Immunonutrition enhances the expression and secretion of mouse intestinal defensins

The nutrient rich lumen of the small intestine provides a favourable environment for microbial cell growth. Secretion of defensins from Paneth cells constitutes a key source of antimicrobial peptide activity in the crypt lumen.¹ In addition, defensins also recruit the adaptive immune system.²,³ Chronic disease, major surgery, trauma, or malnutrition results in immune compromise and can increase the risk of infection and sepsis.⁴ Several clinical studies have demonstrated that patients fed an immune enhancing formula had significantly fewer infectious complications, needed less antibiotics, and had a shorter hospital stay than patients fed standard diets.⁵ However, the mechanism by which these formulas bolster the immune system is unclear.

We hypothesised that a nutrient enriched diet bolsters the immune system by upregulating defensin expression in the jejunum and ileum, the two parts where cryptdins (mouse intestinal defensins) are highly expressed. To study the effect of an immune enhancing diet (Perative; Ross Products, Abbott Laboratories, USA) on intestinal defensin expression, we used mice fed either regular chow or Perative for a week. The volume of Perative (complete balanced nutrition containing partially hydrolysed peptide based high protein (sodium caseinate, lactalbumin, 66.7 g/l), arginine at 2.5% of total calories, dietary nucleotides, canola oil, medium chain triglyceride oil, corn oil, soy lecithin, vitamins, minerals, fructooligosaccharides, lactose, gluten, and growth factor free) consumed by the mice was similar throughout the week (15 ml/day). Body weight did not differ between the Perative fed and chow fed groups. Northern blot analysis⁶ of total RNA extracted from the jejunum and ileum, using an oligo probe specific to cryptdin 1, cryptdin 2, and cryptdin 3 (>93% nucleotide identity), or cryptdin 4, revealed that levels of cryptdins 1–3 (fig 1A) and cryptdin 4 (fig 1B) increased by 1.6‐fold (Student's t test, p<0.05).

Figure 1 Expression levels of cryptdins 1–3 and cryptdin 4 after Perative supplementation. (A) Levels of cryptdins 1–3 in the jejunum and ileum of chow fed versus Perative fed mice. (B) Levels of cryptdin 4 in the jejunum and ileum of chow fed versus Perative fed mice. (C) Levels of cryptdins 1–3 and cryptdin 4 in primary Paneth cells treated with isoleucine (Ile), arginine (Arg), and Perative. In (A) and (B), total RNA was extracted and cryptdin levels analysed by Northern blot analysis and normalised to β‐actin. In (C), cells were treated for six hours, total RNA was extracted, and cryptdin levels were analysed by quantitative real time polymerase chain reaction and normalised to Gapdh. Each bar represents the mean (SEM) of three independent experiments. CM, complete medium. **p<0.005 significantly different from control (Student's t test).

To further study the direct effect of Perative on Paneth cells, we isolated primary Paneth cells⁷ from mouse jejunum and tested the effect of different concentrations of Perative (0.0025%, 0.01%, 0.025%), isoleucine, and arginine (25, 100, 250 μg/ml) on cryptdin expression by real time polymerase chain reaction.⁶ Whereas different concentrations of arginine and isoleucine did not stimulate cryptdin expression (Student's t test, p> 0.05), 0.025% Perative increased significantly expression of cryptdins 1–3 (Student's t test, p = 0.003) (fig 1 C) and cryptdin 4 (Student's t test, p<0.0001) (fig 1C). The ability of similar concentrations of isoleucine and arginine to induce human β‐defensin in HCT‐116 cells⁸ stems, most probably, from the difference in the mechanism regulating expression of cryptdins (α‐defensins) versus β‐defensins. Different control of α‐ and β‐defensin expression is further reiterated by the fact that spleens challenged with Listeria monocytogenes, lungs infected with influenza A, and kidneys challenged with Candida albicans, all β‐defensin expressing tissues, revealed no differences in the cure rate when mice were fed Perative or other immune enhancing formulas.⁹

To further determine whether expression of cryptdin mRNA was concomitant with protein secretion in primary Paneth cells, we analysed the medium of primary Paneth cells stimulated with the aforementioned concentrations of isoleucine, arginine, and Perative. Cryptdins were isolated from the medium using a method to denature, renature, and concentrate small cationic proteins¹⁰ followed by reverse phase high performance liquid chromatography.⁸ Protein data of the primary Paneth cell media correlated with that of RNA (that is, higher concentrations of secreted cryptdins were found in the 0.025% Perative treatment—data not shown). Furthermore, eluting fractions exhibited defensin characteristics, such as a size of 3 kDa on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and activity against Escherichia coli DH5α (data not shown).

In summary, our results demonstrate that immune enhancing formulas, such as Perative, can upregulate cryptdin expression in Paneth cells. Further study is needed to delineate the molecular pathways by which nutrients lead to cryptdin upregulation. As defensins display antimicrobial activity as well as recruit the adaptive immunity by cytokine secretion and recruitment of lymphocytes,²,³ their secretion could be the first step in a cascade that leads to a general bolstering of the immune system.