This study was undertaken to characterize the mucosal pattern of effector cytokines in CD at different stages of the disease. To this end, we considered as “initial lesions” those developing in the neo-terminal ileum of patients after a curative ileo-colonic resection and “established lesions” those seen in patients with a long-history of disease requiring intestinal resection. More than one third of CD patients did not show endoscopic signs of recurrence within the time-frame of 1 year after the ileocolonic resection, in line with previously published studies. 
Immunofluorescence analysis of biopsies taken from this subgroup of patients showed a marked infiltration of the mucosa with both CD3+ and CD68+ cells, reinforcing the notion that T cells and macrophages drive inflammatory events necessary for the development of mucosal lesions. 
Moreover, we found a distinct pattern of cytokines at this early stage of disease. In particular, the macroscopically unaffected neo-terminal ileum contained high levels of IFN-γ and IL-21, two cytokines which are produced by Th1 cells in humans. 
These findings are consistent with the demonstration that the macroscopically unaffected neo-terminal ileum expressed high IL-12, a strong inducer of IFN-γ and IL-21 production in the gut. 
In the same biopsies, we found a slight increase in IL-17A and elevated levels of TNF-α, a cytokine involved in the positive regulation of IL-17A synthesis 
and supposed to play a pathogenic role in the recurrence after intestinal resection in CD. 
In biopsies taken from areas with endoscopic lesions, expression of Th1 cytokines remained elevated and there was marked up-regulation of IL-17A and induction of IL-23 and IL-6, two cytokines which enhance IL-17A production. 
A major strength of our study is that all patients who underwent ileocolonic resection were taking mesalamine only at the time of biopsy sampling. Thus we think it is fair to conclude that the different pattern of cytokines found in the neo-terminal ileum of CD patients with or without endoscopic lesions is not due to medical therapy.
In samples taken from mucosal areas with established lesions there were elevated levels of IFN-γ, IL-17-A, IL-4 and IL-5
as compared to normal controls. However, analysis of the cytokine expression at protein level by flow-cytometry revealed that the percentages of LPMC secreting IFN-γ or IL-17A were markedly higher than the percentage of IL-4-producing cells, reinforcing the concept that, in CD, the tissue-damaging immune response is associated with a predominant synthesis of Th1/Th17 cell-type cytokines. 
A different Th1/Th17 cytokine ratio was however seen in the subgroups of CD patients. Indeed, the immune response in the neo-terminal ileum without endoscopic lesions was mainly polarized along the Th1 pathway while it was dominated by both Th1/Th17 cytokines in areas with either early or established lesions. These findings support previous studies in murine models of CD showing that the initial phase of the inflammation is driven by Th1 cytokines while the later phases are associated with mixed Th1/Th17 cell responses. 
Along the same line is the Kugathasan‘s study showing that IFN-γ is over-produced in the gut of patients with CD at the first attack but not with long-standing CD. 
Our data are however partly conflicting with those published by Kugathasan et al because we found elevated levels of IFN-γ in samples taken from patients with both early and established lesions. It is likely that this discrepancy may simply reflect differences in the methods and cell sources of cytokines used in these studies, since Kugathasan et al analysed IFN-γ in mucosal T cell clones following IL-12 stimulation while our cytokine analysis was focused on fresh biopsy and cell samples. In this context it is also noteworthy that Kugathasan’s study was performed in children and not adults and this could help explain discrepancy because it is well known that the mucosal immunological response of children may differ from that of adults 
Surprisingly, TNF-α was not increased in the CD mucosal specimens with established lesions, despite histopathology confirmed the severity of inflammation in all samples. If this decline in TNF-α production reflects a functional change in the immunological pathways activated during this stage of the disease or is simply secondary to the immunosuppressive therapy taken by patients remains to be ascertained.
The discovery that mucosal cytokines are temporally regulated in CD could have some potential applications that merit further investigation. For example analysis of cytokine expression at specific time points could help direct the choice of therapy and ascertain whether a patient is responding to therapy in the case the mucosal levels of cytokine change. Moreover, determining the cytokine cell sources and mechanisms involved in the control of cytokine synthesis at the different stages of the disease could provide insight into the pathophysiology of CD.
One limitation of this study is its relatively small sample size, despite a noticeable difference between CD patients and controls. However, this is the largest dataset available for patients with early CD lesions. Additionally, we should remain cautious when interpreting the physiologic implications of the Th1/Th2/Th17 imbalance in early and late CD because we analysed cytokines in whole biopsies and mucosal CD3+ T cells and not in purified CD4+ T cells. Thus, we cannot exclude the possibility that cytokines measured in our samples may derive from CD8+ T cells other than Th cells. Although we feel that prospective studies on larger numbers of patients will be needed to confirm data of this study, the cytokine expression results presented here provide evidence that there are potentially different immune mechanisms driving the early and late mucosal lesions in CD. A better understanding of such mechanisms could contribute to optimize therapeutic strategies in this disease.