1. Innate Immunity: The First Line Against Infections Innate Immunity: The First Line Against Infections Juan Pablo Horcajada. Unidad de Enfermedades Infecciosas Hospital Universitario Marqués de Valdecilla Santander. Spain.

2. In adults there are important differences in In adults there are important differences in – susceptibility to infections – outcome of infections under treatment Innate immune system is the “third column” of the immun system Innate immune system is the “third column” of the immun system There are new therapeutical possibilities There are new therapeutical possibilities In adults there are important differences in In adults there are important differences in – susceptibility to infections – outcome of infections under treatment Innate immune system is the “third column” of the immun system Innate immune system is the “third column” of the immun system There are new therapeutical possibilities There are new therapeutical possibilities Relevance

3. CellularImmunity HumoralInmunity Innate immunity

4. The innate immune system The innate immune system Mannose-binding lectin Mannose-binding lectin MBL deficiency and infections: susceptibility and severity MBL deficiency and infections: susceptibility and severity Special populations: Special populations: – Bone marrow transplant patients – HIV-infected patients The innate immune system The innate immune system Mannose-binding lectin Mannose-binding lectin MBL deficiency and infections: susceptibility and severity MBL deficiency and infections: susceptibility and severity Special populations: Special populations: – Bone marrow transplant patients – HIV-infected patients Index

5. Immunity InnateAdaptative Specific Generates memory HumoralresponseCellularresponse AntibodiesLymphocytes Non-specific Does not generate memory Humoralfactors Cells External barriers Complement Acute phase proteins NeutrophilsMonocytes NK Cells

6. CLASSIC PATHWAY Ag-Ab Complexes CLASSIC PATHWAY Ag-Ab Complexes MBL-MASP1MBL-MASP1 C3bC3b C3C3 C1qC1rC1qC1r C4C4C2C2 C2aC2a C3bC3b C3a, C5a C5b-C9C5b-C9 Membrane attack complex, pathogen lysis Membrane attack complex, pathogen lysis Inflamation, fagocyte recruitment recruitment Opsonization, elimination of immunocomplexes Opsonization, elimination of immunocomplexes MBL PATHWAY Microbial surfaces MBL PATHWAY Microbial surfaces ALTERNATIVE PATHWAY Microbial surfaces ALTERNATIVE PATHWAY Microbial surfaces MBL-MASP2MBL-MASP2 C1sC1s C4bC4b THE COMPLEMENT SYSTEM

7. MBL TETRAMER N-terminal collagen first region DRC Bacterial surface Proteases (masp) hexose Disulfur bond Activation C’ collagen second region

8. BB AA CC DD Structure of MBL polipeptidic chain Carbohidrate Recognition Domain (CRD) Alpha helix region. Interacts with CRD and determines its spatial orientation and determines its spatial orientation Alpha helix region. Interacts with CRD and determines its spatial orientation and determines its spatial orientation Collagen region. Functions: fagocytosis, opsonization and protease binding for complement activation Collagen region. Functions: fagocytosis, opsonization and protease binding for complement activation Terminal NH2 segment. Oligomerization through Terminal NH2 segment. Oligomerization through N-terminal cisteins by disulfur bonds Terminal NH2 segment. Oligomerization through Terminal NH2 segment. Oligomerization through N-terminal cisteins by disulfur bonds

10. MBL gen polymorphisms PromotorPromotor Exon 1

11. Serum MBL levels related with different haplotipes High (>1000 ng/ml) HYPA LYQA LYPA Homozygous Sufficient High (>1000 ng/ml) HYPA LYQA LYPA Homozygous Sufficient Medium (500-1000 ng/ml) HYPA LYQA LYPA LXPA Heterozygous sufficient Medium (500-1000 ng/ml) HYPA LYQA LYPA LXPA Heterozygous sufficient Low (200-500 ng/ml) HYPD LYQC LYPB HYPA LYQA LYPA Heterozygous Sufficient-insufficient Low (200-500 ng/ml) HYPD LYQC LYPB HYPA LYQA LYPA Heterozygous Sufficient-insufficient Very Low (<200 ng/ml) HYPD LYPB LYQC LXPA Homozygous insufficient Very Low (<200 ng/ml) HYPD LYPB LYQC LXPA Homozygous insufficient

