Talaromyces marneffei, an opportunistic fungal pathogen, causes a life-threatening deep-seated systemic infection in patients with acquired immune deficiency syndrome(AIDS) in Southeast Asian countries[2, 14]. Due to the lack of effective antifungal agents, talaromycosis is well known as a severe disease that can cause disseminated infection, and treatment of this disease still remains a challenge[12, 15].
In this research, we demonstrated that cotrimoxazole can exert direct fungicidal effects on T.marneffei by inhibiting DHPS, DHFS, DHFR which were evolved in folate biosynthesis, and enhance the indirect fungicidal effect of macrophages by regulating the Dectin-1 signaling pathway.
Since the antimicrobial effect of cotrimoxazole (CTX) comes from its blocking activity of folic acid biosynthesis pathway which are important for the survival of many bacterial, protozoa, and fungi. It has been long assumed that trimethoprim and sulfamethoxazole synergistically block microbial synthesis of folic acid, a vital cofactor in the manufacture of thymidine and purines. Sulfamethoxazole is a competitive inhibitor of dihydrofolic acid synthesis, and trimethoprim acts downstream of sulfamethoxazole to inhibit production of the physiologically active tetrahydrofolic acid. Based on this information, we hypothesized that cotrimoxazole directly inhibiting the growth and proliferation of T.marneffei was through blocking DHPS, DHFR directly and DHFS indirectly, by which SMX inhibits DHPS, thereby inhibiting DHFS and TMP inhibits DHFR respectively. Our results confirmed this hypothesis showing that the concentration of DHPS, DHFS and DHFR were significantly decreased in T.marneffei adding effective concentrations of TMP/SMX group. Our results further found that DHPS and DHFS showed obvious declining tendency in T.marneffei adding effective concentrations of SMX group. But the results suggests that DHFR was not significantly decreased in T.marneffei adding ineffective concentrations of TMP group, which led us to propose that DHFR was only inhibited at an effective inhibiting T.marneffei concentration of TMP/SMX, while the corresponding concentration of TMP only serves as a sulfonamide sensitizer and cannot block DHFR separately. Our data therefore provided a complete chain of evidence that cotrimoxazole inhibiting T.marneffei directly in vitro perhaps by blocking DHPS, DHFS and DHFR at effective concentrations of TMP/SMX, of which SMX plays an essential role in blocking DHPS and DHFR, while TMP serves as a sulfonamide sensitizer, thereby enhancing the fungicidal effect of SMX. This finding reveals a novel mechanism by which cotrimoxazole can block DHPS, DHFS, DHFR and SMX can inhibit DHPS and DHFS separately to inhibit the folate biosynthesis pathway. But TMP cannot inhibit DHFR at an ineffective concentration separately which confirmed the synergistic role of TMP/SMX to kill T.marneffei in vitro.
