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Reduced Gene Expression of Intestinal α-Defensins Predicts Diarrhea in a Cohort of African Adults

  1. Paul Kelly1,2,5,
  2. Mona Bajaj-Elliott1,3,
  3. Max Katubulushi5,
  4. Isaac Zulu5,
  5. Richard Poulsom4,
  6. Roger A. Feldman1,
  7. Charles L. Bevins6 and
  8. Winnie Dhaliwal1
  1. 1Centre for Adult and Paediatric Gastroenterology, Institute of Cell and Molecular Science, Barts and The London School of Medicine,
  2. 2Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine,
  3. 3Infectious Diseases and Microbiology, Institute of Child Health, and
  4. 4Cancer Research UK, Lincoln’s Inn, London, United Kingdom;
  5. 5University of Zambia School of Medicine, Lusaka, Zambia;
  6. 6Department of Medical Microbiology and Immunology, University of California, Davis
  1. Reprints or correspondence: Dr. Paul Kelly, Centre for Adult and Paediatric Gastroenterology, Institute of Cell and Molecular Science, Barts and The London School of Medicine, Queen Mary, University of London, Turner St., London E1 2AD (m.p.kelly{at}qmul.ac.uk); or, Dr. Paul Kelly, Tropical Gastroenterology and Nutrition Group, Dept. of Medicine, University of Zambia School of Medicine, University Teaching Hospital, PO Box 50398, Ridgeway, Lusaka, Zambia (guts{at}coppernet.zm)
 
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Abstract

BackgroundHuman defensin (HD) 5 and HD6, both Paneth cell α-defensins, contribute to the antimicrobial barrier against intestinal infection. We have previously demonstrated that levels of both HD5 and HD6 mRNA were reduced in adults living in urban Zambia, compared with those in adults living in London. The aim of the present study was to determine, during 2 years of follow-up, whether α-defensin expression in Zambian adults is related to susceptibility to diarrhea

MethodsWe analyzed intestinal biopsy samples from a longitudinal cohort study conducted in 83 Zambian adults by quantitative reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and in situ hybridization, and we measured the incidence of diarrhea

ResultsLevels of HD5 and HD6 mRNA in Paneth cells varied between participants, over time, and seasonally and were strongly correlated with mucosal architecture. Gene expression was almost exclusively restricted to Paneth cells. The median (interquartile range) HD5 mRNA level was 6.0 (5.6–6.7) log10 transcripts/μg of total RNA among 18 participants who experienced diarrhea within 2 months after biopsy-sample collection, compared with 6.8 (6.2–7.3) log10 transcripts/μg of total RNA among 94 participants who did not (P=.006). A similar observation was made for HD6

ConclusionsThese data indicate that intestinal α-defensin expression is dynamic and seasonal and suggest that susceptibility to intestinal infection is related to α-defensin expression

Intestinal infectious disease is a major contributor to morbidity and mortality in tropical countries and has been estimated to have caused 1.5 million deaths in children in developing countries in the year 2002 [1]. Although the rate of death from acute diarrheal disease in children has decreased because of the widespread use of oral rehydration therapy, the more refractory problem of persistent diarrhea-malnutrition syndrome remains a public health problem [2]. A similar syndrome has emerged in adults with HIV-related immunosuppression, and both adults and children with these disorders have high mortality rates [3, 4]. In AIDS, in primary malnutrition, and during immunosuppressive therapy, a failure of cell-mediated immunity occurs, so there is an urgent need to elucidate the pathways of intestinal host defense. An improved understanding of innate immunity may lead to new avenues for the treatment of persistent intestinal infections

Antimicrobial peptides [5, 6] secreted into the intestine may constitute an important barrier to colonization of enterocytes (i.e., infection) and to the translocation of bacteria from the lumen into blood and lymph. Paneth cells are specialized intestinal epithelial cells that contain a rich armamentarium of antimicrobial peptides and proteins. In addition, cells of other lineages also secrete molecules with antimicrobial properties. The intestinal α-defensins, human defensin (HD) 5 and HD6, are small cationic peptides synthesized by Paneth cells [7, 8] and probably make a major contribution to intestinal defense [9–11]. Mice with a deletion of the matrilysin gene cannot process α-defensins to their active form and are more susceptible to colonization with Salmonella species [12]. Also, transgenic mice that express HD5 are protected from lethal challenge with Salmonella typhimurium [13], indicating that HD5 can function as an antimicrobial molecule in vivo

