The data presented here suggest that ICD in juvenile rhesus macaques is an IBD-like disease in which the mucosal barrier is compromised, allowing increased bacterial attachment that contributes to persistent inflammation and dysbiosis of the mucosal microbiota. This study revealed that T. trichiura
treatment induces a TH
2-type immune response in the intestinal mucosa of ICD subjects that was associated with symptomatic improvement. This positive clinical response was inversely correlated with cellular markers of mucosal T cell inflammation, such as Ki67+
T cells. In contrast to the TH
2-type response, Treg
expansion was noted under conditions of mucosal inflammation and was reduced by T. trichiura
treatment, suggesting that the presence of Tregs
in the intestinal tract is more indicative of ongoing intestinal inflammation than of helminth exposure. Based on these results, we speculate that T. trichiura
promotes mucosal healing in the setting of ICD by activating mucus production and epithelial cell turnover, aspects of TH
2-type immunity aimed at expelling the parasite 
, thereby reducing the attachment of immunostimulatory bacteria to the colonic epithelium and restoring diversity to the mucosal micobiota (). However, additional studies are needed to determine the mechanism of action for Trichuris
-mediated amelioration of colitis, and functional studies to establish causation would have to be performed in a suitable mouse model to confirm these hypotheses.
Working model of the immunologic mechanisms underlying the amelioration of colitis in the setting of Trichuris sp. treatment.
One weakness of this study is the absence of a control arm to ensure that the macaques with ICD did not undergo spontaneous remission. Future studies to confirm the findings presented here should have a sham-treated control arm, which would also enable a blinded assessment of stool frequency. The association of clinical improvement with weight gains, which is an objective measure, supports the observation that T. trichiura treatment did lead to improvement in symptoms. Additionally, our transcriptional profiling experiments clearly show that clinical improvement is associated with dramatic changes in mucosal gene expression patterns. The number of biopsies that could be collected from these juvenile macaques also limited our study. Histopathological observations that could be correlated with gene expression patterns would have provided additional insights into the relationship between cellular infiltrates and mucosal responses. While ICD shares some similar features with UC, inflammation is typically microscopic and cannot be determined objectively by visual inspection during endoscopy.
The mucosal gene expression patterns (e.g., REG1, REG3, NOS2, TFF1, SAA1, and SAA3) that distinguish ICD and healthy macaques are strikingly similar to signatures observed in microarray studies of UC patients 
as well as in studies of germ free mice repopulated with various bacterial taxa 
, suggesting that a response to gut bacteria may be the predominant driving force for this inflammatory signature. Importantly, differences in gene expression patterns were apparent between the four clinical responders and the single non-responder in this study. Of note, the non-responder showed lower expression of immunoglobulin-related genes (Figure S4
), suggesting that the etiology of disease in this animal may have stemmed from a defect in the mucosal B cell compartment, as has been shown previously to precipitate colitis through the absence of regulatory B cells 
. Recently, MyD88 signaling in B cells was found to prevent the lethal dissemination of intestinal bacteria after dextran sulfate sodium (DSS) treatment in mice 
The IL-23, IL-17 and IL-22 network plays a critical role in intestinal homeostasis and IBD pathogenesis 
. We previously reported that IL-22-producing CD4+
cells were a prominent feature of T. trichiura
infection in a UC patient. Unfortunately, our efforts to stain for IL-17- and IL-22-producing CD4+
cells from the macaque pinch biopsies were unsuccessful (data not shown). When we investigated the expression levels of the cytokines in these pathways pre- and post-T. trichiura
treatment, we found no significant trends (Figure S7
). Since the role of these cytokines in ICD of macaques is completely unknown, this may represent an important difference with human IBD, but it is difficult to draw any conclusions from these results at the moment.
The effects of T. trichiura
treatment on improving symptoms of colitis occurred despite the absence of a chronic and active infection. Ova were never detected in fecal samples from any of the macaques, indicating the lack of a patent infection, and adult worms were not seen during the post-treatment colonoscopy. The lack of patency is most likely due to a species barrier, since the T. trichiura
ova used for inoculation were from a human subject 
. Notably, this parallels the use of T. suis
ova (TSO) as a therapeutic intervention in human subjects with autoimmune diseases, which is dependent on a species barrier to eliminate the parasites after dosing. After hatching, Trichuris
larvae molt several times in the intestines before maturing into adult worms. Thus, the larval stages could have elicited a mucosal response leading to a positive clinical outcome. The absence of egg production does not preclude the presence of adult forms or mature larval forms, although none were observed during the endoscopies. Despite the absence of adult forms in humans, TSO clearly induces a strong TH
2 response in treated subjects, as indicated by increased serum IL-4, eosinophilia, and IgE antibodies 
. Although we did not detect more IL-4-producing CD4+
T cells by intracellular cytokine staining in post-treatment PBMCs, this is not indicative that a systemic Th2 response was absent in the macaques. In our previous study 
, responses in PBMCs could only be detected after antigen-specific expansion of CD4+
cells with T. trichiura
antigen. We propose that the treatment of colitis (but not necessarily other autoimmune diseases) with Trichuris sp.