12. MBL levels in relation with haplotypes Homozygous defficient

13. MBL binding to different microorganisms +++ Candida Aspergillus S. aureus S. pyogenes Bifidobacterium Veillonella +++ Candida Aspergillus S. aureus S. pyogenes Bifidobacterium Veillonella ++ E. coli Klebsiella Haemophilus influenza B ++ E. coli Klebsiella Haemophilus influenza B + S. agalactiae S. pneumoniae S. epidermidis Pseudomonas Enterococcus Clostridium Bacterioides + S. agalactiae S. pneumoniae S. epidermidis Pseudomonas Enterococcus Clostridium Bacterioides

14. MBL defficiency and susceptibility to bacterial infections MBL defficiency and susceptibility to bacterial infections Meningococcal Infection Frequency of homozigous MBL-variants alleles in hospitalized patients 7,7% vs. 1,5% in non-infectious controls OR 6,5 p = 0.0006 Frequency in general population: 8,3% vs. 2,3% in healthy controls OR 4,5 p = 0.06 Hibberd ML. Lancet 1999;353:1049

15. Pneumococal infection Defficient homozygous Controls OR p 28/229 (12%)18/353 (5%)2,590.002 11/108 (10%)36/679 (5%)---0.046 MBL defficiency and susceptibility to bacterial infections MBL defficiency and susceptibility to bacterial infections Roy S. Lancet. 2002;360:1176.

16. MBL levels in elective abdominal surgery and incidence of bacterial infections MBL levels in elective abdominal surgery and incidence of bacterial infections N=172 patientsN infections: 10 (0,58%) M. Siassi. Biochem Soc Tras 2003;31:774

17. MBL defficiency associated with recurrent bacterial infections MBL defficiency associated with recurrent bacterial infections Gomi K. Chest 2004; 126:95–99

18. Recurrent vaginal candidiasis Babula CID 2003 Sep 1;37(5):733 MBL defficiency and susceptibility to fungal infections MBL defficiency and susceptibility to fungal infections

19. Chronic necrotizing pulmonary Aspergillosis Defficients Haplotypes In Controlsp in CNPA 7/10 (70%)20/82 (25%)0,004 7/10 (70%)20/82 (25%)0,004 Crosdale JID 2001 MBL defficiency and susceptibility to fungal infections MBL defficiency and susceptibility to fungal infections

20. MBL defficiency and severity of infections Low MBL (n=13) 9 (33.3)4 (11.4) Normal MBL (n=49)18 (67.6)31 (88.6) Bacteraemic Pyelonephritis N = 27 Non Bacteraemic Pyelonephritis N = 35 P=0.036  2 test Smithson A. 2005 ECCMID. P-1824

21. MBL defficiency and severity of infections Low MBL (n=13) 4 (57)9 (16.3) Normal MBL (n=49) 3 (43)46 (83.7) Pyelonephritis with septic shock N = 7 Pyelonephritis without septic shock N = 5 5 P=0.030 Fisher exact test Smithson A. 2005 ECCMID. P-1824

22. CellularImmunity HumoralInmunity Innate immunity

23. Prospective study (feb-oct 2005) BMT and infections Follow-up 6 months Periodic MBL serum levels determinations RESULTS - - 12 (50%) autologous and 12 (50%) alogenic. - - 55% of infectious episodes: during neutropenic period. - - 63% bacterial; 26% viral, 9% fungal - - 6 (25%) died because an infectious complication MBL Serum levels and Susceptibility to opportunistic Infections in bone marrow transplant patients

24. MBL<1000 *gram positive inf Crosstabulation Count 8311 729 15520 no si MBL<1000 Total noyes gram positive infection Total MBL<1000 * gram negative infec Crosstabulation Count 4711 369 71320 no si MBL<1000 Total noyes gram negative infection Total P = 1 P = 0.16 MBL<1000 * infeccion viral Crosstabulation Count 4610 82 12820 no si MBL<1000 Total n0si infeccion viral Total P = 0,16 10 P = 1

25. Maximum MBL serum levels ng/mL Confirmed fungal infection -Pulmonary Aspergilosis - - Pulmonary Mucormicosis - - Systemic Candidiasis MBL<1000 fungal infection no/yes Crosstabulation Count 11 639 17320 no si MBL<1000 Total noyes fungal infection no/yes Total P = 0,07

26. Polymorphisms of the Mannose-Binding Lectin Gen and Susceptibility to Opportunistic Infections in HIV-Infected Patients 0/0 n=39 527 (252) 36579 (152237) CD4 count, mean (SD) Viral load, mean (SD) A/A or A/0 n=151 460 (304) 48831 (154112) p 0.21 0.66 Genotypes JP Horcajada et al. ICAAC 2004

