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Using a mouse model of coxsackievirus B4 (CVB4-V)-induced chronic pancreatitis; we investigated whether cytokines are involved in the progression of acute disease to chronic inflammatory disease. We show that IL-10 contributed to the development of chronic pancreatitis since acute disease resolved when IL-10 was absent or when IL-10 signaling was disrupted. We explored the underlying mechanisms by which IL-10 affected disease progression, using a novel approach to assess immunological events occurring in situ. Multiple markers that define functional innate immune responses and functional T cell responses were monitored over the course of CVB4-V infection of wild-type and IL-10 knockout mice, using a multiplex transcriptional profiling approach. We show that high levels of IL-10 early during infection were associated with delayed innate and T cell responses. Furthermore, high IL-10 production correlated with altered kinetics of T regulatory responses indicating a disruption in the balance between effector and regulatory T cell responses.
Chronic pancreatitis is a painful and debilitating disease, in which a progressive, destructive inflammatory process destroys the exocrine pancreas (Mergener & Baillie, 1997; Stevens, Conwell et al., 2004). Although gallstones and excessive alcohol consumption are important risk factors in adults, idiopathic disease represents a fairly high percentage (22%) of total cases (Lankisch, Burchard-Reckert et al., 1996; Lankisch, Schirren et al., 1991). Chronic pancreatitis is also a complex disease involving both genetic predisposition and environmental factors and develops from one episode of severe acute pancreatitis or from recurrent episodes of acute disease (Stevens, Conwell et al., 2004). In order to address fundamental questions of pathogenesis, we have developed unique mouse models of coxsackievirus B4-induced acute and chronic pancreatitis that share many pathological features with the corresponding clinical disease (Ramsingh, 2008).
Coxsackieviruses are small RNA viruses belonging to the genus enterovirus of the family Picornaviridae and are capable of inducing a wide range of diseases (Pallansch & Roos, 2007). Although infections with the group B coxsackieviruses (CVBs) in humans are generally asymptomatic, the group B viruses can occasionally cause inflammatory diseases of the pancreas, heart, and central nervous system. These viruses have been implicated in both acute and chronic inflammatory diseases of the pancreas (pancreatitis) and heart (myocarditis) (Ramsingh, 2008; Huber & Ramsingh, 2004; Chapman & Kim, 2008). Our model system relies on the use of two variants designated CVB4-P and CVB4-V. Both variants cause acute infections which result in the development of acute pancreatitis. The CVB4-V variant induces a severe pancreatitis while the CVB4-P variant induces a mild pancreatitis. The severity of acute pancreatitis is determined by the viral genotype (Caggana, Chan et al., 1993) and can be modulated by cytokines such as IL-12 and IFN-γ (Potvin, Metzger et al., 2003; Ramsingh, Lee et al., 1999). Cytokines can also modulate acute disease caused by other coxsackieviruses. Cytokines that are beneficial in the CVB3 model include IL-10 and IFN-γ. Recombinant CVB3 expressing IL-10 or IFN-γ protects mice against challenge with lethal CVB3 (Henke, Zell et al., 2001). While IL-10 and IFN-γ are beneficial during CVB3 infection, TNF-α and IL-1 are deleterious during the acute phase of infection. Exogenous administration of TNF-α and IL-1 during CVB3 infection induces myocarditis in otherwise disease resistant mice (Lane, Neumann et al., 1992).
While infection with CVB4-P results in a self-limiting disease, infection with CVB4-V progresses to chronic pancreatitis. Chronic pancreatitis, in this model, develops in the absence of viral persistence. The chronic phase of disease is characterized by prolonged inflammation, extensive tissue destruction, and pathological changes in the absence of infectious virus or viral RNA. Since cytokines are able to modulate CVB-induced acute disease, we investigated whether cytokines are also involved in the progression of acute pancreatitis to chronic pancreatitis. This was accomplished by initially comparing the profiles of 20 proteins (measured by Luminex) in pancreatic tissues from BALB/c mice infected with either CVB4-P or CVB4-V. The analysis allowed us to identify IL-10 as a potentially pathogenic cytokine during CVB4-V infection of BALB/c mice. We then showed that IL-10 must be contributing to the development of chronic inflammatory disease after CVB4-V infection of BALB/c mice infection, since acute pancreatitis resolved in the absence of IL-10 or when IL-10 signaling was disrupted. We explored the underlying mechanisms by which IL-10 affected disease progression using a novel approach to assess immunological events occurring in situ during CVB4-V infection of BALB/c and IL10 knockout (KO) mice. Multiple markers that define functional immune responses, such as TLR activation, Th17 responses, Th1 responses, Th2 responses, and regulatory T cell responses (Treg), were monitored over the course of CVB4-V infection in the two mouse strains, using a multiplex transcriptional profiling approach.
