Skin cancer induction by the antimycotic drug voriconazole is caused by impaired DNA damage detection due to chromatin compaction.

Phototoxicity and skin cancer are severe adverse effects of the anti-fungal drug Voriconazole (VOR). These adverse effects resemble those seen in xeroderma pigmentosum (XP), caused by defective DNA nucleotide excision repair (NER), and we show that VOR decreases NER capacity. We show that VOR treatment does not perturb the expression of NER, or other DNA damage-related genes, but that VOR localizes to heterochromatin, in complexes containing histone acetyltransferase GCN5. Impairment of GCN5 binding to histone H3 reduced acetylation of H3, restricting damage-dependent chromatin unfolding, thereby reducing NER initiation. Restoration of H3 histone acetylation using histone deacetylase inhibitors (HDACi), rescued VOR-induced NER repression, thus offering a preventive therapeutic option. These findings underline the importance of DNA damage-dependent chromatin remodeling as an important prerequisite of functional DNA repair.

Innate Responses to the Former COVID-19 Vaccine Candidate CVnCoV and Their Relation to Reactogenicity and Adaptive Immunogenicity.

Vaccines are highly effective at preventing severe coronavirus disease (COVID-19). With mRNA vaccines, further research is needed to understand the association between immunogenicity and reactogenicity, which is defined as the physical manifestation of an inflammatory response to a vaccination. This study analyzed the immune response and reactogenicity in humans, post immunization, to the former SARS-CoV-2 mRNA investigational vaccine CVnCoV (CV-NCOV-001 and CV-NCOV-002 clinical trials). Immunogenicity was investigated using whole-blood RNA sequencing, serum cytokine levels, and SARS-CoV-2-specific antibodies. The T cell responses in peripheral blood were assessed using intracellular cytokine staining (ICS) and high-dimensional profiling in conjunction with SARS-CoV-2 antigen-specificity testing via mass cytometry. Reactogenicity was graded after participants' first and second doses of CVnCoV using vaccine-related solicited adverse events (AEs). Finally, a Spearman correlation was performed between reactogenicity, humoral immunity, and serum cytokine levels to assess the relationship between reactogenicity and immunogenicity post CVnCoV vaccination. Our findings showed that the gene sets related to innate and inflammatory immune responses were upregulated one day post CVnCoV vaccination, while the gene sets related to adaptive immunity were upregulated predominantly one week after the second dose. The serum levels of IFNα, IFNγ, IP-10, CXCL11, IL-10, and MCP-1 increased transiently, peaking one day post vaccination. CD4+ T cells were induced in all vaccinated participants and low frequencies of CD8+ T cells were detected by ex vivo ICS. Using mass cytometry, SARS-CoV-2 spike-specific CD8+ T cells were induced and were characterized as having an activated effector memory phenotype. Overall, the results demonstrated a positive correlation between vaccine-induced systemic cytokines, reactogenicity, and adaptive immunity, highlighting the importance of the balance between the induction of innate immunity to achieve vaccine efficacy and ensuring low reactogenicity.

Candida albicans commensalism in the oral mucosa is favoured by limited virulence and metabolic adaptation.

As part of the human microbiota, the fungus Candida albicans colonizes the oral cavity and other mucosal surfaces of the human body. Commensalism is tightly controlled by complex interactions of the fungus and the host to preclude fungal elimination but also fungal overgrowth and invasion, which can result in disease. As such, defects in antifungal T cell immunity render individuals susceptible to oral thrush due to interrupted immunosurveillance of the oral mucosa. The factors that promote commensalism and ensure persistence of C. albicans in a fully immunocompetent host remain less clear. Using an experimental model of C. albicans oral colonization in mice we explored fungal determinants of commensalism in the oral cavity. Transcript profiling of the oral isolate 101 in the murine tongue tissue revealed a characteristic metabolic profile tailored to the nutrient poor conditions in the stratum corneum of the epithelium where the fungus resides. Metabolic adaptation of isolate 101 was also reflected in enhanced nutrient acquisition when grown on oral mucosa substrates. Persistent colonization of the oral mucosa by C. albicans also correlated inversely with the capacity of the fungus to induce epithelial cell damage and to elicit an inflammatory response. Here we show that these immune evasive properties of isolate 101 are explained by a strong attenuation of a number of virulence genes, including those linked to filamentation. De-repression of the hyphal program by deletion or conditional repression of NRG1 abolished the commensal behaviour of isolate 101, thereby establishing a central role of this factor in the commensal lifestyle of C. albicans in the oral niche of the host.

Immunomodulatory role of reactive oxygen species and nitrogen species during T cell-driven neutrophil-enriched acute and chronic cutaneous delayed-type hypersensitivity reactions.

Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production. Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91phox-/-), myeloperoxidase-deficient (MPO-/-), and inducible nitric oxide synthase-deficient (iNOS-/-) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences. Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO-/- mice and unaffected ROS/RNS production in the ears of iNOS-/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91phox-/- mice with chronic DTHR, while the inflamed ears of MPO-/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR. Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes.

