Recurrent candidiasis and early-onset gastric cancer in a patient with a genetically defined partial MYD88 defect.

Gastric cancer is caused by both genetic and environmental factors. A woman who suffered from recurrent candidiasis throughout her life developed diffuse-type gastric cancer at the age of 23 years. Using whole-exome sequencing we identified a germline homozygous missense variant in MYD88. Immunological assays on peripheral blood mononuclear cells revealed an impaired immune response upon stimulation with Candida albicans, characterized by a defective production of the cytokine interleukin-17. Our data suggest that a genetic defect in MYD88 results in an impaired immune response and may increase gastric cancer risk.

An anti-inflammatory property of Candida albicans ß-glucan: Induction of high levels of interleukin-1 receptor antagonist via a Dectin-1/CR3 independent mechanism.

BACKGROUND: Candida albicans is an opportunistic fungal pathogen that induces strong proinflammatory responses, such as IL-1ß production. Much less is known about the induction of immune modulatory cytokines, such as the IL-1 receptor antagonist (IL-1Ra) that is the main natural antagonist of IL-1, by C. albicans. METHODS: Peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with C. albicans and different components of the fungal cell wall. The role of pathogen recognition receptors (PRRs) for the induction of IL-1ß and IL-1Ra was investigated by using specific blockers or in PBMC from Dectin-1 deficient patients. RESULTS: C. albicans induced a strong IL-1Ra response, and this induction was primarily induced by the cell-wall component ß-glucan. Blocking IL-1Ra significantly increased C. albicans ß-glucan hyphae induced IL-1ß and IL-6 production. Surprisingly, blocking the ß-glucan receptor Dectin-1 or the downstream Syk or Raf-1 pathways only marginally reduced C. albicans-induced IL-1Ra production, while blocking of the complement receptor 3 (CR3), TLR2 or TLR4 had no effect. In line with this, blocking MAP kinases had little effect on Candida-induced IL-1Ra production. PBMC isolated from Dectin-1 deficient patients produced normal IL-1Ra amounts in response to C. albicans stimulation. Interestingly, the IL-1Ra synthesis induced by ß-glucan was blocked by inhibitors of the Akt/PI3K pathway. CONCLUSIONS: ß-glucan of C. albicans induces a strong IL-1Ra response, which is independent of the ß-glucan receptors dectin-1 and CR3. These data strongly argue for the existence of an unknown ß-glucan receptor that specifically induces an Akt/PI3K-dependent anti-inflammatory IL-1Ra response upon recognition of C. albicans.

Candida albicans primes TLR cytokine responses through a Dectin-1/Raf-1-mediated pathway.

The immune system is essential to maintain homeostasis with resident microbial populations, ensuring that the symbiotic host-microbial relationship is maintained. In parallel, commensal microbes significantly shape mammalian immunity at the host mucosal surface, as well as systemically. Candida albicans is an opportunistic pathogen that lives as a commensal on skin and mucosa of healthy individuals. Little is known about its capacity to modulate responses toward other microorganisms, such as colonizing bacteria (e.g., intestinal microorganisms). The aim of this study was to assess the cytokine production of PBMCs induced by commensal bacteria when these cells were primed by C. albicans. We show that C. albicans and ß-1,3-glucan induce priming of human primary mononuclear cells and this leads to enhanced cytokine production upon in vitro stimulation with TLR ligands and bacterial commensals. This priming requires the ß-1,3-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. In addition, although purified mannans cannot solely mediate the priming, the presence of mannosyl residues in the cell wall of C. albicans is nevertheless required. In conclusion, C. albicans is able to modify cytokine responses to TLR ligands and colonizing bacteria, which is likely to impact the inflammatory reaction during mucosal diseases.

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

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 ß-glucans induced functional reprogramming of monocytes, leading to enhanced cytokine production in vivo and in vitro. The training required the ß-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. Monocyte training by ß-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.

The classical CD14⁺⁺ CD16⁻ monocytes, but not the patrolling CD14⁺ CD16⁺ monocytes, promote Th17 responses to Candida albicans.

In the present study, we investigated the functional differences between cluster of differentiation (CD)14(++) CD16(-) and CD14(+) CD16(+) monocytes during anti-Candida host defense. CD14(++) CD16(-) are the "classical" monocytes and represent the majority of circulating monocytes in humans, while CD14(+) CD16(+) monocytes patrol the vasculature for maintenance of tissue integrity and repair. Both monocyte subsets inhibited the germination of live Candida albicans, and there was no difference in their capacity to phagocytose and kill Candida. Although production of IL-6 and IL-10 induced by C. albicans was found to be similar between monocyte subsets, IL-1ß and prostaglandin E2 (PGE2) production was higher in CD14(++) CD16(-) compared with CD14(+) CD16(+) monocytes. In line with the increased production of IL-1ß and PGE2, central mediators for inducing Th17 responses, CD14(++) CD16(-) monocytes induced greater Th17 responses upon stimulation with heat-killed C. albicans yeast. The percentage of cells that expressed mannose receptor (MR) was higher in the CD14(++) CD16(-) monocyte subset, and MR-specific stimulation induced higher Th17 responses only in co-cultures of CD14(++) CD16(-) monocytes and CD4 lymphocytes. In conclusion, both monocyte subsets have potent innate antifungal properties, but only CD14(++) CD16(-) monocytes are capable of inducing a potent Th17 response to C. albicans, an important component of antifungal host defense.

