Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing

Wei, Wudi; Wang, Gang; Zhang, Hong; Bao, Xiuli; An, Sanqi; Luo, Qiang; He, Jinhao; Chen, Lixiang; Ning, Chuanyi; Lai, Jingzhen; Yuan, Zongxiang; Chen, Rongfeng; Jiang, Junjun; Ye, Li; Liang, Hao

doi:10.1038/s42003-023-05409-6

Download PDF

Article
Open access
Published: 16 October 2023

Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing

Wudi Wei ORCID: orcid.org/0000-0001-6703-4529^1,2^na1,
Gang Wang ORCID: orcid.org/0000-0001-7264-2457¹^na1,
Hong Zhang²^na1,
Xiuli Bao¹^na1,
Sanqi An^1,2,
Qiang Luo²,
Jinhao He²,
Lixiang Chen¹,
Chuanyi Ning^2,3,
Jingzhen Lai^2,4,
Zongxiang Yuan¹,
Rongfeng Chen^1,2,
Junjun Jiang ORCID: orcid.org/0000-0003-4905-9348^1,2,
Li Ye ORCID: orcid.org/0000-0001-7688-4867^1,2 &
…
Hao Liang ORCID: orcid.org/0000-0001-7534-5124^1,2,4

Communications Biology volume 6, Article number: 1046 (2023) Cite this article

1086 Accesses
1 Altmetric
Metrics details

Subjects

Abstract

Talaromyces marneffei (T. marneffei) immune escape is essential in the pathogenesis of talaromycosis. It is currently known that T. marneffei achieves immune escape through various strategies. However, the role of cellular alternative splicing (AS) in immune escape remains unclear. Here, we depict the AS landscape in macrophages upon T. marneffei infection via high-throughput RNA sequencing and detect a truncated protein of NCOR2 / SMRT, named NCOR2-013, which is significantly upregulated after T. marneffei infection. Mechanistic analysis indicates that NCOR2-013 forms a co-repression complex with TBL1XR1 / TBLR1 and HDAC3, thereby inhibiting JunB-mediated transcriptional activation of pro-inflammatory cytokines via the inhibition of histone acetylation. Furthermore, we identify TUT1 as the AS regulator that regulates NCOR2-013 production and promotes T. marneffei immune evasion. Collectively, these findings indicate that T. marneffei escapes macrophage killing through TUT1-mediated alternative splicing of NCOR2 / SMRT, providing insight into the molecular mechanisms of T. marneffei immune evasion and potential targets for talaromycosis therapy.

Lnc-EST12, which is negatively regulated by mycobacterial EST12, suppresses antimycobacterial innate immunity through its interaction with FUBP3

Article 30 May 2022

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection

Article 25 March 2024

Identification of a PadR-type regulator essential for intracellular pathogenesis of Burkholderia pseudomallei

Article Open access 17 May 2021

Introduction

Talaromycosis (formerly penicilliosis) is caused by a thermodimorphic fungus Talaromyces marneffei (T. marneffei) and is one of the important causes of death among AIDS patients in epidemic areas. T. marneffei, the only known dimorphic species in the genus, exhibits filamentous growth at 25 °C and a yeast phase at 37 °C, but only the yeast-like form has pathogenic potential¹. Talaromycosis is a regional high-incidence opportunistic infection that is endemic throughout southern China, southeast Asia, and northeastern India^2,3. Due to the HIV epidemic, the number of talaromycosis cases has rapidly increased and ranked 3rd as the most common HIV-associated opportunistic infections, accounting for up to 16% of HIV hospital admissions, and the leading cause of death among patients with advanced HIV in Thailand, Vietnam, and southern China^2,3,4. By the end of 2018, 288,000 cases have been reported in 33 countries, with an estimated 17,300 cases (95% CI 9900–23,700) and 4900 deaths (95% CI 2500–7300) each year⁵. It is worth mentioning that with the wide application of bone marrow (organ) transplantation technology and immunosuppressive therapy, the incidence of HIV-negative talaromycosis patients is also increasing. Exploring differences in pathobiology between HIV-negative and positive talaromycosis is another important direction in the future research. As a result, talaromycosis has become a serious endemic health problem and has been proposed to be included in the “List of Priority Fungal Pathogens” by the World Health Organization, which is recognized as a neglected tropical infectious disease^6,7.

Immune evasion of T. marneffei is an important reason for the poor prognosis of talaromycosis. Previous studies have shown that T. marneffei can survive in host macrophages. Ellett et al found that zebrafish embryo macrophages protect T. marneffei from neutrophil fungicidal activity during infection⁸. It is clear that T. marneffei has a set of macrophage-based immune evasion strategies in the humans. As a facultative intracellular fungus, T. marneffei is easily phagocytosed by resident macrophages after invasion^2,9. Paradoxically, as one of the major cells in the innate immune line of defense against pathogen infection, macrophages also provide a niche for T. marneffei to evade immune killing, which is known as the “macrophage paradox”¹⁰. In fact, several pathogens have similar strategies, such as Mycobacterium tuberculosis (M. tuberculosis) and Candida albicans (C. albicans)^10,11,12. Therefore, understanding the molecular mechanism of T. marneffei immune evasion is of great significance for the treatment of talaromycosis patients and the reduction of mortality. Previous studies have demonstrated that T. marneffei may escape macrophage killing by inducing M2-like macrophage polarization via regulating SOCS3-STAT6, TLR9, Jun1/2, and p38 signaling pathways, as well as LncRNAs^13,14,15,16. However, there is still an urgent need for a deeper understanding of these mechanisms.

Alternative splicing (AS) is a ubiquitous mechanism for regulating gene expression, which allows a single gene to produce multiple unique mRNAs. It has been previously reported that 90–95% of human genes undergo multiple levels of AS, of which 37% have been shown to generate multiple protein isoforms that may play distinct biological functions^17,18,19,20. In general, AS includes five categories of events, namely, retained intron (RI), exon skipping (ES), alternative 5’ splice site (A5SS), alternative 3’ splice site (A3SS), and mutually exclusive exon splicing (MXE), to generate multiple mature mRNA isoforms from the same pre-mRNA²¹. Thus, AS plays an important role in the complexity and functional diversity of the proteome. Bacterial pathogens are known to strategically modulate specific host factors via regulating AS to facilitate their replication in host cells^22,23,24. For instance, M. tuberculosis can survive in macrophages by limiting the phagosome maturation by producing an abundance of truncated RAB8B variants²³. As a highly dynamic and complex process, AS is regulated by a broad array of RNA-binding proteins (RBPs). Mechanistically, RBP binds to target pre-mRNA through specific motifs to mediate the occurrence of AS^17,19. Intriguingly, global changes in gene AS events in macrophages after T. marneffei infection have not been understood, and there is no clear description of whether specific isoforms components contribute to T. marneffei immune evasion.

In this study, we performed high-throughput RNA sequencing and depicted a global view of the changes in gene AS events in human macrophages upon T. marneffei infection. Moreover, we identified a TUT1-modulated AS event of NCOR2-013 involved in facilitating T. marneffei immune evasion.

Results

Profiles of alternative splicing in T. marneffei-infected THP-1 macrophages

To investigate the potential function of AS events involved in T. marneffei-infected macrophages, we performed RNA sequencing (RNA-seq) in T. marneffei-infected or -uninfected THP-1 macrophages at 24 h post-infection. Overall, 75,939 AS events were found in T. marneffei-infected macrophages, of which, 1757 were statistically significant. The proportion of ES in significant AS events was 60.33%, which was the highest compared with other types of AS events (Fig. 1a). The UpSet plot showed that 90.60% genes (1234/1362) were involved in only one type of AS event, among which ES type (763) was the largest group, followed by RI (166), A3SS (119), A5SS (95) and MXE (91), and the rest of genes were involved in 2 (8.96%, 122/1362) or 3 (0.44%, 6/1362) AS events simultaneously (Fig. 1b). Since ES type was the most common AS type in T. marneffei-infected macrophages, we next selected ES-type AS genes with FDR < 0.05 and |Lnclevel| > 0.5 for further KEGG enrichment analysis. Finally 6 genes (NCOR2, NR1H3, PPARA, IRF3, MAP2K3, and TLR6) were enriched in 3 immune-related pathways, including toll-like receptor signaling pathway, EB virus infection, and hepatitis C (Fig. 1c, Supplementary Fig. 1a). Therefore, we focused on these 6 genes for the further study.

