INTRODUCTION
Pneumocystis jirovecii is the causative organism of
Pneumocystis pneumonia (PCP) in humans. The mortality rate of PCP is 5 to 40% in patients with treatment and close to 100% in those without treatment (
1).
Pneumocystis organisms also infect other mammalian species.
Pneumocystis infection is very species specific. The organism that infects mice is
P. murina.
Currently, the most effective drug for treatment of PCP is the combination of trimethoprim and sulfamethoxazole (TMP-SMX). Unfortunately, TMP-SMX has adverse effects such as rash, fever, neutropenia, thrombocytopenia, or transaminase elevation in some people (
2), and many AIDS patients with PCP do not respond to therapy with TMP-SMX (
3). We have previously found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs of mice and rats with PCP (
4). Treatment of
P. murina-infected mice and rats with all-
trans-retinoic acid (ATRA) was found to clear the infection in 5 weeks, with the disappearance of MDSCs and increased number of alveolar macrophages (AMs) in the lungs (
4). These findings suggest that ATRA treatment stimulates MDSCs to differentiate to AMs, allowing the host to effectively defend the infection. These results also suggest that ATRA can be used as a supplemental therapy for PCP. To test this hypothesis, we treated mice having PCP with ATRA in combination with 2 mg/kg/day of primaquine (PMQ) and found that this ATRA-PMQ combination was as effective as TMP-SMX for PCP therapy and cleared the infection in 2 weeks (
5).
Since ATRA also has significant adverse effects (
6), we sought alternatives to replace it and found that vitamin D
3 (VitD3) (300 IU/kg/day) had a synergistic effect with a higher dose of PMQ (5 mg/kg/day) for therapy of PCP (
7). This VitD3-PMQ combination was found to clear
P. murina infection, reduce the severity of inflammation, and increase the CD11b
low CD11c
high alveolar macrophage population in the lungs of mice with PCP within 3 weeks. However, those studies were performed on mice with mild PCP, i.e., mice infected with
P. murina for 4 weeks. In this study, we investigated the efficacy of VitD3-PMQ for treatment of mice with severe PCP. The mechanisms of action of VitD3 as supplemental therapy for PCP were also investigated. We hypothesized that VitD3 treatment reduces lung inflammation and enhances host innate immunity by affecting the expression of some of its target genes.
DISCUSSION
A major symptom of PCP is severe inflammation in the lung. Our observations that
P. murina infection greatly increased the levels of proinflammatory cytokines (TNF-α, IFN-γ, and IL-6) and reduced the levels of antimicrobial peptides (cathelicidin) and proteins (GR and GCLM) related to antioxidation and autophagy (ATG5 and BCLN1) implicate these actions as mechanisms of pathogenesis of PCP. Therefore, clearing
P. murina infection is critical in PCP therapy. We had previously found that PMQ alone was not completely effective in the clearance of
P. murina infection, but the combination of VitD3 and PMQ (VitD3-PMQ) was as effective as TMP-SMX (
7). In this study, we investigated the mechanisms by which VitD3 enhances the efficacy of PMQ in PCP therapy. Major efforts were devoted to comparing the effects of PMQ alone and the VitD3-PMQ combination in PCP therapy. The results showed that treatment of mice having PCP with VitD3-PMQ increased the number of alveolar macrophages, enhanced body weight recovery, and reduced
P. murina organism load, levels of proinflammatory cytokines, and severity of inflammation in the lungs. VitD3-PMQ also decreased the expression of iNOS and increased the expression of GR, GCLM, CAMP, TLR4, TLR2, NF-κB, ATG5, and BCLN1 in lung cells of mice with PCP. Since these changes were not significantly observed in mice with PCP treated with PMQ alone, they were attributed to the action of vitamin D.
Vitamin D has been shown to reduce the expression of TNF-α by decreasing NF-κB-p65 mRNA expression and increasing IκB-α mRNA expression (
9). In a recent study of 118 women, those who were deficient in vitamin D had higher serum levels of TNF-α (
10). These observations indicate that vitamin D negatively regulates the expression of TNF-α, as we have observed in this study.
We also found that the IFN-γ level was very low in BAL fluids from uninfected mice but was very high in those from
P. murina-infected mice (
Fig. 3B). This increase in IFN-γ levels is likely a host defense mechanism against
P. murina infection, as treatment of mice having PCP with TMP-SMX, PMQ, or VitD3-PMQ reduced their IFN-γ levels (
Fig. 3B). Although IFN-γ can enhance the ability of alveolar macrophages to kill
P. murina (
11), excessive IFN-γ is harmful, as it is proinflammatory. Similar to what we observed in this study, vitamin D was found to significantly attenuate lipopolysaccharide (LPS)-induced elevation of IFN-γ in mice (
12). Furthermore, treatment with vitamin D resulted in significant downregulation of IFN-γ in patients with recent-onset type 1 diabetes (
13). Among the 3 regimens (TMP-SMX, PMQ, and VitD3-PMQ) tested, VitD3-PMQ was most effective in reducing IFN-γ levels (
Fig. 3B).