Ghilchik et al has mentioned that cotrimoxazole has both immunomodulatory and antimicrobial properties, suggesting that cotrimoxazole can modulate both innate and adaptive immune cells, including enhancing phagocytosis of microorganism such as bacteria, fungi and intracellular killing by macrophages[16–18].An in-vitro study of alveolar macrophage function in a few adults with advanced HIV infection showed enhanced phagocytosis and killing of Staphylococcus aureus in those taking cotrimoxazole, compared with untreated individuals[16, 19].Our results have confirmed that T.marneffei inside macrophages can be inhibited by TMP/SMX, SMX and TMP at high intracellular concentrations respectively. However, the interaction between cotrimoxazole and T.marneffei-infected macrophages remains largely unknown. To investigate whether cotrimoxazole influences the Dectin-1 signaling pathway of T.marneffei-infected macrophages, PMA-induced THP-1 macrophages stimulating with T.marneffei conidia were used, and the mRNA expression levels of Dectin-1 and downstream IL-6, IL-10, IL-23A, CXCL8 and TNF-α inflammatory cytokines were determined by RT-qPCR. In the present study, it was revealed that increased levels of Dectin-1, IL-6, IL-10, IL-23A and decreased levels of CXCL8 and TNF-α were observed in macrophages in response to T.marneffei infection. Our results also revealed that decreased levels of Dectin-1, IL-6, IL-10, IL-23A and increased levels of CXCL8 and TNF-α were observed in T.marneffei-infected macrophages after adding TMP/SMX for 24h. Although the role of Dectin-1 in mediating the expression levels of cytokines released by macrophages in T.marneffei infection remains poorly understood, several studies have mentioned its essential role in antifungal immunity. Dectin-1 is a member of the C-type lectin family and functions as an innate PRR involved in antifungal immunity[12, 14, 20]. Nakamura et al demonstrated that Dectin-1 is essential in sensing T.marneffei for the activation of bone marrow-derived DCs[12, 14].Sun et al demonstrated that Dectin-1 may play a crucial role in Aspergillus-induced innate immune responses in human bronchial epithelial cells[12, 21].These findings suggested that Dectin-1 may be a critical component of the anti-T.marneffei immune response in macrophages. The role and mechanism of cotrimoxazole influencing the anti-T.marneffei ability of macrophages perhaps by regulating the Dectin-1 signaling pathway and downstream inflammatory cytokines.
It is well known that inflammatory cytokines play a critical role in the development of fungal infectious diseases. Monocytes/macrophages serve as an essential role in the innate immunity of host resistance to invasive fungi infection. T.marneffei infection can activate innate immune cells, such as macrophages and the activated macrophages can secrete cytokines, and they primarily produce pro-inflammatory cytokines such as IL-1,IL-6, IL-12,IL-18,IL-23, IL-1β, CXCL8, TNF-α and anti-inflammatory cytokines such as IL-10, IL-38 and so on[22–24].
Previous studies have revealed that Dectin-1 couple to spleen tyrosine kinase (Syk) and the adaptor molecular CARD-9 to induce production of IL-6, IL-23 and TNF-α[25, 26].It was also observed that patients with Dectin-1 deficiency had reduced IL-6 and IL-17 production in monocytes and macrophages challenged with either β-glucan or heat-killed C.albicans, and patients with CARD-9 deficiencies exhibited defects in Th17 differentiation, while IL-23 is required for the activity of Th17 cells and their maintenance in vivo[25, 27].Previous investigations have proved that the T.marneffei-induced activation of JNK1/2 pathway increased the production of IL-10, thereby inhibiting the anti-T.marneffei ability of human macrophages.An in-vitro research using mouse macrophages as cell model, suggesting that incubation of T.marneffei promoted phagocytosis of T.marneffei by macrophages and high levels of pro-inflammatory and anti-inflammatory cytokine production by macrophages, which was consistent with our results, referring to high mRNA expression levels of IL-6, IL-10, IL-23A secreted by macrophages in response to T.marneffei.