CVB4-V is a viral variant that was selected after multiple passages of the JVB strain of CVB4 in mouse pancreas (Ramsingh, Slack et al., 1989). Unlike the prototypical JVB strain of CVB4 which causes a transient acute pancreatitis, CVB4-V causes a severe acute pancreatitis that can progress to chronic pancreatitis. A large-scale stock of plaque-purified virus was grown in LLC-MK2(D) cells and viral infectivity was measured by plaque assay. Two strains of mice were used in this study. BALB/c mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were housed in a Specific Pathogen Free (SPF) facility at the Wadsworth Center (Albany, NY). IL-10 KO (IL-10−/−) mice on the C57BL/6 genetic background were bred onto the BALB/c background by Dr. William Lee (Wadsworth Center) for 11 generations. After 10 generations of traditional backcrossing, the recipient genome is 99.9% (www.criver.com). Thus, after 11 generations, the IL-10 KO mice have greater than 99.9% of the BALB/c genetic background. The IL-10 KO (BALB/c) strain was maintained in a SPF facility by Dr. David Lawrence (Wadsworth Center). Breeder pairs were kindly provided by Dr. Lawrence and IL-10 KO mice were bred and maintained in an SPF environment.
Five-to-six week old mice (15-18g) were used in the study. Mice were infected intraperitoneally with CVB4-V and were allowed to eat and drink ad libitum. Because male mice develop a more severe acute pancreatitis than female mice (Ramsingh, Lee et al., 1999), male mice were infected with 103 pfu of virus while female mice were given 104 pfu of virus. Mice were sacrificed at various time points after infection and organs were removed. Pancreatic tissues were fixed in Bouin's solution (Sigma-Aldrich, St. Louis, MO), processed for routine histology, and sections were stained with hematoxylin and eosin. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wadsworth Center.
Six-week old BALB/c mice (n=4) were infected intraperitoneally with 104 pfu of CVB4-V. Infected mice were injected intraperitoneally with a rat anti-mouse IL-10R antibody (1B1.3a; IgG1), kindly provided by Dr. Stephen Smiley (Trudeau Institute), on the day of infection (0.5 mg/mouse) and every three days (0.25 mg/mouse) thereafter, until 18 days post-infection (dpi). CVB4-V-infected mice in the control group were treated with an isotype control antibody. Disease severity was determined from body weight measurements and histological assessment of pancreatic tissues. Body weight measurements were done every other day. Mice were sacrificed 21 dpi and pancreatic tissues were processed for routine histology as described above.
Pancreatic tissues, harvested at various time points after infection, were frozen immediately on dry ice and placed in TRI reagent (Molecular Research Center, Inc. Cincinnati, OH) which combines phenol and guanidine thiocyanate in a monophase solution to inhibit RNase activity. Tissues were homogenized in a mini-beater (Biospec Products, Bartlesville, OK) using 1 mm Zirconia beads (Biospec Products). After a clarifying spin at 10,000×g for 10 min at 4°C, the homogenate was separated into aqueous and organic phases by the addition of 1-bromo-3-chloropropane (BCP) (Molecular Research Center) and centrifugation. RNA was precipitated from the aqueous phase by the addition of isopropanol, washed with ethanol, and resuspended in water. Residual DNA was digested with DNase (Promega, Madison, WI) and purified RNA was obtained using an RNeasy column (Qiagen, Valencia, CA).
Viral RNA in pancreatic tissues was monitored by RT-PCR using a TaqMan Gene Expression Assay, and copy number was determined by the absolute quantification assay. A standard curve was generated using viral RNA transcribed from a linearized plasmid DNA containing the full-length CVB4-V sequence. After treatment with DNase 1 and proteinase K, the concentration of viral RNA was measured with a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE) and viral copy number was calculated. In vitro transcribed viral RNA (3 ug) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Invitrogen, Carlsbad, CA) and random hexamers, in a total volume of 25 μl. Serial dilutions of viral cDNAs were prepared to generate the standard curve. Total pancreatic RNA (3 μg) from test samples was reverse transcribed under the same conditions as were used for the viral RNA standard. Serially diluted viral cDNA standards and pancreatic test cDNA samples were loaded onto a single 96-well plate. A region of the VP1 sequence was amplified with two unlabeled PCR primers (5’-TGAGCAAATCCCAGCTCTGA, 5’-TGGATCCACCTGGGAAGTATG) and a FAM-dye-labeled TaqMan probe (5’-AGCTGTGGAGACTGG) in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) using the Absolute Quantification software. A standard curve (threshold cycle number (Ct) vs viral copy number), generated from the serially diluted viral cDNAs, was used to determine the viral copy number in the pancreatic test samples.
Viral load is shown as copy number per pancreas instead of per gram tissue because the isolation of intact pancreatic RNA requires rapid harvesting and processing in order to overcome the inherent problem of high RNase levels in the pancreas. Pancreases that are processed for RNA isolation are not weighed because any delay in processing activates pancreatic RNases, resulting in degraded RNA samples.