**Fig. 1: Landscape of alternative splicing (AS) in *T. marneffei*-infected THP-1 macrophages.**

T. marneffei induces the expression of NCOR2-013 isoforms in macrophages

Previous studies have shown that truncated transcripts produced by ES-type AS events play an important role in pathogen immunity^22,23,24. Therefore, we aimed to identify specific truncated transcripts in the 6 ES-related AS genes (NCOR2, NR1H3, PPARA, IRF3, MAP2K3, and TLR6) that may influence the cellular responses to T. marneffei infection. We developed a strategy for the screening of particular truncated transcript. Firstly, the truncated transcripts generated by ES-type AS events predicted by rMATS should match the currently known transcripts. Secondly, the truncated transcript is differentially highly expressed in T. marneffei-infected macrophages, but not detected in uninfected cells. Due to the expression in uninfected cells or / and insignificant difference in transcript expression between T. marneffei-infected and -uninfected macrophages, 5 genes were excluded (Supplementary Table 1, Supplementary Fig. 1b–k) and only NCOR2-013 was selected for in-depth study. Considerable differences were observed in the number of reads corresponding to exon-exon junctions between the T. marneffei-infected and -uninfected macrophages, as shown in Sashimi plots and track plots (Fig. 2a, b). Among different NCOR2 isoforms, NCOR2-013 exhibited the complete loss of exon 14 and partial loss of exons 11 and 15 (Fig. 2c). Subsequently, RNA-seq results revealed that NCOR2-13 isoforms were not detected in control cells and significantly up-regulated T. marneffei-infected cells at 24 h post-infection (padj = 0.0015) (Fig. 2d, Supplementary Table 2). The RT-qPCR results confirmed that the NCOR2-013 isoforms increased 2-fold at the mRNA level in T. marneffei-infected THP-1 macrophages at 24 h post-infection (Fig. 2e). In addition, the same trend was exhibited in T. marneffei-infected human monocyte-derived macrophages (hMDMs), with an approximately 1.5-fold increase (Fig. 2f). Interestingly, the maternal protein of NCOR2, namely SMRT, was not altered after T. marneffei infection, indicating that the biologically functional protein may not be the NCOR2 / SMRT, but more likely NCOR2-013 isoforms (Fig. 2g). Therefore, we focused on the functional roles and related mechanisms of NCOR2-013 in T. marneffei-infected macrophages.

NCOR2-013 inhibits macrophage inflammatory response and antifungal activity

To determine the subcellular localization and function of NCOR2-013, we successfully constructed a Flag-NCOR2-013 overexpressing THP-1 macrophage cell line using lentiviruses vectors and validated the cell line by RT-qPCR, RNA-seq and WB (Fig. 3a–c). We used the Hum-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/hum-multi-2/) to predict subcellular localization of NCOR2-013, indicating that the protein is mainly located in the nucleus (Fig. 3d). Furthermore, we extracted cytoplasm and nucleus proteins of Flag-NCOR2-013 overexpressing macrophages, and found that Flag-NCOR2-013 was mainly localized in the nucleus and almost undetectable in the cytoplasm (Fig. 3e), which was also confirmed by immunofluorescence assay (Fig. 3f). CFUs assay revealed that more T. marneffei were detected in NCOR2-013 overexpressing macrophages compared with T. marneffei-infected control cells (Fig. 3g), suggesting that NCOR2-013 inhibits the ability of macrophages to clear T. marneffei. Similar results of CFUs assay were also observed in hMDMs (Fig. 3h). We further explored the effect of NCOR2-013 overexpression on the inflammatory response of macrophages, and found that T. marneffei infection and NCOR2-013 overexpression down-regulated the levels of TNF-α and IL-1β (NC vs. NC + T. marneffei, NC vs. NCOR2-013 overexpression). Meanwhile, with the premise of T. marneffei infection, the overexpression of NCOR2-013 can further suppress the expression of pro-inflammatory factors (NC + T. marneffei vs. NCOR2-013 overexpression+T. marneffei) (Fig. 3i, j).

NCOR2-013 suppresses activation of JunB by forming a transcriptional regulatory complex with HDAC3 and TBL1XR1 / TBLR1

To explore the molecular mechanism of NCOR2-013 inhibiting the inflammatory response, we performed RNA-seq analysis on T. marneffei-infected NCOR2-013 overexpressing macrophages and control cells at 24 h post-infection. KEGG enrichment analysis showed the DEGs were significantly involved in NF-kappa B signaling pathway and TNF signaling pathway (Supplementary Fig. 2a), which was consistent with the GSEA results (Supplementary Fig. 2b), indicating that NCOR2-013 may inhibit the activation of NF-kappa B and TNF signaling pathways.

Previous studies have shown that NCOR2 / SMRT is a nuclear receptor that forms a co-repressor with HDAC3 and TBL1XR1 / TBLR1 to inhibit transcription factor activation²⁵. Since the NCOR2-013 is predominantly located in the nucleus, we hypothesized that the regulatory mechanism of NCOR2-013 is similar to that of NCOR2 / SMRT. Therefore, we first identified 8 differentially expressed transcription factors (TWIST1, RB1, STAT1, RELA, HDGF, PPARG, E2F1 and JUN)²⁶ via TRRUST analysis combined with DEGs (Supplementary Fig. 2c, d). We then infected NCOR2-013 overexpressing THP-1 macrophages with T. marneffei for 24 h, and screened for proteins binding to NCOR2-013 using IP and mass spectrometry (Fig. 4a). A total of 254 proteins were screened, and after intersecting with DEGs (NC + T. marneffei vs. NCOR2-013 overexpression + T. marneffei), 6 transcription factors were obtained (Fig. 4a). Combined with the results in Supplementary Fig. 2d, JunB was finally selected as the most potential transcription factor for subsequent functional research. To test the hypothesis above, we first verified the interaction between NCOR2-013 (Flag-tagged), TBL1XR1 / TBLR1, HDAC3, and JunB via Co-IP. The results showed that the 4 proteins could bind together in T. marneffei-infected NCOR2-013 overexpressing macrophages at 24 h post-infection (Fig. 4b–e). We also investigated whether NCOR2 / SMRT was involved in inhibition of JunB and performed Co-IP to explore the interaction of NCOR2 / SMRT with these 4 proteins. Interestingly, Co-IP results showed that NCOR2 / SMRT interacts with HDAC3, TBL1XR1 / TBLR1 (Fig. 4b–e). However, it does not interact with NCOR2-013 and JunB (Fig. 4b–e). In addition, because JunD and c-Jun are both homologous proteins of JunB²⁷, we further verified whether NCOR2-013 could bind to JunD and c-Jun via Co-IP. The results showed that, in addition to JunB, NCOR2-013 also binds to c-Jun but hardly interaction with JunD (Fig. 4f, g). Next, we detected the activation levels of JunB and c-Jun by WB, respectively. The results showed that, only at 48 h post infection, not at 24 h, T. marneffei up-regulated the phosphorylation level of JunB, while NCOR2-013 overexpression inhibited the elevation of p-JunB, but there was no significant difference in p-c-Jun under T. marneffei infection or NCOR2-013 overexpression (Fig. 4h, i). Collectively, NCOR2-013 forms the NCOR2-013 - HDAC3 - TBL1XR1 / TBLR1 transcription-regulating complex and inhibits JunB activation.

