bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.253518; this version posted August 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 NLRP3 inflammasome contributes to host defense against 2 Talaromyces marneffei infection 3 4 Haiyan Ma1, Jasper FW Chan2, Yen Pei Tan2, Lin Kui1, Chi-Ching Tsang2, Steven LC Pei1, Yu- 5 Lung Lau1, Patrick CY Woo2, Pamela P Lee1* 6 7 1.Department of Pediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The 8 University of Hong Kong, Hong Kong SAR, China. 9 2.Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Hong Kong SAR, China 11 12 *Correspondence author. E-mail: [email protected] 13 14 Abstract 15 Talaromyces marneffei is an important thermally dimorphic pathogen causing disseminated 16 mycoses in immunocompromised individuals in southeast Asia. Previous study has 17 suggested that NLRP3 inflammasome plays a critical role in antifungal immunity. However, 18 the mechanism underlying the role of NLRP3 inflammasome activation in host defense 19 against T. marneffei remains unclear. We show that T. marneffei yeasts but not conidia 20 induce potent IL-1 response, which is differentially regulated in discrete immune cell types. 21 Dectin-1/Syk signaling pathway mediates pro-IL-1 production, and NLRP3 inflammasome is 22 activated to trigger the processing of pro-IL-1 into IL-1. The activated NLRP3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.253518; this version posted August 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 inflammasome partially promotes Th1 and Th17 immune responses against T. marneffei 24 yeasts. In vivo, mice with NLRP3 or caspase-1 deficiency exhibit higher mortality rate and 25 fungal load compared to wild-type mice. Herein, our study provides the first evidence that 26 NLRP3 inflammasome contributes to host defense against T. marneffei infection, which may 27 have implications for future antifungal therapeutic designs. 28 29 Introduction 30 Talaromyces marneffei is a dimorphic fungus geographically restricted to Southeast Asia. It 31 causes a form of endemic mycosis that predominantly affects individuals with HIV infection 32 and ranks the third most common opportunistic infection in acquired immunodeficiency 33 syndrome (AIDS), following tuberculosis and cryptococcosis (Sirisanthana & Supparatpinyo, 34 1998; Supparatpinyo et al, 1994; Vanittanakom et al, 2006; Cao et al, 2019). In highly 35 endemic regions such as Chiang Mai in Thailand and Haiphong in Vietnam, T. marneffei is in 36 fact the most common systemic opportunistic fungal infection in AIDS (Vanittanakom et al, 37 2006; Son et al, 2014). Compared with HIV-negative individuals, fungemia, hepatomegaly 38 and splenomegaly occur much more commonly in HIV-positive patients, supporting 39 profound T-lymphopenia as the key predisposing factor for disseminated talaromycosis 40 (Kawila et al, 2013). 41 42 Less commonly, talaromycosis occurs in other immunocompromised conditions such as 43 solid organ or hematopoietic stem cell transplantation, autoimmune diseases or primary 44 immunodeficiency diseases (PID) (Chan et al, 2016; Lee et al, 2012; Lee et al, 2014; Lee & 45 Lau, 2017; Luo et al, 2010; Stathakis et al, 2015). T. marneffei shares some common 46 characteristics with other phylogenetically related dimorphic fungi such as Histoplasma and bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.253518; this version posted August 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 47 Coccidioides. They exist naturally in the soil environment as conidia which enter the human 48 body via inhalation route, and are converted to the yeast form after being phagocytosed by 49 tissue-resident macrophages. These dimorphic fungal pathogens commonly result in 50 systemic infection when immunity is suppressed, and the uncontrolled proliferation of 51 yeasts in the macrophages eventually lead to dissemination via the reticuloendothelial 52 system. Paracoccidioides brasiliensis and P. lutzii, another dimorphic fungi which is endemic 53 in Latin America, is prevalent in immunocompetent individuals and co-infection with HIV is 54 uncommon, whereas talaromycosis, histoplasmosis and coccidioidomycosis are regarded as 55 AIDS-defining conditions in their respective endemic regions (Limper et al, 2017). They have 56 also been reported in patients with autoantibodies against interferon gamma (anti-IFN), 57 and PID characterized by impaired