1. Immunomodulatory Function of Extracellular Hsp60, Hsp70, and Hsp90

Hsp60, Hsp70, and Hsp90 interact with the immune system in many ways and thereby have an impact on neurodegenerative diseases. Extracellular Hsp60, Hsp70, and Hsp90 influence both the innate and the adaptive immune responses. Generally, extracellular Hsp–receptor interaction involves specific receptors expressed on macrophages and dendritic and microglia cells, including toll-like receptors (TLRs), scavenger receptors (SR), and other molecules [16]. For example, Hsp70 and Hsp90 can interact with the SR LOX-1 [17], and Hsp70 interacts also with multiple members of the SR family [18]. The SR are expressed on different types of cells and they are involved in the binding and internalization of stress proteins [18]. Extracellular Hsp60, Hsp70, and Hsp90 can modulate the innate immune response, causing the secretion of proinflammatory cytokines by APCs [19]. This interaction elicits a proinflammatory response that involves mainly nuclear factor-kappa B (NF-kB). These chaperones are endogenous ligands for TLRs, and by interacting also with CD14 molecules, they can induce the production of cytokines (e.g., interleukin 1 beta (IL-1β), IL-6, inducible isoform of nitric oxide synthase (iNOS)) [20,21]. TLR4 is a receptor expressed on the microglia plasma cell membrane with a key role in the generation of immune responses in the nervous system, responses that are implicated in the development of neurodegenerative disorders [22]. For instance, Hsp60 can mediate neuroinflammation through a MyD88-dependent pathway by interacting with TLR4 on the microglia surface [21] and by inducing the production of proinflammatory factors via microglial LOX-1 [23]. Intrathecal injection of Hsp60 lead to neurodegeneration and demyelination by the activation of TLR4-MyD88 signaling in microglial cells [24]. Hsp70 can interact with microglia, dendritic cells, and macrophages through TLR2 and TLR4, leading to proinflammatory NF-kB activation and its associated pathways [25]. Hsp90 interacts with an extensive list of key mediators involved in pathways regulating inflammatory and immune responses. For example, among the protein clients of Hsp90, there is the receptor-interacting protein (RIP) kinase, which is involved in the innate immune response and in the cell-death signaling pathway. [26] RIP, following TLR4 activation, induces the expression of proinflammatory cytokines by NF-kB signaling [27] (Figure 1).

Figure 1. Heat shock protein (Hsp)60, Hsp70, and Hsp90 modulate inflammatory reactions by interacting with factors involved in the regulation of innate and adaptive immune responses. Stressors can activate the immune system and, in turn, promote neurodegeneration by inducing Hsps in brain tissue as a mechanism of protection. Extracellular Hsp60, Hsp70, and Hsp90 interact with receptors present on the surface of cells of the neural tissue’s immune compartment (e.g., microglia) and elicit pro- or anti-inflammatory responses, depending on the local cellular status. The interaction of extracellular Hsp60, Hsp70, and Hsp90 with toll-like receptor (TLR)2/4 induces the activation of the nuclear factor-kappa B (NF-kB) inhibitor protein, which in turn triggers the activation of the NF-kB pathway, promoting an inflammatory response. Hsp60, Hsp70, and Hsp90 form complexes with antigens (represented by triangles) mediating their presentation via the CD91 cell surface receptor on antigen-presenting cells (APCs) [28]. Hsp90 plays a proinflammatory role through the interaction with its client proteins, such as members of the receptor-interacting protein (RIP) kinases and, thereby, activates the NF-kB pathway. Under physiological conditions, intracellular Hsp90, by blocking heat shock factor (HSF)1, prevents the transcription of Hsp genes, such as Hsp70, or other genes that code for anti-inflammatory molecules. The pharmacological inhibition of Hsp90 can lead to upregulation of the transcription of intracellular Hsp70 and of anti-inflammatory molecules by its release. Abbreviations: MMP, matrix metalloproteinase; TLR, toll-like receptor; IKK, inhibitor of κB kinase; RIP, receptor interaction protein; CD, cluster of differentiation; lkB, inhibitory subunit I kappa B-alpha; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; HSF1, heat shock factor 1; P, phosphate; Hsp, heat shock protein; iNOS, inducible isoform of nitric oxide synthase.

