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Originally published online as doi:10.1189/jlb.0606396 on April 24, 2007

Published online before print April 24, 2007
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(Journal of Leukocyte Biology. 2007;82:311-319.)
© 2007 by Society for Leukocyte Biology

Maturation of the mucosal immune system underlies colitis susceptibility in interleukin-10-deficient (IL-10–/–) mice

Michele R. Etling*, Sarah Davies{dagger}, Melanie Campbell{dagger}, Raymond W. Redline*, Pingfu Fu{ddagger} and Alan D. Levine*,{dagger},{ddagger},§,1

* Departments of Pathology,
{dagger} Medicine, and
§ Pharmacology and the
{ddagger} Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA

1 Correspondence: Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4952, USA. E-mail: alan.levine{at}case.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated mucosal IL-12/23p40 and IFN-{gamma} accompany early inflammation in IL-10-deficient (IL-10–/–) mice and then later decline while inflammation persists. This report addresses whether this cytokine profile reflects disease progression or inherent, age-related changes in mucosal immunity. IL-10–/– and wild-type (WT) mice were maintained in an ultrabarrier facility or transferred to conventional housing at 3, 12, or 30 weeks of age. Weight, stool changes, and histologic features were followed. Lamina propria mononuclear cells were cultured for cytokine analysis by ELISA. Ultrabarrier-housed IL-10–/– mice are statistically indistinguishable from WT mice by weight, disease activity index, and histologic inflammation. IL-10–/– mice but not WT, transferred at 3 weeks, develop colitis gradually, reaching a significant, sustained maximum by 15 weeks of age. Transfer at 12 weeks induces rapid disease onset in both strains, maximal at 15 weeks of age. Inflammation persists in IL-10–/–, and WT recover. IL-10–/– and WT mice transferred at 30 weeks demonstrate transient diarrhea and weight loss but no chronic inflammation. Probiotics delay symptom onset only in the 12-week-old group. IFN-{gamma} production from ultrabarrier-housed IL-10–/– mice is elevated at 12 weeks of age, and older animals have decreased IFN-{gamma} and increased IL-4. IL-10 is important for suppressing inflammation after transfer at 3 weeks of age and limiting inflammation after transfer at 12 weeks but has little influence at 30 weeks of age. Colitis onset, progression, and response to probiotic therapy vary with immune system age, suggesting that a distinct, Th1-driven, age-dependent cytokine profile may contribute to increased colitis susceptibility in otherwise healthy mice.

Key Words: aging • cytokine • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although a variety of host immune factors interacting with microbial, dietary, and other environmental agents has been implicated, the precise mechanisms underlying the development and perpetuation of inflammatory bowel disease (IBD) have not been elucidated [1 ]. A number of animal models have proven useful to study immune and nonimmune host responses leading to chronic intestinal inflammation [2 , 3 ]. Among them, the IL-10-deficient (IL-10–/–) mouse model was characterized initially as a chronic model of enterocolitis caused by an unregulated Th1 response, with high levels of IL-12 driving the production of IFN-{gamma} to maintain chronic inflammation [4 5 6 ]. In many of these animal models, IL-12 was implicated as a major mediator of colitis based on the ability of neutralizing antibody to the p40 subunit of IL-12 to prevent or reverse intestinal inflammation. As IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAb used in mouse colitis models neutralized the activities of IL-12 and IL-23, the importance of IL-23, relative to IL-12, in chronic intestinal inflammation in the IL-10–/– mouse model and others had remained under-reported [7 8 9 ]. The disease is also dependent on the presence of enteric flora, as animals maintained in a sterile, gnotobiotic facility do not develop inflammation [10 ]. It is interesting that although prophylactic administration of exogenous IL-10 or anti-IL-12/23p40-blocking antibody was shown to prevent colitis in young IL-10–/– mice, the inability of either to reverse established disease suggests another mechanism may be involved in disease maintenance [6 , 11 , 12 ]. Further studies have revealed that IL-10–/– mice demonstrate a biphasic disease course, with elevated mucosal levels of IL-12/23p40 and IFN-{gamma} during the early disease phase, followed by a late phase of chronic inflammation during which IL-12/23p40 and IFN-{gamma} return to basal levels concurrent with an increase in mucosal IL-4 and IL-13 [12 ]. It is not known whether this transition from a Type 1 to a Type 2 cytokine expression pattern is related to the duration of intestinal inflammation or to inherent changes in the aging systemic and mucosal immune systems.

