Myeloid cell deficiency of p38γ/p38δ protects against candidiasis and regulates antifungal immunity

Abstract

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.

Keywords: Candida albicans; infection; kinase inhibitor; p38MAPK; signalling.

Figure 1. Cytokine production and ERK1/2 activation is impaired in p38γ/p38δ‐null BMDM in response to TLRs and Dectin‐1 ligands

1. BMDM from WT or p38γ/δ^−/− mice were exposed to HK‐Ca (1 × 10⁶ CFU/ml) for the indicated times. Relative mRNA expression was determined by qPCR for TNFα, IL‐6, IL‐1β, IL‐10, KC, MIP‐2 and CCL2. Results were normalized to β‐actin mRNA expression, and x‐fold induction was calculated relative to WT expression at 0 h. Data show mean ± SEM from one representative experiment of two in triplicate, with similar results. Only significant results are indicated, *P ≤ 0.05 relative to WT BMDM exposed to HK‐Ca, at each time point. Parametric, unpaired t‐test.

2. BMDM from WT or p38γ/δ^−/− mice were stimulated as in (A). Cell lysates (50 μg) were immunoblotted with antibodies to active phosphorylated ERK1/2 (P‐ERK1/2), p38α (P‐p38α) or JNK1/2 (P‐JNK1/2), or to phosphorylated p105 NF‐κB1 (P‐p105). Total protein levels for the above proteins and for TPL2 were also measured as loading controls. Representative immunoblots from three independent experiments are shown.

3. BMDM were stimulated 200 ng/ml Pam3Cys or 100 ng/ml LPS. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots from three independent experiments are shown.

4. BMDM were exposed for 1 h to 10 μg/ml Curdlan. Relative mRNA expression was determined by qPCR for IL‐1β. Results were normalized and fold induction calculated as in (A). Data show mean ± SEM from one representative experiment of two in triplicate, with similar results. Only significant results are indicated, *P ≤ 0.05 relative to WT BMDM exposed to Curdlan. Parametric, unpaired t‐test.

5. BMDM were stimulated with 10 μg/ml Curdlan and cell lysated immunoblotted as shown. Representative immunoblots from three independent experiments are shown.

Source data are available online for this figure.

Figure 2. ERK1/2 activation is mediated by TPL2 in Dectin‐1 signalling

1. BMDM from TPL2^+/+ or TPL2^−/− mice were stimulated with 1 × 10⁶ CFU/ml HK‐Ca, 10 μg/ml Curdlan or 50 μg/ml Zymosan for 1 h, or with 100 ng/ml LPS for 30 min. Cell lysates were immunoblotted with the indicated antibodies. Representative immunoblots are shown. Bands from three experiments were quantified using the Odyssey infrared imaging system (bottom), and data show mean ± SEM from two experiments in duplicate. ***P ≤ 0.001. Parametric, unpaired t‐test.

2. WT BMDM were incubated for 1 h with or without 5 μM C34 or 2 μM PD184352, and then stimulated for 1 h with 10 μg/ml Curdlan. Representative immunoblots from two independent experiments are shown.

3. BMDM were incubated for 1 h with DMSO or with 5 μM C34, and then exposed for 1 h to 10 μg/ml Curdlan. Relative mRNA expression was determined by qPCR for IL‐1β. Results were normalized and fold induction calculated. Data show mean ± SEM from one representative experiment of two in triplicate, with similar results. ns, not significant, *P ≤ 0.05 relative to WT BMDM exposed to Curdlan. Parametric, unpaired t‐test.

4. WT BMDM were incubated for 1 h with or without 5 μM C34 or 10 μM BI605906 and then stimulated with Curdlan as in (B). Representative immunoblots from four independent experiments are shown.

5. WT BMDM were incubated for 1 h with DMSO or with 2 or 5 μM NG25 and then stimulated with HK‐Ca, Curdlan or LPS as in (A). Representative immunoblots from two independent experiments are shown.

6. WT BMDM were incubated for 1 h in the absence or the presence of 5 μM C34 or 10 μM BI605906, and then stimulated for 1 h with 1 × 10⁶ CFU/ml HK‐Ca. Cell lysates were immunoblotted as indicated. Representative blots from two independent experiments are shown.

