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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 3  |  Page : 268-273

Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy


1 Department of Immunology, National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico
2 Department of Psychoimmunology, National Institute of Psychiatry, Mexico City, Mexico
3 Department of Immunology, Technologic University, Campus Los Reyes, Mexico City, Mexico

Date of Submission20-Feb-2020
Date of Decision20-Feb-2020
Date of Acceptance04-May-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Oscar Rojas-Espinosa
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, Colonia Santo Tomás, 11340 Ciudad de México
Mexico
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_31_20

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  Abstract 


Background: Murine leprosy is a chronic granulomatous disease caused by Mycobacterium lepraemurium (MLM) in mice and rats. The disease evolves with the development of cellular anergy that impedes the production of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and nitric oxide (NO) required to kill the microorganism. In this study we investigated whether histone deacetylase inhibitors (HDACi) (valproic acid and sodium butyrate [NaB]) and the immunomodulator transfer factor in dialyzable leukocyte extracts (DLE) can prevent anergy in murine leprosy. Methods: Five groups of six Balb/c mice were intraperitoneally inoculated with 2 × 107 MLM. Thirty-days post inoculation, treatment was started; one group received no treatment, one was treated with rifampicin-clofazimine (R-C), one with sodium valproate (VPA), one with NaB, and one with DLE. The animals were monitored for the evidence of disease for 96 days. After euthanasia, their spleens were removed and processed for histologic, bacteriologic, and cytokine studies. Results: R-C completely controlled the ongoing disease. DLE and NaB significantly reduced the development of lesions, including granuloma size and the number of bacilli; VPA was less effective. DLE, NaB, and VPA reverted the anergic condition in diverse grades and allowed the expression of IFNγ, TNFα, and inducible NO synthase, also in diverse grades. Conclusion: Anergy in leprosy and murine leprosy allows disease progression. In this study, anergy was prevented, in significant degree, by DLE (an immunomodulator) and NaB (HDACi). VPA was less effective. These results suggest potential beneficial effects of DLE and NaB in the ancillary treatment of leprosy.

Keywords: Anergy, dialyzable leukocytes extract, murine leprosy, sodium butyrate, valproic acid


How to cite this article:
Rojas-Espinosa O, Moreno-García S, Arce-Paredes P, Becerril-Villanueva E, Juárez-Ortega M. Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy. Int J Mycobacteriol 2020;9:268-73

How to cite this URL:
Rojas-Espinosa O, Moreno-García S, Arce-Paredes P, Becerril-Villanueva E, Juárez-Ortega M. Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy. Int J Mycobacteriol [serial online] 2020 [cited 2020 Oct 31];9:268-73. Available from: https://www.ijmyco.org/text.asp?2020/9/3/268/293543




  Introduction Top


Murine leprosy is a chronic infectious disease of mice and rats reminiscent of human leprosy.[1],[2] It is caused by Mycobacterium lepraemurium (MLM), a hardly cultivable microorganism, parasite of macrophages. The disease evolves with the development of cellular anergy whose mechanisms are not completely understood.[3],[4]In vitro, MLM infects murine, unprimed macrophages without stimulating the expression of tumor necrosis factor-alpha (TNFα), nitric oxide (NO), or reactive oxygen species at significant levels.[5],[6] We considered the possibility that the lack of TNFα and NO production in MLM-infected macrophages might obey to histone deacetylase (HDAC) overactivation or histone acetyltransferase inhibition. HDAC overactivation can be investigated using the histone deacetylase inhibitors (HDACi): sodium valproate (VPA) and sodium butyrate (NaB) in thein vivo model of murine leprosy. Functioning as HDACi, VPA, and NaB might revert the anergy, allowing reexpression of TNFα and inducible nitric oxide synthase (iNOS), essential for macrophage activation and bactericidal activity.[7] In this study, an anti-leprosy cocktail (rifampicin-clofazimine [R-C]) and an immunomodulator (dialyzable leukocyte extract) were used as reference treatments.


  Methods Top


Reagents

Unless otherwise indicated, reagents were purchased from Sigma-Aldrich, Toluca, México.

Rodents

The mice were Balb/c, 8-week-old females obtained from the National Polytechnic Institute Medical School (ESM) in Mexico City, México. They were housed under appropriate conditions (stress-free environment, 25°C, 12 h light/darkness periods) and fed Purina chow and purified water ad libitum. The mice were handled under the regulations of the Institutional Committee for the Human Handling of Experimental Animals, and the project was evaluated and approved by the institutional Postgraduate Research Secretary (SIP 2019-0107/ENCB, IPN).