Although numerous studies in aging humans and animal models have demonstrated changes in overall immunologic patterns over time, including a shift from predominantly Th1 to Th2 responses [13 14 15 16 17 ], little information is available about age-related changes in the intestinal mucosa. Increased susceptibility to many infectious diseases and neoplasms is well documented and believed to be largely a result of global alterations in immune function [18 ]. Risk and severity of many autoimmune disorders decline with age, and elderly patients often have an atypical presentation of inflammatory disorders when compared with their younger counterparts [19 ]. IBD exhibits distinctive, age-related phenomena, including the observation that new onset of symptoms is most common during the second and third decades of life [20 ]. In light of this, it is possible that the cytokine changes previously observed in the IL-10–/– colitis model may reflect immunological changes of an aging mucosa [12 ].

In this study, we first modified the IL-10–/– colitis model to investigate a distinction between the age of disease onset from the duration of intestinal inflammation. We demonstrate underlying changes in the immune response of aging mice, which could contribute to the shift between Th1 and Th2 predominant immune responses in early versus late colitis. In addition, we show that the prevailing immunologic tone at different ages affects the onset, response to probiotic therapy, and perpetuation of intestinal inflammation in wild-type (WT) and IL-10–/– mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/10 WT and IL-10–/– mice (Jackson Laboratories, Bar Harbor, ME, USA) were bred in a limited access, shower-in, microisolator-housed ultrabarrier facility (UBF) at our institutional animal resource center. Mice remained in the UBF or were transferred at specified ages to conventional, open-cage housing. Sentinel animals in the UBF and conventional facility were negative for Helicobacter hepaticus. To prevent genetic drift in the breeding colony, IL-10–/– mice were backcrossed every 12 generations with C57BL/10J mice (Jackson Laboratoriees). Genotyping was performed by PCR analysis of DNA isolated from tail tissue [21 ]. Product sizes for the WT and disrupted IL-10 genes are 700 bp and 600 bp, respectively. The Institutional Animal Care and Use Committee approved all experimental protocols.

Probiotic treatment
The probiotic treatment administered was VSL#3 (Sigma-Tau, Pomezia, Italy), a lyophilized preparation of eight strains (Lactobacillus casei, L. plantarum, L. acidophilus, L. delbrueckii subsp. bulgaricus, Bifidobacterium longum, B. breve, B. infantis, and Streptococcus salivarius subsp. thermophilus). Packets were reconstituted at 108 cfu/ml in water and aliquoted into 50 ml conical tubes (Corning, Acton, MA, USA) equipped with stoppers and sipper tubes, which were placed on cage racks and used in place of standard drinking water. VSL water was changed every 2–3 days according to consumption. Treatment was started 2 weeks prior to transfer from the UBF to conventional housing and then discontinued upon transfer.

Weight and disease activity index (DAI)
Animals were weighed on a balance, and measurements were recorded to the nearest 0.1 g. The DAI was recorded as a composite score of stool consistency (0–2) and fecal blood (0–1). Animals were given a score of 0 for hard stools, 1 for soft-formed stools, and 2 for frank diarrhea. The absence (no points) or presence (+1 point) of fecal blood was determined using the Hemoccult® Sensa® card (Beckman Coulter, Miami, FL, USA).

