Abstract

Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach’s myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development.

Key words
Chagas disease; interstitial cells of Cajal; megacolon; Trypanosoma cruzi

INTRODUCTION

Trypanosoma cruzi was identified as the causative protozoan of Chagas disease (CD) more than 100 years ago. Yet, it remains a social and public health problem, mainly in endemic areas of 21 Latin American countries (World Health Organization 2010WORLD HEALTH ORGANIZATION. 2010. First WHO report on neglected tropical diseases: working to overcome the global impact of neglected tropical diseases. WHO. https://www.who.int/publications/i/item/9789241564090.
https://www.who.int/publications/i/item/... ). The disease was a disease of Latin American rural populations for centuries. The exodus from rural towards urban areas has expanded the reach of illness. The non-vectorial transmission channels such as blood transfusion, congenital transmission, and organ transplants, have become critical to other countries and continents affecting international communities. In previous decades, Chagas disease was increasingly detected in the United States of America and Canada and many European and some Western Pacific countries due mainly to population mobility, mostly migration (World Health Organization 2019WORLD HEALTH ORGANIZATION. 2019. World Chagas Disease Day: raising awareness of neglected tropical diseases. https://www.who.int/news/item/24-05-2019-world-chagas-disease-day-raising-awareness-of-neglected-tropical-diseases.
https://www.who.int/news/item/24-05-2019... ).

Chagas disease usually comprises an acute and a chronic phase. A proliferanting parasite in blood and tissue marks the acute phase. It presents asymptomatic, in most cases, or with mild symptoms, such as fever, fatigue, body aches, headache, rash, loss of appetite, diarrhea, and vomiting. Severe acute disease occurs in less than 5% of patients presenting acute myocarditis, pericardial effusion, and meningoencephalitis (Centers for Disease control and prevention 2022CENTERS FOR DISEASE CONTROL AND PREVENTION. 2022. Parasites - American Trypanosomiasis (also known as Chagas Disease). https://www.cdc.gov/parasites/chagas/index.html.
https://www.cdc.gov/parasites/chagas/ind... ). Untreated patients remain chronically infected, most people never develop symptoms (indeterminate form) or show visceral involvement characterized by cardiomyopathy or mega viscera (megaoesophagus, megacolon, or both) (Higuchi et al. 1993HIGUCHI ML, DE BRITO T, MARTINS REIS M, BARBOSA A, BELLOTTI G, PEREIRA-BARRETO AC & PILEGGI F. 1993. Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: Light microscopy and immunohistochemical findings. Cardiovasc Pathol 2: 101-106., Koberle & Nador 1955KOBERLE F & NADOR E. 1955. Etiology and pathogenesis of megaesophagus in Brazil. Rev Paul Med 47: 643-661., Pérez-Molina & Molina 2017PÉREZ-MOLINA JA & MOLINA I. 2017. Chagas disease. Lancet. https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(17)31612-4/abstract.
https://www.thelancet.com/journals/lance... ).

Megacolon is characterized by an increase in the thickness, dilation, and sometimes elongation of the intestinal wall. These changes include considerable damage to the enteric nervous system (ENS) and subsequent ganglionic and intramuscular denervation (da Silveira et al. 2007aDA SILVEIRA A, D’AVILA REIS D, DE OLIVEIRA E, NETO S, LUQUETTI A, POOLE D, CORREA-OLIVEIRA R & FURNESS J. 2007b. Neurochemical coding of the enteric nervous system in chagasic patients with megacolon. Dig Dis Sci 52: 2877-2883., Koberle 1956KOBERLE F. 1956. Pathologische Befunde an den muskularen Hohlorganen bei der experimentellen Chagaskrankheit. Zbl allg Path path Anat 95: 321-329., Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25.). The thickening of its wall accompanies the permanent dilation of a (colonic) gut segment. Initially, thickening predominates over dilation, whereas later, as a sign of decompensation, the gut wall distends with the muscle layers becoming thinner (Koberle 1968KOBERLE F. 1968. Chagas’ Disease and Chagas’ Syndromes: The Pathology of American Trypanosomiasis. Adv Parasitol 6: 63-116., Côbo et al. 2012CÔBO EC, SILVEIRA TP, MICHELETTI AM, CREMA E & ADAD SJ. 2012. Research on Trypanosoma cruzi and Analysis of Inflammatory Infiltrate in Esophagus and Colon from Chronic Chagasic Patients with and without Mega. J Trop Med 2012: 232646.). Muscle layer thickening is not only due to increased muscle tissue but also to the proliferation of connective tissue. Indeed, massive fibrosis has also been found in chagasic megacolon (Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25., Campos et al. 2016CAMPOS CF, CANGUSSÚ SD, DUZ ALC, CARTELLE CT, NOVIELLO ML, VELOSO VM, BAHIA MT, ALMEIDA-LEITE CM & ARANTES RME. 2016. Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection. PLoS ONE 11: e0153038., da Silveira et al. 2007bDA SILVEIRA ABM, LEMOS EM, ADAD SJ, CORREA-OLIVEIRA R, FURNESS JB & D’AVILA REIS D. 2007a. Megacolon in Chagas disease: a study of inflammatory cells, enteric nerves, and glial cells. Hum Pathol 38: 1256-1264., Iantorno et al. 2007IANTORNO G, BASSOTTI G, KOGAN Z, LUMI CM, CABANNE AM, FISOGNI S, VARRICA LM, BILDER CR, MUNOZ JP, LISERRE B, MORELLI A & VILLANACCI V. 2007. The enteric nervous system in chagasic and idiopathic megacolon. Am J Surg Pathol 31: 460-468., Jabari et al. 2014JABARI S, DE OLIVEIRA EC, BREHMER A & DA SILVEIRA ABM. 2014. Chagasic megacolon: enteric neurons and related structures. Histochem Cell Biol 142: 235-244.).

