Keywords
homeopathy - fungus - microsporidiosis - cytokines - chemokines
Introduction
The microsporidian Encephalitozoon cuniculi is an extremely important pathogen with zoonotic potential. This pathogen infects
rabbits, causing severe and chronic granulomatous interstitial nephritis with fibrosis
and granulomatous encephalitis.[1]
[2] Encephalitozoonosis in humans can cause local infections to spread systemically.
The competence of the host's immune response and the fungus species involved are factors
that determine the pathogenicity of the disease.[3]
[4]
[5] Rabbits can be infected with E. cuniculi via ingestion or inhalation of spores as well as by a trans-placental route. During
the acute phase of the disease, which persists for an average of 30 days, the microsporidian
replicates in the lungs, liver, and kidneys.[6] The clinical signs are classified into three groups: neuronal signals with vestibular
syndrome (including head tilt), kidney failure, and eye injuries. Among the neuronal
signals, vestibular syndrome, weakness, paresis of the hind limbs, and ataxia are
often observed. The diagnosis is crucial to determine its possible zoonotic risks,[6]
[7] and there is no specific treatment for encephalitozoonosis.[7] Considering the difficulty associated with establishing an effective therapeutic
protocol, the search for alternatives is critical. Several studies have demonstrated
specific and relevant effects of homeopathic medicines in animals of different species
under various nosological conditions;[8]
[9]
[10] nevertheless, the understanding of the general mechanisms through which such agents
exert their effects is still very poor.[10]
Some previous clinical experience of one of the co-authors of this article was the
main motivation for this study. Forty-two rabbits (Oryctolagus cuniculus), in which a diagnosis of encephalitozoonosis was established according to standard
clinical parameters,[11]
[12] were treated daily with Phosphorus (Phos) 200 cH based on the traditional concept of “genus epidemicus” in which the three
most important symptoms, ocular changes (including cataract, uveitis, hypopyon, and
blindness), weakness of limbs and torticollis,[11]
[12] were used as the basis for this therapeutic choice. Based on the evolution of neuronal
and renal signs, very good clinical outcome was obtained in all cases after 3 weeks
of treatment. Of the 42 treated animals, 27 presented neurological signs (defined
as head tilt, opisthotonus and tremors), and 18 presented renal signs such as hematuria,
weight loss, and polyuria. Symptom remission was observed in 40/42 animals, with no
mortality.
The second motivation for performing this in-vitro study was the previous establishment of a model for studying the effects of highly
diluted substances in immune cells. Infection of macrophages in-vitro can provide a sensitive translational model for understanding the effects of highly
diluted substances on the immune response. This model was previously validated by
the results obtained in Leishmania amazonensis-infected macrophages treated with various homeopathic protocols,[13]
[14]
[15] which are related closely to observations obtained in-vivo. Using various models of macrophage challenge with other micro-organisms, our team
has previously shown that highly diluted medicines, instead of killing the micro-organisms,
facilitate the organization of a more effective pattern of macrophage response and
arrest the parasite's life cycle.[8]
[9]
[10]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23] For this reason, a similar model was designed herein to explain the clinical results,
and avoiding new in-vivo experiments.
Materials and Methods
Preparation of Homeopathic Medicine, Quality Control, Treatment and Blinding
Phos matrix (Phos 5 cH, 29 cH or 199 cH) was prepared in a commercial pharmacy registered with the
National Agency for Health Surveillance (ANVISA) according to the guidelines described
in the Brazilian Homeopathic Pharmacopoeia, 3rd edition, 2011;[24] that is, sequential 1:100 dilutions followed by 100 succussions (vertical agitations)
each using 30% ethanol solution and a mechanical Denise automatic arm (Autic; São
Paulo, Brazil). In the terminology, the number represents the step of the sequential
dilution, and cH refers to “centesimal Hahnemannian” or 1:100 dilution (proportion
between active principle and solvent) followed by agitation according to the method
described by Samuel Hahnemann.
The final working dilutions (Phos 6 cH, 30 cH and 200 cH) were prepared in our own laboratory 1 day prior to conducting
the cell assays using pure water under sterile conditions. The control was the vehicle
itself (30% ethanol) prepared at the same time using the same procedures, including
a final 1:100 dilution in sterile pure water; thus, the final alcohol concentration
was 0.06%. Before use in the cell cultures, the final dilution was filtered through
a 0.22-µm filter (Millipore; Saint Louis, Missouri, USA), and the flasks were coded
by staff not involved in the experimental laboratory to permit blinded procedures
until the statistical analyses were completed.