Isolation of lamina propria mononuclear cells (LPMC)
Animals were killed by carbon dioxide asphyxiation, and colons were removed and rinsed with calcium- and magnesium-free HBSS (BioWhittaker, Walkersville, MD, USA) to remove contents. The intestines were opened longitudinally, and the luminal surface was scraped gently to remove mucus. The tissue was then chopped vigorously for 15–20 min and washed twice for 15 min at 37°C in 25 ml HBSS with 0.05 mM EDTA, pipetting the tissue every 5 min to aid in the dissociation of the epithelial layer. After each wash, the suspension was filtered through a 100-µm mesh filter (Cat. #2360, Falcon, Bedford, MA, USA), the filtrate discarded, and the remaining tissue transferred carefully to fresh buffer. After the second EDTA wash, the tissue was washed once in 25 ml RPMI-1640 media with 10% FCS and 10 mM HEPES (all from BioWhittaker) for an additional 15 min, filtered, and transferred to 25 ml of the same medium with 10 Wünsch units of Blendzyme 2 (Roche Molecular Biochemicals, Indianapolis, IN, USA). The tissue was incubated in the Blendzyme solution for 1 h at 37°C with vigorous pipetting every 15 min. After the tissue was digested, the suspension was forced first through a 100-µm and then a 40-µm mesh filter (Cat. #2340, Falcon). Cells were washed twice in HBSS and resuspended in complete RPMI media (RPMI 1640, 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, all from BioWhittaker), 50 µM β-ME (Sigma-Aldrich, St. Louis, MO, USA). Yield was determined by counting viable cells using trypan blue exclusion. For the initial experiments, whose data were not included, herein, an additional percoll step gradient purification was used as described when using a combination of collagenase and DNase in the previous purification procedure [12 ]. After careful evaluation, we confirmed that LPMC purity from the murine colon was similar when Blendzyme 2 was used in the absence or presence of the percoll step, and LPMC yield was reduced greater than 50% after gradient centrifugation.

Cell activation
Mononuclear cells were plated at a final concentration of 1 million cells/mL in 96-well round-bottom plates (Falcon), without stimulation or stimulated with one of the following: 50 ng/ml PMA with 500 ng/ml ionomycin (both from Sigma-Aldrich), 10 µg/ml LPS (Escherichia coli 0111:B4, Sigma-Aldrich), or 500 ng/ml soluble anti-CD3 antibody (Clone 145-2C11, American Type Culture Collection, Manassas, VA, USA). Cultures were maintained for 48 h at 37°C in 5% CO2 before harvesting the spent media. Supernatants were stored at –80°C until examination of secreted cytokine levels by ELISA.

IFN-{gamma} ELISA
Paired rat anti-mouse IFN-{gamma} antibodies (BD PharMingen, San Diego, CA, USA) were used as recommended by the manufacturer. Ninety-six-well ELISA plates (Dynatech, Chantilly, VA, USA) were coated for 1 h at 37°C with 4 µg/mL capture antibody (#551216) in 0.1 M Na2PO4. The wells were washed five times with PBS/0.05% Tween buffer and blocked at room temperature for 2 h with 200 µL 5% BSA in PBS (Sigma-Aldrich). After five washes with PBS/Tween, 100 µL culture supernatant from triplicate culture wells or recombinant murine (rm)IFN-{gamma} standard in duplicate (4000–63 pg/mL in twofold serial dilutions, R&D Systems, Minneapolis, MN, USA) was added. If necessary, culture supernatants were diluted up to 1:50 with complete RPMI to achieve a signal within the standard curve. The plates were incubated for 2 h at room temperature, followed by five washes with PBS/Tween. The biotinylated secondary antibody (100 µL; #554410) was added, incubated 1 h at room temperature, and washed five times with PBS/Tween. A 1:2000 dilution of a HRP-streptavidin conjugate (Zymed, Carlsbad, CA, USA) was added, and the plates were incubated for 30 min at room temperature, followed by eight washes with PBS/Tween. Plates were developed with 1:1 vol:vol of 2,29-azino-di-(3-ethylbenzthiazoline-6-sulfonate) peroxidase substrate and H2O2 (Kirkegaard and Perry Labs, Gaithersburg, MD, USA) for 20 min at room temperature. The absorbance was read at 405 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

IL-4 ELISA
Expression was determined using a DuoSet® ELISA development system, according to the manufacturer’s specifications (R&D Systems). Twofold serial dilutions of rmIL-4 were used as a standard (1000–16 pg/mL). Plates were developed using a 1:1 mixture of H2O2 and 3,3',5,5' tetramethylbenzidine (BD PharMingen), followed by an acid stop (2 N H2SO4), and read at 450 nm.

