Talaromyces marneffei infection - bioRxiv · 2020-08-17 · 84 dependent on the Dectin-1/Syk...

NLRP3 inflammasome contributes to host defense against 1

Talaromyces marneffei infection 2

3

Haiyan Ma1, Jasper FW Chan2, Yen Pei Tan2, Lin Kui1, Chi-Ching Tsang2, Steven LC Pei1, Yu-4

Lung Lau1, Patrick CY Woo2, Pamela P Lee1* 5

6

1.Department of Pediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The 7

University of Hong Kong, Hong Kong SAR, China. 8

2.Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 9

Hong Kong SAR, China 10

11

*Correspondence author. E-mail: [email protected] 12

13

Abstract 14

Talaromyces marneffei is an important thermally dimorphic pathogen causing disseminated 15

mycoses in immunocompromised individuals in southeast Asia. Previous study has 16

suggested that NLRP3 inflammasome plays a critical role in antifungal immunity. However, 17

the mechanism underlying the role of NLRP3 inflammasome activation in host defense 18

against T. marneffei remains unclear. We show that T. marneffei yeasts but not conidia 19

induce potent IL-1 response, which is differentially regulated in discrete immune cell types. 20

Dectin-1/Syk signaling pathway mediates pro-IL-1 production, and NLRP3 inflammasome is 21

activated to trigger the processing of pro-IL-1 into IL-1. The activated NLRP3 22

.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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

The copyright holder for this preprintthis version posted August 23, 2020. ; https://doi.org/10.1101/2020.08.17.253518doi: bioRxiv preprint

https://doi.org/10.1101/2020.08.17.253518

http://creativecommons.org/licenses/by-nc-nd/4.0/

inflammasome partially promotes Th1 and Th17 immune responses against T. marneffei 23

yeasts. In vivo, mice with NLRP3 or caspase-1 deficiency exhibit higher mortality rate and 24

fungal load compared to wild-type mice. Herein, our study provides the first evidence that 25

NLRP3 inflammasome contributes to host defense against T. marneffei infection, which may 26

have implications for future antifungal therapeutic designs. 27

28

Introduction 29

Talaromyces marneffei is a dimorphic fungus geographically restricted to Southeast Asia. It 30

causes a form of endemic mycosis that predominantly affects individuals with HIV infection 31

and ranks the third most common opportunistic infection in acquired immunodeficiency 32

syndrome (AIDS), following tuberculosis and cryptococcosis (Sirisanthana & Supparatpinyo, 33

1998; Supparatpinyo et al, 1994; Vanittanakom et al, 2006; Cao et al, 2019). In highly 34

endemic regions such as Chiang Mai in Thailand and Haiphong in Vietnam, T. marneffei is in 35

fact the most common systemic opportunistic fungal infection in AIDS (Vanittanakom et al, 36

2006; Son et al, 2014). Compared with HIV-negative individuals, fungemia, hepatomegaly 37

and splenomegaly occur much more commonly in HIV-positive patients, supporting 38

profound T-lymphopenia as the key predisposing factor for disseminated talaromycosis 39

(Kawila et al, 2013). 40

41

Less commonly, talaromycosis occurs in other immunocompromised conditions such as 42

solid organ or hematopoietic stem cell transplantation, autoimmune diseases or primary 43

immunodeficiency diseases (PID) (Chan et al, 2016; Lee et al, 2012; Lee et al, 2014; Lee & 44

Lau, 2017; Luo et al, 2010; Stathakis et al, 2015). T. marneffei shares some common 45

characteristics with other phylogenetically related dimorphic fungi such as Histoplasma and 46



https://doi.org/10.1101/2020.08.17.253518


Coccidioides. They exist naturally in the soil environment as conidia which enter the human 47

body via inhalation route, and are converted to the yeast form after being phagocytosed by 48

tissue-resident macrophages. These dimorphic fungal pathogens commonly result in 49

systemic infection when immunity is suppressed, and the uncontrolled proliferation of 50

yeasts in the macrophages eventually lead to dissemination via the reticuloendothelial 51

system. Paracoccidioides brasiliensis and P. lutzii, another dimorphic fungi which is endemic 52

in Latin America, is prevalent in immunocompetent individuals and co-infection with HIV is 53

uncommon, whereas talaromycosis, histoplasmosis and coccidioidomycosis are regarded as 54

AIDS-defining conditions in their respective endemic regions (Limper et al, 2017). They have 55

also been reported in patients with autoantibodies against interferon gamma (anti-IFN), 56

and PID characterized by impaired Th17 immune response such as autosomal dominant (AD) 57

hyper-IgE syndrome caused by loss-of-function STAT3 defect and AD chronic 58

mucocutaneous candidiasis (CMC) caused by gain-of-function STAT1 defect, as well as 59

CD40L deficiency and IFN- receptor deficiency. These acquired defects and inborn errors of 60

immunity provide evidence on the critical role of Th1 and Th17 immune response in host 61

defense against these dimorphic fungi (Lee & Lau, 2017; Lee et al, 2019). However, 62

knowledge about host-pathogen interaction in endemic mycoses is much less 63

comprehensive than other common opportunistic fungal pathogens of worldwide 64

distribution. In particular, little is known about how T. marneffei is recognized by innate 65

immune cells and the ensuing cytokine signaling that triggers adaptive immune response. 66

67

Immune responses against fungal infection are mounted by fungal pathogen-associated 68

molecular patterns (PAMPs) that can be recognized by pathogen recognition receptors (PRRs), 69

including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs) 70



https://doi.org/10.1101/2020.08.17.253518


(Erwig & Gow, 2016). CLRs are central for fungal recognition, uptake and induction of innate and 71

adaptive immune responses (Vautier et al, 2012). Dectin-1 specifically recognizes -1,3-glucans 72

in fungal cell wall (Taylor et al, 2007), while Dectin-2 and macrophage inducible C-type lectin 73

(Mincle) recognize -mannan and -mannose, respectively (Vautier et al, 2012). Dectin-1, 74

Dectin-2 and Mincle signal through the Syk-CARD9 pathway which induces NF-kB activation as 75

well as cytokine and chemokine gene expression (Mócsai et al, 2010). NLRP3 is a member of 76

NLRs family, which is able to indirectly sense danger signals, e.g. PAMPs (Schroder & Tschopp, 77

2010). Upon recognition, NLRP3 interacts with adaptor protein ASC that can further recruit pro-78

caspase-1, leading to the assembly of multiprotein complex known as NLRP3 inflammasome 79

(Latz et al, 2013). Thereafter, pro-caspase-1 can be activated through auto-proteolysis, resulting 80

in the release of caspase-1 p10 and p20 (Schroder & Tschopp, 2010). Active caspase-1 not only 81

processes pro-IL-1 and pro-IL-18 into bioactive IL-1 and IL-18, but also mediates pyroptosis 82

(Man & Kanneganti, 2016). The activation of IL-1 transcription and NLRP3 inflammasome is 83

dependent on the Dectin-1/Syk signaling pathway in human macrophages (Kankkunen et al, 84

2010). The activation of Dectin-1 also triggers the induction of IL-23 through the Syk-CARD9 85

signaling pathway and drives robust Th17 response (LeibundGut-Landmann et al, 2007). The IL-86

17/IL-23 axis has been shown to promote immunity to C. albicans, Aspergillus and dimorphic 87

fungi such as H. capsulatum (Deepe Jr. & Gibbons, 2009; Chamilos et al, 2010; Wu et al, 2013) 88

and Paracoccidioides brasiliensis (Tristão et al, 2017). In a murine model of disseminated 89

candidiasis, NLRP3 inflammasome exerts antifungal activity through driving protective Th1 90

and Th17 immune responses (van de Veerdonk et al, 2011). NLRP3 inflammasome has also 91

been shown to mediate IL-1 and IL-18 signaling in murine DC and macrophages infected by 92

P. brasiliensis (Tavares et al, 2013; Feriotti et al, 2015; Ketelut-Carneiro et al, 2015), and is 93



https://doi.org/10.1101/2020.08.17.253518


essential for the induction of protective Th1/Th17 immunity against pulmonary 94

paracoccidioidomycosis (Feriotti et al, 2017; de Castro et al, 2018). 95

96

Previous study showed that IL-1 gene transcription is upregulated in human monocyte-97

derived DC and macrophages stimulated by T. marneffei (Ngaosuwankul et al, 2008). 98

However, the mechanism of IL-1 induction has not been explored, and whether NLRP3 99

inflammasome plays a role in protective immunity against T. marneffei is unknown. From 100

our earlier work, impaired Th1 and Th17 effector functions emerges as the common 101

pathway in various types of PIDs rendering susceptibility to talaromycosis (Lee et al, 2012; 102

Lee et al, 2014; Lee et al, 2019; Lee & Lau, 2017). In this study, we sought to investigate the 103

role of NLRP3 inflammasome in the induction of Th1 and Th17 immune response to T. 104

marneffei. Our findings show that Dectin-1/Syk signaling pathway mediates pro-IL-1 105

production, and NLRP3 inflammasome is assembled to enhance the processing of pro-IL-1 106

and pro-IL-18 into mature IL-1 and IL-18 in response to T. marneffei yeasts. T. marneffei 107

induces Th1 and Th17 cell differentiation, which is attenuated by caspase-1 inhibition. 108

Furthermore, our in vivo studies show that NLRP3 and caspase-1 confer protection against 109

disseminated T. marneffei infection. 110

111

Results 112

T. marneffei yeasts, but not conidia, induce potent IL-1 response in human PBMCs 113

To dissect the production of cytokines elicited by T. marneffei, freshly isolated human 114

peripheral blood mononuclear cells (PBMCs) were co-cultured with T. marneffei yeasts or 115

conidia at 0.5 multiplicity of infection (MOI) for indicated incubation time points. As shown 116

in Fig 1A, IL-1became detectable at 8 hr, peaked at 18 hr, and remained plateaued 117



https://doi.org/10.1101/2020.08.17.253518


thereafter till day 5 post infection in the culture supernatant of human PBMCs infected with 118

live T. marneffei yeasts. On the other hand, TNF- production was detectable as early as 4 119

hr, and rapidly peaked at 18 hr, and subsequently showed a slight falling trend up to day 5 120

post infection (Fig 1B). These data demonstrated that T. marneffei yeasts were a potent 121

inducer of IL-1 and TNF-a production. In contrast to T. marneffei yeasts, only minimal levels 122

of IL-1 was detected in PBMCs co-cultured with conidia at 5 days post infection (Fig 1C), 123

whereas TNF- production began to increase steadily after 2 days of incubation (Fig 1D). 124

This suggests that T. marneffei conidia were able to trigger TNF- production in PBMCs but 125

failed to induce IL-1response. 126

127

To examine whether the viability of T. marneffei is a prerequisite for the induction of IL-1 128

in PBMCs, yeasts and conidia were inactivated by heat (100C for 40 min) or 4% 129

paraformaldehyde (PFA) treatment for 10 min. Our results showed that there was no 130

significant difference in IL-1 production in PBMCs infected with live, heat-killed or PFA-131

treated T. marneffei yeasts (Fig EV1A), indicating that the viability of T. marneffei yeasts was 132

not necessary for the induction of IL-1 response. In contrast, IL-1 production in the 133

PBMCs stimulated with heat-killed conidia was significantly higher compared to live and 134

PFA-treated conidia (Fig EV1B), suggesting that heat treatment might promote the exposure 135

of some pro-inflammatory PAMPs on the conidia cell wall to induce IL-1 response. In 136

addition, both yeast and pseudo-hyphal forms of C. albicans were capable of inducing IL-1 137

production in human PBMCs, and no differential IL-1 response was observed for heat- and 138

