The Mechanism of Talaromyces Marneffei Influencing Macrophage RAW264.7 Polarization and Sterilization Ability via Arginine Metabolism Pathway


 Background

The opportunistic fungi T. marneffei is an intracellular pathogen that causes systemic and lethal infection through the mononuclear phagocyte system. Macrophages act as pacesetters to resist pathogen invasion and communicators to activate adaptive immunity. During infection, macrophages induce iNOS to generate NO, which kills invading pathogenic microorganisms. Arginase competes with the iNOS for a common substrate L-arginine. We tried to investigate the effect of arginine metabolism on the escape of T. marneffei from macrophage killing.
Results

We performed qPCR, immunobolt detection, immunofluorescent staining, arginase activity assay and NO production assay when macrophages were co-cultured with T. marneffei. We conducted immunobolt detection, arginase activity assay, NO production assay, phagocytic activity assay and T. marneffei killing assay after adding the arginase specific inhibitor nor-NOHA to the co-culture system. T. marneffei decreased the NO production in macrophages by increasing macrophage arginase activity. With lastingness of infection, T. marneffei induced macrophages towards M2 phenotype polarization, thereby reducing antimicrobial activity and promoting fungal survival. Moreover, inhibiting the T. marneffei-induced macrophage arginase activity with nor-NOHA restored NO synthesis and strengthened the function of fungal killing.
Conclusions

 T. marneffei potentiated its own survival via regulating arginine metabolism pathway in macrophages during an infection.


Background
Penicilliosis marneffei is a fungal disease caused by Talaromyces marneffei (T. marneffei) which is regarded as a temperature-dependent dimorphic opportunity pathogenic fungus. Penicilliosis marneffei was rare in the past. Since the increasing number of population with HIV, and the wide use of glucocorticoid and immunosuppressor, T. marneffei infection has been the most common systemic fungal infection in Southeast Asia and southern China [1]. Up to now, the pathway of T. marneffei infection has been controversial. Hamilton et al thought T. marneffei infection was presumed to originate in the lung after inhalation of the airborne conidia into alveoli, which were engulfed by macrophages after combining with laminin [2]. Via the phagocyte system, T. marneffei causes the fatal disseminated infection in immunocompromised/healthy individuals. However, the mechanism that T. marneffei successfully replicates and escapes from the immune system has not been completely clear. The treatment of T. marneffei infection is still in the exploratory stage. We fail to nd a better way to ght the infection except anti-fungal drugs.
Macrophages participate in the innate and adaptive immunity, and play an essential role in internal environmental homeostasis. Under different pathophysiological conditions, they present distinct functional polarization phenotypes [3]. Classically activated M1 macrophages, induced by IFN-γ or LPS, express the pro-in ammatory mediators (TNF-α, IL-1, IL-6, NO and ROS), which are the key effectors against the microbicidal and tumoricidal activity. In contrast, the alternatively activated M2 macrophages, induced by IL-4 or IL-13, express the anti-in ammatory mediators which mediate the Th2 response and tissue repair.
The focus of macrophage polarization is the arginine metabolism pathway [4]. NOS hydrolyzes the arginine as NO and L-ornithine. As a key component of innate immune response in macrophages, NO possesses a strong sterilization ability against pathogens, especially the intracellular pathogens like mycobacterium tuberculosis, leishmania and salmonella. Arginase is a competitive inhibitor of NOS, which hydrolyzes the arginine into urea and L-ornithine, limiting the excessive production of NO. Many pathogens affect the macrophage polarization by interfering with the arginine metabolism pathway as their survival strategy. They increase the arginase activity in themselves or affect the host arginase activity, thereby consuming the substrate arginine, competitively inhibiting the production of NO, inducing macrophages towards M2 phenotype, and mediating the persistence of infection. When the speci c Arg inhibitor N ω -hydroxy-nor-arginine (nor-NOHA) was used, the synthesis of NO in macrophages was restored and the antibiotic activity was enhanced [5][6][7]. Therefore, the arginine metabolism pathway is a key regulator of innate immune response and targeting it may better control the infection.
Although the mechanism in which T. marneffei evades the host immunity remains unknown, it might be a new method to treat T. marneffei infection by intervening the arginine metabolism pathway of macrophages.