may be more dependent on a TH
2 response than immunoregulation. Since we did not detect ova in the feces of the treated macaques, it is also possible that the clinical improvements were spontaneous and not related to T. trichiura
treatment. However, we find this unlikely since the TH
2 response in the intestinal mucosa at a cellular and molecular level is remarkably consistent among all of the treated macaques.
It is important to note the changes in absolute bacterial quantities as well as the composition of mucosal microbiota following helminth exposure. With both measures, post-treatment samples were much more similar to samples from healthy controls. Perhaps the most consistent observation made in studies on the microbiota of IBD patients is the overall reduction in microbial diversity 
. There is also reduced diversity of the intestinal microbiota in rhesus macaques with colitis, as noted previously 
as well as in this study, supporting the use of rhesus macaques with ICD as a preclinical model for IBD.
We noted a striking expansion of Cyanobacteria in three ICD subjects, which disappeared post-treatment. Cyanobacteria have been previously noted to reside in the intestinal tract of mice and humans 
as well as that of macaques 
. However, there have not been any previous reports, including studies on the mucosal microbiota from IBD patients 
, where this taxon has represented an abundance of up to 32.9% of 16S sequences. This expansion may reflect a unique environment on the intestinal mucosa of juvenile rhesus monkeys with chronic diarrhea. Notably, a recent publication (including data from the Human Microbiome Project) indicated that, in healthy human beings, the taxon Cyanobacteria
Streptophyta was enriched in non-mucosal sites relative to mucosal body sites and also enriched under conditions with high oxygen availability 
. Like Salmonella typhimurium
, perhaps the activation of neutrophils to produce reactive oxygen species favors the expansion of a Cyanobacterium more adapted to an aerobic environment or to alterations in redox conditions. It is also quite possible that these sequences are merely a representation of plant organisms in poorly digested food, which could be more abundantly attached to the mucosa of monkeys suffering from diarrhea.
Since Bacteroidetes is the most predominant phylum in the normal gut, the reduction in proportional abundance in ICD subjects may reflect an expansion of non-Bacteroidetes phyla amongst the mucosal microbiota during ICD. Indeed, quantification of absolute Bacteroidetes abundance by RT-PCR clearly shows a reduction post-treatment. These results reflect a typical problem for this type of 16S compositional data of microbial communities. What is more interesting is the unexpected expansion of Tenericutes after helminth exposure, even relative to control macaques, indicating that bacteria of this phylum could be preferentially expanded in a TH
2-type mucosal environment. Little is known about the role of Tenericutes in the intestinal tract. In one previous study with mice, dextran sodium sulfate (DSS)-driven colitis was shown to decrease the abundance of this phylum 
. Further studies on the relationship between intestinal helminths and Tenericutes are warranted.
ICD-afflicted juvenile rhesus macaques may represent a useful preclinical model in which to further study the pathology, diagnosis, and treatment of UC in humans under certain circumstances. Most notably, the transcriptional signature of the inflamed mucosa in these macaques closely resembles that of UC patients. Given the prevalence of ICD at primate research centers, this could be an important resource to develop new therapeutics for IBD in humans, especially when addressing the need of a non-rodent model during preclinical testing. However, the costs and ethical issues of using non-human primates may limit the usefulness of this animal model for IBD research towards very specific circumstances when an outbred non-rodent model is required. While very similar, there are also distinct differences between the macaque immune system and the human immune system 
that may limit the use of ICD afflicted rhesus macaques as a model for IBD research.
Our findings describe the immune mechanisms that may mediate the therapeutic effect of Trichuris sp. in the setting of colitis and highlight the role of TH2-type immunity in promoting mucosal repair. Instead of acting through an immunoregulatory mechanism, Trichuris sp. may trigger a TH2-driven expulsion mechanism related to increased mucus production and turnover of epithelial cells, thereby reducing the quantity of attached bacteria among the mucosal microbiota. As part of this process, the dysbiosis observed in the mucosal environment during colitis could also revert back to a normal equilibrium.