The interplay between pathogen-associated and ...

Immunology and Cell Biology (2013) 91, 601–610 & 2013 Australasian Society for Immunology Inc. All rights reserved 0818-9641/13 www.nature.com/icb

REVIEW

The interplay between pathogen-associated and danger-associated molecular patterns: an inflammatory code in cancer? Monica Escamilla-Tilch1, Georgina Filio-Rodrı´guez1, Rosario Garcıá-Rocha1, Ismael Mancilla-Herrera1, Nicholas Avrion Mitchison2, Juan Alberto Ruiz-Pacheco1, Francisco Javier Sańchez-Garcıá1, Daniela Sandoval-Borrego1 and Ernesto Antonio Va´zquez-Sańchez1 There is increasing evidence of a close link between inflammation and cancer, and at the core of inflammation there are both pathogen-associated molecular patterns (PAMPs) and danger (or damage)-associated molecular patterns (DAMPs). Microorganisms harbor molecules structurally conserved within groups called PAMPs that are recognized by specific receptors present on immune cells, such as monocytes and dendritic cells (DCs); these are the pattern recognition receptors (PRRs). Activation through different PRRs leads to production of pro-inflammatory cytokines. A robust immune response also requires the presence of endogenous molecules that pose ‘danger’ to self-tissues and are produced by damaged or stressed cells; these are the DAMPs, which act also as inducers of inflammation. PAMPs and DAMPs are each recognized by a limited set of receptors that in number probably do not exceed 100. PAMPs and DAMPs interact with each other, and a single PRR can bind to a PAMP as well as a DAMP. Within this framework, we propose that PAMPs and DAMPs act in synchrony, modifying the activation threshold of one another. Thus, the range of PAMP–DAMP partnerships defines the course of inflammation, in a predictable manner, in an ‘inflammatory code’. The definition of relevant PAMP–DAMP complexes is important for the understanding of inflammatory disorders in general, and of cancer in particular. Here, we review relevant findings that support the notion of a PAMP–DAMP-based inflammatory code, with emphasis on cancer immunology and immunotherapy. Immunology and Cell Biology (2013) 91, 601–610; doi:10.1038/icb.2013.58; published online 8 October 2013 Keywords: cancer; DAMPs; inflammatory response; PAMPs; PRRs

CANCER AND INFLAMMATION Rudolph Virchow noted in 1863 that infectious diseases show signs of a ‘tumor process’, and that inflammatory cells, such as macrophages, are frequently present in tumor biopsies.1 More recently, it has been shown that inflammation and cancer share other common characteristics such as angiogenesis and infiltration with lymphocytes, macrophages and mast cells.2 Inflammation is associated with the various phases of tumor development, from susceptibility to initiation, progression, dissemination and perhaps resolution. In this regard, human malignancies are accompanied by sterile chronic inflammation, similar to the inflammation that follows viral, bacterial or parasite infections when the infective agent is no longer present.3 Furthermore, chronic and prolonged subclinical inflammation may increase the risk of cancer. Patients with ulcerative colitis or Crohn’s disease are at increased risk for colorectal adenocarcinoma.4 The risk

1Departamento

of esophageal cancer, pancreatic cancer and gallbladder cancer are increased in inflammatory diseases, such as esophagitis, Barrett’s metaplasia and chronic pancreatitis.5 Obesity-associated inflammation has also been related to cancer. Calle et al.6 found that the more obese members of a study cohort (with a body mass index 440 kg m !2) were 52–62% more likely to die of cancer than individuals of normal weight. The metabolic inflammatory state associated with obesity has been termed ‘metaflammation’, defined as a low-grade inflammatory response initiated by excess nutrients.7 Significantly from the present standpoint, obesity is associated with alterations in intestinal microbiota and therefore with the array of pathogen-associated molecular patterns (PAMPs).8 Intestinal microbiota and their host’s response mediate weight gain, insulin sensitivity and the inflammatory state of the gut as well as peripheral organs.7 An association between metaflammation and

de Inmunologıá, Escuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico Nacional, Carpio y Plan de Ayala, Col. Santo Toma´s, Me´xico D.F., Mexico and of Infection and Immunity, University College London, London, UK Correspondence: Professor NA Mitchison, Division of Infection and Immunity, Cruciform Building, University College London, Gower Street, London WC1 6BT, UK. E-mail: [email protected]. or Dr FJ Sańchez-Garcıá, Departamento de Inmunologıá, Escuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico Nacional, Carpio y Plan de Ayala, Col. Santo Toma´s, C.P. 11340, Me´xico D.F., Mexico. E-mail: [email protected] or [email protected] Received 13 May 2013; revised 2 September 2013; accepted 2 September 2013; published online 8 October 2013 2Division

Pathogen-associated and danger-associated molecular patterns M Escamilla-Tilch et al 602

PAMPs is evident in loss of Toll-like receptor-4 (TLR4) function studies in macrophages, which decreases obesity-induced inflammation and insulin resistance.9 The suggestion is that as obesity increases, tissues become less vascularized, and the cellular stress response and cell death release cytokines and excess fatty acids; in turn, these are sensed by Toll-like receptors (TLRs), inflammatory kinases (JNK, PKR or IKK) or other components of the inflammasome, and this initiates an inflammatory cascade.7 Inflammatory cells secrete, while necrotic cells passively release, a variety of danger (or damage)-associated molecular patterns (DAMPs) such as high mobility group box 1 (HMGB1), thus adding DAMPs to the overall course of inflammation and obesity. Several inflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-1b and CCL2 have been found in obese tissues.10 On the other hand, infectious agents such as human papilloma viruses, Hepatitis B and C viruses, Epstein–Barr virus, human immunodeficiency virus together with human herpes virus 8, human T-cell lymphotropic virus type I, liver flukes, Helicobacter pylori and schistosomes play a role in infection-attributable cancers.3 In addition, bacterial infections following surgical removal of primary tumors can promote metastatic growth.11 Hanahan and Weinberg systematically list the hallmarks of cancer. They suggest that the vast catalog of cancer cell phenotypes reflect six essential alterations in cell physiology, which collectively dictate malignant growth. They comprise self-sufficiency in growth signals, insensitivity to anti-growth signals, resisting cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis, plus two other ‘emerging hallmarks’, disregulation of cellular energetics and avoiding immune destruction. In addition, neoplasia facilitates the acquisition of further hallmarks: genome instability and mutation, and the tumor-promoting consequences of inflammation.12,13 Regarding self-sufficiency in growth signals, cancer cells often synthesize the growth factors to which they are themselves responsive (autocrine stimulation). Inflammatory cells associated with the tumor may also enhance tumor growth by producing cytokines and chemokines that act as survival and proliferation factors. This leads to increased tumor cell survival, motility and invasiveness.14 Another mechanism of self- sufficiency in growth signals is the disruption of negative-feedback pathways that attenuate proliferative signaling.12 An example is loss-of-function mutations in the tumor suppressor gene phosphatase and tensin homolog that lead to amplification of the PI3K-Akt signaling axis and consequent tumor genesis.12,14 Normally, active Akt regulates cell survival, cell proliferation and metabolism, in particular glucose metabolism, by phosphorylating substrates such as nuclear factor (NF)-kB and glycogen-synthasekinase 3. Pattern recognizing receptor (PRR) activation through interaction with PAMPs, DAMPs or PAMP–DAMP complexes leads to activation of NF-kB, a transcription factor associated with septic and aseptic inflammation that has emerged as a crucial tumor promoter.11,15,16 However, PAMPs and DAMPs may also activate the PI3K/Akt and Ras/MAPK pathways involved, respectively, in cell survival and mitogenic signaling, and there is recent evidence of cross-talk between the survival PI3K/Akt pathway and the mitogenic Ras/MAPK pathway. Positive influence of the PI3K pathway on the MAPK pathway is most effective at low concentrations of growth factors, whereas negative influence of the MAPK pathway on the PI3K pathway is mostly pronounced at high concentrations of growth factors. Combined inhibition of PI3K and MAPK proves more effective in suppressing cancer cell growth and viability than does targeting each pathway separately.16 Immunology and Cell Biology

