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Nutritional Immunology

The Effect of Vitamin A Supplementation on the Intestinal Immune Response in Mexican Children Is Modified by Pathogen Infections and Diarrhea^1,2

Kurt Z. Long^*,3, Teresa Estrada-Garcia, Jorge L. Rosado^**, Jose Ignacio Santos, Meredith Haas, Mathew Firestone, Jui Bhagwat^*, Cheryl Young, Herbert L. DuPont^#, Ellen Hertzmark^* and Nanda N. Nanthakumar

^* Department of Nutrition, Harvard School of Public Health, Boston MA; Department of Molecular Biomedicine, CINVESTAV-IPN, Mexico City, Mexico; ^** Universidad Autonoma de Queretero, Queretero, Mexico; Hospital Infantil de Mexico Federico Gomez, Mexico City, Mexico; DePauw University Greencastle, IN; Department of Anthropology, Harvard University, Boston MA; ^# University of Texas Medical School, and School of Public Health, Houston, TX; and Harvard Medical School, Boston, MA

³ To whom correspondence should be addressed. E-mail: klong{at}hsph.harvard.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Vitamin A supplementation has consistently reduced infant mortality and the severity of pathogen-induced diarrhea. The mechanism by which vitamin A modulates the mucosal immune response to produce these effects remains poorly defined. To address this issue, stools collected during the summer months from 127 Mexican children 5–15 mo old enrolled in a larger, randomized, double-blind, placebo-controlled, vitamin A supplementation trial were screened for interleukin (IL)-4, IL-6, interferon- (IFN-), and gastrointestinal pathogens. Fecal cytokine values were categorized into 3 levels (undetectable, <median, median). Multinomial regression models were used to determine the probability that vitamin A–supplemented children had higher categorical values of a cytokine than children in the placebo group. Differences in categorical values were also analyzed after stratification by gastrointestinal pathogen infections and diarrheal symptoms. Overall, fecal cytokine categorical levels did not differ between children randomized to the 2 arms. Vitamin A–supplemented children infected with enteropathogenic E. coli (EPEC) had reduced IL-4 and IFN- levels [odds ratio (OR) = 0.3, 95% CI 0.13–0.67 and OR = 0.34, 95% CI 0.14–0.83, respectively] compared with children in the placebo group. Vitamin A–supplemented children had increased IL-4 levels when infected with A. lumbricoides (OR = 12.06, 95% CI 0.95–153.85). In contrast, IL-4 levels increased (OR = 2.14, 95% CI 0.94–4.87) and IFN- levels decreased (OR = 0.51, 95% CI 0.26–0.99) among vitamin A–supplemented children with diarrhea compared with children in the placebo group. These findings suggest that the regulation of the mucosal immune response by vitamin A may depend on the type of enteric pathogen infecting the child and the presence of clinical symptoms.

KEY WORDS: • Vitamin A • cytokine • diarrheal pathogen • children

Vitamin A supplementation has consistently reduced overall infant mortality, diarrhea-associated infant mortality, and the severity of diarrheal disease episodes (1,2). As a result, supplementation has been promoted as an important cost-effective intervention for improving childhood survival in developing countries. However, vitamin A does not have a consistent effect on the incidence of diarrheal disease (3–5). Currently, there is no known mechanism that can explain why supplementation reduces infant mortality but has no effect on prevalence. Diarrheal disease is caused by a large group of bacterial, viral, and parasitic enteric pathogens each of which can elicit a distinct mucosal immune response and have distinct mechanisms for inducing pathogenesis (6,7). Specific components of the innate and adaptive gastrointestinal immune response, in turn, are important in reducing both the load of pathogen infections and pathogen-induced clinical symptoms. The inconsistent effect of vitamin A supplementation on different childhood health outcomes may be due to an underlying heterogeneity of the immune response elicited during specific pathogen infections and pathogenesis. Clarification of how vitamin A modulates specific components of the mucosal immune response would allow the targeting of specific subgroups of children who are infected with specific pathogens.

