Strain-Dependent Modulation of Iron Homeostasis Gene Expression in Caco-2 Intestinal Epithelial Cells by Novel Probiotic Strains

• Frank Segreto, Agriculture and Natural Resources, University of Delaware

• Shafeekh Muyyarikkandy, CANR, University of Delaware

Anemia affects 1.92 billion people worldwide, with dietary iron deficiency the leading cause,

and its treatment is complicated by inflammatory suppression of dietary iron absorption.

Interleukin-6 (IL-6) signaling induces hepcidin (HAMP), which drives degradation of the

basolateral iron exporter ferroportin (SLC40A1/FPN1) and traps iron within enterocytes,

rendering oral supplementation ineffective. Cell-free supernatants (CFS) from lactic acid bacteria

have been proposed as modulators of intestinal iron handling, but strain-level effects remain

poorly characterized. We screened three cottage cheese isolates, Leuconostoc

pseudomesenteroides MLS3, Lactococcus lactis MLS12, and Leuconostoc lactis MLS22, for

their capacity to alter iron homeostasis transcripts in intestinal epithelium. Caco-2 cells were

seeded at 1.25 × 10⁵ cells per well in 12-well plates, differentiated for 7 days post-confluence,

serum-starved for 16 h, challenged with 20 ng/mL IL-6 for 6 h, and treated for 15hours with

strain-specific CFS at 50% (v/v) or an equivalent cell-free MRS vehicle. TFRC, SLC40A1, and

HAMP abundance was quantified by RT-qPCR (2-ΔΔCt, GAPDH) across 3 independent

experiments and compared by one-way ANOVA with Sidak’s test against the IL-6-only arm. IL-

6 alone induced HAMP approximately 13-fold, SLC40A1 approximately 12.5-fold, and TFRC

approximately 7.6-fold versus untreated control, confirming inflammatory iron restriction.

Postbiotic effects were strain dependent. MLS3 CFS did not significantly alter any target, though

HAMP trended lower (approximately 5.5-fold) with sustained TFRC elevation (approximately 9-

fold). MLS12 CFS likewise showed no significant change but produced the lowest HAMP

abundance (approximately 2-fold) with retained FPN1 (approximately 8.3-fold), an export-

permissive direction. MLS22 CFS was the only strain producing statistically significant changes

(p<0.05), though the magnitude and coordinate direction of these effects require independent

confirmation. MLS3 and MLS12 trended toward hepcidin attenuation without reaching

significance. However, ongoing experiments would provide more insights. Because ferroportin is

regulated post-translationally, functional validation requires ferroportin immunoblotting and

polarized transwell iron-flux studies.