Genetic Variation in PFKFB3 Impairs Antifungal Immunometabolic Responses and Predisposes to Invasive Pulmonary Aspergillosis.

Activation of immune cells in response to fungal infection involves the reprogramming of their cellular metabolism to support antimicrobial effector functions. Although metabolic pathways such as glycolysis are known to represent critical regulatory nodes in antifungal immunity, it remains undetermined whether these are differentially regulated at the interindividual level. In this study, we identify a key role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the immunometabolic responses to Aspergillus fumigatus. A genetic association study performed in 439 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) and corresponding donors revealed that the donor, but not recipient, rs646564 variant in the PFKFB3 gene increased the risk of invasive pulmonary aspergillosis (IPA) after transplantation. The risk genotype impaired the expression of PFKFB3 by human macrophages in response to fungal infection, which was correlated with a defective activation of glycolysis and the ensuing antifungal effector functions. In patients with IPA, the risk genotype was associated with lower concentrations of cytokines in the bronchoalveolar lavage fluid samples. Collectively, these findings demonstrate the important contribution of genetic variation in PFKFB3 to the risk of IPA in patients undergoing HSCT and support its inclusion in prognostic tools to predict the risk of fungal infection in this clinical setting. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. Activation of glycolysis is essential for innate immune cells to mount effective antifungal responses. In this study, we report the contribution of genetic variation in the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) to the risk of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation. The PFKFB3 genotype associated with increased risk of infection was correlated with an impairment of the antifungal effector functions of macrophages in vitro and in patients with IPA. This work highlights the clinical relevance of genetic variation in PFKFB3 to the risk of IPA and supports its integration in risk stratification and preemptive measures for patients at high risk of IPA.

Polymorphisms within the ARNT2 and CX3CR1 Genes Are Associated with the Risk of Developing Invasive Aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection that affects an increasing number of patients undergoing chemotherapy or allo-transplantation, and recent studies have shown that genetic factors contribute to disease susceptibility. In this two-stage, population-based, case-control study, we evaluated whether 7 potentially functional single nucleotide polymorphisms (SNPs) within the ARNT2 and CX3CR1 genes influence the risk of IA in high-risk hematological patients. We genotyped selected SNPs in a cohort of 500 hematological patients (103 of those had been diagnosed with proven or probable IA), and we evaluated their association with the risk of developing IA. The association of the most interesting markers of IA risk was then validated in a replication population, including 474 subjects (94 IA and 380 non-IA patients). Functional experiments were also performed to confirm the biological relevance of the most interesting markers. The meta-analysis of both populations showed that carriers of the ARNT2rs1374213G, CX3CR1rs7631529A, and CX3CR1rs9823718G alleles (where the RefSeq identifier appears as a subscript) had a significantly increased risk of developing IA according to a log-additive model (P value from the meta-analysis [PMeta] = 9.8 · 10-5, PMeta = 1.5 · 10-4, and PMeta =7.9 · 10-5, respectively). Haplotype analysis also confirmed the association of the CX3CR1 haplotype with AG CGG with an increased risk of IA (P = 4.0 · 10-4). Mechanistically, we observed that monocyte-derived macrophages (MDM) from subjects carrying the ARNTR2rs1374213G allele or the GG genotype showed a significantly impaired fungicidal activity but that MDM from carriers of the ARNT2rs1374213G and CX3CR1rs9823718G or CX3CR1rs7631529A alleles had deregulated immune responses to Aspergillus conidia. These results, together with those from expression quantitative trait locus (eQTL) data browsers showing a strong correlation of the CX3CR1rs9823718G allele with lower levels of CX3CR1 mRNA in whole peripheral blood (P = 2.46 · 10-7) and primary monocytes (P = 4.31 · 10-7), highlight the role of the ARNT2 and CX3CR1 loci in modulating and predicting IA risk and provide new insights into the host immune mechanisms involved in IA development.

Genetic defects in fungal recognition and susceptibility to invasive pulmonary aspergillosis.

The interindividual variability in the onset and clinical course of invasive pulmonary aspergillosis (IPA) raises fundamental questions about its actual pathogenesis. Clinical and epidemiological studies have reported only a few examples of monogenic defects, however an expanding number of common polymorphisms associated with IPA has been identified. Understanding how genetic variation regulates the immune response to Aspergillus provides critical insights into the human immunobiology of IPA by pinpointing directly relevant immune molecules and interacting pathways. Most of the genetic defects reported to increase susceptibility to infection were described or suggested to impair fungal recognition by the innate immune system. In this review, we discuss the contribution of host genetic variation in pattern recognition receptors to the development of IPA. An improved understanding of the molecular and cellular processes that regulate human susceptibility to IPA is ultimately expected to pave the way toward personalized medical interventions based on host-directed risk stratification and individualized immunotherapy.