Tumor cells have evolved a variety of mechanisms that help them evade apoptosis, a characteristic that was initially regarded as a hallmark of cancer.15 However, necrosis, another form of cell death, also occurs in tumors, where necrotic cells release pro-inflammatory signals such as DAMPs into the surrounding microenvironment; this recruits inflammatory cells, which in turn promote tumor growth by fostering angiogenesis, cancer cell proliferation and invasiveness.17 Radiotherapy and/or chemotherapy may induce yet another form of cell death called ‘immunogenic cell death’ and trigger the uptake of cancer cell antigens by dendritic cells (DCs), thus stimulating antigenspecific cytotoxic T lymphocytes and the production of tumorspecific antibodies in murine models of cancer. Key mediators of this process include HMGB1 and calreticulin.18 HMGB1 is a prototypic DAMP, and calreticulin is a Ca þ 2-binding lectin chaperone that resides mostly within the lumen of the endoplasmic reticulum and when prematurely exposed on the cell membrane of dying tumor cells, following radiation, forms a complex with ERp57, providing a ‘devour me’ signal that enhances their uptake by DCs. There is emerging evidence that chemoradiation could induce tumor antigen-specific T lymphocyte responses in a clinical setting and that this correlates with HMGB1 levels but not with calreticulin expression,18 showing a discrepancy between the murine model and clinical setting in terms of calreticulin, but highlighting the importance of HMGB1. In this regard, tumor antigen-specific cytotoxic T lymphocytes were found in 6 out of 16 esophageal squamous cell carcinoma patients receiving chemoradiotherapy and, furthermore, the level of HMGB1 after chemoradiation was higher in the patients with tumor antigen-specific cytotoxic T lymphocytes (CTLs) than in the patients without them.18 There is evidence of both autophagy-dependent cell survival under conditions of stress, and autophagy-dependent cell death or type II cell death on the grounds that cell death is preceded by massive autophagic vacuolization; autophagy is a process in which biomolecules and cell organelles are recycled, and its role in tumorigenesis is controversial.19 From an evolutive perspective, it is likely that this process was selected as a way to maintain cell homeostasis following starvation and later it helped to limit the release of DAMPs. It has been shown that phagocytosis of human cancer cells dying by autophagy activates the NLRP3 inflammasome, as well as maturation of IL-1b, in macrophages,20 and there is evidence that cancer cells that have died by autophagy are more immunogenic than their living counterparts20,21 and therefore are better inducers of inflammation.22 In a murine tumor model, oxaliplatina and mitoxantrona treatment induced autophagy in cancer cells and immunogenic cell death, which in turn activated the inflammasome and the release of ATP (a DAMP), besides the recruitment of DCs and T lymphocytes to the tumor.21,22 During cellular stress, autophagy is the main regulator of HMGB1 localization, whereas endogenous and exogenous HMGB1 regulate autophagy.23 Endogenous HMGB1 compete with Bcl-2 for binding to Beclin-1, thus regulating the formation of autophagosomes.24 In addition, HMGB1-mediated immune responses are dependent on its redox state.25 Binding of HMGB1 to RAGE (the receptor for advanced glycation end products) induces Beciclin-1-dependent autophagy as well as resistance to chemotherapy and ionizing radiation.26,27 On the other hand, oxidized HMGB1 increases the cytotoxic effect of chemotherapeutic agents as well as mitochondrialdependent apoptosis, thus suggesting that the redox state of HMGB1 modifies the tumor susceptibility of anti-cancer drugs.27 The interaction between HMGB1 and p53 in colorectal cancer regulates apoptosis and autophagy.28 Loss of p53 increases cytosolic


HMGB1 and therefore its binding to Beciclin-1 and autophagy, and in consequence diminishes apoptosis. On the other hand, loss of HMGB1 increases cytosolic p53 and apoptosis and diminishes autophagy.28 Chemotherapy of esophageal cancer induces cancer cells to undergo autophagy while inhibiting apoptosis. Once DAMPs are released by dying tumor cells, they can be degraded in the extracellular space by proteolitic enzymes, and DAMP residues may not be able to signal PRRs, lowering the inflammatory potential of immunogenic cell death. This could explain in part the lack of immune response against some cancer cells.29 Regarding angiogenesis, together with tissue invasion and metastasis as hallmarks of cancer, it is worth noting that HMGB1 is implicated in tumor formation, progression and metastasis. High levels of HMGB1 have been observed in solid tumors, including melanoma, colon cancer, prostate cancer, pancreatic cancer and breast cancer.30 In addition to stimulating inflammatory cells, HMGB1 also activate vascular endothelial cells promoting neovascularization.30,31 Cancer invasion may be facilitated by inflammatory cells that accumulate at the boundaries of tumors. These produce extracellular matrix-degrading enzymes and other factors that allow invasive growth.32 In addition to their role in tissue remodeling and tumor metastasis, extracellular matrix-degrading enzymes may also provide DAMPs in the tumor environment. The breakdown products of the extracellular matrix such as hyaluronic acid fragments function as DAMPs.33 Table 1 lists some PAMPs and DAMPs associated with specific types of cancer. In some instances, microorganisms such as Listeria monocytogenes and Mycobacterium tuberculosis associate with specific cancers; the associated PAMPs have still to be identified. These microorganisms are jointly listed here as PAMPs. THE PAMPS AND DAMPS CONCEPT Two theories underlie the molecular specificity of the innate immune response. In 1989, Charles Janeway Jr34 proposed that PAMPs are common to groups of pathogens and are recognized by specific receptors on innate immune cells (PRRs). In 1994, Polly Matzinger proposed the concept of the ‘danger signal’, pointing out that for an immune response to take place, in addition to an antigen (self or nonself), the cells engaged must also recognize endogenous molecular patterns associated with danger to self-tissue. The molecules involved were termed DAMPs.35 Support for these theories came from identification of the first mammalian PRR, the TLR4 that recognizes lipopolysaccharide (LPS), a molecule common to Gram-negative bacteria. In addition to TLRs, other PRR families now include NOD-like receptors, RIG-like receptors, C-type lectin receptors and absence in melanoma 2-like receptors, and their ligands (PAMPs) such as peptidoglycan and muramyl dipeptide, viral RNA, b-glucans, cytosolic bacteria and viral DNA have been identified. Equally, heat-shock proteins (hsp), uric acid, HMGB1, nucleic acids, heparan sulfate, ATP and other molecules have been identified as DAMPs, and some of their receptors (PRRs) have also been identified.36,37 DEFINING THE INFLAMMATORY RESPONSE BY THE INTERPLAY BETWEEN PAMPS AND DAMPS Based on current information, we suggest that PAMPs and DAMPs act in synchrony to regulate the inflammatory response and that the various PAMP–DAMP partnerships determine the course of inflammation, as defined by a PAMP–DAMP-mediated inflammatory code. The code would define inflammation in quantitative, qualitative and temporal terms. As new PAMP–DAMP partnerships are found and

Table 1 PAMPs and DAMPs associated with cancer PAMP

DAMP

EBV, HHV-4

Cancer association Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodkin’s lymphoma, immunosuppressive related whit Hodkin’s lymphoma, T cell lymphoma/NKL98 Hepatocellular carcinoma98 Leukemia and T-cell lymphoma98

HBV HTLV-1 High-risk HPV

HMBG1, S100A8/A9

Cancer of cervix, vagina, vulva, penis, anus, oral cavity and oropharynx tonsil98,100,111 Hepatocellular cancer and