Before the medicines were used in the experiments, rapid quality control testing was
performed. Samples of each dilution were examined by scanning electron microscopy
to permit the identification of suspended microparticles; the chemical nature of the
samples was then analyzed using an energy dispersion spectroscopy system. Since contaminant
micro- and nanoparticles are expected to be present in water,[25]
[26]
[27]
[28]
[29] this analysis was useful in eliminating any possibility that a predominant contaminating
element was responsible for the effects described. The identification of suspended
contaminant microparticles present in the tested solutions was performed using the
micro-evaporation method followed by scanning electron microscopy under 10-kV working
conditions and energy dispersive X-ray spectroscopy (JSM 6510; JEOL Ltd., Tokyo, Japan).
Traces of Cl, Pr, Ca, Na, Zn, Si, Au, Pb and Al were randomly distributed among the
analyzed particles (n = 30 per sample); the results showed that there was no predominant particulate contaminant
that could produce false-positive results.
Following these steps, treatment of infected cells was performed according to the
following design. To verify the step-wise internalization of fungi by macrophages,
two experimental series were performed, the first after 1 hour of infection and the
second after 24 hours of infection. These times were chosen due to the rapid digestion
activity of RAW 264.7 cells. In 24-well plates, 4 wells were treated with vehicle,
Phos 6cH, Phos 30cH, and Phos 200cH, and 4 wells were untreated; 2 wells contained only macrophages, with no infection,
these being data used to define the baseline values of each studied parameter and
not included in the statistical analysis; 2 well plates were not used. The volume
of the medicines and vehicle was equal to 20% of the total liquid volume in each well
(500 µL); the total volume included 34 µL of E. cuniculi suspension + 100 µL medicine + 366 µL Roswell Park Memorial Institute (RPMI) medium
enriched with 10% bovine fetal serum (BFS). The calculated final ethanol concentration
was 0.06% in all treated wells. The proportion between medium and medicines was defined
in a pilot study as a limit that did not cause osmotic stress in the cells.
Macrophage Culture and Infection
RAW 264.7 macrophages were purchased from the Cell Bank of Rio de Janeiro and kept
frozen in a −80°C freezer. After thawing, they were expanded in cell culture bottles
in RPMI medium (Cutilab; Campinas, Brazil) enriched with 10% BFS. When the confluence
of the macrophage cultures reached 90%, the cells were washed with serum-free RPMI
medium to remove any dead cells present in the supernatant. Then, 5 mL of RPMI medium
was added, and the cells were detached using a cell scraper. A Pasteur pipette was
used to remove the detached cells completely from the bottles. The cells were transferred
to a Falcon tube, and their viability was determined using a Countess automatic counting
system (Thermo Fisher; Camarillo, California, USA) after staining with 0.4% trypan
blue dye. For plating the cells, round coverslips were placed in the 24-well sterile
plates using one coverslip for each well.
The cells were held on ice and then centrifuged for 3 minutes at 333 g. The supernatant
was discarded, and the pellet was homogenized in RPMI. After suspension, the cells
were plated on coverslips (5 × 105 macrophages per coverslip) in 50 µL of RPMI. The cells were dispensed in the center
of the coverslip and gently spread so that they were homogeneously distributed. Prior
to incubation, 250 µL of RPMI enriched with 10% BFS was added to each well. The plate
was incubated at 37°C for at least 2 hours to allow complete adherence of the macrophages.
E. cuniculi spores were then added at a 2:1 ratio. The cells were treated at the moment of infection
according to the protocol described in the section above.
Preparation of E. cuniculi Spores
E. cuniculi spores were previously cultured in RK-13 (rabbit kidney) cells. RK-13 cells were
maintained in Eagle's medium (Cultilab; Campinas, Brazil) supplemented with penicillin-streptomycin
(Sigma-Aldrich; Saint Louis, Missouri, USA) plus 10% BFS, 10% non-essential amino
acids, and 10% pyruvate (Sigma-Aldrich; Saint Louis, Missouri, USA) and incubated
at 37°C in 5% CO2. Every 7 days, the culture supernatants were collected and centrifuged for 30 minutes
at 500 g to obtain spores, which were stored at 4°C in 1x phosphate-buffered saline
(PBS). Prior to infecting the cell cultures, the supernatants obtained from the cultures
were pooled and purified. Briefly, 50% Percoll (Sigma-Aldrich; Saint Louis, Missouri,
USA) diluted in PBS was added to the pellet pool, and the mixture was centrifuged
for 20 minutes at 500 g. The supernatant containing the cell debris was discarded,
and the pellet containing the spores was washed twice in PBS. E. cuniculi spores were counted in a Neubauer chamber[8] before their addition to macrophage cultures.