Deep sequencing profiles MicroRNAs related to Aspergillus fumigatus infection in lung tissues of mice.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a severe opportunistic infection with high mortality in patients with compromised immunity. The full repertoire of microRNAs (miRNAs) involved in the regulation of IPA infection remains to be established. METHODS: We established a mouse IPA model and analyzed small RNA transcriptomes in lung tissues of immunodeficient IPA mice (IPA group) and matched immunodeficient control mice (control group) through next-generation sequencing. RESULTS: A total of 3759 known miRNAs were detected, in which 23 miRNAs were identified to be related to IPA. IPA-associated miRNAs include upregulated mmu-let-7b-3p, mmu-miR124-3p, mmu-miR21a-3p, mmu-miR29c-5p, mmu-miR3473b and mmu-miR3473e, and downregulated mmu-miR-150-3p and mmu-miR-503-5p. The expression levels of eight identified miRNAs were quantified in a validation cohort (n = 40) by qRT-PCR, and results revealed the same change patterns. MiRNA target prediction revealed that all IPA-related miRNAs possibly engage a cooperative regulation of key elements in the NF-kappa B signaling pathway. CONCLUSION: We conclude that deep-sequencing small RNAs can uncover miRNA pool-regulating IPA. Our results may lead to further understanding IPA pathogenesis and gain insight into the complexity and diversity of small RNA molecules that regulate immunodeficient IPA.

Aspergillus fumigatus conidial metalloprotease Mep1p cleaves host complement proteins.

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

Predicting Invasive Aspergillosis in Hematology Patients by Combining Clinical and Genetic Risk Factors with Early Diagnostic Biomarkers.

Personalized medicine provides a strategic approach to the management of IA. The incidence of IA in high-risk hematology populations is relatively low (<10%), despite unavoidable Aspergillus exposure in patients with a potentially similar clinical risk. Nonclinical variables, including genetic mutations that increase susceptibility to IA, could explain why only certain patients develop the disease. This study screened for mutations in 322 hematology patients classified according to IA status and developed a predictive model based on genetic risk, established clinical risk factors, and diagnostic biomarkers. Genetic markers were determined by real-time PCR and, with clinical risk factors and Aspergillus PCR results, subjected to multilogistic regression analysis to identify a best-fit model for predicting IA. The probability of IA was calculated, and an optimal threshold was determined. Mutations in dectin-1 (rs7309123) and DC-SIGN (rs11465384 and rs7248637), allogeneic stem cell transplantation, respiratory virus infection, and Aspergillus PCR positivity were all significant risk factors for developing IA and were combined in a predictive model. An optimal threshold requiring three positive factors generated a mean sensitivity/specificity of 70.4%/89.2% and a probability of developing IA of 56.7%. In patients with no risk factors, the probability of developing IA was 2.4%, compared to >79.1% in patients with four or more factors. Using a risk threshold of 50%, preemptive therapy would have been prescribed for 8.4% of the population. This pilot study shows that patients can be stratified according to risk of IA, providing personalized medicine based on strategic evidence for the management of IA. Further studies are required to confirm this approach.

Nucleic acid amplification methodologies for the detection of pulmonary mold infections.

INTRODUCTION: The detection of pulmonary mold infections has historically required technically demanding methods obtained through invasive procedures. Nucleic acid amplification assays have the potential to circumvent the technical hurdles associated with diagnosis, but are not without potential pitfalls. Areas covered: In this paper, the authors review new assays for the diagnosis of pulmonary mold infections due to aspergillosis, mucormycosis, and hyalohyphomycoses as well as uncommon infections caused by dematiaceous molds. Expert commentary: Nucleic acid amplification assays have the potential to rapidly identify patients with invasive mycoses and could shorten the time to implementation of appropriate antimicrobial therapy. However, selection of appropriate patient populations will be crucial to ensure the highest Bayesian positive predictive value for any novel diagnostic platform.

Genomic Analysis of Single Nucleotide Polymorphisms Asp299Gly and Thr399Ile in Japanese Patients with Invasive Aspergillosis.

Single nucleotide polymorphisms (SNPs) 1063A/G (Asp299Gly) and 1363C/T (Thr399Ile) in the gene encoding Toll-like receptor 4 (TLR4) increase susceptibility to invasive aspergillosis. However, limited information is available on the prevalence of these SNPs in Japan. Therefore, we surveyed these TLR4 SNPs by using formalin-fixed and paraffin-embedded tissue blocks obtained from autopsies of patients with invasive pulmonary aspergillosis. Tissue samples of approximately 30% patients were included in genomic analysis. However, none of these samples showed the presence of TLR4 Asp299Gly and Thr399Ile polymorphisms. Thus, the present study provided information on the prevalence of TLR4 SNPs in Japanese patients with invasive aspergillosis and indicated that these SNPs played a minor role in increasing the susceptibility of Japanese individuals to invasive aspergillosis.

