All-Trans Retinoic Acid in Combination with Primaquine Clears Pneumocystis Infection
et al. (2013) All-Trans Retinoic Acid in Combination with Primaquine Clears Pneumocystis Infection. PLoS
ONE 8(1): e53479. doi:10.1371/journal.pone.0053479
All-Trans Retinoic Acid in Combination with Primaquine Clears Pneumocystis Infection
Guang-Sheng Lei 0
Chen Zhang 0
Shoujin Shao 0
Hsin-Wei Jung 0
Pamela J. Durant 0
Chao-Hung Lee 0
Chad Steele, University of Alabama at Birmingham School of Medicine, United States of America
0 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine , Indianapolis, Indiana , United States of America
Pneumocystis pneumonia (PcP) develops in immunocompromised patients. Alveolar macrophages play a key role in the recognition, phagocytosis, and degradation of Pneumocystis, but their number is decreased in PcP. Our study of various inflammatory components during PcP found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs of mice and rats with Pneumocystis pneumonia (PcP). We hypothesized that treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, may effectively control Pneumocystis (Pc) infection by inducing MDSCs to differentiate to AMs. In rodent models of PcP, we found that 5 weeks of ATRA treatment reduced the number of MDSCs in the lungs and increased the number of AMs which cleared Pc infection. We also found that ATRA in combination with primaquine was as effective as the combination of trimethoprim and sulfamethaxazole for treatment of PcP and completely eliminated MDSCs and Pc organisms in the lungs in two weeks. No relapse of PcP was seen after three weeks of the ATRA-primaquine combination treatment. Prolonged survival of Pc-infected animals was also achieved by this regimen. This is the very first successful development of a therapeutic regimen for PcP that combines an immune modulator with an antibiotic, enabling the hosts to effectively defend the infection. Results of our study may serve as a model for development of novel therapies for other infections with MDSC accumulation.
Funding: The study was supported by grants R01 AI062259 and R03 AI091418 from the National Institutes of Health. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Pneumocystis pneumonia (PcP) is a common opportunistic disease
in immunocompromised hosts, such as patients with AIDS [1,2]
and those with other predisposing immune deficiencies including
acute lymphoblastic leukemia, severe combined immunodeficiency
syndrome, Hodgkins disease, rhabdomyosarcoma, Wegeners
granulomatosis, collagen vascular disease, and primary metastatic
tumor in the central nervous system . The use of
immunosuppressive drugs in organ transplantation patients  and
anti-TNFa monoclonal antibodies, such as infliximab  and adalimumab
, in patients with rheumatoid arthritis may also result in PcP.
Drugs that are commonly used to treat PcP include the
combination of trimethoprim and sulfamethoxazole (TMP-SMX,
also referred to as Septra, Bactrim, and Co-trimoxazole),
pentamidine, dapsone, atovaquone, and the combination of
clindamycin and primaquine. Among these, TMP-SMX and
pentamidine are considered first line drugs . TMP-SMX is the
most effective drug for treatment and prevention of PcP.
Unfortunately, many patients fail therapy with TMP-SMX due
to toxicity or resistance. In a study of 38 AIDS patients with PcP,
only five patients completed TMP-SMX therapy. Twenty-nine
(76%) of these patients had drug toxicity, and 19 (50%) had to be
switched to other therapies. The adverse effects of TMP-SMX
found in this study included rash (86%), fever (77%), neutropenia
(66%), thrombocytopenia (26%), and transaminase elevation
(31%) . In a study of 962 European AIDS patients with PcP
treated with TMP-SMX, 22% of the patients were switched to
other regimens .
The mechanisms of PcP pathogenesis are largely unknown.