Analysis of Parasite Internalization
The number of parasites internalized by endocytosis and phagocytosis was measured
from digital photomicrographs obtained using a fluorescence microscope (Olympus 60i;
Tokyo, Japan). Non-fixed cells adhering to coverslips were stained using Calcofluor
White stain (Sigma-Aldrich; Saint Louis, Missouri, USA) (10 µL per coverslip) 1 and
24 hours after infection and treatment.[30]
[31] In this method, the spores become blue and fluorescent due to the interaction of
the dye with their chitin walls. Ten microscopic fields on each coverslip were recorded
using a 40x objective; the captured images were analyzed using the manual counting
tool in Offline MetaMorph software (Molecular Devices; San José, California, USA),
and the number of internalized spores per macrophage was calculated. Two coverslips
were used per treatment.
Determination of Cytokines and Chemokines
Culture supernatants were harvested at various times, centrifuged, and frozen at −80°C.
The cytokine levels were measured using the MAGPIX—Luminex method (Kit e-Bioscience;
Millipore, Saint Louis, Missouri, USA) according to the manufacturer's instructions.
The kit permits the detection of the following cytokines: interleukin 1α (IL-1α),
IL-1 β, interferon gamma (IFNγ), IL-10, IL-6, vascular endothelial growth factor A
(VEGFA), tumor necrosis factor alpha (TNF-α), RANTES (regulated on activation, normal
T cell-expressed and secreted) or CCL5, granulocyte-macrophage colony-stimulating
factor (GM-CSF), IL-12-p40, IL-12-p70, macrophage inflammatory protein 1 (MIP-1β or
CCL4), and monocyte chemoattractant protein-1 (MCP-1 or CCL2). Peptides that were
present at levels below the detection limits of the method (defined in the manual
provided by the manufacturer) were excluded from the statistical analysis. These samples
were performed in quadruplicate.
Assessment of Lysosome Activity by Fluorescence Microscopy
After 1 and 24 hours, coverslips containing adherent macrophages infected with E. cuniculi were incubated with acridine orange dye for 20 minutes at 37°C to permit measurement
of lysosome activity in real time. After incubation, the coverslips were washed in
PBS and mounted on slides, using a drop of PBS as adherent. Quantitative analysis
was performed automatically using offline MetaMorph software (Molecular Devices, USA);
in this method, the intensity of the orange fluorescence exhibited by the vacuoles
was measured. To ensure reliable results, the color filter of the software was standardized
and adjusted to reveal only fluorescence in the orange region (color model: HIS; hue:
19–20; intensity: 89–90). The principle of this method is based on the increase in
the intensity of the orange fluorescence of the dye in acid medium. Ten microscopic
fields on each coverslip were randomly measured using a 200x objective. This sample
corresponds to 50% of the total area occupied by the adherent cells. This method was
carried out in duplicate, two coverslips per treatment.
Transmission Electron Microscopy
When the experimenters were no longer blinded to the identity of the samples, a replication
of the experiment was conducted, and two treatments (vehicle and Phos 200 cH) were chosen to be examined by transmission electron microscopy (TEM). In
this experiment, the treatment was applied to cells in culture bottles rather than
to cells in plates; after 24 hours of treatment, the cells were detached from the
bottles using a cell scraper, fixed in 2% glutaraldehyde in cacodylate buffer (0.2 M,
pH = 7.2) for 10 hours at 4°C and pooled in two microtubes, according to the treatment.
After centrifugation (1,500 rpm for 5 minutes), the pellet at the bottom of each microtube
was post-fixed in buffered 1% OsO4 for 2 hours, treated with increasing concentrations of ethanol and embedded in Epon
resin. The ultrastructural images were captured using a Tecnai G20 electron microscope
(FEI Co., Hillsboro, Oregon, USA) with tomographic capacity and equipped with a high-resolution
Eagle 4k x 4k camera.
Prior to electron microscopy, the material was examined in an optical microscope after
the preparation of semi-thin sections stained with toluidine blue, aqueous uranyl
citrate, and lead citrate. Thirty-five digital photomicrographs were registered from
each treatment and the number of apoptotic cells was computed by MetaMorph software,
using the manual counting option operated by two independent observers. Crescent-shaped
nuclei, membrane blebs, and apoptotic bodies were the hallmarks used to identify them.
Statistical Analysis
Statistical analysis was performed using IBM SPSS version 21.0 and GraphPad Prism
version 6.0 for Windows. Normality was assessed using the Shapiro–Wilk test and evaluated
by inspection of quartile–quartile plots (Q–Q plots). The homogeneity of the variances
was evaluated using the Levene test, and Welch correction was applied to analysis
of variance (ANOVA) in cases of non-homogeneity. Outliers were evaluated by Q–Q plot
inspection and removed if necessary. Each treatment was analyzed in duplicate or quadruplicate
for all parameters analyzed, except the apoptotic cells counting, in which 35 fields
per slide were used from pooled samples.