PFA-treated C. albicans (Fig EV1C and D). 139

140



https://doi.org/10.1101/2020.08.17.253518


We further evaluated the production of other pro-inflammatory cytokines in T. marneffei 141

yeasts-infected human PBMCs. As shown in Fig 1E-J, IFN- and IL-17A could be detected only 142

after 3 days of co-culture, whereas IL-6, MCP-1, IL-8 and IL-18 were detectable as early as 18 143

hr. Specifically, IFN-production reached its maximum at 3 days and slightly reduced at 5 144

days, while IL-17A production greatly increased from 3 days to 5 days of co-culture (Fig 1E 145

and F). IL-6 and MCP-1 production peaked at 18 hr and remained at similar levels at 3 days 146

and 5 days of co-culture (Fig 1G and H). IL-8 and IL-18 production was maximal at 18 hr and 147

showed a downward trend thereafter (Fig 1I and J). These results suggested that T. 148

marneffei yeasts sequentially activated innate and adaptive immune responses. 149

150

Differential requirement for IL-1 response to T. marneffei yeasts in various human 151

immune cell types 152

It has been suggested that IL-1 production can be mediated by inflammasome activation 153

which requires two signals: the triggering of pro-IL-1 production (signal 1) and the 154

processing of pro-IL-1 into mature IL-1 (Signal 2) (Latz et al, 2013). To investigate whether 155

there was any differential requirement for IL-1 response to yeasts in different cell types, 156

human PBMCs, monocytes, monocytes-derived macrophages and monocytes-derived DCs 157

were primed with or without lipopolysaccharide (LPS) prior to T. marneffei yeasts 158

stimulation. As shown in Fig 2A and B, T. marneffei yeasts alone were able to induce 159

abundant IL-1 production in PBMCs and monocytes, indicating that yeasts provided ‘signal 160

1’ and ‘signal 2’ for inflammasome activation in these cell types. In contrast, LPS priming 161

was required for IL-1 response to T. marneffei yeasts in macrophages and DCs (Fig 2C and 162

D), suggesting that T. marneffei yeasts only provided ‘signal 2’, and the pro-IL-1induced by 163

LPS (‘signal 1’) was essential in triggering inflammasome activation in these two cell types. 164



https://doi.org/10.1101/2020.08.17.253518


On the other hand, high level of TNF- could be detected in human PBMCs (Fig 2E), 165

monocytes (Fig 2F) and DCs (Fig 2H), but not in macrophages (Fig 2G), after T. marneffei 166

yeast stimulation. 167

168

Dectin-1/syk signaling mediates IL-1 response to T. marneffei yeasts in human 169

monocytes 170

Dectin-1 recognizes -1,3-glucans in fungal cell wall, which leads to pro-inflammatory 171

immune responses (Hardison & Brown, 2012). To elucidate the involvement of Dectin-1 in 172

the initiation of IL-1 response to T. marneffei yeasts, human CD14+ monocytes were co-173

cultured with heat-killed T. marneffei yeasts in the presence of anti-human Dectin-1 or TLR2 174

neutralizing antibodies or isotype controls (mouse IgG or human IgA). As shown in Fig 3A, 175

Dectin-1 blockade resulted in a marked reduction of IL-1 in T. marneffei yeasts-stimulated 176

monocytes compared with mouse IgG treatment group. TNF- production was slightly 177

reduced by Dectin-1 blockade, though not significantly different (Fig 3B). These data 178

suggested that Dectin-1 mediated the production of IL-1 and TNF- in response to T. 179

marneffei yeasts in human monocytes. Conversely, blockade of TLR2 resulted in significantly 180

elevated IL-1 secretion and had no effect on TNF- production in response to yeasts (Fig 181

3C and D), suggesting that TLR2 negatively regulated IL-1 production in the context of T. 182

marneffei stimulation. 183

184

To study the role of Dectin-1/Syk signaling pathway in the induction of IL-1 response to T. 185

marneffei yeasts, we measured Syk phosphorylation in T. marneffei stimulated human 186

CD14+ monocytes by flow cytometry. As expected, T. marneffei yeasts induced Syk 187

phosphorylation compared to the unstimulated cells (Fig 3E). Pre-treatment of CD14+ 188



https://doi.org/10.1101/2020.08.17.253518


monocytes with R406 (Syk inhibitor) prior to T. marneffei yeasts stimulation resulted in 189

significantly reduced expression of pro-IL-1 in cell lysates (Fig. 3F) as well as IL-1 in culture 190

supernatant (Fig 3G), in a dose-dependent manner. A similar trend of dose-dependent 191

inhibition of TNF- production in monocytes co-cultured with T. marneffei yeasts was also 192

observed (Fig 3H). Collectively, our data suggested that Syk, the molecule that acts 193

downstream to Dectin-1 in the initiation of antifungal immunity (Drummond et al, 2011), 194

played an important role in eliciting IL-1 response to T. marneffei yeasts in human 195

monocytes. 196

197

To verify our hypothesis that caspase-1 activation mediates IL- and IL-18 release in human 198

monocytes infected with T. marneffei yeasts, we pre-treated human CD14+ monocytes with 199

caspase-1 inhibitor (Z-YVAD, 10µM and 20µM) prior to co-culture with heat-killed T. 200

marneffei yeasts. IL-1 and IL-18 secretion was significantly attenuated in Z-YVAD-treated 201

monocytes (Fig 3I and J), whereas inhibition of caspase-1 activity had no obvious effect on 202

TNF-production (Fig 3K), demonstrating that caspase-1 activation contributed to IL-1 and 203

IL-18 release in response to T. marneffei. 204

205

T. marneffei yeasts trigger IL-1 production via the NLRP3 inflammasome 206

To delineate whether IL-1release induced by T. marneffei yeasts was mediated by NLRP3 207

inflammasome, murine bone marrow-derived DCs (BMDCs) from WT, Nlrp3-/-, Casp1-/- mice 208

were co-cultured with heat-killed yeasts, and then NLRP3, caspase-1 p20 and p45 209

expression were analyzed by immunoblots. As shown in Fig 4A, we found that NLRP3 210

expression was obviously upregulated by yeasts in BMDCs from WT and Casp-1-/- mice. 211

Moreover, pro-caspase-1 (p45) expression in cell lysates was comparable between 212



https://doi.org/10.1101/2020.08.17.253518


unstimulated and yeasts-stimulated cells in either WT or Nlrp3-/- mice. Active caspase-1 p20 213

was detectable in the concentrated supernatant of yeasts-stimulated BMDCs from WT mice, 214

but it was absent in the supernatant from cells deficient in NLRP3 or caspase-1, suggesting 215

that caspase-1 activation is dependent on NLRP3. LPS plus nigericin, which are considered as 216

the activators of classical NLRP3 inflammasome, could activate caspase-1 since active 217

caspase-1 p20 was observed in both cell lysates and supernatant of WT BMDCs. 218

219

To determine whether T. marneffei yeasts could induce ASC specks to mediate IL-1 220

processing, BMDCs from WT and Nlrp3-/- mice were co-cultured with yeasts, and the 221

formation of ASC pyroptosomes (“specks”) was examined. Confocal microscopy revealed 222

that ASC pyroptosomes, appearing as a single cytoplasmic speck, was present in WT cells 223

but not in Nlrp3-/- cells stimulated with T. marneffei yeasts or LPS plus nigericin (Fig 4B). We 224

also quantified the percentage of cells containing ASC specks and observed that around 8% 225

of WT cells formed ASC specks while it was undetectable in Nlrp3-/- cells after T. marneffei 226

yeasts stimulation (Fig 4C). Similarly, LPS plus nigericin could induce about 29% of WT cells 227

containing ASC specks (Fig 4C). Our results indicated that T. marneffei yeasts induced ASC 228

speck formation, which was dependent on NLRP3, to induce IL-1 processing. 229

230

Next, to further test the hypothesis that IL-1 response to T. marneffei yeasts was 231

dependent on NLRP3 inflammasome, BMDCs from WT, Casp-1-/-, and Nlrp3-/- mice were co-232

cultured with heat-killed T. marneffei yeasts, and IL-1 and TNF- in the supernatant were 233

examined. As shown in Fig 4D, LPS induced a small amount of IL-1 in these BMDCs, 234

whereas there was remarkably lower IL-1in Casp-1-/- and Nlrp3-/- BMDCs than WT cells 235

after stimulation with LPS plus nigericin. More importantly, we observed that BMDCs from 236



https://doi.org/10.1101/2020.08.17.253518


Casp-1-/- and Nlrp3-/- mice had significantly impaired IL-1 response to T. marneffei yeasts 237

when compared with WT mice (Fig 4D). Furthermore, an intriguing observation was that IL-238

1 production in BMDCs from Nlrp3-/- mice was slightly lower than those from Casp-1-/-mice 239

after T. marneffei yeasts stimulation with or without LPS priming (Fig 4D), indicating that IL-240

1 response to T. marneffei yeasts was more dependent on NLRP3 than caspase-1. In 241

contrast to IL-1 production, BMDCs from WT, Casp-1-/-, and Nlrp3-/- mice produced 242

comparable levels of TNF- when co-cultured with T. marneffei yeasts (Fig 4E). Collectively, 243

these results corroborated that NLRP3 and caspase-1 were required for IL-1 response to T. 244

marneffei yeasts in murine BMDCs. 245

246

T. marneffei yeasts elicit differential Th1 and Th17 immune responses in human CD14+ 247

monocytes and monocytes-derived DCs. 248

We have observed that T. marneffei yeasts triggered IFN- and IL-17A production in human 249

PBMCs (Fig 1E and F). To further validate our hypothesis that T. marneffei yeasts would 250

induce Th1 and Th17 immune responses, isolated human CD4+ T cells were co-cultured with 251

autologous monocytes or monocytes-derived DCs with T. marneffei yeasts or C. albicans for 252

7 and 12 days, and intracellular IFN-and IL-17A production in CD4+ T cells was quantified by 253

flow cytometry. Upon co-culture of CD4+ T cells with T. marneffei yeasts, no IFN-- or IL-17A-254

producing cells could be detected (Figs 5A-C, and EV2A and B). However, when CD4+ T cells 255

were co-cultured with T. marneffei yeasts for 7 and 12 days in the presence of monocytes, 256

robust IFN- and/or IL-17A production was evident (Figs 5A,B, G, and EV2C and D), while no 257

statistically significant differences were observed for IL-17A-producing cells on day 12 (Fig 258

5B) and IFN-+IL-17A+ -producing cells on day 7 and 12 (Fig 5C) after stimulation with T. 259

marneffei yeasts. These findings suggested that the induction of Th1 and Th17 response by T. 260



https://doi.org/10.1101/2020.08.17.253518


marneffei yeasts required the presence of antigen presenting cells (APCs) such as 261

monocytes. We also found that C. albicans yeasts induced much stronger Th1 and Th17 262

immune responses than T. marneffei in the presence of monocytes (Figs 5A-C, and G, and 263

EV2C and D). Consistent with IFN- and IL-17A production, increased T-bet and RORt, the 264

master transcription factors of respective Th1 and Th17 cells, were observed in CD4+ T cells 265

after co-culture with TM yeasts-primed autologous monocytes for 7 days (Fig EV2G and H). 266

Distinct from CD14+ monocytes, monocytes-derived DCs stimulated with T. marneffei or C. 267

albicans yeasts induced delayed Th1 and Th17 cell differentiation; the remarkable increase 268

in the percentage of IFN- -and/or IL-17A-producing CD4+ T cells could mostly be detected 269

on day 12 when T cells were co-cultured with T. marneffei or C. albicans yeasts in the 270

presence of DCs (Figs 5D-F, and EV2E and F). 271

272

In order to further dissect the difference between monocytes and DCs in promoting the 273

differentiation of CD4+ T cells after fungal stimulation, we next assessed the release of IL-274