Mycobacterium lepraemurium isolation

Because MLM is a noncultivable microorganism, bacilli were isolated from the spleen of a mouse bearing a 3-month infection, following the sucrose–KCl–percoll gradient procedure described in detail elsewhere.[8] Purity and viability were assessed by the Ziehl–Neelsen stain and the hydrolysis of fluorescein diacetate, respectively.[9]

Infection and treatment

Mice were intraperitoneally inoculated with 2 × 107 bacilli. Four weeks after infection, five groups of six mice each were treated by the intragastric route with (a) nothing, (b) R-C, (c) dialyzable leukocyte extract (DLE), (d) NaB, and (e) sodium VPA, at the doses shown in [Table 1]. Infection was monitored by weighing the animals every other day (murine leprosy evolves with continuous weight loss). Doses of each drug (R-C, NaB, and VPA) were calculated from their bioavailability and pharmacologic properties[10] and from the previous studies on dose-effect trials (DLE).[3]
Table 1: Groups of mice and treatment

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Sacrifice and tissue processing

At the end of the experiment (96-day post infection), mice were euthanized by CO2 inhalation. Their spleens were removed, fixed in 4% neutral formalin, and paraffin processed for histopathologic (H and E stain), bacteriologic (Ziehl–Neelsen stain), and immunohistochemical analysis looking for the expression of interferon gamma (IFNγ) (sc-74108 Santa Cruz Biotechnology, Dallas, TX), TNFα (sc52746), and iNOS (sc7271). All stains were standard technique,[11] while the immunohistochemical staining was performed following the Santa Cruz Biotechnology Protocols for immunoperoxidase stain, with concentrations of primary and secondary antibodies properly adjusted by proof and error assays.

Statistic treatment of data

The data were analyzed using the one-way and two-way ANOVA with Dunnett's multiple comparisons test. For each parameter, one family of data with five comparisons per family, a total of 36 values and an alpha value of 0.05 were used in the analysis (one-way ANOVA), and each family, mean was compared with the control family mean (two-way ANOVA). The software used was GraphPad InStat, Version 3.05 (GraphPad Inc., La Jolla, CA, USA).


  Results Top


Dialyzable leukocyte extract, sodium butyrate, and Valproic acid prevent cachexia in Mycobacterium lepraemurium infected mice

Except for the untreated MLM-infected group, the mice showed a steady increase in body weight compatible with the animals' normal growth. Mice infected with MLM that did not receive treatment showed a gradual loss of body weight noticeable from day 52 after infection until the end of the study [Figure 1]. Body weight loss in murine leprosy (cachexia) has been described as a consequence of tissue lipid consumption in favor of bacillus proliferation.[12]
Figure 1: Body weight. Mice with an ongoing infection with MLM exhibit a progressive loss of body weight (cachexia). Cachexia was observed in the untreated group (MLM) of mice infected with murine leprosy (MLM group) but not in the MLM groups treated with MLM-R-C, MLM-DLE, MLM-NaB or MLM-VPA. n = 6 mice per group. *P values for each group compared with the murine leprosy group (MLM). Standard deviations are omitted for graph simplicity. Two-way ANOVA Dunnett's multiple comparisons test. MLM: Mycobacterium lepraemurium, DLE: Dialyzable leukocyte extract, NaB: Sodium butyrate, VPA: Valproate, R-C: Rifampicin/clofazimine

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Dialyzable leukocyte extract and sodium butyrate reduce the extent of the granuloma fraction

Granuloma fraction (GF) is the area in a tissue occupied by the granuloma which, in murine (and human lepromatous) leprosy, is made of bacilli-laden macrophages intermixed with variable numbers of lymphocytes. The GF was maximal in the MLM-infected untreated group and minimal in the R-C (a standard anti-leprosy drug combination) treated group. Of the non anti-leprosy drugs used, DLE and NaB were the most effective drugs in reducing the size of the granuloma; VPA had a lesser effect [Figure 2].
Figure 2: GF in the spleen of mice subjected to different treatments. The size of the GF followed the order: MLM-infected untreated group > MLM-VPA >, MLM-NaB > MLM-DLE-MLM, and > MLM-R-C-treated groups. Results are representative of each group (n = 6). Arrows point at the granuloma fractions. (H and E, ×100). GF: Granuloma fraction, MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine

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Bacilli load following treatment

As expected from the GF size, the number of bacilli was maximal in the MLM-infected untreated mice and minimal in the R-C treated group. Again, the DLE treatment had the best anti-leprosy effect followed by NaB and VPA [Figure 3].
Figure 3: Mycobacterial load in granulomas. The highest number of bacilli was found in the MLM-infected untreated group, and the lowest in the MLM-DLE group, followed by the MLM-NaB group and then by the MLM-VPA group. Mice of groups MLM-R-C and MLM-DLE contained the smallest numbers of bacilli. Results are representative of each group (n = 6). (Ziehl–Neelsen, ×100). MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine

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Expression of interferon gamma following treatment