Gene expression was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a TaqMan Low Density Array (LDA), a 384-well micro fluidic card, preloaded with appropriate PCR primers and probes (Applied Biosystems). Total pancreatic RNA was reverse-transcribed with M-MLV RT and random hexamers in a reaction volume of 25 μl using standard conditions. Briefly, random hexamers (Invitrogen, Carlsbad, CA) were added to 3 μg total RNA which was denatured at 90°C. Samples were frozen immediately on dry ice and then thawed slowly on ice. After the addition of a cocktail containing dNTPs (1mM) (Roche, Basel, Switzerland), RNasin (Promega, Madison, WI), DTT (0.1M), and M-MLV RT, the reaction was incubated at 37°C, heat-inactivated at 70°C, and cDNAs were stored at −80°C. 100 ng of cDNA were mixed with Taqman Fast Universal PCR Master Mix (Applied Biosystems) to a final 1X concentration and approximately 2 ng of the cDNA solution were applied to each of 48 wells in the LDA card. PCR amplification was carried out in an Applied Biosystems 7900HT Fast Real-Time PCR System. Eight pancreatic samples were analyzed simultaneously for the expression of 47 genes and 18S ribosomal RNA in one 384-well LDA card. The LDA card contained three possible controls, 18S RNA, Egf, and Slc2a2/Glut-2. The expression of Egf remained unchanged during CVB4-V infection while the expression of both 18S RNA and Slc2a2 varied over the course of the infection. As a result, expression of test genes was normalized to that of Egf. The expression of one additional gene, RORγt, not present on the LDA card, was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR in a single-tube format, with the Applied Biosystems 7500 Real-Time PCR System. The expression of RORγt was again normalized to that of Egf.
The study focused on pancreatic gene expression at multiple time points in two treatment groups, CVB4-V-infected BALB/c mice and CVB4-V-infected IL-10 KO mice. Three to five mice per strain were analyzed at each time point and gene expression was quantified by a comparative Ct method (Applied Biosystems). The Ct value of each test gene in each organ was normalized to that of the control gene, Egf. The resulting [delta] Ct values for samples from each time point were then averaged. Since the goal of the study was to evaluate the magnitude of gene expression over time within a single treatment group and between treatment groups, a reference group was needed. The reference group chosen for all except one test gene was CVB4-V-infected BALB/c mice, 2 dpi, because gene expression was very low or undetectable prior to this time point. For the one other test gene, RORγt, the reference group was CVB4-V-infected BALB/c mice at 4 dpi. The change in gene expression is represented as a fold-change which was calculated by determining 2–ddCt (ddCt = averaged [delta]Ct (specific time point)- averaged [delta]Ct BALB/c 2dpi). As a result, for all genes except RORγt, the relative expression in CVB4-V-infected BALB/c mice at 2 dpi is 1. For RORγt, the relative expression in CVB4-V-infected BALB/c mice at 4 dpi is 1.
Organs, harvested at various time points after infection, were frozen immediately on dry ice and placed in 1ml of lysis buffer containing protease inhibitors (50mM Tris, 150mM NaCl, 2mM EDTA, 1% NP40, 30μM PMSF, 420μM leupeptin, 0.02% aprotinin, 3mM NaN3). Organs were homogenized in a mini-beater (Biospec Products, Bartlesville, OK) using 1 mm Zirconia beads (Biospec Products). After a clarifying spin at 10,000×g for 10 min at 4°C, the supernatants were stored frozen at −80°C. Cytokines and chemokines in pancreatic homogenates were measured using a multiplex bead-based assay, Luminex (Invitrogen), according to the manufacturer's protocol. Three to six organs were analyzed individually at each time point.
For some genes, the kinetics of peak mRNA expression differed between CVB4-V-infected BALB/c mice and CVB4-V-infected IL-10 KO mice. The variable nature of the kinetics of gene expression was captured by clustering cytokines on an arbitrary weighting of three characteristics of the profile during the early viral phase of disease (1-8 dpi): time of peak occurrence (P, 10%); percent area of the peak (A, 20%); and shift in day of maximal response (S, 70%). The results are insensitive to changes in the weightings. The resulting cytokine scores (0.1P+0.2A+0.7S) were then subjected to an agglomerative hierarchical clustering algorithm (Kaufman, L. et al., 1990) using a Euclidean distance metric between the scores. The clustering analysis allowed us to identify four kinetically distinct groups of genes in infected BALB/c mice (Table 1).
