Immunology

1. Induced Cellular Innate Responses
• Microbes
– trigger their own uptake and killing by phagocytic cells
– In addition, induce a broad spectrum of cellular innate
immune responses by a wide variety of cell types
• Several families of pattern recognition receptors –PRRs–
present (other than those mediating phagocytosis) on these
immune cells play major role in innate immunity
• These PRRs bind to
– Pathogen associated molecular patterns –PAMPs– (on
pathogens)
– some endogenous (self) DAMPs also and
– trigger signal-transduction pathways that turn on expression
of genes with important functions in innate immunity

2. • Proteins encoded by these genes are antimicrobial
molecules such as
– antimicrobial peptides and interferons,
– chemokines and cytokines
• These proteins recruit and activate
– other cells,
– enzymes such as iNOS that generate antimicrobial
molecules, and
– proinflammatory mediators (i.e., components that
promote inflammation).

3. • Some PRRs are expressed on the plasma
membrane
• Other PRRs are actually found inside our cells,
(either in endosomes/ lysosomes or in the
cytosol).
• This ensures that the cell can recognize PAMPs
on both extracellular and intracellular
pathogens.
• DAMPs released by cell and tissue damage also
can be recognized by both cell surface and
intracellular PRRs.

4. • Many cell types in the body express these
PRRs:
– all types of myeloid white blood cells (monocytes,
macrophages, neutrophils, eosinophils, mast cells,
basophils, dendritic cells) and
– subsets of the three types of lymphocytes (B cells,
T cells, and NK cells)
– the skin, mucosal and glandular epithelial cells,
– vascular endothelial cells that line the blood
vessels, and
– fibroblasts and stromal support cells in various
tissues.

5. Cell wall components of Gram-negative and Gram-positive bacteria.

6. • There are four main families of mammalian PRRs that
activate signaling pathways leading to protective
responses.
1. Toll-like receptors (TLRs)
• the first family of PRRs to be discovered;
• best-characterized in terms of their structure, how
they bind PAMPs and activate cells, and the
extensive and varied set of innate immune
responses that they induce.
• recognize many types of pathogen molecules
• As of 2011, 13 TLRs that function as PRRs have
been identified in mice and humans
• TLRs 1-10 are conserved between mice and humans;
TLR10 is not functional in mice, TLRs 11-13 are
expressed in mice but not in humans.

7. • These TLRs can detect a wide variety of PAMPs from
bacteria, viruses, fungi, and parasites, as well as
DAMPs from damaged cells and tissues
• Each TLR has a distinct repertoire of specificities for
conserved PAMPs; the TLRs and some of their known
PAMP ligands are listed in Table 5-4.
• TLRs do not promote phagocytosis

9. • TLRs are membrane spanning proteins that share a
common structural element in their extracellular
region called leucine-rich repeats (LRRs);
• Multiple LRRs make up the horseshoe-shaped
extracellular ligand-binding domain of the TLR
polypeptide chain (Figure 5-11a).

10. FIGURE 5-11 Toll-like receptor (TLR) structure - Structure of a TLR polypeptide chain.
Each TLR polypeptide chain is made up of a ligand-binding exterior domain that contains
many leucine-rich repeats (LRRs, repeating segments of 24-29 amino acids containing the
sequence LxxLxLxx, where L is leucine and x is any amino acid), a membrane-spanning
domain (blue), and an interior Toll/IL-1R (TIR) domain, which interacts with the TIR
domains of other members of the TLR signal-transduction pathway. Two such polypeptide
chains pair to form Toll/IL-1R (TLR) dimers, the form that binds ligands.

11. Cellular location of TLRs. TLRs that interact with extracellular ligands reside in the plasma membrane;
TLRs that bind ligands generated from endocytosed microbes are localized to endosomes and/or
lysosomes. Upon ligand binding, the TLR4/4 dimer moves from the plasma membrane to the
endosomal/lysosomal compartment, where it can activate different signaling components.

12. 2. C-Type Lectin Receptors
– second family of cell surface PRRs
– activate innate and inflammatory responses
– CLRs are plasma membrane receptors expressed
variably on monocytes, macrophages, dendritic cells,
neutrophils, B cells, and T-cell subsets.
– CLRs bind carbohydrates on the surfaces of
extracellular pathogens; generally recognize
carbohydrate components of fungi, mycobacteria,
viruses, parasites, and some allergens (peanut and dust
mite proteins).
– Humans have at least 15 CLRs that function as PRRs
– CLRs have a variety of functions:
• some CLRs function as phagocytic receptors (see Table 5-3),
and
• all CLRs trigger signaling pathways that activate transcription
factors that induce effector gene expression.

14. 3. Retinoic Acid-Inducible Gene-I-Like Receptors
(RLRs)
– are soluble PRRs that
– reside in the cytosol of many cell types
– play critical roles as sensors of viral infection
– bind viral RNA in the cytosol of infected cells

15. 4. Nod-Like Receptors
– Final family of PRRs is the NLRs.
– large family of cytosolic proteins
– activated by a variety of intracellular PAMPs and
substances that alert cells to damage or danger
(DAMPs and other harmful substances).
– Play major roles in activating beneficial innate
immune and inflammatory responses;
– some NLRs also trigger inflammation that causes
extensive tissue damage and disease.