12p70 and IL-23, which are considered to be the key cytokines driving Th1 and Th17 cell 275

differentiation respectively, from human CD14+ monocytes and monocyte-derived DCs. We 276

found that the induction of IL-12p70 and IL-23 by T. marneffei was negligible in monocytes 277

(Fig 5I and J). In contrast, IL-23 was induced by T. marneffei in DCs at a level comparable 278

with C. albicans (Fig 5J). 279

280

Taken together, our data showed that T. marneffei yeasts elicited Th1 and Th17 immune 281

responses in the presence of APCs. CD14+ monocytes exhibited earlier induction of IFN--282

producing and IL-17A-producing CD4+ T cells than monocyte-derived DCs when co-cultured 283

with T. marneffei or C. albicans. 284



https://doi.org/10.1101/2020.08.17.253518


285

Caspase-1 inhibition attenuates Th1 and Th17 immune response to T. marneffei yeasts 286

To characterize the role of activated NLRP3 inflammasome in the Th1 and Th17 immune 287

responses towards T. marneffei yeasts, human PBMCs were co-cultured with T. marneffei 288

yeasts for 5 days in the presence of caspase-1 inhibitor, Z-YVAD. Blocking of caspase-1 289

activation resulted in significantly reduced IFN- and IL-17A release in response to T. 290

marneffei yeasts (Fig 6A). Next, we determined the percentage of IFN-- and/or IL-17A-291

producing CD4+ T cells upon co-culture of T. marneffei yeasts-stimulated monocytes and 292

CD4+ T cells in the presence of Z-YVAD. Our results showed that caspase-1 inhibition led to 293

approximately 2-fold reduction in overall IL-17A-producing CD4+ T cells (Fig 6B and D) and 294

IFN-/IL-17A-producing CD4+ T cells (Fig 6B and E), although there was no significant effect 295

on the overall intracellular IFN- production (Fig 6B and C). Our data indicated that NLRP3 296

inflammasome activation partially enhanced T. marneffei yeasts-induced Th1 and Th17 297

immune responses in human immune cells in vitro. 298

299

To clarify the role of caspase-1 and NLRP3 in the induction of Th1 and Th17 response to T. 300

marneffei, we isolated murine splenocytes from wild-type, Casp-1-/-, Nlrp3-/- mice and 301

incubated them with T. marneffei yeasts for 2 days or 6 days, and the supernatant was 302

collected for the quantification of IFN- and IL-17A. As shown in Fig 6F, it was observed that 303

splenocytes from Casp-1-/- and Nlrp3-/- mice had impaired IFN- and IL-17A production 304

compared to WT mice at 2 days post stimulation, while there was no statistically significant 305

effect on IL-17A. On day 6 of incubation, IFN- and IL-17A production by Casp-1-/- and Nlrp3-306

/- splenocytes was sharply increased and reached a similar level as splenocytes from WT 307



https://doi.org/10.1101/2020.08.17.253518


mice. Our findings suggested that NLRP3 inflammasome activation promoted early stage of 308

IFN- production in murine splenocytes. 309

310

The NLRP3 inflammasome controls antifungal immunity in vivo 311

Our in vitro study has demonstrated that T. marneffei yeasts activated NLRP3 312

inflammasome that promoted Th1 and Th17 immune responses. To examine the protective 313

role of NLRP3 inflammasome in defense against T. marneffei infection in vivo, WT, Casp-1-/-, 314

Nlrp3-/- mice were infected with 5x105 CFU of T. marneffei yeasts per mouse by intravenous 315

injection and their survival was monitored. As shown in Fig 7A, Casp-1-/- and Nlrp3-/- mice 316

were more susceptible to T. marneffei yeasts infection than WT mice. Of note, Casp-1-/- mice 317

showed more resistance to fungal infection than Nlrp3-/- mice. These observations 318

suggested that NLRP3 and caspase-1 conferred host protection against T. marneffei 319

infection, in particular, NLRP3 played a more protective role than caspase-1. To further 320

investigate the factors resulting in the differential survival rates among WT, Casp-1-/-, Nlrp3-321

/- mice, murine spleens and livers were collected at 7 and 14 days post infection (dpi) for 322

fungal load detection. There was comparable fungal load in spleens from WT, Casp-1-/-, 323

Nlrp3-/- mice at 7 dpi (Fig 7B). Nevertheless, significantly elevated fungal load was observed 324

in spleens from Nlrp3-/- mice when compared to WT mice at 14 dpi (Fig 7B). Similarly, liver 325

specimen from Nlrp3-/- mice exhibited the highest fungal load, followed by Casp-1-/- mice, 326

and WT mice showed the least fungal load in livers at 7 and 14 dpi (Fig 7C). It was worth 327

noting that the fungal load in spleens and livers from WT mice at 14 dpi was similar to that 328

at 7 dpi, whereas the fungal load in these organs from Nlrp3-/- mice at 14 dpi was 329

significantly higher than that 7 dpi (Fig 7B and C), indicating that NLRP3 inflammasome 330

played a pivotal role in the control of T. marneffei proliferation. 331



https://doi.org/10.1101/2020.08.17.253518


332

Histological studies of liver sections from WT, Casp-1-/- and Nlrp3-/- mice at 7 dpi showed a 333

great number of granulomas (Fig 7D, upper panel). By day 14, granulomas were 334

considerably enlarged and almost replaced the liver parenchyma in Casp-1-/- and Nlrp3-/- 335

mice when compared to those at 7 dpi, but liver sections from WT mice exhibited a smaller 336

number of granulomas (Fig 7D, lower panel). HE staining of livers revealed that WT mice had 337

relatively fewer cytoplasmic fungal yeasts within macrophages in the central granulomas 338

than Casp-1-/- and Nlrp3-/- mice (Fig 7D, insets of lower panel). Tissue necrosis was not 339

observed in all three strains of mice. GMS staining further verified that fungal yeasts were 340

present in the center of granulomas (Fig 7E). Slightly fewer yeasts were observed in WT 341

mice than Nlrp3-/- and Casp-1-/- mice at 7 dpi (Fig 7E, upper panel). Importantly, massive 342

number of T. marneffei yeasts were seen in the granulomas of Casp-1-/- and Nlrp3 -/- livers, 343

while T. marneffei yeasts were sparse in the granulomas of WT mice at 14 dpi (Fig 7E, lower 344

panel). Spleen sections of Nlrp3-/- mice showed a more severe loss of follicular structure in 345

the white pulp than WT and Casp-1-/- mice at 7 dpi (Fig EV3A, upper panel). No granulomas 346

were observed in the spleens (Fig EV 3A). We failed to detect T. marneffei in the spleens at 7 347

dpi, but scattered fungal yeasts could be seen in the spleens of WT, Nlrp3-/- and Casp-1-/- 348

mice at 14 dpi by GMS staining (Fig EV3B). In conclusion, these histopathological 349

examinations further demonstrated that NLRP3 inflammasome was essential for host 350

defense against T. marneffei infection in vivo. 351

352

Discussion 353

T. marneffei is an important form of endemic mycoses causing major morbidity and 354

mortality in patients with HIV infection, as well as those with primary and secondary 355



https://doi.org/10.1101/2020.08.17.253518


immunodeficiencies. However, innate immune recognition of T. marneffei and the induction 356

of adaptive immunity, is largely unknown. For the first time, our study characterizes Syk- 357

and caspase-1 mediated IL-1 response towards T. marneffei in human myeloid cells, and 358

the role of caspase-1 in the induction of Th1 and Th17 response in human lymphocytes. 359

Importantly, our murine model of disseminated talaromycosis demonstrates increased 360

fungal load in the liver and spleen of Casp-1-/- and Nlrp3-/- mice, and correlates with reduced 361

survival. 362

363

Human T. marneffei infection is initiated by inhalation of conidia existing in the environment. 364

After phagocytosis by pulmonary alveolar macrophages, T. marneffei conidia undergo 365

morphological switch and germinate into the pathogenic yeast cells, which readily disseminate 366

throughout the body causing systemic infection. Our findings reveal differential cytokine 367

response towards T. marneffei conidia and yeasts in human PBMCs (Fig 1A-D). These 368

interesting findings indicate that conidia are less immunological than yeasts, as shown by 369

negligible IL-1 and TNF- production in PBMCs during the first 24-48 hours of co-culture. 370

The delayed rise in TNF-, and IL-1 to a lesser extent, after 3 days of co-culture probably 371

represents the switch of conidia to yeast form in vitro, suggesting that the transformation of 372

conidia into yeasts would be required for IL-1 response. The change in cell wall composition 373

and architecture during different life stages of fungi can lead to differential exposure of PAMPs 374

in the inner cell wall, causing varying degree of PRR activation which determines the level of 375

pathogenicity (Cheng et al, 2011). 376

377

Our work demonstrates that IL-1 response to T. marneffei yeasts is partially mediated by 378

Dectin-1/Syk signaling pathway. Blockade of Dectin-1 with neutralizing antibodies impairs 379



https://doi.org/10.1101/2020.08.17.253518


the production of IL-1 and TNF- to a lesser extent, in response to T. marneffei yeasts in 380

human CD14+ monocytes. This suggests that Dectin-1 plays a role in initiating downstream 381

signaling that leads to pro-IL-1 and TNF- production, which has been demonstrated in 382

other fungal pathogens such as C. albicans and H. capsulatum (Chang et al, 2017; Hise et al, 383

2009). We further show that T. marneffei yeasts are capable of inducing Syk kinase 384

phosphorylation, and blockade of Syk kinase significantly inhibits both pro-IL-1 and IL-1 as 385

well as TNF- in a dose-dependent fashion (Fig 3F-H). Acquired immunity to other 386

dimorphic fungi such as Blastomyces dermatitidis, Histoplasma capsulatum and Coccidioides 387

posadasii infection variably depends on innate sensing by Dectin-1, Dectin-2 and Mincle 388

(Wang et al, 2014), and the role of these CTL receptors in cytokine response against T. 389

marneffei will require further studies. 390

391

TLR2 heterodimerizes with either TLR1 or TLR6 and recognizes triacylated and diacylated 392

lipoprotein. TLR2 and dectin-1 physically associate with one another and synergize to 393

augment anti-fungal response by modulating cytokine production (Dennehy et al, 2009; 394

Gantner et al, 2003; Hise et al, 2009). Our results show that blockade of TLR2 leads to 395

increased IL-1 production but has no impact on TNF- production in T. marneffei yeasts-396

stimulated human monocytes (Fig 3C and D), suggesting that TLR2 ligation might exert a 397

negative effect on IL-1-mediated immune response against T. marneffei. TLR2-deficient 398

mice infected with P. brasiliensis have preferential activation of Th17 response and lower 399

fungal load, while Treg expansion is diminished and aggravates lung inflammation (Loures et 400

al, 2010). A similar role of TLR2 was also observed in a murine model of disseminated 401

candidiasis, where TLR2 exerts anti-inflammatory effect by promoting IL-10 production and 402

Treg cell proliferation (Netea et al, 2004). 403



https://doi.org/10.1101/2020.08.17.253518


404

It was previously shown that IL-12p40 production is induced by T. marneffei in murine 405