Resistant invasive aspergillosis in an autosomal recessive chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by severe bacterial and fungal infections. Invasive aspergillosis and other rare mold diseases are the leading causes of mortality. We report one case of CGD revealed by retropharyngeal abscess. On evolution, the patient developed an invasive aspergillosis resistant to treatment.

Interleukin-12 and interleukin-2 alone or in combination against the infection in invasive pulmonary aspergillosis mouse model.

Aspergillus fumigatus is an intracellular opportunistic fungus causing invasive pulmonary mycosis, characterised by hyphal invasion and destruction of pulmonary tissue. Th1 cytokines could enhance fungicidal activity. The effects from the combination of interleukin-12 (IL-12) and IL-2 are rarely known in invasive pulmonary aspergillosis infection. To assess the cleaning of A. fumigatus infection in the pulmonary tissues by IL-12 and IL-2, interferon-γ (IFN-γ) was detected in the sera using ELISA, quantification of IFN-γ mRNA using real-time RT-PCR and lung Colony-forming unit was assayed by cultivation. Morphology was analysed by histopathological examination. Our results showed that IL-12 and/or IL-2 could enhance the IFN-γ expression in the pulmonary tissue, reduce the colony load in the pulmonary tissue and increase the survival rate of mouse. The combination of IL-12 and IL-2 could assist in increasing the IFN-γ expression in the pulmonary tissue, but neither reduce colony load in the pulmonary tissue nor increase the survival rate of mouse significantly. It was demonstrated that IL-12 and IL-2 were strong immunomodulatory cytokines as a prerequisite for protecting the host from infectious agents.

Azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012.

A new azole resistance mechanism in Aspergillus fumigatus consisting of a TR46/Y121F/T289A alteration in the cyp51A gene was recently described in the Netherlands. Strains containing these mutations are associated with invasive infection and therapy failure. This communication describes the first case of fatal invasive aspergillosis caused by TR46/Y121F/T289A outside the Netherlands, in the neighboring country of Belgium, suggesting geographical spread. TR46/Y121F/T289A leads to a recognisable phenotypic susceptibility pattern which should trigger cyp51A genotyping to monitor further spread.

Recent advances in invasive pulmonary aspergillosis.

PURPOSE OF REVIEW: This review highlights the most important and salient recent developments with regards to invasive pulmonary aspergillosis (IPA), currently the most common opportunistic fungal pneumonia in patients with hematological malignancies. RECENT FINDINGS: Besides patients in hematology units, other immunosuppressed and critically ill patients are also at risk of IPA. Identification of patients who possess specific polymorphisms of Toll-like receptor 4 and dectin-1, both of which are involved in the immune sensing of Aspergillus spp., may facilitate risk-stratification. The use of the galactomannan assay in bronchoalveolar fluid to improve diagnosis of IPA is undergoing validation. Trending galactomannan and other biomarker results may prognosticate clinical outcomes. During intensive chemotherapy for leukemia, posaconazole and aerosolized liposomal amphotericin B (L-AMB) have demonstrated efficacy as prophylaxis against invasive fungal infection. However, fluconazole remains an effective prophylactic agent in the setting of hematopoietic stem cell transplantation despite availability of newer antifungal agents. Although voriconazole is still the drug of choice for IPA, both caspofungin and L-AMB are viable alternatives. SUMMARY: Despite increasing knowledge of IPA and availability of newer antifungal agents, clinical management remains a challenge in the setting of a compromised host defense system that is unable to mount an appropriate immune response against the pathogen.

Susceptibility of mice genetically deficient in SP-A or SP-D gene to invasive pulmonary aspergillosis.

Pulmonary surfactant proteins, SP-A and SP-D, are carbohydrate pattern recognition molecules of innate immunity, which significantly enhance phagocytosis and killing of Aspergillus fumigatus, a pathogenic fungus, by neutrophils and macrophages. The present study examined the susceptibility of immunosuppressed SP-A gene deficient (SP-A(-/-)) or SP-D gene deficient (SP-D(-/-)) mice to A. fumigatus conidia challenge compared to wild-type (WT) mice. A. fumigatus-challenged SP-A(-/-) (SP-A(-/-) IPA) mice showed less mortality (40%) than the WT-IPA mice (100%) and increased mortality (60%) following administration of SP-A with decreased TNF-alpha and IFN-gamma to IL-4 ratio than SP-A(-/-) IPA mice. The SP-D(-/-) IPA mice (57.14%) showed similar mortality as WT-IPA mice (60%). However, the SP-D (-/-) IPA mice (42.86% mortality on day 2) died earlier than the WT-IPA mice (20% mortality on day 2), showed a higher hyphal density and tissue injury in lungs. Treatment with SP-D or a recombinant fragment of human SP-D rhSP-D reduced the mortality to 50% and 33%, respectively, concomitant with higher IFN-gamma to IL-4 ratios in treated SP-D(-/-) mice, compared to untreated control group. The results showed that SP-D gene deficient mice are more susceptible to IPA while SP-A gene deficient mice acquire resistance to IPA.