The results were evaluated through one-way ANOVA and Dunnett or Games–Howell's post-tests
according to the case. The partial eta-square (η
2), a measure of effect size (from 0.1 to 1.0), has also been reported. This parameter
is particularly useful for variables that present large deviation values and p-value analyses that could lead to type I errors (rejection of a null hypothesis when
it is true) or type II errors (acceptance of a null hypothesis when it is not true).
Values of partial η
2 higher than 0.4 were considered as relevant. Student “t” test was used to compare the number of apoptotic cells in control and Phos 200 cH treated cells, from the semi-thin sections.
The results are presented as the mean ± standard error, and values of p < 0.05 are considered to be significant. The figures were prepared using Prism 5.0
software.
Results
The time-dependent evolution of internalized microorganisms presented different patterns
as a function of the treatment. As seen in [Fig. 1], the number of internalized fungi was significantly reduced in all treated cultures
after 1 and 24 hours of exposure, except in the case of samples treated with Phos 200 cH, in which reduction was significant only after 24 hours. Morphological analysis
of the same samples after 24 hours of treatment showed a marked reduction in the number
and size of internalized spores, reinforcing this finding ([Fig. 2]). Moreover, the cultures treated with Phos 30 cH and 200 cH presented non-internalized spores ([Fig. 2]).
Fig. 1 Number of internalized Encephalitozoon cuniculi spores per macrophage measured using the Calcofluor White staining method and fluorescence
microscopy. Counting was performed using Offline MetaMorph software and a manual counting
system. After 1 hour: *ANOVA, Games–Howell, F (4, 25.422) = 9.663, p < 0.001, η
2 = 0.486 in relation to control. After 24 hours: *ANOVA, Games–Howell, F (4, 21.232) = 9.387, p < 0.001, η
2 = 0.744. The values shown are represented by a box-plot, with maximum and minimum
limits. Control = untreated infected macrophages. ANOVA, analysis of variance.
Fig. 2 Photomicrograph of coverslip-adherent macrophages 24 hours after infection with Encephalitozoon cuniculi. The samples shown include an untreated control, cells treated with vehicle, and
cells treated with Phos at various dilutions. Narrow arrows: internalized spores; large arrows: non-internalized spores. The spores were stained by the blue fluorescent dye Calcofluor
White due to their chitin walls. Objective: 200x.
The TEM and semi-thin toluidine-stained sections prepared after 24 hours of treatment
corroborated these findings ([Figs. 3] and [4]), showing two different patterns of parasite digestion in the cytoplasm. The vehicle-treated
cells contained many easily recognized spores and debris within phagocytic vacuoles
([Fig. 4A]). Apoptotic cells were seen in both the vehicle and Phos 200 cH-treated cultures; these cells presented a classical degeneration pattern including
cell shrinkage, half-moon-shaped nuclei, chromatin condensation ([Fig. 3]), and apoptotic body formation ([Fig. 4B]). In the case of the Phos 200 cH cultures, much thinner and more delicate pathogen debris was found than in
the control; moreover, empty vacuoles were frequently found ([Fig. 4A], [B]). An intermediate pattern was seen in vehicle-treated cells ([Fig. 4C]). The quantitative analysis performed from the semi-thin sections revealed 23.00 ± 0.65
apoptotic cells per field in the vehicle treated cells and 17.28 ± 0.57 apoptotic
cells in the Phos 200-cH treated cells (p < 0.001; degrees of freedom = 70).
Fig. 3 Photomicrographs of semi-thin sections of infected macrophages after 24 hours. (A) Vehicle; (B) Phos 200 cH-treated cells. Arrows: apoptotic cells showing cell shrinkage, half-moon-shaped nuclei, and membrane blebs
(apoptotic bodies). Toluidine stain. Objective: 100x.
Fig. 4 Ultra-micrographs of macrophages infected with Encephalitozoon cuniculi and treated with vehicle or Phos 200 cH or not treated. (A) Vehicle-treated macrophages containing digestive vacuoles with spores undergoing
lysis (narrow arrow) or without spores (large arrow); (B) Macrophages treated with Phos 200 cH containing digestive vacuoles (large arrow). (C) Vehicle-treated macrophages with digestive vacuoles (large arrow); an apoptotic
cell (AC) is visible.
