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surface HSP molecules, which may activate APCs. Li et al. observed that physical contact of tumor cells

artificially engineered to express cell surface HSPs with immature DCs elicits a powerful maturation of

DCs.244

Other HSP Receptors

CD40 was reported as a receptor for Hsp70 when anti-CD40 antibodies were observed to inhibit

macrophage chemokine secretion in response to mycobacterial Hsp70. Subsequently, Becker noted the

association of recombinant GST-tagged CD40 with murine Hsp70.245,246 They further demonstrated

enhanced binding after the APCs were stimulated with LPS. As LPS induces expression of a number of

cell surface molecules, the authors concluded that CD40 mediates recognition and binding of Hsp70.247

CD36 has been implicated as a receptor for gp96. Transfection of CD36 into CD36-negative cells has

been shown to enhance gp96 binding. In addition, CD36−/− macrophages have a 52% reduction in gp96

binding compared with wild-type controls, suggesting that some role for CD36 as an Hsp receptor.247

LOX-1, a member of the same scavenger superfamily as CD91 and CD36 has been postulated to be an

additional receptor for endocytic uptake of Hsp70 and chaperoned peptides by human DCs. Anti-LOX-1

antibodies and LOX-1 ligand-acetylated albumin competed with Hsp70 for binding to DCs.114

Both TLRs and CD14 have been implicated as receptors for HSPs, such as Hsp70.248 Hsp70 stimulates

macrophage IL-12p40 production that is partially abrogated by inhibiting downstream TlR signaling

cascades. In addition, human Hsp60 activated NFκB and TNFα production by 293T cells transfected with

either TLR-2 or TLR-4 and MD-2.249 Hsp60 also activated JNK kinase and IKK kinase signaling and

TNFα production by macrophages, an effect that was partially eliminated by knocking out MyD88 or

TRAF6. Similar functional studies have proposed that CD14 might serve as an Hsp70 receptor in APCs.

All of these studies heavily suggest a role of TLRs or CD14 in HSP binding, although several

investigators call into question the potential for contamination with LPS to be mediating much of these

effects.247

AUTOPHAGY

Autophagy is an ancient cytoplasmic homeostatic process governing cellular biomass quantity, quality,

and distribution through the recycling of cytoplasmic proteins and organelles to support vital functions

during periods of stress (i.e., starvation).250,251 It literally originates from the Greek words auto

meaning “self” and phagein meaning “to eat.” Fundamental for embryological development and survival,

it integrates extensively with apoptosis and may itself function as a form of programmed (type II) cell

death.251–253 Hence, it is not surprising that it is preserved in all eukaryotic organisms, from yeast to

humans.

Macroautophagy is coordinated by specialized autophagy-related proteins (ATGs) that sequester large

protein aggregates, dysfunctional organelles, and even microorganisms and target them for lysosomal

degradation. In the setting of nutrient deprivation, the mechanisms orchestrating this dynamic process

involve the main growth rheostat mammalian target of rapamycin (mTOR) and adenosine

monophosphate (AMP) kinase, a sensor of cellular energy status.251,252,254–258 Prototypically, AMP

kinase, sensing reduced cellular energy (i.e., elevated AMP) inhibits mTOR, a negative regulator of

autophagy, thereby inducing autophagy.252,254,259,260

Autophagy is initiated by complex formation of ULK1 and ULK2, the class III phosphatidylinositol-3-

phosphate kinase VPS34, and ATG14-like protein, ATG14L. This initiates the ATG conjugation cascade in

which ATG5 and ATG12 complex with ATG16L1 to form an isolation membrane around the target (Fig.

7-12). The complex facilitates the addition of a phosphatidylethanolamine group to LC3-1 to form LC3-

II. LC3-II together with other factors leads to elongation and closure of the autophagosome, which is

then targeted for fusion with the lysosome, forming the autolysosome (Fig. 7-12).

Though the quintessential function of autophagy is to digest and recycle portions of the cytoplasm

during starvation, it has recently been described as an important effector arm of immune cells

responding to exogenous (i.e., PAMP: LPS)261,262 and endogenous (DAMP: HMGB1) stimuli.250,259,262–266

It is proposed that competition with microorganisms for nutrients, and thus nutrient deprivation, may

have served as a sign of invading organisms and thereby supported the emergence of eukaryotic

autophagic mechanisms of pathogen elimination. However, a host of other PAMPs and DAMPS and

mediators may induce autophagy. In Mφ, LPS induces autophagy through mechanisms dependent on

TLR4, TRIF, RIP1, and p38 MAPK.261,267 Cytosolic pathogens, such as viruses, can induce autophagy, or

rather xenophagy. DAMPS, such as DNA, ATP, and HMGB1 may also induce autophagy, the later

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through RAGE. The induction of autophagy by the inflammatory cytokine IL-1β is critical for control of

Mycobacterium tuberculosis. Similarly, TNFα stimulates autophagy in restricting intracellular bacteria. By

contrast, TH2 cytokines such as IL-4 and IL-13 and NO inhibit autophagy.