HCV

non-Hodkin’s lymphoma98 KSHV

Kaposi’s sarcoma, Hodkin’s primary infection98

Propionibacterium acnes

S100A8/A9

Prostate100,111

HPV and EBV H. pylori

S100A8/A9

Breast cancer100,111,112 Gastric cancer113,114 Gastric cancer115

Listeria monocytogenes Candida spp. Helicobacter hepaticus

HMBG1

Oral cancer112 Liver cancer56,112

Chlamydia trachomatis EBV

Ovarian cancer112 Testicular cancer, nasopharyn-

Schistosoma spp.

geal cancer112 Bladder cancer112

Mycobacterium tuberculosis, Streptococcus

Lung cancer112

pneumoniae, Chlamydia pneumonidae CMV

Brain tumors112

In some instances, a microorganism along with a DAMP has been associated with a specific type of cancer. The identity of the corresponding PAMP(s) still awaits investigation. Meanwhile, for the sake of simplicity, the microorganism are listed as PAMPs.

the PAMP–DAMP-mediated inflammatory code grows, so will its usefulness increase. The following four recent discoveries seem particularly relevant. (1) PAMPs and DAMPs form sets that interact with one another: thus LPS from Gram-negative bacteria (a PAMP) can bind to any of the several different DAMPs, such as HMGB1, hsp70, hsp90, surfactant protein A and a-defensins. Similarly, lipoteichoic acid (a PAMP) can bind to plasma fibronectin (a DAMP).37 These interactions are expected to affect the activation threshold as well as the course of the inflammatory response. (2) DAMPs can act as PAMP transporters: thus HMGB1 protein binds to LPS through the lipid A fragment and transports the LPS to CD14 molecules on the cell membrane of monocytes. This in turn transfers the LPS to TLR4.38 In addition, HMGB1 may also bind single-stranded DNA (ssDNA), and the HMGB1–ssDNA complex can then be recognized by either TLR9 or RAGE (both are thus PRRs).39 The HMGB1–ssDNA complex activates monocytes through TLR9, an intracellular PRR that depends on CD14 for transfer of DNA from the inner cell microenvironment to the intracellular space. In this process, HMGB1 transports DNA to CD14 and CD14 to TLR9 for activation to take place.40 (3) PAMPs and DAMPs sometimes bind to the same receptors: thus TLRs, a group of PRRs originally described as PAMPs receptors, Immunology and Cell Biology


can also bind DAMPs,41 and DAMP-mediated activation through TLRs induces the synthesis of cytokines.42 Thus, the association between a given PAMP and a given DAMP quantitatively and qualitatively determines the inflammatory response. This PAMP– DAMP association constitutes an additional level of immune regulation that depends on the repertoire of PAMPs and DAMPs, the conformation of the PAMP–DAMP complexes and the diversity of the PAMPs’ and DAMPs’ interactions (one receptor in a PAMP–DAMP complex or two receptors acting at the same time, one for the PAMP and one for the DAMP, and so on). (4) PAMPs can induce the release of DAMPs: thus infection processes, such as infection of airway epithelial cells by respiratory syncytial virus, induce the release of hsp27, activating neutrophils via TLR4,43 and DCs infected with M. tuberculosis undergo caspaseindependent cell death that allows the release of inflammatory DAMPs, not impeded by caspase neutralization.44 Owing to similarities to IL-1a and HMGB1 in regard to constitutive nuclear expression and passive release, it has been suggested that IL-33 is a member of the alarmin (or DAMPs) family,45 and the active release of IL-33 by macrophages can be triggered by PAMPs, such as LPS.46 Taking all this into account, we assemble a tentative PAMP–DAMPbased inflammatory code. Table 2 shows, for instance, that the outcome of LPS (a PAMP) stimulation is qualitatively different whether LPS acts alone or in partnership with HMGB1 (a DAMP), in which case there is an enhanced production of TNF-a, IL-6, and IL-18.47,48 Inhibitory signals are also possible as a result of PAMP– DAMP interactions, as has been shown for the LPS–(PAMP)–hsp70 (DAMP) partnership, which inhibits LPS-induced responses.49 Table 2 includes only the 10 best-studied PAMPs and 15 DAMPs. As many as 150 different PAMP–DAMP partnerships would be possible, if only one PAMP and one DAMP configuration is considered in each case. Our literature search found 14 such partnerships. New PAMP– DAMPs interactions will surely be discovered in the near future,

making the range of these interactions wider than the current data would suggest (only about 10% of the potential partnerships). Our hope is that the ‘PAMP–DAMPs-mediated inflammatory code’ will contribute to a better understanding of the processes of inflammation, and that this may lead to new therapeutic approaches. Figure 1 depicts a PAMP or PAMP–DAMP-mediated configuration required for cell activation. When a different response mechanism operates, it is likely that functional differences could be involved right from the initial formation of a cluster. However, the effect of PAMPs or PAMP–DAMP partnerships on cluster size, duration, signaling and how all these tune the inflammatory response still awaits investigation. Some PAMP–DAMP complexes increase inflammation in the course of an infection process to an extent that induces pathological damage. Thus, administration of lethal doses of endotoxin activates biphasic responses of cytokines with early and late cytokine profiles. The classic pro-inflammatory cytokine response of TNF-a, IL-1b and IL-6 peaks in the first few hours, whereas release of HMGB1 peaks some 16–32 h after endotoxemia.50,51 The delayed release of HMGB1 is required for the lethal inflammation characteristic of endotoxemia. The administration of non-toxic quantities of HMGB1 along with low doses of LPS synergizes for the induction of lethal toxicity, and the administration of anti-HMGB1 antibodies to endotoxemic animals, a few hours after the peak of TNF-a, confers protection from lethality.52 The PAMP-carrier property of DAMPs and the role of DAMPs as enhancers or inhibitors of PAMPs signaling bear on the regulation of inflammatory responses triggered by infection, and how inflammatory processes not associated with infection result from illicit PAMP–DAMP partnerships. By definition, PAMPs are absent from non-infected tissues. However, it has been shown that intestinal microbe-derived LPS reaches serum concentrations as high as 1–10 pg/ml, under physiological conditions such as meat ingestion, and that asymptomatic and transitory bacteremia is common in healthy individuals.53 This suggests that PAMPs could be transitorily present

Table 2 PAMP–DAMPs-based inflammatory code PAMPs

HMGB1

1.

Differences in IL-6 production, enhanced production of TNF, IL-6, and IL-8 47, 48

DNA

2.

Enhances monocyte activation, promotes autoreactive B cell activation65, 66

Hsp60

3.

Synergistic activity to produce NO 103, 104

Hsp70

4.

Enhances the expression of MHC-I and increases the number of specific CD8 cells 105

Hsp90

5.

Hsp70 inhibits LPS-mediated response 49, 106

Gp96

6.

LL-37 inhibits LPS-mediated response107

LL-37

7.

Plasmacytoid dendritic cells sense self DNA coupled with antimicrobial peptides108

11 8

B-defensins-3

8.

LL-37 and cationic peptides enhance TLR-3 signaling by viral double-stranded RNAs 109

12

Fibronectin

9.

LL-37 and its truncated derivatives potentiate pro-inflammatory cytokine induction by lipoteichoic acid in whole blood 108

Hyaluronic acid

10. Fibronectin enhances in vitro LPS-priming110

Surfctantprotein A

11. Interaction between fibronectin and DNA95

Tenascin-C

12. Interaction between fibronectin and HIV-1 proteins 97

Oxidized LDL

13. LTA form Staphylococcus pyogenes binds to fibronectin 96

MRP8

14. Initiates activation of TLR4, enhanced expression of TNF-α 99

1 3 2

LPS

5

CpG DNA

4

RNA/DNA

6

PGN 10

Tri-acyl-lipoprotein Lipoarabinomannan Viral Glycoproteins Bacterial Toxins Zymosan Lipoteichoic acid

9

Effect

DAMPs

+

13 14

7

MRP14 The absence of lines between a PAMP and a DAMP imply potential PAMP-DAMP interactions that have not yet been experimentally proved

The intersections between 15 DAMPs and 10 PAMPs represent all the 150 potential interactions in a one PAMP plus one DAMP configuration. Annotated interactions (1–14) represent already experimentally identified interactions, and in some cases the immunological consequences of such interactions. The absence of lines between a PAMP and a DAMP imply potential PAMP–DAMP interactions that have not yet been experimentally proved.