**Fig. 4: NCOR2-013 suppresses activation of JunB by forming a transcription-regulatory complex with HDAC3 and TBL1XR1/TBLR1.**

NCOR2-013 inhibits transcription of pro-inflammatory cytokines by inhibiting acetylated histone H3

To explore whether NCOR2-013 transcription-regulating complex directly regulates transcription of pro-inflammatory cytokines, we performed ChIP-qPCR analysis using Flag, HDAC3, TBL1XR1/TBLR1 and JunB antibodies, respectively. The level of transcription factor occupancy of the promoter region of TNF-α and IL-1β in T. marneffei-infected NCOR2-013 overexpressing macrophages at 24 h post-infection were detected. The results showed that NCOR2-013, HDAC3, TBL1XR1 / TBLR1 and JunB were enriched in the TNF-α and IL-1β promoter regions, indicating that these proteins could act on the promoter regions of TNF-α and IL-1β genes (Fig. 5a, b).

In epigenetics, histone deacetylation via HDAC3 is a common mechanism associated with transcriptional activation by modulating chromatin condensation^28,29,30. To further explore the molecular mechanism of NCOR2-013 suppressing the expression of pro-inflammatory cytokines, NCOR2-013-overexpressing macrophages and control cells were infected with T. marneffei for 24 h, and ChIP-qPCR assays were conducted using HDAC3 and H3K27Ac antibodies (Fig. 5c, d). The results showed that NOCR2-013 up-regulated the enrichment level of HDAC3 in promoter regions of TNF-α and IL-1β, and down-regulated the enrichment level of H3K27Ac, indicating that NOCR2-013 inhibited the histone H3 acetylation, which did not favor the binding of transcriptional factors to the corresponding promoter regions, thereby suppressing the expression of TNF-α and IL-1β (Fig. 5c, d).

JunB overexpression or IFN-γ has little effect on NCOR2-013-mediated inhibition of pro-inflammatory response in macrophages, while LPS has partial effect

To explore the role of JunB in NCOR2-013-mediated inhibition of pro-inflammatory response, we overexpressed JunB in NCOR2-013-overexpressing THP-1 macrophages. However, JunB overexpression did not overcome the inhibitory effect of NCOR2-013, evidenced by the fact that JunB overexpression had no significant changes in cytokine expression and antifungal ability (Fig. 6a–c). Similarly, a parallel phenomenon was observed in NCOR2-013-untransfected cells (Supplementary Fig. 3). Subsequently, we conducted exploration experiments using IFN-γ and LPS, respectively. Interestingly, LPS, but not IFN-γ, was able to significantly upregulate the expression of TNF-α and IL-1β (Fig. 6d, e), while significantly increasing the killing ability of macrophages against T. marneffei (Fig. 6f, g), suggesting that distinct roles for IFN-γ and LPS in modulating the inflammatory and antifungal responses in NCOR2-013-overexpressing THP-1 macrophages, with LPS exerting a notably enhancing effect.

**Fig. 6: JunB overexpression or IFN-γ has little effect on NCOR2-013-mediated inhibition of inflammatory response in macrophages, while LPS has partial effect.**

TUT1-mediated generation of the NCOR2-013 isoforms suppresses inflammatory response in T. marneffei-infected THP-1 macrophages

Generally, AS is mainly regulated by a series of RBPs. To identify the upstream RBPs that regulate NCOR2 for AS, we used rMATS to predict splicing regulators, which were then intersected with DEGs derived from RNA-seq analysis, indicating that TUT1 is the likely RBP that regulates AS of NCOR2 (Fig. 7a), with the motif of [AC][AG]ATACT (Fig. 7b). RT-qPCR and WB results confirmed that T. marneffei up-regulated the expression of TUT1 in THP-1 macrophages at 24 h post-infection (Fig. 7c). Subsequently, we performed the visualization of origin genome profiles via IGV, showing that the TUT1 motif can bind NCOR2 between exon 14 and 15 (Fig. 7d), suggesting that TUT1 may be involved in the AS event of exon 14 of NCOR2 and mediate the generation of NCOR2-013 isoforms (Fig. 2). To determine whether the effect on NCOR2 AS was mediated by direct binding of TUT1, we performed RIP assay in T. marneffei-infected TUT1-overexpressing THP-1 macrophages at 24 h post-infection, and found that TUT1 binds to NCOR2 pre-mRNA (Fig. 7e). Moreover, at 24 h post infection, the expression of NCOR2-013 isoforms was significantly increased in the T. marneffei-infected TUT1-overexpressing THP-1 macrophages, but significantly decreased in the T. marneffei-infected TUT1-knockdown THP-1 macrophages (Fig. 7f–i). These results indicated that TUT1 plays an important role in the occurrence of AS in NCOR2 upon T. marneffei infection.

We then investigated the effect of TUT1 overexpression or knockdown on T. marneffei clearance in THP-1 macrophages. As a result, more T. marneffei were detected in TUT1-overexpressing THP-1 macrophages at 24 h post-infection (Fig. 7j), and pro-inflammatory factors (TNF-α, IL-1β) were down-regulated at 24 h post-infection (Fig. 7l). However, the opposite trend was observed in TUT1-knockdown macrophages (Fig. 7k, m), suggesting that TUT1 mediates T.marneffei-impaired macrophage clearance of fungi. Collectively, these data suggest that T. marneffei suppresses macrophages inflammation by producing the truncated protein NCOR2-013 via TUT1-regulated AS (Fig. 8).

**Fig. 8: Schematic diagram: TUT1-mediated alternative splicing produces a truncated protein NCOR2-013 that suppresses *T. marneffei*-stimulated macrophage inflammation.**

Discussion

The diverse products resulting from AS play a crucial role in biological functions, particularly in the regulation of cellular immune responses, which is supported by increasing evidence^31,32. In this study, we reported drastic changes in AS event profile of macrophage genes upon T. marneffei infection by high-throughput RNA sequencing, and found that NCOR2-013 transcript, a truncated variant of NCOR2 / SMRT, was significantly upregulated during T. marneffei infection. Mechanistically, we found that NCOR2-013 inhibits JunB-mediated transcriptional activation of pro-inflammatory factors TNF-α and IL-1β. Furthermore, we identified an AS regulator RBP, TUT1, which is involved in promoting T. marneffei immune evasion via regulation of NCOR2-013 production. Taken together, these findings elucidate for the first time how T. marneffei escapes macrophage killing from the perspective of cellular AS.

During the evolution of pathogens, some evasion strategies have been evolved to evade the host innate immunity. The most common strategy is to inhibit innate immune pathways through the interactions between pathogen virulence proteins and host proteins^11,33,34,35. In recent years, growing evidence supports the important role of AS in regulating innate immunity^31,32. Thousands of AS events have been detected in human dendritic cells and macrophages caused by the bacterial challenges^23,36,37. To date, little is known about the landscape of AS events associated with T. marneffei-infected macrophages. Therefore, we performed a comprehensive analysis of AS events in human THP-1 macrophage challenged by T. marneffei infection, and found 1757 different AS events in T. marneffei-infected macrophages compared to control cells, which is comparable to bacterial infections, such as M. tuberculosis²³, suggesting increased AS occurs upon pathogen-host interactions. Subsequently, host genes that harbor differential AS events were highly enriched in pathogen immune-related signaling pathways, indicating AS of these genes may serve as another mechanism for macrophages to resist T. marneffei infection or T. marneffei immune escape. Finally, 6 immune-related regulators were found to have the significantly different AS events, suggesting that AS is involved in the key steps of regulating T. marneffei immune response.

