Innate Immunity Innate immunity is an evolutionary ancient form of host defense that serves to limit infection from the 1st minutes to hours after exposure to microorganisms.

Innate Immunity Epithelial barriers Soluble molecules The major components of innate immunity are the epithelial barriers; soluble molecules (anti-microbial peptides, complement, cytokines, ab); phagocytes (macrophages and neutrophils); pattern-recognition receptors (TLRs, NLRs, scavenger receptors and C-type lectin receptors. Works in concert to form network of host defense mechanisms. Provides almost complete protection against almost all invading pathogens. NLRs – in cytosol nuclear-binding oligomerization domain – nfkb, caspases. Stress recognition. Recognize proteoglycans, nod2 – muramyl dipeptide. Scavenger receptors: recognize anionic polymers, acetylated low-density lipoproteins. Some recognize structures which are shielded by sialic acid on host cells. C type lectin domain type 9 – dying cells. Cross-presentation of necrotic cells. Pattern-recognition receptors: TLRs, NLRs, scavenger Rs, C-type lectin Rs Macrophages Neutrophils

Mannose-Binding Lectin (MBL) Circulating in blood C-type lectin, contains multiple CRDs (carbohydrate recognition domain); Member of collectin family of proteins, which contain both collagen-like and lectin (sugar-binding) domains. Other members of the family: surfactant proteins A and D in lung; Binds to glycans displayed on the surfaces of microorganisms (particular orientation of certain sugar residues and their spacing are important); Can initiate the lectin pathway of complement activation; Once formed, the MBL-pathogen complex is bound by phagocytes; Recognizes cryptic self-antigens, that are exposed by cell-death or reperfusion injury, or are modified by disease states. structurally heterogeneous proteins and several types of protein domains have been found to be involved in pattern recognition. One such protein domain is the carbohydraterecognition domain (CRD) in C-type lectin receptors [e.g. mannose receptor, dectin-1, DNGR-1, and mannose-binding lectin (MBL)], which is responsible for binding to polysaccharide (i.e. glycan) structures presented by pathogens. One key example of polysaccharide-recognizing protein in the innate immune system, and the focus of this review, is MBL.

Control of MBL Production Further Reading: Teillet et al. The Journal of Immunology, 2005, 174: 2870-2877 MBL is primarily synthesized in liver. Predominantly found in blood serum, can be detected in middle ear fluid, synovial fluid of inflamed joint, nasopharyngeal secretion, amnyotic fluid. Intracellular pools of MBL have been described: coat protein complex II vesicles, ER – protein quality control. Expression of functional MBL is largely genetically determined. 3 common polymorphisms are found in the codon 52, 54, 57, which cause amino acid replacements in the collagen domain, causing the alteration in oligomerization. These mutations are also called D, B, and C allele, respectively (Fig. 1). In addition, two common polymorphisms (H⁄ L and Y ⁄ X) identified in the upstream promoter region (Fig. 1) also determine serum levels by regulating protein expression. Different combinations of these polymorphisms result in a wide range of serum levels. MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit . tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for carbohydrates. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs .

MBL Structure and Its Selective Binding to Carbohydrates MBL binds to certain bacterial surfaces that display particular spatial arrangement of mannose or fucose residues, N-acetyl-D-glucosamine. But not galactose and sialic acid (marks animal surfaces). The presence is not enough, the correct spacing is important, that MBL is able to bind. High-avidity is provided by multiple CRDs.

MBL Interactions with Microorganisms MBL binds to wide range of microorganisms: Gram-positive, Gram-negative bacteria, fungi, viruses, protozoa. Ca2+ dependent binding.

MBL Interactions with Altered Self MBL binds to apoptotic and necrotic cells. Binds to stressed or damaged cells (after ischemia reperfusion injury). Binds to some transformed human tumor cell lines, that have abberant glycosylation patterns (colon adenocarcinoma, colorectal carcinoma). Binds galactose isoforms on antibodies in rheumatoid joints, polymeric IgA, and certain glycoforms of IgM. Dying cells might have altered pattern of exposed sugars, which may lead to MBL binding to them.

MBL and Complement Activation MBL is able to activate Complement by co-opting with MBL-associated serine proteases: MASP1, 2, 3 and sMAP/Map19. MASP 1 and 2 are encoded by different genes, whereas masp 3 and smap are splicing products of masp1 and masp 2 respectively. only masp 1 and 2 have serine protease domain. the other two might be the regulators of this pathway. in circulation MBL and MASP are present as complexes, binding between them occurs via collagen domain. Current understanding: MBL binding to microbial surfaces initiates auto-activation of MASP2. it is then able to cleave c2 and c4 to form c3 convertase. masp1 is able to cleave c2 and directly c3. MBL may lead to bacterial inactivation without activation of C3. involves phagocytosis.in contrast, ischemia rperfusion injury appears to be complement dependent.