The main symptom associated with megacolon is chronic constipation (Lopes et al. 1988LOPES ER, ROCHA A, MENESES AC, LOPES MA, FATURETO MC, LOPES GP & CHAPADEIRO E. 1988. Prevalence of visceromegalies in necropsies carried out in Triângulo Mineiro from 1954 to 1988. Rev Soc Bras Med Trop 22: 211-215., Bern et al. 2007BERN C, MONTGOMERY SP, HERWALDT BL, RASSI A, MARIN-NETO JA, DANTAS RO, MAGUIRE JH, ACQUATELLA H, MORILLO C, KIRCHHOFF LV, GILMAN RH, REYES PA, SALVATELLA R & MOORE AC. 2007. Evaluation and Treatment of Chagas Disease in the United States. JAMA 298: 2171., Meneghelli 1985MENEGHELLI UG. 1985. Chagas’ disease: a model of denervation in the study of digestive tract motility. Braz J Med Biol Res 18: 255-264., De Oliveira et al. 1998DE OLIVEIRA RB, TRONCON LEA, DANTAS RO & MENEGHELLI UG. 1998. Gastrointestinal manifestations of Chagas’ disease. Am J Gastroenterol 93: 884-889.). Through manometry studies, it was shown that colonic basal motility is lower in chagasic patients compared to normal subjects. Some studies showed a reduced relaxation of the internal sphincter of the anus in chagasic patients (Adad et al. 2001ADAD SJ, CANÇADO CG, ETCHEBEHERE RM, TEIXEIRA VP, GOMES UA, CHAPADEIRO E & LOPES ER. 2001. Neuron count reevaluation in the myenteric plexus of chagasic megacolon after morphometric neuron analysis. Virchows Arch 438: 254-258., Cavenaghi et al. 2008CAVENAGHI S, FELICIO OCS, RONCHI LS, CUNRATH GS, MELO MMC & NETINHO JG. 2008. Prevalence of rectoanal inhibitory reflex in chagasic megacolon. Arq Gastroenterol 45: 128-131., Meneghelli et al. 1982MENEGHELLI UG, DE GODOY RA, MACEDO JF, DE OLIVEIRA RB, TRONCON LE & DANTAS RO. 1982. Basal motility of dilated and non-dilated sigmoid colon and rectum in Chagas’ disease. Arq Gastroenterol 19: 127-132., Meneghelli 1985MENEGHELLI UG. 1985. Chagas’ disease: a model of denervation in the study of digestive tract motility. Braz J Med Biol Res 18: 255-264., Salvador et al. 2015SALVADOR F, MEGO M, SÁNCHEZ-MONTALVÁ A, MORÍS M, RAMÍREZ K, ACCARINO A, MALAGELADA J-R, AZPIROZ F & MOLINA I. 2015. Assessment of rectocolonic morphology and function in patients with Chagas disease in Barcelona (Spain). Am J Trop Med Hyg 92: 898-902.). However, the mechanisms involved in CD-related intestinal dysmotility are poorly understood. In this scenario, peristaltic motor activity studies are essential to understanding the contractility activity coordination in the intestine (Huizinga & Lammers 2009HUIZINGA JD & LAMMERS WJEP. 2009. Gut peristalsis is governed by a multitude of cooperating mechanisms. Am J Physiol Gastrointest Liver Physiol 296: G1-G8., Dickson et al. 2007DICKSON EJ, SPENCER NJ, HENNIG GW, BAYGUINOV PO, REN J, HEREDIA DJ & SMITH TK. 2007. An enteric occult reflex underlies accommodation and slow transit in the distal large bowel. Gastroenterology 132: 1912-1924.).