Immunology and Cell Biology

Pathogen-associated and danger-associated molecular patterns M Escamilla-Tilch et al 605 PAMP

PAMP/DAMP TLR4

TLR4

RAGE

CD14

CD14

LOW

¿?

HIGH

¿?

MICRODOMAIN

CLUSTER

CLUSTER

DIFFERENT IMMUNE RESPONSE? Figure 1 PAMP- vs PAMP–DAMP-induced cell activation. Cell activation by a PAMP or by a PAMP–DAMP partnership uses a different set of receptors and may induce different immune responses by mechanisms that may include differences in receptor clustering at the cell membrane.

in the blood and thus could interact with DAMPs, triggering inflammation. In addition, the presence of normal microbiota, and therefore of PAMPs, in healthy mucosal tissues such as in the gut has to be taken into account.54 Tissue damage and cell death favor the release of cellular molecules to the extracellular milieu, some of which have been recognized as DAMPs. Their cellular origin includes plasma membrane, nuclei, endoplasmic reticulum, mitochondria and cytoplasm. Necrosis, apoptosis or netosis cell death, as well as increases in cell metabolism or DNA hypermethylation, induce the release of DAMPs.55,56 The association of PAMPs such as LPS with DAMPs such as HMGB1 increases the synthesis of pro-inflammatory cytokines such as IL-6 by peripheral blood mononuclear cells.42 There is evidence that TLRs engagement is one of the mechanisms by which immune cells are constantly activated and that this is at the basis of the onset of a wide array of inflammatory diseases such as lupus, atherosclerosis, asthma, type I diabetes, multiple sclerosis and rheumatoid arthritis. Interestingly, elevated concentrations of DAMPs have also been found in those patients,41 thus suggesting a PAMP– DAMP interplay. There is growing evidence on the association of some TLRs, PAMPs and DAMPs with cancer. For instance, H. pylori stimulates gastric epithelial cells through TLR2/TLR9, thus inducing a tumor-promoting inflammatory process57 and, in a murine model of breast cancer, TLR4 stimulation by LPS induces tumor metastasis by promoting angiogenesis.58 DAMP-mediated activation of TLRs induces inflammation-related and tissue-repairing gene expression, and DAMPs have also been implicated in settings in which excessive inflammation favors pathogenesis, as TLR activation by DAMPs initiates rounds of tissue damage and inflammation, leading to chronic inflammation associated with cancer and other diseases.37 TLR2 and TLR4 act synergistically in response to hsp70 in HEK293 cells, potentiating the production of IL-6 by the CD14-dependent MyD88/NF-kB activation pathway,59 two small hsp family members (aB crystallin, and hspB8) induce maturation of DCs and the production of cytokines through TLR4 engagement.

Peritoneal macrophages obtained from TLR4 !/ ! mice and stimulated with aB crystallin and hspB8 produce a lesser amount of IL-6 than wild-type mice.60 Likewise, HMGB1 interact with TLR4 and TLR2 generating an inflammatory response similar to that induced by LPS.61 We speculate that specific PAMP–DAMP partnerships determine the extent of synthesis of the cytokines that are associated with these physiological inflammatory diseases. HMBG1 can transport soluble CD14 monomers and also aggregates of LPS to membrane-bound CD14 and then to TLR4-MD2, thus leading to TNF-a production.38 On the other hand, HMGB1 acquires pro-inflammatory properties following interaction with mediators such as IL-1b, IFN-g and TNF-a, enhancing the synthesis of TNF-a and MIP-2.62 LPS is a potent inducer of dendritic cell maturation through the signaling of the TLR4/CD14/MD2 complex.63,64 Following our hypothesis, the interaction of LPS with a DAMP, before engagement with the TLR4/CD14/MD2 complex, would change the signal threshold required for dendritic cell maturation, with the consequences on both the innate and adaptive immune responses. HMGB1 is required for an inflammatory reaction to proceed, even in the absence of infection (sterile inflammation), such as in autoimmune arthritis, brain ischemia, haemorrhagic shock, hepatic necrosis and other conditions that involve inflammation and tissue damage,65 and it is an important factor for the formation of complexes that signal pro-inflammatory responses.55 There is evidence that HMGB1 participates in the triggering and resolution of inflammation, which follows infection or trauma. Moreover, some DAMPs, including HMGB1, promote muscular, nervous, and heart tissue regeneration through the recruitment of stem cells to the affected tissue.66 The presence of high concentrations of DAMPs is not always related to poor prognosis. A lower concentration of HMGB1 has been found in the sera of septic patients that later died, as compared with the levels found in septic patients that survived the infectious process.67 Immunology and Cell Biology


TLRs selectively recognize PAMPs but can also recognize DAMPs.41 The release of DAMPs alerts the immune system through their recognition by TLRs, triggering the synthesis of pro-inflammatory cytokines.42 In addition to the finding of PAMP–DAMP association, DAMPs have also been found to be associated with pro-inflammatory cytokines such as IL-1b, TNF-a and IFN-g and, in doing so, stimulate a stronger immune response.42 TLRs have a central role in the recognition of pathogens by the recognition of PAMPs. For instance, TLR4 recognizes LPS, TLR1 in combination with either TLR2 or TLR6 recognizes peptidoglycan, lipoarabinomannan, or lipoteichoic acid, and TLR5 recognizes flagellin, whereas TLR7, TLR8 and TLR9 recognize nucleic acids, and TLR2, TLR4 and TLR9 can also recognize HMGB1.36,37 In addition, PAMPs and DAMPs can be recognized by the same PRR, as gathered in more detail in other works, see Miyaji et al.68 For instance, the surfactant protein A (a DAMP) can diminish peptidoglycan- and zymosan-induced activation of NFkB and the secretion of TNF-a by binding to the extracellular domains of TLR2 in RAW 264.7 cells and alveolar macrophages.69,70 Also, hsp90 (a DAMP) has been implicated in the recognition of CpG DNA (a PAMP) by TLR9 (a PRR) and the binding of HMGB1 to nucleic acids is required for the efficient recognition of TLR3, TLR7 and TLR9.71,72 DAMPs may require different PAMP co-receptors and accessory molecules. For instance, a group of DAMPs requires CD14 as well as MD2; this includes hsp60, hsp70, bi-glycans and oxidized lipids.59,73 A second group of DAMPs only requires CD14 as an accessory molecule. This group includes surfactant proteins A and D and lactoferrin.74–76 A third group of DAMPs only requires MD2 for appropriate recognition by TLRs and includes Gp96 and HMGB1 that activate TLR2 and TLR4,61,77 whereas a fourth group of DAMPs use co-receptors and accessory molecules other than CD14 and MD2.78 Although there is some molecular machinery overlap, there are different outcomes whether TLRs are activated by PAMPs or by DAMPs,59 and perhaps this is also the case for PAMP–DAMP complex interactions with TLRs or other PRRs. The crystal structures for at least three PRR–ligand complexes have been solved, the three of them showing TLR–PAMP interactions. These studies suggest that even a minor modification in the ligand structure may generate different responses. In contrast, and as far as we know, no crystal structures of TLR–DAMPs have been solved.37 However, there is circumstantial evidence that some PAMPs and DAMPs may occupy the same or closely binding sites on TLRs and also that some DAMPs may utilize different binding sites. For instance, D299G and T399I mutations on TLR4 prevent activation by LPS while conferring enhanced activation by fibrinogen, and also there is evidence that DAMPs require different co-receptors and accessory molecules than PAMPs.37 So, on this basis it is still difficult to assess the affinity of PAMPs and DAMPs for TLRs, not to mention the affinity of these molecular patterns for other PRRs and the affinity of PAMPs-DAMPs complexes for PRRs. TLR signaling pathways induced by DAMPs in different cell types are poorly studied, but there is some evidence that DAMPs use distinct adaptor molecules and induce distinct signaling pathways downstream of TLRs than PAMPs. For instance, TLR4 activation by LPS induces TRIF and MyD88-dependent pathways, whereas tenascin-C signals via MyD8837 and although a detailed comparative analysis of the biological outcomes of TLRs and other PRRs signaling by PAMPs and DAMPs is still missing, some differences between host responses to PAMPs versus DAMPs have been identified. It has been shown, for instance, that HMGB1 and LPS induce distinct patterns of gene expression in neutrophils. For instance, there is an increase in Immunology and Cell Biology