T. marneffei utilized the arginine metabolism pathway of macrophages
We hypothesized that T. marneffei promoted its survival in macrophages by utilizing the arginine metabolism pathway. Therefore, we analyzed the Arg1, iNOS mRNA and protein expressions in Mφ and LPS-induced macrophages co-cultured with T. marneffei (MOI = 10) for 12 h, 24 h and 72 h. T. marneffei signi cantly increased the iNOS mRNA expression in the co-culture system at 24 h, 48 h and 72 h. With the extension of co-culture time, the iNOS mRNA level increased initially and then decreased (Fig. 1A, C). However, the Arg1 mRNA expression in the co-culture system increased only at 72 h (Fig. 1B, D).
Interestingly, T. marneffei signi cantly increased the iNOS protein expression similarly at 24 h, 48 h and 72 h ( Fig. 2A-C). But T. marneffei decreased the Arg1 protein level in Mφ + TM group at 72 h, and in Mφ + LPS + TM group at 48 h and 72 h (Fig. 2D-F).
Simultaneously, we examined the arginase activity of macrophages and the concentration of NO in the supernatant. T. marneffei markedly increased the arginase activity at 24 h, 48 h and 72 h. The longer the co-culture period was, the higher the arginase activity was in the Mφ + LPS + TM group (Fig. 3A, C). The NO synthesis decreased at 24 h, 48 h and 72 h, but there was no difference among the three time points (Fig. 3B, D).

T. marneffei affected the macrophage polarization
To explore the effect of T. marneffei on macrophage polarization, we analyzed the mRNA expressions of TNF-α, IL-1β,CD301 and IL-10 in Mφ or LPS-activated macrophages co-cultured with T. marneffei (MOI = 10) for 12 h, 24 h and 72 h. T. marneffei signi cantly increased the mRNA expressions of TNF-α and IL-1β in co-culture system at 24 h, 48 h and 72 h. With the extension of co-culture time, the TNF-α and IL-1β mRNA expressions in the Mφ + TM group gradually increased (Fig. 4A, B). Moreover, the TNF-α mRNA expression in the Mφ + LPS + TM group showed a trend of fall-rise (Fig. 4E), while the IL-1β mRNA expression increased and then decreased (Fig. 4F). In addition, T. marneffei signi cantly increased the IL-10 mRNA level in the Mφ + TM group at 24 h, 48 h and 72 h, while there was no difference among 3 time points (Fig. 4C). T. marneffei also elevated the IL-10 mRNA level in the Mφ + LPS + TM group at 24 h and 48 h (Fig. 4G). The CD301 mRNA expression in co-culture system increased at 48 and 72 h. With the extension of co-culture time, the CD301 mRNA level in the Mφ + LPS + TM group gradually increased (Fig. 4D, H).
Simultaneously, we also analyzed the CD86, IL-4R and CD206 protein expressions. CD86 is a marker of M1 polarization. IL-4R and CD206 are markers of M2 polarization. The results showed that T. marneffei reduced the CD86 protein expression at 72 h and increased the IL-4R protein expression at 72 h ( Fig. 5A-D). Moreover, the uorescence intensity of CD206 in macrophages co-cultured with T. marneffei (MOI = 10) for 72 h was greater than the Mφ/Mφ + LPS group (Fig. 5E).
Effect of nor-NOHA on the arginine metabolism pathway in co-culture system To explore the effect of nor-NOHA on macrophages against T. marneffei infection, we studied the effect of nor-NOHA on the arginine metabolism pathway in co-culture system. The Arg1 and iNOS protein expressions of macrophages, which were pretreated with 20 µM nor-NOHA and then co-cultured with T. marneffei for 24 h (MOI = 10), were detected by immunoblotting. The results indicated that nor-NOHA signi cantly decreased the Arg1 protein expression and increased the iNOS protein expression (Fig. 6A, B). Furthermore, we analyzed the arginase activity of macrophages and NO production in the co-culture supernatant. 20 µM nor-NOHA markedly inhibited the arginase activity in the co-culture system (Fig. 6C), and rescued the NO production up to nearly 50% (Fig. 6D).
Nor-NOHA promoted the antimicrobial function of macrophages Next, we determined the impact of nor-NOHA on the antimicrobial function of macrophages. Inhibition of arginase activity with 20μM nor-NOHA signi cantly increased the intake of T. marneffei which was killed by LPS-activated macrophages ( Fig.7A-C). The colony count was obviously higher in the co-cultured system than that in the T. marneffei group (Fig.7B, D). Besides, the colony count was lower in the cocultured system with 20μM nor-NOHA than that in the T. marneffei group (Fig.7B, E).