We have previously observed that adults living in a crowded township in Lusaka, Zambia, had lower levels of both HD5 and HD6 mRNA than did adults living in London [14], yielding an ∼10-fold lower quantity of specific mRNA per microgram of total RNA. This result is surprising, because it might be expected that increased expression of antimicrobial peptides would confer a survival advantage in populations in tropical regions, where exposure to intestinal pathogens is frequent and intense. It has been known for many years that healthy members of tropical populations have a background environmental enteropathy. This “tropical enteropathy” [15] appears to be a consequence of high exposure to enteropathogens, given that it is largely reversible in visitors to the tropics [16], is more closely related to economic circumstances than to climate [17], and shows seasonal variation [18]. Tropical enteropathy is characterized by reduced villous height, increased crypt depth, and increased T cell activation relative to those in populations living in temperate climates, in both adults [19] and children [20]. This pattern of crypt hyperplastic enteropathy also characterizes many infective states [21, 22], but in our Zambian population villous height and crypt depth are positively correlated [18], as occurs during starvation [23]. We studied here the pattern of Paneth cell α-defensin expression in adults in this tropical population and in relation to the individual risk for diarrhea during 2 years of follow-up in a longitudinal cohort study

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Methods

Study groups and tissue collectionWe studied 83 Zambian adults, chosen at random from a cohort study, who were fully representative of the population of a crowded shanty compound in Lusaka in which we previously analyzed small-intestinal architecture and function [18, 24]. HIV seroprevalence in Lusaka is relatively stable at 25%–30% [25], so HIV status was established in those participants willing to undergo testing. Jejunal biopsy samples were obtained by enteroscopy approximately annually during this period and were used for quantification of HD5 and HD6 mRNA [14] and for localization of mRNA and peptides. Morphometrical analysis for villous height, crypt depth, villous width, epithelial surface area, and villous compartment volume was done as described elsewhere [18], but only the first 2 measurements were taken for biopsy samples obtained after baseline. Paneth cells were counted as described elsewhere [26]. Biopsy samples for morphometrical analysis, immunohistochemical analysis, and in situ hybridization were collected into formal saline; biopsy samples for reverse-transcription polymerase chain reaction (RT-PCR) and peptide analysis were immediately snap-frozen in liquid nitrogen, stored at −80°C, and analyzed within 6 months. Approval for these experiments was obtained from the research ethics committees of the University of Zambia and the London School of Hygiene and Tropical Medicine

Competitive RT-PCR for HD5 and HD6We have described elsewhere a quantitative assay for HD5 and HD6 mRNA [14]. Briefly, biopsy samples were treated with Trizol (Invitrogen) for RNA extraction, then treated with DNase (Promega), and subsequently co–reverse transcribed with known quantities of a standard synthetic RNA before PCR amplification. The threshold of detection was determined to be 1×104 (log10 4.0) transcripts/μg of total RNA

Immunohistochemical analysis and in situ hybridization Immunohistochemical analysis was used to define expression of HD5, as described elsewhere [26]. No antibody to HD6 was available. HD5 and HD6 mRNA distribution was defined using in situ hybridization [26]

Peptide isoformsTo determine whether there was variation in the stored forms of HD5, tissue extracts of cationic peptides were analyzed by acid-urea gel electrophoresis followed by Western blotting, as described elsewhere [27]. Recombinant HD5 and pro-HD5 peptides were used as markers of migration, and an antibody from animals injected with vehicle only was used as a negative control

Analysis of diarrhea incidenceParticipants of the Lusaka cohort study were interviewed every 2 weeks, to ascertain whether they had experienced diarrhea during the previous 2 weeks. Participants who had experienced diarrhea were not invited for endoscopy until 1 month had elapsed; otherwise, dates of appointment were allocated randomly at any time of the year except for July and August, to detect seasonal variation

Data analysisHD5 and HD6 mRNA levels were expressed as log10 transcripts per microgram of total RNA. The levels were not normally distributed, so results are presented as medians and interquartile ranges (IQRs), and nonparametric statistical tests—the Kruskal-Wallis test, the Wilcoxon matched-pair&rank sum test, and Spearman’s&rank correlation coefficient—were used in statistical comparisons. For purposes of analysis, when an RT-PCR result was below the threshold of detection (4.0 log10 transcripts/μg of total RNA), the result was designated to be 4.0, which would not affect the nonparametric test results. Seasonality was assessed by grouping data on villous height and mRNA level from all years of data collection into 2-month periods. Data on diarrhea incidence were used to estimate incidence as a dichotomous (yes/no) variable for the 2 months before or after the date of biopsy. Analysis was performed using STATA (version 8.0; StataCorp)

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Results

Dynamics of α-defensin expression in Zambian adultsWe analyzed the biopsy samples obtained from 83 participants at baseline and then analyzed the biopsy samples subsequently obtained from those participants who remained under follow-up for some portion of the next 2 years; 46 were still under follow-up after 1 year, and 40 were still under follow-up after 2 years, so 169 biopsy samples in total were available for analysis. The characteristics of the study participants at baseline are shown in table 1, including HIV status (when known), CD4+ and CD8+ cell counts, results of the morphometrical analysis of the biopsy samples, and α-defensin mRNA levels