Th17 immune response such as autosomal dominant (AD) 58 hyper-IgE syndrome caused by loss-of-function STAT3 defect and AD chronic 59 mucocutaneous candidiasis (CMC) caused by gain-of-function STAT1 defect, as well as 60 CD40L deficiency and IFN- receptor deficiency. These acquired defects and inborn errors of 61 immunity provide evidence on the critical role of Th1 and Th17 immune response in host 62 defense against these dimorphic fungi (Lee & Lau, 2017; Lee et al, 2019). However, 63 knowledge about host-pathogen interaction in endemic mycoses is much less 64 comprehensive than other common opportunistic fungal pathogens of worldwide 65 distribution. In particular, little is known about how T. marneffei is recognized by innate 66 immune cells and the ensuing cytokine signaling that triggers adaptive immune response. 67 68 Immune responses against fungal infection are mounted by fungal pathogen-associated 69 molecular patterns (PAMPs) that can be recognized by pathogen recognition receptors (PRRs), 70 including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs) bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.253518; this version posted August 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 71 (Erwig & Gow, 2016). CLRs are central for fungal recognition, uptake and induction of innate and 72 adaptive immune responses (Vautier et al, 2012). Dectin-1 specifically recognizes -1,3-glucans 73 in fungal cell wall (Taylor et al, 2007), while Dectin-2 and macrophage inducible C-type lectin 74 (Mincle) recognize -mannan and -mannose, respectively (Vautier et al, 2012). Dectin-1, 75 Dectin-2 and Mincle signal through the Syk-CARD9 pathway which induces NF-kB activation as 76 well as cytokine and chemokine gene expression (Mócsai et al, 2010). NLRP3 is a member of 77 NLRs family, which is able to indirectly sense danger signals, e.g. PAMPs (Schroder & Tschopp, 78 2010). Upon recognition, NLRP3 interacts with adaptor protein ASC that can further recruit pro- 79 caspase-1, leading to the assembly of multiprotein complex known as NLRP3 inflammasome 80 (Latz et al, 2013). Thereafter, pro-caspase-1 can be activated through auto-proteolysis, resulting 81 in the release of caspase-1 p10 and p20 (Schroder & Tschopp, 2010). Active caspase-1 not only 82 processes pro-IL-1 and pro-IL-18 into bioactive IL-1 and IL-18, but also mediates pyroptosis 83 (Man & Kanneganti, 2016). The activation of IL-1 transcription and NLRP3 inflammasome is 84 dependent on the Dectin-1/Syk signaling pathway in human macrophages (Kankkunen et al, 85 2010). The activation of Dectin-1 also triggers the induction of IL-23 through the Syk-CARD9 86 signaling pathway and drives robust Th17 response (LeibundGut-Landmann et al, 2007). The IL- 87 17/IL-23 axis has been shown to promote immunity to C. albicans, Aspergillus and dimorphic 88 fungi such as H. capsulatum (Deepe Jr. & Gibbons, 2009; Chamilos et al, 2010; Wu et al, 2013) 89 and Paracoccidioides brasiliensis (Tristão et al, 2017). In a murine model of disseminated 90 candidiasis, NLRP3 inflammasome exerts antifungal activity through driving protective Th1 91 and Th17 immune responses (van de Veerdonk et al, 2011). NLRP3 inflammasome has also 92 been shown to mediate IL-1 and IL-18 signaling in murine DC and macrophages infected by 93 P. brasiliensis (Tavares et al, 2013; Feriotti et al, 2015; Ketelut-Carneiro et al, 2015), and is bioRxiv preprint doi: https://doi.org/10.1101/2020.08.17.253518; this version posted August 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 94 essential for the induction of protective Th1/Th17 immunity against pulmonary 95 paracoccidioidomycosis (Feriotti et al, 2017; de Castro et al, 2018). 96 97 Previous study showed that IL-1 gene transcription is upregulated in human monocyte- 98 derived DC and macrophages stimulated by T. marneffei (Ngaosuwankul et al, 2008). 99 However, the mechanism of IL-1 induction has not been explored, and whether NLRP3 100 inflammasome plays a role in protective immunity against T. marneffei is unknown. From 101 our earlier work, impaired Th1 and Th17 effector functions emerges as the common 102 pathway in various types of PIDs rendering susceptibility to talaromycosis (Lee et al, 2012; 103 Lee et al, 2014; Lee et al, 2019; Lee & Lau, 2017). In this study, we sought to investigate the 104 role of NLRP3 inflammasome in the induction of Th1 and Th17 immune response to T.