H&E staining and histologic colitis scoring
Intestines of IL-10–/– mice were opened longitudinally, rinsed gently in HBSS, and placed into histology cassettes in a serpentine manner. The tissue was fixed in buffered Formalde-Fresh (10% formalin; Fisher, Pittsburgh, PA, USA) for 24 h and embedded in paraffin blocks. Tissue slices (5 µm) were placed onto glass slides and stained with H&E for analysis. Slides were given a composite score by a blinded pathologist based on the presence and degree of five different markers of inflammation: inflammatory infiltrate, mucosal hyperplasia and gland distortion, mucosal ulceration, polymorphonuclear accumulation, and depth of inflammation. Each subscore had a range of 0–3 points, with a total range of 0–15 points.

Statistical analysis
The temporal pattern of weight and disease activity score was summarized by scatter-plot, superimposed with a lowess smoother [22 ]. DAI (score) was dichotomized into dummy variables, namely, no disease or mild disease (DAI ≤1) and severe disease (DAI ≥2) or no disease (DAI =0) and disease (DAI ≥1), depending on the frequency of DAI in certain time periods. Logistical regression on the binary outcomes was used to compare the severity of disease between groups of mice over different time periods. As the scores of disease activity from the same mouse during follow-up were usually correlated, generalized estimating equations with autoregressive correlation structure were used for the inference [23 ]. The odds ratio for experiencing severe disease in one group compared with another group as well as the rate of disease activity were estimated. For histologic analysis, colitis scores of transferred groups were compared with age-matched UBF groups at each time-point using a Student’s t-test. Similarly, cytokine production was analyzed using the Student’s t-test. All tests were two-sided, and a P value less than or equal to 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-10–/– mice develop normally in the UBF
Animals maintained in the UBF at our institution are well-colonized with enteric flora, as evaluated by denaturing gradient gel electrophoresis of DNA extracted from fecal contents (data not shown). It is striking that although all mice contained a robust variety of microbiota, differences in the pattern of colonization were observed between those maintained in the UBF or conventional housing, as well as between individuals of different ages in the same environment. We asked whether the presence of this microbiota in IL-10–/– mice maintained in the UBF would initiate intestinal inflammation. IL-10–/– mice in UBF housing gained weight normally and exhibited minimal disease activity, similar to that observed in WT mice (Fig. 1a and 1b ). In addition, there was no histologic evidence of inflammation, and all animals in the UBF displayed a well-developed, mucosal architecture upon microscopic examination (Fig. 1c 1d 1e 1f) .


Figure 1
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  		Figure 1. IL-10–/– mice do not develop colitis in the UBF. IL-10–/– ({diamondsuit}; n≥20) and WT (shaded circle; n≥10) mice were housed in the UBF and observed weekly for (a) weight and (b) DAI. Data shown are the mean ± SEM. No significant differences between the two groups for either parameter were observed, as determined by two-sided, logistical regression. Representative colonic sections stained with H&E, obtained from 15-week-old IL-10–/– (c) and WT (e) and 30-week-old IL-10–/– (d) and WT mice (f) show no signs of colonic inflammation.

