Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice

Abstract

Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle, necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

Keywords: Cryptosporidium parvum; Gut; Malnutrition; Myeloperoxidase; Neuroinflammation.

Fig. 1

Cryptosporidium colonizes and induces impairment of growth in protein-deficient mice. (A) Percentual of body weight loss (mean ± SEM) of C57BL/6 mice receiving a nourished diet (N = 10) or protein-deficient diet (dPD) (N = 10) before C. parvum infection. (B) Percentual of body weight loss (mean ± SEM) of C57BL/6 mice receiving a nourished diet (n = 10) or protein-deficient diet (dPD) (N = 10) after Cryptosporidium infection. *p < 0.05, **p < 0.01 and ****p < 0.0001. (C) Tail and (D) body length (mean ± SEM) of uninfected (nourished or dPD; N = 9) and infected mice (dPD + Crypto; N = 9) on day 14 of the experimental protocol. *p < 0.05 and ***p < 0.01. (E) Quantification of C. parvum burden in stools of infected mice. (F) Quantification of C. parvum (mean ± SEM) burden in ileum tissues on day 14 of experimental protocol (N = 5 per group). (G) Representative ileal H&E histology from uninfected (nourished or dPD) and infected mice (dPD + Crypto) at day 7 post-infection (p.i.). C. parvum induces ileal epithelial damage (black arrow) and inflammatory cell infiltration (red arrow).

Fig. 2

C. parvum infection promotes systemic inflammation increasing plasma SAA and MPO in protein-deficient mice. Levels of (A) SAA and (B) MPO (mean ± SEM) measured by ELISA in plasma of uninfected (nourished or dPD; N = 7) and infected (dPD + Crypto; N = 7) C57Bl/6 mice on day 14 of the experimental protocol. *p < 0.05.

Fig. 3

C. parvum infection results in inflammation in the prefrontal cortex of mice. (A) Representative western blots of NF-kB and β-actin levels in prefrontal cortex tissues from C57BL/6 mice uninfected (nourished or dPD; N = 5) and infected (dPD + Crypto; N = 5) on day 14 of the experimental protocol. Bars represent (mean ± SEM) the quantification of western blot bands. #p < 0.03. (B) Photomicrographs of Iba-1 (green) immunostaining in prefrontal cortex tissues from C57BL/6 mice uninfected (nourished or dPD) and infected (dPD + Crypto) on day 14 of the experimental protocol. (C) MPO levels (mean ± SEM) measured by ELISA in prefrontal cortex tissues from uninfected (nourished or dPD; N = 7) and infected (dPD + Crypto; N = 7) C57Bl/6 mice on day 14 of the experimental protocol. *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)