In this study, we have systematically characterized regional differences of intestinal lamina propria CD4+ T cell populations by flow cytometry in a healthy population and identified gene expression patterns that correlate with their frequencies. We found the cecum to have significantly elevated frequencies of TH17, TH22, and TReg lymphocyte populations (but not TH1 or TH2 cells). Interestingly, these are the CD4+ subsets that are most important in regulating intestinal homeostasis. While TH17 cells are enriched in the terminal ileum in mice, we find here that the human cecum has a higher frequency of TH17 cells than does the ileum, reflecting important differences between mice and humans. Since intestinal mucosal CD4+ T cells are being increasingly studied under disease conditions (e.g. inflammatory bowel disease and HIV), it is important to rigorously investigate the baseline mucosal immune environment throughout the gut in healthy individuals. When mucosal biopsies are collected from different parts of the human intestine, there must be an awareness that inherent regional differences among CD4+ T cell populations are present in order to interpret study findings.
The results of our study are consistent with a previous histopathology study showing that the cecum is a site of inherent inflammation even in a normal healthy population without gastrointestinal symptoms 
. Compared to the rectum, increased microscopic inflammation (especially greater cellularity of the lamina propria) in a significant proportion of cecal biopsies taken from endoscopically normal mucosa in healthy patients has been reported 
The cecum is generally considered to play a digestive role, being enlarged in herbivores to aid in the breakdown of cellulose 
. However, there have been recent suggestions that it may have evolved in some species to perform an immunological function. In combination with the appendix, it has been proposed that the cecum could serve as a “safe house” for reconstituting commensal microbiota after an acute infectious diarrheal illness 
. This hypothesis was partly driven by the finding that microbial biofilms are more abundant in the appendix than other parts of the human colon 
. Secreted IgA and mucins in the cecum and appendix may support the growth of these microbial biofilms 
. More recently, it was reported that prior appendectomy was positively associated with increased risk of Clostridium difficile
recurrence, suggesting that the appendix may play a protective role against infection. In contrast, previous appendectomy has been shown to inversely correlate with risk of colectomy, relapse rate, and extent of disease in ulcerative colitis (UC) patients in a series of case-control studies 
. Prophylactic appendectomy has even been prospectively evaluated as a potential therapy for UC with promising results 
. Taken together, this literature suggests that microbial biofilms and immune activation in the cecum and appendix play a role in regulating homeostasis of intestinal microbiota, which could in turn impact homeostasis of the intestinal tissues. Based on this hypothesis, it would not be surprising that TH
22, and TReg
populations are enriched in the cecum to maintain an appropriate homeostasis between microbes and the intestinal mucosa.
Recently, innate lymphoid cells (ILCs) have emerged as an important non-CD4+
source of effector cytokines that may be particularly important at mucosal barrier surfaces 
. IL-17 and IL-22 producing human ILCs are responsive to IL-23 signaling and could be important mediators of inflammatory bowel diseases 
. Of additional interest was our observation that the increase in TH
17 cells in the cecum (compared to the terminal ileum and sigmoid colon) was offset by having relatively fewer numbers of IL-17 producing CD3−
cells in this region. This inverse relationship raises the possibility of regulatory feedback between innate and adaptive sources of IL-17 in the healthy human intestinal tract.
We attempted to determine if the biopsies collected from the cecum had a greater bacterial load attached to the intestinal wall compared with those obtained from the terminal ileum and the sigmoid colon, but no significant differences were observed based on qPCR analysis of several bacterial ribosomal 16S DNA sequences (relative to host DNA). It is certainly conceivable that a deep sequencing analysis of bacterial ribosomal 16S units may lead to the identification of novel bacterial species or operational taxonomic units (OTUs) that are more abundant on the cecal wall compared to other parts of the intestine. Micro-heterogeneity between sample sites has been reported based on the very limited number of individuals who have been evaluated by deep sequencing studies of the colonic mucosa 
, but large variations in bacterial populations among intestinal regions has not been found. However, the cecum was not analyzed in detail in these studies. It would be extremely interesting to conduct deep sequencing analyses to determine the nature of the cecal microbiota attached to the mucosa using paired biopsy samples for which the gene expression profiles, as well as flow cytometry profiles of CD4+
T cell populations, is well characterized.