IL-6 is another proinflammatory cytokine. Serum IL-6 levels were found to be higher in AIDS patients who died of PCP than in those who survived PCP (
14). Non-AIDS patients with PCP also had significantly higher levels of IL-6 in their BAL fluids than non-PCP patients (
15,
16). Vitamin D has been shown to decrease IL-6 production by human renal proximal tubular epithelial cells (
17) and LPS-stimulated human periodontal ligament cells (
18). These observations are consistent with our finding that treatment of mice having PCP with VitD3-PMQ greatly reduced the levels of IL-6 in their BAL fluids (
Fig. 3C).
Another difference between PMQ alone and VitD3-PMQ in the treatment of PCP is the effect on the expression of iNOS.
P. murina infection greatly increased iNOS mRNA levels in AMs (
Fig. 6A). Shellito et al. found that both iNOS mRNA and protein levels are increased during PCP but that elevated iNOS levels do not result in clearance of
P. murina infections (
19). Based on this observation, they postulated that both iNOS and nitric oxide play no role in defense against
P. murina infection. We had found that
P. murina is susceptible to killing by nitric oxide. The reason why elevated iNOS levels do not correlate with
P. murina clearance is that iNOS dimerization, which is required for production of a functional iNOS, is defective during PCP because of decreased levels of calmodulin in AMs (
20). As a result, the production of nitric oxide is diminished. In this study, we found that treatment of mice having PCP with PMQ alone reduced iNOS mRNA levels in lung tissue and that VitD3-PMQ treatment caused a further reduction in iNOS mRNA levels (
Fig. 6A). This is likely due to
P. murina clearance by these treatments, thus eliminating the stimulation in iNOS expression.
Glutathione reductase (GR) converts glutathione disulfide (GSSG) to glutathione (GSH) (
21), which scavenges hydroxyl radicals, reactive oxygen species (ROS), and various electrophiles, thus preventing oxidative stress in cells (
22). GSH deficiency is implicated in many diseases, such as cardiovascular diseases, immune disorders, diseases related to aging, and diabetes (
22–28). AIDS patients with PCP were found to have elevated levels of GSSG in BAL fluids (
29), suggesting that patients with PCP suffer from ROS toxicity in their lungs. Our finding that VitD3-PMQ treatment resulted in greatly increased expression of GR is an indication that VitD3 promotes the conversion of GSSR to GSH, which in turn reduces the oxidative stress caused by
P. murina infection. As PMQ alone did not affect GR expression (
Fig. 6B), the GR upregulation seen in mice with PCP treated with VitD3-PMQ is very likely due to the action of VitD3. This result agrees with that of a previous study showing that vitamin D upregulates the expression of GR and decreases ROS levels in U937 cells (
30).
The glutamate-cysteine ligase (GCL) also plays a key role in the production of GSH. It consists of two subunits, a catalytic subunit and a modifier subunit. The GCLM gene encodes the modifier subunit of GCL. GCL condenses
l-glutamate and
l-cysteine to become gamma-glutamyl cysteine, which is the precursor of glutathione. The effect of
P. murina infection on the expression of GCL or the production of gamma-glutamyl cysteine has never been reported. In this study, we discovered that
P. murina infection dramatically decreased the levels of GCLM. Treatment of PCP with TMP-SMX slightly increased its levels. PMQ had no effect, but VitD3-PMQ treatment returned GCLM levels close to those in uninfected mice (
Fig. 6C).
A major function of vitamin D is inducing the production of antimicrobial peptides such as β-defensin and cathelicidin (
31,
32). Patients with tuberculosis (TB) infection who are deficient in vitamin D produce very low levels of cathelicidin LL37 (
33), and
Camp gene knockout is found to block the antimicrobial effect of vitamin D (
34). In this study, we found that
P. murina infection greatly suppressed the expression of cathelicidin and that treatment with TMP-SMX or VitD3-PMQ restored its expression (
Fig. 6D).