Vitamin A has been shown to regulate both the innate and adaptive immune responses. The adaptive immune response consists of subgroups of T helper lymphocyte populations: the T helper type 1 (Th1),⁴ T helper type 2 (Th2) and T regulatory helper cells. Different pathogens can induce either a Th1 or Th2 type response, causing a bias in the mucosal immune response. Helminths, for example, induce a Th2 response, whereas bacterial pathogens are thought to induce a Th1 response (8,9). There is an extensive literature reporting that vitamin A deficiency induces important changes in the adaptive immune response such as reduced T lymphocyte proliferation and functionality(10), and reduced response against specific antigens (11,12). These changes may be due to vitamin A deficiency inducing a polarized Th1 response and a reduced Th2 response (13,14) because these 2 arms regulate many humoral and cellular immune functions. These regulatory effects of vitamin A may be responsible for the inconsistent clinical outcomes of supplementation on diarrheal diseases because the effect of vitamin A on the immune response may be pathogen-specific.

To date, no community-based randomized clinical trials have been carried out to investigate the differential effect of vitamin A supplementation on the gastrointestinal mucosal immune response among young children and how pathogen infections and the onset of pathology modify these effects. Accordingly, we conducted a randomized, placebo-controlled, double-blind trial to evaluate the effect of vitamin A supplementation on pathogen-induced immune response among children from periurban communities of Mexico City, Mexico. The primary goal of the research reported here was to determine the effect of vitamin A on the overall proinflammatory, Th1, and Th2 fecal cytokines as a measure of its effect on the gastrointestinal mucosal immune response among a subsample of study children during the summer diarrheal season. A secondary goal of this analysis was to determine whether vitamin A supplementation modified the enteropathogenic E. coli (EPEC)- and A. lumbricoides-induced proinflammatory and adaptive Th1 and Th2 cytokine responses. EPEC and A. lumbricoides are the most common bacterial and helminth gastrointestinal pathogens in this setting and induce clear Th1 and Th2 responses, respectively (8,15). Additionally, we analyzed the effect of vitamin A on the cytokines representing these responses during diarrheal episodes to determine how the onset of pathology may modify the effect of vitamin A supplementation.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Study population. Community health workers first carried out a census of all children <2 y old living within the periurban community of La Magdalena Atlicpac located along the eastern perimeter of Mexico City. This community is made up of a number of neighborhoods (colonias) populated by recent immigrants to the Valley of Mexico. Households in older, more established colonias within La Magdalena Atlicpac have greater access to piped water and sanitary facilities, whereas households in the more recently founded colonias have little or no access to these facilities. Mothers of all children from 5 to 15 mo old in the community were invited to participate in the study. We targeted this population so that we could determine the effect of the supplement on the children's developing immune response during and immediately after weaning. Children were excluded from the study if they had diseases that caused immunosuppression or any congenital or acquired alteration of the digestive tract that could alter the absorption of micronutrients. Children who were taking vitamin supplements were also excluded from the study. Overall, 200 children were enrolled in the study after their parents had given informed consent for their participation. The study protocol was approved by the human subjects committee of the National Institute of Public Health of Mexico.

Each child, once enrolled, was randomly assigned to receive vitamin A or a placebo using a random number table by personnel at the National Institute of Public Health. Block randomization was used so that the regimens were balanced every 20 children. Children <12 mo old assigned to the vitamin A group were administered a solution containing 20,000 IU of retinol [3.3 IU = 1 retinol activity equivalent (µg)] at baseline and subsequently every 2 mo until the end of the study; children >12 mo old received a solution containing 45,000 IU of retinol every 2 mo. This design allows the evaluation of the effect of frequent low doses of vitamin A on the pathogen-specific immune response and diarrheal disease. Testing was carried out at the National Institute of Nutrition to ensure that the placebo and vitamin A water miscible solution were similar in taste, viscosity, and color. Personnel at the National Institute of Nutrition carried out the preparation of the supplements to ensure that field personnel and the principal investigator were unaware of the regimens. The field team was in charge of administering the supplements and placebos to children using identical opaque plastic droplet bottles.