The acridine orange assay showed increased size, distribution, and brightness intensity
of fluorescent cytoplasmic vacuoles in cells treated with Phos 6 cH, 30 cH and 200 cH after 1 hour and in cells treated with Phos 200 cH after 24 hours relative to the respective controls. The quantitative analysis
is shown in [Fig. 5]. A time-dependent increase in fluorescence intensity occurred after all treatments;
greater variation was observed in cultures treated with Phos 6 cH and 30 cH, whereas treatment with Phos 200 cH resulted in a more stable effect. Illustrations of these differences are shown
in [Figs. 6] and [7].
Fig. 5 Average fluorescence intensity (pixels) of macrophages stained with acridine orange
and observed by fluorescence microscopy. The measurements were made using MetaMorph
offline software and an automatic counting system. (A) After 1 hour: *ANOVA, Games–Howell, F (4, 23.653) = 6.060, p = 0.002, η
2 = 0.390 in relation to control. (B) After 24 hours: *ANOVA, Games–Howell, F (4, 21.150) = 53.624, p < 0.001, η
2 = 0.522. The values shown are represented by a box-plot, with maximum and minimum
limits. ANOVA, analysis of variance.
Fig. 6 Photomicrographs of coverslip-adherent macrophages 1 hour after infection with Encephalitozoon cuniculi: untreated control, cells treated with vehicle, and cells treated with Phos at various dilutions. The cytoplasm of the cells exhibits different degrees of brightness
due to the orange fluorescence of lysosomes (arrow) after staining with acridine orange dye for 20 minutes at 37°C. Objective: 200x.
Fig. 7 Photomicrographs of coverslip-adherent macrophages 24 hours after infection with
Encephalitozoon cuniculi: untreated control, cells treated with vehicle and cells treated with Phos at various dilutions. The cytoplasm of the cells exhibits different degrees of brightness
due to the orange fluorescence of lysosomes (arrow) after staining with acridine orange dye for 20 minutes at 37°C. Objective: 200x.
The observed changes in cytokine and chemokine production were also time- and dilution-dependent.
After 1 hour, there was a significant increase in IL-6 and TNFα in vehicle-treated
cells, and the levels of these cytokines increased further after 24 hours of treatment
(though the differences were not statistically significant due to the increase in
variability). MCP-1, however, showed a very marked increase in all treated groups,
including vehicle-treated cells, at both analyzed times; its concentration in the
supernatant was nine-fold higher after 24 hours ([Tables 1] and [2]). RANTES showed an important and significant increase in Phos 200 cH-treated cells after the first hour of treatment and a 10-fold increase after
24 hours. Treatment with lower dilutions resulted in intermediary values of RANTES
at both times. The η
2 did not present relevant levels for RANTES at any tested potency after 24 hours ([Tables 1] and [2] and [Fig. 8]).
Fig. 8 Concentrations of relevant peptides in the culture supernatants of macrophages infected
with Encephalitozoon cuniculi and treated for 1 hour (A) or 24 hours (B) with various homeopathic dilutions of phosphorus. (A) *ANOVA, Tukey, F (4, 11) = 3.372, p = 0.05, η
2 = 0.574 in relation to control. (B) ANOVA, Games–Howell, F (4, 4.027) = 4.585, p = 0.084, η
2 = 0.378. The values shown represent the mean and standard error. ANOVA, analysis
of variance.