Typically, protein-coded genes can produce alternatively spliced variants of multiple transcripts, including some non-functional and/or truncated variants of the proteins that mediate different biological functions. The reasons for the significant biological differences of these alternately spliced variants may include that the truncated regions have certain biological functions. In immune cells, the expression levels of different alternative spliced variants may have significant effects on the activation of signaling pathways, immune status, and antibacterial capacity. For instance, MyD88L and MyD88S are long isoforms and short isoforms of MyD88 proteins, which play a role in activating and inhibiting innate immunity, respectively^38,39. Dengue virus deregulates innate immune response via inducing SAT1 exon 4 skipping to produce an isoform of SAT1²². In addition, up-regulation of the RAB8B truncated variant helps M. tuberculosis evade killing effect of macrophage phagosome²³. However, in the field of fungal-host interactions, spliced variants have been poorly studied.

In the present study, we identified an alternative spliced variant of NCOR2/SMRT, NCOR2-013, in T. marneffei-infected macrophages. Of note, normal THP-1 macrophages barely express NCOR2-013 (Fig. 3b), suggesting NCOR2-013 may not be a protein that maintains the normal cell functions. In this case, T. marneffei may use NCOR2-013 to implement some biological processes that favor its survival in macrophages. NCOR2/SMRT is a large nuclear receptor that form a co-repressor in the nucleus with a molecular weight up to 274 kDa²⁵. Interestingly, as one of the 25 isoforms of NCOR2/SMRT, NCOR2-013 has a molecular weight of 17 kDa, which is only one-sixteenth of NCOR2/SMRT^40,41. Structurally, NCOR2-013 retains only 4 exons compared with NCOR2/SRMT. Nevertheless, NCOR2-013 still retains some biological functions similar to NCOR2/SMRT. NCOR2-013 acts as part of a multisubunit complex that includes TBL1XR1/TBLR1 and HDAC3 to inhibit chromatin unfolding and specifically block JunB-mediated basal transcriptional activity of pro-inflammatory genes^25,42,43. The T. marneffei-induced short isoform functions similarly to the long isoform, but differently from those of the other pathogens mentioned above^23,36,37. Our data demonstrate that NCOR2-013 has a highly effective anti-inflammatory function at such a small molecular weight. Co-IP results showed that NCOR-013 did not bind to NCOR2/SMRT, indicating that NCOR2-013 had an anti-inflammatory effect independent of NCOR2/SMRT. More interestingly, even overexpression of JunB did not reverse the anti-inflammatory response of NCOR2-013, which implies a strong affinity of NCOR2-013 for JunB or a role for JunB in transcriptional regulation through phosphorylation of JunB (p-JunB). Furthermore, LPS, but not IFN-γ, partially overcomes NCOR2-013-mediated inhibition of inflammatory response in macrophages, suggesting that NCOR2-013 may only mediate inhibition of specific inflammatory pathways. Previous studies have demonstrated that the IFN-γ-induced signaling pathway selectively targets SMRT through the MAPK signaling pathway. Upon activation of MAPK/ERK kinase, SMRT undergoes phosphorylation and is subsequently redifferentiated from the nucleus to the cytoplasm. This process may lead to the relief of repression on target genes that are specific to SMRT and NCoR-SMRT response. In contrast, TLR4 induces the removal of NCoR from target genes through the p65 subunit of NF-κB²⁵.. Collectively, from our results, NCOR2-013 may have similar biological effects as NCRO2/SMRT, but with its own regulatory features. It will be an interesting topic for future studies to uncover the specific molecular mechanisms responsible for the action of NCOR2-013.

In terms of epigenetic mechanism, the NCOR2-013 regulatory complex depends on HDAC3 to inhibit histone acetylation, thereby inhibiting the transcriptional activation response of pro-inflammatory factors²⁵. HDAC3 is a chromatin-modifying enzyme that silences transcription via inhibition of histones acetylation. Traditionally, HDAC3 requires interacts with NCRO2/SMRT to engage its catalytic activity^44,45,46. Interestingly, we found that HDAC3 can interact with NCOR2-013 rather than NCOR2 / SMRT to exert an inhibitory effect, which is similar to the results of a recent study indicating that HDAC3 is recruited to ATF2-bound sites in the absence of NCOR2 / SMRT⁴⁷. In this study, we found that T. marneffei-infected NCOR2-013-overexpressing macrophages have more HDAC3 protein enrichment in promoter regions of pro-inflammatory factors, and conversely, significantly reduced histone acetylation levels in these regions, which are similar to results of other studies⁴⁸, demonstrating that HDAC3 mediates inflammatory reactivity in macrophages.

AS processes are controlled by splicing regulators, mainly RBPs. In this study, TUT1 was identified as an anti-inflammatory factor that was important for NCOR2-013 production and T. marneffei immune evasion. TUT1 is a nucleotidyl transferase that functions as both a terminal uridylyltransferase and a nuclear poly(A) polymerase, which can specifically add and remove nucleotides from the 3’ end of small nuclear RNAs as well as select mRNAs, thereby affecting gene expression^49,50. However, little is known about the role of TUT1-mediated AS in immune responses. The RIP result demonstrated that TUT1 is capable of interacting with pre-NCOR2 mRNA, indicating that TUT1 is involved in the regulation of NCOR2-013 production. TUT1 overexpression increased the expression of NCOR2-013 and decreased the expression of pro-inflammatory factors as well as antifungal capacity of macrophages, partly by regulating AS of NCOR2. On the contrary, TUT1 knockdown led to the opposite results, suggesting that TUT1 acts as a splicing regulatory factor that promotes T. marneffei immune escape.

T. marneffei is a dimorphic pathogenic fungi that grows filamentously at 25 °C and yeast at 37 °C. The transformation process from conidia to yeast may be an important virulence mechanism of T. marneffei against killing by host phagocytes. Previous studies have demonstrated that conidia replicate intracellularly by binary fission and express yeast-phase specific antigen within 24 h of phagocytosis^51,52,53. In contrast, in the absence of macrophages, T. marneffei conidia take at least 3 weeks to fully convert to the yeast form⁵⁴. Given that it is unclear whether the yeasts produced by these two transformation processes are different, we prioritized the use of a conidia-cell co-culture for at least 24 h approach, which more closely mimics the in vivo environment. Nonetheless, the potential that this phenomenon of T. marneffei suppressing the host macrophage inflammatory response could be affected by conidia-dependence or conidia-yeast intermediate stages cannot be disregarded and warrants further investigation.

In addition, this study has several limitation that need to be considered. First, our current work leans on overexpression systems to investigate the function of NCOR2-013, this approach does not provide direct evidence that the intact form suppresses cytokine production and bactericidal activity. Future research endeavors will seek to employ more precise techniques to provide a more comprehensive understanding of the biological role of NCOR2-013. Second, how T. marneffei infection affects TUT1 expression still needs to be systematically investigated. Third, HIV infection was not considered in this study, but NCOR2-013-mediated T. marneffei survival may be different in the context of HIV infection, which should be an interesting issue for the future studies. Finally, while preliminary data indicate that LPS, a component of the bacterial cell wall, enhances the expression of NCOR2-013 and TUT1 in macrophages (Supplementary Fig. 4), suggesting that such an induction is not specific to T. marneffei alone. Thus, it remains essential to explore which pathogens can specifically induce TUT1-mediated NCOR2-013 AS events and to understand the underlying molecular mechanisms.

In summary, we systematically analyzed AS events in T. marneffei-infected macrophages and identified TUT1 as the splicing regulator that was important for the NCOR2-013 production and T. marneffei immune evasion. TUT1 inhibits inflammatory response via inducing AS of NCOR2 to produce a novel transcript, NCOR2-013. The truncated protein NCOR2-013 acts as an important T. marneffei immune escape-related factor in macrophages through recruiting TBL1XR1/TBLR1 and HDAC3 to form a regulatory complex to inhibit histone acetylation modification, which specifically blocks JunB-mediated transcriptional activity of pro-inflammatory genes (Fig. 8). TUT1 and NCOR2-013 represent the potential targets for the development of mechanism-based strategies for the prevention of talaromycosis.