The Aspergillus fumigatus P-type Golgi apparatus Ca2+/Mn2+ ATPase PmrA is involved in cation homeostasis and cell wall integrity but is not essential for pathogenesis.

The Aspergillus fumigatus DeltapmrA (Golgi apparatus Ca(2+)/Mn(2+) P-type ATPase) strain has osmotically suppressible basal growth defects and cationic tolerance associated with increased expression of calcineurin pathway genes. Despite increased beta-glucan and chitin content, it is hypersensitive to cell wall inhibitors but remains virulent, suggesting a role for PmrA in cation homeostasis and cell wall integrity.

[Association between genetic polymorphism in the promotor region of CD209 and propensity to develop invasive pulmonary aspergillosis]. / Asociación de un polimorfismo genético en la región promotora del gen CD209 y propension a la aspergilosis pulmonar invasiva.

Fungi of the genus Aspergillus are found everywhere in the natural environment; they cause invasive pulmonary aspergillosis (IPA), an infectious complication common in immunocompromised individuals, which has a mortality rate of up to 90% in patients with hematological malignancy. The first line of defense of innate immunity is the recognition of Aspergillus conidia by dendritic cells or alveolar macrophages. DC-SIGN is an integrin directly involved in this recognition; its degree of expression in immune cells and its functionality may be partly determined by genetic variations. The objective of this study was to determine whether the presence of polymorphisms of a single nucleotide in the DC-SIGN gene increases the risk of invasive pulmonary aspergillosis. For this purpose, the variants DC-SIGN-139A/G (rs2287886) and DC-SIGN+11C/G (rs7252229) were analyzed In 314 subjects (152 patients with hematologic malignancy and 162 healthy controls). Of the 152 hematologic cancer patients, 81 were diagnosed with demonstrated invasive pulmonary aspergillosis per EORTC/IFICG criteria, and the remaining 71 patients had no symptoms of the infection. An association was found between the variant DC-SIGN-139(A/G) and resistance to IPA. Carriers of the allele A (A/A + A/G) were significantly more resistant to the infection than patients with the G/G genotype (p = 0.0574). Analysis of the serum concentration of the galactomannan antigen supported the hypothesis that this polymorphism may be implicated in the susceptibility to suffer invasive pulmonary aspergillosis. Although the difference was not statistically significant, carriers of the allele G had a higher frequency of positive galactomannans than subjects with the genotype A/A (p = 0.1921). These results suggest that the variant DC-SIGN-139(A/G) in the DC-SIGN gene promoter influences the risk of invasive pulmonary aspergillosis and may therefore be used as a genetic biomarker to stratify patients according to risk.

Potential of lung surfactant proteins, SP-A and SP-D, and mannan binding lectin for therapy and genetic predisposition to allergic and invasive aspergillosis.

A significant proportion of bronchial asthma patients have underlying pulmonary fungal infections that contribute to persistent inflammation and allergic reactions. Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen causing a spectrum of allergic and infectious diseases. Currently, oral corticosteroids form the first line of treatment for allergic aspergillosis and use of antifungals such as itraconazole has been indicated in non-responders. In view of the protective role of innate immunity in host defense against Aspergillus fumigatus, we aimed to identify the relevant innate immune proteins In a series of studies, we identified and established the therapeutic potential of pulmonary collectins SP-A and SP-D and serum collectin MBL in murine models of allergic and invasive aspergillosis. Use of SP-D for diagnosis and therapy of lung disorders and MBL for therapy of various infections including invasive aspergillosis has been patented. Genetic polymorphisms in these genes may result in partial or total loss of function and may increase the host's susceptibility to aspergillosis. Candidate gene association studies showed SNPs in SP-A2 and MBL significantly associate with patients of allergic bronchopulmonary aspergillosis and bronchial asthma with rhinitis. The patients carrying either one or both of GCT and AGG alleles of SP-A2 and patients with A allele at position 1011 of MBL had markedly higher eosinophilia, total IgE antibodies and lower FEV1 (the clinical markers of ABPA). These SNPs may be useful for predicting susceptibility to allergic aspergillosis and bronchial asthma with allergic rhinitis and have been patented. Elucidation of the immunoregulatory role of SP-A, SP-D and MBL in mechanisms of allergy and inflammation suggests that they may also be potentially useful for predisposition diagnosis and therapy of non-fungal bronchial asthma.