Bacteriology & Immunology of Leprosy

Presentation made by Dr. Jerriton Brewin on the bacteriology and immunology of Leprosy.

1. BACTERIOLOGY & IMMUNOLOGY of M. leprae Dr. Jerriton, DVL PG, SVMCH, Pondy 18.5.20

2. BACTERIOLOGY

3. TAXONOMY CLASSIFICATION KINGDOM - Bacteria PHYLUM - Actinobacteria ORDER - Actinomycetales SUBORDER - Corynebacterineae FAMILY - Mycobacteriaceae GENUS - Mycobacterium SPECIES - Leprae

4. ORGANISM DESCRIPTION Obligate Intracellular Acid Fast – Mycolic Acid Aerobic Microaerophilic Non-motile Non-spore forming Pyridine extractable Pathogenic

5. MEASUREMENTS & SPECIAL FEATURES Length – 1.8 μm Diameter – 0.3 μm Length = 5 x Breath Parallel sides Rounded ends Bright Pink on ZN stain Bundle of cigars appearance

6. Non - Cultivable Generation Time: 11-13 days Temperature: 27 - 30 ° C Can remain dormant in humidity for 5 months Undergone reductive evolution – Catabolic pathways ↓ Unique host: Schwann cells Principle host: Macrophages

7. MODES OF TRANSMISSION Exact method unknown By direct contact Respiratory droplet spread Insects NOT SPREAD BY… Shaking hands or hugging Sitting next to each other on the bus Sitting together at a meal Mother to child during pregnancy Sexual contact Source: WHO & CDC

8. ANIMAL MODELS MOUSE ARMADILLO Immunocompetent mice Immunodeficient mice Inoculum size: upto 10 4 Bacilli Obtained: 10 6 Inoculum size: > 10 5 Bacilli Obtained: > 10 6 Inoculum size: 10 8 Bacilli Obtained: 10 10

9. CELLULAR MORPHOLOGY

10. M. leprae smears on treating with pyridine, lose the ability to stain subsequently with carbon fuchsin and thereby appear non-acid fast. It is unique to M. leprae. PYRIDINE EXTRACTION

11. VIRULENCE FACTORS Phenolic Glycolipid I (PGL – I) Lipoarabinomannan (LAM)

12. PATHOGENESIS OF INFECTION SCHWANN CELL (SC) Initial Target: Laminin α 2 –> PGL 1 of M. leprae binds to it Laminin α 2 seen in Schwann cell, Striated muscle, Placenta H1p/LBP21 –> potentiates interaction of M. leprae with SC SC processes antigen & presents it through MHC – II CD4+ T Cells then get activated & releases Ils –> leads to Macrophage activation –> kills bacteria Concurrent nerve demyelination occurs due to inflammatory events

13. SC membrane has laminin 2 and a laminin 2 receptor (α-dystroglycan) Laminin 2 has a G domain on the α2 chain PGL-1 of M. leprae binds to this domain. This PGL-Laminin-2 complex interacts with α-dystroglycan, leading to uptake of M. leprae. Laminin binding protein 21 (LBP21) of M. leprae also binds to α-DG od SC membrane, leading to its entry. MECHANISM OF ENTRY INTO NERVE

14. It is a major glycolipid antigen of M. leprae. Is unique to M. leprae. It is part of lipid capsule. Accounts for 2% of mass of bacilli. PGL-1

15. Has an antigenically distinct trisaccharide linked to phenol, which is linked to 29C phthiocerol, which are attached 2 mycoserosic acids. Specific IgM antibodies develop to it, more at lepromatous spectrum. Antigen specificity resides in terminal sugars, which has been exploited for serodiagnosis. PGL-1 (contd.)

16. Helps in entry and colonization within phagocytes. Once inside phagocytes, it can scavenge ROS and helps the bacteria survive intracellularly. PGL-1 (contd.)

17. STAINS Haematoxylin – Eosin stain Job-Chacko Modification of Fite Faraco stain Gomori – Grocott Methenamine Silver Immunochemical stain S100 stain Fluorescent microscopy FDA – EB stain Other stains

18. AFB STAIN

19. INDICES BACTERIAL INDEX - Determines bacterial load (live + dead) - Score: +6 = Over 1,000 bacilli + globi on an average field +5 = Over 100 bacilli but less than 1,000 in an average field +4 = Over 10 bacilli but less than 100 in an average field +3 = 1 – 10 bacilli in an average field +2 = 1 – 10 bacilli in 10 fields +1 = 1 – 10 bacilli in 100 fields = No bacilli in 100 fields

20. INDICES BACTERIAL INDEX Sum of values from all sites Total number of sites ZN stain with Acid Fast bacilli

21. INDICES MORPHOLOGICAL INDEX Percentage of live bacilli from 200 bacilli Solid staining = Live bacilli Solid stain: - Entire organism must be uniformly stained - Longitudinal sides are parallel - Both ends are rounded - Length is 5 times width

22. Although M. leprae has been extensively studied, and complete genome sequencing has been done, there are still grey areas to be explored, including the inability to grow in vitro. The knowledge of M. leprae, when integrated with the knowledge of immunological responses in the host, will provide a better understanding for diagnosis and treatment. CONCLUSION

