Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

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.

Chocolate consumption modulates cytokine production in healthy individuals.

Epidemiological studies suggest that chocolate increases the incidence and severity of acne. Here we demonstrate that chocolate consumption primes human blood mononuclear cells from volunteers to release more interleukin-1ß (IL-1ß) and IL-10 upon stimulation with Propionibacterium acne or Staphylcoccus aureus, the two microorganisms involved in the pathogenesis of acne. In contrast, production of the Th17-derived cytokine IL-22 was inhibited by chocolate. Modulation of inflammation could represent an important mechanism through which chocolate consumption influences acne.

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.

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.

In vitro induction of trained immunity in adherent human monocytes.

A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using ß-glucan (from Candida albicans) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016).

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors.

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

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.

A New Model to Calibrate a Reference Standard for Bovine Tuberculin Purified Protein Derivative in the Target Species.

Since 1986, use of a Bovine International Standard (BIS) for bovine tuberculin has been required to ensure national and international uniformity regarding the potency designation of bovine tuberculin Purified Protein Derivative (PPDb) preparations produced by multiple manufacturers. The BIS is the unique golden standard in the guinea pig potency assay, representing 100% potency, where potencies of production batches are calculated as relative potencies in comparison with the potency of the BIS which was set at 32,500 international Unit (IU) per mg. The stock supply and lifetime of the BIS is limited.The aim of this study was to develop a model to determine the potency of a newly produced in-house Reference Standard (RS) for PPDb with great accuracy in the target species (cattle) and to prove its precision and accuracy in the guinea pig potency test. First simulations were done to estimate the required number of cattle needed. Then, 30 naturally bTB infected cattle were subjected to a tuberculin skin test using multiple injections of both the RS and the BIS. Both were applied randomly in the same volume and concentration (1 dose). The potency of the RS against the BIS was directly derived from the least square means (LSMEANS) and was estimated as 1.067 (95% CI: 1.025-1.109), equal to a potency of 34,700 ± 1,400 IU/mg. In six guinea pig potency assays the RS was used to assign potencies to production batches of PPDb. Here, precision and accuracy of the RS was determined according to the parallel-line assay. Relative potencies were estimated by exponentiation of the common slope. The corresponding 95% confidence intervals were obtained according to Fieller's theorem. In sensitized guinea pigs, the relative potency of the RS against the BIS was 1.115 (95% CI: 0.871-1.432), corresponding to an absolute potency of 36,238 IU/mg (95% CI: 28,308-46,540).In conclusion: the method used to determine the potency of the RS against the BIS in naturally bTB infected cattle, resulted in a highly accurate potency estimate of the RS. The RS can be used in the guinea pig test to assign potencies to PPDb production batches with high precision and accuracy.

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.

Validation of a LightCycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus.

A specific reverse transcription polymerase chain reaction (RT-PCR) for the detection of the polymerase gene (3D) of foot-and-mouth disease virus (FMDV) was developed and validated with an analytical sensitivity of equal to, to 1,000 times higher than that of a single passage virus isolation. The performance of the RT-PCR was determined in 180 runs. After implementation, 5.3% of the tests had to be rejected due to invalid controls (e.g. cross-contamination of negative controls). The diagnostic sensitivity, determined using 124 samples from experimentally infected animals, was 91.9% for RT-PCR and 84.7% for virus isolation. Diagnostic specificity, determined by testing 258 samples from uninfected animals, was 100% by both tests. Of the 627 samples tested by RT-PCR and virus isolation, 85 reacted positively in both tests (13.5%) and 447 negatively in both tests (71.3%). One sample was positive by virus isolation and negative by RT-PCR (0.2%), 94 samples were positive by RT-PCR and negative by virus isolation (15%). The majority (84 of 94) of the 15% RT-PCR positive and virus isolation negative samples were among other samples from farms that reacted positively by both tests. The new RT-PCR is a robust, reliable and sensitive test, provided that adequate measures are taken to prevent cross-contamination. A possible preventive measure is to exclude ELISA positive samples from the RT-PCR testing.

Detection of economically important viruses in boar semen by quantitative RealTime PCR technology.

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests. All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage. In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.

Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors.

Upon priming with Candida albicans or with the fungal cell wall component ß-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed "trained immunity." In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

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.

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.

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.

Functional consequences of the Asp299Gly Toll-like receptor-4 polymorphism.

Toll-like receptor-4 (TLR4) is a pattern-recognition receptor not only for exogenous ligands such as lipopolysaccharide (LPS) of Gram-negative bacteria, but also for endogenous ligands such as fibronectin, heat shock proteins and hyaluronan oligosaccharides. The Asp299Gly allele of the TLR4 gene has been associated with increased risk for severe infections, but reduced progression of atherosclerosis. We have investigated the consequences of the presence of Asp299Gly polymorphism after stimulation of mononuclear cells with lipopolysaccharide (LPS), the non-LPS TLR4 microbial stimuli Aspergillus fumigatus and Cryptococcus neoformans, and the endogenous TLR4 ligand heat shock protein 60. No differences in either production of the proinflammatory cytokine TNF or the antiinflammatory cytokine interleukin-10 were observed between volunteers with the wild-type allele, volunteers heterozygous for the Asp299Gly allele and one volunteer homozygous for the TLR4 variant. In conclusion, the presence of the Asp299Gly TLR4 polymorphism does not result in defective pro and antiinflammatory cytokine production after stimulation with either exogenous (LPS and non-LPS) or endogenous TLR4 ligands, and alternative explanations are likely to be responsible for the epidemiological data showing associations with inflammatory conditions. In addition, this is the first study to demonstrate that even homozygosity for the Asp299Gly mutation does not confer hyporesponsiveness to stimulation with TLR4 stimuli.

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.