the expression of the anti-apoptosis protein Bcl-xL when neutrophils are activated by HMGB1 but not by LPS, as well as a slower induction of TNF-a mRNA when cells are stimulated by LPS, as compared to HMGB1.37 For a discussion on a possible PAMP–DAMP-based inflammatory code it has to be taken into account that some DAMPs have been put into question. It has been suggested, for instance, that the immunostimulatory activity of hsps might be due to contamination with LPS on the grounds that purified hsp preparations are sometimes not immunostimulatory.79 However, as pointed out by Seong and Matzinger, the methods used for hsp purification can remove many aggregates and might easily remove the denatured/disregulated and aggregated hsps, leaving only the non-stimulatory water-soluble complexes that lack the exposed hydrophobic portions of those molecules or ‘hyppos’ that confer DAMPs properties.80 Also, the capacity of HMGB1 to trigger immune responses has been questioned on the grounds that recombinant HMGB1 failed to induce cytokine production in vitro. This lack of activity has been explained by the redox state of cysteine residues, as commercially available preparations of HMGB1 may contain reducing agents.81 In this regard, two new levels of immune regulation arise. One of these is the redox state of HMGB1 and perhaps of other DAMPs and PAMPs. Tissue injury in the absence of concomitant infection as well as some types of cancer induce an inflammatory state characterized by the recruitment of leukocytes, especially neutrophils and monocytes, and their activation to release pro-inflammatory cytokines. This type of inflammation is dubbed ‘sterile inflammation’, and has been associated with the release of HMGB1. Recent studies demonstrate that the chemoattractant and cytokine-stimulating activities of HMGB1 are mutually exclusive and depend on the redox state of three cysteins (Cys23, Cys45 and Cys106). Cys23 and Cys45 induce conformational changes in response to oxidative stress, whereas Cys106 is critical for HMGB1 translocation from the nuclei to the cytoplasm.82 The formation of a Cys23–Cys45 disulfide bond inhibits the chemoattractant function of HMGB1 but not its cytokineinducing activity, and when HMGB1 is terminally oxidized by reactive oxygen species both functions are lost.83 For this reason, HMGB1 has been proposed as a sensor of oxidative stress. On the other hand, the chemoattractant and cytokine-stimulating functions of HMGB1 require different receptors, that is, CXCR4 and TLR4, respectively. For recruitment of inflammatory cells, HMGB1 has to form a complex with CXCL12 and signal through CXCR4, and for cytokine release by macrophages, HMGB1 has to engage with TLR4,83–85 thus providing a link between redox balance, HMGB1 conformation, ability to interact with other biomolecules and receptors, and proinflammatory function. The other level of immune regulation arises from the fact that some DAMPs may bind to a sialoglycoprotein, and the DAMP– sialoglycoprotein complex can then bind to a sialic acid-binding Ig-like lectin or Siglec, attenuating DAMP-mediated inflammation.83 A subset of CD24 glycoforms has the appropriate forms of Siglec ligands, and CD24 bound to HMGB1 can associate with Siglec-10. The ligation of Siglecs by DAMP–CD24 complexes presumably prevents uncontrolled inflammation in the context of tissue damage by reducing NFkB activation.83 The proposal of a PAMP–DAMP–sialoglycoprotein–Siglec-based inflammatory code would complicate matters. Let us, for the time being, just consider a PAMP–DAMP-based inflammatory code. If our hypothesis is correct, the experimental exposure to different PAMP–DAMPs combinations in in vitro or in vivo models should differentially activate cell signaling pathways and induce different cell


responses in terms of gene expression profiles, proteomes, secretomes and so on. This information would provide a better definition of the PAMP–DAMP-based inflammatory code. IMPLICATIONS OF A PAMP–DAMP-BASED INFLAMMATORY CODE FOR CANCER IMMUNOTHERAPY The innate immune system, as distinct from acquired immunity, has long been recognized as providing scope for cancer immunotherapy and has now become a major focus of new developments in cancer research. NK cells are one of these systems’ oldest-established and most familiar components, where at present it is the TLRs that occupy center stage. These pattern-recognition receptors play an important role in host defense against pathogens by recognizing a wide variety of PAMPs and thus initiating the response of the innate immune system.85 PAMPs act as a double-edged sword: uncontrolled TLR signaling in cancer provides a microenvironment that enables tumor cells to proliferate, while at the same time enhancing the immune response.84 Therefore, fine-tuning of TLR signaling in immune-based anti-cancer intervention would be desirable. Since TLRs recognize and are signaled for by PAMPs, DAMPs and PAMP–DAMP complexes with differential functional outcomes, as mentioned in the previous sections, PAMP–DAMP configurations should have to be defined for appropriate immunotherapy. We suggest that systemizing incoming information in a PAMP–DAMP-based inflammatory code would be a suitable starting point to achieve this. For example, tumor antigen-loaded DC therapies are proving promising.86 In this regard, on 29 April 2010, Sipoleucel-T, a cellbased immunotherapy for prostate cancer, was approved by the FDAUSA (www.cancer.gov/cancertopics/druginfo/fda-sipuleucel-T) that takes advantage of in vitro differentiation of patient-derived monocytes into DCs and the up-take of prostate cancer-specific antigens, so that when these antigen-loaded DCs are injected back into the patient, antigen is efficiently presented to specific T lymphocytes. This type of therapy can be improved by the combined use of TLR agonists such as poly I:C87,88 DCs that play a key role in the induction and maintenance of effective immune responses, express multiple Siglecs, and tumor cell-derived mucins act as ligands for Siglec-9, downregulating the synthesis of IL-12 but not of IL-10 by these cells.89 In addition, dying tumor cells release DAMPs, such as HMGB1,90 and some ligands for Siglecs can also interact with DAMPs.83,91,92 Therefore, tumor cell-derived mucins and DAMPs, and perhaps mucin–DAMP complexes, regulate DCs activity. The exact mechanisms and timing of these interactions remain to be analyzed. On the other hand, death of cancer cells as a result of radiotherapy or chemotherapy causes the release of DAMPs, which augment the presentation of tumor antigens by DCs, and therefore anti-tumor immunity.93 Necrotic as well as apoptotic cells release HMGB1 (a DAMP) and, in vivo, HMGB1 transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines.90 In addition, recombinant human HMGB1 exerts strong cytotoxic effects on malignant tumor cells by a mechanism that involves recombinant human HMGB1 translocation to mitochondria, the formation of giant mitochondria and a rapid depletion of mitochondrial DNA, and thus it has been suggested that recombinant human HMGB1 may offer therapeutic applications for the treatment of patients with malignant brain tumors.93 Less is known about the effect of HMGB1 associated with either LPS or ssDNA (PAMPs) on this process. Continuous stimulation of NF-kB and STAT3 has been found in many tumors.1 NFkB-dependent pro-inflammatory cytokines in turn

