Enteric ganglionitis in rhesus macaques infected with simian immunodeficiency virus

Abstract

Gastrointestinal (GI) disease is a debilitating feature of human immunodeficiency virus (HIV) infection that can occur in the absence of histopathological abnormalities or identifiable enteropathogens. However, the mechanisms of GI dysfunction are poorly understood. The present study was undertaken to characterize changes in resident and inflammatory cells in the enteric nervous system (ENS) of macaques during the acute stage of simian immunodeficiency virus (SIV) infection to gain insight into potential pathogenic mechanisms of GI disease. Ganglia from duodenum, ileum, and colon were examined in healthy and acutely infected macaques by using a combination of routine histology, double-label immunofluorescence and in situ hybridization. Evaluation of tissues from infected macaques showed progressive infiltration of myenteric ganglia by CD3+ T cells and IBA1+ macrophages beginning as early as 8 days postinfection. Quantitative image analysis revealed that the severity of myenteric ganglionitis increased with time after SIV infection and, in general, was more severe in ganglia from the small intestine than in ganglia from the colon. Despite an abundance of inflammatory cells in myenteric ganglia during acute infection, the ENS was not a target for virus infection. This study provides evidence that the ENS may be playing a role in the pathogenesis of GI disease and enteropathy in HIV-infected people.

FIG. 1.

(A) H&E staining of duodenum from a healthy macaque. (B) Peripherin immunohistochemistry on duodenum from a healthy macaque demonstrating the myenteric and submucosal plexuses which make up the ENS. Note also the numerous peripherin-positive nerve fibers throughout the muscle layers. (C and D) Higher magnification views of myenteric ganglia from the ileum of a healthy macaque (C) and from an SIV-infected macaque at 21 dpi (D). Note the presence of numerous mononuclear cells in the ganglia from the infected animal compared to the healthy animal.

FIG. 2.

(A to F) Double-label immunofluorescence staining for peripherin (green) and IBA1 (red) on myenteric ganglia from duodenum, ileum, and colon from a healthy macaque (A, B, and C, respectively) and an SIV-infected macaque at 21 dpi (D, E, and F, respectively). (G) The inflammatory index, i.e., the ratio of the area occupied by macrophages (IBA1) to area of the ganglia (peripherin), was calculated for ganglia at each level of intestine in uninfected macaques and in macaques sacrificed during acute SIV infection. Bars represent mean ± the SEM of at least five representative ganglia per animal at each level of intestine. (H) Fold change in the inflammatory index from uninfected control values for each level of intestine in SIV-infected macaques. Bars represent the mean of the two animals evaluated at each time point.

FIG. 3.

(A to F) Double-label immunofluorescence staining for peripherin (red) and CD3 (green) on myenteric ganglia from the duodenum, ileum, and colon from a healthy macaque (A, B, and C, respectively) and an SIV-infected macaque at 21 dpi (D, E, and F, respectively). (G) Mean numbers of CD3⁺ T cells per ganglia are shown for each level of intestine in uninfected macaques and in macaques sacrificed during acute SIV infection. Bars represent mean ± the SEM of 10 sequential ganglia at each level of intestine. (H) Fold change in T-cell numbers from uninfected controls values for each level of intestine in SIV-infected macaques. Bars represent the mean of the two animals evaluated at each time point.

FIG. 4.

(A and B) Double-label immunofluorescence staining for peripherin and either CD3 (A) or IBA1 (B) in myenteric ganglia from the ileum of an SIV-infected macaque at 14 dpi. Inflammatory macrophage and T cells appear to colocalize within the same ganglion in the small intestine.

FIG. 5.

(A and B) Double-label immunofluorescence staining for peripherin and either CD3 (A) or IBA1 (B) in the duodenum of an SIV-infected macaque at 14 dpi. Note the perivascular localization of inflammatory macrophages and T cells within the smooth muscle layers of the small intestine. “V” denotes a small blood vessel containing one to two autofluorescent red blood cells. The table summarizes the distribution of mural lesions in the GI tract of healthy and SIV-infected macaques. Symbols: +, presence of at least one mural lesion in the representative level of intestine; −, no lesions detected.

FIG. 6.

(A and B) Immunofluorescence staining for HuC/D antigen expression on myenteric ganglia from ileum of a healthy macaque (A) and an SIV-infected macaque (B) at 21 dpi. (C) Mean numbers of HuC/D⁺ neurons per ganglia at each level of intestine in uninfected macaques and in macaques sacrificed during acute SIV infection. Bars represent mean ± the SEM of 25 sequential ganglia. (D) Fold change in neuronal numbers from uninfected controls values for each level of intestine in SIV-infected macaques. Bars represent the mean of the two animals evaluated at each time point.