We undertook a comparative analysis of multiple cytokines in pancreatic tissues to identify cytokines whose profiles differed during infection with CVB4-P and CVB4-V. Twenty proteins were measured by Luminex at different times after infection with either CVB4-V or CVB4-P and the data is shown on a separated log scale in Fig. 1A. A distance metric was calculated to indicate differences in levels of cytokine expression during CVB4-P and CVB4-V infection. Based on the score of the distance metric, the expression patterns of the twenty proteins were sorted into four groups (A, B, C, and D). The group A proteins (IL-4, VEGF, IFN-γ, IL-13, IL-6, IL-1b, and GM-CSF) had scores of less than 2.5 indicating that expression profiles during CVB4-P and CVB4-V infection were similar. The group B proteins (FGF, MIG, TNF-α, IP-10, KC, and IL-2) had scores between 2.5 and 5.0 indicating that expression profiles during CVB4-P and CVB4-V infection were different. The group C proteins (MIP-1α, MCP-1, IL-17, IL-1a) had scores of between 5.0 and 10.0 indicating that expression profiles during CVB4-P and CVB4-V infection were highly divergent. The most divergent group, group D, contained three proteins, IL-12, IL-5, and IL-10. Of these three proteins, the profiles for IL-10 were the most different with a distance metric score of 23.8. IL-10 production during viral infection was also plotted on a linear scale (Fig. 1B). During CVB4-V infection, IL-10 production was high and sustained. IL-10 was detected as early as 1 dpi, increased to very high levels from 2 to 6 dpi, and subsequently decreased by 8 dpi. IL-10 production during CVB4-P infection was transient and relatively low. Peak expression was observed at 4 dpi and baseline IL-10 levels were restored by 6 dpi.
This study, using male BALB/c mice, identified IL-10 as a potentially pathogenic cytokine during CVB4-V-induced disease. Since both CVB4-V-infected male and female mice progress to chronic pancreatitis, the prediction is that IL-10 production in infected female mice would be similar to that observed in infected male mice. IL-10 production in CVB4-V-infected female mice was also high and sustained (data not shown). Subsequent studies used female mice since they show less morbidity than male mice after CVB4-V infection.
The differential production of IL-10 during infection with CVB4-P or with CVB4-V suggested that IL-10 is pathogenic during CVB4-V infection. To test this hypothesis, we evaluated the course of pancreatitis in CVB4-V-infected IL-10 KO (BALB/c genetic background) mice. Disease severity was monitored by body weight and by histological assessment of pancreatic tissues since weight loss and destroyed exocrine tissues are reliable indicators of pancreatitis in this model. Unlike CVB4-V infection of BALB/c mice, CVB4-V infection of IL-10 KO mice resulted in an acute pancreatitis which subsequently resolved (Fig. 2). At 7 dpi, pancreatic tissues from both IL-10 KO and BALB/c mice showed a generalized inflammation, widespread coagulative necrosis, loss of acinar cells, and acinoductular metaplasia. Acinar cell necrosis is characterized by pyknotic nuclei, eosinophilic cytoplasmic content and cellular debris. At 7 dpi, the main difference between the two strains was a less extensive loss of acinar cells in the IL-10 KO mice (75% loss) than in the BALB/c mice (100% loss). By 14 dpi, the exocrine tissues of IL-10 KO mice were beginning to recover and showed an increase in acini. At this time point, about 50% of the exocrine tissues in the IL-10 KO mice appeared normal and contained intact acini. Fat cells appeared to replace destroyed acini. At the same time point, BALB/c mice showed no signs of recovery. By 21 dpi, the exocrine tissues of IL-10 KO mice had almost completely recovered and contained abundant intact acini. Pockets of residual fat cells were observed among morphologically normal acini. At the same time point, signs of recovery were notably absent in BALB/c mice. Pathological changes (coagulative necrosis, generalized inflammation, acinoductular metaplasia) in the pancreas were observed against a background of fibrosis. Disease severity as determined by histological assessment of pancreatic tissues was corroborated by measurements of body weight. During the 3-week follow-up period, CVB4-V-infected IL-10 KO mice, which had less overall pancreatic tissue damage, gained weight while CVB4-V-infected BALB/c mice, which had extensive pancreatic tissue damage, lost weight (Fig. 3).
We then validated the data from the IL-10 KO mouse model by blocking the signaling of IL-10, via its receptor, in CVB4-V-infected BALB/c mice, by means of an anti-IL-10R antibody. The number of mice treated with the antibody was small (n=4) because of the relatively large amount of total antibody (2 mg) needed per mouse. As was observed with CVB4-V-infected IL-10 KO mice, anti-IL-10R-treated, infected BALB/c mice showed an initial weight loss during the first week of infection followed by weight gain (Fig. 3). Infected mice treated with a control antibody lost weight during the 3-week follow-up period. Pancreatic tissues, harvested from anti-IL-10R-treated CVB4-V-infected BALB/c mice at 21 dpi, showed signs of recovery. Representative tissue sections are shown in Fig. 2 (panels i and j). Exocrine pancreatic repair, denoted by the presence of morphologically intact acini and the absence of characteristic pathological changes associated with chronic pancreatitis, was evident in anti-IL-10R-treated mice. The extent of tissue repair in anti-IL-10R-treated mice was variable. For example, in some samples, 50% of the exocrine pancreas showed recovery (Fig. 2j) while in other samples, almost 100% of the exocrine pancreas showed recovery (Fig. 2i). Pockets of fat cells and residual focal inflammation were noted within the exocrine tissues that showed 50% recovery, but these characteristics were absent in tissues that showed complete recovery. CVB4 infection in the anti-IL-10R-treated mice was verified by measurement of anti-viral antibodies by ELISA. All anti-IL-10R-treated mice developed anti-CVB4 antibodies (data not shown).