BMDCs, and is mediated by Dectin-1, TLR2 and MyD88 (Nakamura et al, 2008). Our data 406

showed that IL-12p70 could not be induced by T. marneffei yeasts in human CD14+ 407

monocytes and monocyte-derived DCs. This confirms the previous published finding that T. 408

marneffei yeasts do not upregulate IL-12 expression in human monocyte-derived DCs and 409

monocyte-derived macrophages (Ngaosuwankul et al, 2008). Furthermore, our findings 410

provide strong evidence that human Th17 cells are expanded when co-cultured with T. 411

marneffei-stimulated monocytes or monocyte-derived DCs, in contrast to the findings from 412

a recent study showing that murine BMDCs are unable to induce, or actually lower, the 413

percentage of Th17 cells upon co-culture with T. marneffei (Tang et al, 2020). These 414

observations point to the possible difference in Th1 and Th17 immune response towards T. 415

marneffei in human vs murine hosts. 416

417

We demonstrate that IFN- and IL-17A-producing human CD4+ T cells are induced by T. 418

marneffei-primed human CD14+ monocytes (Fig 5A and B and G), albeit less strong as C. 419

albicans-primed monocytes. IL-12 is an important cytokine which induces the differentiation 420

of naïve CD4+ T cell into IFN- secreting Th1 cells, whereas differentiation of naïve CD4+ T-421

cells to Th17 cells is a result of the combined activity of IL-23 and IL-1 (Goncalves et al, 422

2017; Zhou et al, 2009). As neither IL-12 nor IL-23 could be detectable upon co-culture of T. 423

marneffei-stimulated human CD14+ monocytes (Fig 5I and J), this suggests that cytokines 424

other than IL-12 and IL-23 might be secreted by human monocytes for the differentiation of 425

Th1 and Th17 cells in response to T. marneffei infection. For example, it was found that IL-6 426

contributes to IFN- expression in lung tissues in a murine model of systemic P. brasiliensis 427



https://doi.org/10.1101/2020.08.17.253518


infection (Tristão et al, 2017). Alternatively, Th17 cells against T. marneffei could originate 428

from memory T cells that are cross-reactive to C. albicans instead of being differentiated de 429

novo, since Th17 cells cross-reactive to C. albicans have been demonstrated to contribute to 430

Th17 response towards A. fumigatus (Bacher et al, 2019). 431

432

On the other hand, IL-23 could be induced by T. marneffei yeasts in monocyte-derived DCs, 433

but not IL-12 (Fig 5J). The preferential induction of IL-23 over IL-12 response in DCs is 434

possibly related to dectin-1-mediated signaling, as previously shown in H. capsulatum 435

(Chamilos et al, 2010). Furthermore, IL-23 is induced by T. marneffei in human DCs at a level 436

comparable to C. albicans, and correlates with a similar percentage of both IFN- and IL-437

17A-producing CD4+ T-cells upon co-culture with the two fungal pathogens. IL-12 and IL-23 438

are thought to promote mutually exclusive CD4+ Th1 and Th17 cell fates (Teng et al, 2015), 439

However, recent investigations on human monogenic IL12R2 and IL-23R deficiency, which 440

result in defective responses to IL-12 or IL-23 respectively, show that the isolated absence of 441

IL-12 or IL-23 could in part be compensated by its counterpart for the production of IFN-, 442

conferring protection against intracellular pathogen such as mycobacteria (Martínez-443

Barricate et al, 2018). In the absence of IL-12, IL-17 is the requisite for the protective effect 444

induced by IL-23 in pulmonary histoplasmosis (Deepe Jr. & Gibbons, 2009). This might 445

explain how loss-of-function mutation in STAT3, which encodes the transcription factor 446

activated by IL-23, leads to increased susceptibility to dimorphic fungi such as T. marneffei, 447

C. immitis and H. capsulatum in AD hyper-IgE syndrome (Lee & Lau, 2017). 448

449

IL-1 is essential for Th17 differentiation in human and mice (Acosta-Rodriguez et al, 2007; 450

Chung et al, 2009). IL-1 synergizes with IL-23 in controlling the expression of RORt and IL-451



https://doi.org/10.1101/2020.08.17.253518


23 receptor to sustain IL-17 production by effector Th17 cells (Chung et al, 2009). Another 452

report also indicates that NLRP3 inflammasome-derived IL-18 and IL-1 drive protective Th1 453

and Th17 immune responses in disseminated candidiasis (van de Veerdonk et al, 2011). 454

Consistently, treatment of human PBMCs with caspase-1 inhibitor, which suppressed the 455

production of IL-1 and IL-18 production in human monocytes (Fig 3I and J), significantly 456

impaired the secretion of IFN- and IL-17A, and the differentiation of IL-17A-producing-T 457

cells (Fig 6C-E). These findings highlight the important role of caspase-1 in bridging the 458

innate and adaptive immune response, particularly Th17 response towards T. marneffei 459

infection in human cells. 460

461

Furthermore, in line with the role of inflammasome activation in host defense against C. 462

albicans (Hise et al, 2009) and A. fumigatus (Karki et al, 2015), our study defined the critical 463

role of NLRP3 and caspase-1 in the induction of IL-1 response to T. marneffei. In WT 464

BMDCs, T. marneffei yeasts strongly upregulated the expression of NLRP3, cleavage of pro-465

caspase-1 to caspase-1 p20, and the formation of ASC pyroptosomes (Fig 4A and C). Using 466

the murine model of systemic T. marneffei infection, we found that Nlrp3-/- and Casp-1-/- 467

mice had higher mortality rate. Of note, the fungal load in the spleen and liver of WT mice 468

were comparable on 7 dpi and 14 dpi, while an increase in fungal load became obvious on 469

14 dpi in Casp-1-/- and Nlrp3-/- mice, particularly the latter (Fig 7A-C). This suggests that 470

NLRP3/Caspase-1 signaling is crucial in controlling fungal proliferation in vivo. 471

472

An intriguing observation is that the significantly lower survival rate of Nlrp3-/- mice 473

correlated with higher fungal load, particularly in the liver at 14 dpi, compared with Casp-1-/- 474

mice (Fig 7A-C). These in vivo data correlate well with IL-1 response to T. marneffei in 475



https://doi.org/10.1101/2020.08.17.253518


murine BMDCs in vitro, reinforcing the critical role of IL-1 in protective immunity against T. 476

marneffei. The residual IL-1 response to T. marneffei in BMDCs from Casp-1-/- and Nlrp3-/- 477

mice (Fig 4D), and particularly the lower fungal load and better survival in Casp-1-/- mice 478

compared to Nlrp3-/- mice (Fig 7A-C), suggests possible contributions from alternate 479

signaling pathways. For example, the engagement of Dectin-1 by P. brasiliensis activates 480

caspase-8 which drives the transcriptional priming and post-translational processing of pro-481

IL-1. Interestingly, the canonical caspase-1 inflammasome pathway normally restricts the 482

noncanonical caspase-8 inflammasome activation, therefore in the context of caspase-1/11 483

deficiency, the lack of inhibition from caspase-1 enables caspase-8-dependent IL-1 484

processing (Ketelut-Carneiro et al, 2018). A recent study show that IL-1 release via 485

caspase-11-mediated pyroptosis induced by P. brasiliensis reprograms Th17 lymphocytes to 486

produce high levels of IL-17 and enhances effector mechanisms by innate cells (Ketelut-487

Carneiro et al, 2019). Further studies are required to investigate alternative molecular 488

pathways in the induction of inflammasome activation against T. marneffei infection. 489

490

DCs from patients with HIV was previously shown to exhibit poor activation of NLRP3 491

inflammasome in response to PAMPs and DAMPs, which could be the consequence of 492

increased basal expression and activation of NLRP3 induced by HIV in chronic infection 493

leading to immune exhaustion (Pontillo et al, 2012), as well as the inhibitory effect on 494

NLRP3 inflammasome activity by type I interferon which is commonly elevated in plasma of 495

HIV-infected patients (Reis et al, 2019; Hardy et al, 2013). In response to LPS stimulation, 496

PBMCs from HIV patients express relatively lower levels of activated caspase-1 (Ahmad et al, 497

2002), and IL-1 production in DCs fails to be upregulated by either LPS (Pontillo et al, 2012) 498

or ATP which classically induces canonical activation of NLRP3 (Reis et al, 2019). It is possible 499



https://doi.org/10.1101/2020.08.17.253518


that functional defect of NLRP3 could represent an additional contributory factor to 500

increased susceptibility to T. marneffei infection in HIV-positive individuals. Further studies 501

on functional evaluation of NLRP3 and caspase-1 activation in HIV-positive patients infected 502

with T. marneffei will be required. 503

504

This study enhances our understanding of host defense mechanisms against T. marneffei. 505

Knowledge about human immune response towards T. marneffei will have therapeutic 506

implications in managing patients suffering from this fatal infection. Delineation of the roles 507

of various cytokines towards protection against T. marneffei will provide important 508

information to the treatment of patients who have secondary immunodeficiency resulting 509

from the use of biologics for immunological disorders. For example, with the increasingly 510

wide clinical use of IL-1receptor antagonist (e.g. Anakinra, Rilonacept) or neutralization 511

antibodies (Canakinumab) for patients with auto-inflammatory or auto-immune diseases, 512

and therefore clinicians should be alerted to the susceptibility and signs of talaromycosis 513

when treating patients who reside in endemic regions where there is increased risk of 514

environmental exposure to T. marneffei. Finally, the study of functional cellular response 515

towards T. marneffei may provide breakthroughs for the discovery of monogenic immune 516

defects in patients with talaromycosis which is not otherwise explained. It is possible that 517

genetic defects in molecules involved in Dectin-1/Syk signaling pathway, NLRP3 518

inflammasome activation and induction of Th1 and Th17 immune responses might 519

predispose to T. marneffei infection. 520

521

To conclude, in this study, we first demonstrate that T. marneffei yeasts rather than conidia 522

induce IL-1response, which is differentially regulated in distinct cell types. Our findings 523



https://doi.org/10.1101/2020.08.17.253518


show that Dectin-1/Syk signaling pathway mediates pro-IL-1 production upon fungal 524

stimulation. T. marneffei yeasts also trigger the assembly of NLRP3-ASC-caspase-1 525

inflammasome to facilitate IL-1 maturation. The activated inflammasome partially 526

enhances Th1 and Th17 immune responses against fungal infection. Nlrp3-/- and Casp1-/- 527

mice are more susceptible to T. marneffei yeasts with higher fungal load as compared to WT 528

mice. Collectively, our study highlights the importance of NLRP3 inflammasome in host 529

defense against T. marneffei infection and sheds new light on the interaction between host 530

and fungal cells. 531

532

Materials and Methods 533

Fungi 534

T. marneffei and C. albicans were isolated from patients by the Department of Microbiology, 535

Queen Mary Hospital. T. marneffei conidia were cultured on Sabouraud Dextrose Agar (SDA) 536

plates at 25C for 7 days, and then collected in sterile water by wet cotton swabs, washed 3 537

times and re-suspended in sterile water, and finally filtered through 40µm nylon filters. To 538

prepare T. marneffei yeasts, T. marneffei conidia were cultured on SDA plates at 37C for 10 539

days, and then collected in sterile 0.1% PBST by wet cotton swabs, washed 3 times and re-540

suspended in RPMI 1640 with shaking for 24 hr at 37C, and finally filtered through 40µm 541

nylon filters. C. albicans were cultured on SDA plates at 30℃ for 48 hr. Then, they were 542

transferred to Sabouraud Dextrose (SD) broth and incubated at 37C with shaking for 16–24 543

hr to obtain the yeast form of C. albicans. Simultaneously, C. albicans were cultured with 544

shaking in enriched media consisting of RPMI+10% fetal bovine serum (FBS) at 37C for 24 545

hr to obtain the pseudo-hyphae form of C. albicans. For fixed fungal preparation, fungi were 546



https://doi.org/10.1101/2020.08.17.253518


washed in sterile PBS, fixed in 4% paraformaldehyde (PFA) solution for 10 min, and then 547

washed three times in sterile PBS. For heat-killed fungal preparation, fungi were boiled at 548

