Antibacterial responses

Question 1

Explain the mechanisms of antibacterial immunity from prior knowledge about bacterial pathogenesis

Answer 1

Innate Immunity Mechanisms:

1) Physical and Chemical Barriers:

• Skin serves as a physical barrier, while mucosal surfaces in respiratory, GI, and genitourinary tracts secrete mucus to trap bacteria
• Secretions such as saliva, tears and sweat contain antibacterial enzymes like lysozymes,
• Gastric juice with its low pH can destroy bacteria ingested in food
• The normal flora, non-pathogenic bacteria that colonise the body surface occupy niches to prevent pathogen invasion

2) Phagocytic Cells:

• Neutrophils and macrophages are the first cells to respond to a bacterial infection.
• They engulf and destroy bacteria. Upon phagocytosis, these cells form phagosomes (vesicles) which fuse with lysosomes containing hydrolytic enzymes leading to bacterial degradation
• Macrophages and dendritic cells play a key role in antigen presentation, they process bacterial antigens and present them on their surface using MHC molecules priming adaptive immune system
• Macrophages also produce pro-inflammatory cytokines

3) Natural Killer Cells:

• Recognise and kill cells infected with bacteria, especially those that escape intracellular destruction

4) Complement system:

• Series of proteins cascades in the blood and tissue fluids that can be activated to clear pathogens.
• Can directly kill bacteria through formation of the membrane attack complex (MAC) causing bacterial lysis, and it also helps to opsonise bacteria, making them easier for phagocytes and promoting inflammation

5) Inflammatory Responses:

• Tissue damage or infection triggers inflammation, characterised by redness, heat, swelling and pain
• The release of inflammatory mediators such as histamines, prostaglandins and cytokines increases blood flow and vascular permeability, allowing more immune cells and molecules to reach the site of infection

Adaptive Immunity Mechanisms

1) T-cells:

• Th1: They produce interferon-gamma, enhancing macrophage bactericidal activity and aiding in the defence against intracellular bacteria
• Th2: They produce IL-4, IL-5, and IL-13, playing a key role in the humoral immune response against extracellular bacteria
• Th17: produce IL-17, promoting neutrophil recruitment and activation, defending against extracellular bacteria
• Cytotoxic T cells: Destroy cells infected with intracellular bacteria

2) B Cells and Antibodies:

• Produce antibodies (immunoglobulins) upon activation by antigen and Th cells
• Neutralise bacteria, prevent them from adhering to cells, and opsonise them for phagocytosis. Also activate the complement system
• IgM: First antibody to be produced in response to infection, part of the BCR
• IgG: Most abundant, provides long-lasting immunity
• IgA: Found in mucosal areas, it protects against bacterial invasion
• IgE: allergic reactions and defence against parasites

3) Memory Cells:

• B and T cells that have responded to bacterial antigens differentiate into memory cells.
• These cells can recognise the same antigen and mount a faster and stronger immune response during subsequent infections, affinity maturation in the case of B cells

Question 2

Identify the main features of the immune responses against extracellular and intracellular bacteria

Answer 2

Vs Extracellular Bacteria:

1) Antibody production:

• Neutralisation: antibodies can bind and block toxins or adherence factors on the bacteria, preventing them from damaging host tissues or establishing an infection
• Opsonisation: The bound antibodies serve as tags that is recognised by phagocytic cells, which then engulf and destroy the bacteria
• Complement activation: The binding of antibodies to bacteria trigger the complement system, a series of protein reactions leading to the formation of a membrane attack complex (MAC) that can directly lyse bacteria

2) Complement system:

• Beyond being activated by antibodies, the complement system can also be activated directly by bacterial surface components in the alternative pathway.
• This cascade results in inflammation, opsonisation and direct bacterial lysis

3) Phagocytosis:

• Neutrophils and macrophages
• Recognition enhanced by opsonisation
• Once inside the phagocyte, bacteria are killed and digested in phagolysosomes

4) Inflammation:

• Damaged tissues and certain immune cells release inflammatory mediators that increase vascular permeability and recruit more immune cells to the site of infection

Vs Intracellular Bacteria:

1) Macrophage activation:

• When macrophages engulf bacteria, they process them and present the bacterial antigen via MHC class 2 molecules
• These antigen-MHC 2 complexes are recognised by Th1 cells, which release interferon-gamma (IFN-γ)
• IFN-γ is a potent activator of macrophages, increasing their ability to destroy the bacteria they have engulfed

2) Cytotoxic T-cell response:

• In cases where intracellular bacteria infect non-phagocytic cells, these cells present bacterial antigens on their surface using MHC class 1 molecules
• Cytotoxic T cells recognise these antigens and release cytotoxic molecules that induce apoptosis
• Granule-mediated = perforin and granzyme B
• Death receptor-mediated = TRAIL and Fas ligand (FasL)

3) Granuloma formation

• In response to certain persistent intracellular bacterial infections (such as tuberculosis), the immune system can form granulomas
• Granulomas are tightly organised clusters of immune cells (primarily macrophages) that serve to contain and isolate the bacteria, preventing their spread

4) Interferon response:

• Type 1 interferons (IFN-α and IFN-β) are produced by many cell types in response to infection and promote cell resistance to infection, activating NK cells, and macrophages and shaping adaptive immune responses

5) Antibody production:

• Bind to bacteria as they exit cells to prevent their spread and opsonisation

Question 3

Describe and explain the role of complement in antibacterial immunity

Answer 3

1) Complement Activation Pathways:

• The classical pathway is initiated by the binding of C1q to antibody-antigen complexes. This triggers a cascade involving C2 and C4, leading to the formation of the C3 convertase (C4b2a), which cleaves C3 into C3a and C3b
• The lectin pathway is similar but is initiated by the binding of mannose-binding lectin (MBL) or ficolins to specific carbohydrate patterns present on microbial surfaces
• The alternative pathway is initiated by the spontaneous hydrolysis of C3, allowing it to bind factor B, which is then cleaved by Factor D. The resulting complex (C3bBb) is a C3 convertase, which cleaves more C3
• C3b is a major opsonin which enhances phagocytosis. It binds covalently to microbial surfaces, marking them for recognition by phagocytes
• These phagocytes express complement receptors (CR1) which recognise C3b-oponised targets

2) Formation of Membrane Attack Complex (MAC):

• Directly kill certain bacteria through the formation of MAC
• MAC is a pore-forming structure made up of complement proteins C5b, C6, C7, C8 and multiple C9
• This complex inserts into the bacterial cell membrane, creating pores in the cell wall and causing cell lysis. Important against gram-negative bacteria

3) Chemotaxis and Anaphylatoxins:

• Cleavage of C3, C4 and C5 during complement activation generates small fragments (C3a, C4a and C5a) known as anaphylatoxins that have powerful pro-inflmmatory effects. C5a being the most potent
• Chemotaxis: C5a attracts neutrophils, monocytes and other immune cells to the site of inflammation
• Increased vascular permeability: C5a acts on the endothelial cells of blood vessels to increase their permeability, allowing more
• Attracting immune cells like neutrophils and monocytes to the infection site, facilitating the inflammatory response
• Activation of leukocytes: C5a can act on neutrophils and monocytes to increase their phagocytic activity.

4) Linking Innate and Adaptive Immunity

• Complement helps bridge innate and adaptive immunity
• C3d, a cleavage product of C3b, bind to complement receptor 2 (CR2) on B cells
• When a BCR and CR2 recognise their respective ligands (antigen and C3d) on the same complex, it greatly enhances B cell activation
• Ensures that antibodies are produced against antigens that have triggered an innate immune responses, avoid unnecessary immune reactions

Question 4

Differentiate between recognition of pathogens that leads to inflammation and recognition leading to phagocytosis

Answer 4

Recognition leading to Inflammation:

• When innate immune cells, such as macrophages and dendritic cells, recognise pathogens, they often respond by initiating an inflammatory response
• This recognition involves the binding of a pattern recognition receptor (PRR), such as a Toll-like receptor on the immune cell to a pathogen-associated molecular pattern (PAMP) on the pathogen
• This interaction triggers a signalling cascade within the immune cell that activates the transcription factor nuclear factor-kappa B (NF-kB)
• NF-kB then enters the nucleus, where it prompts the transcription of pro-inflammatory genes
• These genes encode proteins such as cytokines (IL-1, IL-6 and TNF-ɑ), chemokines and other signalling molecules
• These factors increase vascular permeability, recruit additional immune cells to the site of infection, and stimulate those cells to combat infection

Recognition leading to Phagocytosis:

• The process begins when a pathogen is recognised, either directly via binding to a PRR or indirectly through opsonisation
• In opsonisation, the pathogen is coated in opsonins (antibodies and components of the complement system).
• When a pathogen-specific antibody binds to a pathogen, the other end of the antibody - the Fc region - can bind to an Fc receptor on a phagocytic cell promoting phagocytosis
• Similarly, the complement component C3b can bind to pathogens and promote their phagocytosis via complement receptors on phagocytic cells
• Once the pathogen has been recognised and bound, the phagocytic cell engulfs it, enclosing it in a vesicle called a phagosome
• The phagosome then fuses with a lysosome, release enzymes that degrade the pathogen

Question 5

Describe the role of antibodies in antibacterial humoral responses

Answer 5

1) IgM:

• First antibodies to be produced during a primary immune response.
• Effective at activating the complement pathway
• The pentameric structure of IgM allows it to bind multiple antigens simultaneously, making it particularly effective at agglutinating bacteria

2) IgG:

• The most abundant type of antibody in the blood and are produced in large quantities during a secondary immune response
• They are extremely versatile and can neutralise toxins, opsonise bacteria for phagocytosis, and activate the classical complement pathway
• IgG antibodies can also cross the placenta to provide passive immunity to the fetus

3) IgA:

• Mucosal immunity
• Found in secretions such as tears, saliva and mucus, where they prevent bacterial colonisation by binding to and neutralising bacteria and their toxins
• In the gut, secretory IgA can bind to bacteria, preventing their interaction with epithelial cells and promoting their removal in faeces
• IgE:
• Allergic reactions and parasites
• Trigger mast cells to degranulate to release inflammatory mediators

Neutralisation: Antibodies bind to bacterial toxins, preventing them from interacting with host cell

Opsonisation

Complement activation

Agglutination: Each antibody can bind to multiple antigens, antibodies can cross-link bacteria, causing them to clump together - agglutination

Antibody-Dependent Cellular Cytotoxicity (ADCC): Certain immune cells, like NK cells, can recognise the Fc region of antibodies bound to bacteria. Upon recognition, these cells release cytotoxic substances to eliminate the bacterium

Question 6

Describe the role of CD4 helper T cells in antibody production and cellular immunity

Answer 6

CD4 Helper T cells and Humoral Immunity:

1) Antigen Presentation and T-cell Activation:

• B cells encounter an antigen, internalise, process and present it on B cell surface via MHC 2 molecules
• CD4+ T cells with a TCR specific for the same antigen can recognise and bind to this antigen-MHC 2 complex
• This interaction is stabilised by the CD4 molecule on the T cell, along with co-stimulatory signals (CD40-CD40L interaction), leading to T cell activation

2) Differentiation into Tfh cells:

• IL-4 promotes Th2 differentiation, while IL-12 promotes Th1
• Tfh cells differentiation under the influence of IL-6 and IL-21
• These Tfh cells migrate to follicles in the lymph node or spleen, where they engage in long-lasting interactions with antigen-specific B cells

3) Germinal centre reaction and B cell Help:

• Tfh cells provide critical help to B cells within germinal centres (GCs), structures within the lymphoid follicles
• This interaction involves CD40L on T cell and CD40 B cell engagement and cytokine signals
• In return, B cells present antigen to Tfh cells, further enhancing their activation and differentiation

4) Class Switch Recombination (CSR )and Affinity Maturation:

• Tfh cells produce IL-4, IFN-γ, TGF-β, etc. which guides the CSR in B cells, resulting in B cells switching from producing IgM to other antibody isotypes based on the nature of the pathogen
• Simultaneously, B cells undergo somatic hypermutation (SHM) in their immunoglobulin genes, which increases the diversity of the BCR repertoire and allows the selection of B cells with higher-affinity BCRs for the antigen