7. Schematic representation of the Dectin‐1 signalling pathways involved in ERK1/2 activation, which is controlled by TAK1‐IKK‐TPL2 in HK‐Ca and Curdlan‐stimulated macrophages. The activation of the TAK1 complex (TAB 1‐TAK1‐TAB 2/3) and of the IKK pathway (Cohen, 2014) might be mediated by TRAF6, which binds to the CARD9/BCL‐10/MALT1 complex downstream of Syk (Geijtenbeek & Gringhuis, 2009). TLR stimulation by HK‐Ca also triggers the activation of TAK1‐IKK‐TPL2 via MyD88. p38γ and p38δ regulate TPL2 steady‐state levels, which is in a complex with ABIN‐2 and p105 (Gantke et al, 2011).

Source data are available online for this figure.

Figure 3. p38γ/p38δ deletion decreases Candida albicans infection in mice

1. A, B

   WT, p38γ/δ^−/− and LysM‐p38γ/δ^−/− mice were infected with 1 × 10⁵ CFU C. albicans. (A) Survival monitored as indicated. Data are presented as a Kaplan–Meier plot from two independent experiments (n = 20 mice per genotype). Two‐way ANOVA using GraphPad Prism software. (B) Kidney fungal burden at day 3 post‐infection. Data are expressed as CFU/g kidney (mean ± SEM). Each symbol represents an individual mouse. ns, not significant, *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 relative to WT mice. Parametric, unpaired t‐test.

2. C

   Representative PAS‐haematoxylin staining of kidney sections from mice at day 3 post‐infection. Bottom panels are high magnification of the area marked by a dotted square in the top panels. Scale bars are 100 μm.

3. D, E

   TPL2^+/+ and TPL2^−/− mice were infected with C. albicans as in (A). (D) Death was monitored. Data are a summary of two independent experiments (n = 12 mice per genotype). (E) Kidney fungal burden at day 3 post‐infection with 1 × 10⁵ CFU. Each symbol represents an individual mouse. Data are expressed as CFU/g kidney (mean ± SEM). *P ≤ 0.05 relative to WT mice. Two‐way ANOVA using GraphPad Prism software.

Figure 4. Reduced inflammation in p38γ/δ^−/− mice in response to Candida albicans infection

1. WT, p38γ/δ^−/− and LysM‐p38γ/δ^−/− mice were intravenously infected with 1 × 10⁵ CFU C. albicans and at days 1 and 3 post‐infection, relative TNFα, IL‐6 and IL‐1β mRNA expression in the kidney was determined by qPCR and normalized to β‐actin mRNA. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 5–8). ns, not significant, *P ≤ 0.05, **P ≤ 0.01 relative to WT mice. Parametric, unpaired t‐test.

2. Kidney cells from 0‐, 1‐ and 3‐day C. albicans‐treated WT, p38γ/δ^−/− and LysM‐p38γ/δ^−/− mice as in (A) were stained with anti‐CD45, ‐Ly6G and ‐F4/80 antibodies and positive cells analysed by flow cytometry. CD45⁺ cells were gated and ‐F4/80⁺ and ‐Ly6G⁺ cells analysed by flow cytometry. Representative profiles are shown. Each symbol represents an individual mouse (two to three independent experiment). Figure shows mean ± SEM (n = 5–14 mice/condition), ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, relative to WT kidney cells, at each time point. Parametric, unpaired t‐test.

3. Mice were intraperitoneally infected with 5 × 10⁶ CFU C. albicans, and at day 1 post‐infection, peritoneal cells were stained and analysed as in (B). Representative profiles are shown. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 4), ns not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, relative to WT kidney cells. Parametric, unpaired t‐test.

4. Mice were infected with C. albicans as in (A), relative MIP‐2, KC and CCL2 mRNA expression in the kidney was determined by qPCR as in (A). Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 3–5). ns, not significant, *P ≤ 0.05, **P ≤ 0.01 relative to WT mice. Parametric, unpaired t‐test.