100C for 40 min. 549

550

Cell isolation and culture 551

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats of healthy 552

donors according to the principles of the Declaration of Helsinki and were approved by the 553

responsible ethics committee (Institutional Review Boards of the University of Hong 554

Kong/Hospital Authority Hong Kong West Cluster). Human primary CD14+ monocytes and 555

CD4+ T cells were isolated from PBMCs by magnetic labeling with CD14 or CD4 microbeads 556

respectively, followed by positive selection with MACS columns and separators (Miltenyi 557

Biotec). For preparation of monocyte-derived macrophages, PBMCs were cultured in 10cm 558

dish, and medium was changed gently at 2 hr after cell seeding. Cells were left overnight, 559

and adherent cells were treated with cold 5mM EDTA for 10 min, scrapped and cultured in 560

24-well plates for 12 days. During this period, medium was changed on day 7 and day 9 to 561

remove non-adherent cells. Human PBMCs, CD14+monocytes, monocyte-derived 562

macrophages were cultured in RPMI 1640 supplemented with 5% heat-inactivated 563

autologous sera and 1% penicillin/streptomycin. For monocyte-derived dendritic cells (DCs), 564

CD14+ monocytes were cultured for 5 days in RPMI1640 containing 10% FBS, 1% 565

penicillin/streptomycin, human IL-4 (40ng/ml, PeproTech) and GM-CSF (50ng/ml, 566

PeproTech). Non-adherent or loosely adherent cells were collected. 567

568

Murine bone marrow-derived dendritic cells (BMDCs) were prepared as previously 569

described (Helft et al, 2015). Briefly, bone marrow cells were isolated from the femurs and 570



https://doi.org/10.1101/2020.08.17.253518


tibias of mice. Red blood cells were lysed on ice for 5 min by using 1x RBC Lysis Buffer 571

(BioLegend, 420301). Bone marrow cells (3x106 per well) were cultured in 6-well plates in 3 572

ml of complete medium (RPMI 1640, 1% penicillin/ streptomycin, 10% FBS, 1% Glutamax, 1% 573

sodium pyruvate, and 55M -mercaptoethanol) supplemented with murine GM-CSF (20 574

ng/ml, Peprotech). Half of the medium was removed on day 2 and fresh medium 575

supplemented with murine GM-CSF (40 ng/ml) was added. The culture medium was entirely 576

aspirated on day 3 and replaced by fresh medium containing GM-CSF (20 ng/ml). Non-577

adherent cells in the culture supernatant and loosely adherent cells were harvested on day 578

6. The experiments that were performed for Western blot analysis of cell culture 579

supernatant were carried out in Opti-MEMTM serum free medium (Gibco). Murine 580

splenocytes were prepared by homogenization of murine spleen, and subsequent lysis of 581

red blood cells, and cultured in the complete medium. 582

583

Cell stimulation 584

PBMCs (4x106/ml) were co-cultured with live, PFA-treated or heat-killed T. marneffei, as 585

well as live, heat-killed or PFA-treated C. albicans yeasts or pseudohyphae at 0.5 multiplicity 586

of infection (MOI) for indicated time points. PBMCs (4x106/ml), CD14+monocytes (2x106/ml), 587

monocyte-derived macrophages (1x106/ml), or monocyte-derived DCs (1x106/ml) were 588

primed with LPS (10ng/ml for PBMCs and monocytes, 100ng/ml for monocyte-derived 589

macrophages and monocyte-derived DCs, Invivogen) for 3 hr and then stimulated with heat-590

killed T. marneffei yeasts at 0.5 MOI for 18 hr. Human CD14+ monocytes (2x106/ml) were 591

pre-incubated with human anti-Dectin-1 (10g/ml, Invivogen), anti-TLR2 blocking antibodies 592

(10g/ml, Invivogen), or corresponding isotype controls (10g/ml, Invivogen), or different 593

concentrations of R406 (Invivogen), or Z-YVAD(OMe)-FMK (ALX-260-074, Enzo) for 1 hr 594



https://doi.org/10.1101/2020.08.17.253518


before co-culture with heat-killed T. marneffei yeasts for 18 hr. Murine BMDCs (2x106/ml) 595

were primed with or without LPS (100 ng/ml) for 3 hr, and then co-cultured with heat-killed 596

T. marneffei yeasts at 1 MOI for additional 18 hr. Alternatively, Nigericin (10 M) was added 597

into LPS-primed cells in the last 40 min for positive control. Total CD4+ T cells (1x105/well) 598

were co-cultured for 7 days or 12 days with monocytes (1x105/well)) or monocyte-derived 599

DCs (0.2x105/well) that were pre-pulsed for 3 hr with heat-killed T. marneffei yeasts 600

(1x105/well) or C. albicans yeasts (1x105/well) in 96-well plates. Moreover, murine 601

splenocytes (2x106/ml) were co-cultured with heat-killed T. marneffei yeasts at 1 MOI for 0, 602

2, 6 day(s). 603

604

Cytokine assays 605

Cytokine concentration in culture supernatant was measured by commercial ELISA kits: 606

human IL-1 (ebioscience), human TNF-, IL-18, and IFN-, IL-17A, IL-12p70, and IL-23 (R&D 607

systems), and murine IL-1, TNF-, IFN- and IL-17A (R&D systems) according to 608

manufacturer’s instructions. IL-6, MCP-1, IL-8, IL-18 and IFN- from human PBMCs were 609

measured by LEGENDplex™ Human Inflammation Panel (Biolegend) according to 610

manufacturer’s instructions. 611

612

Western blot 613

Total cell lysates were prepared using sample buffer containing 5% -mercaptoethanol. 614

Protein concentration was determined by bicinchoninic acid (BCA) assay. Concentrated 615

serum free culture supernatant was prepared as previously described (Hornung et al., 2008). 616

Briefly, cell culture supernatant was precipitated by adding an equal volume of methanol 617

and 0.25 volumes of chloroform. It was vigorously vortexed and centrifuged at 20,000 g for 618



https://doi.org/10.1101/2020.08.17.253518


10 min, and the upper phase was aspirated and discarded, and 500 l methanol was added 619

and centrifuged at 20,000 g for 10 min. Then protein pellet was dried in 55℃ water baths 620

for 5 min. Proteins from cell lysates (20-50 g) or concentrated supernatant were boiled, 621

separated on SDS-PAGE, and transferred onto PVDF membranes (Bio-Rad Laboratories). 622

Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) 623

with 0.1% Tween 20 for 1 hr at room temperature, incubated overnight at 4℃ with the 624

following primary antibodies diluted 1:1000 in 5% BSA/TBST: anti-pro-IL-1 (#12703, Cell 625

Signaling Technology) and anti-NLRP3 (AG-20B-0014-C100, Adipogen Life Sciences), and 626

anti-mouse caspase-1 (AG-20B-0042-C100, Adipogen Life Sciences), and then incubated for 627

1 hr with goat anti-rabbit and goat anti-mouse secondary antibodies, respectively, at room 628

temperature. Membranes were washed and exposed by adding enhanced 629

chemiluminescence (ECL) in dark room. When necessary, stained membranes were 630

incubated in stripping buffer at 65℃ for 10 min, and washed 3 times with 0.1% TBS-Tween 631

20, and blocked with 5% BSA for 1 hr, and stained with anti--actin rabbit mAb (4970S, Cell 632

Signaling Technology) followed by goat anti-rabbit secondary antibody staining and film 633

exposure. 634

635

Confocal imaging of ASC specks 636

Murine BMDCs (2x106/ml) were seeded on the chamber slides (154941, Nalge Nunc 637

International) and were co-cultured with heat-killed T. marneffei yeasts at 1 MOI for 18 hr, 638

or were stimulated with LPS (100 ng/ml) for 18 hr with addition of Nigericin (10 M) in the 639

last 40 min. BMDCs were fixed with 4% PFA for 20 min and permeabilized with 0.1% Triton-640

X-100 for 10 min. Cells were washed 3 times and blocked with 5% BSA for 1 hr, followed by 641

incubation with anti-ASC rabbit polyclonal antibody (SC-22514-R, Santa Cruz Biotechnology) 642



https://doi.org/10.1101/2020.08.17.253518


overnight at 4C. After washing, cells were incubated with AlexaFlour-488 conjugated goat 643

anti-rabbit secondary antibody (Life Technologies) at room temperature for 1 hr. 644

Subsequently, cells were stained with Alexa Fluor 647 phalloidin (ThermoFisher Scientific, 645

A22287) for 30 min at room temperature avoiding light. Finally, slides with cells were 646

mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen) and analyzed by 647

confocal microscopy (Zeiss LSM 710). For quantification of ASC speck, 40-150 monocytes per 648

field under microscopy with the magnification of 40x objective were manually analyzed, and 649

the ratio of ASC speck positive cells to total cells was calculated. At least 3 fields per sample 650

were counted. 651

652

Flow cytometry 653

To detect Syk phosphorylation, CD14+ monocytes (2x106/ml) were co-cultured with heat-654

killed T. marneffei yeasts at 0.5 MOI for 18 hr. Cells were fixed with BD Phosflow Fix Buffer I 655

for 10 min at 37℃, followed by permeabilization with BD Phosflow Perm Buffer III for 30 656

min on ice. After washing, cells were stained with PE-conjugated phospho-Syk (Tyk525/526) 657

antibody (#6485, Cell Signaling Technology) for 30 min on ice, and analyzed by flow 658

cytometer (LSR II, BD). For intracellular staining of IFN- and IL-17A, CD4+ T cells were co-659

cultured with monocytes or monocyte-derived DCs in the presence of T. marneffei yeasts or 660

C. albicans yeasts for 7 days or 12 days. Cells were then stimulated with PMA and ionomycin 661

for 5 hr and Golgi plug (BD Biosicence) was added in the last 4 hr. Subsequently, cells were 662

stained for 30 min at 4C with FITC-conjugated anti-human CD3 antibody (Biolegend), 663

followed by fixation and permeabilization of cells with BD Cytofix/Cytoperm™ (BD 664

Bioscience). After washing, cells were stained with Alexa Fluor® 647-conjugated anti-human 665



https://doi.org/10.1101/2020.08.17.253518


IFN- antibody (502516, Biolegend) and PE-conjugated anti-human IL-17A antibody (512305, 666

Biolegend) for 30 min at 4℃. Results were analyzed by flow cytometer (LSR II, BD). 667

668

Murine systemic talaromycosis model 669

For in vivo T. marneffei yeasts infection, WT, Nlrp3-/-, Casp1-/- mice were intravenously 670

injected with 100 l of suspension containing 5x105 colony forming units (CFUs) live T. 671

marneffei yeasts in sterile PBS. Mice survival was daily monitored following infection and 672

mice were sacrificed once they showed signs of the humane endpoints. In the second batch 673

of infection experiments, mice were sacrificed, and spleens and livers were harvested at 7 674

or 14 days post infection (dpi) of T. marneffei yeasts. Part of spleen and liver were weighed 675

and homogenized in sterile PBS, and a series of diluted solutions of cell suspensions were 676

plated onto SDA plates. Fungal load was assessed after culturing for 2 days at 25℃. Part of 677

spleen and liver were fixed in 4% paraformaldehyde (PFA) and paraffin slides were prepared 678

for hematoxylin and esosin (HE) staining and Grocott’s methenamine silver (GMS) staining. 679

The stained sections were mounted and observed under microscope for histopathological 680

assessment. 681

682

Data analysis 683

Statistical analysis was carried out using GraphPad Prism v6.0 software. Figure 5G and H 684

were ploted by R software, version 3.6.1. Data were represented as mean ± SEM. Statistical 685

significance was determined by two-tailed Student’s t test, one-way ANOVA or two-way 686

