ORIGINAL RESEARCH published: 14 April 2021 doi: 10.3389/fimmu.2021.627638 Protective Effect of Intestinal Helminthiasis Against Tuberculosis Progression Is Abrogated by Intermittent Food Deprivation Edited by: Cristina Garrido-Amaro 1†, Paula Cardona 2,3†, Diana Gassó 1,4, Lilibeth Arias 2,3, Mario Alberto Flores-Valdez, Roser Velarde 1, Asta Tvarijonativiciute 5, Emmanuel Serrano 1 and Pere-Joan Cardona 2,3* CONACYT Centro de Investigación y Asistencia en Tecnologı´a y Diseño del 1 Wildlife Ecology & Health Group (WE&H) and Servei d’Ecopatologia de Fauna Salvatge (SEFaS), Universitat Autònoma de Estado de Jalisco (CIATEJ), Mexico Barcelona (UAB), Bellaterra, Spain, 2 Unitat de Tuberculosi Experimental, Institut Germans Trias i Pujol, UAB, Badalona, Reviewed by: Spain, 3 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain, 4 Departament of Animal Science, Agrifood, Forestry and Veterinary Campus, University of Lleida, Lleida, Spain, Roland Lang, 5 Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Regional Campus of International Excellence Campus Mare University Hospital Erlangen, Germany Katarzyna Łysoniewska, Nostrum, Universidad de Murcia, Murcia, Spain Military Institute of Hygiene and Epidemiology, Poland Julieta Luna-Herrera, Background: Tuberculosis (TB) is still a major challenge for humankind. Because regions Instituto Politécnico Nacional (IPN), with the highest incidence also have a high prevalence of helminthiasis and nutritional Mexico scarcity, we wanted to understand the impact of these on TB progression. *Correspondence: Pere-Joan Cardona Methods: We have developed an experimental murine model for active TB in C3HeB/FeJ, pj.cardona@gmail.com coinfected with Trichuris muris and Heligmosomoides polygyrus nematodes, and † These authors have contributed exposed to an environmental mycobacterium (M. manresensis) and intermittent fasting. equally to this work Cause-effect relationships among these factors were explored with Partial Least Squares Specialty section: Path modelling (PLSPM). This article was submitted to Microbial Immunology, Results: Previous parasitization had a major anti-inflammatory effect and reduced a section of the journal systemic levels of ADA, haptoglobin, local pulmonary levels of IL-1b, IL-6, TNF-a, Frontiers in Immunology CXCL-1, CXCL-5 and IL-10. Oral administration of heat-killed M. manresensis resulted Received: 09 November 2020 in a similar outcome. Both interventions diminished pulmonary pathology and bacillary Accepted: 26 March 2021 Published: 14 April 2021 load, but intermittent food deprivation reduced this protective effect increasing stress and Citation: inflammation. The PLSPM revealed nematodes might have protective effects against Garrido-Amaro C, Cardona P, TB progression. Gassó D, Arias L, Velarde R, Tvarijonativiciute A, Serrano E and Conclusions: Significantly higher cortisol levels in food-deprivation groups showed it is a Cardona P-J (2021) Protective stressful condition, which might explain its deleterious effect. This highlights the impact of Effect of Intestinal Helminthiasis Against Tuberculosis food security on TB eradication policies and the need to prioritize food supply over Progression Is Abrogated by deworming activities. Intermittent Food Deprivation. Front. Immunol. 12:627638. Keywords: co-infection, tuberculosis, Trichuris muris, Heligmosomoides polygyrus, environmental mycobacteria doi: 10.3389/fimmu.2021.627638 Mycobacterium manresensis, fasting, C3HeB/FeJ Frontiers in Immunology | www.frontiersin.org 1 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB INTRODUCTION Surprisingly, our data shows a protective effect of intestinal helminthiasis related to its anti-inflammatory properties, which Tuberculosis (TB) is an infectious disease caused by is abrogated by intermittent food deprivation, thus reinforcing Mycobacterium tuberculosis (Mtb) and is a major global health the importance of food security in any TB eradication program. problem. In 2018, 10 million people fell ill with TB and it caused 1.4 million deaths worldwide (1). Most TB infection occurs in regions where parasitization by helminths is highly prevalent (2). MATERIALS AND METHODS Soil-transmitted intestinal parasites are also a global health problem, with Ascaris lumbricoides, Trichuris trichiura and Animals Necator americanus infecting an estimated 804, 477 and 472 A total of 24 female and 36 male C3HeB/FeJ specific-pathogen- million people respectively worldwide (3). The response to free mice (8–10 weeks old) were obtained from Germans Trias i helminth infection can be summarized as causing a bias Pujol Research Institute stock. All procedures were conducted in toward Th2 and an increased Treg response, which modulates a BSL-3 facility, according to protocol DMAH6119. This was both Th1 