Lung damage during PcP is mainly due to inflammatory responses
mediated by CD8 T-cells . CD4 T-cells have also been
shown to play such role in Pc-related immune reconstitution
inflammatory syndrome . A characteristic feature of PcP is
that alveolar macrophages (AMs) are defective in phagocytosis
. The expression of the antizyme inhibitor is greatly
increased in AMs . Since antizyme inhibitor stabilizes
ornithine decarboxylase, the key enzyme in polyamine synthesis,
and promotes the import of exogenous polyamines, polyamine
levels in AMs are elevated leading to increased rate of apoptosis
and decreased number of AMs [16,17]. Pc infection also causes
reduced production of calmodulin, resulting in a defect in the
dimerization of iNOS and thus decreased production of nitric
oxide by AMs . The expression of the PU.1 gene in AMs is
also decreased . Since PU.1 regulates the expression of many
macrophage receptors , this finding partially explains the
defect in phagocytosis of AMs during PcP. Very recently, we found
that myeloid-derived suppressor cells (MDSCs) accumulate in the
lung during PcP .
MDSCs are a heterogeneous population of bone
marrowderived myeloid progenitor cells and immature myeloid cells and
are immunosuppressive . All-trans retinoic acid (ATRA), one
form of vitamin A-derived retinoids, has been shown to stimulate
MDSCs to differentiate to dendritic cells and macrophages
[26,27], and administration of therapeutic concentrations of
ATRA can substantially decrease the number of MDSCs in
tumor-bearing mice and in patients with cancer, improving their
antigen-specific response of T-cells [28,29]. We hypothesize that
ATRA treatment can push the MDSCs in the lungs to differentiate
to functional AMs which may clear Pc infection. In this study, we
tested the effects of ATRA alone and in combination with an
8aminoquinoline primaquine for treatment of PcP.
Materials and Methods
Rodent Models of PcP
C57BL/6 mice and Sprague Dawley rats were obtained from
Harlan (Indianapolis, IN). All animals used in this study were
female, with body weights of 1820 g in mice and 120140 g in
rats. Animal studies were approved by the Indiana University
Animal Care and Use Committee and carried out under the
supervision of veterinarians. Immunosuppression of mice was
achieved by intraperitoneal injection of 0.3 mg anti-CD4 mAb
(clone GK1.5, Harlan, Indianapolis, IN) once a week until the
mice were sacrificed. Three days after the initial injection, mice
were transtracheally instilled with 26106 of Pc organisms in 50 ml
sterile PBS. Rats were immunosuppressed with 1.8 mg/ml
dexamethasone in drinking water. One week after initiation of
immunosuppression, rats were transtracheally instilled with 26106
of Pc organisms in 200 ml sterile PBS. The Pc organisms used as
inoculum were obtained from heavily infected lungs and isolated
as previously described . Tetracycline (0.74 g/L) was added to
the drinking water to prevent bacterial infections.
Immunosuppressed-uninfected animals were used as controls. For treatment,
TMP-SMX (TMP, 50 mg/kg/day and SMX, 250 mg/kg/day),
ATRA (5 mg/kg/day in 8% DMSO), and primaquine (PMQ)
(2 mg/kg/day in water) were given orally once a day starting from
3 weeks post Pc inoculation for mice and 2 weeks post Pc
inoculation for rats. ATRA at 5 mg/kg/day is equivalent to
15 mg/m2/day . TMP-SMX (Septra) was purchased from
HiTech Pharmacal (Amityville). Both ATRA and PMQ were
obtained from Sigma-Aldridge (St. Louis, MO).
Histopathology Examination and Evaluation of Infection
Animals were anesthetized by intramuscular injection of
ketamine cocktail (ketamine hydrochloride, 80 mg/ml;
acepromazine, 1.76 mg/ml; atropine, 0.38 mg/ml) and then sacrificed by
cardiac exsanguination. The left lung was removed from each
animal, inflated with buffered 10% formalin, fixed overnight, and
then embedded in paraffin. Five-micrometer histologic sections
were stained with H&E for evaluation of lung inflammation and
architecture. Histologic lung sections stained with Grocotts
methenamine silver (GMS) and modified Wright-Giemsa stained
lung impression smears were examined under light microscope to
determine organism burden.