During innate immunity, autophagy has been shown to be important in the elimination of

intracellular microbes, through LC3-associated phagocytosis and xenophagy. This latter process of

selectively degrading intracellular pathogens involves sequestosome 1–like receptors (SLRs) that

recognize molecular motifs on invading pathogens. These SLRs help to clear microorganisms that gain

entry to the cytoplasm if they escape the defenses that are controlled by conventional PRRs. Microbial

polymers, such as DNA, that are present in the cytoplasm might function as autophagy-inducing PAMPS,

as in the case of M. tuberculosis infections. The critical importance of autophagy to cell survival is

perhaps best made evident by the defenses several microbes (Listeria, Shigella, Legionella) exhibit to

combat autophagy machinery.

Autophagy can assist PRR by delivering cytosolic PAMPs to endosomal TLR (i.e., TLR7) and stimulate

their activity.268–270 Nucleic acid receptors called RIG (retinoic acid inducible gene)-I-like receptors

(RLRs) recognize viral RNA, and induce the production of type I IFN to thwart viral invasion. The

interaction between TLRs and autophagy may also serve to augment antigen presentation by DCs.

Autophagy limits the activation of the inflammasome, a cytoplasmic complex that responds to PAMPs

and DAMPs by inducing the proteolytic processing and secretion of IL-1β and IL-18. It does so by

clearing the cytoplasm of debris, such as damaged or depolarized mitochondria that can function as

endogenous agonists. Inhibition of autophagy leads to the release of the products, such as mitochondrial

DNA and ROS.268–270 Autophagy also affects the secretion of pro- and anti-inflammatory mediators. In

senescent cells, it regulates the secretion of IL-6 and IL-8. It reduces immunoglobulin secretion from

plasma cells, and thus, may be important in negatively regulating antibody production.

During adaptive immunity, autophagy contributes to MHC class II presentation. Classically this is

described in the context of presenting cytoplasmic antigen (e.g., virus or self antigen) to endosomal

receptors, such as TLR7 (see above). Thus, autophagy is implicated in autoimmune disorders such as

autoimmune colitis and Crohn’s disease. The link between autophagy and inflammatory diseases has

also been reported. Polymorphisms in ATG16L1 and IRGM are linked to Crohn’s disease. IRGM

polymorphisms may be a risk factor for SLEs, and rheumatoid arthritis has been associated with

variations in the ATG5 gene. In summary, autophagy influences adaptive immune responses through its

effects of antigen presentation, naïve T cell–repertoire selection, T-cell homeostasis and TH-cell

polarization.268–270

Interestingly, nonimmune cells, such as hepatocytes and renal tubular cells also exhibit TLR signaling

and respond to the stress of sepsis or lipopolysaccharide by inducing autophagy.32,264,271–274 Studies

suggest that these mechanisms are similarly induced in vivo in the early response to sepsis, and regulate

bronchoalveolar cytokine/chemokine production and neutrophil accumulation. Others have shown that

sepsis induces similar AMPK- and mTOR-dependent mechanisms of autophagy in the Mϕ, kidney, and

liver.267,271,275–277 This mechanism may be critically important for cell survival, as it has been shown

that augmenting mTOR- or AMPK-dependent autophagy during CLP sepsis attenuates mitochondrial

injury and reduces acute kidney injury (AKI) (Fig. 7-6).274 In summary, recent data support a

fundamental role of autophagy in nearly every arm of immunity as an antimicrobial effector of

TLR.253,264,265,267,268

Mitophagy, autophagy directed at the removal of damaged mitochondria, is considered to of

particular importance in protecting against organ injury, such as AKI.278 At least two mechanisms are

involved in the sequestration and elimination of defective mitochondria: Atg7 and PINK1/Parkin. Atg7

is an autophagy protein that is required for the initiation of ubiquitin-like conjugation pathways of

autophagy. During erythroid maturation, Atg7 serves a nonredundant role in mitochondrial clearance

through canonical autophagosomal pathways.279,280 Alternatively, PINK1 accumulation at the outer

mitochondrial membrane of dysfunctional mitochondria selectively recruits Parkin, which in turn

promotes their selective degradation by mitophagy.281–284 Conditions that induce mitochondrial

dysfunction and depolarization strongly induce this pathway, which is important for cell survival.285

Indeed, endogenous Parkin production and exogenous Parkin overexpression have been found

cytoprotective in different conditions of stress, including sepsis. Not surprising, due to the potential for

release of cytochrome C and ROS, it is vital that a cell possess a programmatic mechanism for the

elimination of defective mitochondria.