Methods

Reagents

Human recombinant IFN-γ and GM-CSF antibodies were purchased from Solarbio Science & Technology (China), and the LPS (from E. coli) was from Sigma-Aldrich (USA). The IRDye 680RD donkey anti-mouse and IRDye 800CW donkey anti-rabbit antibodies were purchased from LI-COR Biosciences (USA). Anti-rabbit IgG (H + L), F(ab’)2 fragment (Alexa Fluor® 647 Conjugate), anti-FLAG (D6W5B), anti-PCNA (D3H8P), anti-β-actin (8H10D10), anti-normal Rabbit IgG, anti-HDAC3 (D2O1K), anti-TBL1XR1/TBLR1 (D4J9C), anti-JunB (C37F9), anti-phospho-JunB (Thr102 / Thr104) (D3C6), anti-JunD (D17G2), anti-c-Jun (60A8), anti-phospho-c-Jun (Ser73), anti-acetyl-histone H3 (Lys27) (D5E4), and anti-GAPDH (D16H11) antibodies were purchased from Cell Signaling Technology (USA). Anti-NCOR2 antibody was obtained from Novus Biologicals (USA). Anti-TUT1 (PA5-50151) antibody was obtained from Invitrogen (USA). For western blot, all primary antibodies were diluted at 1 : 1000 and secondary antibodies at 1 : 20,000. For immunofluorescence, the primary antibody was diluted 1 : 200. For IP, RIP, and ChipIP, 1 µg of primary antibody was used to precipitate proteins.

T. marneffei strain and culture conditions

T. marneffei strain (ATCC18224) was obtained from American Type Culture Collection (ATCC) and used in all infection experiments. T. marneffei was cultivated on potato dextrose agar (Sigma-Aldrich, USA) and grew at 27 °C for 7–10 days. The conidia were harvested as previously described^52,53,55. Briefly, colonies of T. marneffei were flooded with PBS, and the conidia were filtered through sterile glass wool (Corning, USA) to remove clumps and fragments. The conidia-enriched supernatant was collected by centrifugation at 14,000 × g for 3 min and enumerated using a hemacytometer. Conidia were resuspended in sterile PBS at 10⁷/mL and stored at 4 °C until use. The collected conidia were tested for viability after each collection and used within 2 weeks.

THP-1 macrophages and human monocyte-derived macrophages (hMDMs)

THP-1 cells were obtained from ATCC and cultured in RPMI 1640 medium (Solarbio Science & Technology, China) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin / streptomycin (Solarbio Science & Technology, China). The cells were allowed to differentiate in macrophages with the treatment of 100 ng/mL phorbol myristate acetate (Sigma-Aldrich, USA) for 48 h. Human monocyte-derived macrophages (hMDMs) were obtained from human peripheral blood mononuclear cells (PBMCs) of healthy adult donors. Briefly, PBMCs were isolated by density gradient centrifugation (Ficoll 1.077 g/mL, Sigma, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Solarbio Science & Technology, China) supplemented with 10% FBS and 1% penicillin / streptomycin. To induce differentiation to macrophages, PBMCs were cultured for 7 days in the presence of 25 ng/mL recombinant human macrophage colony-stimulating factor (rh-MCSF, Solarbio Science & Technology, China). The cells were > 95% CD14⁺ cells, as determined by flow cytometry with an anti-CD14 antibody (BD Biosciences, USA). All cells were cultured at 37°C containing 5% CO₂. In this study, the hMDMs were differentiated from PBMCs isolated from three healthy blood donors.

T. marneffei infection of macrophages

T. marneffei infection of macrophages followed the method described in previous studies (56). Briefly, macrophages were seeded at a density of 2 × 10⁶ cells/well in 6-well plates, 1 × 10⁶ cells/well in 12-well plate, or 1 × 10⁵ cells/well in 96-well plates, and then were infected with T. marneffei conidia at a multiplicity of infection (MOI) of 10. The infection lasted for 2 h, after which the medium was discarded and the cells were washed with pre-warmed PBS (37 °C). The infected cells were then cultured in 10% DMEM until the time required for the subsequent experiments.

Lentiviral transfection

Lentiviral siRNA vector system GV248 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) and lentiviral overexpression vector system GV208 (Ubi-MCS-3FLAG-EGFP) were constructed, packaged, and purified by GeneChem (China), and manipulated according to the protocols provided by the manufacturer. Briefly, THP-1 cells were seeded in 96-well plates and cultured overnight prior to transfection, and hMDMs were differentiated from PBMCs in 96-well plates. Subsequently, siRNA-TUT1 lentivirus, Flag-TUT1-overexpressing lentivirus, Flag-NCOR2-013-overexpressing lentivirus and their negative control (NC) empty lentivirus vector were added to serum-free RPMI 1640 medium (for THP-1 cells) or DMEM medium (for THP-1 macrophages and hMDMs) media, respectively. The THP-1 cells or THP-1 macrophages were infected with an MOI of 30 and hMDMs were infected with an MOI of 50 according to the manufacturer’s protocols. The transfection lasted for 48 h, and the medium containing lentivirus was then removed and fresh complete medium was added. The sequences of siRNA-TUT1 were designed as follows: siRNA-TUT1-F: 5’-CCGGGATCTTGACCTCTTCTTGGACTCGAGATCCAAGAAGAGGTCAAGATCTTTTTG-3’, siRNA-TUT1-R: 5’-AATTCAAAAAGATCTTGACCTCTTCTTGGATCTCGAGATCCAAGAAGAGGTCAAGATC-3’.

Reverse transcription quantitative PCR (RT-qPCR)

Total cellular RNA was extracted using Trizol and chloroform reagents (Invitrogen, USA), followed by precipitation in isopropyl alcohol and 75% ethanol. Reverse transcription was performed using a reverse transcription kit (TAKARA, Japan). RT-qPCR was performed using SYBR Premix ExTaq^TM (TAKARA, Japan) by StepOnePlus real-time PCR system (Thermo Fisher Scientific, USA). Data were analyzed with 2^{−ΔΔ Ct} method. Relative gene expression was calculated as the ratio of target gene expression versus reference GAPDH gene expression. Primers for NCOR2 were designed using primer-BLAST and Primer Premier 5 software to amplify the exon region of NCOR2. Primers for IL-1β, TNF-α, etc were from Primer Bank. All primers targeted exon region and were optimized before RT-qPCR experiments. Primer sequences are provided in Supplementary Table 3. The primer design principles and primer sequences for calculating NCOR2-013 expression levels are shown in Supplementary Fig. 5 and Supplementary Table 4.

Western blot

Total proteins, nuclear proteins, or cytoplasmic proteins were extracted using the cell lysis buffer (Cell Signaling Technology, USA), nuclear protein and cytoplasmic protein extraction kit (Beyotime, China), respectively, according to the manufacturer’s instructions. Protein concentration was measured using a BCA protein assay kit (Beyotime, China). Total proteins, nuclear proteins, or cytoplasmic proteins were separated by 10–12% SDS-PAGE (BOSTER, China) and transferred to PVDF membranes (Bio-Rad, USA). After blocking with 5% nonfat milk (BD Biosciences, USA) for 1 h, the membranes were incubated with primary antibody and 5% nonfat milk overnight at 4 °C (the dilution ratio of primary antibodies is 1 : 1000). The membranes were then washed three times with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05%V/V Tween-20) and incubated with the corresponding secondary antibody for 1 h (the dilution ratio of secondary antibodies is 1 : 20,000). Images were performed using the Odyssey CLX two-color infrared laser imaging system (LI-COR Biosciences, USA). The densitometric analysis of blots was performed by Image Studio Ver 5.2 software (LI-COR Biosciences, USA). The values were normalized to those of control β-actin or GAPDH (for total protein, cytoplasmic protein), or PCNA (for nuclear protein).