and Th2, a bias that may be transmitted to infants in reviewed by the Animal Experimentation Ethics Committee of utero (4). Due to the importance of Th1 responses in controlling the Hospital Universitari Germans Trias i Pujol (registered as Mtb infection, it is constantly questioned whether to advocate for B9900005) and approved by the Departament d’Agricultura, a deworming policy or not as a coincidental factor to improve TB Ramaderia, Pesca, Alimentació i Medi Natural of the Catalan vaccination programs. Equally, it is presumed that people in Regional Government, according to current national and these regions have a high level of contact with environmental European Union legislation regarding the protection of mycobacteria, which has a clear impact in the immune response experimental animals. Mice were supervised daily following a against Mtb. In fact, it is one of the reasons believed to explain strict monitoring protocol to ensure animal welfare and the failure of the protection induced by BCG vaccination in euthanized, if required, by cervical dislocation after anesthesia several settings (5), either because exposure to environmental with isoflurane. mycobacteria provides some protective immunity to TB (6, 7) or because it blocks the replication of BCG and thus its protective Experimental Design effect (7, 8). Animals were divided into 6 experimental groups (Figure 1), 10 Additionally, food insecurity is also common in these regions mice per group evenly matched in sex and weight. (9). Body mass index (BMI) below 18.5 is a well-known risk Four groups were orally infected by gavage, twice, with 0.2 mL factor for TB progression (10) due to the reduction of essential of a solution containing 30 Trichuris muris eggs and 200 nutrients required to build an appropriate immune response. Heligmosomoides polygyrus larvae as previously done (11, 12). However, little is known about the effect of intermittent food Once the parasitization had been confirmed by coprologic deprivation, as occurs in a state of food insecurity, and the impact examination, 6 weeks after parasite infection, all animals of the stress it causes, even when an overall malnutrition has not were infected with 2×104 CFU (colony-forming units) of been reached. M. tuberculosis H37Rv Pasteur strain via the caudal vein (13). FIGURE 1 | Experimental design. Six groups of 10 animals were used to evaluate the influence of intestine parasitization, intermittent fasting and oral exposure to environmental mycobacteria in an active TB model. Frontiers in Immunology | www.frontiersin.org 2 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB Environmental mycobacterial treatment started one day after Tridelta Development, Ireland, respectively) in an automated Mtb infection. Mice received oral treatment with 105 CFU of biochemistry analyzer (Olympus AU600, Beckman Coulter, heat-killed M. manresensis (hkMm) (14) in a galenic formulation Brea, USA). Cortisol was analyzed following Arantes-Rodrigues with mannitol. Control groups received mannitol in the same et al. (21) and using a commercially available solid-phase, concentration. A total of 7 doses were administered, every other competitive chemiluminescence enzyme immunoassay (COR day, for 14 days. Cortisol, Siemens Health Diagnostics, Deerfield, IL, USA) Two groups of animals were submitted to intermittent fasting in the automatic analyzer (Immulite 1000, Siemens throughout the experiment, with a weekly regime of 5 days of HealthcareDiagnostic, Deerfiels, IL, USA). normal diet and 2 days of food deprivation (15). Otherwise, mice received food and water ad libitum. Lung Immune Response Animal weight was recorded every 2-3 days. Fecal samples A cytokine profile study was performed in lung homogenates were obtained from each cage weekly to quantify parasite eggs. from right cranial and middle lobes. The following cytokines were After 3 weeks from Mtb infection, mice were anesthetized with measured by Luminex xMAP® technology: IFN-g, TNF-a, IL-1b, isoflurane inhalation and blood samples were obtained through IL-6, IL-10, IL-12(p40), IL-13, IL-17, CXCL-1 and CXCL-5. cardiac puncture. They were then euthanized by cervical Results are expressed as pg per mL of homogenate. The assay dislocation. Lungs, spleen, liver, kidneys and intestines were was performed with the MILLIPLEX® MAP kit (EMD Millipore retrieved. Kidneys and liver were weighed in a precision Corporation, Billerica, MA, USA) following the manufacturer’s balance with 0.0001 g accuracy. instructions and analyzed with xPONENT Software (Luminex Corporation, Austin, TX, USA). Bacillary Load (BL) Spleen and left lobe lung samples from each animal were collected, Data Analysis homogenized and 10-fold serial dilutions were made. 100 µL of Multiple Comparisons these dilutions were