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 inflammation and concurrently mediated the anti-inflammatory response after being engulfed by macrophages. It was a dynamic process. As the immune response progressed, the inflammatory cytokines increased while the anti-inflammatory factors decreased. CD86 is a member of immunoglobulin superfamily, and its co-stimulatory 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 specific 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 finding 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 [13–16]. Some scholars have proposed that the use of arginase inhibitors could reverse the pathology of diseases. As a specific 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 [17–20]. However, in the cases of fungal infection, there were few studies on using nor-NOHA as a means of fighting 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-deficient 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 CO2 and NH3 through urea amidolyase. CO2 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 identified 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 deficiency, such as Human Immunodeficiency 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.