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THE STRESS RESPONSE

The stress response is the cellular reaction to any perturbation or disruption in equilibrium and serves to

restore homeostasis. Inducers of the stress response include physical stresses (burns, radiation, trauma),

chemical agents and mediators (toxin, heavy metals, cytokines, ROS), infectious agents (bacteria,

viruses, parasites), and allergens. It is often referred to as the heat-shock response after the

identification in the 1960s of the HSPs, a group of genes expressed after exposure to heat.286 However,

subsequent investigations delineated additional cellular proteins that are expressed in response to a

wide variety of insults. Clinically, expression of HSP has been observed under conditions in which

oxygen delivery is compromised, as in hemorrhage or ischemia.287

Activation of the stress response is characterized by both morphologic and metabolic cellular

alterations. Morphologic alterations include the accumulation of unprocessed forms of mRNA in the

nucleolus, and increased numbers of actin microfilaments in the cytoplasm. Changes in cellular

metabolism include a rapid reduction in intracellular ATP levels, most likely correlated with alterations

in the integrity of mitochondria. The stress response is characterized by transient downregulation of

most cellular products and by the upregulation of stress proteins.288 It is the induction of stress proteins

that confers the primary adaptive and protective effects of the stress response.

After expression of stress genes, cells become resistant to subsequent stresses. Members of the stress

protein family include HO (see above); the multiple-drug resistance gene product P-glycoprotein;

ubiquitin, involved in targeting proteins for degradation; scavengers such as superoxide dismutase,

ferritin, and metallothioneins; and the glycolytic enzymes enolase and glyceraldehyde 3-phosphate

dehydrogenase. The most extensively characterized are the HSPs.39

HSPs are molecular chaperones that may either be constitutively expressed or induced upon cellular

stress.289 Classification is based upon their molecular mass and degree of homology. The most

extensively studied is the Hsp70 family, members of which possess a mean molecular mass of 70 kD and

greater than 70% homology. Members of the Hsp70 family bind ATP, and are induced under conditions

of energy depletion and stress. Hsp70 is integral in cellular adaptation to and survival during

environmental stresses. Both Hsp72 and Hsp73 are present in the cytosol and nucleus. The former is

constitutively expressed, whereas, expression of Hsp72 is exclusively induced. In most studies Hsp72 is

used as a marker of HSP induction.39,287,290,291

The Hsp60 family members are also referred to as chaperones. The glucose-regulated protein group of

HSP is induced with glucose starvation, inhibitors of N-glycosylation, and calcium ionophores. The

decrease in glucose content may affect the pool of sugar donors during protein glycosylation. The low–

molecular-weight HSP (molecular masses of 20 to 30 kD) may be important regulatory components of

the actin-based cytoskeleton.287

HSP regulation of transcription occurs through the activation of heat-shock elements in the gene

promoters. Two heat-shock transcriptional factors (HSF) have been identified, HSF1 and HSF2. HSF1

activates transcription of the Hsp72 gene in response to heat, heavy metals, and other inducers of the

stress response. With stimulation, unbound HSF1 oligomerizes, translocates to the nucleus, and binds to

the HSP promoter to activate the transcription of the gene. HSF2 is not activated by the classic inducers

of heat-shock genes, but may be important in controlling the activities of HSP gene expression in the

normal or unstressed cell.39,287,290,291

HSP can play multiple roles in modulating the inflammatory response. A number of conditions such as

rheumatoid arthritis, ARDs, and asthma have been shown to benefit experimentally from increased HSP

expression.286–288,291 Functions of HSP include enhancement of immune responses, thermotolerance,

regulation of apoptosis, hemostasis, and cytoprotection against ROS and other inflammatory mediators.

HSP-CD91 interaction is integral to the processing and representation of antigen by APCs. HSP may

shift the balance between TH1 and TH2 toward an increase in more anti-inflammatory TH2 cells. ROS,

including H2O2

, hydroxyl radical, and peroxynitrite, activate HSP synthesis. In the presence of iron, ROS

also induce the oxidation-specific stress proteins HO and ferritin, which afford protection against

oxidative stress by binding iron and preventing it from participating in the Fenton reaction. Mechanisms

of HSP-mediated cytoprotection from the toxic effects of ROS include the maintenance of cellular GSH

levels (Hsp27) and mitochondrial protection (Hsp70). Hence, ROS induce a cytoprotective response that

counteracts their own toxicity. Other inflammatory mediators such as NO have also been shown to

induce expression of HSP.39

HSP may participate in intracellular signaling pathways that modulate the production or function of

inflammatory mediators. For example, Hsp90 has been shown to facilitate signaling that leads to NO

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