16. • The PRR-activated signaling pathways
– induce the transcription of genes that encode an
arsenal of proteins that help us to mount
protective responses.
– Some of the induced proteins are antimicrobial
and directly combat pathogens,
– While others serve key roles in activating and
enhancing innate and adaptive immune
responses.
– Some of the most common proteins and peptides that are secreted by
cells following PAMP activation of PRRs and that contribute to innate
and inflammatory responses are listed in Table 5-5.
PRR signaling induce Expression of innate immunity proteins

17. Antimicrobial Peptides
• Defensins and cathelicidins
– are important in barrier protection, such as on the skin and
the epithelial layers connected to the body’s openings (see
Table 5-2).
– Some cells and tissues constitutively (i.e., continually,
without activation) express these peptides.
– For example, α-defensins and some β-defensins are
constitutively expressed in human intestinal Paneth epithelial
cells
– some defensins and the cathelicidin LL-37 are constitutively
synthesized and packaged in the granules of neutrophils,
ready to kill phagocytosed bacteria, fungi, viruses, and
protozoan parasites.

18. • In histology, an intestinal gland (also crypt of
Lieberkühn and intestinal crypt) is a gland found
in the intestinal epithelium lining of the small
intestine and large intestine (colon). The glands
and intestinal villi are covered by epithelium,
which contains multiple types of cells:
enterocytes (absorbing water and electrolytes),
goblet cells (secreting mucus), enteroendocrine
cells (secreting hormones), cup cells, tuft cells,
and at the base of the gland, Paneth cells
(secreting anti-microbial peptides) and stem cells.

19. – In some other cell types, the expression of these
antimicrobial peptides is induced or enhanced by
signaling through PRRs, in particular TLRs and the
NLRs.
• such as mucosal and glandular epithelial cells, skin
keratinocytes, and NK cells
– Macrophages do not produce these antimicrobial
peptides following PRR activation; there is an
indirect pathway by which microbes induce
cathelicidin in macrophages, which then can help
the macrophages kill the pathogens.

21. Type I Interferons ( IFN-α,β)
• Another major class of antimicrobial proteins
transcriptionally induced directly by PRRs
• Their production generally activated by those cell surface
TLRs and intracellular TLRs, RLRs, and NLRs that recognize
viral nucleic acids and other components and activate the
transcription factors.
• One particular type of dendritic cell, called the
plasmacytoid dendritic cell (pDC) because of its shape, is
a particularly effective producer of Type I IFNs.
• key roles in controlling viral infections; other immune-
related activities: activate NK cells and regulate activities
of macrophages and T cells.

22. • Plasmacytoid dendritic cells (pDCs) are innate immune
cells that circulate in the blood and are found in peripheral
lymphoid organs.
• As components of the innate immune system, these cells
express intracellular Toll-like receptors 7 and 9 which
detect ssRNA and unmethylated CpG DNA sequences,
respectively.
• Upon stimulation and subsequent activation, these cells
produce large amounts (up to 1,000 times more than other
cell type) of type I interferon (mainly IFN-α(alpha) and IFN-
β (beta)), which are critical pleiotropic anti-viral compounds
mediating a wide range of effects.
• The number of circulating pDCs are found to be decreased
during chronic HIV infection as well as HCV (Hepatitis C
virus) infection.

23. Cytokines
• Several key cytokines are among the proteins transcriptionally
induced by PRR activation— not directly antimicrobial —which
activate and regulate a wide variety of cells and tissues
involved in innate, inflammatory, and adaptive responses.
• Cytokines function as the protein hormones of the immune
system, produced in response to stimuli and acting on a
variety of cellular targets.
• Three of the most important cytokines are IL-1, IL-6, and TNF-
α, the major proinflammatory cytokines that act locally on
blood vessels and other cells to increase vascular permeability
and help recruit and activate cells at sites of infection; they
also have systemic effects.
• IL-1, IL-6, and GM-CSF also feed back on bone marrow
hematopoiesis to enhance production of neutrophils and other
myeloid cells that will contribute to pathogen clearance.

24. • Cytokines are a broad and loose category of small proteins (~5–20 kDa)
that are important in cell signaling.
• Their release has an effect on the behavior of cells around them. It can be
said that cytokines are involved in autocrine signaling, paracrine
signaling and endocrine signaling as immunomodulating agent
• may include chemokines, interferons, interleukins, lymphokines,
and tumour necrosis factors but generally not hormones or growth
factors (despite some overlap in the terminology).
• Cytokines are produced by a broad range of cells, including immune cells
like macrophages, B lymphocytes, T lymphocytes and mast cells, as well
as endothelial cells, fibroblasts, and various stromal cells; a given cytokine
may be produced by more than one type of cell.
• They act through receptors, and are especially important in the immune
system; cytokines modulate the balance between humoral and cell-
based immune responses, and they regulate the maturation, growth, and
responsiveness of particular cell populations.
• Some cytokines enhance or inhibit the action of other cytokines in complex
ways.