IL-23 supports host defense against systemic Candida albicans infection by ensuring myeloid cell survival.

The opportunistic fungal pathogen Candida albicans can cause invasive infections in susceptible hosts and the innate immune system, in particular myeloid cell-mediated immunity, is critical for rapid immune protection and host survival during systemic candidiasis. Using a mouse model of the human disease, we identified a novel role of IL-23 in antifungal defense. IL-23-deficient mice are highly susceptible to systemic infection with C. albicans. We found that this results from a drastic reduction in all subsets of myeloid cells in the infected kidney, which in turn leads to rapid fungal overgrowth and renal tissue injury. The loss in myeloid cells is not due to a defect in emergency myelopoiesis or the recruitment of newly generated cells to the site of infection but, rather, is a consequence of impaired survival of myeloid cells at the site of infection. In fact, the absence of a functional IL-23 pathway causes massive myeloid cell apoptosis upon C. albicans infection. Importantly, IL-23 protects myeloid cells from apoptosis independently of the IL-23-IL-17 immune axis and independently of lymphocytes and innate lymphoid cells. Instead, our results suggest that IL-23 acts in a partially autocrine but not cell-intrinsic manner within the myeloid compartment to promote host protection from systemic candidiasis. Collectively, our data highlight an unprecedented and non-canonical role of IL-23 in securing survival of myeloid cells, which is key for maintaining sufficient numbers of cells at the site of infection to ensure efficient host protection.

Proteolytic Degradation of reduced Human Beta Defensin 1 generates a Novel Antibiotic Octapeptide.

Microbial resistance against clinical used antibiotics is on the rise. Accordingly, there is a high demand for new innovative antimicrobial strategies. The host-defense peptide human beta-defensin 1 (hBD-1) is produced continuously by epithelial cells and exhibits compelling antimicrobial activity after reduction of its disulphide bridges. Here we report that proteolysis of reduced hBD-1 by gastrointestinal proteases as well as human duodenal secretions produces an eight-amino acid carboxy-terminal fragment. The generated octapeptide retains antibiotic activity, yet with distinct characteristics differing from the full-length peptide. We modified the octapeptide by stabilizing its termini and by using non-natural D-amino acids. The native and modified peptide variants showed antibiotic activity against pathogenic as well as antibiotic-resistant microorganisms, including E. coli, P. aeruginosa and C. albicans. Moreover, in an in vitro C. albicans infection model the tested peptides demonstrated effective amelioration of C. albicans infection without showing cytotoxity on human cells. In summary, protease degradation of hBD-1 provides a yet unknown mechanism to broaden antimicrobial host defense, which could be used to develop defensin-derived therapeutic applications.

Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression.

Controlled immune activation in response to commensal microbes is critical for the maintenance of stable colonization and prevention of microbial overgrowth on epithelial surfaces. Our understanding of the host mechanisms that regulate bacterial commensalism has increased substantially, however, much less data exist regarding host responses to members of the fungal microbiota on colonized surfaces. Using a murine model of oropharyngeal candidiasis, we have recently shown that differences in immune activation in response to diverse natural isolates of Candida albicans are associated with different outcomes of the host-fungal interaction. Here we applied a genome-wide transcriptomic approach to show that rapid induction of a strong inflammatory response characterized by neutrophil-associated genes upon C. albicans colonization inversely correlated with the ability of the fungus to persist in the oral mucosa. Surprisingly, persistent fungal isolates showed no signs of a compensatory regulatory immune response. By combining RNA-seq data, genetic mouse models, and co-infection experiments, we show that attenuation of the inflammatory response at the onset of infection with a persistent isolate is not a consequence of enhanced immunosuppression. Importantly, depletion of regulatory T cells or deletion of the immunoregulatory cytokine IL-10 did not alter host-protective type 17 immunity nor did it impair fungal survival in the oral mucosa, indicating that persistence of C. albicans in the oral mucosa is not a consequence of suppressed antifungal immunity.

Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4.

Neutrophils are the most abundant innate immune cells and the first line of defense against many pathogenic microbes, including the human fungal pathogen Candida albicans. Among the neutrophils' arsenal of effector functions, neutrophil extracellular traps (NETs) are thought to be of particular importance for trapping and killing the large fungal filaments by means of their web-like structures that consist of chromatin fibers decorated with proteolytic enzymes and host defense proteins. Peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histones in activated neutrophils correlates with chromatin decondensation and extrusion and is widely accepted to act as an integral process of NET induction (NETosis). However, the requirement of PAD4-mediated histone citrullination for NET release during C. albicans infection remains unclear. In this study, we show that although PAD4-dependent neutrophil histone citrullination is readily induced by C. albicans, PAD4 is dispensable for NETosis in response to the fungus and other common NET-inducing stimuli. Moreover, PAD4 is not required for antifungal immunity during mucosal and systemic C. albicans infection. Our results demonstrate that PAD4 is dispensable for C. albicans-induced NETosis, and they highlight the limitations of using histone citrullination as a marker for NETs and PAD4-/- mice as a model of NET-deficiency.