upregulate the expression of DAMPs and RAGE, leading to a pathological cycle of inflammation, necrosis and tumor genesis.1 Perhaps, this process should be taken into account in vaccination against cancer strategies.94 The transcription factor NF-kB is at the crossroads of anti-cancer therapy, anti-apoptotic signaling and resistance to cancer treatment. NF-kB can be induced by irradiation and by chemotherapeutic agents such as etoposide, vinca alkaloids and taxol,95 and it has been shown that it suppresses the apoptotic potential of chemotherapy.96,97 Therefore, it has been suggested that activation of NF-kB in response to chemotherapy could protect cancer cells from apoptotic stress. On the other hand, the anti-cancer property of some chemotherapeutic drugs is associated with the activation of the tumor suppressor gene p53, which results in limited cell proliferation and mitochondrial-dependent apoptosis by activating p53-target gene products such as Bax and Bak, which are involved in the release of the apoptosome-forming cytochrome c.98,99 The NF-kB signaling pathway can also suppress mitochondrial-dependent apoptotic pathway through transcriptional induction of antiapoptotic genes, including Bcl-2 and subunit p65. NF-kB null MEF cells were found to be much more resistant to cell death than wild-type cells, thus suggesting that NF-kB plays a role in blocking the efficacy of chemotherapy with DNA-damaging agents.100 PAMPs have been used on their own in anti-tumor therapies long before the term PAMP was coined. In 1891, Coley treated an inoperable sarcoma by injecting a live culture of Staphylococcus pyogenes in the tumor of a man’s neck, which resulted in an infection and complete regression of the tumor.93 As pointed out by Ludgate, the infection caused by Staphylococcus causes cancer necrosis, thus releasing cancerspecific antigens as well as DAMPs that in turn regulate adaptive immune responses, particularly cytotoxic T lymphocytes that would be responsible for tumor regression and systemic abscopal (from Latin ab, position away from; and scopus, mark or target) response.93 As thus shown, cancer therapy based on PAMPs and DAMPs offers promise but should be taken with caution, since in addition to PAMPs DAMPs are also a double-edged sword: HMGB1 is implicated in the transformation of mesothelial cells and establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival.101 MicroRNAs have been implicated in the regulation of malignant progression of cancer, and the overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 has been associated with an increase in malignancy scores in primary cultures of excised papillary lesions and in established papillary cancer cell lines.102 Again, only a good definition of PAMP–DAMP interactions and functional outcomes (PAMP–DAMPs-based inflammatory code) could provide a basis for precise PAMP and DAMP-based therapy of cancer, and a PAMP–DAMP-based inflammatory code would also help diagnosis and prognosis. Here, we began the setting of that code based on available information (Table 2) and forecast the addition of new PAMP–DAMP partnerships still to be discovered. High levels of DAMPs are found in a number of inflammatory diseases including TLR activators such as hsp, HMGB1, fibrinogen and tenascin-C,37 and thus they can be used as disease biomarkers for diagnosis. Likewise, high levels of DAMPs such as HMGB1 have been found in the sera of cancer patients.18 However, at present, there is a paucity of information regarding PAMP–DAMPs complexes in the sera of cancer patients. We think that this information will refine the role of DAMPs as biomarkers in cancer. Levels of endogenous TLR activators are in some cases indicative of disease activity. Accordingly, elevated levels of extracellular HMGB1 specifically localize to active lesions of multiple sclerosis patients and Immunology and Cell Biology


correlate with active inflammation, and also the members of the S100 family of calcium-binding proteins are considered reliable biomarkers of inflammation in a wide array of diseases.59 Endogenous TLR activators are also overexpressed in tumor cells, for instance, fibrinogen in osteosarcoma and breast cancer; HMGB1 in breast, lung, colorectal and pancreatic cancer, as well as in melanoma; hsps in breast cancer and lung cancer and so on,37 thus helping prognosis. In addition, in esophageal squamous cell carcinoma, the presence of HMGB1 within the tumor microenvironment is significantly related to pre-operative chemoradiotherapy and the levels of HMGB1 positively correlate with survival. Interestingly, the amount of tumor-infiltrating cytotoxic T lymphocytes was comparatively higher in patients with high intratumoral levels of HMGB1. Therefore, it has been proposed that HMGB1-related immune responses after CRT may play a critical role in the clinical outcome of cancer patients.18 Paradoxically, it has also been shown that increased levels of HMGB1 could support tumor growth and invasiveness in malignant mesothelioma.18 Regarding treatment, pyruvate, stearoyl lysophosphatidylcholine and nicotine, for instance, have been shown to ameliorate experimental sepsis by preventing HMGB1 release, and the release of other DAMPs could also be affected by these compounds.37 Perhaps, in the near future the pharmacological inhibition or stimulation of specific DAMP release could be used along with anticancer drugs for the treatment of some types of cancer. To achieve this, we need to know exactly in which settings DAMPs are beneficial (it has been mentioned earlier that the effect of some anticancer drugs is boosted by the release of DAMPs, and also the direct effect of some DAMPs such as HMGB1 on cancer cells) or detrimental (inflammation-induced cancer), and also the consequences of having PAMP– DAMP complexes in the outcome of cancer treatments. In addition, for the cancer-specific antigen-based treatments, we should keep in mind the adjuvant properties of DAMPs,103 but also their potential to provoke pathological inflammation (high tissue concentrations) or tissue repair (low tissue concentrations).37 No doubt, as new PAMPs, DAMPs and PAMP–DAMP partnerships are revealed, molecules capable of specifically inhibiting or fostering PAMP–DAMPs interactions will be designed and will open new avenues for treatment of inflammatory disorders, cancer included.

6

7 8 9

10 11 12 13 14 15 16 17 18 19

20

21

22

23

24

25

26 27 28

CONFLICT OF INTEREST The authors declare no conflict of interest.

29

ACKNOWLEDGEMENTS

30

We thank the two anonymous reviewers for their insightful criticism that greatly contributed to improve this work. Authors other than Nicholas Avrion Mitchison and Francisco Javier Sańchez-Garcıá are recipients of Consejo Nacional de Ciencia y Tecnologıá (CONACYT) studentships. Francisco Javier Sańchez-Garcıá is a COFAA/EDI/SNI fellow.

31

32 33

34 1

2 3 4

5

Srikrishna G, Freeze HH. Endogenous damage-associated molecular pattern molecules at the crossroads of inflammation and cancer. Neoplasia 2009; 11: 615–628. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986; 315: 1650–1659. Trinchieri G. Cancer and inflammation: an old intuition with rapidly evolving new concepts. Annu Rev Immunol 2012; 30: 677–706. Triantafillidis JK, Nasioulas G, Kosmidis PA. Colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies. Anticancer Res 2009; 29: 2727–2737. Lu H, Ouyang W, Huang C. Inflammation, a key event in cancer development. Mol Cancer Res 2006; 4: 221–233.