Although it is widely known that IL-10 deficient mice develop chronic enterocolitis (Pestka, Krause et al., 2004; Kuhn, Lohler et al., 1993), an important point is that non-pathogenic enteric bacteria play a key role in the development of enterocolitis in these mice (Werner, Shkoda et al., 2007). The IL-10 KO mice used in this study were reared in an SPF facility and did not develop enterocolitis during the course of the study which ended when mice were three months old. The intestines from mock-infected mice and from CVB4-V infected mice (35 dpi) appeared similar and showed characteristic features of healthy, non-inflamed tissues (Fig. 2, panels k and l). The mucosa, sub-mucosa, and muscularis externa appeared intact. Villi that project into the lumen were tightly opposed and showed no signs of damage. Inflammation was notably absent in the small intestine of both mock-infected- and CVB4-V-infected IL-10 KO mice.
In the current study, viral RNA copy number was used to monitor viral load in the pancreatic tissues of infected mice (Fig.4). The results of the viral load data for the IL-10 KO mice were confirmed by monitoring the infectivity of selected samples by plaque assay. Viral titers peaked between 2 dpi and 4 dpi in both BALB/c and IL-10 KO mice. Although the kinetics of viral replication was similar in the two strains of mice, there were two notable differences. First, overall viral replication was lower in the IL-10 KO mice. Second, viral clearance was faster in IL-10 KO mice than in BALB/c mice. Infectious virus (limit of detection is 60 pfu per organ) was not detected at 14 dpi in IL-10 KO mice. In BALB/c mice, infectious virus is cleared by 21 dpi (Ramsingh, Lee et al., 1999).
Pancreases from CVB4-V-infected BALB/c mice decrease in weight between 4 and 7 dpi. By 7 dpi, pancreases from CVB4-V infected BALB/c mice are, on average, one-third the weight of those from uninfected mice or mice undergoing pancreatic repair. If viral load were reported as copy number per gram of tissue, the difference in viral load between BALB/c and IL-10 KO mice would be even more pronounced, but the two main observations would remain unchanged. Of interest is the correlation between viral load (Fig. 4A) and IL-10 production during CVB4-V infection of BALB/c mice (Fig. 1B). IL-10 production was highest during the early viral phase of disease (2-6 dpi) when viral loads were high. A summary of the characteristic features of CVB4-V infection of BALB/c mice, resulting in the development of chronic pancreatitis, and of CVB4-V infection of IL-10 KO mice, resulting in disease resolution, is shown in Figure 4B.
A question that arises from this study is whether faster viral clearance is responsible for disease resolution in the IL-10 KO mice. We have analyzed viral replication in several KO mouse models, namely, CD4 KO, IL-4 KO, and IFN-γ KO mice (Ramsingh, Lee et al., 1999) and we have shown that viral clearance is faster in the KO strains than in wild-type BALB/c mice. The main difference noted among the KO strains is that, whereas the IL-10 KO mice were able to resolve virus-induced acute inflammatory disease, the other KO strains were unable to resolve acute pancreatitis; instead, they progressed to chronic inflammatory disease similar to that observed in wild-type BALB/c mice. The combined data indicate that factors other than viral load and clearance must control disease progression.
The goal of this portion of the study was to explore the effects of IL-10 on various immune responses by monitoring the expression of specific markers associated with functional responses. Since IL-10 is generally considered to be an anti-inflammatory cytokine that affects both innate and T cell responses, we examined the expression of genes associated with innate immune responses (TLRs, CC chemokines, CXC chemokines, and markers expressed by macrophages, neutrophils, and NK cells) and genes associated with T cell responses (cytokines, cytokine receptors, transcription factors). The approach relied on monitoring gene expression using a PCR-based assay in a low-density microarray format (44 test genes) and in a single-sample (1 test gene) format. The expression of 45 test genes in pancreatic tissues was evaluated at various time points after CVB4-V infection of BALB/c and IL-10 KO mice. The impact of high IL-10 on gene expression profiles during the early and late viral phases of disease was evaluated by comparing expression profiles between CVB4-V-infected BALB/c mice and CVB4-V-infected IL-10 KO mice, from 1 dpi to 14 dpi.
