Immunology of tuberculosis

1. IMMUNOLOGY OF TUBERCULOSIS Dr. Amith S reedharan DEPT OF PULMONARY MEDICINE SCB MCH,CUTTACK

2. DISCUSSION HEADINGS INTRODUCTION STAGES OF PATHOGENESIS HOST IMMUNE RESPONSE INNATE AND ACQUIRED IMMUNITY PRO INFLAMMATORY & ANTI INFLAMMATORY MEDIATORS IN TB REFERENCES

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 .

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9. 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.

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

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

12. 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. I ncreased production of hydrogen peroxide by alveolar macrophages is not specific for TB. Moreover, the alveolar macrophages produced less H2O2 than the corresponding blood monocytes

13. 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

14. 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

15. 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

16. 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

17. Host immune mechanisms in TB Innate immune response Acquired immune response Humoral Cell mediated CD 8 CD 4 Th1 Th2

18. 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.

19. 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 Fe ᶧᶧ 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

20. 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.

21. 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 ). 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 κ B (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. γ / δ T cells Large granular lymphocytes with dendritic morphology in lymphoid tissue Non MHC restricted Function as cytotoxic T cells for monocytes pulsed with mycobacterial antigen. Secrete cytokines involved in granuloma formation.

31. NK T CELLS CD 1 molecules are antigen presenting molecules that present lipids or glycolipids to T cells. Usually found on dendritic cells present in the lungs. Stimulate CD 1 restricted T(NK T cells) in the granuloma that can have a bystander effect on infected M φ .

32. T reg cells T cells with suppressing (other T cells) capability CD4 cells expressing CD 25 FOXp3,transcription factor essential for development of T reg cells (also MARKER) CTLA4 expressed on T reg induce negative signalling (contact dependent CD80/CD86)) It secrete IL 10,TGF β which supress activated T cells It requires Expansion & recruitment(CCR1,CCR4) at pathologic sites for the above actions Express TLR2,TLR 4 On its surface TLR2 engagement –ELIMINATION → Th1 response TLR 4 engagement – EXPANSION →immunosuppression

33. Th1/Th2 dichotomy T h1 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.

34. 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. Systemic spill over of TNF- may account for unwanted inflammatory effects like fever and wasting. ( double edged sword ) Clinical deterioration early in treatment is associated with a selective increase of TNF α in plasma , and quick recovery is associated with a rapid decrease of TNF α in plasma . To limit the deleterious effects of TNF α , systemic production of TNF- is downregulated and soluble TNF- receptors which block TNF- activity are increased . In line with this, the use of potent monoclonal anti-TNF- antibodies in Crohn’s disease and rheumatoid arthritis has been associated with increased reactivation of tuberculosis ( including miliary and extrapulmonary disease .

35. 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

36. 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.

37. IL 12 IL-12 is a key player in host defense against M. tuberculosis . IL-12 is produced mainly by phagocytic cells(M φ ,DC) 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

38. 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 . IL-18 also stimulates the production of other proinflammatory cytokines, chemokines , and transcription factors . 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

39. IL 2 Pivotal in generating immune response Induce expansion of pool of lymphocytes Secreted by CD4 Th1 subtype lymphocytes Influence the course of Tuberculosis

40. IFN γ The protective role of IFN- in tuberculosis is well established, primarily in the context of antigen-specific T-cell immunity. Produced by both CD4,CD8 T cells & NK cells Augment antigen presentation leading to recruitment of Lymphocytes potent activator of infected macrophage resulting in lytic mechanisms can be used as a surrogate marker of infection with M. tuberculosis Ability of cells to respond to this cytokine is more predictive than the amount . Severely depressed in far advanced cases of TB

41. 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 )

42. IL 10 Produced by M φ after phagocytosis of M.Tb Downregulation of IFN γ ,TNF α ,IL 12 Macrophage deactivation Directly inhibits CD4 T cell by inhibiting APC function of M.Tb infected cells M.Tb induced IL 10 suppresses an effective immune response

43. TGF β Produced by monocytes and dendritic cells after stimulation with M.Tb or LAM Inhibits IFN γ production and proliferation In M φ ,it antagonises antigen presentation,proinflammatory cytokine production & cellular activation Involved in TISSSUE DAMAGE & FIBROSIS by promoting macrophage collagenases and deposition of collagen matrix

44. IL 4 Deleterious effects of IL 4 in intracellular infections including TB ascribed to suppression of IFN γ & macrophage activation Progressive disease & reactivation were asso with an ↑ IL 4 Overexpression of IL 4 intensified tissue damage Conversely,Inhibition of IL 4 didn’t seem to promote cellular immunity So, IL 4 in TB is subject of controversy !

45. REFERENCES TUBERCULOSIS – WILLIAM ROM,STUART GARAY TUBERCULOSIS – SURENDRA SHARMA,ALLADI MOHAN IMMUNOLOGY OF TUBERCULOSIS- IJMR Dr.ALAMELA RAJU,TRC,CHENNAI IMMUNOLOGY OF TUBERCULOSIS: FROM BENCH TO BEDSIDE- RESPIROLOGY JOURNAL

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