Discussion
The objective of our study was to dissect the arginine metabolism pathway on regulating the immune function of macrophages against T. marneffei infection. According to the results, we found that T. marneffei limited the NO production in macrophages by enhancing the activity of arginase, thereby attenuating the antimicrobial function of macrophages. The mRNA expressions of TNF-α, IL-1β, IL-10 and CD301 in the co-culture system were higher than the control group. With the extension of co-culture time, the protein expression of CD86 in the co-culture system was lower than the control group while the CD206 and IL-4R protein expressions were higher. It indicated that T. marneffei caused the acute in ammation and concurrently mediated the anti-in ammatory response after being engulfed by macrophages. It was a dynamic process. As the immune response progressed, the in ammatory cytokines increased while the anti-in ammatory factors decreased. CD86 is a member of immunoglobulin superfamily, and its costimulatory signal pathway with CD80 promotes the proliferation of T lymphocytes, which play a vital role in clearing the pathogens and maintaining the balance of immune system [8]. Pathogenic microorganism inhibits the T lymphocyte function by down-regulating the expression of CD86, thus disrupting the immunoreaction, making the host unable to eliminate the pathogens effectively, and leading to the persistent infection. CD206 is known as the mannose receptor which mediates the internalization of viruses, bacteria and fungi, facilitating antigen uptake and processing in the adaptive immune response, as well as mediating direct uptake of pathogens in the innate immune response [9]. The activation of CD206 could affect the maturation of phagosomes and provide the favorable condition for the survival of pathogens in macrophages by restricting the fusion of phagosomes and lysosomes. [10,11]. IL-4R is a speci c receptor of interleukin-4. After binding to IL-4R, IL-4 exerts its biological function via the Ras pathway, PI3K pathway, IRS1/2 pathway and Jak-STAT pathway. A study showed that viruses caused the transfer of CD8 + T cells by encoding various types of cytokines. CD8 + T cells produced IL-4/IL-3 which suppressed the anti-viral immune response of Th1 cells [12]. According to the results, we inferred that when T. marneffei was engulfed by macrophages, macrophages transformed to M2 phenotype polarization by increasing the activity of arginase, so as to protect T. marneffei from immune killing. The severity of T. marneffei infection depended on its virulence and the host immune state. T. marneffei not only used the arginine metabolism pathway to escape the oxidative killing, but also affected the macrophage polarization balance of M1/M2. The polarization imbalance provided the favorable condition for its sustainable survival in host. This nding might provide a new theory for understanding the mechanism of T.marneffei resisting the host immune response.
In addition, we noticed that there was a difference between the Arg1 mRNA expression and the Arg1 protein expression when macrophages were co-cultivated with T.marneffei. The co-culture period of our study was 24-72 h while the co-culture period of other researches was generally less than 12 h, which might lead to the different results from ours. Besides, we speculated that it might be the excessive increase of arginase activity, causing the arginine to react with substrate rapidly and to be consumed, thus reducing the protein expression level. The immune regulation of macrophages was complex, and there might be other mechanisms of anti-T.marneffei infection, so that the increased Arg1 mRNA could not successfully convert into protein, maintaining the M1/M2 polarization balance.
In recent years, it has been found that the expression of arginase in macrophages was up-regulated in the studies of fungal infection, parasite infection, bacterial infection and other diseases [13][14][15][16]. Some scholars have proposed that the use of arginase inhibitors could reverse the pathology of diseases. As a speci c inhibitor of arginase, nor-NOHA can effectively inhibit the activity of arginase without inhibiting the up-regulation of iNOS. The application of nor-NOHA has been studied in some patients with hypertension, coronary disease, heart failure and familial hypercholesterolemia, and has presented great achievements [17][18][19][20]. However, in the cases of fungal infection, there were few studies on using nor-NOHA as a means of ghting infection.