Isolation of Total BAL Cells
Lungs were lavaged with sterile saline (5 ml for rats and 1 ml for
mice at a time) through an intratracheal catheter until a total of
50 ml of lavage fluid from each rat or 10 ml from each mouse was
recovered as described previously . The bronchoalveolar
lavage fluid (BALF) was centrifuged at 3006g for 10 min to pellet
BAL cells. The pelleted cells were washed twice with PBS and then
resuspended in PBS at 16106 cells per ml.
Morphological Analysis of BAL Cells
One hundred microliters of a cell suspension (36104 cells/ml)
was loaded into a cytospin chamber and spun for 5 min at
500 rpm (Cytospin 2, Shandon). Slides were air-dried at room
temperature for 5 min and stained with Giemsa using the
LeukoStat staining kit (Fisher Scientific).
Flow Cytometry Analysis
BAL cells were obtained from uninfected or Pc-infected
animals. After incubating in 5% bovine serum albumin for 1 hr,
the cells were stained with specific fluorescence-labeled antibodies
including anti-mouse CD11b, Gr-1, and CD11c; and anti-rat
CD11bc and His48 (BioLegend) on ice for 1 hr. Separate sets of
cells were stained with phycoerythrin (PE) or FITC-labeled IgG
isotype control antibody. After washing twice with 4 ml PBS, the
stained cells were examined with a BD FACSCalibur flow
cytometer (BD Biosciences), and the flow cytometry data thus
generated were analyzed with the FlowJo software (Tree Star,
Comparisons were made between the mean values of the
treatment and control groups or between two treatment groups by
the unpaired Students t test with a two-tail distribution.
Comparisons between three or more treatment groups were made
by one ANOVA. Survival rates between groups were compared by
Mantel-Cox test and Gehan-Breslow-Wilcoxon test. A p
value,0.05 was considered significant.
ATRA Eliminated MDSC and Cleared Pc Infection in Mice
after 5 Weeks of Treatment
To investigate the potential therapeutic effect of ATRA on PcP,
immunosuppressed C57BL/6 mice were transtracheally
inoculated with Pc and then treated with ATRA at 5 mg/kg/day starting
3 weeks post Pc inoculation. This group of mice was referred to as
the PcP/ATRA group (Fig. 1). A separate group of Pc-infected
mice, referred to as the PcP/DMSO group, were treated with 8%
DMSO, which was the vehicle used to dissolve ATRA, in the same
manner to serve as controls. Three mice each in uninfected, PcP/
DMSO, and PcP/ATRA groups were sacrificed and lavaged every
week for 5 weeks after initiation of ATRA treatment. The number
of MDSCs in the BALF of each mouse was determined by flow
cytometry and by morphological examinations of BAL cells
cytospun on slides. The severity of Pc infection was determined by
examining lung sections stained with H&E for histology and GMS
for Pc organism load.
As shown in Fig. 1A, cells with ring-like nuclei characteristic of
MDSCs were seen in the BALF from untreated PcP mice (PcP/
DMSO group). These cells were not present in the BALF from
uninfected mice and were greatly reduced in numbers in the BALF
from Pc-infected mice after 4 weeks of ATRA treatment. Few such
cells were observed in the BALF from uninfected mice or
Pcinfected mice treated with ATRA for 5 weeks.
Flow cytometry studies of BAL cells revealed the presence of
a population of Gr-1+/CD11b+ MDSCs in the BAL samples from
Pc-infected mice but not in those from uninfected mice. The
percentage of these cells in PcP mice with 3 weeks or less ATRA
treatment (28.1%) was similar to that (23.6%) in PcP mice treated
with DMSO only (Fig. 1B and Fig. 1C). While the percentage of
MDSCs increased by approximately 20% 5 weeks post DMSO
treatment, it decreased by 26% after Pc-infected mice had been
treated with ATRA for 5 weeks.
sample is shown in the right upper quadrant of each flow cytogram. (C)
Percentage of Gr-1+/CD11b+ cells in BALF, as determined by flow
cytometry. Data are means 6 S. D. of 36 mice in each group at each
time point. (D) Histologic sections of the lungs stained with GMS (left)
and H&E (right). Red arrows indicate GMS-stained Pc organisms. Images
are representative of 36 mice in each group at each time point.