Classified as “pacemakers” of the gut, the interstitial cells of Cajal (ICCs) play critical roles in gastrointestinal motility and have been associated with a pathogenic role in CD. The ICCs are located in the lamina propria, submucosal and myenteric plexus, and the different muscle layers associated with the ENS (Figure 1). Several studies show the reduction of the ICCs population in megacolon of chagasic patients using immunohistochemistry and immunofluorescence technique (Adad et al. 2013ADAD SJ, SILVA GB & JAMMAL AA. 2013. The development of chagasic megacolon requires severe denervation and the reduction in interstitial cells of Cajal number might be a contributing factor. Virchows Arch 462: 127., Alonso Araujo et al. 2012ALONSO ARAUJO SE, DUMARCO RB, RAWET V, SEID VE, BOCCHINI SF, NAHAS SC & CECCONELLO I. 2012. Reduced population of interstitial cells of Cajal in Chagasic megacolon. Hepatogastroenterology 59: 2147-2150., Jabari et al. 2014JABARI S, DE OLIVEIRA EC, BREHMER A & DA SILVEIRA ABM. 2014. Chagasic megacolon: enteric neurons and related structures. Histochem Cell Biol 142: 235-244.). Interestingly, this such as Hirschsprung’s disease, inflammatory bowel disease, and slow transit constipation (He et al. 2000HE CL, BURGART L, WANG L, PEMBERTON J, YOUNG-FADOK T, SZURSZEWSKI J & FARRUGIA G. 2000. Decreased interstitial cell of cajal volume in patients with slow-transit constipation. Gastroenterology 118: 14-21., Wang et al. 2009WANG H, ZHANG Y, LIU W, WU R, CHEN X, GU L, WEI B & GAO Y. 2009. Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung’s disease and total colonic aganglionosis. Neurosci Lett 451: 208-211., Farrugia 2008FARRUGIA G. 2008. Interstitial cells of Cajal in health and disease. Neurogastroenterol Motil 20 Suppl 1: 54-63.) ICCs also have distinct profiles based on the expression of specific markers, such as mature ICCs (CD117⁺CD44⁺CD34^-) and progenitors ICCs (CD117^lowCD44⁺CD34⁺) (Lorincz et al. 2008LORINCZ A, REDELMAN D, HORVÁTH VJ, BARDSLEY MR, CHEN H & ORDÖG T. 2008. Progenitors of interstitial cells of cajal in the postnatal murine stomach. Gastroenterology 134: 1083-1093.). The phenotype profiles of progenitors ICCs are observed in the intestine in some experimental models (Lorincz et al. 2008LORINCZ A, REDELMAN D, HORVÁTH VJ, BARDSLEY MR, CHEN H & ORDÖG T. 2008. Progenitors of interstitial cells of cajal in the postnatal murine stomach. Gastroenterology 134: 1083-1093., Li et al. 2019LI L, ZOU C, ZHOU Z, WANG X & YU X. 2019. Phenotypic changes of interstitial cells of Cajal after intestinal obstruction in rat model. Braz J Med Biol Res 52: e8343.), and in human pathologies such as Hirschsprung’s disease (Chen et al. 2014CHEN Z-H, ZHANG Y-C, JIANG W-F, YANG C, ZOU G-M, KONG Y & CAI W. 2014. Characterization of Interstitial Cajal Progenitors Cells and Their Changes in Hirschsprung’s Disease MIAO X (Ed). PLoS ONE 9: e86100.). Also, progenitor ICCs descriptions have been found in human CD studies and, other gastrointestinal motor disorders (Geraldino et al. 2006GERALDINO RS, FERREIRA AJ, LIMA MA, CABRINE-SANTOS M, LAGES-SILVA E & RAMIREZ LE. 2006. Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity. Pathophysiology 13: 71-74., de Lima et al. 2008DE LIMA MA, CABRINE-SANTOS M, TAVARES MG, GEROLIN GP, LAGES-SILVA E & RAMIREZ LE. 2008. Interstitial cells of Cajal in chagasic megaesophagus. Ann Diagn Pathol 12: 271-274., Jabari et al. 2013JABARI S, DA SILVEIRA ABM, DE OLIVEIRA EC, QUINT K, WIRRIES A, NEUHUBER W & BREHMER A. 2013. Interstitial cells of Cajal: crucial for the development of megacolon in human Chagas’ disease? Colorectal Dis 15: e592-e598.).