Table 1
Concentrations of cytokines, chemokines and VEGF in the culture supernatants of macrophages
infected with Encephalitozoon cuniculi and treated for 1 hour with various homeopathic dilutions of phosphorus
|
Peptide
|
Mean ± standard deviation
|
p-Value and η
2
|
|
Untreated control
|
Vehicle
|
Phos 6 cH
|
Phos 30 cH
|
Phos 200 cH
|
|
IL-1α
|
14.08 ± 5.41
|
30.08 ± 7.80
|
2859.0 ± 16.6
|
25.95 ± 15.75
|
26.94 ± 17.52
|
0.786
|
0.135
|
|
IFNγ
|
4.94 ± 0.39
|
5.33 ± 0.56
|
4.45 ± 0.68
|
4.21 ± 0.76
|
5.50 ± 2.88
|
0.464
|
0.162
|
|
IL-6
|
3.52 ± 0.71
|
6.83 ± 1.69[a]
|
5.79 ± 0.65
|
5.55 ± 1.29
|
5.13 ± 0.46
|
0.035
|
0.582
|
|
IL-10
|
21.47 ± 8.40
|
51.98 ± 26.02
|
49.57 ± 19.93
|
45.78 ± 24.26
|
50.09 ± 29.08
|
0.602
|
0.191
|
|
IL-12p40
|
6.23 ± 1.04
|
6.61 ± 2.46
|
5.70 ± 0.48
|
4.96 ± 0.55
|
5.95 ± 3.06
|
0.502
|
0.134
|
|
IL-12p70
|
8.10 ± 3.05
|
17.43 ± 13.12
|
16.69 ± 8.04
|
14.34 ± 8.32
|
5.04 ± 2.14
|
0.332
|
0.319
|
|
MCP-1
|
717.5 ± 21.92
|
1816.0 ± 128.2[a]
|
1942.0 ± 228.9[a]
|
1633.75 ± 131.20
|
1774.33 ± 721.21[a]
|
< 0.001
|
0.629
|
|
MIP-1β
|
6036.0 ± 1193.5
|
10959.0 ± 816.6
|
10087.75 ± 732.64
|
8896.75 ± 633.61
|
7624.0 ± 5719.3
|
0.062
|
0.358
|
|
RANTES
|
8.49 ± 1.55
|
14.17 ± 4.09
|
14.17 ± 4.09
|
11.64 ± 2.85
|
21.65 ± 4.01[a]
|
0.054
|
0.574
|
|
VEGF
|
2.65 ± 0
|
3.06 ± 0.41
|
3.13 ± 0.13
|
2.93 ± 0.33
|
3.01 ± 0.83
|
0.769
|
0.131
|
|
TNFα
|
1887.0 ± 57.9
|
2320.5 ± 177.4[a]
|
2374.75 ± 331.05
|
2208.75 ± 99.66
|
1865.0 ± 1071.8
|
0.046
|
0.359
|
Abbreviations: IFNγ, interferon gamma; IL-6, interleukin 6; MCP-1, monocyte chemotactic
protein-1; MIP-1β, macrophage inflammatory protein 1β; RANTES, regulated on activation,
normal T-cell expressed and secreted; TNF-α, tumor necrosis factor alpha; VEGF, vascular
endothelial growth factor.
Note: The values shown represent the mean and standard deviation, the level of significance,
and eta-squared (effect size, η
2).
a ANOVA, Games-Howell in relation to control.
Table 2
Concentrations of cytokines, chemokines and VEGF in the culture supernatants of macrophages
infected with Encephalitozoon cuniculi and treated for 24 hours with various homeopathic dilutions of phosphorus
|
Peptide
|
Mean ± standard deviation
|
p-Value and η
2
|
|
Untreated control
|
Vehicle
|
Phos 6 cH
|
Phos 30 cH
|
Phos 200 cH
|
|
IL-1α
|
78.19 ± 4.78
|
56.09 ± 26.46
|
72.69 ± 1.83
|
84.50 ± 11.31
|
90.01 ± 11.46
|
0.332
|
0.562
|
|
IFNγ
|
5.97 ± 0.22
|
4.02 ± 1.86
|
5.58 ± 1.33
|
6.59 ± 2.54
|
7.30 ± 2.56
|
0.406
|
0.332
|
|
IL-6
|
5.40 ± 0.27
|
20.36 ± 5.76
|
10.64 ± 6.30
|
23.04 ± 17.55
|
26.67 ± 9.28
|
0.191
|
0.461
|
|
IL-10
|
48.01 ± 2.20
|
46.59 ± 25.81
|
49.64 ± 16.18
|
54.69 ± 30.09
|
51.73 ± 26.98
|
0.994
|
0.022
|
|
IL-12p40
|
5.56 ± 0.29
|
5.02 ± 1.42
|
5.46 ± 1.40
|
5.92 ± 2.08
|
8.55 ± 2.58
|
0.222
|
0.438
|
|
IL-12p70
|
5.37 ± 0.80
|
3.88 ± 0.88
|
6.82 ± 3.50
|
7.86 ± 3.63
|
6.01 ± 1.25
|
0.428
|
0.321
|
|
MCP-1
|
4981.0 ± 1544.3[a]
|
9025.66 ± 411.07
|
9406.66 ± 1287.16
|
9736.0 ± 1681.3
|
9752.66 ± 1096.79
|
0.014
|
0.717
|
|
MIP-1β
|
16289.5 ± 331.6
|
14912.0 ± 2834.0
|
15956.66 ± 1472.71
|
16640.66 ± 1302.29
|
16252.67 ± 395.93
|
0.752
|
0.175
|
|
RANTES
|
84.28 ± 1.28
|
218.66 ± 163.22
|
113.38 ± 13.50
|
252.66 ± 165.71
|
292.66 ± 122.83
|
0.084
|
0.378
|
|
VEGF
|
2.65 ± 0.26
|
2.86 ± 0.32
|
3.00 ± 0.48
|
2.91 ± 0.47
|
3.03 ± 0.42
|
0.864
|
0.121
|
|
TNFα
|
3154.0 ± 216.3
|
2207.0 ± 936.0
|
7030.0 ± 5963.9
|
2619.0 ± 895.7
|
2735.66 ± 367.45
|
0.450
|
0.384
|
Abbreviations: IFNγ, interferon gamma; IL-6, interleukin 6; MCP-1, monocyte chemotactic
protein-1; RANTES, regulated on activation, normal T-cell expressed and secreted;
TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor.