The number of transcripts in the baseline biopsy samples from 83 different participants varied from 4.0 (i.e., the threshold of detection) to 8.0 log10 transcripts/μg of total RNA, and a similar range was observed during every year of the study. This range of variation was not explained by interexperimental variation and exceeded the variation observed in triplicate biopsy samples from the same individual [14]. The correlation between HD5 and HD6 mRNA level was strong (ρ=0.88; P<.001). No overall differences were observed in HD5 or HD6 mRNA level between the HIV-seropositive participants and the HIV-seronegative participants (table 1). No correlation was observed between mRNA levels and CD4+ cell count among the HIV-seropositive participants. There was also substantial longitudinal variation among the values obtained for each participant during the 2 years of follow-up (figure 1). In the group as a whole, the longitudinal variation displayed a seasonal effect (table 2)

Figure 1
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Figure 1

Longitudinal changes in human defensin (HD) 5 (solid lines) and HD6 (dashed lines) mRNA levels at 3 time points during 2 years of follow-up. Data from 12 Lusaka study participants (identification nos. are given above the graphs) are shown to demonstrate the range of variation observed. mRNA levels are log10 transcripts per microgram of total RNA

This variation in the α-defensin mRNA levels was not due to changes in the cell types expressing HD5 and HD6, as assessed by in situ hybridization and immunohistochemical analysis. HD5 and HD6 were almost exclusively expressed by Paneth cells (figure 2), with only 1 or 2 cells higher up the crypt staining by either technique among all 60 sections examined, and the numbers of Paneth cells varied little or not at all. Because HD5 is stored as propeptide [27], we looked for evidence of variation in the quantity of stored propeptide or in the posttranslational processing accompanying the changes in the mRNA levels. Western blots of cationic peptide extracts from these biopsy samples showed the same profile of peptide isoforms that has been previously observed in human intestinal tissue [27], including propeptide and an O-linked glycan modification of the HD5 (aa 20–94) propeptide (C.L.B., unpublished data) (figure 3). Very little variation was observed in either the quantity or the migration pattern of HD5 propeptide isoforms stored in Paneth cell granules (figure 3). Because no antibody to HD6 was available, peptide analyses were performed only for HD5

Figure 2
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Figure 2

In situ hybridization and immunohistochemical analysis. A In situ hybridization using antisense riboprobe to human defensin (HD) 5. B Immunostaining using monoclonal anti-HD5 antibody. C In situ hybridization using antisense riboprobe to HD6. Labeling of mRNA and peptide was almost exclusively localized to the Paneth cell compartment

Figure 3
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Figure 3

Western blot of tissue extracts from jejunal biopsy samples probed with anti–human defensin (HD) 5 antibody. Gel electrophoresis was performed by acid-urea polyacrylamide gel electrophoresis, in which peptides are separated by both size and charge. Recombinant peptides are, therefore, used as markers of migration. Lane 1 Recombinant pro-HD5 (aa 20–94) (pro-rHD5); lane 2 recombinant HD5 (rHD5); lanes 3–7 individual biopsy samples; lane 8 control ileal tissue from a healthy patient. The quantity and isoform distribution of propeptide shows little variation. The diffuse, slow migrating band corresponds to an O-linked glycan modification of the HD5 (aa 20–94) propeptide (C.L.B., unpublished data)

Variations in HD5 and HD6 mRNA levels correspond tochanges in mucosal architectureAmong the HIV-seronegative participants, both HD5 and HD6 mRNA expression at baseline were inversely correlated with villous height, epithelial surface area, and villous compartment volume; no correlation was observed with crypt depth (table 3). During 2 years of follow-up, the changes in α-defensin mRNA expression showed strong inverse correlations with the changes in villous height, and significant correlations were also observed with the changes in crypt depth (table 3). The seasonal variation in α-defensin mRNA expression was inversely correlated with the seasonal variation in villous height (table 2). Among the HIV-seropositive participants, no correlations between α-defensin mRNA expression and determinants of mucosal architecture were observed

Table 1
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Table 1

Demographic and clinical characteristics of the Lusaka study participants at baseline

Table 2
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Table 2

Seasonal variation in α-defensin expression

Table 3
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Table 3

Correlations between mucosal architecture and α-defensin mRNA levels in the HIV-seronegative participants, at baseline and during 2 years of follow-up

Inverse correlation between α-defensin mRNA expression and incidence of diarrheaDefensin mRNA expression in biopsy samples was compared between those participants who experienced diarrhea in the 2 months following sample collection and those who did not. The median (IQR) HD5 mRNA level was 6.0 (5.6–6.7) log10 transcripts/μg of total RNA among 18 participants who experienced diarrhea, compared with 6.8 (6.2–7.3) log10 transcripts/μg of total RNA among 94 participants who did not (P=.006). Similarly, the median (IQR) HD6 mRNA level was 6.0 (5.6–7.3) log10 transcripts/μg of total RNA among 20 participants who experienced diarrhea, compared with 6.6 (6.2–7.5) log10 transcripts/μg of total RNA among 99 participants who did not (P=.04)