5) Memory Response:

• Tfh aid in the development of long-lived plasma cells and memory B cells

CD4 Helper T cells and Cellular Immunity:

1) Th1 and Th17 responses:

• IL-12 = Th1 differentiation and IL-6 + TGF-β for Th17
• Th1 cells produce IFN-γ, which activates macrophages
• Th17 produce IL-17 which recruits neutrophils and promotes barrier integrity against extracellular pathogens

2) Th2 responses:

• Th2 cells produce IL-4, IL-5 and IL-13 for defence against parasite and allergens

3) Activation of CD8+ T Cells:

• Th1 produce IL-2 which promotes CD8+ T cell proliferation and express CD40L, which provide a necessary second signal for CD8+ activation

Question 7

Identify the role of APC in immunity against extracellular and intracellular bacteria

Answer 7

Role of APCs against Extracellular Bacteria:

1) Phagocytosis:

• When an extracellular bacterium enters the body, APC (such as dendritic cells and macrophages) respond by recognising the pathogen-associated molecular patterns (PAMPs) on the bacterial surface via pattern recognition receptors (PRRs) like Toll-like receptors (TLRs)
• This interaction leads to phagocytosis of the bacteria and the production of pro-inflammatory cytokines that help recruit other immune cells to the infection site

2) Antigen Presentation:

• APCs degrade the phagocytosed bacteria into antigenic peptides and present these to their MHC 2 molecules, the antigen-MHC 2 are then presented on APCs surface

3) T-cell activation:

• TCR and antigen-MHC 2 complex interaction, in the presence of co-stimulatory signals (CD40) leads to the activation and proliferation of CD4+ T cells
• Depending on the cytokine, T cells can differentiate into various subsets; Th1 = IL-12, Th2 = IL-4 or Th17 = IL-6 + TGFβ
• Th1 cells secrete IFN-γ which activates macrophages
• Th17 produce IL-17 which recruits neutrophils

Role of APCs against Intracellular Bacteria:

1) Infection of APCs:

• Within the APC, the bacteria can be enclosed in a phagosome and degraded just as extracellular bacteria are.
• The bacterial peptides can then be presented on MHC II molecules for recognition by CD4+ T cells and produce IFN-γ
• Or present on MHC 1 molecule for cytotoxic T cells

2) Cross-presentation:

• Dendritic cells can capture and process antigens from the extracellular environment/dying infected cells and present them on MHC 1 molecules

Question 8

Identify the need for Th-1 T cells and a cytokine response against intracellular bacteria

Answer 8

Th1 Cells: Essential Drivers of Cell-mediated Immunity

1) Activation and Differentiation:

• APC presents bacterial peptides via MHC 2 molecules to TCR on naive CD4+ cells.
• Along with co-stimulatory molecules (CD28 on the T cell with B7 on APC)
• During infection with intracellular bacteria, the microbe-associated molecular patterns (MAMPs) are recognised by PRRs like Toll-like receptors (TLRs) on APC
• Leading to the production of IL-12 which promotes the differentiation of naive CD4+ T cells into Th1 cells

2) Interactions with Macrophages:

• Once activated, Th1 cells interact with macrophages, recognise the bacterial peptide presented on macrophages’ MHC class 2 molecules via their TCR
• This recognition, along with the binding of the CD40 ligand (CD40L) on the Th1 with CD40 on CD40 on the macrophage, results in macrophage activation

Role of Cytokines in Fighting Intracellular Bacteria:

1) Interferon-gamma (IFN-γ):

• IFN-γ activates macrophages, making them more effective in killing phagocytosed bacteria by increasing their production of toxic radicals and enhancing phagocytosis
• IFN-γ promotes further Th1 differentiation in an autocrine and paracrine manner

2) Tumour Necrosis Factor-alpha (TNF-ɑ):

• Inducing inflammation (pro-inflammatory), facilitating leukocyte adhesion and migration, and directly killing infected cells

3) IL-2:

• For growth and proliferation of T cells