Note: The values shown represent the mean and standard deviation, the level of significance
and eta-squared (effect size, η
2).
a ANOVA, Games-Howell in relation to the other groups.
Discussion
Cellular mechanisms related to the effects of homeopathic treatments have been well
described in recent years and are related to the epigenetic control of specific genes,
which depends on the nature of the medicine used and its dilution.[17]
[18]
[29]
[32]
[33]
[34]
[35] Based on our previous experience, macrophages respond to specific homeopathic stimuli
by expressing specific cytokines and chemokines depending on the experimental context.[13]
[15] The choice of Phos to be used in this study was based on the classical homeopathic similia principle,
since the main symptoms of encephalitozoonosis are listed in Phos materia medica and previous clinical observations reinforced the utility of this
medicine for this kind of epidemiological problem.
The results show that the effects of the tested treatments can be classified into
two categories: (1) effects related to the vehicle itself (increased levels of inflammatory
cytokines and chemokines, mainly MCP-1); (2) effects related to treatment with specific
potencies of Phos (changes in lysosome activity after 1 and 24 hours of treatment, reduced apoptosis
rate and a progressive increase in RANTES/CCL5 between 1 hour and 24 hours relative
to the controls), associated with the presence of non-internalized parasites.
The biological meaning of these effects can be understood in the context of recent
findings on the pathogenesis of E. cuniculi infection. The balance between the increased digestion of internalized spores and
the presence of non-internalized spores, even after 24 hours, suggests that macrophages
treated with Phos 200 cH were able to phagocytoze and digest the parasites more efficiently than control
cells, thereby inhibiting the phagocytosis-independent E. cuniculi internalization, which is the primary route of cell infection.[3]
[36]
[37] After ingestion, changes in pH and osmotic pressure cause the polar tube and sporoplasm
to be ejected from the spore, with subsequent penetration of the host cell by the
parasite.[36]
[38]
[39] Another means through which spores gain access to the host cell cytoplasm is endocytosis.
If a spore is phagocytozed by a host cell, the polar tube can pass through the phagocytic
vacuole membrane, delivering the sporoplasm into the host cell cytoplasm. Then, the
parasite can manipulate host-cell death mechanisms as a way of perpetuating its survival.[36]
[37] In our study, the absence of intact spores in cell vacuoles simultaneously with
the marked presence of intercellular forms of the pathogen within macrophages, points
to the efficacy of the microbicidal activity of treated macrophages, and a possible
increase in the vulnerability of free spores to the host immune response, should the
same effect be observed in-vivo.
In this sense, the presence of intact spores within macrophages following treatment
could be related to a possible impairment of cell invasion. Two non-exclusive hypotheses
can be considered: (1) the balance between the enhanced digestion of internalized
spores and the presence of non-internalized spores, even after 24 hours of treatment
with Phos 200 cH, might be caused by an improvement in phagocytosis and digestion of the parasites
by the treatment, causing these processes to be more efficient than in the controls;
(2) the treatment may inhibit the phagocytosis-independent E. cuniculi internalization pathway that represents the main pathway for cell infection.[3]
[36]
[37] The latter hypothesis could be related to a direct effect of the treatment on the
surface structure of the spore that leads to a reduction in its rate of internalization.
Moreover, the reduction in apoptotic cells after treatment with Phos 200 cH in relation to vehicle might represent reduction in macrophage susceptibility
to the parasite mechanisms of corrupting cell functions and shows higher vitality
of these cells, which could impact the intensity of lysosome activity.
With respect to chemokine production, MCP-1 (or CCL2) and RANTES were the most affected.