Microscope magnification: 40X.
Histology examinations showed that the alveoli of Pc-infected
mice were filled with exudates, Pc organisms, and inflammatory
cells at 4 and 5 weeks post DMSO treatment (Fig. 1D). The lung
inflammation and architecture of PcP mice were improved after 4
weeks of ATRA treatment and became close to normal after 5
weeks of ATRA treatment (Fig. 1D). The organism load as
revealed by GMS staining was greatly reduced after 4 weeks of
ATRA treatment. No GMS stained Pc organisms were seen in the
lung sections from PcP mice that had been treated with ATRA for
5 weeks (Fig. 1D).
Combination of ATRA with PMQ Eliminated MDSCs and
Cleared Pc Infection in 2 Weeks
The results described above showed that ATRA treatment
could cure PcP; however, it required at least 5 weeks of treatment.
Since ATRA itself is not known to have any antimicrobial activity,
we tested whether ATRA in combination with PMQ would
control Pc infection more efficiently than ATRA alone.
Immunosuppressed mice with Pc infection for 3 weeks were treated with
ATRA (5 mg/kg/day) plus PMQ (2 mg/kg/day) or TMP-SMX
(TMP, 50 mg/kg/day and SMX, 250 mg/kg/day) for 2 weeks.
BAL cells cytospun on slides and lung impression smears were
examined morphologically for the presence of MDSCs and Pc
organisms, respectively. Surprisingly, the combination of ATRA
and PMQ combination worked as effectively as TMP-SMX; it
cleared the infection and also eliminated MDSCs in the lungs in
two weeks in all PcP mice (Fig. 2).
To determine whether the combination of ATRA and PMQ
also works in other animals, the same regimen was tested in rats.
Immunosuppressed Sprague Dawley rats with Pc infection for 2
weeks were treated with ATRA, ATRA-PMQ, or TMP-SMX for
4 weeks. A group of Pc-infected rats treated with 8% DMSO was
used as untreated control. Since MDSCs in rats are His48+/
CD11bc+, the numbers of MDSCs in the BALF were determined
by flow cytometry using anti-His48 and anti-CD11bc antibodies.
The severity of Pc infection was determined by examining lung
sections stained with H&E for histology and GMS for Pc organism
load. As shown in Fig. 3, a significant number of His48+/CD11bc+
MDSCs were present in the BAL fluid from untreated PcP rats.
MDSCs were greatly reduced in number (10.7%) after 4 weeks of
ATRA treatment, and were completely disappeared after 4 weeks
treatment with ATRA-PMQ or TMP-SMX. Histological
examination of lung sections revealed that lung inflammation and
architecture of Pc-infected rats were greatly improved after 4
weeks of ATRA treatment and were close to normal after 4 weeks
treatment with ATRA-PMQ or TMP-SMX, similar to those
observed in mice (data not shown).
ATRA and ATRA-PMQ Treatments Increased the Number
As described above, the number of AMs is decreased during
PcP. To investigate whether these drug treatments would increase
the number of AMs, Pneumocystis-infected mice (36 mice per
group) were treated with DMSO, ATRA, ATRA-PMQ, or
TMPSMX for 5 weeks as described above. BAL cells of untreated and
Figure 2. Effects of ATRA-PMQ combination on MDSCs and Pc infection in mice. Immunosuppressed, Pc-infected mice were given vehicle
control 8% DMSO, ATRA-PMQ, or TMP-SMX for 2 weeks. Treatment was initiated 3 weeks after Pc inoculation. (A) Lung impression smear stained with
Giemsa. Red arrows indicate nuclei of Pc organisms. Magnification: 100X. (B) Giemsa-stained BAL cells cytospun on slides. Black arrows indicate
MDSC-like cells. Microscope magnification: 40X. Images are representative of 36 mice of each group.
treated PcP mice were collected and analyzed by flow cytometry
with fluorescence-labeled anti-mouse CD11c antibody, as CD11c
has been shown to be a marker of AMs [12,33]. As seen in Fig. 4A
and 4B, the number of CD11c+ cells was greatly increased after
treatment of PcP mice with ATRA, ATRA-PMQ, or TMP-SMX.