Recruited children were followed prospectively for up to 15 mo. During this follow-up period, households were visited twice a week at which time the child's mother or caretaker was interviewed to determine the presence of diarrhea in the child, the number and consistency of evacuations, fever, and the presence of blood and mucus in stools. A stool sample was collected every 2 wk among healthy children, whereas 2 samples were collected from children with diarrhea: one during the acute phase of the episode and a second within 1 wk of the onset of the episode. No initial and final blood samples were taken from the study children because parents of the study children did not consent for blood to be drawn. Results from screened stools collected from children during the months of June, July, and August are included in the fecal cytokine study reported here.

The endpoints for this analysis were changes in the proinflammatory and adaptive fecal cytokines in response to vitamin A supplementation, as well as changes in the cytokine profile after infections by diarrheal pathogens and/or after the onset of diarrheal episodes. Work in animal models showed that mucosal cytokine levels in the gastrointestinal tract correlate with cytokine levels in stools (16). Accordingly, we used fecal cytokines as a measure of the effect of vitamin A supplementation on the mucosal cytokine response.

    Laboratory methods. Fresh stools collected from children during the summer months were placed in sealed test tubes in ice and then frozen within 4 h after their collection at –20°F. Samples were extracted by homogenization and centrifugation (15 min, 10,000 x g) at 4°C with PBS containing protease inhibitors [benzenesulfonyl, pepstatinA, leupetin and aprotinin, 1:10 wt:vol; (Sigma)]. The supernatants were collected, frozen, and stored at –70°C until assayed for the proinflammatory cytokine IL-6, the Th1 cytokine IFN-, and the Th2 cytokine IL-4 by an ELISA using paired ELISA specific capture and biotinylated detecting antibody (Ab) (Pierce-Endogen and R&D Systems). Peroxidases conjugated to steptavidin (Pierce-Endogen) were used to detect the capture of Ab; peroxidase activity was measured using ABTS substrate and read at a wavelength of 405 nm. Recombinant cytokines were used to generate a standard curve, and levels of these cytokines from the stool extracts were determined using the standard curve in 96-well plates according to the manufacturer's protocol. Cytokine concentrations (ng/L) were normalized to stool protein concentration (g/L). The detection limit for these cytokine assays was 10 ng/L.

Stool samples were plated onto SS, MacConkey, and MacConkey-tellurite agars for the identification of Salmonellae spp., Shigella, and Escherichia coli (17). Ova and parasites in stool were determined using the Kato-Katz technique to identify gastrointestinal infections by A. lumbricoides (18). Lactose-fermenting colonies (n = 5) with morphology resembling that of E. coli were selected from MacConkey agar plates (when present) and speciated biochemically. Selected E. coli were characterized as EPEC through the detection of the pathogenic genes intimin (eaeA) and plasmid-mediated bundle-forming pilus (bfp) using a previously described single multiplex technique (19).

    Data analysis. Stools collected from children followed during the summer months were used in this analysis. Data were entered in Visual Fox Pro 6.0 (Microsoft), verified, and checked for range and consistency. Because an important proportion of samples had no detectable levels of cytokines, conventional analytic techniques could not be used. Accordingly, we used multinomial regression analysis, which models the probability distributions of cytokine values categorized into 3 levels ordered from lowest to highest: undetectable, <median of positives, median of positives (20). The inclusion of the vitamin A variable in the model tests the hypothesis that the probability distributions of categorized cytokine values in the supplemented group will differ from the distributions in the placebo group expressed as an OR. These analyses were carried out in 3 stages. We first modeled the probability that categorical levels of IL-6, IFN-, and IL-4, as markers of the proinflammatory, Th1, and Th2 responses, respectively, among children randomized to the vitamin A group were different from levels among children in the placebo group. IL-6 was selected for this analysis because it was thought to be the best marker of the inflammatory response in the infected gut. It is secreted apically into the lumen during the acute phase response; after infections, it is responsible for macrophage differentiation, recruitment, and degranulation of neutrophils, thus contributing greatly to the pathogenesis of disease. Second, nonrandomized comparisons of these cytokine levels were then conducted on children stratified by the presence or absence of infections due to EPEC and A. lumbricoides, irrespective of whether the infection was asymptomatic or symptomatic. A pathogen infection was defined as encompassing at least one pathogen-positive stool and stools collected 3 wk after that positive stool. Finally, nonrandomized comparisons of fecal IL-6, IFN-, and IL-4 categorical levels were then conducted stratified by the presence or absence of diarrheal symptoms. A diarrheal episode was defined as the mother's reporting of symptoms in the child and confirmed by the passage of 3 liquid stools in 1 d. A period of 3 symptom-free days was used to define the end of an episode. An interaction term was included in these stratified analyses to test whether the effect of vitamin A was significantly different between strata. Statistical significance was set at a probability level of < 0.05 and < 0.1 for interactions. Data were analyzed using the GENMOD procedure in the Statistical Analysis System (SAS Institute, version 8.2) software.