Extracellular Hsp60, Hsp70, and Hsp90 can also help antigen presentation in the adaptive immune responses by upregulating the expression of major histocompatibility complex (MHC) molecules and their load [29,30]. Extracellular Hsp70 and Hsp90 complexed with antigens elicit the responses of cluster of differentiation (CD)8+ or CD4+ T cells by adaptive receptors [16], while Hsp60 by itself can stimulate regulatory CD4+ and CD 25+ T cells (Tregs), leading to an immunosuppressive adaptive response without APC participation [16,31]. In addition, the chaperone–peptide complexes can also recognize the CD91 receptor of macrophages/dendritic cells and facilitate antigen presentation [28] (Figure 1). The activation of the adaptive response via Hsp70 might represent a negative reaction for the cell, but it could be considered advantageous for the development of immunological memory in preparation for rapid reaction against subsequent insults [32]. In contrast to the proinflammatory function of extracellular Hsp70, intracellular Hsp70 has an anti-inflammatory effect in the brain, especially when overexpressed following brain damage. Thus, Hsp70 can be anti-inflammatory because it can block the expression of proinflammatory molecules, such as matrix metalloproteinases [33], and it can also promote the reduction or the inhibition of NF-kB activity [34,35] (Figure 1). In addition, intracellular Hsp70 interferes also with genes involved in various neuronal pathways such as transmission of nerve impulses [36]. Therefore, extracellular Hsp70 could in principle have anti-inflammatory and neuroprotective effects similar to those of the intracellular counterpart [37]. Consequently, it is likely that an increase of intracellular Hsp70 will lead to an increase of functional extracellular Hsp70, contributing to the reduction of the inflammation associated with neurodegeneration. Pharmacological increase of the Hsp70 level in neurons and microglia by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) reduced the hemorrhagic volume in a mouse model of traumatic brain injury [38]. Likewise, 17-AAG inhibition of Hsp90 induced the expression of Hsp70 and Hsp60 [39]. Thus, it may be said that Hsp70, Hsp60, and Hsp90 promote inflammatory responses and, consequently, neuronal damage and are implicated in neuroinflammation and neurotoxicity. Cytosolic Hsp60 has been shown to directly interact with the inhibitor of κB kinase (IKK), promoting activation of NF-kB-dependent gene transcription by tumor necrosis factor-α (TNFα) [40] (Figure 1). Hsp90 can induce a proinflammatory response in different ways, for example, by sequestering the regulator transcriptional factor heat shock factor (HSF)1 and thereby inhibiting the expression of Hsps (e.g., Hsp70) or activating the NF-kB pathway through the activation of its protein clients RIP [41] (Figure 1). In view of these results, Hsp60 and Hsp90 modulators appear as potentially useful agents for controlling inflammation in the nervous system [42,43]. Currently, numerous compounds have been designed to inhibit Hsp90 activity, but few have been developed for Hsp60 [15]. Hsp90 inhibitors have been developed to directly act on the chaperone or on its client proteins. Some inhibitors block the Hsp90 folding activity linked to adenosine triphosphate (ATP)-dependent conformation changes [44], while others inactivate its client proteins via proteasomal degradation [45]. For example, geldanamycin induces the degradation of Hsp90 client proteins of the RIP family with the consequent inhibition of TNF-mediated IkB kinase and NF-kB activation [41]. Furthermore, Hsp90 forms a complex with HSF1, blocking its translocation to the nucleus and, thereby, impedes the upregulation of Hsp70 and other anti-inflammatory molecules [46] (Figure 1). Most of the compounds that inhibit Hsp70 function by targeting its ATP hydrolysis activity or specific cysteine residues [47].

2. Extracellular Hsp60, Hsp70, and Hsp90 in Acute Nervous System Injury and Chronic Neurodegenerative Diseases

Neurodegenerative diseases are accompanied by inflammatory responses aimed at eliminating dead and damaged neuronal cells to restore the compromised area to its normal status [7]. It should be borne in mind that while short-lived inflammatory responses generally have a beneficial effect, excessive and persistent release of inflammatory mediators can be harmful to brain tissue [9]. Moreover, prolonged activation of microglia and astrocytes could also lead to the alteration of their beneficial functions, which they display under normal conditions [48]. Therefore, it is not surprising that neuroinflammation contributes to CNS diseases [49]. Although different in their origins, many neurodegenerative conditions are characterized by shared cellular responses that promote the upregulation of molecular chaperones as the first line of defense against misfolded, dysfunctional, and aggregation-prone proteins [50]. There is increasing evidence for the release of Hsp60, Hsp70, and Hsp90 into the extracellular environment, with functions that are complementary or independent of those of their intracellular counterparts. Since these chaperones lack a secretion signal in their sequences, the mechanisms by which they are released are poorly understood. In vitro and in vivo studies with Hsp60 have unveiled secretion pathways, involving lipid rafts and exosomes, which would explain the presence of Hsp60 in extramitochondrial sites such as interstitial space, cellular membrane, and biological fluids [51]. Similarly, nontraditional secretion mechanisms participate in the membrane delivery and release of Hsp70, involving lipid rafts [52] and lysosomes [53], in line with its role as a lysosomal stabilizer [54]. Secretion of Hsp90 via exosomes depends on its ATPase function and on the open or closed conformational state of the Hsp90 dimer: the open state promotes Hsp90 release via exosomes, whereas the closed state blocks this process [55]. Different types of CNS cells, including neurons and glial cells, can release exosomes with their cargo of specific molecules that could affect the function of acceptor cells [56]. At the extracellular level, Hsp60 is known to contribute to neuroinflammation with possible negative implications: this chaperone is highly expressed in activated microglia, and when released extracellularly, it induces neuroinflammation with neuronal cell death [57]. For this reason, inhibition of Hsp60 expression and its release represents a possible therapeutic mechanism applicable to neurodegenerative diseases. The pro- and anti-inflammatory effects of extracellular Hsp60, Hsp70, and Hsp90 in AD, PD, ALS, HD, and MS are summarized in Table 1 and discussed in the following paragraphs.