IFNγ is a key cytokine for the control of intracellular pathogens. Expression of this cytokine was marked in the MLM-infected group treated with DLE, and it was less manifest in the groups treated with NaB or VPA [Figure 4]. Healthy and untreated MLM-infected mice showed no expression of this cytokine, while mice treated with R-C expressed only vestigial amounts of it.
Figure 4: Expression of interferon gamma. The presence of interferon gamma appears as a fine brown precipitate whose expression is maximal in the MLM-DLE group, less in the MLM-NaB group, and minimal in the MLM-VPA group (arrows). No expression of this cytokine appears in the uninfected (HLT), MLM-untreated, and MLM-R-C groups. Results are representative of each group (n = 6) (Immunoperoxidase and hematoxylin and eosin, ×100). MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine

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Expression of tumor necrosis factor-alpha in the granuloma fraction

TNFα is a cytokine produced by macrophages that plays a critical role in the granuloma development. It was observed that although less marked than the expression of IFNγ, MLM-infected mice treated with DLE and NaB showed significant expression of TNFα, and its expression was the lowest in the group treated with valproic acid. Untreated MLM-infected mice did not show the expression of TNFα and mice treated with R-C showed only residual traces of it [Figure 5].
Figure 5: Expression of tumor necrosis factor-alpha. TNFα appears as a weak brown precipitate whose intensity is maximal in the MLM-DLE followed by the MLM-NaB and MLM-VAP groups. No expression of this cytokine appears in the remaining (HLT, MLM, and MLM-R-C) groups. Results are representative of each group (n = 6). Immunoperoxidase and hematoxylin and eosin, ×100. MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine, TNFα: Tumor necrosis factor-alpha

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Inducible nitric oxide synthase expression

In line with the expression of IFNγ and TNFα, the expression of iNOS was higher in the group treated with DLE, followed by the NaB group, and finally, the VPA-treated group. As expected, iNOS expression was not detected in the MLM untreated mice nor in the R-C treated group [Figure 6].
Figure 6: Expression of inducible nitric oxide synthase. Although dim, expression of iNOS was higher in the MLM-DLE group, followed by the MLM-NaB group, and then the MLM-VPA group. No expression of iNOS was observed in the MLM-infected untreated group nor in the MLM-R-C group, the HLT group, and the R-C group. Results are representative of each group (n = 6). Immunoperoxidase and hematoxylin and eosin, ×100. MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine, iNOS: Inducible nitric oxide synthase

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Global changes in the granuloma of murine leprosy

[Figure 7] is a graphic illustration of the changes mentioned in [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], which are representative of each experimental group (n = 6 mice per group). The figure was constructed with the data calculated from the images shown, but the P values are for the complete sets of data in each group (n = 6).
Figure 7: A composite image of the results observed. (a) The average granuloma area in pixels, (b) the average number of bacilli in pixels, (c) the average amount of IFNγ in pixels, (d) the average amount of TNFα in pixels, and (e) the average amount of iNOS in pixels. Six × 40 microscopic fields per tissue section were analyzed. Bars correspond to the images shown but P values were calculated from the average values of the parameters compared to those values in control MLM-infected group. ImageJ. One-way ANOVA post-Dunnett's multiple comparison test. MLM: Mycobacterium lepraemurium, VPA: Valproate, NaB: Sodium butyrate, DLE: Dialyzable leukocyte extract, R-C: Rifampicin/clofazimine, IFNγ: Interferon gamma, TNFα: Tumor necrosis factor-alpha, iNOS: Inducible nitric oxide synthase

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We found that DLE was the most beneficial treatment, because it significantly contained the development of GF (mean 9,286,699 pixels in the MLM group vs. mean 31,174 pixels in the DLE-treated group, P < 0.0001) and reduced the bacillary load (mean 105,436 pixels in the MLM group vs. mean 211 pixels in the DLE-treated group, P < 0.0001), and simultaneously increased the expression of IFNγ (mean 907 pixels in the ML group vs. mean 368,740 pixels in the DLE-treated group, P = 0.008), TNFα (mean 183,556 pixels in the MLM group vs. mean 1,368,740 pixels in the DLE-treated group, P = 0.01), and iNOS (mean 30,468 pixels in the MLM group vs. mean 426,340 pixels in the EDL group, P = 0.003). The second best beneficial treatment was NaB, while VPA had only a positive effect on the size of the GF and the number of bacilli without leading to meaningful expression of IFNγ, TNFα, or iNOS. As expected, the R-C cocktail was the best microbicidal treatment.