The three main observations from the mRNA expression data are (a) all 45 test genes were expressed at higher levels in infected BALB/c mice than in infected IL-10 KO mice, (b) the pattern of gene expression, monophasic or biphasic, was similar between the two strains of mice, and (c) the kinetics of peak expression of some genes differed between the two strains of mice. Higher expression of immune markers probably reflects the increased viral replication observed in CVB4-V-infected BALB/c mice. The magnitude of gene expression in CVB4-V-infected IL-10 KO mice is probably underestimated in this study. The accumulation of mRNA at a given time point reflects the dynamic balance between transcriptional control and mRNA stability. In addition, steady-state levels of mRNAs are influenced by the events occurring in the affected organ, such as inflammation and tissue repair of the exocrine pancreas. During CVB4-V infection of BALB/c mice, the mRNA contribution from the pancreas must have remained constant over the 2-week period of study, given that the exocrine tissues are completely destroyed 2 days after infection. During CVB4-V infection of IL-10 KO mice, the mRNA contribution from the pancreas changed because of decreased tissue damage (75% in the KO versus 100% in BALB/c mice at 7 dpi) and tissue repair (50% tissue recovery at 14 dpi). The temporal change in mRNA contribution from the pancreas in infected IL-10 KO mice affects the magnitude of expression profiles. As a result, peak expression of immune markers during the early viral phase of disease in the IL-10 KO mice is probably underestimated by roughly 25% while peak expression during the late viral phase of disease is probably underestimated by roughly 50%.
Since the kinetics of peak expression of some genes differed between infected BALB/c and IL-10 KO mice, a clustering algorithm was used to identify groups of genes with similar kinetic profiles. The analysis identified four groups of genes (Table 1) which were also sorted according to function (Table 2). Group 1 contains 17 genes whose peak expression occurred at the same time (4 dpi) in the two strains of mice. The group 1 genes consist of markers of TLR, Th17 and Th1 responses. Group 2 contains 22 genes whose peak expression is delayed in BALB/c mice. The group 2 genes consist of markers of TLR, Th17, Th1, and Th2 responses. Group 3 contains 4 genes (exhibiting a monophasic pattern) whose peak expression is earlier in BALB/c mice than in IL-10 KO mice. The group 3 genes consist of markers of Th17 responses. Group 4 contains 2 genes (exhibiting a biphasic pattern) whose peak expression is earlier in BALB/c mice than in IL-10 KO mice. The two genes, foxp3 and il13, are markers of regulatory responses. Representative expression profiles of genes in each of the four groups are depicted in Figure 5. To determine if the RNA expression profiles correlated with protein profiles, we analyzed representative cytokines and chemokines in CVB4-V infected BALB/c mice. Representatives of the group 1 genes, TNF-α and CXCL10, yielded protein profiles that correlated with their respective RNA profiles, peaking at 4 dpi (Fig. 5). A representative of the group 2 genes, CXCL9, also yielded a protein profile that correlated with the RNA profile, peaking at 6 dpi. A representative of the group 3 genes, IL-6, also yielded a protein profile that correlated with the RNA profile, peaking at 2 dpi. The overall data show that high IL-10 is associated with delays in maximal expression of markers for inflammatory and T cell responses during CVB4-V infection of BALB/c mice. In addition, high IL-10 is associated with alterations in the kinetics of regulatory responses.
The present study shows, for the first time, that IL-10 must play a critical role in the development of chronic inflammatory disease in the pancreas, subsequent to CVB4-V infection, since the absence of IL-10 or disruption of signaling via IL-10 resulted in the resolution of acute disease. We also show that high levels of IL-10 early during CVB4-V infection were associated with delayed innate and T cell responses. Furthermore, high IL-10 production correlated with altered kinetics of Treg responses indicating a disruption in the balance between effector T cell responses and regulatory T cell responses.
A key question in the study of CVB4-V-induced chronic pancreatitis is whether viral persistence contributes to the development of chronic disease. Viral persistence may be due to continuous production of low levels of infectious virus or to the prolonged presence of viral RNA, capable of generating low levels of viral proteins that continue to stimulate T cells. The question is relevant in light of studies with certain strains of LCMV that cause long-term persistence with viremia. When the IL-10 pathway is disrupted, LCMV strains that would normally establish persistence are cleared from blood and organs of infected mice (Brooks, Trifilo et al., 2006; Ejrnaes, Filippi et al., 2006). The evidence suggesting that viral persistence does not contribute to the development of chronic pancreatitis in our model is as follows. Firstly, amplified VP1 sequences in pancreatic tissues of CVB4-V-infected mice are not detected after infectious virus is cleared (A. Ramsingh, unpublished observations). Secondly, the target cells for CVB4-V in the pancreas are the acinar cells which are completely destroyed by 14 days after infection (Ostrowski, Reilly et al., 2004), resulting in the absence of a reservoir in which CVB4-V can persist. Finally, disruption of IL-10 signaling in CVB4-V-infected BALB/c mice prevented the development of chronic disease. The combined data indicate that, in this model, IL-10, and not viral persistence, is critical in the development of chronic pancreatitis.
