Tb - pathogenesis, immunohhhhhlogy.pptx

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1. Tb - pathogenesis, immunology

2. DISCUSSION HEADINGS • INTRODUCTION • STAGES OF PATHOGENESIS • HOST IMMUNE RESPONSE • INNATE AND ACQUIRED IMMUNITY • PRO INFLAMMATORY & ANTI INFLAMMATORY MEDIATORS IN TB TYPE 4 HYPERSENSITIVITY GRANULOMATOUS INFLAMMATION

3. Route and site of infection Mycobacterium tuberculosis is an obligatory aerobic, intracellular pathogen, which has a predilection for the lung tissue rich in oxygen supply. The tubercle bacilli enter the body via the respiratory route . The bacilli spread from the site of initial infection in the lung through the lymphatics or blood to other parts of the body,the apex of the lung and the regional lymph node being favoured sites. Extrapulmonary TB of the pleura, lymphatics, bone, genito -urinary system,meninges , peritoneum, or skin occurs in about 15 percent of TB patients

4. Events following entry of bacilli Stage1: Phagocytosis of M.Tb by Alveolar macrophage Destruction of M.Tb,but some evade destruction & continue to multiply inside them & infect Bystander macrophages Stage 2: Influx of PMN’s,recruitment of Monocytes , differentiate into Macrophage, but fail to eliminate completely Logarithmic growth of bacilli, little tissue destruction

5. Stage 3: • Ag specific T-cells are recruited to the site that activate monocytoid cells & differentiate into two types of Giant cells • EPITHELIOID, LANGHANS’ GIANT CELL • Walling off infection from rest of the body & prevent dissemination of bacilli.

6. Stage 4: • Stage of Latency ( Granuloma ) disrupts under conditions of failing immune surveillance & leads to ENDOGENOUS RE ACTIVATION of dormant foci • Characterised by CASEATION NECROSIS

7. Binding of M. tuberculosis to monocytes /macrophages • Complement receptors (CR1, CR2,CR3 and CR4), mannose receptors (MR) and other cell surface receptor molecules play an important role in binding of the organisms to the phagocytes. • The interaction between MR on phagocytic cells and mycobacteria seems to be mediated through the mycobacterial surface glycoprotein lipoarabinomannan (LAM). • Prostaglandin E2 (PGE2) and interleukin (IL)-4, a Th2-type cytokine,upregulate CR and MR receptor expression and function • interferon-g (IFN-g) decreases the receptor expression, resulting in diminished ability of the mycobacteria to adhere to macrophages. • There is also a role for surfactant protein receptors, CD14 receptor7 and the scavenger receptors in mediating bacterial binding.

8. Phagolysosome fusion • Phagocytosed microorganisms are subject to degradation by intralysosomal acidic hydrolases upon phagolysosome fusion. • This highly regulated event constitutes a significant antimicrobial mechanism of phagocytes. • Prevention of phagolysosomal fusion is a mechanism by which M. tuberculosis survives inside macrophages. • mycobacterial sulphatides,derivatives of multiacylated trehalose 2-sulphate,have the ability to inhibit phagolysosomal fusion. • studies demonstrated that M. tuberculosis generates copious amounts of ammonia in cultures,which is thought to be responsible for the inhibitory Effect.

9. Fate of engulfed M.Tb Anti mycobacterial effector functions ROI RNI Other mechanisms (IFN- γ , TNF- α) MACROPHAGE APOPTOSIS

10. MACROPHAGE ACTIVATION • IFN ,TNF (Th1 cells) Most well documented • Vitamin D • SLC11A1 (formerly Nramp1)

11. Reactive oxygen intermediates (ROI) • Hydrogen peroxide (H2O2), one of the ROI generated by macrophages via the oxidative burst, was the first identified effector molecule that mediated mycobactericidal effects of mononuclear phagocytes. • M.tuberculosis infection induces the accumulation of macrophages in the lung and also H2O2 production. • Similar local immune response in tuberculous ascitic fluid has also been demonstrated. • Increased production of hydrogen peroxide by alveolar macrophages is not specific for TB. • Moreover, the alveolar macrophages produced less H2O2 than the corresponding blood monocytes

12. Reactive nitrogen intermediates (RNI) • Phagocytes upon activation by IFN and TNF generate NO & related RNI via iNOS2 • 1,25 dihydroxy Vit D3 reported to induce the expression of NOS2 • High level expression of NOS 2 detected immunohistochemically in macrophages obtained by BAL in individuals with active PTB

13. Other mechanisms of growth inhibition/killing • IFN and TNF mediated antimycobacterial effects • VIT D3 alone or in combination with IFN and TNF able to activate macrophage to inhibit/kill M.Tb