ANOVA with multiple comparison tests, log-rank test or Gehan-Breslow-Wilcoxon test for 687

comparison of survival rate. p<0.05 was considered statistically significant. 688



https://doi.org/10.1101/2020.08.17.253518


689

Acknowledgements 690

We gratefully acknowledge the Laboratory Animal Unit, the Faculty of Core Facility, and the 691

Histopathological Service Center at the Department of Pathology, the University of Hong 692

Kong, for their professional technical supports. This work was supported by the Edward and 693

Yolanda Wong Fund, the RGC General Research Fund (GRF) (HKU project code: 17111814), 694

and the Health and Medical Research Fund (No. HKM-15-M07 [commissioned project]). 695

696

Author contributions 697

HM, JC, YL, PW, PL designed the experiments and analyzed the results. HM, YT, LK, CT, SP 698

performed the experiments. HM and PL wrote and revised the manuscript. 699

700

Conflict of interest 701

The authors declare that they have no conflict of interest. 702

703

References 704

Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F (2007) Interleukins 1beta and 705

6 but not transforming growth factor-beta are essential for the differentiation of 706

interleukin 17-producing human T helper cells. Nat immunol 8: 942-9 707

Ahmad R, Sindhu ST, Toma E, Morisset R, Ahmad A (2002) Elevated levels of circulating 708

interleukin-18 in human immunodeficiency virus-infected individuals: role of peripheral 709

blood mononuclear cells and implications for AIDS pathogenesis. J Virol 76: 12448-56 710

Bacher P, Hohnstein T, Beerbaum E, Rocker M, Blango MG, Kaufmann S, Rohmel J, 711

Eschenhagen P, Grehn C, Seidel K, Rickerts V, Lozza L, Stervbo U, Nienen M, Babel N, 712



https://doi.org/10.1101/2020.08.17.253518


Milleck J, Assenmacher M, Cornely OA, Ziegler M (2019) Human anti-fungal Th17 713

immunity and pathology rely on cross-reactivity against Candida albicans. Cell 176: 1-16 714

Cao C, Xi L, Chaturvedi V (2019) Talaromycosis (Penicilliosis) Due to Talaromyces (Penicillium) 715

marneffei: Insights into the Clinical Trends of a Major Fungal Disease 60 Years After the 716

Discovery of the Pathogen. Mycopathologia 184: 709-720 717

Chamilos G, Ganguly D, Lande R, Gregorio J, Meller S, Goldman WE, Gilliet M, Kontoyiannis 718

DP (2010) Generation of IL-23 producing dendritic cells (DCs) by airborne fungi regulates 719

fungal pathogenicity via the induction of T(H)-17 responses. PLoS One 5: e12955 720

Chan JF, Lau SK, Yuen KY, Woo PC (2016) Talaromyces (Penicillium) marneffei infection in 721

non-HIV-infected patients. Emerg Microbes Infect 5: e19 722

Chang TH, Huang JH, Lin HC, Chen WY, Lee YH, Hsu LC, Netea MG, Ting JP, Wu-Hsieh BA 723

(2017) Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic 724

cell response to Histoplasma capsulatum. PLoS Pathog 13: e1006485 725

Cheng SC, van de Veerdonk FL, Lenardon M, Stoffels M, Plantinga T, Smeekens S, Rizzetto L, 726

Mukaremera L, Preechasuth K, Cavalieri D, Kanneganti TD, van der Meer JW, Kullberg BJ, 727

Joosten LA, Gow NA, Netea MG (2011) The dectin-1/inflammasome pathway is 728

responsible for the induction of protective T-helper 17 responses that discriminate 729

between yeasts and hyphae of Candida albicans. J Leukoc Biol 90: 357-66 730

Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, Ma L, Watowich SS, Jetten 731

AM, Tian Q, Dong C (2009) Critical regulation of early Th17 cell differentiation by 732

interleukin-1 signaling. Immunity 30: 576-87 733

de Castro LF, Longhi LNA, Paião MR, Justo-Júnior ADS, de Jesus MB, Blotta MHSL, Mamoni 734

RL (2018) NLRP3 inflammasome is involved in the recognition of Paracoccidioides 735



https://doi.org/10.1101/2020.08.17.253518


brasiliensis by human dendritic cells and in the induction of Th17 cells. J Infect 77: 137-736

144. 737

Deepe GS Jr, Gibbons RS (2009) Interleukins 17 and 23 influence the host response to 738

Histoplasma capsulatum. J Infect Dis 200: 142-51 739

Dennehy KM, Willment JA, Williams DL, Brown GD (2009) Reciprocal regulation of IL-23 and 740

IL-12 following co-activation of Dectin-1 and TLR signaling pathways. Eur J Immunol 39: 741

1379-1386 742

Drummond RA, Saijo S, Iwakura Y, Brown GD (2011) The role of Syk/CARD9 couple C-type 743

lectins in antifungal immunity. Eur J Immunol 41: 276-281 744

Erwig LP, Gow NA (2016) Interactions of fungal pathogens with phagocytes. Nat Rev 745

Microbiol 14: 163-76 746

Feriotti C, Bazan SB, Loures FV, Araújo EF, Costa TA, Calich VL (2015) Expression of dectin-1 747

and enhanced activation of NALP3 inflammasome are associated with resistance to 748

paracoccidioidomycosis. Front Microbiol 6: 913 749

Feriotti C, de Araújo EF, Loures FV, da Costa TA, Galdino NAL, Zamboni DS, Calich VLG (2017) 750

NOD-Like Receptor P3 Inflammasome Controls Protective Th1/Th17 Immunity against 751

Pulmonary Paracoccidioidomycosis. Front Immunol 8: 786 752

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM (2003) Collaborative 753

Induction of Inflammatory Responses by Dectin-1 and Toll-like Receptor 2. J Exp Med 754

197: 1107-1117 755

Goncalves AC, Ferreira LS, Manente FA, de Faria C, Polesi MC, de Andrade CR, Zamboni DS, 756

Carlos IZ (2017) The NLRP3 inflammasome contributes to host protection during 757

Sporothrix schenckii infection. Immunology 151: 154-166 758



https://doi.org/10.1101/2020.08.17.253518


Hardison SE, Brown GD (2012) C-type lectin receptors orchestrate antifungal immunity. Nat 759

Immunol 13 (9): 817-822 760

Hardy GA, Sieg S, Rodriguez B, Anthony D, Asaad R, Jiang W, Mudd J, Schacker T, Funderburg 761

NT, Pilch-Cooper HA, Debernardo R, Rabin RL, Lederman MM, Harding CV (2013) 762

Interferon-α is the primary plasma type-I IFN in HIV-1 infection and correlates with 763

immune activation and disease markers. PLoS One 8: e56527 764

Helft J, Bottcher J, Chakravarty P, Zelenay S, Huotari J, Schraml BU, Goubau D, Reis e Sousa C 765

(2015) GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of 766

CD11c(+)MHCII(+) Macrophages and Dendritic Cells. Immunity 42: 1197-211 767

Hise AG, Tomalka J, Ganesan S, Patel K, Hall BA, Brown GD, Fitzgerald KA (2009) An essential 768

role for the NLRP3 inflammasome in host defense against the human fungal pathogen 769

Candida albicans. Cell host microbe 5: 487-97 770

Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E (2008) 771

Silica crystals and aluminum salts mediate NALP-3 inflammasome activation via 772

phagosomal destabilization. Nat Immunol 9 (8): 847-856 773

Kankkunen P, Teirila L, Rintahaka J, Alenius H, Wolff H, Matikainen S (2010) (1,3)-beta-774

glucans activate both dectin-1 and NLRP3 inflammasome in human macrophages. J. 775

Immunol 184: 6335-42 776

Karki R, Man SM, Malireddi RK, Gurung P, Vogel P, Lamkanfi M, Kanneganti TD (2015) 777

Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host 778

protection against Aspergillus infection. Cell host microbe 17: 357-68 779

Kawila R, Chaiwarith R, Supparatpinyo K (2013) Clinical and laboratory characteristics of 780

penicilliosis marneffei among patients with and without HIV infection in Northern 781

Thailand: a retrospective study. BMC Infect Dis 13: 464 782



https://doi.org/10.1101/2020.08.17.253518


Ketelut-Carneiro N, Ghosh S, Levitz SM, Fitzgerald KA, da Silva JS (2018) A Dectin-1-Caspase-783

8 Pathway Licenses Canonical Caspase-1 Inflammasome Activation and Interleukin-1β 784

Release in Response to a Pathogenic Fungus. J Infect Dis 217: 329-339 785

Ketelut-Carneiro N, Souza COS, Benevides L, Gardinassi LG, Silva MC, Tavares LA, Zamboni 786

DS, Silva JS (2019) Caspase-11-dependent IL-1α release boosts Th17 immunity against 787

Paracoccidioides brasiliensis. PLoS Pathog 15: e1007990 788

Latz E, Xiao TS, Stutz A (2013) Activation and regulation of the inflammasomes. Nat Rev 789

Immunol 13: 397-411 790

Lee PP, Chan KW, Lee TL, Ho MH, Chen XY, Li CH, Chu KM, Zeng HS, Lau YL (2012) 791

Penicilliosis in children without HIV infection--are they immunodeficient? Clin Infect Dis 792

54: e8-e19 793

Lee PP, Mao H, Yang W, Chan KW, Ho MH, Lee TL, Chan JF, Woo PC, Tu W, Lau YL (2014) 794

Penicillium marneffei infection and impaired IFN-gamma immunity in humans with 795

autosomal-dominant gain-of-phosphorylation STAT1 mutations. J Allergy Clin Immunol 796

133: 894-6 e5 797

Lee PP, Lau YL (2017) Cellular and Molecular Defects Underlying Invasive Fungal Infections-798

Revelations from Endemic Mycoses. Front Immunol 8: 735 799

Lee PP, Lao-Araya M, Yang J, Chan KW, Ma H, Pei LC, Kui L, Mao H, Yang W, Zhao X, 800

Trakultivakorn M, Lau YL (2019) Application of Flow Cytometry in the Diagnostics 801

Pipeline of Primary Immunodeficiencies Underlying Disseminated Talaromyces 802

marneffei Infection in HIV-Negative Children. Front Immunol 10: 2189 803

LeibundGut-Landmann S, Gross O, Robinson MJ, Osorio F, Slack EC, Tsoni SV, Schweighoffer 804

E, Tybulewicz V, Brown GD, Ruland J, Reis e Sousa C (2007) Syk- and CARD9-dependent 805



https://doi.org/10.1101/2020.08.17.253518


coupling of innate immunity to the induction of T helper cells that produce interleukin 806

17. Nat Immunol 8: 630-8 807

Limper AH, Adenis A, Le T, Harrison TS (2017) Fungal infections in HIV/AIDS. Lancet Infect Dis 808

17: e334-e343 809

Loures FV, Pina A, Felonato M, Araújo EF, Leite KR, Calich VL (2010). Toll-like receptor 4 810

signaling leads to severe fungal infection associated with enhanced proinflammatory 811

immunity and impaired expansion of regulatory T cells. Infect Immun 78: 1078-88 812

Luo DQ, Chen MC, Liu JH, Li Z, Li HT (2010) Disseminated Penicillium Marneffei Infection in 813

an SLE Patient: A Case Report and Literature Review. Mycopathologia 171: 191-19 814

Man SM, Kanneganti TD (2016) Converging roles of caspases in inflammasome activation, 815

cell death and innate immunity. Nat Rev Immunol 16: 7-21 816

Martínez-Barricarte R, Markle JG, Ma CS, Deenick EK, Ramírez-Alejo N, Mele F, Latorre D, 817

Mahdaviani SA, Aytekin C, Mansouri D, Bryant VL, Jabot-Hanin F, Deswarte C, Nieto-818