 
Exposure to conventional flora induces colitis; onset and course vary with age
Upon transfer of weanling mice from the UBF to conventional housing at 3 weeks of age, IL-10–/– mice exhibited a delayed, gradual onset of disease, requiring 8 weeks before the appearance of an immune pathology, which was statistically different from UBF controls (P<0.001 by 11 weeks of age; Fig. 2a ). The slight increase in DAI at 7 weeks of age is not accompanied by histologic evidence of colonic inflammation. In contrast, IL-10–/– and WT mice, transferred to conventional housing at 12 weeks of age, showed a rapid onset of disease activity, which was statistically different from UBF controls within 3 weeks (P<0.001; Fig. 2b ). However, disease activity in the WT animals was self-limited, returning to normal by 18 weeks of age, and IL-10–/– mice maintained significant statistical differences in DAI when compared with UBF-housed IL-10–/– (P<0.001) or transferred WT mice (P<0.001 after 17 weeks).


Figure 2
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  		Figure 2. Age at exposure to conventional flora alters disease course in IL-10–/– and WT mice. IL-10–/– ({diamondsuit}; n≥12) and WT (shaded circle; n≥6) mice were transferred from UBF to conventional housing at (a) 3 weeks or (b) 12 weeks of age and followed for disease activity. Data shown are the mean ± SEM. Representative colonic sections stained with H&E were isolated from (c) 8-, (d) 15-, and (e) 30-week-old IL-10–/– mice, as well as (f) 8-, (g) 15-, and (h) 30-week-old WT mice, all of which were transferred at 3 weeks of age. Additional H&E-stained representative colonic sections were obtained from (i) 15- and (j) 30-week-old IL-10–/– and (k and l) WT mice transferred at 12 weeks of age. I, Leukocyte infiltrate; E, epithelial hyperplasia; G, loss of goblet cells; T, transmural infiltration; D, disruption of mucosal architecture; U, ulceration.

 
Histologic examination of transferred IL-10–/– and WT animals
Upon histologic examination of IL-10–/– and WT mice transferred at 3 weeks, we saw no evidence at 8 weeks of age of the colonic inflammation, which was present at 15 and 30 weeks of age (Fig. 2c 2d 2e) , and there was no evidence of inflammation in the WT animals at any age (Fig. 2f 2g 2h) . In contrast, IL-10–/– and WT mice, transferred at 12 weeks of age, demonstrated obvious colonic inflammation at the 15-week time-point (Fig. 2i and 2k) . By 30 weeks, IL-10–/– mice still showed signs of inflammation, which was no longer evident in WT mice (Fig. 2j and 2l) . H&E-stained colonic sections were scored based on the presence and degree of lymphocytic infiltrates, polymorphonuclear accumulations, mucosal ulceration, gland distortion, and depth of inflamed area (Table 1 ). As expected, histologic scoring demonstrated a lack of inflammation in colons of UBF-housed mice at all ages. Scoring of sections from IL-10–/– mice transferred at 3 weeks showed the first evidence of progressive colitis at 12 weeks of age, and no inflammation was observed in WT mice at any age. In contrast, sections from IL-10–/– and WT mice transferred at 12 weeks demonstrated the presence of indistinguishable inflammation at 15 weeks of age, corresponding to the elevated DAI observed in both genotypes. This inflammation persists in older IL-10–/– mice in the 12-week transfer group, and sections from older WT mice appeared normal, corresponding with the lack of any recordable DAI in this group (Fig. 2i 2j 2k 2l , and Table 1 ).


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  		Table 1. IL-10–/– Mice Transferred to Conventional Housing at 3 Weeks of Age Show a Gradual Disease Course, and Those Transferred at 12 Weeks Develop Maximal Symptoms within 3 Weeks

 
Probiotic pretreatment delays onset of colitis in 12-week-old IL-10–/– mice
Animals were given a short course of treatment with VSL#3, a probiotic preparation, which has been shown effective in treating active ulcerative colitis (UC) and preventing pouchitis, as well as preventing colitis when given long-term to young IL-10–/– mice [24 25 26 ], which in the UBF, were treated with VSL#3 between 10 and 12 weeks of age and then transferred to conventional housing with no further probiotic treatment. Analysis of weight and DAI demonstrated a delay of several weeks in the onset of symptoms, when compared with untreated mice transferred at 12 weeks (Fig. 3a and 3b ). These results are consistent with an earlier report, in which VSL#3 treatment in IL-10–/– mice between 8 and 12 weeks of age with developing colitis also reduced the level of inflammation, cytokine production, and epithelial cell permeability [26 ].