Our experimental results indicated that nor-NOHA could enhance the phagocytosis of T.marneffei conidia by macrophages. Nor-NOHA inhibited the increase of arginase activity and up-regulated the expression of iNOS, so as to promote the production of abundant NO and to strengthen the sterilization ability of macrophages. Compared with the untreated T.marneffei group, T.marneffei pretreated with 20 µM nor-NOHA showed the lower colony count. It demonstrated that the arginase inhibitor nor-NOHA could suppress T.marneffei proliferation. Some studies reported that Spirillum, Mycobacterium, Salmonella, Schistosoma, Trypanosoma, Leishmania and Candida could code their arginase [21]. The arginase of Leishmania increased after treatment of IL-4, which promoted the growth of Leishmania. Moreover, the growth of Leishmania was also inhibited by the inhibition of arginase, and this growth inhibition was attributed to the reduction of ornithine availability [22]. The arginase encoded by rocF of Helicobacter pylori could protect it from acid stress. In a mouse model, the arginase-de cient strain of Helicobacter pylori showed a weakened colonization [23]. The arginase of Helicobacter pylori could also damage the function of T cells by reducing the expression of CD3 ζ chain [24]. Candida albicans yeast cells rapidly up-regulated the arginase encoded by Car1p after being phagocytosed by macrophages, which metabolized L-arginine to ornithine and urea, and then the urea was degraded into CO 2 and NH 3 through urea amidolyase. CO 2 activated adenylate cyclase and cAMP-dependent protein kinase A pathway (PKA), thus activating Efg1p and inducing Candida albicans to transform from yeast to hypha, so that fungi could penetrate the cell membrane and escape killing by macrophages [25]. These studies showed that some pathogenic microorganisms could increase their arginase, thus promoting their proliferation and survival in cells. T.marneffei whole-genome sequencing showed that there were some genes encoding arginase, urea amidolyase and adenylate cyclase. T.marneffei is an asexual reproductive fungi. The key factor of asexual development is GasA and the activation of GasA signal may involve the regulation of cAMP-PKA pathway [26]. Therefore, we speculated that T.marneffei might up-regulate its arginase and urea amidolyase to produce a large amount of CO2, which activated cAMP-PKA to regulate the GasA signal, thereby stimulating the T.marneffei asexual development. Nor-NOHA suppressed the arginase of T.marneffei and blocked this pathway, thus inducing a low colony count.
It was worth noting that the single cultured T.marneffei (with or without nor NOHA) showed a lower colony count, compared with the co-culture group (T.marneffei co-cultured with macrophages). This suggested that the co-culture system promoted the growth of T.marneffei. In a study on Leishmania, it was found that Leishmania enhanced the host's arginase to obtain polyamines, and also used its own arginase to obtain polyamines, which were the necessary component for its growth [27]. The biosynthesis of polyamines in eukaryotes and protozoa is initiated by the hydrolysis of L-ornithine into putrescine via ornithine decarboxylase (ODC). Putrescine is converted into spermidine through the continuous transfer of amino propyl, and then into spermine. Putrescine, spermidine and spermine are the most common polyamines in bacteria and fungi. Except for two archaea, polyamines are identi ed in each organism [28,29]. The universal existence proves its importance in organism. Polyamines are involved in many cellular processes, such as gene expression, cell proliferation, survival and stress response. The reverse-genetic study of microorganisms showed that the gene deletion or polyamine consumption in polyamine metabolism pathway had a negative effect on cell survival and proliferation [29]. In a study on the mutant T.marneffei strain of S-adenosylmethionine decarboxylase (SAMDC), which was the key enzyme of polyamine synthesis, it was found that the mutant strain had poor growth and sporulation capacity on the basic culture medium. However, the addition of spermidine in culture medium recovered the sporulation capacity and promoted the yeast development at 37℃ [30]. We speculated that, on the one hand, T.marneffei could induce the increase of arginase activity of macrophages, inhibit the production of NO by the L-arginine pathway, thus weakening the killing capacity of macrophages, on the other hand, T.marneffei could regulate the increase of polyamine concentration in the environment to promote its proliferation.
T.marneffei is an intracellular fungi, mainly affecting the people with immune de ciency, such as Human Immunode ciency Virus (HIV) positive people, IFN-γ antibody positive people, CD20 antibody positive people and kinase inhibition people [31,32]. It has been reported that the Arg1 expression in lymph nodes and peripheral T cells of HIV patients was negatively correlated with the peripheral CD4 + T cell count and positively correlated with the viral load. The expression of iNOS was positively related with the peripheral CD4 + T cell count and negatively related with the viral load [33]. Perhaps the environment of high arginase in HIV patients was one of the reasons why they were susceptible to T.marneffei.