plated on nutrient Middlebrook 7H11 agar The scores of the first dimension (PC1) of a principal component (BD Diagnostics, Sparks, USA). Visible CFU were counted after analysis (PCA) on liver and kidneys weights were used as proxy incubation for 28 days at 37°C. Data was analyzed as CFU per mL. for body condition in mice. Likewise, the PC1 of a PCA for the antioxidant (TEAC1, TEAC2, FRAP, CUPRAC, Thiol and Lung Pathology PON1) and oxidant (AOPP and ROS) biomarkers was used as Right caudal lung lobe samples were fixed in 10% buffered proxy for the oxidative stress profile. The same approach was formalin, embedded in paraffin and 5-mm sections stained with followed for lungs immune response (analytes detailed in section hematoxylin-eosin for microscopic observation. Two paraffin 2.6). Differences in the PCA scores among experimental groups blocks per group were obtained, each containing samples from were explored with ANOVA and a Holm-Sidak post-hoc 5 animals. Four recuts of every block were used to determine the multiple comparison tests (22, 23). damaged area as a percentage of total lung area. This analysis was On the other hand, a Mann-Whitney test was used to compare done using the NISElements D version 3.0x software package damage area and BL among experimental groups. The same (Nikon Instruments Inc., Tokyo, Japan). statistical test was used to compare differences among treatments in concentration of oxidative stress mediators in serum and Oxidative Stress, Inflammatory Mediators cytokines/chemokines in lungs. and Cortisol in Serum PCA was performed using the package “FactomineR” version Blood samples were extracted in tubes containing clotting 2.0 (24) of the statistical software R 3.6.2 version (25). Graphpad activator (Sarstedt, Nümbrecht, Germany). After centrifugation, Prism (GraphPad software v7.0, La Jolla, California, USA) was used sera were obtained and stored at -20°C until analysis. for graphics and simple statistics, with differences of p<0.05 being Total antioxidant capacity was determined by four methods: considered statistically significant. Statistically significant differences trolox equivalent antioxidant capacity (TEAC1 and TEAC2), are summarized as follows: *p<0.05, **p<0.01, ***p<0.001. ferric reducing ability of plasma (FRAP) and cupric reducing antioxidant capacity (CUPRAC), as previously reported (16). PLS Path Modeling Thiol was determined using method reported by Jocelyn (17). We used Partial Least Square Path Modelling (PLS-PM) (26–29) PON1 activity was analyzed following previously described to explore the association of intermittent fasting, helminth method (18) and advanced oxidation protein product (AOPP) infection and hkMm with Mtb load. Briefly, this approach concentrations as described by Witko-Sarsat (19). All procedures quantifies the network relationship between a set of were performed in an automated biochemistry analyzer unobservable latent variables (LV) and a set of parameters (Olympus AU600, Beckman Coulter, Brea, USA). Reactive directly measured (manifest variables or MV). The LVs are oxygen species (ROS) were estimated by luminol-mediated conceptual variables defined by one or several MVs and chemiluminescence assay (20) using a microplate reader organized in a network of relationships where the connections (Victor 2 1420 Multilabel Counter; PerkinElmer, Finland). among LVs are assumed to represent a cause-effect process called Total adenosine deaminase (ADA), albumin and haptoglobin inner model. The links among LV are quantified through path were measured using commercially available kits (Diazyme coefficients while the links between LV and MV are quantified Laboratories, Poway, CA, USA; Beckman Coulter, USA and through weights (30). Frontiers in Immunology | www.frontiersin.org 3 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB Our PLS-PM included 28 MVs organized into 10 LVs, was evidenced in liver and kidney weight at the final endpoint. described in Supplementary Table 1. The initial inner PLS- This data was used to perform a PCA, showing that both PM model is shown in Supplementary Figure 1. The assumed fasting groups had statistically significant lower scores of PC1 direct effects for the model structure are detailed in (Figures 2B, C). Supplementary Table 2. Even though PLS-PM is distribution Albumin was also measured in sera, but there were no statistically free, some variables (IFN-g, IL-10, IL-12, IL-17, parasite burden significant differences between groups (Supplementary Figure 2). and BL) were log-transformed to improve the model fit. After The number of eggs per gram of feces was determined weekly fitting the first model including all the variables, we performed a for samples from each cage. At the endpoint of the experiment, simplification removing those MVs uncorrelated