Determination of colony-forming units (CFU)

CFU method was used to quantify the amount of T. marneffei in macrophages as previously described^56,57,58,59. Briefly, the culture supernatant was discarded, and the macrophages were washed twice with PBS and then lysed with sterile water to release the fungus. The intracellular T. marneffei were collected by centrifugation (14,000 g, 5 min) and suspended with sterile water to 200 μL. Four gradient serial dilutions (10⁰, 10⁻¹, 10⁻², 10⁻³) were performed, and 5 μL of each diluted sample was plated onto YPD agar. CFUs were counted after 24 h incubation at 30 °C. The fungal load was calculated as number of colonies × dilution gradient × 40, where 40 is the ratio of 200 μL to 5 μL.

Immunofluorescence

Immunofluorescence was performed to detect the subcellular localization of NCOR2-013. Flag-NCOR2-013-overexpressed THP-1-derived macrophages were plated onto a cover slip. The cells were fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, and then incubated with 1% Triton X-100 PBS solution for 20 min at room temperature and washed 3 times with PBS. After blocking with 5% nonfat milk for 1 h, the cells were incubated with 1 : 200 primary anti-Flag antibody at 4 °C overnight, washed with PBS, and then incubated with specific fluorescence-conjugated secondary antibody for 1 h at room temperature. Finally, DAPI (4, 6-diamidino-2-phenylindole) was added to label nuclei. Immunofluorescence signals were detected using fluorescence microscopy (Nikon, Japan).

Cytometric bead array (CBA)

CBA was used to determine the levels of secreted TNF-α and IL-1β according to the manufacturer’s instructions (BD Biosciences, USA). Briefly, the culture supernatants were collected, and 50 µL of supernatant was mixed with 50 µL capturing beads and 50 µL detection reagents of the kit, then incubated for 2 h at room temperature. The beads were washed and re-suspended in 300 µL wash buffer and analyzed by flow cytometry (Beckman Coulter, USA).

Immunoprecipitation (IP) and Co-Immunoprecipitation (Co-IP)

Cell lysates were prepared in cell lysis buffer (for immunoprecipitation, Cell Signaling Technology, USA) or Co-IP buffer (for co-immunoprecipitation, Cell Signaling Technology, USA) supplemented with phosphatase and protease inhibitors. Lysates were pre-cleared with anti-mouse or anti-rabbit IgG agarose beads for 30 min. Proteins were precipitated with 1 µg of primary antibodies for 1 h and subsequently collected with anti-mouse or anti-rabbit IgG agarose beads by overnight incubation. Bead-protein complex was washed 3 times with PBS, and then the immunoprecipitates were subsequently eluted and separated by SDS-PAGE gel electrophoresis, followed by western blot.

Chromatin immunoprecipitation (ChIP)

Chromatin immunoprecipitation was carried out using agarose ChIP kit (Thermo Scientific, USA) according to the manufacturer’s instructions. Immunoprecipitation was performed with 1 µg of each antibody or negative control IgG. Real-time PCR was performed on the purified DNA and input DNA using SYBR Premix ExTaq^TM (TAKARA, Japan) by StepOnePlus real-time PCR system (Thermo Fisher Scientific, USA). The primer sequences are provided in Supplementary Table 5. The results were computed as percent antibody bound per input and data were displayed after subtracting control IgG values. Data from three independent experiments were collected, and mean value and standard deviation (SD) were presented.

RNA-binding protein Immunoprecipitation (RIP)

RIP assay was performed using RIP RBP Immunoprecipitation kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol. Briefly, cells were collected by using RIP lysis buffer and incubated with beads and antibody complexs overnight. After washing 3 times, RNAs that binding to protein were eluted and qRT-PCR was used to detect target RNAs co-immunoprecipitated with the antibody. Meanwhile, The quantification of target RNAs was carried out by agarose gel electrophoresis. Specifically, a 1% agarose gel in 1X TAE buffer was prepared and loaded with cDNA samples that were reverse transcribed from the target RNAs and mixed with loading buffer. The gel was then subjected to electrophoresis at 100 V for 45 min. Ethidium bromide was used to stain the gel, which was subsequently visualized using a UV transilluminator.

Liquid chromatography/tandem mass spectrometry (LC-MS / MS)

Protein samples were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue. The gel bands were manually excised and and decolorize with decolorizing solution. A solution of tryptic peptides were then obtained by proteolysis and transferred to vials for LC-MS / MS analysis. LC-MS / MS analysis was performed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) in tandem with a a nano Acquity UPLC system (Waters, USA). A total of 3 μl was loaded (Thermo Fisher Scientific Acclaim PepMap trap column, C18, 100 μm × 25 cm, and Acclaim PepMap self-packed analytical column, C18, 75 μm × 25 cm), and the eluted peptides were introduced directly into the linear ion trap mass spectrometer at a flow rate of 200 nL/min and a spray voltage of 2.0 kV. The MS and MS/MS data were acquired using Xcalibur (Thermo Fisher Scientific, USA).

RNA-seq and function enrichment analysis

Total RNA was extracted using Trizol regent (Invitrogen, USA) according to the manufacture’s protocol. RNA quality was assessed on Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Then, mRNA was enriched by Oligo (dT) beads, while mRNA was enriched by removing rRNA by Ribo-ZeroTM magnetic kit (Epicenter, USA). Subsequently, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNA. The cDNA was purified with QIAquick PCR extraction kit (Qiagen, Netherlands) and ligated to Illumina sequencing adapters. The library preparation and RNA-seq was performed by GeneChem (China). In this study, a total of 200 ng of RNA in 50 μL was used for preparation of paired-end sequencing libraries. The RNA samples were subjected to quality control, cluster generation and sequencing. The reads were de-multiplexed and converted to FASTA format. Finally, alignment and QC were performed. Transcript counts were normalized using conditional quantile normalization for transcript length and GC content. Paired-end clean reads were mapped to the reference genome using HISAT2. 2.4 to calculate read counts for each unigene. The differential expression between T. marneffei uninfected and infected macrophages using a two-sided Wald test in DESeq2 package. The statistical significance was set to |log2 fold of change| > 0.4 and adjusted P value < 0.05. The heatmap was constructed using the pheatmap R package to show the expression intensity and direction of the DEGs. The potential function of DEGs with different infection times and modules enrichment analyses were predicted using Clusterprofiler R package. Genes with |log2 fold of change| > 0.4 and adjusted P value < 0.05 were selected for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. Gene set enrichment analysis (GSEA) was performed using the ‘clusterProfiler’ R package.

Statistics and reproducibility

The results are expressed as the mean ± standard deviation (SD). The level of statistical significance was set at P < 0.05 using an unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01. All statistical analyses were performed using GraphPad Prism software. The sample and replicate size were indicated in the figure legends.

Ethics statement

The studies involving human participants were reviewed and approved by Ethics and Human Subjects Committee of Guangxi Medical University (Ethical Review No. 20220018). The patients/participants provided their written informed consent to participate in this study. All ethical regulations relevant to human research participants were followed.

Data availability

Source data for Figures and Supplementary Figures are provided as Supplementary Data File (Supplementary Data 1) and uncropped Western blots are provided in Supplementary Fig. 6 along with this article. We deposited the raw fastq files in the Sequence Read Archives (SRA) of the National Center for Biotechnology Information (NCBI) under accession number GSE200512, GSE154779 of Bioproject PRJNA824858, PRJNA647412, respectively. Mass spectrometry proteomics data have been deposited on figshare under the following https://doi.org/10.6084/m9.figshare.24160839. All other data are available from the corresponding author on reasonable request.