MBL and Phagocytosis Opsonin-mediated Phagocytosis Ligand binds to Rs, changes in actin cytoskeleton, significant membrane deformation, extension of pseudopods. Once internalized phagosome binds to late phagosomes and lysosomes to become bacteriostatic phagolysosome. Depends on the cargo of the phagolysosome which scenario it undergoes. Particles can also bind indirectly to soluble PR molecules that function as opsonins to facilitate receptor-mediated engulfment. MBL is an example of pattern recognition molecules. It can facilitate particle uptake through direct opsonization as well as through complement activation. It is not clear how MBL can function as an opsonin in the absence of C3. Investigayors are trying to define the receptor for collectins‘ collagenous tails. Candidate: calreticulin in complex with CD91. what is surprising, calreticulin is known to be ER protein – acts as chaperone during protein assembly. It was shown that small amount of ER can be recruited to phagocytic cup, providing the mechanism for intracellular molecule to participate in particle and collectin recognition. NUmerous molecules were shown to increase binding of collectins: CD93/C1qRp, CD35/CR1. Phagocytosis without opsonization Opsonin-mediated Phagocytosis

Other Receptors for Inflammatory Signaling In addition to phagocytic receptors, the immune system also relies on canonical PRRs to trigger inflammatory responses. These are membranous TLRs and cytosolic NLRs (NOD1/2). None of them mediate microbial uptake by phagocytosis. 1.TLRs – transmembrane Rs, essential for detecting the pathogen. TLR signaling is induced by homo- or hetero-dimerization of the molecules after ligand encounter. They have extracellular leucine rich repeat region and intracellular domain which is shared with IL-1R, called Toll-IL-1R (TIR) domain. This domain interacts with downstream adapters including myeloid differentiation response gene 88 (MyD88), TIR domain containing adapter protein (TIRAP), TLR adapter molecule 2 (TRIF/TRAM) to initiate complex cascade of events. These events lead to activation of NfkB and interferon responsive genes leading to production of pro-inflammatory cytokines (pro-IL-1b, pro-IL-18 – substrates for caspases, activated by NLRs). 2. NLRs – located in cytosol. Divided into 2 classes: NODs which activate NFkB and NALPs which assemble a complex inflammasome, which activates caspases and cleavage of pro-inflammatory cytokines, eading to production of their active forms. Have 3 domains: N-terminus effector caspase-recruitment domain (CARD), nucleotide-binding domain (NBD) and c-terminus composed of leucine-rich repeats (LRR). Ligand stimulation recruits activation of RICK=RIP2 – activation of NFkB-IkB complex. Phagolysosome was also shown to activate inflammasome complex. Especially in case of leak in lysosome.

MBL and Phagosome-dependent TLR Signaling MBL can regulate proinflammatory cytokine release from phagocytes. Before, MBL was not appreciated in regulating PRR signaling. It was demonstrated that MBL cooperates with TLR2/TLR6 to increase NFkB activation and cytokine response to S. Aureus. It was not just due to increased bacterial uptake. MBL was found with TLR2 in a s.aureus recognition complex in phagosome. It was shown that LTA can block MBL binding to s.auerus, suggesting LTA being a very important ligand. MBL acts to increase delivery of LTA to TLR2/TLR6 in a manner similar to LPS-binding protein that delivers LPS to TLR4. MBL can also modify phagosome function. It was shown that it accelerates maturation of phagosomes containing N.meningitides. MBL opsonization also redirects S.aureus from tight to spacious phagosomes. From this we can say that MBL facilitates ligand liberation or R clustering to alter subsequent inflammatory signaling. The authors suggest that opsonins function not only to mediate uptake but also to coordinate ligand delivery and receptor engagement in the phagosome.

Physiological Role of MBL Early effect in mammals during lag period, that is required to develop an antibody response against infectious agents. MBL alone can account for susceptibility to infection. There’s no evolutionary advantage, nor disadvantage to different MBL alleles. MBL can act as a disease modifier in the context of other defects in innate immunity. Key role in containing and preventing the systemic spread of the bacterial infection. MBL contributes to the pool of genes that together comprise to the “innate immune haplotype”, that ultimately defines the immunological fitness of the host.

Summary MBL is a pattern-recognition molecule that contains CRD for glycan interaction and is capable of activating complement via lectin pathway. MBL is able to recognize carbohydrate structures from both infectious agents and altered self. MBL mediates defense against microbes via phagocytosis or extracellular complement mediated effector functions. MBL acts as a TLR2/6 co-receptor within the cell in directing intracellular signaling. Molecules such as MBL coordinate internalization, ligand delivery and ligation of pattern recognition receptors, which ultimately define the response of the phagocytic cell to the ingested organism.