with their own the total number of parasites was determined in the intestines of LVs (Supplementary Table 3), following Sanchez (26). Finally, each animal. Neither measurement showed significant we also estimated the partial contribution of each LV to the final differences between groups (Supplementary Figure 3). PLS-PM as well as the goodness of fit [GOF, (31)]. PLS-PM analysis was performed using the package ‘plspm’ version 0.4.9 Intestinal Parasites and Oral hkMm (26) of the statistical software R 3.6.2 version (25). Reduced Lung BL and Pathology The damaged area in lungs was reduced with parasitization and oral hkMm (Figure 3A). Representative lung sections of each RESULTS group are shown in Supplementary Figure 4. In agreement with these results, the Mtb load in lungs was significantly reduced in Intermittent Fasting Had a Low Impact on the same groups (Figure 3B). This protective effect against the Body Condition progression of active TB was not observed in fasting animals. Fasting mice reduced their body weight in a transitory manner There were no differences between groups for BL in spleen (Figure 2A). Nonetheless, the effect of the intermittent fasting (Supplementary Figure 5). A B C FIGURE 2 | Change of animals’ body condition. (A) Median weight of mice as infection progresses, infection day being time 0. (B) PCA based on liver and kidneys weight at end-point time. (C) PC1 scores, each circle represents an animal and lines are means; ANOVA p-value=0.0004, Holm-Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01). hkMm, heat-killed M. manresensis. Frontiers in Immunology | www.frontiersin.org 4 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB A B FIGURE 3 | Damage area (A) and pulmonary bacillary load (B) at week 3 post-infection. Each circle represents a “recut” (A) or an animal (B) and lines show medians. Mann-Whitney test: *p<0.05, **p<0.01, ***p<0.001. hkMm, heat-killed M. manresensis. A B C FIGURE 4 | Oxidative stress analysis at week 3 post-infection. (A) PCA based on mediator concentration in serum. (B) PC1 scores, each circle represents an animal and lines are means; ANOVA p-value=0.0001, Holm-Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Mediators contribution to PC1. hkMm, heat-killed M. manresensis; TEAC, trolox equivalent antioxidant capacity; CUPRAC, cupric reducing antioxidant capacity; FRAP, ferric reducing antioxidant power; PON1, paraoxonase 1; ROS, reactive oxygen species; AOPP, advanced oxidation protein products. Fasting Induced Antioxidant Activity (Figure 4B), which was mainly explained by TEAC1, CUPRAC, Oxidative stress was measured in different ways in sera TEAC2, FRAP and thiol variation (Figure 4C). (Supplementary Figure 6). When compared with control animals, parasitized mice had a significant increase in TEAC2, Intestinal Parasites and Oral hkMm CUPRAC and FRAP. This was also the case in parasitized- Reduced Inflammation fasting, hkMm and hkMm-parasitized-fasting groups. Fasting ADA, haptoglobin and cortisol were measured in sera (Figure 5). animals also showed increased TEAC1 and reduced PON1, ADA was significantly reduced when compared with control regardless of the oral treatment, and increased thiol and animals in all groups except parasitized-fasting. The reduced ROS when not receiving hkMm. The hkMm group administration of hkMm in parasitized groups also showed a was the only one that presented lower levels of AOPP than reduction in ADA levels when compared with their control control mice. The hkMm-parasitized group had no statistically counterparts. Parasitization induced a reduction of haptoglobin significant differences from controls. levels, but only under normal feeding conditions. Cortisol was These results were used to perform a PCA (Figure 4A). The significantly increased in parasitized-fasting animals. first two components explained 71.1% of the variation in the Lung homogenates were used to quantify various cytokines data set. Fasting induced a significant increase in the PC1 score and chemokines (Supplementary Figure 7). Parasitized animals Frontiers in Immunology | www.frontiersin.org 5 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB A B C FIGURE 5 | Adenosine deaminase (ADA) (A), haptoglobin (B) and cortisol (C) levels in serum at week 3 post-infection. Each circle represents an animal and lines are medians, Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001). hkMm, heat-killed M. manresensis. showed a statistically significant reduction in TNF-a, IL-10, IL- cortisol. Body condition is affected by helminths and fasting. 