Figure 1
Ilustration of the distribution of ICCs in the lamina propria, submucosa and myenteric plexus in different muscle layers associated with the enteric nervous system (ENS). Created by https://www.biorender.com/.

Injury in the ICCs associated with enteric nervous damage has been considered the cause of uncoordinated contractility activity in megacolon CD (Negreanu et al. 2008NEGREANU LM, ASSOR P, MATEESCU B & CIRSTOIU C. 2008. Interstitial cells of Cajal in the gut--a gastroenterologist’s point of view. World J Gastroenterol 14: 6285-6288., Hasler 2003HASLER WL. 2003. Is constipation caused by a loss of colonic interstitial cells of Cajal? Gastroenterology 125: 264-265.). Also, altered distribution of ICCs has been demonstrated in human chagasic megacolon (Adad et al. 2012ADAD SJ, SILVA GBE & JAMMAL AA. 2012. The significantly reduced number of interstitial cells of Cajal in chagasic megacolon (CM) patients might contribute to the pathophysiology of CM. Virchows Arch 461: 385-392., Geraldino et al. 2006GERALDINO RS, FERREIRA AJ, LIMA MA, CABRINE-SANTOS M, LAGES-SILVA E & RAMIREZ LE. 2006. Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity. Pathophysiology 13: 71-74., Hagger et al. 2000HAGGER R, FINLAYSON C, KAHN F, DE OLIVEIRA R, CHIMELLI L & KUMAR D. 2000. A deficiency of interstitial cells of Cajal in Chagasic megacolon. J Auton Nerv Syst 80: 108-111.). However, these human studies were based on small, limited samples of megacolon and performed with only descriptive assays using immunohistochemistry technique. Here, we have focused on studying the profile of ICCs in the chronic phase using the flow cytometry platform. This powerful tool allows us to separate and characterize isolated ICC profiles in T. cruzi infection since the visualization of these cells is not easily done in the histological sections. Finally, this work may partly explain the initial functional changes in the murine model of chagasic megacolon previously published by our group (Campos et al. 2016CAMPOS CF, CANGUSSÚ SD, DUZ ALC, CARTELLE CT, NOVIELLO ML, VELOSO VM, BAHIA MT, ALMEIDA-LEITE CM & ARANTES RME. 2016. Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection. PLoS ONE 11: e0153038.). In non-infected (control animals), we do not expect any changes in ICCs as documented in mice and human intestines by Klüppel et al. (1998)KLÜPPEL M, HUIZINGA JD, MALYSZ J & BERNSTEIN A. 1998. Developmental origin and Kit-dependent development of the interstitial cells of cajal in the mammalian small intestine. Dev Dyn 211: 60-71. Thus, accurately identifying and quantifying the ICC profile in CD remains essential. We performed the first flow cytometry characterization to describe the ICC profile in a murine model of acute and chronic megacolon chagasic disease. A better description of these cells provides mechanistic insights into effective interventions to prevent morbidity associated with late manifestations of megacolon in CD.

MATERIALS AND METHODS

Ethics

In vivo, animal experiments complied with the principles established by guidelines and the Brazilian Practice Directive for the Care and Use of Animals for Scientific and Didactic Purposes (Notice MCTI No. 1 - CONCEA / MCTI). Animal studies were approved under protocol numbers 262/2016 and 31/2017.

Mice

Female Swiss mice, 4-week old, supplied by the Bioterium of the Institute of Biological Sciences of UFMG, kept in plastic cages in a room with controlled dry temperature (24°C) under a light / dark cycle of 14/10 h and access to water and conventional mouse food (Nuvilab® Nuvital, Brazil).

T. cruzi infection experimental protocols

Mice infection was performed by intraperitoneal (IP) injection of 50,000 blood trypomastigotes of T. cruzi Y strain, as described before (Brener 1962BRENER ZBZT. 1962. Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi. Rev Inst Med Trop São Paulo 4: 389-396.). The efficacy of the infection was evaluated by the presence of parasites in fresh blood four days after inoculation.