Figure 3
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  		Figure 3. Probiotic treatment delays disease onset in 12-week transfer mice. UBF-housed IL-10–/– were given 108 cfu/ml VSL#3 in the drinking water for 2 weeks prior to transfer (arrow) to conventional housing ({square}; n≥9) or transferred with no pretreatment ({blacktriangleup}; n≥12). Mice remaining in the UBF are shown for reference ({diamondsuit}; n≥20). Mice were observed weekly for (a) weight and (b) DAI. Data shown are the mean ± SEM.

 
Age-related changes in mucosal cytokine profile in the absence of colitis
To identify a molecular mechanism to explain the gradual versus rapid onset of colitis in 3- and 12-week-old mice, we examined cytokine expression by anti-CD3-stimulated LPMC isolated from the colons of UBF-housed IL-10–/– mice. Overall expression of IFN-{gamma} and IL-4, when compared with colitic mice, was low at all ages (N.B., the difference in scale between Fig. 4a and 4b ). It is interesting that IFN-{gamma} secretion is elevated at 12 weeks, with an IFN-{gamma}:IL-4 ratio of 35:1. As the UBF-housed mice age, the IFN-{gamma} level decreases, and IL-4 increases, suggesting a Type 2 cytokine bias in older, healthy mice. This Th2 bias is confirmed by the decrease in the IFN-{gamma}:IL-4 ratio to 3:1. Consistent with the presence of disease as indicated in Figure 2 , LPMC, isolated from IL-10–/– mice transferred to conventional housing at 12 weeks of age, secrete significantly higher levels of IFN-{gamma} in spontaneous and anti-CD3-stimulated cultures after disease onset (Fig. 3b , P<0.05 at 20, 24, and 30 weeks compared with the time of transfer at 12 weeks of age). The decline in spontaneous IFN-{gamma} production at 36 weeks is also statistically significant (P=0.03), and IL-4 production in late-phase colitis was below the level of detection. The age at maximal, spontaneous, mucosal IFN-{gamma} and the subsequent decline in IFN-{gamma} synthesis after a 12-week transfer are delayed by 8–10 weeks, relative to each event in the 3-week transfer model (Fig. 4b) . These results are consistent with our previous report, in which mice are weaned into conventional housing at 3 weeks of age [12 ].


Figure 4
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  		Figure 4. Age- and disease-dependent changes in cytokine expression in IL-10–/– mice. LPMC were isolated from the large intestines of a pair of IL-10–/– mice and cultured for 48 h. Conditioned media were harvested at 48 h, and cytokine levels were detected by sandwich ELISA. (a) IFN-{gamma} (solid bars) and IL-4 (shaded bars) production by soluble, anti-CD3-stimulated LPMC from 5-, 12-, 30-, and 36-week-old, UBF-housed mice. (b) IFN-{gamma} production by unstimulated (open bars) and soluble, anti-CD3-stimulated (shaded bars) LPMC from mice transferred to conventional housing at 12 weeks of age. Data shown are the mean ± SD of three to four pairs of mice. *, P < 0.05, versus unstimulated, 12-week LPMC; **, P < 0.05, versus anti-CD3-stimulated, 12-week LPMC; #, P < 0.05, versus unstimulated, 20-week LPMC.