Conclusions
To conclude, T.marneffei can promote the expression of arginase in itself or the host to maintain survival through the arginine metabolism pathway. Nor-NOHA, an arginase inhibitor, can play an important role in macrophages against T.marneffei infection.

Cell medium and reagents
The murine macrophage cell line RAW264.7, obtained from American Type Culture Collection (ATCC, VA, USA) were maintained in complete Dulbecco's modi ed Eagle's medium containing 10% fetal bovine serum, 100U/ml penicillin and 100mg/ml streptomycin at 37°C in a humidi ed atmosphere containing 5% CO 2 . M1-polarized macrophages were induced by 500ng/ml LPS (Invitrogen, USA) for 24h.

T. marneffei strain and conidia preparation
The T. marneffei strain FRR2161 was cultured in the hypha phase for 2 weeks at 25℃ onto PDA medium (6g potato extract, 20g dextrose, 20g agar). Collecting the colonies into 1×PBS buffer solution and ltering the hypha through sterile gauzes, then the conidia suspension was obtained.
Arginase activity assay Arginase activity was determined using the arginase activity assay kit (Sigma-Aldrich). Samples were performed as the manufacture introduction. Activity NO production assay The production of NO was determined with assay kit for nitrite determination (Nanjing Jiancheng Bioengineering Institute, China). The co-cultured precipitation was centrifuged and discarded. The supernatant was collected. Different reagents were added according to the manufacture instruction. Reactions were then measured at 550nm using a microplate reader.

Immuno uorescent staining
Macrophages were seeded onto coverslips in a 12-well plate and cultivated overnight. After co-culturing with T. marneffei conidia for 72h, macrophages were washed in PBS. Cells were xed in 4% paraformaldehyde for 15 minutes, permeabilized in 0.1% Triton X-100 and 1% bovine serum albumin for 15 minutes. All samples were incubated with primary antibody against CD206 (1:200, Abcam) at 4°C overnight and then incubated with secondary antibody for 1 hour at room temperature. Nuclei were counterstained in DAPI (1:500, Sigma) for 10 minutes.
Phagocytic activity assay T. marneffei conidia suspension was prestained with Calco uor white (Sigma, USA) and 10% KOH for 10 min. Macrophages were added on coverslips, pretreated with 20μM nor-NOHA for 2h. Following coincubation with prestained conidia suspension (MOI=10) for 2h at 37°C, samples were washed gently to remove non-phagocytized conidia. Then the cells were xed in 0.5% paraformaldehyde. Fluorescence was observed with a uorescencemicroscope. Phagocytic activity was calculated as a phagocytic index (PI) using the following formula: PI= Number of intracellular T. marneffei conidia/Number of macrophages containing T. marneffei conidia.
T. marneffei killing assay The T. marneffei killing was evaluated by colony-forming unit (CFU) measurement. Pretreat LPS-activated macrophages with 20μM nor-NOHA for 2h. Then, the cells were co-cultured with T. marneffei conidia for 24h (MOI=1). After 24h of incubation, the supernatant was collected. Sterile water was added to each well and the plate was shaken adequately. Combine well contents into the supernatant. Centrifugation, abandonment of supernatant, suspension of sediment with sterile water. Then the fungi suspension was serially diluted in sterile water, and eventually plated (quintuplicate samples) on solid YPD agar plates.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. As to the raw data, please refer to the Additional le1.

Competing Interest
No con ict of interest to declare.

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