References

Yilmaz, N., Visagie, C. M., Houbraken, J., Frisvad, J. C. & Samson, R. A. Polyphasic taxonomy of the genus Talaromyces. Stud. Mycol. 78, 175–341 (2014).
CAS PubMed PubMed Central Google Scholar
Vanittanakom, N., Cooper, C. R. Jr., Fisher, M. C. & Sirisanthana, T. Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects. Clin. Microbiol. Rev. 19, 95–110 (2006).
CAS PubMed PubMed Central Google Scholar
Limper, A. H., Adenis, A., Le, T. & Harrison, T. S. Fungal infections in HIV/AIDS. Lancet Infect. Dis. 17, e334–e343 (2017).
PubMed Google Scholar
Jiang, J. et al. Effects of talaromyces marneffei infection on mortality of HIV/AIDS patients in southern China: a retrospective cohort study. Clin. Microbiol. Infect. 25, 233–241 (2019).
CAS PubMed Google Scholar
Ning, C. et al. The Global Distribution, Drivers, and Burden of Talaromycosis 1964-2017. Boston, MA, USA., (abstr 749) (2020).
WHO. First Meeting of the WHO Antifungal Expert Group on Identifying Priority Fungal Pathogens. World Health Organization, https://www.who.int/publications/i/item/9789240006355 (2020).
Narayanasamy, S. et al. A global call for talaromycosis to be recognised as a neglected tropical disease. Lancet Glob. Health 9, e1618–e1622 (2021).
CAS PubMed PubMed Central Google Scholar
Ellett, F. et al. Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo. PLoS Pathog. 14, e1007063 (2018).
PubMed PubMed Central Google Scholar
Chan, J. F., Lau, S. K., Yuen, K. Y. & Woo, P. C. Talaromyces (Penicillium) marneffei infection in non-HIV-infected patients. Emerg. Microbes Infect. 5, e19 (2016).
PubMed PubMed Central Google Scholar
Price, J. V. & Vance, R. E. The macrophage paradox. Immunity 41, 685–693 (2014).
CAS PubMed Google Scholar
Wang, L. et al. Host-mediated ubiquitination of a mycobacterial protein suppresses immunity. Nature 577, 682–688 (2020).
CAS PubMed Google Scholar
Wagener, J., MacCallum, D. M., Brown, G. D. & Gow, N. A. Candida albicans Chitin increases Arginase-1 activity in human macrophages, with an impact on macrophage antimicrobial functions. mBio 8, e01820–16 (2017).
CAS PubMed PubMed Central Google Scholar
Wei, W. et al. Talaromyces marneffei promotes M2-like polarization of human macrophages by downregulating SOCS3 expression and activating the TLR9 pathway. Virulence 12, 1997–2012 (2021).
CAS PubMed PubMed Central Google Scholar
Chen, R. et al. Activation of autophagy and IL-10 production are regulated by Jun N-terminal kinase 1 and 2 and p38 mitogen activated protein kinase signaling pathways during Talaromyces marneffei infection within dendritic cells. Microb. Pathog. 139, 103891 (2020).
CAS PubMed Google Scholar
Chen, R. et al. Role of intracellular free calcium in killing penicillium marneffei within human macrophages. Microb. Pathog. 83-84, 29–34 (2015).
CAS PubMed Google Scholar
Li, Y. et al. LncSSBP1 functions as a negative regulator of IL-6 through interaction with hnRNPK in bronchial epithelial cells infected with talaromyces marneffei. Front. Immunol. 10, 2977 (2019).
CAS PubMed Google Scholar
Wang, E. T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).
CAS PubMed PubMed Central Google Scholar
Melé, M. et al. Human genomics. The human transcriptome across tissues and individuals. Science 348, 660–665 (2015).
PubMed PubMed Central Google Scholar
Pan, Q., Shai, O., Lee, L. J., Frey, B. J. & Blencowe, B. J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 40, 1413–1415 (2008).
CAS PubMed Google Scholar
Kim, M. S. et al. A draft map of the human proteome. Nature 509, 575–581 (2014).
CAS PubMed PubMed Central Google Scholar
Ma, Y. et al. Arabidopsis exoribonuclease USB1 interacts with the PPR-domain protein SOAR1 to negatively regulate abscisic acid signaling. J. Exp. Bot. 71, 5837–5851 (2020).
CAS PubMed PubMed Central Google Scholar
Pozzi, B. et al. Dengue virus targets RBM10 deregulating host cell splicing and innate immune response. Nucleic Acids Res. 48, 6824–6838 (2020).
CAS PubMed PubMed Central Google Scholar
Kalam, H., Fontana, M. F. & Kumar, D. Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection. PLoS Pathog. 13, e1006236 (2017).
PubMed PubMed Central Google Scholar
Gokhale, N. S. et al. Altered m(6)A modification of specific cellular transcripts affects flaviviridae infection. Mol. cell 77, 542–555 (2020).
CAS PubMed Google Scholar
Glass, C. K. & Saijo, K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nat. Rev. Immunol. 10, 365–376 (2010).
CAS PubMed Google Scholar
Han, H. et al. TRRUST v2: an expanded reference database of human and mouse transcriptional regulatory interactions. Nucleic Acids Res. 46, D380–d386 (2018).
CAS PubMed Google Scholar
Lukey, M. J., Greene, K. S., Erickson, J. W., Wilson, K. F. & Cerione, R. A. The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy. Nat. Commun. 7, 11321 (2016).
CAS PubMed PubMed Central Google Scholar
Sanchez, H. N. et al. B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids. Nat. Commun. 11, 60 (2020).
CAS PubMed PubMed Central Google Scholar
Massimino, M. et al. Non ABL-directed inhibitors as alternative treatment strategies for chronic myeloid leukemia. Mol. Cancer 17, 56 (2018).
PubMed PubMed Central Google Scholar
Park, S. Y. et al. Structural basis of the specific interaction of SMRT corepressor with histone deacetylase 4. Nucleic Acids Res. 46, 11776–11788 (2018).
CAS PubMed PubMed Central Google Scholar
Su, Z. & Huang, D. Alternative splicing of Pre-mRNA in the control of immune activity. Genes (Basel) 12, 574 (2021).
CAS PubMed Google Scholar
Chang, M. X. & Zhang, J. Alternative Pre-mRNA splicing in mammals and teleost fish: a effective strategy for the regulation of immune responses against pathogen infection. Int. J. Mol. Sci. 18, 1530 (2017).
PubMed PubMed Central Google Scholar
Yan, D., Wang, X., Luo, L., Cao, X. & Ge, B. Inhibition of TLR signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs. Nat. Immunol. 13, 1063–1071 (2012).
CAS PubMed Google Scholar
Wang, Z. et al. PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection. PLoS Pathog. 17, e1009940 (2021).
CAS PubMed PubMed Central Google Scholar
Jiang, H. W. et al. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cell. Mol. Immunol. 17, 998–1000 (2020).
CAS PubMed Google Scholar
Rodrigues, R., Grosso, A. R. & Moita, L. Genome-wide analysis of alternative splicing during dendritic cell response to a bacterial challenge. PLoS One 8, e61975 (2013).
CAS PubMed PubMed Central Google Scholar
Liu, H. et al. Alternative splicing analysis in human monocytes and macrophages reveals MBNL1 as major regulator. Nucleic Acids Res. 46, 6069–6086 (2018).
CAS PubMed PubMed Central Google Scholar
Pizzolla, A., Smith, J. M., Brooks, A. G. & Reading, P. C. Pattern recognition receptor immunomodulation of innate immunity as a strategy to limit the impact of influenza virus. J. Leucoc. Biol. 101, 851–861 (2017).
CAS Google Scholar
De Arras, L. & Alper, S. Limiting of the innate immune response by SF3A-dependent control of MyD88 alternative mRNA splicing. PLoS Genet. 9, e1003855 (2013).
PubMed PubMed Central Google Scholar
Sande, S. & Privalsky, M. L. Identification of TRACs (T3 receptor-associating cofactors), a family of cofactors that associate with, and modulate the activity of, nuclear hormone receptors. Mol. Endocrinol. 10, 813–825 (1996).
CAS PubMed Google Scholar
Chen, J. D. & Evans, R. M. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377, 454–457 (1995).
CAS PubMed Google Scholar
Ogawa, S. et al. A nuclear receptor corepressor transcriptional checkpoint controlling activator protein 1-dependent gene networks required for macrophage activation. PNAS 101, 14461–14466 (2004).
CAS PubMed PubMed Central Google Scholar
Ghisletti, S. et al. Cooperative NCoR/SMRT interactions establish a corepressor-based strategy for integration of inflammatory and anti-inflammatory signaling pathways. Genes Dev. 23, 681–693 (2009).
CAS PubMed PubMed Central Google Scholar
Guenther, M. G., Barak, O. & Lazar, M. A. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol. 21, 6091–6101 (2001).
CAS PubMed PubMed Central Google Scholar
Watson, P. J., Fairall, L., Santos, G. M. & Schwabe, J. W. Structure of HDAC3 bound to co-repressor and inositol tetraphosphate. Nature 481, 335–340 (2012).
CAS PubMed PubMed Central Google Scholar
You, S. H. et al. Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo. Nat. Struct. Mol. Biol. 20, 182–187 (2013).
CAS PubMed PubMed Central Google Scholar
Nguyen, H. C. B., Adlanmerini, M., Hauck, A. K. & Lazar, M. A. Dichotomous engagement of HDAC3 activity governs inflammatory responses. Nature 584, 286–290 (2020).
CAS PubMed PubMed Central Google Scholar
Ghiboub, M. et al. HDAC3 mediates the inflammatory response and LPS Tolerance in human monocytes and macrophages. Front. Immunol. 11, 550769 (2020).
CAS PubMed PubMed Central Google Scholar
Yashiro, Y. & Tomita, K. Function and regulation of human terminal Uridylyltransferases. Front. Genet. 9, 538 (2018).
CAS PubMed PubMed Central Google Scholar
Yamashita, S., Takagi, Y., Nagaike, T. & Tomita, K. Crystal structures of U6 snRNA-specific terminal uridylyltransferase. Nat. Commun. 8, 15788 (2017).
CAS PubMed PubMed Central Google Scholar
Pruksaphon, K. et al. Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei. Sci. Rep. 10, 21169 (2020).
CAS PubMed PubMed Central Google Scholar
Cogliati, M. et al. Development of an in vitro macrophage system to assess penicillium marneffei growth and susceptibility to nitric oxide. Infect. Immun. 65, 279–284 (1997).
CAS PubMed PubMed Central Google Scholar
Pruksaphon, K., Nosanchuk, J. D., Ratanabanangkoon, K. & Youngchim, S. Talaromyces marneffei infection: virulence, intracellular lifestyle and host defense mechanisms. J. Fungi (Basel) 8, 200 (2022).
CAS PubMed Google Scholar
Lau, S. K. et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid identification of mold and yeast cultures of Penicillium marneffei. BMC Microbiol. 16, 36 (2016).
PubMed PubMed Central Google Scholar
Andrianopoulos, A. Laboratory maintenance and growth of Talaromyces marneffei. Curr. Protoc. Microbiol. 56, e97 (2020).
PubMed Google Scholar
Kim, T., Zhang, Q., Li, J., Zhang, L. & Jokerst, J. V. A gold/silver hybrid nanoparticle for treatment and photoacoustic imaging of bacterial infection. ACS nano 12, 5615–5625 (2018).
CAS PubMed PubMed Central Google Scholar
Fan, D. et al. Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization. Nat. Med. 21, 808–814 (2015).
CAS PubMed PubMed Central Google Scholar
Waack, U. et al. CpaA is a Glycan-Specific Adamalysin-like protease secreted by acinetobacter baumannii that inactivates coagulation factor XII. mBio 9, e01606–e01618 (2018).
PubMed PubMed Central Google Scholar
Duvenage, L. et al. Inhibition of classical and alternative modes of respiration in candida albicans leads to cell wall remodeling and increased macrophage recognition. mBio 10, e02535–18 (2019).
CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

