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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: ppwlee@hku.hk 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
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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
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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
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(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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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The copyright holder for this preprintthis version posted August 23, 2020. ; https://doi.org/10.1101/2020.08.17.253518doi: bioRxiv preprint
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
.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
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
.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
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
.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
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
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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
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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
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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
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The copyright holder for this preprintthis version posted August 23, 2020. ; https://doi.org/10.1101/2020.08.17.253518doi: bioRxiv preprint
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
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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
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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
.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
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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
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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
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1
2
Figure 1. T. marneffei yeasts, but not conidia, induce potent IL-1b response in human 3
PBMCs 4
5
6
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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
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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
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28
29
Figure 4. T. marneffei yeasts trigger IL-1b production via the NLRP3 inflammasome. 30
31
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32
Figure 5. T. marneffei yeasts elicit differential Th1 and Th17 immune responses in human 33
CD14+ monocytes and monocyte-derived DCs. 34
35
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36
Figure 6. Caspase-1 inhibition attenuates Th1 and Th17 immune responses to T. marneffei 37
yeasts. 38
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The copyright holder for this preprintthis version posted August 23, 2020. ; https://doi.org/10.1101/2020.08.17.253518doi: bioRxiv preprint
39
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Figure 7. The NLRP3 inflammasome controls antifungal immunity in vivo 41
42
43
44
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The copyright holder for this preprintthis version posted August 23, 2020. ; https://doi.org/10.1101/2020.08.17.253518doi: bioRxiv preprint
45
46
47
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Figure EV1. The viability of T. marneffei yeasts is for IL-1b response in human PBMCs 49
50
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53
54
55
56
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57
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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
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Figure EV3. Histopathological analysis of murine spleens after fungal infection 67
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Graphical abstract 76
77
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