Nutrition & Gut Immunity

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

% Survival Survival vs. Malnutrition (after septic peritonitis with E. coli) 100 75 70% 50 60% 25 10% 0% 5% Well Nourished Depleted Depleted + Re-fed TPN TPN + Fat

% Survival Survival After Septic Peritonitis 100 80 60 IG-TPN Chow 40 IV-TPN 20 1 Day 2

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

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

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

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.

What is it? OFFENSE? (the bacteria)

Bacterial Response to Stress

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

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

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

or the defense?

DEFENSE: Mucosal immunity Innate immunity

Cells and molecules that defend the intestinal mucosal surface

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

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

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

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

IgA Levels 300 250 200 150 100 50 0 Intestinal * * * 0 1 2 3 4 5 TPN days 300 250 200 150 100 50 0 * p < 0.05 vs Day 0 Respiratory * * * 0 1 2 3 4 5 TPN days Arch Surg. 1997;132:1303-1309.

% Viral Shedding % Mortality at 48hrs (Ps. Pneumonia) Route of Diet and Established Respiratory Immunity 100 100 80 60 40 20 80 60 40 20 0 Chow CED IV-TPN 0 Chow CED Ann Surg. 1996;223(6):629-638. Ann Surg. 1999;229(2):272-278. TPN Nonimmune

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

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

Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells 100 80 60 40 α 4 β 7 20 L-selectin 0 Chow Blockade

Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells 100 80 60 40 α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells 100 80 60 40 α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells 100 80 60 40 α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

Entry into GALT: Cell/ Peyer s Patch Interactions 120 (%) Peyer s patch cells 100 80 60 40 α 4 β 7 20 L-selectin 0 Chow α 4 β 7 Blockade

MAdCAM-1 Expression (mgab/ Total PP) 0.06 MAdCAM-1Expression in PP 0.05 0.04 * * 0.03 0.02 0.01 0 0 4 8 12 24 48 72 Hrs of TPN 0 4 8 12 24 48 Hrs of Refeeding ± SEM *p<.04 vs 0 and 4 hrs ± SEM p<.02 vs 0 hrs Surgical Forum. 2001;52:194-196.

100 80 60 40 20 Lymphocyte Mass: PP & LP 100 % of Chow 80 60 40 20 0 Chow 1 2 3 4 5 0 1 2 3 4 TPN Day Chow Recovery Arch Surg. 1997;132:1303-1309.

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

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

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

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

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

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

Small Intestinal pigr expression (%) Intestinal pigr Expression ~ 120 kda ~ 94 kda 100 80 * p<0.05 vs. Chow p<0.05 vs. CED 60 40 * * 20 Sano Y,et al.jpen 2007; 31:(5) 351-357. 0 Chow CED IG-PN IV-PN

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

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

Does any of this happen in injured humans?

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

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

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

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

Respiratory IgA (ng) Mouse Respiratory IgA Response to Trauma * p<0.05 vs no blockade 200 * p<0.05 vs all other groups * 150 100 * 50 0 h 4 h 8 h 24 h 8 h w/ TNF blockade

Murine IgA response to injury after PN? Completely stopped!

DEFENSE: Mucosal immunity Innate immunity

Cells and molecules that defend the intestinal mucosal surface

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.

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

spla2 activity (FI/min/μL) SIWF spla2 activity 14 p<0.05 12 10 8 6 4 2 0 Chow PN Unpaired t-test, Means ± SE

Intestinal Fluid spla2 Activity 5 4 * * * vs. Chow, p < 0.001 vs. PN, p < 0.001 Activity 3 2 * 1 0 Chow CED IG-PN PN Concentration spla2-iia

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) 25 20 15 10 5 0-5 -10-15 * * Chow Chow -LPS0-LPS10 HBSS * PN PN -LPS0-LPS10

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

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?

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

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

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

% of lymphocytes 35 30 Chow PN PN-BBS P<.05 T cell phenotype changes P<.05 LP CD4 + CD25 + LPAM-1 + with PN/DES 25 20 to Chow levels with bombesin 15 10 No CD8 + T cell changes anywhere 5 0 LP Almost all increase is Tregs

% of lymphocytes LP B cell phenotype changes 45 40 P<.05 P<.05 P<.05 P<.05 Unactivated naïve (IgD + ) with PN/DES 35 Chow to Chow levels with bombesin 30 25 20 PN PN-BBS Ag-stimulated (IgD - LPAM-1 + ) with PN/DES 15 P<.05 P<.05 to Chow levels with bombesin 10 5 0 IgD + IgD - LPAM-1 + CD44 + LP Effector/memory (CD44 + ) with PN/DES to Chow levels with bombesin

spla2 activity (FI/min/μL) SIWF spla2 activity 14 p<0.05 12 10 8 6 4 2 0 Chow PN Unpaired t-test, Means ± SE

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

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

Systemic effects with lack of enteral stimulation?

Cytokines and GALT Function Mucosa GALT Endothelium o IgA IL-4 IL-10 + + o ICAM-1 o o IgA IFN ICAM-1 o o

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

Gut PMN Priming (hemorrhagic shock) Lung PMN Localization 2 ND Insult Pulmonary injury Moore EE et al. J Trauma 1994; 37: 881-887

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

Route of Feeding: Effects After Gut I/R Injury 100 90 80 70 15 min I/R Mortality at 72 hrs 0.5 0.4 PERMEABILITY Lung Liver * * 60 0.3 50 40 * 0.2 30 20 0.1 10 0 0 Chow IG-PN IV-PN Chow IG-PN IV-PN Chow IG-PN IV-PN

Lung Stain for CD18 (activation) after I/R Injury IV-TPN Chow IG-TPN Fukatsu et al.ann Surg. 2001; 233 (5):660-668.