The finding that IL-10 plays a critical role in the development of chronic inflammatory disease in the pancreas is surprising, given that IL-10 is an anti-inflammatory cytokine that regulates innate and adaptive Th1 and Th2 responses (Pestka, Krause et al., 2004; Couper, Blount et al., 2008). IL-10 plays a key immunoregulatory role during infections: this cytokine functions to suppress proinflammatory responses during an infection to reduce immunopathology. Ablation of IL-10 signaling generally results in severe, often fatal immunopathology in several infections, including Toxoplasma gondii (Gazzinelli, Wysocka et al., 1996; Wilson, Wille-Reece et al., 2005), malaria (Li, Corraliza et al., 1999; Li, Sanni et al., 2003), and Trypanosoma cruzi (Hunter, Ellis-Neyes et al., 1997). The timing of the production of IL-10 must be well-controlled because mistimed and/or excessive production of this cytokine will suppress the very proinflammatory responses that are needed to clear the pathogen (Couper, Blount et al., 2008). Mistimed and/or excessive IL-10 production can lead to a pathogen escaping immune control and subsequent fulminant and fatal or chronic non-healing infection. One such example is Mycobacterium avium infection of BALB/c mice; this infection is associated with very early IL-10 production and the failure to control the infection (Roque, Nobrega et al., 2007).
A relevant issue is whether early high production of IL-10 is the consequence of a high pathogen burden or whether IL-10 production is a function of pathogen virulence. If IL-10 production were the consequence of a high pathogen burden, then the kinetics of IL-10 production should correlate with the kinetics of viral replication, independently of pathogen virulence. We have shown that the kinetics of replication for both CVB4-P and CVB4-V are similar (Ostrowski, Reilly et al., 2004; Ramsingh, Lee et al., 1997; Ramsingh, Lee et al., 1999). While the kinetics of IL-10 production during CVB4-V infection correlates with the kinetics of viral replication, the kinetics of IL-10 production during CVB4-P infection does not correlate with the kinetics of viral replication. In our model, IL-10 production appears to be a function of pathogen virulence, with early high production of this cytokine during infection with the virulent CVB4-V variant and low transient production during infection with the less virulent CVB4-P variant.
Our study used a novel approach to assess immunological events occurring in situ. Multiple markers that define functional immune responses, such as TLR activation, Th17 responses, Th1 responses, Th2 responses, and Treg responses, were monitored over the course of infection by means of a multiplex transcriptional profiling approach. An important issue is whether conclusions concerning basic immunological mechanisms can be made based on this approach. Two pieces of evidence suggest that a multiplex transcriptional profiling approach is useful for gaining insight into immunological events occurring in situ. Firstly, we showed that representative RNA expression profiles correlate with protein profiles of cytokines and chemokines. Secondly, published data from a variety of experimental approaches (KO mouse models, phenotypic analysis of inflammatory infiltrates, cytokine treatment, in vivo depletion of cytokines, and in vivo depletion of T cell subsets) show that Th1 responses, while beneficial during the viral phase of disease, contribute to disease progression (Ramsingh, Lee et al., 1999; Potvin, Metzger et al., 2003; Ramsingh, Lee et al., 1997). The current study also identified markers of Th1 responses as being important in the disease process; thereby providing support for the idea that a multiplex transcriptional profiling approach is informative for assessing immunological events occurring in situ.
