Dynamics of molecular responses to coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis

Abstract

A coxsackievirus B4 induces acute pancreatitis with different outcomes. The study utilized a systems biology approach to identify molecular immune responses that differentiate between disease resolution and disease progression. The data establish a temporal pattern of host responses that differentiate the resolution of acute pancreatitis from the progression to chronic pancreatitis. A group of twenty-five genes exhibited characteristic expression profiles that were observed during the development of chronic pancreatitis but not during the resolution of disease. We postulate that the temporal dynamics of the twenty-five genes influence the development of pathogenic immune responses associated with chronic pancreatitis. Furthermore, a subset of eleven genes exhibited increased expression as viral titers waned. Of the eleven gene products, five are secreted molecules, TNF-α, IFN-γ, CXCL10, IL-10, and IL-22b, and represent novel potential therapeutic targets since they can be readily modulated with antibodies against the specific cytokine/chemokine or with antibodies against the corresponding receptors.

FIG. 1

Patterns of gene expression profiles after CVB4-V infection of BALB/c and IL-10 KO mice. Three general patterns of gene expression (#1, #2, #3) were identified in the transcriptional profiling studies. Genes displaying profiles #2B, #3A, and #3B were unique to CVB4-V-infected BALB/c mice and were identified as correlates of disease progression.

FIG. 2

Relative change in expression profiles of functional groupings of genes after CVB4-V infection of BALB/c and IL-10 KO mice. A. TLR related grouping; B. A miscellaneous grouping consisting of three genes, Gata3, Socs3, and Il10. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression, for both BALB/c and IL-10 KO mice, is represented as a fold-change relative to the expression level of a reference group which is BALB/c mice at 2dpi. As a result, the relative expression of a gene in BALB/c mice at 2dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.

FIG. 3

Relative change in expression profiles of TH17 related genes after CVB4-V infection of BALB/c and IL-10 KO mice. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression is represented as a fold-change relative to the expression level of a reference group. For all genes except RORγt, the reference group is BALB/c at 2dpi. For RORγt, the reference group is BALB/c at 4 dpi. As a result, the relative expression of a gene, except for RORγt, in BALB/c mice at 2dpi is 1. The relative expression of RORγt in BALB/c mice at 4dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.

FIG. 4

Relative change in expression profiles of TH1 related genes after CVB4-V infection of BALB/c and IL-10 KO mice. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RTPCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression is represented as a fold-change relative to the expression level of a reference group, CVB4-V-infected BALB/c at 2dpi. As a result, the relative expression of a gene in BALB/c mice at 2dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.

FIG. 5

Kinetics of expression of four cytokines, CXCL10, TNF-α, IFN-γ, and IL-10, in the pancreas of CVB4-V-infected BALB/c or IL-10 KO mice. Three-to-five mice per strain were analyzed at each time point and the amount of cytokines/chemokines present in pancreatic homogenates was measured using a Luminex assay. Mean values are shown. The overall kinetics of protein expression in both strains of mice is similar to that of the corresponding RNA. Statistical analysis was done using the Mann-Whitney U test. Statistically significant differences (P < 0.05) in protein expression between the two mouse strains are indicated by asterisks.

FIG. 6

Simultaneous administration of anti-TNF-α and anti-IFN-γ antibodies had a beneficial effect on chronic pancreatitis. Panels a-d depict representative hematoxlyin and eosin-stained pancreatic sections from CVB4-V infected mice. a. Normal pancreatic tissue from mock-infected BALB/c mice. b. Pancreatic tissue from CVB4-V infected BALB/c mice depicting chronic pancreatitis characterized by exocrine tissue destruction and inflammation. c. Pancreatic tissue from CVB4-V infected IL-10 KO showing disease resolution. Exocrine tissues are largely intact and inflammation is absent. d. Pancreatic tissue from CVB4-V-infected BALB/c mice treated with anti-TNF-α and anti-IFN-γ antibodies. Multiple foci of normal acini are present. A, acinus; IL, islet of Langerhans; f, fat replacement; an, acinar cell necrosis; in, inflammatory infiltrates. Magnification, X250.