The animals were randomly divided into two groups, infected and non-infected controls. The non-infected control group was composed of a control acute phase (CAP) and a control chronic phase (CCP) group. The infected group was composed of the infected acute phase (IAP) group euthanized at 11 days post-infection (d.p.i) before manifesting disease signals. The infected chronic phase group (ICP) was treated orally with a single dose of benznidazole (Lafepe, Brazil), 500 mg/kg, at 11 d.p.i. and maintained up to 7 months post-infection (m.p.i.). The animals of ICP group were euthanized at 3 and 7 m.p.i. (ICP3 and ICP7 groups, respectively). The non-infected age-matched chronic control (CAP and CCP) groups were maintained in the same conditions and euthanized at appropriate months indicated as the control chronic phase (CCP3 and CCP7 groups, respectively).

Immunohistochemistry

Colon tissue slides from mice in the chronic phase (7 months) were immunostained. Briefly, the slides were incubated with primary anti-CD117 antibody (human, 1:500) (Abcam, USA) overnight at 4°C (Abdo et al. 2021ABDO W, ELMADAWY MA, ABDELHIEE EY, ABDEL-KAREEM MA, FARAG A, ABOUBAKR M, GHAZY E & FADL SE. 2021. Protective effect of thymoquinone against lung intoxication induced by malathion inhalation. Sci Rep 11: 2498., Pawlicki et al. 2019PAWLICKI P, HEJMEJ A, MILON A, LUSTOFIN K, PŁACHNO BJ, TWORZYDLO W, GOROWSKA-WOJTOWICZ E, PAWLICKA B, KOTULA-BALAK M & BILINSKA B. 2019. Telocytes in the mouse testicular interstitium: implications of G-protein-coupled estrogen receptor (GPER) and estrogen-related receptor (ERR) in the regulation of mouse testicular interstitial cells. Protoplasma 256: 393-408.). Then, the primary antibody were detected using an anti-mouse/anti-rabbit detection system (Novolink Polymer Detection System; Leica Biosystems, Newcastle Upon Tyne, UK) according to the manufacturer’s instructions. The sections were counterstained with diluted Harris Hematoxylin solution and permanently mounted with Entellan (Merck, USA).

Immunofluorescence and Photographic Documentation

Primary cultures of mouse enteric neurons (Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25.) submitted to the above-described conditions and grown on coverslips were fixed with 4% buffered paraformaldehyde. The slides were incubated with primary anti-CD117 antibody (human, 1:500) (Abcam, USA) overnight at 4°C. Identification of cell nuclei in vitro was performed by the fluorescence-emitting probe Hoechst H33342 (Invitrogen, USA), which binds to nuclear DNA and allows the optimal visualization of parasite and host nuclear morphology. The coverslips were analyzed under the Olympus BX51 fluorescence microscope and images were obtained using Image-Pro Express 4.0 software (Media Cybernetics, USA).

Isolation of cells in the inter-muscular layers (ICC-MY) in the murine model of chagasic megacolon

Isolate cells were performed as routinely done (Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25.). Briefly, after removal, the large intestine segment was placed in Krebs solution and sectioned along the mesenteric border and delaminated of the layers (mucosa, submucosa, and serosa) to expose the muscular layer where the Auerbach’s myenteric plexuses were laid (Smith et al. 2013SMITH TH, NGWAINMBI J, GRIDER JR, DEWEY WL & AKBARALI HI. 2013. An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse. J Vis Exp 7: e50688.). The tissue was incubated for 15 minutes at 37 °C in 1mg/ml collagenase type II-S solution (Merck, USA). The fragments were centrifuged at 200 x g for 5 minutes at 4 °C and washed with Hanks/HEPES buffer solution for 5 minutes under stirring, followed by centrifugation at 200 x g for 5 minutes at 4 °C. Trypsin solution (0.25%; Merck, USA) was added at 37 °C, followed by stirring for 15 minutes and manual homogenization. Next, the supernatant was removed and added to 1ml of sterile culture medium (Minimum Essential Medium; 31095-029; Gibco, Invitrogen, USA). The suspended cells were submitted to immunostaining of the cell markers and submitted to a subsequent acquisition of data in a flow cytometer.

Flow cytometry analysis

The cells were centrifuged at 3000 G for 5 min and resuspended in PBS (0.01 M phosphate-buffered saline, pH 7.2) with 0.5% Bovine Serum Albumin (BSA, Inlab, Brazil) and stained with anti-CD117 (BD Bioscience, USA), anti-CD45, anti-CD11b, anti-CD11c, anti-CD34, and anti-CD44 (eBiosciences, USA) (Table I), diluted in PBS with 0.5% BSA mix, fixed by a 45-min incubation at 4°C. The cells were washed two times in PBS-1% bovine serum albumin, fixed with a 4% paraformaldehyde solution (20 min), and examined in a flow cytometer. The data acquisition was performed by FORTESSA LSR cytometer using the DIVA software (BD Biosciences) at the Multi-User Flow Cytometry Platform (Instituto René Rachou/FIOCRUZ/MG). Analysis was performed using the Flow Jo, LLC program. The strategy used to identify the ICCs was using an exclusion gate (FITC) for anti-CD45, anti-CD11b, and anti-CD11c to exclude inflammatory cells and a positive gate for CD34, CD44, and CD117.