1b, IL-6, CXCL-1 and CXCL-5 levels. Mice receiving oral hkMm Antioxidants appear increased in fasted mice and oxidation is had lower levels of TNF-a, IL-1b, IL-6, IL-17, CXCL-1 and reduced by the effect of antioxidants. Proliferative lesions were CXCL-5. The levels of IL-13 were below the detection limit in all stimulated under food restriction. The bacillary load LV was the samples (data not shown). negatively related to hkMm, helminths and cortisol, but The PCA analysis of lung cytokines and chemokines showed positively related to exudative lesions and antioxidants. the first two components accounted for 70.9% of the data set However, only exudative lesions were significantly related to variation (Figure 6A). The PC1 score, based mainly on levels of BL in the lungs. IL-1b, CXCL-1, IL-6 and TNF-a, related to the induction of After removing the non-significant effects, antioxidant and exudative lesions, was significantly reduced in the parasitized oxidant LVs were isolated from the path, so these blocks were animals (Figures 6B, C). excluded from the refined model. The LV proliferative lesions was not related to bacillary load and hence also removed from PLS Path modeling the model. The final refined model shows both hkMm and According to the initial model (Supplementary Table 4), fasting helminths reduce exudative lesions that in turn are responsible increases helminth load, both fasting and helminths increase for higher bacillary loads (Figure 7). A B C FIGURE 6 | Immune mediator analysis at week 3 post-infection. (A) PCA based on cytokines concentration in lung homogenates. (B) PC1 scores, each circle represents an animal and lines are means; ANOVA p-value=0.0067, Holm-Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01). (C) Mediators contribution to PC1. hkMm, heat-killed M. manresensis. Frontiers in Immunology | www.frontiersin.org 6 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB higher levels of Tregs and Th2 cytokine response, helminth coinfection was related with a lower sputum smear-positivity (36). Recent data obtained from healthy migrants from Nepal, shows a negative correlation between hookworm infection and LTBI. This has been linked to the increased mycobacterial growth inhibition in the blood of hookworm-infected individuals, leading to a reduced risk of Mtb infection (37). A protective effect of H. polygyrus infection in air-borne infections, such as respiratory syncytial virus (38) and P. aeruginosa (39), has been reported in mice. Several authors have tried to understand the impact of helminthiasis on the progression of TB using experimental models. The design of these studies should be considered to evaluate their relevance. First, most of them have been carried out in mouse strains (mainly BALB/c and C57Bl/6) that develop a sort of chronic infection based on the induction of proliferative lesions, with a strong Th1 response. Thus, they do not develop exudative lesions, characterized by neutrophilic infiltration, which are the key for developing liquefacted lesions with the capacity to become cavitated (40). So far, the only mouse strain able to FIGURE 7 | Refined partial least square path model. Red arrows indicate a develop such lesions is the C3HeB/FeJ, precisely the one used in negative relation among latent variables (path coefficient bellow 0) and blue our study (13). Also, several studies used M. bovis BCG as a arrows indicate a positive one (path coefficient over 0). surrogate of M. tuberculosis infection, thus developing a very attenuated course of infection. Thirdly, it is important to clarify which helminth species have been used. There are The goodness of fit for the model was 0.39 (39% of fit). The several studies where there is a combination of intestinal LV body condition had the greatest impact (46.1%) on the helminths, with and without a pulmonary phase in their cycle, explained variability, followed by cortisol (29.9%) and bacillary or even filarial worms (affecting mainly the lymphatic system) load (18.8%). thus probably reflecting different scenarios (41–44). Therefore, by changing the coinfection helminth we may have an alternative immunomodulatory effect, resulting in a new outcome than DISCUSSION the one obtained in this experiment. The order of co-infection between nematodes and among nematodes and TB is also a In this work, we aimed to study the relation between three determinant factor (45). important factors affecting global health: tuberculosis, intestinal We have evaluated the parasitization with T. muris and H. helminthiasis and food scarcity. Our data showed a protective polygyrus, which only affect the intestinal mucosa. We decided to effect of parasitization in the outcome of Mtb infection, which use two nematodes since it is a common situation in nature (46, was abrogated by intermittent food deprivation. 