.g., lupus nephritis, MS lesions) extracellular pH can reach pH values as low as 5.5 (Treuhaft and McCarty, 1971; Nedergaard et al., 1991; Simmen and Blaser, 1993).

A significant amount of research on cell functions in acidic conditions comes from the cancer field. Tumor cells tend to use glycolysis for energy generation even when there is adequate oxygen supply – the Warburg effect (Cardone et al., 2005). The inefficiency of glycolytic metabolism, the active secretion of acid, and the poor perfusion in solid tumors is thought to contribute to an acidic microenvironment. Acidic microenvironments increase the invasiveness and metastatic potential of some cancers as a result of remodeling of the ECM (Martinez-Zaguilan et al., 1996; Rofstad et al., 2006) and activation of inflammatory processes that have been shown to promote tumor survival (Grivennikov, 2013).

Several studies have indicated that acidity might act as a danger signal. For example, extracellular acidification can promote activation of the inflammasome and secretion of IL-1β by monocytes (Jancic et al., 2011) and seems to activate neutrophils through the activation of PI3K-Akt and ERK pathways (Trevani et al., 1999; Martinez et al., 2006). Additionally, non-immune cells may also produce inflammatory cytokines in response to extracellular acidification (Shime et al., 2008; Ichimonji et al., 2010) or may alter cell death programs (Jouan-Lanhouet et al., 2012).

Acidity in the extracellular microenvironment was initially proposed to inhibit DC differentiation (Gottfried et al., 2006), but some work suggests that DCs are instead activated by acidosis (Martinez et al., 2006). Other evidence has shown that acidic environments might improve antigen uptake and antigen presentation in DCs, although the mechanisms are still unclear (Vermeulen et al., 2004). Recently, acid-induced activation of DCs was found to be in part due to acid-sensing ion channels (Tong et al., 2011), but our understanding of DC responses to protons remains limited (Lardner, 2001). Low pH may also be sensed by proton-sensing G protein-coupled receptors (Seuwen et al., 2006), as seen in responses from synovial cells (Christensen et al., 2005; Tomura et al., 2005). Acidity is a common feature of inflammation found in many autoimmune diseases (e.g. within the arthritic joint, nephritic kidney, ischemic lesions of MS). However, to our knowledge, it is still unclear whether pathways triggered by acidity are involved in DC immune functions in the context of autoimmune diseases.

Cell volume expansion, as a result of extracellular hypotonicity, may also act as a danger signal. In response to hypotonic osmotic challenge lymphocytes first swell, then undergo a process termed regulatory volume decrease (RVD) (Garber and Cahalan, 1997; Cahalan et al., 2001). A reciprocal process occurs in hypertonic environments called regulatory volume increase (RVI). RVD is largely the result of the activity of potassium and chloride channels, with efflux of KCl giving way to water efflux as a result of decreased intracellular osmolarity. In some cases KCl efflux also leads to activation of other ion channels and cellular depolarization, thus having major implications in fundamental cellular processes like metabolism and cell death. Indeed, a similar process is seen during apoptosis. Apoptotic cell volume decrease seems to be due to the activation of the same potassium and chloride channels (Maeno et al., 2000, 2006). Cellular responses resulting from homeostatic signals are complicated by the observation that ion channels form complexes with integrins and may signal together (Arcangeli and Becchetti, 2006).

Earlier studies demonstrated that hypotonicity influence immune cell function, for example in neutrophil shedding of L-selectin (Kaba and Knauf, 2001). Mammalian target of rapamycin (mTOR), in conjunction with mitochondrial metabolism, may also be an important sensor for osmotic stress, as well as other homeostatic signals (Desai et al., 2002; Kwak et al., 2012). DCs are able to pinocytose, uptake fluids from the extracellular compartment as a way to sample for antigens and danger signals. This process is very efficient in immature/resting DCs which can uptake fluids up to one thousand fold their volume (Sallusto et al., 1995). This large flow of fluids requires a tight control of the DC volume, and therefore DCs express aquaporins for this purpose (de Baey and Lanzavecchia, 2000). Inhibition of aquaporins leads to sudden increase in cell volume, which may ultimately result in DC cell death. The idea that hypotonicity may be regarded as a danger signal has recently gained credence with the finding that hypotonic solutions activate the NLRP3 inflammasome in macrophages via the efflux of K⁺ and Cl⁻ ions and RVD (Compan et al., 2012). The role of alterations of cell volume in DC physiopathology remains largely unclear.