35 36 37 38

39 40

Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 2003; 348: 1625–1638. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol 2011; 29: 415–445. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 2005; 102: 11070–11075. Saberi M, Woods NB, de Luca C, Schenk S, Lu JC, Bandyopadhyay G et al. Hematopoietic cell-specific deletion of toll-like receptor 4 ameliorates hepatic and adipose tissue insulin resistance in high-fat-fed mice. Cell Metab 2009; 10: 419–429. Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resistance. J Clin Invest 2006; 116: 1793–1801. Multhoff G, Molls M, Radons J. Chronic inflammation in cancer development. Front Immunol 2011; 2: 98. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57–70. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646–674. Jiang BH, Liu LZ. PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res 2009; 102: 19–65. Brazil DP, Yang ZZ, Hemmings BA. Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 2004; 29: 233–242. Whiteman EL, Cho H, Birnbaum MJ. Role of Akt/protein kinase B in metabolism. Trends Endocrinol Metab 2002; 13: 444–451. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140: 883–899. Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol 2008; 8: 279–289. Petrovski G, Zahuczky G, Katona K, Vereb G, Martinet W, Nemes Z et al. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Differ 2007; 14: 1117–1128. Petrovski G, Ayna G, Majai G, Hodrea J, Benko S, Madi A et al. Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1beta release in human macrophages. Autophagy 2011; 7: 321–330. Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science 2011; 334: 1573–1577. Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med 2009; 15: 1170–1178. Tang D, Kang R, Livesey KM, Zeh HJ 3rd, Lotze MT. High mobility group box 1 (HMGB1) activates an autophagic response to oxidative stress. Antioxid Redox Signal 2011; 15: 2185–2195. Tang D, Kang R, Cheh CW, Livesey KM, Liang X, Schapiro NE et al. HMGB1 release and redox regulates autophagy and apoptosis in cancer cells. Oncogene 2010; 29: 5299–5310. Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA. Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity 2008; 29: 21–32. Liu L, Yang M, Kang R, Wang Z, Zhao Y, Yu Y et al. HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells. Leukemia 2010; 25: 23–31. Tang D, Kang R, Livesey KM, Cheh CW, Farkas A, Loughran P et al. Endogenous HMGB1 regulates autophagy. J Cell Biol 2010; 190: 881–892. Livesey KM, Kang R, Vernon P, Buchser W, Loughran P, Watkins SC et al. p53/HMGB1 complexes regulate autophagy and apoptosis. Cancer Res 2012; 72: 1996–2005. Ivanov S, Dragoi AM, Wang X, Dallacosta C, Louten J, Musco G et al. A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA. Blood 2007; 110: 1970–1981. Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol 2010; 28: 367–388. Schlueter C, Weber H, Meyer B, Rogalla P, Roser K, Hauke S et al. Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule. Am J Pathol 2005; 166: 1259–1263. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell 2010; 141: 39–51. Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon JC, Gallo RL. Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J Biol Chem 2004; 279: 17079–17084. Janeway CA Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 1989; 54: 1–13. Matzinger P. An innate sense of danger. Semin Immunol 1998; 10: 399–415. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20: 197–216. Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signaling. Mediators Inflamm 2010; 2010: pii: 672395. Youn JH, Oh YJ, Kim ES, Choi JE, Shin JS. High mobility group box 1 protein binding to lipopolysaccharide facilitates transfer of lipopolysaccharide to CD14 and enhances lipopolysaccharide-mediated TNF-alpha production in human monocytes. J Immunol 2008; 180: 5067–5074. Bianchi ME. HMGB1 loves company. J Leukoc Biol 2009; 86: 573–576. Baumann CL, Aspalter IM, Sharif O, Pichlmair A, Bluml S, Grebien F et al. CD14 is a coreceptor of Toll-like receptors 7 and 9. J Exp Med 2010; 207: 2689–2701.

Pathogen-associated and danger-associated molecular patterns M Escamilla-Tilch et al 609 41 Drexler SK, Foxwell BM. The role of toll-like receptors in chronic inflammation. Int J Biochem Cell Biol 2010; 42: 506–518. 42 Hreggvidsdottir HS, Ostberg T, Wahamaa H, Schierbeck H, Aveberger AC, Klevenvall L et al. The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote inflammation. J Leukoc Biol 2009; 86: 655–662. 43 Wheeler DS, Chase MA, Senft AP, Poynter SE, Wong HR, Page K. Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4. Respir Res 2009; 10: 31. 44 Ryan RC, O’Sullivan MP, Keane J. Mycobacterium tuberculosis infection induces nonapoptotic cell death of human dendritic cells. BMC Microbiol 2011; 11: 237. 45 Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ’alarmin’? PLoS One 2008; 3: e3331. 46 Ohno T, Oboki K, Kajiwara N, Morii E, Aozasa K, Flavell RA et al. Caspase-1, caspase8, and calpain are dispensable for IL-33 release by macrophages. J Immunol 2009; 183: 7890–7897. 47 Hreggvidsdottir HS, Lundberg AM, Aveberger AC, Klevenvall L, Andersson U, Harris HE. High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor. Mol Med 2012; 18: 224–230. 48 Wahamaa H, Schierbeck H, Hreggvidsdottir HS, Palmblad K, Aveberger AC, Andersson U et al. High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts. Arthritis Res Ther 2011; 13: R136. 49 Dokladny K, Lobb R, Wharton W, Ma TY, Moseley PL. LPS-induced cytokine levels are repressed by elevated expression of HSP70 in rats: possible role of NF-kappaB. Cell Stress Chaperones 2009; 15: 153–163. 50 Kox M, de Kleijn S, Pompe JC, Ramakers BP, Netea MG, van der Hoeven JG et al. Differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia. Crit Care Med 2011; 39: 1866–1870. 51 Kokkola R, Sundberg E, Ulfgren AK, Palmblad K, Li J, Wang H et al. High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis. Arthritis Rheum 2002; 46: 2598–2603. 52 Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 1999; 285: 248–251. 53 Erridge C. Endogenous ligands of TLR2 and TLR4: agonists or assistants? J Leukoc Biol 2010; 87: 989–999. 54 Rosenstiel P. Stories of love and hate: innate immunity and host-microbe crosstalk in the intestine. Curr Opin Gastroenterol 2013; 29: 125–132. 55 Pisetsky DS, Gauley J, Ullal AJ. Microparticles as a source of extracellular DNA. Immunol Res 2011; 49: 227–234. 56 Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002; 418: 191–195. 57 Chang YJ, Wu MS, Lin JT, Sheu BS, Muta T, Inoue H et al. Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Src-dependent nuclear factor-kappaB activation. Mol Pharmacol 2004; 66: 1465–1477. 58 Harmey JH, Bucana CD, Lu W, Byrne AM, McDonnell S, Lynch C et al. Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion. Int J Cancer 2002; 101: 415–422. 59 Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE et al. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem 2002; 277: 15028–15034. 60 Roelofs MF, Boelens WC, Joosten LA, Abdollahi-Roodsaz S, Geurts J, Wunderink LU et al. Identification of small heat shock protein B8 (HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis. J Immunol 2006; 176: 7021–7027. 61 Park JS, Svetkauskaite D, He Q, Kim JY, Strassheim D, Ishizaka A et al. Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J Biol Chem 2004; 279: 7370–7377. 62 Sha Y, Zmijewski J, Xu Z, Abraham E. HMGB1 develops enhanced proinflammatory activity by binding to cytokines. J Immunol 2008; 180: 2531–2537. 63 Fenton MJ, Golenbock DT. LPS-binding proteins and receptors. J Leukoc Biol 1998; 64: 25–32. 64 Schuster JM, Nelson PS. Toll receptors: an expanding role in our understanding of human disease. J Leukoc Biol 2000; 67: 767–773. 65 Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005; 5: 331–342. 66 Pistoia V, Raffaghello L. Damage-associated molecular patterns (DAMPs) and mesenchymal stem cells: a matter of attraction and excitement. Eur J Immunol 2011; 41: 1828–1831. 67 Sunden-Cullberg J, Norrby-Teglund A, Rouhiainen A, Rauvala H, Herman G, Tracey KJ et al. Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 2005; 33: 564–573. 68 Miyaji EN, Carvalho E, Oliveira ML, Raw I, Ho PL. Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants. Braz J Med Biol Res 2011; 44: 500–513. 69 Murakami S, Iwaki D, Mitsuzawa H, Sano H, Takahashi H, Voelker DR et al. Surfactant protein A inhibits peptidoglycan-induced tumor necrosis factor-alpha secretion in U937 cells and alveolar macrophages by direct interaction with tolllike receptor 2. J Biol Chem 2002; 277: 6830–6837.