14. Macrophage apoptosis   • potential mechanism involved in macrophage defense against M. tuberculosis is apoptosis or programmed cell death. • apoptosis associated with TB is mediated through a downregulation of bcl-2, an inhibitor of apoptosis • Within the granuloma , apoptosis is prominent in the epithelioid cells as demonstrated by condensed chromatin viewed by light microscopy or with the in situ terminal transferase mediated nick end labeling (TUNEL) • Results in reduced viability of M.Tb

15. Evasion of host immune response by M.tuberculosis • Modulation of antigen presentation to avoid elimination by T cells. • Protein secreted by m. Tuberculosis such as superoxide dismutase and catalase are antagonistic to ROI. • Mycobacterial components such as sulphatides , LAM and PHENOLIC- GLYCOLIPID I (PGLI) are potent oxygen radical scavengers. • M.Tuberculosis -infected macrophages appear to be diminished in their ability to present antigens to CD4+T cells, which leads to persistent infection. • Another mechanism by which antigen presenting cells (APCS) contribute to defective T cell proliferation and function is by the production of cytokines, including TGF- Β, IL-10 OR IL-6. • Virulent mycobacteria were able to escape from fused phagosomes and multiply

16. Host immune mechanisms in TB Innate immune response Acquired immune response Humoral Cell mediated CD 8 CD 4

17. Innate immune response • The phagocytosis and the subsequent secretion of IL-12 are processes initiated in the absence of prior exposure to the antigen and form a component of innate immunity. • The other components of innate immunity are natural resistance associated macrophage protein ( Nramp ), neutrophils , natural killer cells (NK) . • PLASMA LYSOZYME and other enzymes may play an important role in the first line defense , of innate immunity to M. tuberculosis. • The role of CD-1 restricted CD8+ T cells and non-MHC restricted T cells (γ/δ cell) have been implicated but incompletely understood.

18. Nramp • Nramp (SLC11A1) is crucial in transporting nitrite from intracellular compartments such as the cytosol to more acidic environments like phagolysosome , where it can be converted to NO. • Integral membrane protein,ion transporter family esp Feᶧᶧ • Defects in Nramp production increase susceptibility to mycobacteria . • NRAMP1 gene might not be associated with the susceptibility to pulmonary and spinal TB in the Indian population

19. NEUTROPHILS • Increased accumulation of neutrophil in the granuloma and increased chemotaxis has suggested a role for neutrophils . • At the site of multiplication of bacilli, neutrophils are the first cells to arrive followed by NK cells, γ/δ cells and α/β cells. • There is evidence to show that granulocytemacrophage - colony stimulating factor (GM-CSF) enhances phagocytosis of bacteria by neutrophils • Human studies have demonstrated that neutrophils provide agents such as defensins , which is lacking for macrophage- mediated killing. • neutrophils can bring about killing of M. tuberculosis in the presence of calcium under in vivo conditions.

20. NK CELL • Effector cells of innate immunity. • Directly lyse the pathogens or can lyse infected monocytes . • Culture with live m. Tuberculosis brought about the expansion of NK cells • During early infection, NK cells are capable of activating phagocytic cells at the site of infection. • A significant reduction in nk activity is associated with multidrug resistant tb (MDR-TB).

21. • Nk activity in BAL has revealed that different types of pulmonary tb are accompanied by varying degrees of depression • Apoptosis is a likely mechanism of NK cytotoxicity . NK cells produce IFN-g and can lyse mycobacterium pulsed target cells. • Lowered NK activity during TB infection is probably the ‘effect’ and not the ‘cause’ for the disease • Augmentation of NK activity with cytokines implicates them as potential adjuncts to TB chemotherapy.

22. TOLL LIKE RECEPTOR • Phylogenetically conserved mediators of innate immunity essential for microbial recognition on macrophages & dendritic cells • M. tuberculosis can immunologically activate cells via either TLR2 or TLR4 in a CD 14- independent, ligand -specific manner • TLR 2 – LAM • TLR 4 – Heat labile factor • TLR 9 – cpg DINUCLEOTIDES

23. • TLR binds to target • Coupling of IRAK -1 signalling molecules & MyD88 (myeloid differentiation) gene • Translocation of NF κB (transcription factor) from cytosol to nucleus • Cytokine production

24. Acquired immune response • Humoral immune response: • Since M. tuberculosis is an intracellular pathogen, the serum components may not get access and may not play any protective role. • Although many researchers have dismissed a role for B cells or antibody in protection against TB, recent studies suggest that these may contribute to the response to TB.