This result suggests that ATRA or ATRA-PMQ treatment
converts MDSCs in the lungs of PcP mice to AMs.
ATRA-PMQ Treatment Completely Eradicated Pc
Organisms in the Lung
To determine whether ATRA-PMQ treatment completely
eradicates Pc organisms from the lung, PcP mice were treated
with the combination for 2 weeks and then examined for signs of
relapse 3 weeks after cessation of the treatment. PcP mice treated
with TMP-SMX were used as controls. Examination of BAL cells
from these animals showed that MDSCs that were seen in infected
mice were not present in the BALF from PcP mice after
ATRAPMQ or TMP-SMX treatment. Similarly, Pc organisms were seen
in lung impression smears of untreated PcP mice, but not in those
of ATRA-PMQ or TMP-SMX treated mice. Since microscopic
examination of Pc organisms may not have sufficient sensitivity,
lung tissues of untreated or ATRA-PMQ or TMP-SMX treated
PcP mice were subjected to mitochondrial rRNA PCR [34,35],
and a 340-bp PCR product was detected from the lungs of
untreated PcP mice but not from those of treated at the end of the
3-week treatment or at 3 weeks after cessation of the treatment
Prolonged Survival in Animals with PcP Treated with
To investigate whether the ATRA-PMQ combination therapy
prolongs the survival of animals with PcP, Pc-infected rats (10 in
each treatment group) were treated with ATRA-PMQ or
TMPSMX daily, starting from 2 weeks post Pc inoculation and
continuing through the entire period of the study. A separate
group of Pc-infected rats treated with DMSO only was used as
untreated control. The experiment was terminated when all
animals in the control group were moribund and sacrificed. As
shown in Fig. 6, PcP rats treated with ATRA-PMQ or TMP-SMX
had significantly prolonged survival, compared with those of
untreated group (p,0.05). At days 40, 58, 60, and 61, the
numbers of untreated PcP rats that survived were 8, 6, 4, and 3,
respectively. At day 62, all the remaining untreated PcP rats had to
be sacrificed, whereas all 10 rats in each of the ATRA-PMQ or
TMP-SMX treatment group survived for the entire 70-day period
of the study.
Figure 3. Effects of ATRA, ATRA-PMQ, and TMP-SMX on MDSCs in rats. Immunosuppressed, Pc-infected rats were given vehicle control 8%
DMSO, ATRA, ATRA-PMQ, or TMP-SMX for 4 weeks. Treatment was initiated 2 weeks after Pc inoculation. BAL cells were examined by flow cytometry.
The percentage of His48+/CD11bc+ MDSCs in each sample is shown in the right upper quadrant of each flow cytogram.
Figure 4. Increased number of CD11c+ BAL cells after ATRA, ATRA-PMQ, or TMP-SMX treatment. Immunosuppressed, Pc-infected mice
were given vehicle control 8% DMSO, ATRA, ATRA-PMQ, or TMP-SMX for 5 weeks. Treatment was initiated 3 weeks after Pc inoculation. BAL cells were
collected and analyzed by flow cytometry with fluorescence labeled anti-mouse CD11c antibody. (A) Representative flow cytograms of BAL cells from
animals of each group. (B) Percentage of CD11c+ BAL cells in BALF, as determined by flow cytometry. Data are means 6 S. D. of 36 mice in each
Most PcP drug developments either target Pc metabolic
pathways or cell wall synthesis. Because Pc cannot be continuously
cultured, our understanding of Pc proliferation and metabolism is
inadequate. Therefore, very little success in the development of
new therapies for PcP has been made. The combination of
clindamycin and PMQ as a regimen for PcP was developed in
1988 ; no new successful treatments have been developed
since then, and current PcP treatment and prophylaxis rely on
drugs developed decades ago.
In this study, we employed a novel approach to treat PcP by
converting immuno-suppressive cells to immuno-protective ones
so that the hosts became able to effectively defend the infection.