27. S. pneumoniae 0/0 n=39 7 (18) 1 (2.5) 7 (18) Pneumococcal bacteremia Recurrent pneumococcal bacteremia Recurrent pneumonia A/A or A/0 n=151 32 (21) 5 (3.3) 17 (11) p 0.65 1 0.28 Genotypes JP Horcajada et al. ICAAC 2004

28. Candidiasis 0/0 n=39 1 (2.5) 5 (13) 1 (2.5) 7 (18) Oral (Muget), n(%) Esophageal, n(%) Vaginal, n(%) Any candidiasis, n(%) A/A or A/0 n=151 7 (4.6) 18 (12) 4 (2.6) 29 (19.2) p 1 0.96 Genotypes JP Horcajada et al. ICAAC 2004

29. Virus 0/0 n=39 1 (2.5) 4 (10.2) 5 (13) 1 (2.5) 0 Cytomegalovirus Herpes Zoster Recurrent Herpes simplex Progressive multifocal leukencephalopathy Molluscum contagiosum A/A or A/0 n=151 7 (4.6) 32 (21) 5 (3.3) 2 (1.3) 4 (2.6) p 0.20 0.15 0.03 0.50 0.58 Genotypes JP Horcajada et al. ICAAC 2004

30. Other OI 0/0 n=39 1 (2.5) 3 (7.7) 1 (2.5) 3 (7.7) 2 (5.1) 0 Toxoplasmosis Pneumocystis carinii MAI Hairy leukoplakia Condiloma Non-TB Mycobacteria A/A or A/0 n=151 6 (3.9) 10 (6.6) 2 (1.3) 3 (1.9) 13 (8.6) 1 (0.6) p 1 0.52 0.50 0.10 0.73 1 Genotypes JP Horcajada et al. ICAAC 2004

31. Tuberculosis 0/0 n=39 1 (2.5) 0 2 (5.1) Pulmonary, n(%) Lymph node, n(%) Bone, n(%) Milliary, n(%) Any TB, n(%) A/A or A/0 n=151 15 (10) 3 (1.9) 1 (0.6) 8 (5.3) 27 (18) p 0.20 1 0.35 0.048 Genotypes JP Horcajada et al. ICAAC 2004

32. MBL is a key protein of the innate immune system MBL is a key protein of the innate immune system MBL serum level is genetically determined MBL serum level is genetically determined Genetic polymorphisms are very prevalent Genetic polymorphisms are very prevalent There is a higher predisposition for some infections in There is a higher predisposition for some infections in MBL-deficient patients MBL is a key protein of the innate immune system MBL is a key protein of the innate immune system MBL serum level is genetically determined MBL serum level is genetically determined Genetic polymorphisms are very prevalent Genetic polymorphisms are very prevalent There is a higher predisposition for some infections in There is a higher predisposition for some infections in MBL-deficient patients Conclusions (I)

33. MBL defficiency is associated with higher severity of MBL defficiency is associated with higher severity ofinfections In bone marrow transplant MBL deficiency is associated In bone marrow transplant MBL deficiency is associated with a higher incidence of invasive fungal infections. No relation between low MBL levels and the incindence No relation between low MBL levels and the incindence bacterial / viral infections and in these patients MBL defficiency is associated with higher severity of MBL defficiency is associated with higher severity ofinfections In bone marrow transplant MBL deficiency is associated In bone marrow transplant MBL deficiency is associated with a higher incidence of invasive fungal infections. No relation between low MBL levels and the incindence No relation between low MBL levels and the incindence bacterial / viral infections and in these patients Conclusions (II)

34. In HIV-infected patients MBL deficiency is associated with In HIV-infected patients MBL deficiency is associated with a higher incidence of recurrent herpes. On the contrary, tuberculosis is more frequent in patients On the contrary, tuberculosis is more frequent in patients With normal or high MBL levels. Milliary tuberculosis is not detected in MBL-deficient Milliary tuberculosis is not detected in MBL-deficient HIV-infected patients. In HIV-infected patients MBL deficiency is associated with In HIV-infected patients MBL deficiency is associated with a higher incidence of recurrent herpes. On the contrary, tuberculosis is more frequent in patients On the contrary, tuberculosis is more frequent in patients With normal or high MBL levels. Milliary tuberculosis is not detected in MBL-deficient Milliary tuberculosis is not detected in MBL-deficient HIV-infected patients. Conclusions (III)