A key finding of the study is that the production of high IL-10 during CVB4-V infection is associated with a delay in the peak expression of several genes that function in innate and adaptive immune responses. Group 2 genes consisting of markers of TLR activation, Th17 responses, Th1 responses, and Th2 responses exhibited delayed maximal expression in infected BALB/c mice when compared to those in infected IL-10 KO mice (Table 1). Markers of TLR activation include tlr4, tlr7, tlr9, and myd88. TLR4 is a surface receptor that binds LPS while TLR7 and TLR9 are endosomal receptors that bind ssRNA and CpG DNA, respectively (Sabroe, Parker et al., 2008; Thompson & Iwasaki, 2008). All three TLRs signal via the adaptor MyD88. TLR signaling via MyD88 can activate NF-κB, leading to a proinflammatory response due to transcription of numerous genes, including those encoding cytokines and chemokines (Liu & Malik, 2006). While TLR4 and TLR7 have been identified by others (Huber, 2008; Triantafilou, Orthopoulos et al., 2005) as initiating the inflammatory response during CVB infection, the present report is the first to link TLR9 to CVB infection. Studies of CVB3 infection of cardiac cells show that TLR8, and to a lesser extent TLR7, initiate the inflammatory response via recognition of ssRNA (Triantafilou, Orthopoulos et al., 2005). TLR4 can also trigger a low-level inflammatory response, indicating that the CVB3 virion itself is recognized by the cell surface receptor (Huber, 2008). A delay in TLR activation during CVB4-V infection of BALB/c mice is expected to delay proinflammatory responses needed to contain viral infection. Consequences of delayed proinflammatory responses are higher viral loads and delayed viral clearance, both of which are observed during CVB4-V infection of BALB/c mice. Markers of various T cell responses, including cytokines, chemokines, and receptors, also exhibited delayed maximal expression during CVB4-V infection of BALB/c mice. In addition, genes encoding transcription factors for specific T cell subsets, such as RORγt (Th17), T-bet (Th1), and GATA-3 (Th2), displayed delays in peak expression. The data support the conclusion that proinflammatory and T cell responses are delayed in the presence of high IL-10 during CVB4-V infection of BALB/c mice. Given that IL-10 production is a function of pathogen virulence in this model, the implication is that the virulent CVB4-V variant induces early high production of IL-10, and this production delays proinflammatory and T cell responses, thereby providing the opportunity for the virus, early in the infection cycle, to replicate rapidly and spread throughout the exocrine pancreas. Generalized infection of the pancreas yields more severe tissue injury and heightened immune responses than limited focal infection and leads to extensive tissue destruction and the development of severe acute pancreatitis. In the absence of IL-10, proinflammatory and T cell responses were not delayed, virus replication was decreased, and less tissue damage was observed during the acute phase of disease.
Another finding of the present study is that high IL-10 production correlated with altered kinetics of Treg responses indicating a disruption in the balance between effector and regulatory T cell responses. Since CVB4-V infection of IL-10 KO mice resulted in viral clearance, tissue repair, and the absence of immunopathology, the implication is that infected IL-10 KO mice mounted effective, well-regulated immune responses against the infection. The assumption is that responses that peaked during the early viral phase of disease (first week after infection) are indicative of effector responses while responses that peaked during the late viral phase of disease (second week after infection) are indicative of regulatory responses. In infected IL-10 KO mice, genes that displayed maximal expression early in infection (4 dpi) were those that are indicative of TLR, Th17, Th1, and Th2 effector responses. Two genes, foxp3 and il13, showed maximal expression later in infection (10 dpi). Foxp3 is a key transcription factor of Treg cells (Shevach, 2009; Zhou, Chong et al., 2009; Curotto de Lafaille & Lafaille, 2009) while IL-13 can induce the development of CD25+ Treg cells (Skapenko, Kalden et al., 2005) and of alternatively activated macrophages (Martinez, Helming et al., 2009). Alternatively activated macrophages have a regulatory function since they suppress Th1 responses and favor tissue repair. High expression of foxp3 and il13 in infected IL-10 KO mice correlated with diminishing effector responses. Unlike the findings in IL-10 KO mice, expression of both foxp3 and il13 in infected BALB/c mice was maximal early in infection (6 dpi) and remained high during the late viral phase of disease. High expression of foxp3 and il13 in infected BALB/c mice did not correlate with diminishing effector responses. The data suggest that the dynamic balance between effector T cell responses and regulatory T cell responses is preserved in infected IL-10 KO mice and is disrupted in infected BALB/c mice. Although one mechanism by which regulatory T cells exert their suppressive function is the production of IL-10, the expression profiles of IL-10 (Fig. 1) and foxp3 (Fig. 5) in infected BALB/c mice do not correlate with one another. Our working hypothesis is that the combination of a virulent lytic virus and high early IL-10 production in pancreatic tissues causes severe acute pancreatitis and a cytokine environment that favors disease progression. The progression of acute disease to chronic disease is due to disruption in the balance between effector and regulatory T cell responses leading to the failure of regulatory T cells to inhibit effector T cells. If this is true for clinical chronic pancreatitis, then immunomodulatory approaches may be useful in halting disease progression. Ongoing studies of our model system are exploring immunomodulatory strategies to restore the balance between effector and regulatory T cell responses during the chronic phase of disease to determine whether damaged pancreatic tissues can be restored.
This work was supported by Public Health Service grant AI066938 from the National Institutes of Health and by research funding from Centocor Inc. (Malvern, PA).
The anti-IL-10R antibody was kindly provided by Dr. Stephen Smiley (Trudeau Institute, Saranac Lake, NY). The technical assistance of Xiaoyan Huang was greatly appreciated. We thank Helen Johnson (Zoonotic Diseases Laboratory, Wadsworth Center) for processing tissue samples for histology. We also acknowledge the secretarial assistance provided by Sandra Lionarons and Kathleen Vindittie.
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