 
Older mice develop only sporadic signs of colitis
To explore the effect of an increasing Type 2 cytokine bias in older, healthy animals, IL-10–/– and WT mice were transferred from the UBF to conventional housing at 30 weeks of age. Individual mice in IL-10–/– and WT groups showed sporadic onset, recovery, and relapse of disease over the 12-week follow-up. Less than 60% of the animals at any given time-point for either genotype demonstrated disease (DAI>1; maximum of 52% for IL-10–/–, 57% for WT), and fewer demonstrated severe disease (DAI=3; 38% for IL-10–/–, 50% for WT). Furthermore, the presence of the immunosuppressive cytokine IL-10 had no impact, as the two groups were not significantly different from each other (P=0.675). In addition to incomplete disease penetrance, the frequency and severity of relapse could not be predicted. Given the unpredictability of disease onset in any particular mouse and the absence for requirement of immunodeficiency, the findings suggest that the inflammatory process is regulated by a different mechanism in these older animals. Histologic evaluation of the mice after transfer showed no significant difference in colitis score (data not shown). Similarly, pretreatment with probiotics for 2 weeks prior to transfer did not impact the sporadic occurrence of colitis symptoms as measured by DAI (data not shown). This observation was in stark contrast to younger animals (Fig. 3 and refs. [26 , 27 ]), where probiotic treatment delays onset. Together, these results indicate that the age of the mucosal immune system alters the kinetics, intensity, and immunobiology of gastrointestinal inflammation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
With increased life expectancy and rising diagnosis of IBD, the number of aging and elderly patients with IBD will continue to climb. In this regard, longitudinal studies, which distinguish effects of long-term, mucosal inflammation from overall age-related changes in immune function, are critically needed. The availability of animal models of colitis, which are mediated by similar immune cells and cytokines as human disease, provides a rapid alternative to long-term human studies. Using the IL-10–/– mouse model, we demonstrate that the mucosal immune response in young adult mice is biased toward IFN-{gamma} synthesis and an accelerated inflammatory response upon exposure to colitigenic flora. It is striking that these distinct changes in the lamina propria are not reflected in the spleen, peripheral lymph nodes, or mesenteric lymph nodes, as we have reported previously (data not shown; ref. [28 ]). As mice age, susceptibility to and severity of chronic colitis wane, the requirement for IL-10 in the regulation of intestinal inflammation is abrogated, and mucosal IL-4 production increases. Corresponding to these age-related changes in disease and mucosal immune response, the efficacy of therapeutic biologicals is diminished in older animals.

A broad range of animal models of colitis and ileitis has been reported in the past decade. Consistent with our proposal that mucosal immune homeostasis is regulated differently in aging subjects, longitudinal studies of intestinal inflammation in mice have shown that the population of infiltrating inflammatory cells as well as production of immune mediators change over time, including a dramatic reduction in the effectiveness of treatment with exogenous IL-10 or blocking anti-IL-12/23(p40) antibody as animals progress into established disease [6 ]. We reported previously in the IL-10–/– model that although IFN-{gamma} and IL-12/23p40 are elevated in young colitic mice, the cytokine profile shifts toward more IL-4 and IL-13 in older animal [12 ]. Subsequently, we reported that neutralizing antibodies to IL-4 and IL-13, alone and in combination, partially abrogate late-phase disease [29 ]. It is likely that age is one of many factors that modulate the mucosal cytokine pattern. For example, the penetrance, severity, and rate of the inflammation, which develop within an IL-10–/– colon, vary with the genetic background yet are always dependent on the presence of microflora [10 ]. IL-10 deficiency on a 129/SvEv or Balb/c background causes severe colitis as early as 3 weeks of age, and the same transgene in a C57BL/6 background produces little inflammation [11 ]. It is interesting that the mucosal cytokine profile in all four strains studied (129xB6, Balb/c, C57/BL6, C56/BL10) during disease progression (noted as early disease in this manuscript) is quite similar [30 ]. Late colitis in IL-10–/– mice has only been studied in this and our previous manuscript [12 ]. In the SAMP1/YitFc ileitis model, IFN-{gamma} is elevated during the initiation phase and IL-4 during the later chronicity stage [31 ]. In addition, the similarity of responses observed in the IL-10–/– and WT mice after transfer at 30 weeks indicates an age-related reduction in the effectiveness of IL-10-mediated regulation. Together, these findings demonstrate the importance of immune changes in the regulation of inflammation in aging mice and highlight the potential significance of age at onset, disease duration, and current patient age in human IBD.

