In the second part of this thesis we investigated the anti-C. albicans host immune response from a genetic point of view. Until now, all research aimed at identifying antifungal host defense mechanisms in humans has relied on candidate-gene approaches that are based on hypotheses extracted from in vitro or animal studies. While this approach has been fruitful in confirming important pathways of antifungal defense, it has lacked the power to identify novel host defense mechanisms against Candida in humans and to provide a hierarchy of the importance of these pathways. In Chapter 6 we have addressed this issue by using a combination of transcriptional analysis, systems biology, and functional genomics to investigate the C. albicans- induced immune response. We identified several known common inflammatory host defense pathways, but we also discovered an unanticipated crucial role of the type I interferon (IFN) pathway in antifungal host defense in humans. The role of type I IFN pathway in humans was confirmed both with classical in vitro immunological assays, as well as in genetic studies in patients with systemic candidiasis and chronic mucocutaneous candidiasis (CMC). Hopefully, this knowledge can be applied in the development of adjuvant immunotherapy.
A primary immunodeficiency syndrome that is associated with defective IFN-γ and IL-17 production and mucosal fungal infections is CMC 5,6. We recently reported mutations in the coiled-coil domain of STAT1 as the genetic cause of autosomal dominant (AD)-CMC) 7. However, the cellular and molecular mechanisms responsible for the increased susceptibility to fungal infections in patients with AD-CMC and STAT1 mutations remained to be deciphered. In Chapter 7 we confirm Arg274Trp mutations in the coiled-coil (CC) domain of STAT1 as the genetic cause of AD-CMC in three additional families of patients. We demonstrate that the Arg274Trp mutation leads to increased phosphorylation of STAT1, with increased IFN-γ signaling, while signaling via the IL-12 and IL-23 receptor is inhibited. This leads to strongly diminished Th1 and Th17 responses, explaining the increased susceptibility to fungal infections of CMC patients.
Recently, it has been demonstrated that there are interactions between the host genome, the immune system and the gut microbiome in healthy volunteers 8,9. However, it is not known how primary immunodeficiencies influence the microbiome, and whether this in turn could influence innate immune responses. In Chapter 8 we investigated the skin microbiome in patients with CMC and hyper IgE syndrome (HIES), which are caused by STAT1 and STAT3 mutations respectively. We demonstrate that CMC and HIES patients have an imbalance in their skin microbiome towards Gram- negative colonization. These Gram-negative bacteria can in turn impair host innate immune responses to Candida albicans and Staphylococcus aureus, the two major pathogens causing infections in these patients. This information can provide the basis for potential microbiome-based therapies in these patients.
The inflammasome is a protein platform that cleaves pro-IL-β into active IL-β. It has been suggested that reactive oxygen species (ROS) are necessary for inflammasome activation, however chronic granulomatous disease (CGD) patients, who cannot produce ROS, have a proinflammatory phenotype 10. Therefore we investigated the role of ROS in inflammation. In Chapter 9 we demonstrate that ROS are not required for inflammasome activation. Monocytes isolated from CGD patients contain cleaved caspase-1, an indication for inflammasome activation, and they produce normal to high levels of IL-1β. Also in cells isolated from healthy individuals, inhibiting ROS production
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with a synthetic inhibitor (DPI) cannot dampen activation of the inflammasome. In fact, DPI even increases the production of pro-inflammatory cytokines. Interestingly, monocytes from CGD patients readily produce IL-1β upon NLRP3 inflammasome stimulation with uric acid crystals alone, while cells from healthy controls need pre- stimulation with lipopolysaccharide in order to be able to do this. This suggests that ROS may even dampen inflammasome activation. These findings may explain why CGD patients are characterized by an inflammatory phenotype in the absence of ROS.
It has been suggested that ROS deficient mice have a defective tryptophan metabolism, and therefore have an increased IL-17 response. We tested this hypothesis in human CGD patients in Chapter 10. Interestingly, we demonstrate that CGD patients, who are susceptible for recurrent bacterial and fungal infections, have low IL-17 production in response to C. albicans and Aspergillus spp., while the production of other pro-inflammatory cytokines is higher. We also demonstrate that in humans NADPH dependent ROS are not necessary for the conversion of L-tryptophan to L-kynurenine. Furthermore, L-kynurenine does not suppress IL-17 production in human cells.
GENERAL CONCLUSIONS AND FUTURE PERSPECTIvES
In this thesis we investigated several aspects of the anti-Candida immune response. In a large, fast-paced research field such as immunology, these findings are modest contributions to our overall understanding of disease and the functioning of the immune system. However, a thorough understanding of the immune system is essential when developing new treatment strategies, and jointly all these pieces of information can form the basis for the development of novel therapies.
The studies presented here have several relevant consequences for our understanding of human fungal diseases on the one hand, and for the improvement of therapy for these severe infections on the other hand. The use of adjunctive immunotherapy, such as recombinant IFN-y, has proven to have a beneficial effect against several fungal infections 11. In addition to that, we have now demonstrated the importance of the type I IFN pathway for human antifungal host defense, and this opens additional new possibilities for adjunctive immunotherapy during these severe infections. In order to improve further the chances of success of these therapies, additional transcriptome study in patients with systemic Candida infections will be performed in the near future.
Furthermore, we have demonstrated that CMC and HIES patients have an altered skin microbiome, which in turn is able to influence host defense mechanisms. This could be a rationale for testing microbiome-based adjuvant therapies in patients with Th17 defects. However, currently there are no treatment options available targeting the skin microbiome. It has been proposed that this should be attempted using emollients or bacterial products in order to promote the growth of specific commensals 12, but these therapies still need to be developed. There are some preliminary studies, mainly case-reports, looking at the effect of targeting the gut microbiome through fecal transplantation. Three patients with multiple sclerosis (MS), an IL-17-driven autoimmune disease resulting in neurological degradation, were successfully treated for constipation with daily fecal transplants for one to two weeks. They also experienced
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Summary & DiscussionMS symptom reversal and remained asymptomatic for two to 15 years after treatment 13. Other studies have demonstrated that fecal transplantation can also be effective against infectious diseases, like diarrhea caused by Clostridum difficile 14. It is however not yet known whether fecal transplantation is also effective in boosting the immune response against fungal pathogens. Moreover, the gut microbiome of CMC and HIES patients would first need to be mapped accurately.
In addition to the aspects discussed in this thesis, other aspects that need to be explored in fungal infections should also be mentioned. Firstly, diagnostic tools need to be improved in terms of accuracy and speed 15. Secondly, since drug resistance is becoming an increasing problem 16, more emphasize should be put on developing new pharmaceutical fungicides. Thirdly, alternative therapies for the most severe forms of acquired or inborn defects, such as adoptive T cell therapy or gene therapy 11 should be considered. Finally, development of active (vaccines) 17 or passive (antibodies) 18 immunizations that have been shown to be effective in animal models, should become a priority in translational studies with the future aim to be able to use such approaches in a clinical setting.
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