The pathogenesis of cryptoglandular anal fistula: New insight into the immunological profile

Abstract

Aim: The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization.

Method: Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed.

Results: Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1β, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients.

Conclusion: A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients.

Keywords: aetiology; anal fistula; inflammation.

FIGURE 1

Pro‐inflammatory cytokines interleukin (IL)‐1β, IL‐4, IL‐8 and IL‐17, were all significantly overexpressed in anal fistula when compared with control tissue. Tumour necrosis factor‐α (TNFα) was not overexpressed. In the univariate analysis, levels of the tested analytes were not associated with gender, age, fistula complexity or recurrent disease.

FIGURE 2

Anti‐inflammatory cytokines interleukin (IL)‐6 and IL‐10 were both significantly overexpressed in anal fistula when compared with control tissue.

FIGURE 3

Levels of chemokines involved in macrophage recruitment (A, CCL2; B, CCL3; C, CCL4) and growth factors involved in inflammation (D) fibroblast growth factor (FGF), (E) granulocyte monocyte colony‐stimulating factor (GM‐CSF) were significantly higher in anal fistula than in healthy fat.

FIGURE 4

Increased presence of CD8 Tc17 T cells in the inflamed mucosa of patients with anal fistula (AF). The mucosa collected from marginal tissue controls is representative of the normal distribution of immune cells (A) compared with AF marginal zone samples (B). Scale bars: 50 μm. Arrows indicate Th17 cells, short arrows indicate CD8 T lymphocytes, long arrows indicateTc17 CD8 T lymphocytes. (C) Evaluation of the mean number of T lymphocytes counted in three randomly selected high‐power fields (HPFs). (D) Percentage of the different T lymphocyte subsets calculated with regard to the total number of CD3 T lymphocytes counted in three randomly selected HPFs. The data are representative of 20 patients and five controls. *p < 0.05, **p < 0.01.

FIGURE 5

Characterization of M1 and M2 macrophage infiltration in anal fistula (AF) tissue. Immunofluorescence for the pan macrophage marker CD68 and inflammatory marker iNOS for M1 macrophages or CD163 or the M2 anti‐inflammatory macrophages in marginal tissue controls (A) and in AF (B). Long arrows indicate M1 macrophages and short arrows indicate M2 macrophages. Scale bars: 50 μm. (C) Evaluation of the mean number of CD68 infiltrating macrophages counted in three randomly selected high‐power fields (HPFs). (D) Mean number of M1 or M2 macrophages calculated with regard to the total number of CD3 T lymphocytes counted in three randomly selected HPFs. (E) M1/M2 macrophage ratio. The data are representative of 20 patients and five controls. *p < 0.05, **p < 0.01.