23. IMMUNOLOGY

24. CYTOKINE PROFILES

25. INNATE IMMUNITY

26. INNATE IMMUNITY

27. Entry of pathogen Internalization INNATE IMMUNITY

28. Recognition Induction of cytokines INNATE IMMUNITY

29. TT Leprosy Induces CMI INNATE IMMUNITY

30. Tuberculoid Induces CMI INNATE IMMUNITY

31. LL Leprosy Induces humoral immunity - IL 4 (Antibody production) Suppresses CMI - IL 10 Inhibits TLR activation INNATE IMMUNITY

32. Antigen presentation to lymphocytes (Acquired immunity) INNATE IMMUNITY

33. ACQUIRED IMMUNITY

34. TT LL Cytokine Profile Th 1 Th 2 Immunity CMI Humoral CD4 cells Cytolytic cells, more Helper cells, less CD8 cells Cytotoxic, more Suppressor, less Macrophage Absent Present NON-REACTIONAL LEPROSY STATES

35. In lepromatous subjects , there is characteristic lymphopenia. Hence macrophages contain large no. of M. leprae, but cannot mount CMI. There is B cell activation leading to antibody production, which cannot attack intracellular M. leprae, but forms immune complexes with tissue / circulating M. leprae antigens. Antibodies are also exploited for diagnosis / clinical complications (eg. IgM antibodies against PGL-1). CD8 suppressor cells suppress CMI

36. Why is CMI characteristically suppressed in LL? IMMUNOLOGICAL UNRESPONSIVENESS

37. Antibody mediated suppression Presence of CD8 suppressor T cells Macrophages suppress T cell proliferation Macrophage factors: PGE2, thromboxane, leukotrienes, IL10. PGL of M. leprae FOXP3 T-regs - secrete TGF-β and IL10 - leads to suppression Th phenotype paradigm - presence of Th0 phenotype in 50% tuberculoid and 40% LL. TLR2 mutation REDUCED T CELLS IN LL IS DUE TO:

38. IMMUNOLOGY OF REACTIONS

39. Occurs in borderline cases Type 4 DHT T cell activation (lymphocyte responsiveness) Reflects a switch from a Th2 toward a Th1 response Immunological marker: CXCL10 Infiltration of IFN-γ and TNF-secreting CD4+ T cells in skin lesions and nerves, resulting in edema and painful inflammation. Increased Treg activity TYPE 1 REACTION

40. Occurs in LL > BL Type 3 immune complex reaction T cell activation (lymphocyte responsiveness) Increased CD4 cells Switch from Th2 to Th1 profile Massive neutrophil infiltration - TNF production - tissue damage Immune complex deposition in blood vessels (vasculitis), adipocytes (panniculitis) and eye (uveitis) TYPE 2 REACTION

41. NERVE DAMAGE IN LEPROSY

42. Nerve damage is due to immunological and non-immunological events But not due to direct effect of bacteria.

43. Intraneural macrophages produce TNFα - demyelination TLR-2 activation on SC - apoptosis In T1R - antigens released from SC induce DTH reaction In T2R - Immune complex deposition, granulocyte attraction and compliment activation SC process and present M. leprae antigens to CD4 cells - which gets activated, leading to SC lysis. IMMUNOLOGICAL MECHANISMS OF NERVE DAMAGE

44. Contact dependent demyelination NO producing demyelination Protective role of myelin NON-IMMUNOLOGICAL MECHANISM OF NERVE DAMAGE

45. Chromosome 6q25 - linked to PARK2/PCRG gene regulatory region Chromosome 6p21 - linked to lymphotoxin-α gene Chromosome 10p13 - candidate gene not identified, susceptible to PB (Tuberculoid) IMMUNOGENETICS

46. TLR 1, 2, 4 polymorphisms TLR 2 mutation increases IL-10, an anti-inflammatory cytokine that suppresses Th1 response Mutations of Mannose Receptor 1, a phagocytic receptor that mannose capped LAM of M. leprae Vitamin D polymorphism (Vitamin D enhances Th2 T-cell responses at the expense of Th1 responses) Laminin 2 polymorphism

47. IL-10 polymorphism leads to increased production (IL-10, an anti-inflammatory cytokine, inhibits Th1 response) IL-12 polymorphism (susceptibility to LL) TNF-α mutation (TNF is important for resistance to leprosy) IFN-γ polymorphism

48. Both innate and adaptive immune responses play a role in leprosy Lymphopenia is a characteristic feature of LL. Others include absent CMI, presence of humoral immunity with a Th2 cytokine profile. Several genetic polymorphisms make a person susceptible to specific spectrums in leprosy, which gets reflected in their immunological profiles. More understanding is needed for diagnostics, vaccine development and therapy for a given population. CONCLUSION

49. IAL Textbook of Leprosy Jopling’s Hnadbook of Leprosy Bhat RM, Prakash C. Leprosy: an overview of pathophysiology. Interdiscip Perspect Infect Dis. 2012;2012:181089. REFERENCES