MCP-1 attracts cells of the monocyte lineage to sites of inflammation and is tightly
related to the pathogenesis of E cuniculi infection.[38]
[40] Instead, RANTES plays a critical role in determining the direction of inflammation
after infection. RANTES is expressed by cells that play a crucial role in the homing
and migration of memory T-cells during acute infections. Modifications in the function
of RANTES (CCL5) and the RANTES receptor (CCR5) can lead to changes in prognosis due
to dysfunctions in the CD8 T-cell response.[41] RANTES–CCR5 interaction promotes diapedesis and transmigration of Th1 cell types
(but not Th2 cell types) through the endothelial layer at sites of inflammation and
determines the type of immune response that occurs.[42] In the context of E. cuniculi infection, RANTES, among other chemokines (CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8),
plays an important role in granulocyte and T cell migration.[40] Patients infected with HIV1 who exhibit higher serum levels of RANTES are partially
protected against the infection, indicating a better prognosis.[40]
[43] Herein, the specific increase in RANTES observed after treatment with Phos 200 cH is consistent with the biological context of E. cuniculi infection and could explain the clinical success of this therapy, as mentioned in
“Introduction”.
Analysis of the infected cells using a quantitative automatic method (MetaMorph) allowed
us to determine the level of lysosome activity in the cells with high sensitivity.
This finding, together with the ultrastructural findings, indicates the level of parasite
digestion induced by the treatments. Thus, the vehicle-induced decrease in the number
of internalized spores is probably related to increased parasite digestion, in contrast
to the findings for Phos 200 cH-treated cells, in which complete parasite digestion was associated with reduction
in the number of apoptotic cells and increase in non-internalized spores, suggesting
changes in parasite pathogeny. The administration of vehicle alone also increased
the levels of important inflammatory mediators such as IL-6 and MCP-1. The reason
that the vehicle itself can produce pro-inflammatory responses could be related to
the presence of the oxygen-rich nanobubbles that are found in highly agitated liquids,
including homeopathic preparations;[44] thus, it could represent a general non-specific effect on cultured cells. All statistically
significant data were related to a high “partial η
2” value (higher than 0.4), which reinforces the biological meaning of the results.
The effects of homeopathic medicines in relation to the vehicle (placebo) may be seen
in studies performed in-vivo or in standard clinical trials.[8]
[9]
[14]
[15]
[16]
[19]
[20]
[21]
[22]
[23] However, characterizing the specificity of such effects is even more important in
refined studies designed in-vitro,[44] since it is known that the succussion itself can promote changes in the solvent
organization, including the generation of different oxygen-derived radicals, mainly
when water is used.[45]
[46]
[47] Macrophage cultures are very sensitive for detecting the effect of high dilutions
but are also sensitive for detecting such solvent changes as well, concerning elements
related to cell oxidative metabolism.[48]
[49]
[50] Such biochemical interference could lead to non-specific results—that is, not related
to the Similia principle itself, but to the physico-chemical changes of the solvent
due to agitation. Similar solvent-dependent effects can also be easily seen in vegetal
models.[51]
[52] Thus, the comparison between the effects of succussed vehicle and the active homeopathic
preparations in relation to a negative control is usually elucidative.
In the present work, differences comparing Phos 200cH and controls, in regard to parasite–phagocyte interaction, were shown by three
complementary results: (1) the presence of higher numbers of non-internalized parasites
for Phos 200cH than for the untreated control; (2) the acute production of RANTES in Phos 200cH in relation to the untreated control; and (3) the increase in lysosome activity,
which presented clear statistical difference between Phos 200cH and vehicle itself, as shown in [Fig. 5], associated with cell preservation or reduced apoptosis. These last findings are
particularly important, indicating that eventual oxidative stress related to lysosomal
function was clearly different between the treatments and cannot be related to a non-specific
and biased outcome, secondary to the succussion of water. The findings (1) and (2)
above are parasite and/or macrophage features, whose mechanisms need to be elucidated
in further studies, though they are probably related to the specific expression of
certain genes, as already seen in other similar studies using macrophages in-vitro.[17]
[18]
[48]
[49]
[50]
From a practical point of view, putative overlapping between the pro-inflammatory
effects of succussed water, including the induction of inflammatory cytokines and
chemokines (such as MCP-1, MIP-1β and TNFα) and the specific effects of Phos 200cH (acute expression of RANTES and lysosome activation), would also be present
in clinical situations. Thus, an important consideration emerges: perhaps homeopathy
acts through different and simultaneous mechanisms, with diverse degrees of specificity,
which could be in accordance with the systemic effects observed in clinical practice.
In short, this study reveals some of the cell mechanisms involved in the action of
Phos 200 cH on the experimental infection of macrophages with E. cuniculi, which would help to explain its clinical success in rabbits, as described above.
Conclusions
The in-vitro results presented in this work indicate putative mechanisms that underlie the clinical
benefits of Phos 200 cH, as previously observed clinically in rabbits. Although non-specific, pro-inflammatory
mechanisms that are related to the vehicle itself were observed, the specific action
of Phos 200 cH was clearly related to increase in RANTES production, to lysosome activity,
and to reduction in apoptosis of infected cells.
