Faecal zonulin, calprotectin and the infant microbiome in early life

Here, we present novel data associating faecal calpro-tectin and zonulin, biomarkers of intestinal inflammation and permeability respectively, and the gut microbiome during early life.

functional pathways using HuMaNn3.Statistical comparisons were done at each timepoint separately with one sample per subject using MaAsLin2 for differential abundance of taxa and pathways, and Kruskal-Wallis tests for α-diversity.See Supporting information for full method details.
We found that zonulin peaked at 6 m, while calprotectin was elevated at birth, with both subsequently decreasing over time (Figure 1A,B).At 2 m, higher zonulin levels were associated with vaginally delivered infants (p = .036),while at 6 m they were associated with no maternal antibiotic exposure (prenatal and/or peripartum) (p = .007)(Figure 1C).Increased calprotectin levels were associated at 2 and 6 m with no maternal antibiotic exposure (p = .042and.034, respectively) (Figure 1D).Additionally, calprotectin was elevated in females at birth (p = .0002).
Shannon α-diversity was positively associated with zonulin at 2 m (p = .002);there was no association between α-diversity and calprotectin.β-diversity did not differ with any biomarker quartiles.
Several bacterial taxa at the genus level and above were associated with these biomarkers (Figure 2A).At the species-level, Clostridioides difficile positively associated with zonulin at 6 m (p = .001).Zonulin also positively associated with Klebsiella quasipneumoniae (p = .001)and negatively with Rothia mucilaginosa (p = .003)at 2 m, both of which stratified by delivery mode and/or no maternal antibiotic exposure (Figure 2B).Additionally, at the species-level, calprotectin positively associated with several species of Enterocloster and Clostridium (p < .005)stratifying by the delivery mode and/or maternal antibiotic exposure at 6 m (Figure 2C).These associations were not seen at later timepoints.Regarding metabolic potential, zonulin positively associated with the following pathways at 6 m (p < .0005)stratifying by the delivery mode and maternal antibiotic exposure: pyruvate fermentation to butanoate (CENTFERM-PWY), L-glutamine biosynthesis III (PWY-6549), and the superpathway of Clostridium acetobutylicum acidogenic fermentation (PWY-6590) (Figure 2D).Building on the previous work, we observed that vaginal delivery and lack of maternal antibiotic exposure, which are known to positively influence the microbial and immune environment, were associated with higher zonulin and/or calprotectin levels at early timepoints only. 4,5This suggests that these biomarkers play a different role during early life and may be related to healthy gut and immune development.It is worth noting that delivery mode and maternal antibiotic exposure are not mutually exclusive, as all caesarean sections in this study included the peripartum antibiotic treatment.Further, there are reports regarding low specificity of commercial ELISA kits for detecting faecal zonulin.
Furthermore, previous work uncovered that calprotectin was higher in vaginally delivered infants during the first week of life. 5We found a similar trend after birth but without statistical significance.However, zonulin was higher in vaginally delivered infants at the 2 m timepoint, supporting the idea that these biomarkers may indicate healthy gut development during early life.Interestingly, elevated calprotectin in females was also observed in a study of pre-term infants during the first week of life. 6Our results suggest that the sex-difference in calprotectin levels may not be restricted to pre-term infants and resolve by 2 m of age.
Our novel use of shotgun metagenomics identified species-level and metabolic pathway associations at early timepoints with zonulin and/or calprotectin which is previously unreported.Notably, Clostridioides difficile was positively associated with zonulin at 6 m.Although toxigenic C. difficile has pathogenic potential later in life, it is a normal component of the microbiome in infancy. 7he association with zonulin early in life could be postulated to support the immune maturation process.Moreover, Rothia mucilaginosa was negatively associated with zonulin at 2 m.R. mucilaginosa is associated with the oral microbiome and can produce microbiome altering enterobactin. 8Thus, R. mucilaginosa may alter the gut environment triggering reduced zonulin levels.Additionally, zonulin positively correlated at 6 m with short-chain fatty acid (SCFA) fermentation pathways and stratified by delivery mode and maternal antibiotic exposure.SCFA fermentation is an indicator of gut health and shapes the gut mucosal immune system. 9,10We hypothesise that the positive correlation with zonulin may contribute to immune education at this early life timepoint.
In conclusion, elevated zonulin and calprotectin were associated with various bacterial taxa, as well as early life clinical factors known to have an important influence on health outcomes later in life.Taken together, we F I G U R E 2 Bacterial taxa associations with zonulin and calprotectin levels at different timepoints.(A) Table of bacterial taxa at genus-level or higher that associated with zonulin and calprotectin with a false discovery rate (FDR q-value) less than.01 at various timepoints.The unadjusted p-value is reported as asterisks.Taxa level is indicated as follows: p: phylum; c: class; o: order; f: family; and g: genus.(B) Graphs of centred log-ratio of abundance of taxa at a species level found to significantly associate with fecal zonulin levels.(C) Graphs of centred log-ratio of abundance of taxa at a species level found to significantly associate with fecal calprotectin levels.(D) Graphs of relative abundance for pathways found to significantly associate with fecal zonulin levels.
hypothesise that these faecal biomarkers may be related to healthy microbiome and immune development during this critical period as opposed to being associated with pathology as in adults, 2,3 with exposure to certain microbes and their inflammatory mediators influencing immune system education.Further investigation to examine this is warranted given this preliminary observation.

S U P P O R T I N G I N F O R M AT I O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.

F
Zonulin and calprotectin are elevated during early life.Tukey box-and-whisker plots of (A) Zonulin over the first 2 years of life.(B) Calprotectin over the first 2 years of life.(C) Zonulin at 2 and 6 m stratified by the delivery mode and maternal antibiotic exposure.(D) Calprotectin at birth stratified by sex, and calprotectin at 2 and 6 m stratified by maternal antibiotic exposure.*p < .05;**p < .01;***p < .001;****p < .0001.