The authors gratefully acknowledge the financial supports by Guangxi Youth Science Fund Project (2021GXNSFBA196004, to W.W.), National Natural Science Foundation of China (NSFC;82302550, 81971934), Guangxi Bagui Scholar (to J.J.), Thousands of Young and Middle-aged Key Teachers Training Program in Guangxi Colleges and Universities (to J.J.), Guangxi Medical University Training Program for Distinguished Young Scholars (to J.J.), China Postdoctoral Science Foundation (2020M683212, to W.W.), and Guangxi Postdoctoral Special Foundation (to W.W.).

Author information

These authors contributed equally: Wudi Wei, Gang Wang, Hong Zhang, Xiuli Bao.

Authors and Affiliations

Guangxi Key Laboratory of AIDS Prevention and Treatment, School of Public Health, Guangxi Medical University, Nanning, 530021, Guangxi, China
Wudi Wei, Gang Wang, Xiuli Bao, Sanqi An, Lixiang Chen, Zongxiang Yuan, Rongfeng Chen, Junjun Jiang, Li Ye & Hao Liang
Guangxi-ASEAN Collaborative Innovation Center for Major Disease Prevention and Treatment, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
Wudi Wei, Hong Zhang, Sanqi An, Qiang Luo, Jinhao He, Chuanyi Ning, Jingzhen Lai, Rongfeng Chen, Junjun Jiang, Li Ye & Hao Liang
Nursing College, Guangxi Medical University, Nanning, 530021, Guangxi, China
Chuanyi Ning
Guangxi Biobank, Life Sciences Institute, Guangxi Medical University, Nanning, 530021, Guangxi, China
Jingzhen Lai & Hao Liang

Authors

Wudi Wei
View author publications
You can also search for this author in PubMed Google Scholar
Gang Wang
View author publications
You can also search for this author in PubMed Google Scholar
Hong Zhang
View author publications
You can also search for this author in PubMed Google Scholar
Xiuli Bao
View author publications
You can also search for this author in PubMed Google Scholar
Sanqi An
View author publications
You can also search for this author in PubMed Google Scholar
Qiang Luo
View author publications
You can also search for this author in PubMed Google Scholar
Jinhao He
View author publications
You can also search for this author in PubMed Google Scholar
Lixiang Chen
View author publications
You can also search for this author in PubMed Google Scholar
Chuanyi Ning
View author publications
You can also search for this author in PubMed Google Scholar
Jingzhen Lai
View author publications
You can also search for this author in PubMed Google Scholar
Zongxiang Yuan
View author publications
You can also search for this author in PubMed Google Scholar
Rongfeng Chen
View author publications
You can also search for this author in PubMed Google Scholar
Junjun Jiang
View author publications
You can also search for this author in PubMed Google Scholar
Li Ye
View author publications
You can also search for this author in PubMed Google Scholar
Hao Liang
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

H.L., L.Y., J.J., W.W. and G.W. provided research ideas and designed experiments. C.N., J.L., Z.Y. and R.C. provided reagents and participated in data analyses. W.W., H.Z., X.B., J.H. and L.C. performed the experiments. W.W., G.W., H.Z. S.A. and Q.L. analyzed data. W.W. and G.W. wrote the manuscript.

Corresponding authors

Correspondence to Junjun Jiang, Li Ye or Hao Liang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Communications Biology thanks Samuel Gonçalves and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Si Ming Man and Joao Valente.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data

nr-reporting-summary

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Wei, W., Wang, G., Zhang, H. et al. Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing. Commun Biol 6, 1046 (2023). https://doi.org/10.1038/s42003-023-05409-6

Download citation

Received: 16 May 2023
Accepted: 02 October 2023
Published: 16 October 2023
DOI: https://doi.org/10.1038/s42003-023-05409-6

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.