TLR: Toll Like Receptor PAMPS: Pathogen Associated Molecular Patterns TIR domain: Toll/IL-1 Receptor TRIF: TIR–domain-containing adaptor inducing IFN-B factor MyD88: Myeloid differentiation factor 88 TIRAP: TIR domain containing adaptor protein TRAM: TRIF related adaptor molecule IRF: Interferon regulatory factor TRAF6: TNF receptor associated factor IRAKs: Interleukin-1 Receptor Associated Kinase

TLR signaling pathway

NON-TLR PRR families: 1. C-type lectin receptor (CLR). Plasma membrane receptors found on moncytes, macrophages, neutrophils, B and T cells. Humans have at least 15 CLRs. Examples of CLRs are Mannose receptor, Dectin 1 and DC-SIGN.

2. NOD-like receptors (NLRs). Large family of cytosolic proteins activated by intracellular PAMPS and host –derived danger signals (DAMPS).

3. Retinoic Acid-inducible gene-1 like Receptors (RLRs). Soluble receptors found in the cytosol of many cell types where they play a critical role in sensing viral infections. They are CARD-containing RNA helicases eg are RIG-1, MDA5 and LGP2.

CLR, RLR and NLR signaling pathway

Differential signaling through DC PRRs influences T cell function

•The NLRs recognize PAMPs, as well as host-derived danger signals (danger associated molecular patterns, DAMPs). • The NLRs assemble into high-molecular weight, caspase-1-activating platforms called ‘‘inflammasomes’’ that control maturation and secretion of interleukins such as IL-1b and IL-18, whose potent pro inflammatory activities direct host responses to infection and injury. • Distinguishing feature from TLRs: soluble cytoplasmic proteins

• The NLR family is characterized by the presence of a central nucleotide-binding and oligomerization (NACHT) domain, which is commonly flanked by C-terminal leucine-rich repeats (LRRs) and N-terminal caspase recruitment (CARD) or pyrin (PYD) domains. • LRRs are believed to function in ligand sensing and autoregulation • The CARD and PYD domains mediate homotypic protein- protein interactions for downstream signaling. • The NACHT domain, which is the only domain common to all NLR family members, enables activation of the signaling complex via ATP-dependent oligomerization.

From: The Inflammasomes: Guardians of the body: Annual review Immunology 27: 229,2009

Phylogenetic analysis of NLR family NACHT domains reveals 3 distinct subfamilies within the NLR family: • the NODs (NOD1-2, NOD3/ NLRC3, NOD4/NLRC5, NOD5/NLRX1, CIITA),

• the NLRPs (NLRP1-14, also called NALPs)

• and the IPAF subfamily, consisting of IPAF (NLRC4) and NAIP

Cell: 140:821, 2010.

PYD

The NLRP3 inflammasome and its activators

Models for NLRP3 inflammasome activation.

HMGB1 and RAGE The immune system has evolved to respond not only to pathogens, but also to endogenous danger signals released from dying cells (damage associate molecular patterns DAMPs). Cell death through necrosis induces inflammation, whereas apoptotic cell death provides an important signal for tolerance induction. • High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic DAMP molecule and has been implicated in several inflammatory disorders.

• Receptor for advanced glycation end products (RAGE) is a multiligand receptor binds advanced glycation end products (AGEs) but also binds structurally diverse molecules including HMGB1. RAGE activation has been implicated in sterile inflammation as well as in cancer, diabetes, and Alzheimer’s disease.

How cells die has profound effects on the immune system. When cells undergo necrosis, necrotic debris leads to inflammation and priming for adaptive T cell responses, whereas cell death by apoptosis leads primarily to tolerogenic responses.

DAMPS The recognition of necrotic cell death is mediated by DAMPs, a structurally and sequence-diverse family of endogenous molecules, generally intracellular, often released from necrotic cells, that activate the innate immune system. DAMP superfamily is growing, known members now include breakdown products of the extracellular matrix such as hyaluronan fragments, heat shock proteins (HSPs), S100 family of proteins (S100A8/9), fibrils of amyloid-β, uric acid , cytokines including IL-1α and IL-33, and nuclear-associated proteins such as HMGB1.

Liberation of the nuclear DNA-binding molecule high mobility group box 1 (HMGB1) may play a critical role in mediating the immune responses to damage. HMGB1 is a relatively small protein of 215 amino acid residues. Structurally, the protein is organized into three distinct domains: two tandem HMG box domains (A box and B box), which are spaced by a short flexible linker, and a 30 amino acid–long acidic C-terminal tail. As structural units, HMG boxes are well conserved throughout evolution. The A and B box domains of HMGB1 bind DNA.

HMGB1 and immune homeostasis during cell death.

(a) In necrotic cell death, molecules that include HMGB1 are released, associate with other molecules (including DNA or immune complexes), and activate plasmacytoid DCs, myeloid DCs, and macrophages to induce IFN-α and TNF-α, upregulate costimulatory molecules such as CD80 and CD86, and expand effector T cells. (b) In contrast, during apoptosis, through the generation of reactive oxygen species, molecules including HMGB1 are oxidized and deliver tolerogenic signals to dampen immune activation. ( c) If, however, professional phagocytes have an impaired ability to remove apoptotic cells, cells undergo secondary necrosis and release nucleosomes associated with HMGB1 that can induce inflammatory cytokine production from macrophages and potentially represent an alternative pathway to immune complex–mediated diseases (c).

HMGB1 can bind to DNA and RNA and signal through RAGE. HMGB1 can also bind to IL-1β and signal through the IL-1R/IL-1RAcP complex or associate with lipopolysaccharide and activate TLR4. When HMGB1 is associated with the nucleosome, released during secondary necrosis, TLR2 is preferentially engaged. HMGB1 has also been reported to bind to TREM-1 (triggering receptor expressed on myeloid cells-1). Thrombospondin and CD24 also bind to HMGB1 and may provide important negative regulatory signals to inhibit HMGB1-mediated coagulation and inflammation.

RAGE The receptor for advanced glycation end products was initially identified as a receptor for AGEs. Now known that this receptor has various binding partners. Rather than binding to a single specific ligand or even a group of closely related ligands, RAGE binds to several classes of molecules that lack sequence similarities. These ligands include HMGB1, several members of the calcium-binding S100 family of proteins, some species of AGEs, and β-sheet fibrillar material such as amyloid- β, serum amyloid A, immunoglobulin light chains, transthyretin (transports thyroxine and retinol) , and prions, among others. Consequently, RAGE can be considered a pattern-recognition receptor that binds predominately endogenous molecules that are either generated or released during cellular or physiological stresses

RAGE is a multiligand receptor and the prototypic DAMP receptor. RAGE can bind to structurally diverse molecules that include not only AGEs, but also DNA and RNA that bind in a sequence-independent manner, HMGB1, an array of S100 family member proteins, and fibrillar proteins that include amyloid-β. The association of ligands with RAGE results in a complex signal transduction cascade leading to cell activation.