Substance P is responsible for physiological alterations such as increased chloride ion secretion and glucose malabsorption in cryptosporidiosis

Abstract

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl(-)) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl(-) secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl(-) secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

FIG. 1.

Jejunal SP and NK1 are up-regulated after Cryptosporidium infection in macaques. SP protein was studied by immunohistochemistry using a polyclonal antibody to SP in paraformaldehyde-fixed paraffin-embedded jejunal biopsies derived from a representative macaque (A) before C. parvum challenge and (B) after C. parvum challenge (arrows in A and B represent cells that are stained positively for SP protein) (magnification, ×200). (C) Immunofluorescence deconvolution restoration microscopy using intestinal tissues derived from a representative macaque following C. parvum challenge showing substance P protein staining as detected by anti-SP polyclonal antibody (green and blue indicate substance P and nuclei, respectively). (D) CD3 protein staining as detected by anti-CD3 polyclonal antibody (red and blue indicate CD3 and nuclei, respectively). (E) Merged images showing orange/yellow CD3⁺ lymphocytes expressing substance P. The yellow scale bar in the upper right corner of each panel is 40 μm (magnification, ×400). SP receptor protein NK1 was studied by immunohistochemistry using a polyclonal antibody to NK1 in paraformaldehyde-fixed paraffin-embedded jejunal biopsies derived from a representative macaque (F) before C. parvum challenge and (G) after C. parvum challenge (an arrow in F represents a cell that is faintly positive for NK1 protein, and arrows in G represent strong positive staining for NK1 protein) (magnification, ×200).

FIG. 2.

Quantitation of jejunal SP levels in macaques before and after Cryptosporidium infection. SP protein measurements were studied by competitive ELISA of jejunal biopsies derived from macaques (n = 3) before C. parvum challenge and after C. parvum challenge. Results are expressed as picograms of SP in 1 mg of total protein extract. *, P = 0.011 (Student's paired t test for SP protein level in prechallenge biopsies versus postchallenge biopsies derived from macaques) (error bars represent standard errors of the means).

FIG. 3.

Basal chloride ion secretion is elevated in jejunal tissues derived from macaques infected with C. parvum, and in vitro SP receptor antagonist treatment rectifies this defect. SP induced chloride ion secretion as studied by the Ussing chamber technique using prechallenge jejunal biopsies, postchallenge biopsies without SP receptor antagonist treatment, and postchallenge biopsies with SP receptor antagonist treatment from a representative macaque. Changes in basal I_sc (ΔI_sc) are expressed as μA/0.725 cm².

FIG. 4.

Glucose absorption is decreased in response to C. parvum infection in macaques and is rectified after in vitro SP receptor (SPr) antagonist pretreatment. Glucose absorption was studied by the Ussing chamber technique using prechallenge jejunal biopsies and postchallenge biopsies from macaques, respectively, before and after C. parvum challenge and in postchallenge biopsies with and without SP receptor antagonist treatment. Changes in I_sc (ΔI_sc) induced by glucose are presented as maximal elevations in I_sc and are expressed as μA/0.725 cm². Bars represent mean values derived from data for three macaques. *, P < 0.05 (Student's t test for glucose-induced ΔI_sc in postchallenge biopsies versus prechallenge biopsies); ‡, P < 0.05 (Student's t test for glucose-induced ΔI_sc in postchallenge biopsies with and without SP receptor antagonist treatment).