  Discussion Top


Leprosy and murine leprosy are chronic diseases characterized by the loss of cell-mediated immunity to the offensive microorganisms allowing the unrestricted proliferation of bacteria and progression of the diseases. Although anergy is a well-known condition in leprosy, its mechanisms are not completely understood and may result from different immunologic anomalies.[4] Murine leprosy, although different from human leprosy, shares some of its characteristics, anergy being the most relevant for the present study. Anergy develops as a specific loss of cell-mediated immune response to MLM. Although there are several proposed reasons for the development of anergy in murine leprosy,[2] a unified explanation is still lacking. In this study, we explored the possibility that anergy might result from epigenetic modifications where the bacillus could activate the expression of histone deacetylases (namely HDACi) or inhibit the activity of histone acetylases. The first situation was explored using the HDACi inhibitors NaB and valproic acid (VPA). Rifampicin-clofazimine (R-C) was used as a positive treatment control, as MLM is susceptible to these and other anti-leprosy drugs.[8] The transfer factor (TF) in DLE,[13] which has shown beneficial effects in murine leprosy,[3] was used as an immunomodulatory treatment.

As noted from the results, DLE had the best anti-leprosy effect after R-C, followed by NaB and then by VPA. The action mechanism of the DLE TF is almost completely unknown but is reported to have dual activity (proinflammatory and anti-inflammatory) because of the existence of inducer and suppressor fractions in the same preparation.[14] Activation of inducer or suppressor activity will depend on the tissue microenvironment; pathogen-associated molecular patterns (in the case of infections) will activate the inducer fraction while danger-associated molecular patterns (in autoimmunity and cancer) and will activate the suppressor fraction. Beneficial effects of TF have been described in both infectious[15] and noninfectious[16] diseases, and the factor has been regarded as an immunomodulator.[17] In leprosy, only a few articles have been reported on the use of the TF, but their results were inconclusive.[18],[19] The TF has also been produced from mouse spleen cells.[20] In murine leprosy, a controlled infection model, the murine TF shows a clear proinflammatory effect that restrains or reverses the development of the disease.[3] The target cell of TF is unknown but lymphocytes and antigen-presenting cells are likely candidates. The molecular targets of the TF are also unknown, although some reports point at nuclear factor kappa B as a converging target[21] whose upstream participants are still unidentified, although one report mentions TLR-2 as an initial target.[22] It is obvious that the molecular action mechanism of TFs deserves further investigation.

In relation to valproic acid and NaB, two drugs used for epilepsy treatment, although some articles endow them with antimicrobial properties,[23],[24] their most probable role is as inhibitors of HDAC whose effects eventually upregulate protective immune responses.[25],[26],[27],[28] Based on our group previous reports,[5],[6] our working hypothesis for this study was that MLM is able to induce activation of HDAC or inhibition of histone acetylation, leading to downregulation of the IFNγ and TNFα genes. IFNγ is required to activate TNFα in macrophages, which in turn, is needed to activate iNOS for NO production. Pathogenic mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis, and MLM, usually resist or evade the toxic effects of reactive oxygen intermediaries but succumb to NO derivatives such as nitrites, nitrates, peroxynitrites, and nitrosamines.[29]

In our work, the use of NaB in the treatment of murine leprosy led to significant containment of the infection and simultaneously allowed the expression of IFNγ, TNFα, and iNOS even at 96 days post infection, probably by reversing anergy. VPA was less efficient than NaB, but we believe that it is a matter of dose; the dosage used needs to be readjusted in a future experiment.

To summarize, the nonexpression of the inflammatory mediators such as IFNγ, TNFα, and iNOS in the tissue specimens means that (a) the tissue samples came from healthy animals (the noninfected group), and (b) the samples came from successfully chemotherapy-treated animals (the R-C group), so a protective immune response was no longer necessary, or (c) the animals did not receive treatment to prevent the anergy (the MLM-infected group). IFNγ, TNFα, and iNOS were variably upregulated in groups treated with DLE, NaB, and VPA in that decreasing order.

The present results back up our hypothesis on the possible activation of HDAC by MLM as a mechanism of anergy in murine leprosy. However, to consolidate the hypothesis and determine whether the effects noted were anergy reversion or direct anti-mycobacterial effect of the drugs used, a sequential monitoring study must be performed looking for parameters measured in the present study. In addition, the presence of histone acetylases and deacetylases in the evolving lesions of leprosy must also be ascertained. This research will soon be initiated in our laboratory.


  in Conclusion Top


Murine leprosy is a disease model on which novel mechanisms of pathogenesis can be analyzed to better understand the disease. Anergy in murine leprosy is a state of unresponsiveness due to the lack of activity of IFNγ- and TNFα-producing T-cells, namely Th1/CD4+ and Tc/CD8+ cells. There seem to be excessive histone deacetylation (variably reverted by NaB and VPA) or immune downregulation (upregulated by DLE) in the phenomenon. NaB, VPA, and DLE might be used as complementary medicines in the treatment of leprosy.

Financial support and sponsorship

This study was financially supported by Sección de estudios de posgrado e investigación del Instituto Politécnico Nacional, México.

Conflicts of interest

There are no conflicts of interest.



 
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