This approach is based on our recent finding that Pc infections
result in the accumulation of MDSCs in the lungs . The
observation that these cells suppressed the proliferation of T-cells
 strongly suggest that they are not neutrophils as neutrophils
do not have this function . We found that treatment of PcP
mice with ATRA for five weeks cured the disease as evidenced by
the disappearance of Pc organisms (Fig. 1D) and MDSCs (Fig. 1A
C) in the lungs and greatly reduced lung inflammation (Fig. 1D).
This finding further suggest that the MDSCs we observed are not
neutrophils. If they were neutrophils, ATRA treatment would not
eliminate them as they are terminally differentiated. Since this
treatment was effective in both mice and rats with PcP (Fig. 1 and
3), it is very likely that it will also work for humans although the
existence of MDSCs in the lungs of patients with PcP remains to
Although ATRA treatment of Pc-infected mice or rats resulted
in the clearance of the infection, it took five weeks. In contrast, the
conventional TMP-SMX treatment controlled Pc infection in less
than 3 weeks. In order for ATRA to become useful for treatment
of PcP, a better efficacy is needed. We hypothesized that ATRA in
combination with a certain antibiotic may be more effective and
therefore tested PMQ for its potential synergistic effect with
ATRA. Surprisingly, the combination of ATRA and PMQ was as
effective as TMP-SMX for therapy of PcP and cleared the
infection in two weeks, as compared to five weeks of treatment
with ATRA alone.
Since PMQ by itself has no significant effect on PcP , the
major therapeutic activity of this regimen is likely due to ATRA
which presumably pushes MDSCs to further differentiate to
alveolar macrophages that clear the infection. This postulation is
supported by the result showing that the number of CD11c+ cells
in the BALF from ATRA or ATRA-PMQ treatment of PcP mice
was significantly increased (Fig. 4) and that of MDSCs was greatly
decreased (Fig. 2). MDSCs are a heterogeneous population of
bone marrow-derived myeloid progenitor cells and immature
myeloid cells. In health, these cells quickly differentiate into
granulocytes, macrophages, or dendritic cells. MDSC
differentiation is blocked in certain pathological conditions, such as cancer,
various infectious diseases, sepsis, trauma, bone marrow
transplantation, and some autoimmune diseases . ATRA has been
shown to unblock this differentiation allowing MDSCs to be
converted to macrophages and dendritic cells . The role of
PMQ in this combination therapy may be to prevent the
proliferation of Pc, thus enabling alveolar macrophages to
eradicate them as PMQ can interfere with the microbial electron
transport system via the generation of quinone metabolites and
superoxides . Since alveolar macrophages are known to be
defective in phagocytosis during PcP , it is possible that the
newly formed alveolar macrophages due to ATRA treatment are
fully functional or that ATRA treatment also activates the
phagocytic activity of preexisting alveolar macrophages. These
possibilities remain to be investigated.
Although the combination of ATRA and PMQ can be an
effective regimen for therapy of PCP, it is by no means perfect as
both ATRA and PMQ have adverse effects. In a phase I trial of
ATRA on patients with various cancers including lung, head and
neck, mesothelioma, colon, breast, melanoma, approximately 25%
patients developed cheilitis, skin reactions, headache, nausea,
vomiting, or transient elevation of transaminase and triglyceride
levels . ATRA is being used to treat acute promyelocytic
leukemia (APL). The major adverse effect of ATRA in APL
patients is retinoic acid syndrome, which is characterized by fever,
weight gain, elevated white blood cells, respiratory distress,
interstitial pulmonary infiltrates, pleural and pericardial effusion,
dyspnea, episodic hypotension, or acute renal failure. However,
these adverse effects can be effectively suppressed by steroids .