Patlán A, Surace L, Kerner G, Itan Y, Jovic S, Avery DT, Wong N et al. (2018) Human IFN-γ 819

immunity to mycobacteria is governed by both IL-12 and IL-23. Sci Immunol 3: eaau6759 820

Mócsai A, Ruland J, Tybulewicz VL (2010) The SYK tyrosine kinase: a crucial player in diverse 821

biological functions. Nat Rev Immunol 10: 387-402 822

Nakamura K, Miyazato A, Koguchi Y, Adachi Y, Ohno N, Saijo S, Iwakura Y, Takeda K, Akira S, 823

Fujita J, Ishii K, Kaku M, Kawakami K (2008) Toll-like receptor 2 (TLR2) and dectin-1 824

contribute to the production of IL-12p40 by bone marrow-derived dendritic cells 825

infected with Penicillium marneffei. Microbes Infect 10: 1223-7. 826

Netea MG, Sutmuller R, Hermann C, Van der Graaf CAA, Van der Meer JWM, van Krieken JH, 827

Hartung T, Adema G, Kullberg BJ (2004) Toll-Like Receptor 2 Suppresses Immunity 828



https://doi.org/10.1101/2020.08.17.253518


against Candida albicans through Induction of IL-10 and Regulatory T Cells. J. Immunol 829

172: 3712-3718 830

Ngaosuwankul P, Pongtanalert P, Engering A, Chaiyaroj SC (2008) Differential gene 831

expression profiles of human monocyte-derived antigen presenting cells in response to 832

Penicillium marneffei: roles of DC-SIGN (CD209) in fungal cell uptake. Asian Pac J 833

Allergy Immunol 26: 151-63. 834

Pontillo A, Silva LT, Oshiro TM, Finazzo C, Crovella S, Duarte AJ (2012) HIV-1 induces NALP3-835

inflammasome expression and interleukin-1β secretion in dendritic cells from healthy 836

individuals but not from HIV-positive patients. AIDS 26: 11-8 837

Reis EC, Leal VNC, da Silva LT, Dos Reis MML, Argañaraz ER, Oshiro TM, Pontillo A (2019) 838

Antagonistic role of IL-1ß and NLRP3/IL-18 genetics in chronic HIV-1 infection. Clin 839

Immunol 209: 108266 840

Schroder K, Tschopp J (2010) The inflammasomes. Cell 140: 821-32 841

Sirisanthana T, Supparatpinyo K (1998) Epidemiology and management of penicilliosis in 842

human immunodeficiency virus-infected patients. In 8th International Congress on 843

Infectious Diseases, pp 48-53. Boston, Massachusetts: 844

Son VT, Khue PM, Strobel M (2014) Penicilliosis and AIDS in Haiphong, Vietnam: evolution 845

and predictive factors of death. Med Mal Infect 44: 495-501 846

Stathakis A, Lim KP, Boan P, Lavender M, Wrobel J, Musk M, Heath CH (2015) Penicillium 847

marneffei infection in a lung transplant recipient. Transplant infectious disease : an 848

official journal of the Transplantation Society 17: 429-34 849

Supparatpinyo K, Khamwan C, Baosoung V, Nelson KE, Sirisanthana T (1994) Disseminated 850

Penicillium marneffei infection in Southeast Asia. Lancet 344: 110-13 851



https://doi.org/10.1101/2020.08.17.253518


Tang Y, Zhang H, Xu H, Zeng W, Qiu Y, Tan C, Tang S, Zhang J (2020) Dendritic Cells Promote 852

Treg Expansion but Not Th17 Generation in Response to Talaromyces marneffei Yeast 853

Cells. Infect Drug Resist 13: 805-813 854

Tavares AH, Magalhaes KG, Almeida RD, Correa R, Burgel PH, Bocca AL (2013) NLRP3 855

inflammasome activation by Paracoccidioides brasiliensis. PLoS Negl Trop Dis 7: e25 856

Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, Findon H, Haynes K, Steele C, Botto 857

M, Gordon S, Brown GD (2007) Dectin-1 is required for beta-glucan recognition and 858

control of fungal infection. Nat Immunol 8: 31-8 859

Teng MW, Bowman EP, McElwee JJ, Smyth MJ, Casanova JL, Cooper AM, Cua DJ (2015) IL-12 860

and IL-23 cytokines: from discovery to targeted therapies for immune-mediated 861

inflammatory diseases. Nat Med 21: 719-29 862

Tristão FSM, Rocha FA, Carlos D, Ketelut-Carneiro N, Souza COS, Milanezi CM, Silva JS (2017) 863

Th17-Inducing Cytokines IL-6 and IL-23 Are Crucial for Granuloma Formation during 864

Experimental Paracoccidioidomycosis. Front Immunol 8: 949 865

van de Veerdonk FL, Joosten LA, Shaw PJ, Smeekens SP, Malireddi RK, van der Meer JW, 866

Kullberg BJ, Netea MG, Kanneganti TD (2011) The inflammasome drives protective Th1 867

and Th17 cellular responses in disseminated candidiasis. Eur J Immunol 41: 2260-8 868

Vanittanakom N, Cooper CR, Jr., Fisher MC, Sirisanthana T (2006) Penicillium marneffei 869

infection and recent advances in the epidemiology and molecular biology aspects. Clin 870

Microbiol Rev 19: 95-110 871

Vautier S, MacCallum DM, Brown GD (2012) C-type lectin receptors and cytokines in fungal 872

immunity. Cytokine 58: 89-99 873



https://doi.org/10.1101/2020.08.17.253518


Wang H, LeBert V, Hung CY, Galles K, Saijo S, Lin X, Cole GT, Klein BS, Wüthrich M (2014) C-874

type lectin receptors differentially induce th17 cells and vaccine immunity to the 875

endemic mycosis of North America. J Immunol 192: 1107-1119 876

Wu SY, Yu JS, Liu FT, Miaw SC, Wu-Hsieh BA (2013) Galectin-3 negatively regulates dendritic 877

cell production of IL-23/IL-17-axis cytokines in infection by Histoplasma capsulatum. J 878

Immunol 190: 3427-37 879

Zhou L, Chong MM, Littman DR (2009) Plasticity of CD4+ T cell lineage differentiation. 880

Immunity 30: 646-55 881

882

883

884

885

886

887

888

889

890

891

892

893

894

895

896

897



https://doi.org/10.1101/2020.08.17.253518


Figure Legends 898

Figure 1. T. marneffei yeasts, but not conidia, induce potent IL-1 response in human 899

PBMCs. 900

A, B. Quantification of IL-1 (A) and TNF- (B) by ELISA in human PBMCs (4x106/ml) infected 901

with live T. marneffei yeasts (0.5 MOI) for indicated time points (n=5, mean ± SEM). 902

C, D. ELISA detection of IL-1 (C) and TNF- (D) in human PBMCs (4x106/ml) infected with 903

live T. marneffei conidia (0.5 MOI) for indicated time points (n=5, mean ± SEM). 904

E. Quantification of IFN- by LEGENDplexTM in human PBMCs (4x106/ml) infected with live 905

T. marneffei yeasts (0.5 MOI) for indicated time points (n=5). 906

F. Quantification of IL-17A by ELISA in human PBMCs (4x106/ml) stimulated with heat-907

killed T. marneffei yeasts (0.5 MOI) for indicated time points (n=4). 908

G-J. Quantitative analyses of cytokines by LEGENDplexTM in human PBMCs (4x106/ml) 909

infected with live T. marneffei yeasts (0.5 MOI) for indicated time points (n=5). 910

Data information: In (A-D), “h” and “d” denote “hours” and “days”, respectively. In (E-J), 911

each symbol represents one healthy donor. 912

913

Figure 2. Differential requirement for IL-1 response to T. marneffei yeasts in various 914

human immune cell types. 915

A-D. Quantitative ELISA detection of IL-1 in human PBMCs (4x106/ml), CD14+ monocytes 916

(2x106/ml), human monocytes-derived macrophages (1x106/ml), CD14+ monocytes-917

derived dendritic cells (DCs) (1x106/ml), respectively, after stimulation with heat-killed 918

T. marneffei (TM) yeasts (0.5 MOI) for 18 hr with or without LPS priming. 919

E-H. Quantification of TNF- by ELISA in human PBMCs (4x106/ml), CD14+ monocytes 920

(2x106/ml), human monocytes-derived macrophages (1x106/ml), CD14+ monocytes-921



https://doi.org/10.1101/2020.08.17.253518


derived dendritic cells (DCs) (1x106/ml), respectively, after stimulation with heat-killed 922

T. marneffei (TM) yeasts (0.5 MOI) for 18 hr with or without LPS priming. 923

Data information: In (A-H), each symbol represents one healthy donor, and data are 924

analyzed by one-way ANOVA. ns =not significant, ****p<0.0001. 925

926

Figure 3. Dectin-1/syk signaling mediates IL-1 response to T. marneffei yeasts in human 927

monocytes. 928

A, B. Quantification of IL-1 and TNF- by ELISA in human CD14+ monocytes (2x106/ml) 929

stimulated with heat-killed T. marneffei (TM) yeasts (0.5 MOI) for 18 hr in the presence 930

of anti-hDectin-1 blocking antibodies or mouse IgG (mIgG) (10 g/ml). 931

C, D. Quantification of IL-1 and TNF- by ELISA in human CD14+ monocytes (2x106/ml) 932

stimulated with heat-killed T. marneffei (TM) yeasts (0.5 MOI) for 18 hr in the presence 933

of anti-hTLR2 blocking antibodies or human IgA (hIgA) (10 g/ml). 934

E. Detection of median fluorescence intensity (MFI) of phospho-Syk by flow cytometry in 935

human CD14+ monocytes (2x106/ml) stimulated with heat-killed T. marneffei (TM) 936

yeasts (0.5 MOI) for 18 hr (n=3, mean ± SEM). 937

F. A representative immunoblot of pro-IL- of cell lysates in human CD14+ monocytes 938

(2x106/ml) stimulated with heat-killed T. marneffei (TM) yeasts (0.5 MOI) for 18 hr in 939

the absence or presence of Syk inhibitor, R406 (n=3). 940

G, H. ELISA quantification of IL-1 and TNF- in the culture supernatant of human CD14+ 941

monocytes (2x106/ml) stimulated with heat-killed T. marneffei (TM) yeasts (0.5 MOI) 942

for 18 hr in the absence or presence of Syk inhibitor, R406. 943



https://doi.org/10.1101/2020.08.17.253518


I-K. Quantification of IL-1, IL-18 and TNF-by ELISA in the culture supernatant of human 944

CD14+ monocytes (2x106/ml) stimulated with heat-killed T. marneffei yeasts (0.5 MOI) 945

for 18 hr in the absence or presence of Z-YVAD (caspase-1 inhibitor). 946

Data information: Data are analyzed by paired two-tailed t test (A-E) or by one-way ANOVA 947

(G-K). Each symbol represents one healthy donor. ns =not significant, *p<0.05, **p<0.01, 948

***p<0.001, ****p<0.0001. 949

950

Figure 4. T. marneffei yeasts trigger IL-1 production via the NLRP3 inflammasome. 951

A. Representative immunoblots of NLRP3, caspase-1 p45 and p20 from cell lysates and 952

caspase-1 p20 from concentrated cell supernatant in WT, Nlrp3-/- and Casp-1-/- murine 953

BMDCs stimulated with heat-killed T. marneffei (TM) yeasts (1 MOI) for 18 hr, or LPS 954

plus nigericin as a positive control (n=3). 955

B, C. Representative confocal micrographs (B) and the percentages (C) of ASC specks 956

formation in WT, Nlrp3-/- murine BMDCs (2x106/ml) stimulated with heat-killed T. 957

marneffei (TM) yeasts (1 MOI) for 18 hr, or LPS plus nigericin as a positive control. 958