Statistical analysis

The statistical analyses were performed using the software GraphPad Prism (v 8.0) (GraphPad Software Inc., La Jolla, CA). The Shapiro-Wilk test revealed that the parameters evaluated did not show a significant departure from normal distribution. Comparisons between means were made using unpaired student’s tests (i.e., Control vs. Infected). When a significant F-value was found, we performed a post-hoc test according to the coefficient of variation (CV): Tukey (CV ≤ 15%) or Student-Newman-Keuls (CV > 15%). The α level was set at 0.05. Data are shown as mean ± standard deviation (SD).

RESULTS

In vivo and in vitro evidence for the presence of ICCs

ICCs were observed in the immunohistochemically stained sections from the intestinal wall collected at the chronic phase at 7 months (Figure 2a, b). The ICC-IM (intramuscular) is scattered throughout the muscle and parallel smooth muscle cells in both muscle layers (black arrows). We also observed ICCs in a primary culture of enteric neurons using the immunofluorescence technique (Figure 2c, d; white arrows).

Figure 2
Distribution of ICCs in chronic colon tissue (7 months) and in a primary culture of myenteric neurons. a-b: Immunolabelling of paraffin-embedded tissues from colons at 7 months using anti-CD117⁺ in the muscle and myenteric region (arrows). c-d: Immunofluorescence of primary cultures of mouse myenteric neurons using anti-CD117⁺ (green) and nuclear staining with Hoechst 33342 (blue). Scale bar: 10μm. 20X and 40X objective. Data are representative of two independent experiments.

The description of the profile of Cajal cells in a chronic murine model of chagasic megacolon

Figure 3a shows the selection of ICCs population to be analyzed and the frequency of ICCs obtained by flow cytometry. ICCs were sub-gated into: CD44⁺CD117⁺ (undifferentiated cells), and the subtypes CD44⁺CD117⁺CD34^- (mature ICCs) and CD34⁺CD117^lowCD44⁺ (progenitor ICCs). There was a decrease in CD44⁺CD117⁺ cells in 3 and 7 m.p.i. compared to their respectively controls (p = 0.02) and (p = 0.0286) (Figure 3b). The ICCs were decreased in IAP and ICP3 compared to their respectively controls (p = 0.0286) and (p = 0.006) (Figure 3c). Regarding the ICCs progenitors (CD117^lowCD34⁺CD44⁺), there are no differences between the groups (Figure 3d).

Figure 3
Alterations of the frequency of ICCs in the murine model of chagasic megacolon. a: Representative dot plots from ganglia and intestinal muscle layer of Swiss mice infected with 50,000 T. cruzi strain Y trypomastigotes obtained from animals at the acute (CAP, controls; IAP, infected) and chronic (CCP, controls; ICP3, ICP7, months post-infection, respectively) phases, showing the gating strategy used to identify ICCs subsets. According to CD45, CD11b and c, CD117, CD34 and CD44 expression levels, the cells were sub-gated into: CD44⁺CD117⁺ (total ICCs), and their subtypes, CD44⁺CD117⁺CD34^- (mature ICCs) and CD34⁺CD117^lowCD44⁺ (progenitor ICCs). b, c and d: Frequency of ICCs subsets in total ICCs cells from mice infected, or not with T.cruzi analyzed by Flow cytometry using Flow Jo, LLC. Statistical analysis: Student t-test. Difference in relation to the control group in infected acute phase (p = 0.0286) and infected chronic phase 3 months (p = 0.006) (*) in the mature ICCs. Difference in relation to the control group, infected chronic phase 3 months (p = 0.02) and infected chronic phase 7 months (p = 0.0286) (*) in the ICCs progenitors. Data are representative of two independent experiments, except for the 7 group that were performed one time (n = 4). Data are shown as mean ± standard deviation (SD).