The sample size for the overall study was calculated assuming that the study population had a diarrheal disease rate of 3 episodes/child each year and that the vitamin A supplement would reduce the incidence rate of diarrhea by 20%. A sample size of 100/group was required to detect a 20% difference between the control and treatment group with a power of 80%, a 95% significance level, and an expected loss to follow up of 20%. This calculation allowed for repeated measurements of the outcome and a correlation between measurements at different time points of 0.7 (21). The power for the cytokine analysis for all samples is higher than that for diarrhea due to the high prevalence of each cytokine in the stool samples.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Results from a subsample of 127 children followed during the months of June, July, and August are included in this analysis of fecal cytokines. The remaining 73 children were not enrolled until the fall and thus were not included in the analysis. A total of 505 stool samples were collected from these 127 children, 262 samples from 70 children administered the placebo and 243 samples from 57 children administered the vitamin A supplement. In the overall study, 4 stools/child were collected, whereas 3.4 and 1.5 stools/child were collected from healthy children and children with diarrhea, respectively. The distribution of sociodemographic characteristics of study children and households did not differ between children administered vitamin A and those in the placebo group (Table 1). There was no access to piped water in 42–45% of households, and 65% had no indoor toilets; 25–30% of the children were stunted. The initial vitamin A status of this population was not assayed. However, the most recent National Nutrition Survey carried out in Mexico in 1998 reported that children from this region of Mexico City were not deficient, but that 37% had low serum retinol levels (10–20 µg/dL retinol) (22). There was a total of 87 diarrheal episodes among the 127 children during the 3-mo period of this study, 51 (0.7 per child) in the placebo group and 36 (0.6 per child) in the vitamin A group.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our findings of the effect of vitamin A supplementation on the fecal cytokine profile in children stratified by pathogen infections are very different from the downregulated Th1 and upregulated Th2 responses among retinoid-treated mice (13,14,23,24). Initial conditions that are more Th1- or Th2-like can determine whether all-trans retinoic acid added to T lymphocyte cultures enhances the further development of either a Th1 or Th2 response (25). Similarly, initial conditions can determine whether retinyl acetate increases or inhibits IL-6 secretion by human peripheral blood monocytes (26–28). Infections by the different pathogens may have produced initial conditions among the supplemented children that influenced the subsequent development of the cytokine response (29,30).

In our study, it was not possible to determine how the timing of supplementation relative to the onset of infection may have influenced the cytokine profile. This timing seems important as demonstrated by Iwata et al. (31) and Mohty et al. (32). However, the reduction in IFN- levels and marginally significant reduction in IL-4 levels among vitamin A–supplemented children infected with EPEC, similar to that reported by Iwata et al., suggests that vitamin A supplementation early in an infection or during primary infections may not uniformly induce a polarized Th2 lymphocyte response as was reported in the animal model literature.

Virulence factors produced by different strains of EPEC, such as the plasmid-mediated bundle-forming pilus (bfp) and the attaching and effacing intimin (eaeA) protein (33), may also be inducing the differential effect of vitamin A on T helper cytokine production. Klapproth et al. (34,35), for example, reported that EPEC produces a toxin that specifically inhibits lymphocyte proliferation and IL-2, IL-4, and IFN- production in response to a variety of stimuli. The reduction in IL-4 and IFN- among vitamin A–supplemented children in our study after EPEC infections may be due to the presence of such virulence factors. The operation of such mechanisms suggests that the effect of vitamin A on the Th1-Th2 cytokine profiles could be the consequence of the direct effect of the pathogen and whether the pathogen triggers a vitamin A–dependent immune response.