D, E. Quantitative detection of IL-1 and TNF- by ELISA in the cell supernatant of WT, Casp-959

1-/- and Nlrp3-/- murine BMDCs (2x106/ml) stimulated with heat-killed T. marneffei (TM) 960

yeasts (1 MOI) for 18 hr with or without LPS priming, or LPS plus nigericin as a positive 961

control. 962

Data information: In (C), the percentages of ASC speck positive cells are quantified and 963

depicted as mean ± SEM of n=3 mice for each group, and “ND” denotes “not detectable”. In 964

(D, E), data represent mean±SEM of n=4 mice, and data are analyzed by two-way ANOVA. 965

*p<0.05, **p<0.01, ****p<0.0001. 966

967



https://doi.org/10.1101/2020.08.17.253518


Figure 5. T. marneffei yeasts elicit differential Th1 and Th17 immune responses in human 968

CD14+ monocytes and monocytes-derived DCs. 969

A-C. Analyses of IFN- and/or IL-17A producing CD4 T cells by flow cytometry in human CD4+ 970

T cells (1x105/well) co-cultured with heat-killed T. marneffei- or C. albicans-stimulated 971

autologous CD14+ monocytes (1x105/well) for 7 days and 12 days. 972

D-F. Analyses of IFN- and/or IL-17A producing CD4 T cells by flow cytometry in human CD4+ 973

T cells (1x105/well) co-cultured with heat-killed T. marneffei- or C. albicans-stimulated 974

autologous monocytes-derived DCs (0.2x105/well) for 7 days and 12 days. 975

G. Scatter plot of overall IFN--producing CD4 T cells (y axis) and overall IL-17A-producing 976

CD4 T cells (x axis) in (A and B). 977

H. Scatter plot of overall IFN--producing CD4 T cells (y axis) and overall IL-17A-producing 978

CD4 T cells (x axis) in (D and E). 979

I. Quantitative ELISA analysis of IL-12p70 in the supernatant of human CD14+ monocytes 980

(Monocytes) (2x106/ml) and monocytes-derived DCs (MDDCs) (1x106/ml) stimulated 981

with T. marneffei yeasts or C. albican yeasts for 18 hr. 982

J. Quantitative ELISA analysis of IL-23 in the supernatant of human CD14+ monocytes 983

(Monocytes) (2x106/ml) and monocytes-derived DCs (MDDCs) (1x106/ml) stimulated 984

with T. marneffei yeasts or C. albican yeasts for 18 hr. 985

Data information: In (A-H), data are given as the percentage of total gated CD3+ cells. In (A-F, 986

and I-J), data are analyzed by two-way ANOVA and depicted as means ± SEM. ns =not 987

significant, *p<0.05, **p<0.01, p<0.0001. Each symbol represents one healthy donor in all 988

the data. 989

990



https://doi.org/10.1101/2020.08.17.253518


Figure 6. Caspase-1 inhibition attenuates Th1 and Th17 immune responses to T. marneffei 991

yeasts. 992

A. Quantification of IFN- and IL-17A by ELISA in the supernatant of human PBMCs 993

(4x106/ml) stimulated with heat-killed T. marneffei (TM) yeasts (0.5 MOI) in the 994

absence or presence of caspase-1 inhibitor, Z-YVAD (20M), for 5 days. 995

B. Representative plots of intracellular IFN- and/or IL-17A in the CD4+ T cells (1x105/well) 996

co-cultured with heat-killed T. marneffei (TM) yeasts-stimulated autologous CD14+ 997

monocytes (1x105/well) in the absence or presence of Z-YVAD (20M) for 7 days. 998

C-E. Statistical analyses of overall IFN--producing cells (C) and overall IL-17A-producing cells 999

(D) and IFN-+IL-17A+ -producing cells (E) in co-cultured CD4+ T cells and T. marneffei-1000

primed autologous CD14+ monocytes in the absence or presence of Z-YVAD for 7 days 1001

as described in (B). 1002

F. Detection of murine IFN- and IL-17A by ELISA in the supernatant of WT, Casp-1-/- and 1003

Nlrp3-/- murine splenocytes (2x106/ml) stimulated with heat-killed T. marneffei yeasts 1004

(1 MOI) for indicated time points. 1005

Data information: In (A) and (C-E), each symbol represents one healthy donor, and data are 1006

analyzed by paired two-tailed Student’s t tests. In (F), data are depicted as mean ± SEM of 1007

n=4 mice and are analyzed by two-way ANOVA. ns=not significant, *p<0.05, ***p<0.001, 1008

****p<0.0001. 1009

1010

Figure 7. The NLRP3 inflammasome controls antifungal immunity in vivo 1011

A. Survival plot of WT, Casp-1-/- and Nlrp3-/- mice intravenously injected with live T. 1012

marneffei yeasts (5x105 CFU per mouse). 1013



https://doi.org/10.1101/2020.08.17.253518


B, C. Fungal load analyses of spleens (B) and livers (C) of WT, Casp-1-/- and Nlrp3-/- mice 1014

infected with live T. marneffei yeasts at 7 and 14 days post infection (dpi). 1015

D. Representative HE staining graphs of murine livers of WT, Casp-1-/- and Nlrp3-/- mice 1016

infected with live T. marneffei yeasts (n=4). Scale bar denotes 500 m. The insets 1017

indicate the magnified area of the smaller box containing granulomas, and the asterisk 1018

indicates massive fungal yeasts in the granulomas. 1019

E. Representative GMS staining graphs of murine livers of WT, Casp-1-/- and Nlrp3-/- mice 1020

infected with live T. marneffei yeasts (n=4). Scale bar denotes 50 m. The insets 1021

indicate the magnified area of the smaller box containing T. marneffei yeasts. 1022

Data information: In (A), the log-rank tests are performed when compared between WT and 1023

Nlrp3-/- mice groups, and between Nlrp3-/- and Casp-1-/- groups; the Gehan-Breslow-1024

Wilcoxon test is performed between WT and Casp-1-/- groups. In (B and C), data are 1025

depicted as mean ± SEM of n=5 mice and are analyzed by two-way ANOVA. *p<0.05, 1026

**p<0.01, ***p<0.001. 1027

1028

Figure EV1. The viability of T. marneffei yeasts is dispensable for IL-1 response in human 1029

PBMCs 1030

A, B. Quantification of IL-1 by ELISA in the cell supernatant of human PBMCs (4x106/ml) 1031

stimulated with live, heat-killed or PFA-treated T. marneffei yeasts (0.5 MOI) and 1032

conidia (0.5MOI) (n=5) for 18 hr. 1033

C, D. Quantification of IL-1 by ELISA in the cell supernatant of human PBMCs (4x106/ml) 1034

stimulated with live, heat-killed or PFA-treated C. albicans yeasts (0.5 MOI) and 1035

pseudohyphae (0.5MOI) (n=5) for 18 hr. 1036



https://doi.org/10.1101/2020.08.17.253518


Data information: In (A-D), data are depicted as mean ± SEM, and are analyzed by one-way 1037

ANOVA. ns =not significant, *p<0.05. 1038

1039

Figure EV2. T. marneffei yeasts elicit differential Th1 and Th17 immune responses in 1040

human CD14+ monocytes and monocytes-derived DCs. 1041

A, B. Representative flow cytometric graphs of IFN- and/or IL-17A producing CD4+ T cells 1042

(1x105/well) in human CD4+ T cells stimulated with heat-killed T. marneffei (TM) yeasts 1043

in the absence of autologous CD14+ monocytes or monocytes-derived DCs for 7 days (A) 1044

and 12 days (B). 1045

C, D. Representative flow cytometric plots of IFN- and/or IL-17A producing CD4+ T cells 1046

(1x105/well) in human CD4+ T cells stimulated with heat-killed T. marneffei (TM) yeasts 1047

in the presence of autologous CD14+ monocytes (1x105/well) for 7 days (C) and 12 days 1048

(D). 1049

E, F. Representative flow cytometric plots of IFN- and/or IL-17A producing CD4+ T cells in 1050

human CD4+ T cells (1x105/well) stimulated with heat-killed T. marneffei (TM) yeasts in 1051

the presence of autologous monocytes-derived DCs (0.2x105/well) for 7 days (E) and 12 1052

days (F). 1053

G, H. Flow cytometric analyses of T-bet (G, n=5) and RORt (H, n=4) in CD4+ T cells co-1054

cultured with TM yeasts-primed autologous monocytes for 7 days. 1055

Data information: In (G and H), data are depicted as mean ± SEM, and are analyzed by two-1056

tail Student’s t tests. “MFI” denotes “median fluorescence intensity”. **p<0.01. 1057

1058

Figure EV3. Histopathological analysis of murine livers and spleens. 1059



https://doi.org/10.1101/2020.08.17.253518


A. Representative graphs of HE staining of spleens from WT, Nlrp3-/- and Casp-1-/- mice 1060

intravenously infected T. marneffei yeasts (5x105 CFU per mouse) at 7 days and 14 1061

days post infection (dpi) (n=4). Scale bar denotes 500 m. 1062

B. Representative graphs of GMS staining of spleens from WT, Nlrp3-/- and Casp-1-/- mice 1063

intravenously infected T. marneffei yeasts (5x105 CFU per mouse) at 7 days and 14 1064

days post infection (dpi) (n=4). Scale bar denotes 50 m. Arrows indicate T. marneffei 1065

yeasts, and the insets indicate the magnified areas containing T. marneffei yeasts. 1066



https://doi.org/10.1101/2020.08.17.253518


1

2

Figure 1. T. marneffei yeasts, but not conidia, induce potent IL-1b response in human 3

PBMCs 4

5

6



https://doi.org/10.1101/2020.08.17.253518


7

8

Figure 2. Differential requirement for IL-1b response to T. marneffei yeasts in various 9

human immune cell types. 10

11

12

13

14

15

16



https://doi.org/10.1101/2020.08.17.253518


17

18

Figure 3. Dectin-1/syk signaling mediates IL-1b response to T. marneffei yeasts in human 19

monocytes. 20

21

22

23

24

25

26

27



https://doi.org/10.1101/2020.08.17.253518


28

29

Figure 4. T. marneffei yeasts trigger IL-1b production via the NLRP3 inflammasome. 30

31



https://doi.org/10.1101/2020.08.17.253518


32

Figure 5. T. marneffei yeasts elicit differential Th1 and Th17 immune responses in human 33

CD14+ monocytes and monocyte-derived DCs. 34

35



https://doi.org/10.1101/2020.08.17.253518


36

Figure 6. Caspase-1 inhibition attenuates Th1 and Th17 immune responses to T. marneffei 37

yeasts. 38



https://doi.org/10.1101/2020.08.17.253518


39

40

Figure 7. The NLRP3 inflammasome controls antifungal immunity in vivo 41

42

43

44



https://doi.org/10.1101/2020.08.17.253518


45

46

47

48

Figure EV1. The viability of T. marneffei yeasts is for IL-1b response in human PBMCs 49

50

51

52

53

54

55

56



https://doi.org/10.1101/2020.08.17.253518


57

58

Figure EV2. T. marneffei yeasts elicit differential Th1 and Th17 immune responses in 59

human CD14+ monocytes and monocytes-derived DCs. 60

61

62

63

64



https://doi.org/10.1101/2020.08.17.253518


65

66

Figure EV3. Histopathological analysis of murine spleens after fungal infection 67

68

69

70

71

72

73

74

75



https://doi.org/10.1101/2020.08.17.253518


Graphical abstract 76

77



https://doi.org/10.1101/2020.08.17.253518


Date post:	20-Nov-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Talaromyces marneffei infection - bioRxiv · 2020-08-17 · 84 dependent on the Dectin-1/Syk...

Documents