DISCUSSION

Loss of ICCs were associated with motor disorders of the gut. However, these cells’ physiological and pathophysiological roles have not been clearly defined. Some authors described the reduction of ICCs in colonic biopsies in chagasic patients with megacolon. However, these studies do not discriminate the profile of ICCs (Hagger et al. 2000HAGGER R, FINLAYSON C, KAHN F, DE OLIVEIRA R, CHIMELLI L & KUMAR D. 2000. A deficiency of interstitial cells of Cajal in Chagasic megacolon. J Auton Nerv Syst 80: 108-111., Geraldino et al. 2006GERALDINO RS, FERREIRA AJ, LIMA MA, CABRINE-SANTOS M, LAGES-SILVA E & RAMIREZ LE. 2006. Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity. Pathophysiology 13: 71-74., Iantorno et al. 2007IANTORNO G, BASSOTTI G, KOGAN Z, LUMI CM, CABANNE AM, FISOGNI S, VARRICA LM, BILDER CR, MUNOZ JP, LISERRE B, MORELLI A & VILLANACCI V. 2007. The enteric nervous system in chagasic and idiopathic megacolon. Am J Surg Pathol 31: 460-468.). For the first time, our study characterizes the subtypes of ICCs in the long-term murine model of chagasic megacolon. This work showed that infection with T. cruzi induces a transient loss of mature ICCs (11 d.p.i. and 3 m.p.i). These cells’ decrease is associated with parasite induced acute inflammation (Campos et al. 2016CAMPOS CF, CANGUSSÚ SD, DUZ ALC, CARTELLE CT, NOVIELLO ML, VELOSO VM, BAHIA MT, ALMEIDA-LEITE CM & ARANTES RME. 2016. Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection. PLoS ONE 11: e0153038., Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25.). The Y strain used in our experiments is partially resistant to benznidazole (BZ) (Khare et al. 2015KHARE S, LIU X, STINSON M, RIVERA I, GROESSL T, TUNTLAND T, YEH V, WEN B, MOLTENI V, GLYNNE R & SUPEK F. 2015. Antitrypanosomal Treatment with Benznidazole Is Superior to Posaconazole Regimens in Mouse Models of Chagas Disease. Antimicrob Agents Chemother 59: 6385-6394.). It acts directly against circulating trypomastigotes and intracellular amastigotes, but its effectiveness depends on the length of treatment, dosing, and disease phase (Filardi & Brener 1987FILARDI LS & BRENER Z. 1987. Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans R Soc Trop Med Hyg 81: 755-759., Marin-Neto et al. 2008MARIN-NETO JA, RASSI A, MORILLO CA, AVEZUM A, CONNOLLY SJ, SOSA-ESTANI S, ROSAS F & YUSUF S. 2008. Rationale and design of a randomized placebo-controlled trial assessing the effects of etiologic treatment in Chagas’ cardiomyopathy: The BENznidazole Evaluation For Interrupting Trypanosomiasis (BENEFIT). Am Heart J 156: 37-43., Pedrosa et al. 2001PEDROSA RC, BEM AF, LOCATELLI C, PEDROSA RC, GEREMIAS R & FILHO DW. 2001. Time-dependent oxidative stress caused by benznidazole. Redox Report 6: 265-270.). So, the animals we treated with only a single dose of BZ at 500mg/kg of body weight improved their survival chances but developed a chronic infection. This protocol ensured the control of parasitemia and increased survival so that the animals that presented parasite-induced colonic wall inflammatory and degenerative changes reached the chronic phase. The treatment of mice in the chronic phase with BZ was, therefore, not the aim of our study since we have a model (Campos et al. 2016CAMPOS CF, CANGUSSÚ SD, DUZ ALC, CARTELLE CT, NOVIELLO ML, VELOSO VM, BAHIA MT, ALMEIDA-LEITE CM & ARANTES RME. 2016. Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection. PLoS ONE 11: e0153038., Ricci et al. 2020RICCI MF, BÉLA SR, MORAES MM, BAHIA MT, MAZZETI AL, OLIVEIRA ACS, ANDRADE LO, RADÍ R, PIACENZA L & ARANTES RME. 2020. Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas’ Disease. Front Cell Infect Microbiol 10: 25.) that mimics the chagasic megacolon without any influence of BZ in this phase. Also, the development of megacolon requires severe denervation, preceding the establishment of dilation, and favoring the hypothesis that the reduction in ICC number is, in part, a consequence of similar mechanisms of denervation (Adad et al. 2012ADAD SJ, SILVA GBE & JAMMAL AA. 2012. The significantly reduced number of interstitial cells of Cajal in chagasic megacolon (CM) patients might contribute to the pathophysiology of CM. Virchows Arch 461: 385-392.) and potential plasticity of smooth muscle and Cajal cells, showing the importance of characterizing these subtypes of cells in the context of structural alterations for chagasic megacolon development.