Age-dependent progression of UC and Crohn’s disease (CD) in the human population is widely recognized. In response to clinical intervention or the natural course of disease, patients diagnosed with either disease will experience a relapse for various periods of time [32 , 33 ]. Recent studies demonstrated typically a more severe and more extensive disease course in pediatric IBD patients than those diagnosed in adulthood [34 35 36 ]. In addition, many pediatric patients with IBD do not respond to or tolerate treatment regimens, which are effective typically in adults [37 ]. Previously, we isolated LPMC from pediatric infectious control, UC, and CD patients within 5 years of diagnosis and compared them with LPMC isolated from aged patients for their propensity to secrete IL-4 versus IFN-{gamma}. We reported that IFN-{gamma} secretion declines in the older patients and that this decline correlates with a loss of synthesis of the β2 chain of the IL-12 receptor [38 ]. These observations further support our premise that cytokine production in the mucosa changes with age.

Although it is widely acknowledged that the immune system changes with age, reports attempting to define the specific immunologic changes attributable to normal aging vary widely in their findings [39 ]. Multiple reports indicate a global, age-related decline in IL-2 and shift from a Th1 to Th2 cytokine profile in normal, aged humans [13 , 14 , 17 ] and mice [15 , 16 ]. An experimental model of systemic lupus erythematosus (SLE) demonstrated increased splenic IFN-{gamma} synthesis, which declined subsequently as IL-4 increased [40 ]. Further studies revealed that an age-related decline in IFN-{gamma} and increase in IL-4 production in healthy animals corresponded to a decreased susceptibility to the induction of experimental SLE [41 , 42 ].

A common feature of many models of T cell-dependent colitis is their failure to develop disease in a germ-free environment, highlighting the contribution of enteric flora to intestinal inflammation [10 , 43 44 45 ]. It has also been shown that enteric microbiota are important in the development of a normal mucosal immune response [46 ]. Similarly, age-related differences in bacterial colonization have been reported, particularly, a reduced colonization with the Lactobacillus species in IL-10–/– mice when compared with WT, which is corrected by 16 weeks of age [27 , 47 ]. It is likely that no single organism will be identified as the causative agent for human IBD, yet it is clear that a change in luminal flora may induce colitis in a wide variety of animal models [48 ]. For example, it has been reported that H. hepaticus infection induces colitis in immunodeficient mice [49 , 50 ], an observation that has not been reproducible in our facility. Other laboratories have described combinations of nonpathogenic, enteric organisms, which induce colitis in a variety of immunodeficient but not WT animal models [43 , 45 , 51 ]. In contrast to the concept of colitigenic organisms, probiotic therapy has been shown to be somewhat effective in humans and animal models of colitis [24 25 26 27 ]. As we demonstrate that the character and kinetics of inflammation vary with age at disease onset, the loss of efficacy of probiotic therapy demonstrated in 30-week-old mice is not surprising.

With increased diagnosis of IBD in pediatric patients, as well as increasing numbers of aging patients in the general population, the need to understand the interactions of the aging immune system with chronic inflammation is becoming more urgent. Further characterization of age-dependent changes in an immune response, including not only cytokine expression but also other physiologic factors such as hormones and intestinal epithelial cell plasticity, which affect immunity, commensal flora colonization, and antigen presentation, should allow the tailoring of treatments for IBD patients to accommodate the differences in systemic and mucosal immune status.


   ACKNOWLEDGEMENTS
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases grant P01 DK-57756 (to A. D. L.), National Institute of Environmental Health Sciences grant F30 ES-11931 (to M. R. E.), and National Institute of General Medical Sciences grant T32 GM-07250 (to Case Medical Scientist Training Program). The authors thank Dr. C. Fiocchi for his insight into IBD pathogenesis, Dr. C. DeSimone for generously providing the VSL#3 preparations, D. Spencer for sharing his expertise of the animal model, and K. Jewett for her assistance with cell preparations.

Received June 13, 2006; accepted January 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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