47). Our results within the PLS-PM indicate that, although both Contrary to our findings, most data from cohort studies suggest species induce similar immune response (48), there was a greater an increased susceptibility to developing TB when coinfected. effect of T. muris with respect to the H. polygyrus counterpart, Helminthiasis infection has been linked to pulmonary TB in a evidenced in the higher weight on the helminth LV. A recent cohort study that compared infected individuals with a control study with M. bovis murine coinfection showed that even when group (32). In another study, patients with TB and coinfected also T. muris parasitization led to a Th2 immune background, it did presented a more advanced disease (33). Lower IFN-g and higher not influence M. bovis BCG proliferation and dissemination. IL-10 responses were found in coinfected TB patients after Coinfection increased the percentage of CD4+, CD4+IL4+ and stimulation of whole-blood with mycobacterial antigens. Among CD8+IL4+ cells and decreased TNF-a secretion after BCG pediatric household contacts of smear positive TB patients, a stimulation in the spleen when compared with the BCG positive tuberculin skin test was significantly associated with infection alone. Detection of mRNA in the lung showed that coinfection (34). This was interpreted as helminth infection coinfection decreased the expression of TGF-b, Foxp3 and IFN- increasing the risk of acquiring latent tuberculosis infection g, when compared with BCG infection alone (42). In our study, (LTBI). This is in concordance with the improvement of Mtb- we have not been able to detect the parasitization effect on the specific immune responses in PBMCs after deworming treatment Th2 response. However, matching the lower levels of Mtb load, in LTBI persons (35). All in all, these results are consistent with the these mice did present a reduction in the pro-inflammatory attenuation of Th1 responses caused by helminths. However, even response in lungs. This was also observed with the oral though the host’s defense against Mtb requires a Th1 response, it is administration of hkMm, which additionally reduced IL-17 not a direct correlate of protection (5). In fact, despite presenting levels, as already described (49). PLS pathway modeling Frontiers in Immunology | www.frontiersin.org 7 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB supported the role of helminths and hkMm in the reduction of investigation, however, should be conducted to elucidate the exudative lesions, and thus reduction of the BL. The levels of IL- drivers of this protective effects beyond the immunological 10 were also lower in parasitized animals, suggesting a reduction pathway used in our research. in the regulatory response, as in Nel et al. (42). Furthermore, To our knowledge, this is the first time that the impact of levels of IFN-g did not experience significant differences between parasitization by intestinal helminths in the progression of active experimental groups, thus dismantling the idea of a Th2-based TB has been studied in an experimental model, considering response in parasitization competing for the Th1 immune important factors like food fasting and exposure to environmental response. The acute-phase protein haptoglobin was reduced in mycobacteria. Our data shows an unprecedented protective sera of parasitized animals, once again pointing to an anti- impact of intestinal parasitization, which is abrogated by inflammatory effect. intermittent food fasting. This data should be considered when Recent estimates of hunger and food insecurity are that 821 contemplating deworming policies and highlights the importance million people are undernourished, more than 10% of the world of good nutrition in the fight against TB. population. This appears to be increasing in almost all regions in Africa and South America, while it is stable in most regions of Asia. A more striking fact is that the number of undernourished has been rising since 2014 (9). A low BMI is a risk factor for TB DATA AVAILABILITY STATEMENT development (10), supported by several experimental models. The raw data supporting the conclusions of this article will be BALB/c mice receiving 80% of the usual amount of food made available by the authors, without undue reservation. consumed had an impaired response to a TB vaccine (50). The administration of a low-protein-diet in Mtb infected mice increased lung BL, both in BCG-vaccinated or control animals (51). Another approach to this issue has been the study of Mtb ETHICS STATEMENT infection in mice not expressing leptin, an adipokine reduced in The animal study was reviewed and approved by Animal malnutrition and fasting (52). This Th1 inducer increases with Experimentation Ethics Committee of the Hospital Universitari Mtb infection and mice lacking leptin have higher BL in Germans Trias i Pujol (registered as B9900005). advanced stages of infection (53). This was accompanied by increased numbers of PMNs in