Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes

Abstract

Immunological memory in vertebrates is often exclusively attributed to T and B cell function. Recently it was proposed that the enhanced and sustained innate immune responses following initial infectious exposure may also afford protection against reinfection. Testing this concept of "trained immunity," we show that mice lacking functional T and B lymphocytes are protected against reinfection with Candida albicans in a monocyte-dependent manner. C. albicans and fungal cell wall β-glucans induced functional reprogramming of monocytes, leading to enhanced cytokine production in vivo and in vitro. The training required the β-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. Monocyte training by β-glucans was associated with stable changes in histone trimethylation at H3K4, which suggests the involvement of epigenetic mechanisms in this phenomenon. The functional reprogramming of monocytes, reminiscent of similar NK cell properties, supports the concept of "trained immunity" and may be employed for the design of improved vaccination strategies.

Figure 1. Nonlethal Candidiasis Protects Mice through a Macrophage-Dependent Mechanism

(A) Survival rate of WT, Rag1^−/−, and Ccr2^−/− mice to systemic candidiasis, following vaccination with PBS (control) or a nonlethal dose of live C. albicans (Candida, preinjection) 7 days earlier (n ≥ 8 per group, two independent experiments). (B) Fungal burdens in kidneys from Rag1^−/− mice 14 days after C. albicans infection. (C) Survival rate of WT mice preinjected with anti-asialo GM1 antibody or an unrelated antibody (control) prior to the inoculation of lethal C. albicans (n ≥ 10 per group). (D) In vivo training of mice (n = 5 per group) with heat-inactivated C. albicans (black bars), 7 days prior to the LPS injection, enhanced proinflammatory cytokines release compared to PBS-treated control mice (white bars). *p < 0.05 versus control animals.

Figure 2. Candida albicans and β-Glucans Prime the Production of Proinflammatory Cytokines

(A) Diagram showing the course of the in vitro preincubation experiment. (B and C) C. albicans training in vitro using freshly isolated human PBMCs (B) or monocytes (C) and different PRR ligands for restimulation (P3C, Pam3ys; C.a, C. albicans; M.tb, Mycobacteria tuberculosis). (D) The training effects induced by purified β-glucans (blue), but not with mannans (gray). E. coli preincubation induced immune tolerance (red). (E) CD14⁺-selected monocytes are also primed by β-glucan. (F) The priming effect obtained after 1 week of resting period was also seen after 2 weeks. (G) Candidacidal activity of cell culture medium (control) or β-glucans-preincubated monocytes was assessed after 5 hr of incubation with live opsonized C. albicans (n = 6). In (B)–(G), *p < 0.05 (mean ± SD, n = 5–8). See also Figure S1.

Figure 3. The Role of β-Glucan Receptors for Monocyte Training

(A–D) Cytokine production in supernatants of adherent monocytes primed for 24 hr with either cell culture medium or β-glucans and restimulated with different PRR ligands in healthy volunteers (control) and cells isolated from a dectin-1-deficient volunteer (A), in the presence or absence of anti-CR3 antibody (B), and in the presence or absence of Syk kinase inhibitor (C) or Raf-1 inhibitor GW5074 (D). (E) Survival rate of WT mice to C. albicans infection. All mice were initially treated with a Raf-1 inhibitor. Mice were then preinjected with either PBS (control) or a nonlethal dose of live C. albicans (Candida, preinjection) 7 days prior to inoculation of the lethal C. albicans dose (n ≥ 10 per group). (F) Western blot of total and phosphorylated p38 in cells primed with RPMI (controls) or β-glucan and subjected to 5, 15, or 30 min stimulation with RPMI or Pam3Cys. (G) Quantification of the effect of β-glucan priming on the phosphorylation of p38. (H and I) Fold changes in Tnfa (H) and Il6 (I) mRNA expression obtained by quantitative real-time PCR in adherent monocytes after stimulation with LPS, Pam3Cys, or C. albicans. Cells were primed with either nutrient-rich medium (RPMI, control) or with β-glucan. (J–M) Cytokine production in supernatants of adherent monocytes primed 24 hr with either cell culture medium or C. albicans (J and L) or β-glucans (K and M) in the absence or presence of the histone demethylase inhibitor pargyline (J and K) and the histone methyltransferase inhibitor MTA (5′-deoxy-5′(methylthio) adenosine) (L and M) and restimulated with different PRR ligands. In (A)–(D) and (J)–(M), the ratios of cytokine production by β-glucan primed versus nonprimed monocytes are presented. Data are shown as mean ± SD. n = 6 for (B), (D), and (J)–(M) and n = 5 for (C). Data presented in (A) are means of two independent experiments. n ≥ 6 for (H) and (I). *p < 0.05. See also Figure S2.

Figure 4. β-Glucan Alters the Epigenetic Landscape in Trained Monocytes, and This Correlates with β-Glucan-Induced Transcriptomal Changes

(A) Box plot of log2 tag density of H3K4me3 and H3K27me3 signal in control versus trained monocytes. (B) Box plot of log2 tag density for genome-wide H3K4me3 enriched regions in peritoneal macrophages retrieved from untreated (control, saline) and Candida preinjected mice. (C) Intensity plots showing the normalized tag density for H3K4me3 and H3K27me3 on the 500 H3K4me3 enhanced and decreased regions. (D–I) Genome browser screen shot showing H3K4me3 binding over TNF-α (D), IL-6 (E), IL-18 (F), DECTIN-1 (G), TLR4 (H), and MYD88 promoter regions (I) before and after the treatment with β-glucan. (J) Box plot showing RNA expression (RPKM) of the genes associated with top and bottom 500 regions with H3K4me3 changes. (K) H3K4me3 tag density as well as RNA-seq expression data over SETD7 histone methyltransferase before and after the treatment with β-glucan. (L and M) RPKM values of genes after β-glucan treatment of monocytes. The genes are markers of classically and alternatively activated macrophages (L) and of inflammatory Tip-DCs (M). See also Figure S3.