Nutrition & Gut Immunity

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2 Nutrition & Gut Immunity Kenneth A. Kudsk, MD FACS Professor of Surgery University of Wisconsin - Madison Madison, Wisconsin No conflicts of interest to disclose

3 % Survival Survival vs. Malnutrition (after septic peritonitis with E. coli) % 50 60% 25 10% 0% 5% Well Nourished Depleted Depleted + Re-fed TPN TPN + Fat

4 % Survival Survival After Septic Peritonitis IG-TPN Chow 40 IV-TPN 20 1 Day 2

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6 INFECTIONS Enteral TPN Pneumonia all patients 11.8 % 31 % severely inj. 18 % 56 % Abscess all patients 3.9 % 17.8 % severely inj. 9 % 33 %

7 INFECTIONS Enteral TPN Pneumonia all patients 11.8 % 31 % severely inj. 18 % 56 % Abscess all patients 3.9 % 17.8 % severely inj. 9 % 33 %

8 INFECTIONS Enteral TPN Pneumonia all patients 11.8 % 31 % severely inj. 18 % 56 % WHY????

9 The Principle If nutrition support and enteral feeding really make a difference, there should be a reason... and it should be measurable and it should be testable in clinical populations.

10 What is it? OFFENSE? (the bacteria)

11 Bacterial Response to Stress

12 Bacterial Gene Expression Non-Hostile Environment Virulence Genes Downregulated Stressors Genes Upregulated Adherence Appendages (e.g. Ps Lectins) Attachment

13 Stressors of Host - Microbe Status Antibiotics Vasoactive drugs Block gastric acid Opiates - Motility TPN Starvation

14 Mortality, Stress and Adhesion 100 % Mortality 50 0 Exotoxin A or PA-1 Alverdy, Ann Surg 2000 Exotoxin A and PA-1 Stress (Hepatectomy) with PA-1 or Exotoxin A blocked Stress (Hepatectomy) with PA-1 or Exotoxin A

15 or the defense?

16 DEFENSE: Mucosal immunity Innate immunity

17 Cells and molecules that defend the intestinal mucosal surface

18 Mucosal Immunity Organization Secretory IgA Epithelium PP pigr IgA Th 2 cytokines Plasma cell LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract

19 Feeding Models Complexity, Intermittency Chow CED Complexity ISONIT / ISOCAL IV-TPN IG-TPN Route

20 Feeding Models Complexity, Intermittency Chow CED Complexity ISONIT / ISOCAL IV-TPN IG-TPN Route Degree of Enteral Stimulation Chow > CED > IG-TPN > IV-TPN IV-TPN prevents lethal malnutrition

21 Mucosal Immunity Parenteral Nutrition (No ENT stimulus) Secretory IgA Epithelium PP pigr IgA Th 2 cytokines Plasma cell LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract Secretory IgA

22 IgA Levels Intestinal * * * TPN days * p < 0.05 vs Day 0 Respiratory * * * TPN days Arch Surg. 1997;132:

23 % Viral Shedding % Mortality at 48hrs (Ps. Pneumonia) Route of Diet and Established Respiratory Immunity Chow CED IV-TPN 0 Chow CED Ann Surg. 1996;223(6): Ann Surg. 1999;229(2): TPN Nonimmune

24 Mucosal Immunity: Cell Entry Parenteral Nutrition (No ENT stimulus) Secretory IgA Epithelium PP pigr IgA Th 2 cytokines Plasma cell LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract Secretory IgA

25 Entry into GALT: Cell/ Peyer s Patch Interactions α 4 β 7 L-selectin

26 Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells α 4 β 7 20 L-selectin 0 Chow Blockade

27 Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

28 Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

29 Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

30 Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

31 MAdCAM-1 Expression (mgab/ Total PP) 0.06 MAdCAM-1Expression in PP * * Hrs of TPN Hrs of Refeeding ± SEM *p<.04 vs 0 and 4 hrs ± SEM p<.02 vs 0 hrs Surgical Forum. 2001;52:

32 Lymphocyte Mass: PP & LP 100 % of Chow Chow TPN Day Chow Recovery Arch Surg. 1997;132:

33 GALT T Cells in Small Intestine of Adults: Enteral Fed (21) vs. Without (6) Okamoto, Fukatsu et al JPEN 2005; 29:56

34 GALT T Cells in Small Intestine of Adults: Enteral Fed (21) vs. Without (6) Okamoto, Fukatsu et al JPEN 2005; 29:56

35 What happens to the cytokines? Normal control of IgA by T cell cytokines Th1 Th2 IFN TNF - IgA + IL-4 IL-5 IL-6 IL-10

36 What happens to the cytokines? Control of IgA by T cell cytokines: IV-TPN Th1 Th2 IFN TNF - IgA + IL-4 IL-5 IL-6 IL-10

37 Mucosal Immunity: IgA Transport Parenteral Nutrition (No ENT stimulus) Secretory IgA TRANSPORT Epithelium PP pigr IgA Th 2 cytokines Plasma cell LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract

38 IgA Transport through the Epithelial Cell Lumen Secretory IgA Mucosal epithelial cell pigr IgA-pIgR complex Lamina propria Dimeric IgA Plasma cell

39 Small Intestinal pigr expression (%) Intestinal pigr Expression ~ 120 kda ~ 94 kda * p<0.05 vs. Chow p<0.05 vs. CED * * 20 Sano Y,et al.jpen 2007; 31:(5) Chow CED IG-PN IV-PN

40 Mucosal Immunity Parenteral Nutrition (No ENT stimulus) Epithelium Secretory IgA IgA transport PP pigr IgA Th 2 cytokines Plasma cell LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract

41 Does any of this happen in humans? Okamoto, Fukatsu et al JPEN 2005; 29:56

42 Does any of this happen in injured humans?

43 Human Lung Response After Injury Epithelial Lining Fluid (Vol) IgA / ELF Total IgA Control Control Control

44 Human Lung Response After Injury Epithelial Lining Fluid (Vol) * IgA / ELF Total IgA Control Trauma Control Control

45 Human Lung Response After Injury Epithelial Lining Fluid (Vol) * IgA / ELF * Total IgA Control Trauma Control Trauma Control

46 Human Lung Response After Injury Epithelial Lining Fluid (Vol) * IgA / ELF * Total IgA Control Trauma Control Trauma Control Trauma

47 Respiratory IgA (ng) Mouse Respiratory IgA Response to Trauma * p<0.05 vs no blockade 200 * p<0.05 vs all other groups * * 50 0 h 4 h 8 h 24 h 8 h w/ TNF blockade

48 Murine IgA response to injury after PN? Completely stopped!

49 DEFENSE: Mucosal immunity Innate immunity

50 Cells and molecules that defend the intestinal mucosal surface

51 Gut innate immunity Paneth cells are secretory epithelial cells located at the end of intestinal crypts Paneth cells secret the antimicrobial proteins lysozyme, defensins, and secretory phospholipase A2 type IIA (spla2-iia) into the crypt lumen.

52 Phospholipase A2 5 major families; 30 different enzymes Secretory PLA2 (spla2) ~ 10 spla2 enzymes ; Ca 2+ dependent - Catalytically Phospholipid Release - Inflammatory Regulation (Host) - Bactericidal (Gut Lumen) - Non-Catalytically Prime Neutrophils

53 spla2 activity (FI/min/μL) SIWF spla2 activity 14 p< Chow PN Unpaired t-test, Means ± SE

54 Intestinal Fluid spla2 Activity 5 4 * * * vs. Chow, p < vs. PN, p < Activity 3 2 * 1 0 Chow CED IG-PN PN Concentration spla2-iia

55 ANOVA, Means ± SE, *p<0.05 vs. HBSS, p<0.05 vs. PN-LPS0, p<0.05 vs. PN- LPS10 Comparison of bactericidal activity in chow and PN culture medium (% of HBSS) * * Chow Chow -LPS0-LPS10 HBSS * PN PN -LPS0-LPS10

56 Can we reproduce GALT effects in people who can t be fed enterally by adding something to the TPN?

57 Can we reproduce GALT effects in people who can t be fed enterally by adding something to the TPN? i.e. is it the lack of enteral feeding or is it the TPN itself?

58 Can we reproduce GALT effects in people who can t be fed enterally by adding something to the TPN? Neuropeptides

59 Enteric Nervous System & Mucosal Immunity Bombina bombina Bombesin (BBS) Gastrointestinal neuropeptide Gastrin-releasing peptide analogue Stimulates gut hormone release - gastrin, CCK, neurotensin

60 Mucosal Immunity on Bombesin Secretory IgA Epithelium PP? pigr IgA Plasma cell Th 2 cytokines LPL Naïve T/B lymphocyte MLN Blood stream Thoracic duct Respiratory Tract Secretory IgA

61 % of lymphocytes Chow PN PN-BBS P<.05 T cell phenotype changes P<.05 LP CD4 + CD25 + LPAM-1 + with PN/DES to Chow levels with bombesin No CD8 + T cell changes anywhere 5 0 LP Almost all increase is Tregs

62 % of lymphocytes LP B cell phenotype changes P<.05 P<.05 P<.05 P<.05 Unactivated naïve (IgD + ) with PN/DES 35 Chow to Chow levels with bombesin PN PN-BBS Ag-stimulated (IgD - LPAM-1 + ) with PN/DES 15 P<.05 P<.05 to Chow levels with bombesin IgD + IgD - LPAM-1 + CD44 + LP Effector/memory (CD44 + ) with PN/DES to Chow levels with bombesin

63 spla2 activity (FI/min/μL) SIWF spla2 activity 14 p< Chow PN Unpaired t-test, Means ± SE

64 spla 2 in Paneth Cell Granules: Chow, PN, PN+BBS Chow spla 2 PN spla 2 PN+BBS spla 2

65 BOMBESIN Lamina propria: Increases T (Tregs) & B cells Increases IgA- Airway and SI Reverses PN-induced defect in bacterial and viral immunity Increases spla2 in Paneth cell after PN but not SIWFbethanecol necessary as well

66 Systemic effects with lack of enteral stimulation?

67 Cytokines and GALT Function Mucosa GALT Endothelium o IgA IL-4 IL o ICAM-1 o o IgA IFN ICAM-1 o o

68 Vascular Function + IV-PN Mucosa GALT IL-4 IL-10 + Endothelium o o o o o o o IFN + op + op op op op op op

69 Gut PMN Priming (hemorrhagic shock) Lung PMN Localization 2 ND Insult Pulmonary injury Moore EE et al. J Trauma 1994; 37:

70 Does IV-PN prime the cells to a second insult? Feed Chow, IG-TPN or IV-TPN 5 days Add stress: 15 minutes of gut ischemia and 2 hours of reperfusion

71 Route of Feeding: Effects After Gut I/R Injury min I/R Mortality at 72 hrs PERMEABILITY Lung Liver * * * Chow IG-PN IV-PN Chow IG-PN IV-PN Chow IG-PN IV-PN

72 Lung Stain for CD18 (activation) after I/R Injury IV-TPN Chow IG-TPN Fukatsu et al.ann Surg. 2001; 233 (5):