The recommended ATRA dose for APL is 45 mg/m2/day which
is much greater than the dose (15 mg/m2/day) we used to treat
PcP in mice and rats. In this study, only one dose each of ATRA
(5 mg/kg/day) and PMQ (2 mg/kg/day) was used. Various doses
of ATRA and PMQ are being tested to determine their optimal
therapeutic concentrations, especially when they are used in
Although ATRA has significant adverse effects at the doses used
as an anticancer drug, the proof of the concept that ATRA can be
used to treat PcP will provide a rationale to test other compounds
such as liposomal ATRA which is less likely to cause retinoic acid
syndrome . Another approach is to use compounds such as
vitamin D which also has the ability to promote the differentiation
of MDSCs with much less toxicity than ATRA . There have
also been attempts to develop ATRA analogues that are less toxic
with better or the same efficacy as ATRA. One such example is
the development of Am80 which has been shown to be effective in
APL patients relapsed from ATRA-induced remission and in the
induction of neuronal differentiation . The main adverse effect
of PMQ is hemolytic anemia in patients deficient of glucose-6
phosphate dehydrogenase or glutathione synthase . Another
adverse effect of PMQ is methemoglobinemia due to
autooxidation of the hemoglobin iron core. Similar to ATRA, the
proof of the concept will provide a rationale to test other PMQ
analogues such as aablaquine and tafenoquine that are in the final
stages of clinical trials against Plasmodium vivax and P. falciparum
. An advantage of using PMQ for therapy is that it rarely
induces resistance .
Using ATRA to convert MDSCs to alveolar macrophages is one
form of immune modulation. Although PcP is a disease of immune
dysfunction, treatment of PcP by immune modulation has not
been widely investigated. Bhagwat et al.  showed that PcP
mice treated with anti-CD3 antibody exhibit a rapid and dramatic
reduction in inflammatory lung injury. Wang et al. treated PcP
mice with the anti-inflammatory drug sulfasalazine and found that
it reduced pulmonary inflammation and enhanced CD4+ T
celldependent alveolar macrophage phagocytosis . These
approaches differ from ours in that they target lung inflammation,
whereas our approach converts MDSCs to alveolar macrophages
to clear the organisms, thus eliminating the original cause of lung
In addition to providing an alternative treatment for PcP, our
study can serve as a model for development of new therapies for
other diseases. MDSC accumulation has been reported in many
other chronic infectious disease conditions, such as toxoplasmosis
, leishmaniasis , candidiasis , and helminthiasis
[47,48]. Eliminating MDSCs in these diseases may similarly
improve host defense and help control the infections. It is
conceivable that improving host defense mechanisms alone may
not be completely effective. However, the combination of both
immune enhancement and antimicrobial approaches may prove to
be ideal. In addition, the enhancement of host defense
mechanisms may also reduce the dose of antimicrobials required and thus
minimize their adverse effects. It is also possible that in
combination with ATRA, other drugs such as dapsone and
atovaquone that are less active but less toxic than TMP-SMX will
become ideal for therapy of PcP. This will be an enormous
advancement in PCP therapy, as different patients may require
a different therapy due to genetic variations or adverse reactions.
The use of ATRA and PMQ combination as an alternative
treatment for PCP will eliminate the potential risk of
hypersensitivity to the SMX component of TMP-SMX. This will be
a significant contribution to the treatment of PCP as
approximately 10% people of a general population are allergic to sulfa
In summary, we have developed a novel therapy for PcP using
animal models. This method challenges all the current PcP
therapy paradigms. This is also the first ever regimen that may be
used as an alternative to TMP-SMX which is currently the most
effective drug for PcP. This new approach presumably enables the
hosts to defend against the infection by making MDSCs
differentiate to alveolar macrophages. It is very likely that further
development of this regimen will make it useful for treatment of
PcP in humans. Proving the feasibility of this approach will
transform antimicrobial therapy and encourage more studies on
MDSCs and development of therapies targeting MDSCs that are
also a major component of the pathogenesis of illnesses such as
cancer, autoimmune diseases, and other microbial diseases.
Conceived and designed the experiments: GSL CZ CHL. Performed the
experiments: GSL SS HWJ PJD. Analyzed the data: GSL CZ. Contributed
reagents/materials/analysis tools: CHL. Wrote the paper: GSL CZ CHL.
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