## 2 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-18), 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). 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  
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].