In contrast to hypotonicity, high salt concentrations do not seem to activate inflammasome activation, as hypertonic solutions are not able to induce caspase-1-dependent release of cytokines IL-18 and IL-1β (Compan et al., 2012). However, high salt concentrations may promote development of other pro-inflammatory conditions such as those associated with T helper 17 cells (Th17). Osmotic shock due to high salt was known to induce the production of IL-8 from human peripheral blood mononuclear cells (Shapiro and Dinarello, 1995), but recently two groups have demonstrated that high salt concentrations may promote pathogenic Th17 responses (Kleinewietfeld et al., 2013; Wu et al., 2013) and a high salt diet exacerbates EAE (Wu et al., 2013). These studies suggest that elevated salt concentrations, similar to levels found in tissues of animals on high salt diets, may exacerbate autoimmune reactions. It is still unclear how high salt concentrations affect DCs in vivo in the context of autoimmunity.

Decreased oxygen tension is another characteristic disturbance of inflamed or damaged tissue (Nizet and Johnson, 2009). Innate immune cells are thought to be better than adaptive immune cells at surviving in such low oxygen conditions (Sica et al., 2011), and a number of signaling pathways are relatively well known (Gloire et al., 2006; Semenza, 2009). In normoxic conditions, the α subunits of hypoxia-inducible factor (HIF), HIF-1α, and HIF-2α, are constantly produced and promptly degraded. Under hypoxic conditions, HIF-α accumulates, forms a dimer with the constitutively expressed HIF-1β subunit within the nucleus, and regulates transcription via hypoxia-responsive elements (HREs). Important gene targets are involved in adaptive response to hypoxia and include glycolytic enzymes, erythropoietin, and vascular endothelial growth factor.

A number of lines of evidence suggest that hypoxia acts as a homeostatic danger signal. Low oxygen levels leads to the generation of ROS, affecting both metabolism and mediators of inflammation. Furthermore, it is well established that HIF-1α signaling is closely tied to NF-kB signaling. In neutrophils, survival in hypoxic microenvironments, such as those found in infected tissue, is mediated by HIF-1α-dependent NF-kB activation (Walmsley et al., 2005). In a number of cell types, including DCs, hypoxia-induced HIF-1α prompts expression of TLR2 and TLR6 (Kuhlicke et al., 2007). Additionally, macrophages with a conditional deficiency in HIF-1α are compromised in their ability to clear bacteria (Cramer et al., 2003; Peyssonnaux et al., 2005), and TLR ligands like LPS may induce the HIF-NF-kB axis even under normoxic conditions (Spirig et al., 2010). Indeed, hypoxia-independent HIF induction may occur via other mechanisms, including in response to bacterial siderophores (Hartmann et al., 2008). Finally, hypoxia is also known to modulate cytokine production via HIF expression, inducing production of pro-inflammatory cytokines (Jeong et al., 2003; Nizet and Johnson, 2009) and enhancing the production of IFN-β in response to viral infection (Hwang et al., 2006).

Studies of DC responses to hypoxia are at times conflicting. It is unclear if hypoxia in itself activates DCs (Jantsch et al., 2008; Wang et al., 2010), and hypoxia may enhance (Jantsch et al., 2008) or inhibit (Mancino et al., 2008) LPS-induced costimulatory molecule expression. However, hypoxia does seem to augment DC production of pro-inflammatory cytokines, particularly in response to LPS (Jantsch et al., 2008; Mancino et al., 2008; Blengio et al., 2013). Similar activation has been demonstrated in human DCs associated with the inflamed joints of patients with juvenile idiopathic arthritis (Bosco et al., 2011).

HIF-1α expression is associated with a number of inflammatory and autoimmune diseases, including within the synovium of RA patients (Hollander et al., 2001) and in the muscle from patients with dermatomyositis and systemic sclerosis (Konttinen et al., 2004). HIF-1α is important for the development of B cells, with HIF-1α deficiency in chimeric mice leading to autoimmunity (Kojima et al., 2002). To our knowledge, the role of HIF signaling in DCs in the context of autoimmunity remains relatively unexplored.