In a previous human study, gene expression profiling analysis was performed along the entire proximal-distal axis of the colon, but the terminal ileum was not included 
. This study revealed two distinct patterns of transcript abundance along the proximal-distal axis of the large intestine. The colon was described as having a (i) dichotomous expression pattern consistent with its embryologic origins from both the midgut and the hindgut and hence segregating genes that are proximal or distal to a point that is two thirds the length of the transverse colon measured from the hepatic flexure and (ii) a gradual change in transcript levels from the cecum to the rectum, likely reflecting luminal flow and alterations in commensal microbiota. Our transcriptional profiling analysis of mucosal pinch biopsies confirms these results, with similar segregation of gene transcripts from both the proximal and distal colon and from the colon and terminal ileum. Additionally, we related transcriptional differences with the frequencies of CD4+
subsets along the intestine.
By correlating our flow cytometry analysis with gene expression profiling data from paired biopsy samples we discovered a positive relationship between the expression of resistin (RETN) and the frequency of TH
17 cells. Resistin is a cytokine produced by adipose tissue and was originally discovered as a potential mediator of metabolic functions involved in insulin resistance 
. More recently, it has been characterized as an inflammatory cytokine that is implicated in several pathologic intestinal conditions (e.g. colon cancer and inflammatory bowel disease) likely through induction of IL-1b, IL-6, IL-8, IL-12, TNFα, and other pro-inflammatory cytokines 
. As a pro-inflammatory cytokine the positive association between the frequency of TH
17 cells in the intestinal mucosa and expression levels of RETN is not surprising. PPARγ has been shown to regulate RETN activity in several studies 
. In this study we found that PPARγ expression and TH
17 cell frequencies are both elevated in the human cecum, hence a relationship with RETN function is plausible. In addition, bacterial products like lipopolysaccharide (LPS) have been found to induce RETN expression in human and murine macrophages 
. Future studies to establish a more mechanistic relationship between RETN and TH
17 cells will need to be conducted before any conclusions can be made about the biological significance of this relationship.
We also found a negative relationship between the frequency of TH
17 cells and the expression levels of TFF1. The trefoil factors are mucin-associated peptides associated with a number of human tissues, with TFF1 and TFF2 primarily located in the gastric mucosa and TFF3 predominantly present in the mucus cells of the small and large intestine 
. Up-regulation of all three TFF members occurs at sites of mucosal injury and has been observed in patients with peptic ulcer disease and inflammatory bowel disease 
. TFF1 plays a critical role in mucosal protection and repair, with loss of TFF1 being associated with NF-κB-mediated inflammation and gastric carcinogenesis 
. Based on these observations, it was surprising to observe a negative relationship between the frequencies of TH
17 cells and TFF1, since both factors could be important for maintaining intestinal homeostasis. One possibility could be that the anti-microbial effects of TFF1 are acting through an independent pathway from TH
17 cells. Hence, when bacterial loads are controlled through a TFF1 mediated pathway, fewer TH
17 cells are required. This hypothesis could be tested further through the careful localization of TFF1 expression and presence of TH
17 cells by immunohistochemistry of the intestinal tissues.
Finally, we found that TReg
frequencies were positively associated with expression of FCRL5 (CD307). FCRL3, a closely related FCRL family member, is highly expressed on dysfunctional TRegs
that express high levels of PD-1 and have a memory phenotype 
. While FCRL5 has mostly been characterized in B cells and B cell lymphomas 
, polymorphisms have also been reported to be associated with autoimmune diseases such as ankylosing spondylitis 
, suggesting an immunoregulatory role 
. The relationship between FCRL5 and TReg
cells in regulating intestinal homeostasis warrants further investigation.
This study has important limitations. The majority of the enrolled subjects were older black men and so their mucosal environment may not be representative of younger patients in a disease state. Furthermore, complete colonoscopies requiring a preparative purge were performed to obtain biopsies from the cecum, a feature that may impact the analysis of the intestinal microenvironment. While the pinch biopsy samples obtained were approximately equal in size from all locations and from all subjects, we did not directly measure the absolute numbers of cells per gram of pinch biopsy tissue from each location. Importantly, CD4+ viability was relatively similar independent of biopsy location. Lastly, we recognize that differences in gene expression profiles of the pinch biopsies may be caused by heterogeneity of cellular populations and cellular density between samples. Nonetheless, this study is a step towards understanding location-specific variations in lamina propria CD4+ T cell populations in healthy individuals. This information may provide a basis for comparison when characterizing disease conditions that have characteristic distribution patterns along the intestinal tract, such as Crohn's disease and ulcerative colitis.