25. Cellular immune response • T CELLS • M.TB is a classic example of pathogen with a protective response relying on CMI • Activated T cells migrate to site of infection and interact with APC’s. • Tuberculous Granulomas contain both CD4 & CD 8 T cells & helps to contain the infection within Granuloma and prevent reactivation.

26. CD4 T CELLS • Most important cells in protective response against M.Tb Primary effector function - production of IFN γ & other cytokines to activate Macrophage Also related to NOS2 expression Other unknown functions(protective) which are IFN & NOS2 independent Target for vaccine design

27. CD 8 T CELLS • Play in regulating Th1/Th2 balance • Studies: TB with slow regression was associated with an ↑ CD 8 T cell in BAL F Capable of secreting IFN & IL 4 (M φ activation) Lysis of infected human dendritic cells & M φ By CD8 T cells specific for M.Tb Ag reduces intracellular bacterial numbers(CTL) Killing : Perforin / Granulysin pathway Attractive vaccine candidate

28. T cell APOPTOSIS • Attenuation of CMI by inducing T CELL apoptosis in M.Tb • Leads to diminished M.Tb stimulated IFN γ & IL 2 production • TB infection leads to CD 95 mediated Th1 depletion

29. Non classically restricted CD 8 T cell CD 1 restricted T cells (NK T cells) γ/δ T cells T reg CELLS

30. Th1/Th2 dichotomy • Th1 secrete IL2, IFN γ Protective role in intracellular infections. • Th2 secrete IL4, IL5, IL10. Exert a negative influence on the immune response IL 12 induce Th1 type response .Th1 type response relate directly to the clinical manifestations of the disease Limited TB: alveolar lymphocytosis with high levels of IFN γ. Far advanced / cavitary disease : no Th1 response.

31. Proinflammatory Cytokines TNF Alfa (Stimulation of monocytes , macrophages, and dendritic cells with mycobacteria or mycobacterial products induces the production of TNF-, a prototype proinflammatory cytokine) TNF α plays a key role in granuloma formation, induces macrophage activation, and has immunoregulatory properties. • In tuberculosis patients, TNF- production is present at the site of disease. IL-1 β • produced by monocytes , macrophages, and dendritic cells . In tuberculosis patients, IL-1 β is expressed in Excess and at the site of disease. Acute phase response Fever & cachexia

32. IL-6: IL-6, which has both pro- and anti-inflammatory properties , is produced early during mycobacterial infection and at the site of infection. Role : inflammation,hematpoeisis,differentiation of T cells IL-6 may be harmful in mycobacterial infections, as it inhibits the production of TNF- and IL-1.

33. IL 12 • IL-12 is a key player in host defense against M. tuberculosis. • IL-12 is produced mainly by phagocytic cells • IL-12 has a crucial role in the induction of IFN- γ production. • In tuberculosis, IL-12 has been detected in lung infiltrates, in pleurisy, in granulomas , and in lymphadenitis. • The expression of IL-12 receptors is also increased at the site of disease. • In humans suffering from recurrent nontuberculous mycobacterial infections, deleterious genetic mutations in the genes encoding IL-12p and IL-12R have been identified.These patients display a reduced capacity to produce IFN- γ. • IL-12 is a regulatory cytokine which connects the innate and adaptive host response to mycobacteria and which exerts its protective effects mainly through the induction of IFN- γ

34. IL-18 and IL-15 IL-18, a novel proinflammatory cytokine which shares many features with IL-1, was initially discovered as an IFN--inducing factor, synergistic with IL-12 • Also, M. tuberculosis-mediated production of IL-18 by peripheral blood mononuclear cells is reduced in tuberculosis patients, and this reduction may be responsible for reduced IFN- γ production . • IL-15 resembles IL-2 in its biologic activities, stimulating T-cell and NK-cell proliferation and activation . • Unlike IL-2, however, IL-15 is primarily synthesized by monocytes and macrophages

35. IL 2 : Pivotal in generating immune response • Induce expansion of pool of lymphocytes • Influence the course of Tuberculosis IFN γ • The protective role of IFN- in tuberculosis is well established, primarily in the context of antigen-specific T- cell immunity can be used as a surrogate marker of infection with M. Tuberculosis

36. Anti-Inflammatory Cytokines The proinflammatory response which is initiated by M. tuberculosis is antagonized by anti-inflammatory mechanisms IL-4, IL-10, and transforming growth factor beta (TGF β)

37. Cell-Mediated (Type IV) Hypersensitivity The cell-mediated type of hypersensitivity is initiated by antigen-activated (sensitized) T lymphocytes. It includes the delayed type hypersensitivity reactions mediated by CD4+ T cells, and direct cell cytotoxicity mediated by CD8+ T cells. It is the principal pattern of immunologic response not only to a variety of intracellular microbiologic agents, such as Mycobacterium tuberculosis, but also to many viruses, fungi, protozoa, and parasites. So-called contact skin sensitivity to chemical agents and graft rejection are other instances of cell-mediated reactions. In addition, many autoimmune diseases are now known to be caused by T cell-mediated reactions.