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Discussion

It is likely that innate immunity in the small intestine makes a major contribution to host defense, being an initial barrier to colonization by, or translocation of, potential pathogens. Experiments in animals have suggested that α-defensins play a significant role in intestinal defense in vivo, and, although recent in vitro data have suggested that HD6 does not have antimicrobial activity against a limited number of bacterial species of the intestinal microbiota [28], the investigators speculated that HD6 may have activity against viral or other pathogens. There are few data from studies in humans, and confirmation of such a role would be of great interest. We found that small-intestinal α-defensin expression is constitutive, in as much as it is always detectable. However, mRNA levels varied substantially between individuals, over time, and in a seasonal pattern. Moreover, mRNA levels varied reciprocally over time with both villous height and crypt depth, which previously were found to be positively correlated with each other in the present population [18]

We also found that reduced gene expression was associated with an increased risk of diarrhea. There are 2 possible explanations for this. The first explanation is that α-defensins make such an important contribution to intestinal defense that a 10-fold reduction in mRNA level could significantly increase susceptibility to infection and, therefore, diarrhea. This is plausible, given that mRNA level predicted peptide turnover in experiments in an ex vivo model (W.D., unpublished data). The second explanation is that increased exposure to tropical microbiota could simultaneously down-regulate α-defensin mRNA levels and increase diarrhea incidence. This hypothesis is supported by experiments that suggest that pathogens or their components can suppress expression of antimicrobial molecules [29, 30]. However, because our study design required participants to have been free of diarrhea for the month before the date of biopsy, we favor the first explanation, although the latter cannot be completely ruled out

The strong correlations over time between α-defensin mRNA level and mucosal architecture deserve further exploration. The very strong correlations for HD5 and HD6 mRNA levels made it important to establish whether the changes in the number of α-defensin transcripts might be explained by changes in the number or distribution of Paneth cells or by changes in the lineage of cells expressing α-defensin genes, but no such changes were found. It is well known that T cell activation induces mucosal remodeling, with reduced villous height and increased crypt depth. This may explain the differences observed between temperate and tropical populations, in which T cell activation has been suggested to play a part [19]. However, it cannot explain the mucosal remodeling within the present tropical population, in which villous height and crypt depth were positively correlated [18]. We suspect that nutritional factors may play a role, because starvation is the only context in which such a positive correlation has been observed. Although there was, to our surprise, no difference in either α-defensin mRNA level between the HIV-seropositive participants and the HIV-seronegative participants, the correlation between mRNA level and mucosal architecture was lost in the HIV-seropositive participants. This suggests that there is some dysregulation of α-defensin transcriptional control during HIV infection

Diarrhea incidence was greater in the participants who had reduced levels of α-defensin mRNA. We can find significance in this observation only if we are reasonably confident that exposure to pathogens would be similar from one individual to another. This is highly probable in the present population, because all of the participants were drawn from one small subsection of one shanty compound in periurban Lusaka. This environment is characterized by overcrowding and poor sanitary facilities. Water and food outlets are few in number, and hygiene is invariably poor. A majority of the adults living in this area at baseline participated in our study, and we believe that exposure to pathogens, although not uniform, is likely to have been quite consistent. We can be confident that the reduced α-defensin mRNA levels in the participants who subsequently experienced diarrhea was not confounded by HIV status, which although predictive of diarrhea incidence (data not shown) was not related to α-defensin mRNA levels, as noted above. Future studies in other populations will be needed to confirm the association between reduced α-defensin expression and increased risk of diarrhea. It will also be necessary to establish whether this association is the result of increased frequency of infection (by which we mean colonization), increased intensity of infection, or a permissive effect on virulence-factor expression by pathogens

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Acknowledgments

We are grateful to Emmanuel Kunda, Rosemary Banda, Vera Yambayamba, Coillard Kaunga, John Samson Mbewe, Stayner Mwanamakondo, Rose Soko, and Miriam Banda of the Misisi clinical team, the endoscopy unit of the University Teaching Hospital, and our study participants. Bo Shen and Dipankar Ghosh of the Cleveland Clinic Foundation contributed to the immunostaining and Western blotting, respectively

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Footnotes

  • ↵Presented in part: Gordon Research Conference on Antimicrobial Peptides, Ventura, California, 6–11 March 2005 (poster)

    Potential conflicts of interest: none reported

    Financial support: Wellcome Trust (grant 056481)

  • Received August 19, 2005.
  • Accepted November 17, 2005.
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  1. J Infect Dis. (2006) 193 (10): 1464-1470. doi: 10.1086/503747
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