It has been shown that impaired recognition through TLR4 is responsible for exacerbated PCP (
35). We had previously shown that mouse AMs respond to
P. murina organisms through TLR2, leading to nuclear translocation of NF-κB and production of proinflammatory cytokines TNF-α and MIP-2 (
36). In this study, we found that VitD3-PMQ, but not TMP-SMX or PMQ, significantly increased the expression of both TLR2 and TLR4. Reports on the effect of vitamin D on the expression of TLR4 have been variable. Some studies showed that vitamin D suppresses the expression of TLR4 and thus alleviates inflammation (
37–41), but others demonstrated that vitamin D has no effect on the expression of TLR4 (
42,
43) as it is normally constitutively expressed (
44).
In this study, we found for the first time that
P. murina infection caused a dramatic decrease in the expression of ATG5 and BCLN1 (
Fig. 7). These two proteins play a major role in autophagy, which is a critical process in cell metabolism. Autophagy is mediated by a membrane trafficking pathway in which autophagosomes engulf cytoplasmic materials to form vesicles that are then transported to the lysosome for degradation (
45). ATG5 is involved in the early stages of autophagosome formation (
46), and BCLN1 functions as a scaffolding protein and interacts with the PI3KC3/VPS34 lipid kinase. This kinase complex produces phosphatidylinositol-3-phosphate (PI3P), which is a critical regulator for autophagy induction and membrane trafficking (
47).
PMQ almost completely suppressed the expression of both ATG5 and BCLN1. VitD3-PMQ treatment restored their expression in mice with PCP to a level very close to that in uninfected mice. TMP-SMX treatment had no significant effect on restoring the expression of these two proteins (
Fig. 7). These results suggest that restoration of the expression of these two proteins in mice with PCP treated with VitD3-PMQ was due not to the clearance of
P. murina organisms but to the action of vitamin D. This hypothesis is supported by previous studies showing that vitamin D upregulates the expression of both ATG5 and BCN1 in monocytes and macrophages from healthy individuals or patients with tuberculosis (
48). This action was found to be mediated by cathelicidin (
49,
50). Vitamin D has also been shown to induce autophagy in human macrophages through a phosphatidylinositol-3-kinase-, ATG5-, and beclin-1-dependent mechanism and significantly inhibits HIV-1 replication in a dose-dependent manner (
51,
52).
Based on the results of this study, we postulate that the action of vitamin D is centered on enhancing self-protection of the host. Increased production of cathelicidin and recovery of AMs would lead to a decreased P. murina organism load. Cathelicidin also restores the production of ATG5 and BCLN1, thus activating autophagy to recycle cytoplasmic materials. Decreased expression of iNOS would lead to reduced production of nitric oxide, and the recovery of the expression of GCLM and GR would decrease the levels of reactive oxygen species (ROS); these two actions would reduce cell damage. Although increased expression of TLR2 and TLR4 may trigger inflammation, the diminished production of proinflammatory cytokines would alleviate lung damage. We believe that the combination of these functions makes vitamin D an effective supplementary therapy for PCP.
It should be noted that mice used in this study had very severe PCP (they were infected by
P. murina for 8 weeks and had body weights less than 20 g). Most regimens, including TMP-SMX, often fail, but VitD3-PMQ effectively cured them. It has been predicted that vitamin D can affect the expression of more than 200 genes (
53). Whether genes other than those investigated in this study also contribute to host protection during PCP remains to be investigated. Although we have clearly demonstrated anti-infective and anti-inflammatory functions of vitamin D in mice, it is not known whether similar effects would occur in humans with PCP. It has been reported that 65% (63 of 97) of HIV-infected patients are deficient in vitamin D and that vitamin D supplementation for 24 weeks significantly increased the number of CD4
+ cells in these patients (
54). This observation suggests that vitamin D supplementation would help resolve PCP in humans. To translate the findings of this study to clinical applications, an epidemiological study to determine whether there is a correlation between serum vitamin D levels and the severity of PCP in humans should be conducted. The dose of vitamin D3 required for adjunctive therapy of PCP also needs to be determined. We used 300 IU/kg/day for mice, but it is unknown whether this dose is appropriate for humans. Although the major action of vitamin D in PCP supplemental therapy is anti-inflammation, it is unknown whether vitamin D can replace a corticosteroid, which is usually administered to reduce lung inflammation. PMQ in combination with clindamycin has been used to treat patients with mild to moderately severe PCP (
55). As clindamycin caused severe gastrointestinal problems in mice when it was administered orally, we were unable to compare the effectiveness of VitD3-PMQ to that of clindamycin-PMQ in PCP therapy. Whether VitD3-PMQ is superior to clindamycin-PMQ for treatment of PCP patients is also unknown. All of these questions must be answered before the VitD3-PMQ combination can be tried in patients with PCP.