In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Summary

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease. An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Abstract

Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state.  Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).  The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

Introduction

Mucosal surfaces serve as physical and immunological barriers providing protection against external threats pervasive in the environment^1,2. This protective epithelial barrier can be compromised when pathogenic organisms invade². In the case of a bacterial pathogen, this encounter often instigates an inflammatory process by activating the innate immune system and triggering a rapid mobilization of first responder granulocytes known as neutrophils^2-4. Chemotactic agents facilitating neutrophil recruitment are produced in part by the mucosal epithelial cells seeking to rid the host of the offending pathogen^2-4. ....

Protocol

Steps (1-3) should be performed in a sterile environment under a laminar flow hood.

1. Collagen Coating Transwells

1. Make a 30 µg/ml collagen solution. Dilute 3 mg/ml collagen stock 1:100 in 60% ethanol that has been passed through a 0.2 µm filter unit.
2. Vortex diluted 30 µg/ml solution. Note: It is not necessary to vortex the 3 mg/ml collagen stock solution, as this solution is quite viscous and air bubbles may be introduced, potentially decreasing accuracy when pipetting.
3. Remove 0.33 cm² growth area transwells with a pore size of 3 µm from outside packaging and place th....

Results

Several studies have demonstrated that pathogen-infected epithelial layers facilitate neutrophil trans-epithelial migration^{3,8,19,24-28,31,32}. This occurs via a pathogen-specific induction of an epithelial cell-derived neutrophil chemotactic gradient^3,23. For example, pathogenic P. aeruginosa interacting with the apical surface of lung epithelial cells causes a substantial number of neutrophils to migrate across the epithelial layer^{18,22,25,26,33,34}. This clinically relevant assa.......

Discussion

Neutrophil migration across mucosal epithelial surfaces is a common feature in disease pathology following infection with bacterial pathogens³. The methodology described herein offers a rapid, straightforward approach to experimentally isolate this discrete event using a human cell derived in vitro coculture assay system that models a feature of the inflammatory process triggered by bacterial infections. This system was originally developed using polarized intestinal epithelial cells infected with ent.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NCl-H292 cells	ATCC	CRL-1848
RPMI-1640 medium	ATCC	30-2001
Pseudomonas aeruginosa PAO1	ATCC	#47085
Escherichia coli MC1000	ATCC	#39531
D-PBS (1x) liquid	Invitrogen	14190-144	without calcium and magnesium
Heat Inactivated Fetal bovine serum	Invitrogen	10082-147	10% added to culture medium
Penicillin-Streptomycin	Invitrogen	15140-122	100x: 10,000 units of penicillin and 10,000 µg of streptomycin per ml.
Trypsin-EDTA (0.05%)	Invitrogen	25300-062	50 ml aliquots are stored frozen at -20 ºC.  Aliquot in use can be stored at 4 ºC short-term.
Hank's Balanced Salt Solution - HBSS(-)	Invitrogen	14175-079	Sterile, without calcium and magnesium
Trypan Blue Solution	Invitrogen	15250-061.	Stock = 0.4%
Collagen, Rat Tail	Invitrogen	A10483-01	Can also be isolated in the laboratory directly from the tails of rats using standard protocols
Citric acid	Sigma-Aldrich	C1909-500G	Component of 1 M citrate buffer and acid citrate dextrose (ACD) solution
Sodium Citrate	Sigma-Aldrich	S4641-500G	Component of 1 M citrate buffer
Dextrose anhydrous	Sigma-Aldrich	D8066-250G	Component of acid citrate dextrose (ACD) solution
Ammonium Chloride	Sigma-Aldrich	213330-500G	Component of red blood cell (RBC) lysis buffer
Sodium bicarbonate	Sigma-Aldrich	S6014-500G	Component of red blood cell (RBC) lysis buffer
EDTA	Sigma-Aldrich	ED-100G	Component of red blood cell (RBC) lysis buffer
HBSS(+) powder	Sigma-Aldrich	H1387-10L	Key component of HBSS+
HEPES	Sigma-Aldrich	H3375-500G	Component of HBSS+
Sigmacote	Sigma-Aldrich	SL2-25ML	Follow vendor instructions to coat glass pipette tips
Triton X-100	Sigma-Aldrich	T-9284
2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)	Sigma-Aldrich	A9941-50TAB	Key component of ABTS substrate solution
30% Hydrogen Peroxide Solution	Sigma-Aldrich	H1009-100ML	Component of ABTS substrate solution
N-Formyl-Met-Leu-Phe (fMLP or fMLF)	Sigma-Aldrich	F-3506	A Stock solution of 10 mM in DMSO should be prepared and aliquots stored at -20 ºC.
Gelatin Type B	Fisher Scientific	M-12026
Pseudomonas isolation agar	Fisher Scientific	DF0927-17-1	Follow manufacturer’s instructions to make PIA plates
Ficoll-Paque PLUS	Fisher Scientific	45-001-749	Optional, can improve neutrophil purity
24-well migration plate	Corning Incorporated	#3524
24-well wash plate	Falcon	35-1147	Can be reused if soaked in 70% ethanol and washed thoroughly prior to reuse
96-well plate	Fisher Scientific	#12565501
Transwell Permeable Supports	Corning Incorporated	#3415	Polycarbonate; Diameter: 6.5 mm; Growth area: 0.33 cm2; Dish style: 24-well plate; Pore size: 3.0 µm
Petri dish	Falcon	35-1013	Each Petri dish holds 24 inverted 0.33 cm2 Transwells.
500 ml 0.2 μm filter / flask	Fisher Scientific	09-740-25A	To sterilize acid citrate dextrose (ACD) solution
5-3/4 in glass Pasteur pipette	Fisher Scientific	13-678-20A	Coat tips with Sigmacote prior to use
Hemostat	Fisher Scientific	13-812-14	Curved, Serrated
Invertoskop Inverted Microscope	Zeiss	#342222
Versa-Max Microplate Reader	Molecular Devices	#432789