lungs. Little is known of the impact of intermittent fasting, a circumstance resembling an aspect of food insecurity. Food deprivation experiments have AUTHOR CONTRIBUTIONS been carried out in mice. In our case, 48 hours deprivation induced weight reduction, which was recovered with a normal Conceptualization: ES and P-JC. Data Curation: CG-A, PC, DG, diet for five days, and an increase in oxidative stress. This LA, RV, and AT. Formal Analysis: CG-A, PC, ES, and P-JC. regimen did not cause a reduction in albumin levels, Funding Acquisition: P-JC. Investigation: CG-A, PC, DG, LA, RV, suggesting that reduction of protection was not due to protein and AT. Methodology: ES and P-JC. Project Administration: ES deprivation. Interestingly, intermittent fasting was related to a and PJC. Visualization: PC. Writing – Original Draft Preparation: cortisol increase, probably caused by stress. Elevated levels of CG-A, PC, ES, and P-JC. Writing – Review & Editing: CG-A, PC, cortisol are linked to neutrophilia (54), which can increase DG, LA, RV, AT, ES, and P-JC. All authors contributed to the neutrophilic infiltration in Mtb lesions, as has already been article and approved the submitted version. described in asthma patients (55). However, hkMm appears to balance this reaction as hkMm-Parasitized-Fasting animals had the same levels of cortisol as control mice. FUNDING ADA has a proinflammatory effect, by inducing Th1 response and reducing extracellular levels of adenosine (56). Parasitized- The project leading to these results has received funding from Fasting animals were the only ones that did not present lower la Caixa Foundation (ID 100010434), under agreement LCF/ levels of ADA than control. This matches the results in lung PR/GN16/10290002. This work was supported by the Plan pathology and BL, but once again animals treated with hkMm Nacional I + D + I co-financed by ISCIII-Subdirecció n General had a distinct outcome. It appears that hkMm gave some level of de Evaluació n and Fondo-EU de Desarrollo Regional (FEDER) protection against intermittent fasting, although it was not through PC contract IFI14/00015. LA was supported by the strong enough to be evidenced in the progression of TB in European Commission Horizon 2020 research and innovation this model. program under grant agreement TBVAC2020 No. 64338. Even though the goodness of fit our PLS-PM model could be The Experimental Tuberculosis Unit is accredited by the considered moderate (28, 29), it is enough to evidence the causal Catalan Agency for Management of University and Research relationship among starvation, parasite load, environmental Grants (AGAUR) with code 2017 SGR500 and the IGTP is a mycobacteria, exudative lesions (ExL), and bacillary load. member of the CERCA network of institutes. Heat-killed M. manresensis and helminths contributed equally ES and AT are supported by the Spanish Ministerio de Ciencia to diminish exudative lesions and thus bacillary load as suggested Innovació n y Universidades (MICINN) through a Ramon y Cajal by previous research in mice (57) and cattle (58). Further agreements (RYC-2016-21120; RYC-2017-22992). The research Frontiers in Immunology | www.frontiersin.org 8 April 2021 | Volume 12 | Article 627638 Garrido-Amaro et al. Helminths Protect Against Active TB activities of CG-A are supported by a research fellow linked to the resented in part in “XXIII Jornadas Internacionales sobre RYC-2016-21120. Tuberculosis”, Fundació n de la Unidad de Investigació n sobre Tuberculosis de Barcelona. Barcelona, Spain. ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL We thank the IGTP Cytometry Core Facility, Centre de Medicina Comparativa i Bioimatge de Catalunya (CMCiB), Pathological The Supplementary Material for this article can be found online Anatomy Service from Hospital Germans Trias i Pujol, and staff at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. for their contribution to this publication. This work has been 627638/full#supplementary-material REFERENCES 19. Witko-Sarsat V, Friedlander M, Capeillère-Blandin C, Nguyen-Khoa T, Nguyen AT, Zingraff J, et al. Advanced oxidation protein products as a 1. Global tuberculosis report 2019. Geneva: World Health Organization (2019). novel marker of oxidative stress in uremia. Kidney Int (1996) 49(5):1304–13. 2. Salgame P, Yap GS, Gause WC. Effect of helminth-induced immunity on doi: 10.1038/ki.1996.186 infections with microbial pathogens. Nat Immunol (2013) 14(11):1118–26. 20. Vong L, Lorentz RJ, Assa A, Glogauer M, Sherman PM. 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No use, distribution or reproduction is permitted which administration of heat-killed mycobacterium manresensis delays progression does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 10 April 2021 | Volume 12 | Article 627638