The association of vitamin A supplementation and elevated IL-4 levels and reduced levels of IFN- fecal cytokines among children with diarrhea is similar to the findings reported in the animal model studies. This effect of supplementation during diarrheal episodes is very different from the cytokine patterns found among supplemented children stratified by specific pathogen infections. It is not clear why vitamin A supplementation may be downregulating the Th1 response and upregulating the Th2 response only with the onset of pathology.

The lack of an overall effect of vitamin A on fecal cytokines in our study may be due to the complexity of its effect on individual cytokines after infection with a specific pathogen and the onset of pathological disease. IL-4, for example, is significantly increased among vitamin A–supplemented children after infection by A. lumbricoides as well as after the onset of diarrhea but is significantly decreased among EPEC-infected children. It is important to note, however, that the immune response is associated with many Th1/Th2cytokines and that the pattern of Th1/Th2 cytokines responses is likely to be more informative than the level of any individual cytokine.

A number of factors must be considered when evaluating the cause-effect associations we report here. The relative size of the effect in the stratified analysis was large, as indicated by the magnitude of the OR. In addition, the significant interaction term in the stratified analyses indicated that there was a clear dose-response effect of vitamin A supplementation, and the effect of supplementation among infected children was significantly different from its effect among uninfected children. These findings appear to strengthen the cause-effect associations found.

A number of limitations of this study also have to be addressed. First, a form of censoring exists in this study as a result of the analysis of samples collected only during the summer months and not from the entire study. However, the bias introduced by such censoring may be limited due to the distinct etiologies of summer diarrheas in Mexico, which make this period a distinct and definable analytical unit. A second limitation of the study may be the failure to address whether the lack of detection of cytokines in stool may be due to a number of different factors such as degradation, insensitivity of the assays, and low productivity. Their inclusion in one category may be introducing bias into the analysis. The treatment of samples during collection, processing, storage, and analysis was identical between the vitamin A and placebo groups, suggesting that such systematic bias was minimized. Finally, because this study is the first to determine the effect of vitamin A supplementation on stool cytokine levels in a randomized clinical trial, it is not possible to determine the external validity of our findings. Stool cytokine profiles in our study are consistent with those found among travelers to Mexico after infections by diarrheal E. coli, suggesting that they are valid (36,37).

Overall, we found that vitamin A has a differential regulatory effect on the fecal cytokine response but that this effect is conditioned by whether a child is infected by a pathogen, the type of pathogen infecting the child, and the onset of pathology. This complexity may explain the inconsistent effects of vitamin A on different health outcomes reported in previous studies. These findings are suggestive at this point; thus, further work is required to confirm that vitamin A has this differential effect on the immune response. Such confirmation could then lead to the design of vitamin A supplementation programs that target groups of children who would benefit most from such supplementation. It is most likely that this targeting would involve children in clinical settings in which the identification of enteric pathogens is possible.

	ACKNOWLEDGMENTS

 
We thank Maltilde Juarez of the Clinical Microbiological Laboratory, Hospital La Perla, Secretary of Health, Cda Netzahuaycoytl, Mexico for her assistance in the laboratory.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 04, 17-21 April 2004, Washington DC [Long KZ, Hass M, Young C, Estrada T, Firestone M, Fawzi W, Bhagwat J, Rosado JL, Santos JI. Impact of vitamin A on the intestinal immune response in pathogen induced diarrhea of children in Mexico City, Mexico (abstract). FASEB J. 18:#37.7].

² Supported by Instituto de Nutricion Danone, CONACYT (National Council of Science and Technology of Mexico) and by the National Institutes of Health grant no. K01 DK06142-02.

⁴ Abbreviations used: Ab, antibody; EPEC, enteropathogenic E. coli; IFN-, interferon-, IL, interleukin; OR, odds ratio; Th1, T helper type 1 lymphocyte; Th2, T helper type 2 lymphocyte.

Manuscript received 31 August 2005. Initial review completed 5 October 2005. Revision accepted 30 January 2005.

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TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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