25. • Interleukins are a group of cytokines (secreted proteins and signal
molecules) that were first seen to be expressed by white blood
cells (leukocytes).
• The function of the immune system depends in a large part on interleukins,
and rare deficiencies of a number of them have been described, all
featuring autoimmune diseases or immune deficiency.
• The majority of interleukins are synthesized by helper CD4 T lymphocytes,
as well as through monocytes, macrophages, and endothelial cells. They
promote the development and differentiation of T and B lymphocytes,
and hematopoietic cells.
• Tumor necrosis factor (TNF, tumor necrosis factor alpha, TNFα, cachexin,
or cachectin) is a cell signaling protein (cytokine) involved in
systemic inflammation and is one of the cytokines that make up the acute
phase reaction. It is produced chiefly by activated macrophages, although
it can be produced by many other cell types such as CD4+ lymphocytes, NK
cells, neutrophils, mast cells, eosinophils, and neurons.[5]
• The primary role of TNF is in the regulation of immune cells.
• Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known
as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein
secreted by macrophages, T cells, mast cells, natural killer cells, endothelial
cells and fibroblasts that functions as a cytokine.

26. • some TLRs in monocytes, macrophages, and dendritic cells
after activation also induce production of IL-12 and IL-18;
– these cytokines play key roles in inducing naïve helper T cells to
become TH1 cells, in particular by inducing production of IFN-γ.
– The hallmark cytokine of TH1 cells is IFN-γ, which stimulates cell-
mediated immunity and is an important macrophage-activating
cytokine. Hence IL-12 and IL-18 are also considered
proinflammatory.
• IL-10 is another important cytokine specifically induced by
some TLRs in macrophages, dendritic cells, other myeloid
cells, and subsets of T, B, and NK cells.
– IL-10 is anti-inflammatory, in that it inhibits macrophage
activation and the production of proinflammatory cytokines by
other myeloid cells. IL-10 levels increase over time and
contribute to controlling the extent of inflammation-caused
tissue damage.

27. Chemokines
• These small protein chemoattractants (agents that
induce cells to move toward higher concentrations of
the agent) recruit cells into, within, and out of tissues.
• Some chemokines are responsible for constitutive
(homeostatic) migration of white blood cells throughout
the body.
• Other chemokines, produced in response to PRR
activation, have key roles in the early stages of immune
and inflammatory responses
– they attract cells that contribute both to clearing the infection
or damage and to amplifying the response.

28. – The first chemokine to be cloned, IL-8 (also called CXCL8),
is produced in response to activation by PAMPs, DAMPs, or
some cytokines
– It leads to activation of a variety of cells at sites of
infection or tissue damage, including macrophages,
dendritic cells, epithelial cells, and vascular endothelial
cells.
– Its key roles occurs in the initial stages of infection or tissue
damage; it serves as a chemoattractant for neutrophils,
recruiting them to sites of infection.

29. – Other chemokines are specifically induced by PRR
activation of epithelial cells in certain mucosal tissues
and serve to recruit cells specifically to those sites, where
they generate immune responses appropriate for clearing
the invading pathogen.
– For example, B cells are recruited to the lamina propria,
the lymphocyte-rich tissue under the intestinal epithelium,
by two chemokines, CCL28 and CCL20, produced by
intestinal epithelial cells activated by PAMP binding to
their TLRs.
– These activated epithelial cells (as well as local dendritic
cells activated by PAMPs) also produce cytokines that
stimulate the B cells to produce IgA, the class of antibodies
most effective in protecting against mucosal infections .

30. ……….continued

31. ……continued

32. Enzymes: iNOS and COX2
• Other genes activated in many cell types by PRR activated
signaling pathways are those for two enzymes that contribute
importantly to the generation of antimicrobial and
proinflammatory mediators: inducible nitric oxide synthase
(iNOS) and cyclooxygenase 2 (COX2).
• The iNOS enzyme catalyzes an important step in the formation
of nitric oxide, which kills phagocytosed microbes (see Figure 5-
8).
• COX2, whose synthesis is induced by PRR activation in
monocytes, macrophages, neutrophils, and mast cells, is key to
converting the lipid intermediate arachidonic acid to
prostaglandins, potent proinflammatory mediators.

33. Figure 5-8 Generation of antimicrobial reactive oxygen and nitrogen species. In the
cytoplasm of neutrophils, macrophages, and dendritic cells, several enzymes,
including phagosome NADPH oxidase, transform molecular oxygen into highly reactive
oxygen species (ROS) that have antimicrobial activity. One of the products of this
pathway, superoxide anion, can interact with a reactive nitrogen species (RNS),
generated by inducible nitric oxide synthase (iNOS) to produce peroxynitrite, another
RNS. NO can also undergo oxidation to generate the RNS nitrogen dioxide.