Figure 5. p38γ/p38δ regulate antifungal activity against Candida albicans

1. A

   BMDM from WT or p38γ/δ^−/− mice were exposed for the indicated times to 1 × 10⁶ CFU/ml HK‐Ca. Relative iNOS mRNA expression was determined by qPCR. Data show mean ± SEM from one representative experiment of two in triplicate, with similar results. *P ≤ 0.05, ***P ≤ 0.001 relative to WT BMDM. Parametric, unpaired t‐test.

2. B

   ROS production in BMDM co‐cultured with 1 × 10⁶ CFU C. albicans. Experiments were performed in triplicate. RLU, relative light unit.

3. C

   Candida albicans killing by BMDM. The results are represented as percentage of killing (the percentage of killed fungal cells among the phagocytosed fungus). The C. albicans/BMDM ratio was 1:10. Values are mean ± SEM (n = 6); ***P ≤ 0.001 relative to WT cells. Parametric, unpaired t‐test.

4. D

   ROS production in neutrophils isolated from blood of WT or p38γ/δ^−/− mice after infection. Neutrophils were stimulated with 1.5 × 10⁶/ml HK‐Ca. Experiments were performed in triplicate.

5. E

   Candicidal activity of neutrophils determined as in (C). Values are mean ± SEM (n = 4); *P ≤ 0.05 relative to WT cells. Parametric, unpaired t‐test.

6. F, G

   ROS production without (F) or with (G) re‐stimulation with C. albicans in intraperitoneal immune cell infiltrates of WT or p38γ/δ^−/− mice 1 day after intraperitoneal C. albicans infection (5 × 10⁶ CFU, n = 4 mice per group).

7. H

   WT and p38γ/δ^−/− mice were intravenously infected with 1 × 10⁵ CFU C. albicans. Relative iNOS mRNA expression in the kidney was determined by qPCR and normalized to β‐actin mRNA. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 3–5). Only significant results are indicated, *P ≤ 0.05, relative to WT mice. Parametric, unpaired t‐test.

8. I

   WT and p38γ/δ^−/− mice were infected with 1 × 10⁵ CFU C. albicans and treated with 200 mg/kg body weight per day of N‐acetylcysteine (NAC) from SIGMA or with the same volume of the vehicle PBS for 5 days [WT + CA + NAC (n = 8); p38γ/δ^−/− + CA + NAC (n = 8)]. Control groups of WT and p38γ/δ^−/− mice treated with 200 mg/kg body weight per day of NAC were included to check its toxicity [WT + NAC (n = 10); p38γ/δ^−/− + NAC (n = 10)]. Control groups of WT and p38γ/δ^−/− mice infected with C. albicans were also included for comparison [WT + CA (n = 8); p38γ/δ^−/− + CA (n = 8)]. Survival was monitored as indicated. Data are presented as a Kaplan–Meier plot. ns, not significant, *P ≤ 0.05; **P ≤ 0.01. Two‐way ANOVA using GraphPad Prism software.

Figure 6. Treatment with p38γ/p38δ inhibitor shows antifungal effects in vivo

1. WT mice were intravenously injected with 1 × 10⁵ CFU of Candida albicans and treated with 10 mg BIRB796 or SB203580 per kg body weight per day, or with the same volume of the vehicle DMSO. Kidney fungal load was determined 3 days after infection. ns, not significant, *P ≤ 0.05 (n = 5 mice/condition). Each symbol represents an individual mouse. Parametric, unpaired t‐test.

2. Mice were treated as in (A) and iNOS mRNA levels in the kidney measured 3 days after infection by qPCR. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 5 mice/condition). ns, not significant, *P ≤ 0.05. Parametric, unpaired t‐test.

3. Neutrophil infiltration in the kidney of infected WT mice, treated with BIRB796 or SB203580 inhibitor as in (A) was determined by flow cytometry. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 6 mice/condition), ns not significant; ***P ≤ 0.001. Parametric, unpaired t‐test.

4. Mice were intraperitoneally infected with 5 × 10⁶ CFU C. albicans and treated with 10 mg/kg body weight per day BIRB796 or SB203580, or with the same volume of the vehicle DMSO. Neutrophil infiltration in the peritoneum was measured by flow cytometry at day 1 post‐infection. Each symbol represents an individual mouse. Figure shows mean ± SEM (n = 4 mice/condition), ns, not significant; *P ≤ 0.05, **P ≤ 0.01. Parametric, unpaired t‐test.