In fact, it must be admitted that Dectin-1 as important sensor in recognizing T.marneffei infection in macrophages, the activation of Dectin-1 signaling pathway influencing pro-inflammatory/anti-inflammatory response to T.marneffei, thereby increasing the production of IL-6, IL-10 and IL-23A to inhibit the anti-T.marneffei effect of macrophages. CXCL8, also known as IL-8, is a pro-inflammatory CXC chemokine involved in inflammatory reactions. The biological effects of IL-8 are mediated by two highly related chemokine receptors, CXCR1(IL-8RA) and CXCR2(IL-8RB), which are co-expressed on human neutrophils. Both the beneficial and harmful roles of neutrophils are critically dependent on the capacity of the cell to undergo directed migration from the blood to local tissue sites, including T.marneffei infection. Because the CXC chemokine interleukin-8(IL-8) is a powerful mediator of this process, its two receptors CXCR1 and CXCR2 are reasonable targets for development of treatments for neutrophil-mediated inflammation.Therefore,CXCL8 exerts its function alongside other cytokines and chemokines, is an important activator and chemoattractant for leukocytes to sites of inflammation, recruitment and activation of neutrophils to phagocytosis, and bacterial clearance, and its role have been implicated in a variety of inflammatory diseases[12, 30, 31].Tumour necrosis factor alpha(TNF-α), an inflammatory cytokine produced by macrophages /monocytes during acute inflammation, which belongs to a family of both soluble and cell-bound cytokines that have a wide range of functions such as host defense, inflammation, and is responsible for a diverse range of signaling events within cells, leading to necrosis or apoptosis[12, 32, 33].Previous investigations have showed that TNF-α plays an essential role in the initiation of the innate response in the mouse model of invasive Aspergillus infection. It mentioned TNF-α most likely mediates its protective effect via following mechanisms, including induction of neutrophil-chemotactic chemokines, inducing of adhesion molecules, and neutrophil-independent pathways. Additionally, several recruitment cytokines play critical roles in mediating influx of specific leukocytes to the site of infection in invasive Aspergillosis. A profound study also revealed that the depletion of TNF-α resulted in a reduced lung neutrophil influx in both normal and cyclophosphamide-treated animals, which occurred in association with a decrease in lung levels of the C-X-C chemokine, macrophage inflammatory protein-2 and the C-C chemokines macrophage inflammatory protein-1 alpha. Notably, our research found that cotrimoxazole has the potential to increase the secretion levels of CXCL8 and TNF-α by macrophages in response to T.marneffei infection. Our data led us to propose that cotrimoxazole exerts fungicidal effect by increasing the levels of TNF-α, thereby inducing CXCL8 to exert the intercellular killing activity of macrophages.
Taken together, our results demonstrated the essential recognizing T.marneffei role of Dectin-1 in macrophages, and further confirmed our hypothesis that cotrimoxazole influencing T.marneffei growth through regulating the Dectin-1 signaling pathway. In the current study, we observed that the activation of T.marneffei-induced Dectin-1 pathway increased the production of IL-6, IL-10 and IL-23A while reduced the production of CXCL8 and TNF-α to inhibit the anti-T.marneffei capacity of macrophages. Cotrimoxzole serves its important role by reducing the T.marneffei-induced inflammatory responses and enhance the phagocytosis and intercellular killing role of macrophages.
There exists several limitations in the present study. Firstly, only one cell line, human THP-1 macrophage, was used as cell model in vitro. Secondly, considering the environment in the human body is complicated and changeable, and there exists individual immune differences among the population, so the safe and effective concentration of cotrimoxazole towards THP-1 macrophages in vitro cannot represent blood drug concentration in vivo. Thirdly, our cell experiments in vitro only contain macrophages without stimulating the immune microenvironment in vivo in response to T.marneffei. As we all known, there are various immune cells interacting with each other to coordinate an antifungal response in vivo. Fourthly, our results cannot determine whether cotrimoxazole influence the expression of Dectin-1 receptor of macrophages by inhibiting the proliferation of T.marneffei, or T.marneffei inhibits the expression of Dectin-1 receptor, which was up-regulated by cotrimoxazole afterwards. Therefore, the combination of population research among HIV/AIDS co-infected with T.marneffei treated with cotrimoxazole and animal experiments should be conducted to further explore the role and mechanism of cotrimoxazole resistance to T.marneffei in vivo.
In conclusion, our data led us to propose that T.marneffei was inhibited by cotrimoxazole through blocking DHPS, DHFS and DHFR directly and regulating Dectin-1 signaling pathway indirectly to enhance intracellular killing-T.marneffei ability in human THP-1 macrophages. Our study reveals a novel mechanism by which cotrimoxazole inhibits the growth and proliferation of T.marneffei inside and outside macrophages, which may provide a therapeutic target for inhibition of infection and dissemination in T.marneffei infection, especially in HIV/AIDS patients.