Human dectin-1 deficiency and mucocutaneous fungal infections.

Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

Redundant role of TLR9 for anti-Candida host defense.

The role of Toll-like receptor-9 (TLR9) in the recognition of Candida albicans and anti-Candida host defense was investigated in a murine model of disseminated candidiasis and in human peripheral blood mononuclear cells (PBMC). Blocking TLR9 by a specific inhibitor of human TLR9 or stimulation of cells isolated from TLR9-deficient (TLR9-/-) mice resulted in a 20-30% reduction in cytokine production induced by C. albicans. However, this defect was not accompanied by differences in mortality and organ fungal growth between TLR9-/- and TLR9+/+ mice. In conclusion, TLR9 is a pathogen-recognition receptor for C. albicans, and TLR9 is involved in the induction of cytokines in response to C. albicans. However, the cytokine defect in TLR9-/- mice is compensated by alternative pathways, and the TLR9-dependent pathway seems to be redundant in the disseminated candidiasis model in mice.

Immune recognition of Candida albicans beta-glucan by dectin-1.

Beta (1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal beta (1,3)-glucans and induce innate immune responses. The importance of beta-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of beta-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of beta-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-alpha . In contrast, stimulation of Th1-type cytokines, such as interferon-gamma , by C. albicans was independent of the recognition of beta-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.

Plasmodium falciparum infection causes proinflammatory priming of human TLR responses.

TLRs are a major group of pattern recognition receptors that are crucial in initiating innate immune responses and are capable of recognizing Plasmodium ligands. We have investigated TLR responses during acute experimental P. falciparum (P.f.) infection in 15 malaria-naive volunteers. TLR-4 responses in whole blood ex vivo stimulations were characterized by significantly (p < 0.01) up-regulated proinflammatory cytokine production during infection compared with baseline, whereas TLR-2/TLR-1 responses demonstrated increases in both proinflammatory and anti-inflammatory cytokine production. Responses through other TLRs were less obviously modified by malaria infection. The degree to which proinflammatory TLR responses were boosted early in infection was partially prognostic of clinical inflammatory parameters during the subsequent clinical course. Although simultaneous costimulation of human PBMC with P.f. lysate and specific TLR stimuli in vitro did not induce synergistic effects on cytokine synthesis, PBMC started to respond to subsequent TLR-4 and TLR-2 stimulation with significantly (p < 0.05) increased TNF-alpha and reduced IL-10 production following increasing periods of preincubation with P.f. Ag. In contrast, preincubation with preparations derived from other parasitic, bacterial, and fungal pathogens strongly suppressed subsequent TLR responses. Taken together, P.f. primes human TLR responses toward a more proinflammatory cytokine profile both in vitro and in vivo, a characteristic exceptional among microorganisms.

Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release.

The recognition of peptidoglycan by cells of the innate immune system has been controversial; both TLR2 and nucleotide-binding oligomerization domain-2 (NOD2) have been implicated in this process. In the present study we demonstrate that although NOD2 is required for recognition of peptidoglycan, this leads to strong synergistic effects on TLR2-mediated production of both pro- and anti-inflammatory cytokines. Defective IL-10 production in patients with Crohn's disease bearing loss of function mutations of NOD2 may lead to overwhelming inflammation due to a subsequent Th1 bias. In addition to the potentiation of TLR2 effects, NOD2 is a modulator of signals transmitted through TLR4 and TLR3, but not through TLR5, TLR9, or TLR7. Thus, interaction between NOD2 and specific TLR pathways may represent an important modulatory mechanism of innate immune responses.

Mannose binding lectin enhances IL-1beta and IL-10 induction by non-lipopolysaccharide (LPS) components of Neisseria meningitidis.

