In our recent study22, we showed that experimental infections with N. americanus enabled individuals with CeD to tolerate escalating challenges with dietary gluten. The biological, immunological and molecular mechanisms underlying this outcome are yet to be fully determined; however, we hypothesized that an expansion of regulatory T cells, together with reductions in pro-inflammatory effector T cells in the duodenum, may have been responsible for the restoration of the intestinal homeostasis and, consequently, for the improved gluten tolerance19,22,26. However, other elements within the complex spectrum of host-parasite interactions may have contributed to this cascade of biological events in the study subjects. Here, we evaluated the impact of hookworm infection and gradual re-introduction of gluten into the diet on the microbial communities inhabiting the intestine of the same cohort of volunteers who took part in the clinical study22.

We had hypothesized that experimental hookworm infection, combined with exposure to escalating doses of gluten, would result in significant quantitative and qualitative fluctuations of the gut microbiota populations of the volunteers. However, we detected no significant differences in the overall community structure, Shannon diversity or relative abundance of individual bacterial species over the course of our study, in accordance with the findings of our previous investigation in which a 8-week, acute hookworm infection alone did not result in significant alterations of the fecal microbiota28. However, in the present study, we observed a significantly increased microbial richness (= total number of microbial species present) in fecal samples of Trial subjects collected following hookworm infection and moderate gluten challenge, compared with pre-trial samples. One possible explanation for this discrepancy is that hookworms may actively contribute to the maintenance of bacterial homeostasis in the gut28 and ‘restore’ homeostasis (by promoting an increase in microbial richness) when inflammatory responses are triggered (e.g. by the introduction of gluten in a celiac subject). Alternatively, it is possible that hookworm-induced changes in microbial species richness can only be detected following the establishment of chronic, rather than acute, infections28. The latter theory is also substantiated by the results of a recent investigation, in which a significantly increased species richness was detected in the intestinal microbiota of individuals from indigenous Malaysian communities naturally infected with gastrointestinal helminths (Trichuris and/or hookworms and/or Ascaris sp.) when compared with helminth-free, uninfected subjects from the same areas29. Similarly, a study using a primate model of chronic idiopathic diarrhea showed that experimental infections with whipworms were associated with a significant increase of microbial species richness and evenness (= diversity) which, in turn, had been hypothesized to play a role in the restoration of intestinal homeostasis and symptomatic improvement24. In our study, the increase in species richness that followed hookworm infection was not associated with an increase in Shannon diversity (p > 0.05). This finding may be the result of limited statistical power as direct consequence of our small sample size, which may also have been responsible for the small (albeit significant) differences in microbial species richness observed over the course of the study. Nevertheless, a higher species richness of the gut microbiota has generally been associated with a “healthier” intestinal homeostasis30,31,32. In particular, a study comparing the intestinal microbiota of subjects suffering from Crohn’s Disease and Ulcerative Colitis with that of healthy controls revealed that species richness was significantly higher in the latter30. In addition, the microbiota isolated from non-inflamed tissue from diseased subjects displayed significantly increased species richness when compared with that from inflamed biopsy samples from the same individuals30. Therefore, based on the results of the present and previous studies that associate increased microbial species richness to infections with helminths29 and the amelioration of inflammation24,30,31,32, we hypothesize that the therapeutic properties of hookworms may be partly linked to their ability to promote species richness and restore/maintain microbial (and immune) homeostasis in the gastrointestinal tract28. However, one limitation of our clinical study22 was the unavailability of a hookworm- or gluten-placebo cohort; therefore, larger placebo-controlled trials, together with the definition of the enterotype of each subject33, are necessary to confirm whether hookworms alone, or the gradual reintroduction of dietary gluten, are involved in modulation of the microbiota.

Three fecal samples from volunteers with active CeD (Control) were included in our study and compared with the pre-trial (T0) samples from the Trial subjects. Quantitative differences in the abundance of the main phyla were observed between the two groups; in particular, the Firmicutes dominated the microbiota of the Control subjects with active CeD (albeit this difference was below statistical significance), whereas the Bacteroidetes were more abundant in the healthy Trial subjects. Despite this overall trend, the genus Lachnospira (a Firmicutes) was more abundant in the Trial subjects (but below statistical significance); Clostridia and Ruminococcus (both within the phylum Firmicutes) dominated the fecal microbiota of the Control subjects. A shift in the Bacteroidetes to Firmicutes ratio in association with active CeD has already been documented in a previous study examining the effects of sustained GFD on the composition of the duodenal microbiota of adult CeD volunteers6,13. Given the recently described roles of Firmicutes in the metabolism of gluten34, the abundance of bacteria within this phylum in subjects with active CeD may reflect the dietary habits of these volunteers. In contrast, the abundance of Clostridia and Ruminococcus (at the class and genus level, respectively), in the Control subjects is in disagreement with previous findings reporting Methylobacterium (class Alphaproteobacteria) and Mycobacterium (Actinobacteria) as the prevalent genera of bacteria in the gut microbiota of adults with active CeD6. One possible explanation for this discrepancy is technical and likely to be associated to methodological differences between our study and that by Nistal et al.6; indeed, the latter utilized PCR amplification of the bacterial 16 S rRNA gene, followed by cloning and sequencing of randomly-picked clones, yielding ~50 sequences per sample. Therefore, in our opinion, the vast difference in sequencing depth between our study and that by Nistal et al.6 does not warrant direct comparisons of findings. However, a higher prevalence of Clostridia had been detected in the feces of CeD children with active disease compared with CeD controls on a strict GFD35. In addition, a recent investigation of the fecal microbiota of infants at either high- or low- genetic risk of developing CeD (HLA-DQ2 carriers and non-HLA-DQ2 carriers, respectively) revealed that the former is characterized by significantly higher proportions of Clostridia27. However, the role of this bacterial group in the progression of CeD is yet to be ascertained.

In conclusion, the present study has revealed a potential beneficial consequence of hookworm infection in the context of an autoimmune disease, i.e. supporting the maintenance and diversity of commensal flora. Whether hookworm-mediated changes in the microbiota are causal for the improved gluten tolerance and reduced inflammation in CeD is unknown, but could be examined in greater detail in an animal model of the disease. It also remains possible that hookworm-induced immunoregulation is mediated by additional mechanisms, in particular the direct actions of parasite excretory/secretory proteins on immune cells36. Clearly, helminth-based therapies hold promise for a range of autoimmune or inflammatory conditions and reversal of the dysbiosis associated with these disorders by tissue-resident parasites is one potential mechanism that requires further investigation.