38. The classic example of delayed hypersensitivity is the tuberculin reaction, which is produced by the intracutaneous injection of tuberculin, a protein- lipopolysaccharide component of the tubercle bacillus. In a previously sensitized individual, reddening and induration of the site appear in 8 to 12 hours, reach a peak in 24 to 72 hours, and thereafter slowly subside.

39. Morphologically, delayed type hypersensitivity is characterized by the accumulation of mononuclear cells around small veins and venules , producing a perivascular "cuffing". There is an associated increased microvascular permeability caused by mechanisms similar to those in other forms of inflammation unexpectedly, plasma proteins escape, giving rise to dermal edema and deposition of fibrin in the interstitium . The latter appears to be the main cause of induration , which is characteristic of delayed hypersensitivity skin lesions.

40. In fully developed lesions, the lymphocyte-cuffed venules show marked endothelial hypertrophy and, in some cases, hyperplasia. Immunoperoxidase staining of the lesions reveals a preponderance of CD4+ (helper) T lymphocytes

41. With certain persistent or non degradable antigens, such as tubercle bacilli colonizing the lungs or other tissues, the initial perivascular lymphocytic infiltrate is replaced by macrophages over a period of 2 or 3 weeks. The accumulated macrophages often undergo a morphologic transformation into epithelium-like cells and are then referred to as epithelioid cells. A microscopic aggregation of epithelioid cells, usually surrounded by a collar of lymphocytes, is referred to as a granuloma . This pattern of inflammation that is sometimes seen in type IV hypersensitivity is called granulomatous inflammation.

42. Schematic illustration of the events that give rise to the formation of granulomas in cell-mediated (type IV) hypersensitivity reactions. Note the role played by T cell-derived cytokines

43. When an individual is first exposed to protein antigens of tubercle bacilli, naive CD4+ T cells recognize peptides derived from these antigens in association with class II molecules on the surface of antigen presenting cells. This initial encounter drives the differentiation of naive CD4+ T cells to TH1 cells. The induction of TH1 cells is of central importance because the expression of delayed hypersensitivity depends in large part on the cytokines secreted by TH1 cells. TH1 cells secrete cytokines, mainly IFN-γ, which are responsible for the expression of delayed-type hypersensitivity

44. GRANULOMATOUS INFLAMMATION Granulomatous inflammation is a distinctive pattern of chronic inflammatory reaction characterized by focal accumulations of activated macrophages, which often develop an epithelial-like ( epithelioid ) appearance. It is encountered in a limited number of immunologically mediated, infectious and some noninfectious conditions. Recognition of the granulomatous pattern in a biopsy specimen is important because of the limited number of possible conditions that cause it and the significance of the diagnoses associated with the lesions

45. A granuloma is a focus of chronic inflammation consisting of a microscopic aggregation of macrophages that are transformed into epithelium-like cells surrounded by a collar of mononuclear leukocytes, principally lymphocytes and occasionally plasma cells. In the usual hematoxylin and eosin stained tissue sections, the epithelioid cells have a pale pink granular cytoplasm with indistinct cell boundaries, often appearing to merge into one another. The nucleus is less dense than that of a lymphocyte, is oval or elongate, and may show folding of the nuclear membrane.

46. Older granulomas develop an enclosing rim of fibroblasts and connective tissue. Frequently, epithelioid cells fuse to form giant cells in the periphery or sometimes in the center of granulomas . These giant cells may attain diameters of 40 to 50 μm . They have a large mass of cytoplasm containing 20 or more small nuclei arranged either peripherally ( Langhans -type giant cell) or haphazardly (foreign body-type giant cell).

47. Typical tuberculous granuloma showing an area of central necrosis, epithelioid cells, multiple Langhans -type giant cells, and lymphocytes.

48. Thank you

49. REFERENCES ROBBINS AND COTRAN – 10 TH EDITION Tuberculosis – WILLIAM ROM, STUART GARAV Tuberculosis- SURENDRA SHARMA, ALLADI MOHAN IMMUNOLOGY OF TUBERCULOSIS – IJMR Dr.ALAMELA RAJU,TRC,CHENNAI IMMUNOLOGY OF TUBERCULOSIS – BENCH OF BESIDE- RESPIRATORY JOURNAL