Interestingly, in the later time point, the mature ICCs returned to the same frequency as the control group in the chronic phase of infection (7 m.p.i.), suggesting that mature ICCs have a considerable regenerative capacity in the long-term murine model of chagasic megacolon. These finds are also observed in other pathological conditions, which could restore their networks after partial mechanical obstruction (Chang et al. 2001CHANG IY, GLASGOW NJ, TAKAYAMA I, HORIGUCHI K, SANDERS KM & WARD SM. 2001. Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction. J Physiol 536: 555-568.), inflammation (Wang et al. 2005WANG X-Y, VANNUCCHI M-G, NIEUWMEYER F, YE J, FAUSSONE-PELLEGRINI M-S & HUIZINGA JD. 2005. Changes in interstitial cells of Cajal at the deep muscular plexus are associated with loss of distention-induced burst-type muscle activity in mice infected by Trichinella spiralis. Am J Pathol 167: 437-453.), and pyloric hypertrophy (Vanderwinden & Rumessen 1999VANDERWINDEN JM & RUMESSEN JJ. 1999. Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech 47: 344-360.). Also, it has been suggested that gastrointestinal stromal tumors (GIST) may originate from ICC precursors due to activating mutations of Kit (CD117) (Streutker et al. 2007STREUTKER CJ, HUIZINGA JD, DRIMAN DK & RIDDELL RH. 2007. Interstitial cells of Cajal in health and disease. Part II: ICC and gastrointestinal stromal tumours. Histopathology 50: 190-202.). ICCs depend on stem cell factor (SCF) signaling via c-Kit to develop and maintain their functions (Klüppel et al. 1998KLÜPPEL M, HUIZINGA JD, MALYSZ J & BERNSTEIN A. 1998. Developmental origin and Kit-dependent development of the interstitial cells of cajal in the mammalian small intestine. Dev Dyn 211: 60-71., Wu et al. 2000WU JJ, ROTHMAN TP & GERSHON MD. 2000. Development of the interstitial cell of Cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand. J Neurosci Res 59: 384-401., Rich et al. 2003RICH A, MILLER SM, GIBBONS SJ, MALYSZ J, SZURSZEWSKI JH & FARRUGIA G. 2003. Local presentation of Steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture. Am J Physiol Gastrointest Liver Physiol 284: G313-G320.). Thus, this activation could be a mechanism that matures ICCs in pathological conditions, including Chagas disease.

ICCs play a central role in the gastrointestinal system, generating slow electrical waves and contractile activity, mediating muscle interactions with the nervous system, establishing potential and tone, and developing a “pacemaker” role in various autonomic innervation organs (Farrugia 2008FARRUGIA G. 2008. Interstitial cells of Cajal in health and disease. Neurogastroenterol Motil 20 Suppl 1: 54-63., Sanders et al. 2014SANDERS KM, WARD SM & KOH SD. 2014. Interstitial cells: regulators of smooth muscle function. Physiol Rev 94: 859-907.). This work follows a previous study in which gastrointestinal rhythmicity was affected in a murine model of chagasic megacolon involving the lack of response of chronically infected animals related to changes in the cholinergic responses modified by the installation of the megacolon (Ricci et al. 2022RICCI MF, BÉLA SR, BARBOSA JL, MORAES MM, MAZZETI AL, BAHIA MT, HORTA LS, SANTIAGO HC, CRUZ JS, CAPETTINI LSA & ARANTES RME. 2022. A Potential Role of Cholinergic Dysfunction on Impaired Colon Motility in Experimental Intestinal Chagas Disease. J Neurogastroenterol Motil 28: 483-500.). Altogether, these results show that it is essential to consider the multiple aspects of the complex CD development, including the alterations in the ICC profile that associate with functional disorders in a murine model of chagasic megacolon.

This model is a valuable tool for better characterization of the ENS impairment mechanisms of intestinal CD with an emphasis on ICCs profile role looking for an integrative comprehension of acute and chronic phases pathogenetic mechanisms of Chagas disease.

ACKNOWLEDGMENTS

The Multi-User Flow Cytometry Platform (Instituto René Rachou/Fundação Oswaldo Cruz - FIOCRUZ/MG). This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES/Brazil (Ph.D. and Post-Doctoral fellowships), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq/Brazil (Researcher fellowship 305952/2017 and 305894/2018-8) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG/Brazil (PPM 00346-14, 00304-16), (CBB- APQ-01419-14, APQ-00921-18), and (Rede Mineira de Imunobiológicos; RED-00140-16, and Rede de Investigação em Mucosas e Pele; RED-00096-22).

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