Mannose binding lectin (MBL) is a key molecule in the lectin pathway of complement activation, and likely of importance in our innate defence against meningococcal infection. We evaluated the role of MBL in cytokine induction by LPS or non-LPS components of Neisseria meningitidis, using a meningococcal mutant deficient for LPS. Binding experiments showed that MBL exhibited low, but significant binding to encapsulated LPS+ meningococci (H44/76) and LPS-deficient (LPS-) meningococci (H44/76lpxA). Experiments with human mononuclear cells (PBMCs) showed that MBL significantly augmented IL-1beta production after stimulation with LPS+ and LPS- meningococci, in a dose-dependent fashion. In addition, IL-10 production was enhanced after stimulation with LPS- meningococci. In contrast, TNFalpha, IL-6 and IFNgamma productions were unaffected. No effect of MBL was observed on cytokine induction by meningococcal LPS. MBL enhanced cytokine production at concentrations >10(7) meningococci. It is concluded that MBL interacts with non-LPS components of N. meningitidis and in this way modulates the cytokine response.

Chlamydia pneumoniae stimulates IFN-gamma synthesis through MyD88-dependent, TLR2- and TLR4-independent induction of IL-18 release.

Recent studies suggest that inflammation plays a central role in the pathogenesis of atherosclerosis, and IFN-gamma is a prominent proinflammatory mediator in this context. However, it is unclear what stimuli are responsible for initial stimulation of IFN-gamma synthesis in the vessel wall. In the present study, we demonstrate that Chlamydia pneumoniae is an important stimulus for IFN-gamma synthesis, and this production depends on release of endogenous IL-18, IL-12, and IL-1, but not of TNF. The production of the proinflammatory cytokines TNF and IL-1beta from PBMC by sonicated C. pneumoniae was mediated through TLR2-dependent pathways. In contrast, C. pneumoniae stimulated the production of IL-18 through MyD88-dependent, TLR2-, TLR4-, and CD14-independent pathways, mediated by posttranscriptional mechanisms not involving de novo protein synthesis. In conclusion, C. pneumoniae is a potent stimulus of IFN-gamma production, in addition to the proinflammatory cytokines TNF and IL-1beta, which may contribute to its proatherogenic effects. Most interestingly, C. pneumoniae is also a potent inducer of IL-18 production through pathways independent of TLR2 and TLR4.

Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria.

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria-induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.

Aspergillus fumigatus evades immune recognition during germination through loss of toll-like receptor-4-mediated signal transduction.

Peritoneal macrophages from Toll-like receptor (TLR) 4-deficient ScCr mice produced less tumor necrosis factor, interleukin (IL)-1alpha, and IL-1beta than did macrophages of control mice, when stimulated with conidia, but not with hyphae, of Aspergillus fumigatus, a finding suggesting that TLR4-mediated signals are lost during germination. This hypothesis was confirmed by use of a TLR4-specific fibroblast reporter cell line (3E10) that responded to the conidia, but not to the hyphae, of A. fumigatus. In contrast, macrophages from TLR2-knockout mice had a decreased production of proinflammatory cytokines in response to both Aspergillus conidia and Aspergillus hyphae, and these results were confirmed in 3E10 cells transfected with human TLR2. In addition, Aspergillus hyphae, but not Aspergillus conidia, stimulated production of IL-10 through TLR2-dependent mechanisms. In conclusion, TLR4-mediated proinflammatory signals, but not TLR2-induced anti-inflammatory signals, are lost on Aspergillus germination to hyphae. Therefore, phenotypic switching during germination may be an important escape mechanism of A. fumigatus that results in counteracting the host defense.

Native LDL potentiate TNF alpha and IL-8 production by human mononuclear cells.

Native LDL (nLDL) increases expression of adhesion molecules on endothelial cells through induction of Ca(2+) mobilization. Ca(2+) mobilization is also involved in the induction of proinflammatory cytokines, important mediators involved in atherogenesis. The aim of the study was to evaluate the capacity of nLDL to affect spontaneous and lipopolysaccharide (LPS)-stimulated cytokine production. Preincubation of human peripheral blood mononuclear cells (PBMC) with nLDL for 24 h did not influence spontaneous production of tumor necrosis factor alpha (TNF alpha) or interleukin-8 (IL-8), but significantly potentiated LPS-induced production of these cytokines. nLDL preincubation of PBMC did not increase the expression of the LPS receptors Toll-like receptor-4, CD14, or CD11c/CD18. Potentiation of cytokine production by nLDL was mediated through induction of Ca(2+) mobilization, because: a) nLDL induced a sustained pattern of repetitive Ca(2+) transients in human PBMC; b) the Ca(2+) chelator fura 2-acetoxymethyl ester, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular Ca(2+) chelator, inhibited the potentiating effect of nLDL on LPS-induced cytokine synthesis; c) induction of Ca(2+) mobilization by thapsigargin potentiated LPS-induced cytokine production. nLDL are able to potentiate LPS-induced production of cytokines by human PBMC, and this effect is probably mediated through induction of Ca(2+) mobilization. This may represent an important pathogenetic mechanism in atherogenesis induced by hyperlipoproteinemia.