70 Sato M, Sano H, Iwaki D, Kudo K, Konishi M, Takahashi H et al. Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A. J Immunol 2003; 171: 417–425. 71 Bandholtz L, Guo Y, Palmberg C, Mattsson K, Ohlsson B, High A et al. Hsp90 binds CpG oligonucleotides directly: implications for hsp90 as a missing link in CpG signaling and recognition. Cell Mol Life Sci 2003; 60: 422–429. 72 Yanai H, Ban T, Wang Z, Choi MK, Kawamura T, Negishi H et al. HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 2009; 462: 99–103. 73 Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol 2000; 164: 13–17. 74 Curran CS, Demick KP, Mansfield JM. Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell Immunol 2006; 242: 23–30. 75 Guillot L, Balloy V, McCormack FX, Golenbock DT, Chignard M, Si-Tahar M. Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Tolllike receptor 4. J Immunol 2002; 168: 5989–5992. 76 Ohya M, Nishitani C, Sano H, Yamada C, Mitsuzawa H, Shimizu T et al. Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide. Biochemistry 2006; 45: 8657–8664. 77 Vabulas RM, Braedel S, Hilf N, Singh-Jasuja H, Herter S, Ahmad-Nejad P et al. The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J Biol Chem 2002; 277: 20847–20853. 78 Babelova A, Moreth K, Tsalastra-Greul W, Zeng-Brouwers J, Eickelberg O, Young MF et al. Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors. J Biol Chem 2009; 284: 24035–24048. 79 Seong SY, Matzinger P. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 2004; 4: 469–478. 80 Harris HE, Andersson U, Pisetsky DS. HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol 2012; 8: 195–202. 81 Hoppe G, Talcott KE, Bhattacharya SK, Crabb JW, Sears JE. Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp Cell Res 2006; 312: 3526–3538. 82 Tang D, Kang R, Zeh HJ 3rd, Lotze MT. High-mobility group box 1, oxidative stress, and disease. Antioxid Redox Signal 2011; 14: 1315–1335. 83 Pillai S, Netravali IA, Cariappa A, Mattoo H. Siglecs and immune regulation. Annu Rev Immunol 2012; 30: 357–392. 84 Chow MT, Moller A, Smyth MJ. Inflammation and immune surveillance in cancer. Semin Cancer Biol 2012; 22: 23–32. 85 Tang D, Kang R, Coyne CB, Zeh HJ, Lotze MT. PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol Rev 2012; 249: 158–175. 86 Schuler G, Schuler-Thurner B, Steinman RM. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 2003; 15: 138–147. 87 Galluzzi L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautes-Fridman C et al. Trial Watch: experimental Toll-like receptor agonists for cancer therapy. Oncoimmunology 2012; 1: 699–716. 88 Navabi H, Jasani B, Reece A, Clayton A, Tabi Z, Donninger C et al. A clinical grade poly I:C-analogue (Ampligen) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro. Vaccine 2009; 27: 107–115. 89 Ohta M, Ishida A, Toda M, Akita K, Inoue M, Yamashita K et al. Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9. Biochem Biophys Res Commun 2010; 402: 663–669. 90 Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep 2004; 5: 825–830. 91 Chen GY, Tang J, Zheng P, Liu Y. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science 2009; 323: 1722–1725. 92 Liu Y, Chen GY, Zheng P. CD24-Siglec G/10 discriminates danger- from pathogenassociated molecular patterns. Trends Immunol 2009; 30: 557–561. 93 Ludgate CM. Optimizing cancer treatments to induce an acute immune response: radiation Abscopal effects, PAMPs, and DAMPs. Clin Cancer Res 2012; 18: 4522–4525. 94 Alpizar YA, Chain B, Collins MK, Greenwood J, Katz D, Stauss HJ et al. Ten years of progress in vaccination against cancer: the need to counteract cancer evasion by dual targeting in future therapies. Cancer Immunol Immunother 2011; 60: 1127–1135. 95 Siddiqa A, Ayad SR, Dickinson P. The interaction between fibronectin and DNA. Anticancer Res 1989; 9: 373–381. 96 Nealon TJ, Beachey EH, Courtney HS, Simpson WA. Release of fibronectinlipoteichoic acid complexes from group A streptococci with penicillin. Infect Immun 1986; 51: 529–535. 97 Torre D, Pugliese A, Ferrario G, Marietti G, Forno B, Zeroli C. Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus. FEMS Immunol Med Microbiol 1994; 8: 127–131. 98 Sarid R, Gao SJ. Viruses and human cancer: from detection to causality. Cancer Lett 2010; 305: 218–227.


Pathogen-associated and danger-associated molecular patterns M Escamilla-Tilch et al 610 99 Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van Zoelen MA et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxininduced shock. Nat Med 2007; 13: 1042–1049. 100 Gebhardt C, Nemeth J, Angel P, Hess J. S100A8 and S100A9 in inflammation and cancer. Biochem Pharmacol 2006; 72: 1622–1631. 101 Jube S, Rivera ZS, Bianchi ME, Powers A, Wang E, Pagano I et al. Cancer cell secretion of the DAMP protein HMGB1 supports progression in malignant mesothelioma. Cancer Res 2012; 72: 3290–3301. 102 Mardente S, Mari E, Consorti F, Di Gioia C, Negri R, Etna M et al. HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells. Oncol Rep 2012; 28: 2285–2289. 103 Hung CF, Cheng WF, Hsu KF, Chai CY, He L, Ling M et al. Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 2001; 61: 3698–3703. 104 Gao JJ, Zuvanich EG, Xue Q, Horn DL, Silverstein R, Morrison DC. Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages. J Immunol 1999; 163: 4095–4099. 105 Panja S, Jana B, Aich P, Basu T. In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2 þ . Biopolymers 2008; 89: 606–613. 106 Nicholas SA, Coughlan K, Yasinska I, Lall GS, Gibbs BF, Calzolai L et al. Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions. Int J Biochem Cell Biol 2011; 43: 674–681.


107 Suphasiriroj W, Mikami M, Shimomura H, Sato S. Specificity of antimicrobial peptide LL-37 to neutralize periodontopathogenic lipopolysaccharide activity in human oral fibroblasts. J Periodontol 2013; 84: 256–264. 108 Gustafsson A, Sigel S, Ljunggren L. The antimicrobial peptide LL37 and its truncated derivatives potentiates proinflammatory cytokine induction by lipoteichoic acid in whole blood. Scand J Clin Lab Invest 2010; 70: 512–518. 109 Lai Y, Adhikarakunnathu S, Bhardwaj K, Ranjith-Kumar CT, Wen Y, Jordan JL et al. LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs. PLoS One 2011; 6: e26632. 110 Bortolussi R, Rajaraman K, Qing G, Rajaraman R. Fibronectin enhances in vitro ipopolysaccharide priming of polymorphonuclear leukocytes. Blood 1997; 89: 4182–4189. 111 Salama I, Malone PS, Mihaimeed F, Jones JL. A review of the S100 proteins in cancer. Eur J Surg Oncol 2008; 34: 357–364. 112 Kutikhin AG, Yuzhalin AE. Inherited variation in pattern recognition receptors and cancer: dangerous liaisons? Cancer Manag Res 2012; 4: 31–38. 113 Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420: 860–867. 114 Pinto-Santini D, Salama NR. The biology of Helicobacter pylori infection, a major risk factor for gastric adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2005; 14: 1853–1858. 115 Huang B, Zhao J, Shen S, Li H, He KL, Shen GX et al. Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling. Cancer Res 2007; 67: 4346–4352.