The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant ...isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited. We reasoned that clinically relevant antifungal resistance could derive from transcriptional rewiring, promoting drug resistance without concomitant reductions in pathogenicity. Here we report a genome-wide annotation of transcriptional regulators in A. fumigatus and construction of a library of 484 transcription factor null mutants. We identify 12 regulators that have a demonstrable role in itraconazole susceptibility and show that loss of the negative cofactor 2 complex leads to resistance, not only to the azoles but also the salvage therapeutics amphotericin B and terbinafine without significantly affecting pathogenicity.
Schistosome infection is a major cause of global morbidity, particularly in sub-Saharan Africa. However, there is no effective vaccine for this major neglected tropical disease, and re-infection ...routinely occurs after chemotherapeutic treatment. Following invasion through the skin, larval schistosomula enter the circulatory system and migrate through the lung before maturing to adulthood in the mesenteric or urogenital vasculature. Eggs released from adult worms can become trapped in various tissues, with resultant inflammatory responses leading to hepato-splenic, intestinal, or urogenital disease - processes that have been extensively studied in recent years. In contrast, although lung pathology can occur in both the acute and chronic phases of schistosomiasis, the mechanisms underlying pulmonary disease are particularly poorly understood. In chronic infection, egg-mediated fibrosis and vascular destruction can lead to the formation of portosystemic shunts through which eggs can embolise to the lungs, where they can trigger granulomatous disease. Acute schistosomiasis, or Katayama syndrome, which is primarily evident in non-endemic individuals, occurs during pulmonary larval migration, maturation, and initial egg-production, often involving fever and a cough with an accompanying immune cell infiltrate into the lung. Importantly, lung migrating larvae are not just a cause of inflammation and pathology but are a key target for future vaccine design. However, vaccine efforts are hindered by a limited understanding of what constitutes a protective immune response to larvae. In this review, we explore the current understanding of pulmonary immune responses and inflammatory pathology in schistosomiasis, highlighting important unanswered questions and areas for future research.
The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in ...the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.
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•Pleural immune cells in steady state and nematode infection differ by mouse strain•Th2 cells license the conversion of monocytes to tissue-resident macrophages•Monocyte transition to tissue residency alters macrophage function•The GATA6 tissue-residency program is required for resistance to nematode infection
Current understanding is that, in the steady state, macrophages acquire tissue residency in response to local tissue signals. Finlay et al. show that during nematode infection of the pleural cavity, Th2 cells can accelerate the progression of monocytes into tissue-resident macrophages, which become anti-inflammatory and support nematode killing.
T‐bet is the lineage‐specifying transcription factor for CD4+ TH1 cells. T‐bet has also been found in other CD4+ T cell subsets, including TH17 cells and Treg, where it modulates their functional ...characteristics. However, we lack information on when and where T‐bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T‐bet‐expressing cells using a fluorescent fate‐mapping mouse line. We demonstrate that T‐bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve‐like T‐bet‐experienced cells are polarized to the TH1 lineage, predisposed to produce IFN‐γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage‐specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T‐helper response.
T-bet is the lineage-specifying transcription factor for CD4
T
1 cells. T-bet has also been found in other CD4
T cell subsets, including T
17 cells and Treg, where it modulates their functional ...characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4
T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4
T cells. Naïve-like T-bet-experienced cells are polarized to the T
1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.
We sought further understanding of the association between methotrexate (MTX) therapy and accelerated development of subcutaneous rheumatoid nodules. The objective was to establish expression of ...genes involved in the transport, metabolism, and mechanism of action of MTX within nodule tissue. We also examined for differences in gene expression between nodules from patients actively receiving MTX compared to those not receiving MTX.
Subcutaneous nodule tissues (n=23) were obtained from 21 patients with RA, undergoing elective surgery. Expression of genes important to the transport (SLC19A1, ABCB1, ABCC1, ABCG2), metabolism (FPGS, GGH), and mechanism of action (TYMS, MTR, MTRR) of MTX, including for the adenosine receptors ADORA1, ADORA2A, ADORA2B, ADORA3 and ADORA3variant were quantitated by real-time PCR in each nodule sample.
Transcripts for all genes were found in all nodules. Expression of MTR was significantly reduced in nodules from patients receiving MTX therapy. Patterns of gene expression differed, with those metabolising MTX more prominent in nodules from patients receiving MTX when compared to nodules from those not receiving MTX, where genes involved in MTX transport were more prominent.
Genes involved in MTX handling are expressed in rheumatoid nodules, providing further evidence that metabolism of MTX within nodules could exert a local effect. Furthermore the profile of gene expression in nodules differed from that previously observed in rheumatoid synovial membrane. The significant reduction of MTR expression in nodules has implications for MTR- and MTRR-mediated re-methylation reactions. Our data suggest that in contrast to synovium, downstream methylation reactions involving methionine and the biosynthesis of S-adenosylmethionine (SAM) could be reduced in nodule tissue. This could help explain differing responses to MTX in rheumatoid nodules and synovium and warrants further investigation.
BACKGROUNDA controlled human infection model for schistosomiasis (CHI-S) can speed up vaccine development and provides insight into early immune responses following schistosome exposure. Recently, we ...established CHI-S model using single-sex male-only Schistosoma mansoni (Sm) cercariae in Schistosoma-naïve individuals. Given important differences in antigenic profile and human immune responses to schistosomes of different sex, we pioneered a single-sex female-only CHI-S model for future use in vaccine development.METHODSWe exposed 13 healthy, Schistosoma-naïve adult participants to 10 (n = 3) or 20 (n = 10) female cercariae and followed for 20 weeks, receiving treatment with praziquantel (PZQ) 60 mg/kg at week 8 and 12 after exposure.FINDINGSThe majority (11/13) participants reported rash and/or itch at the site of exposure, 5/13 had transient symptoms of acute schistosomiasis. Exposure to 20 cercariae led to detectable infection, defined as serum circulating anodic antigen levels >1.0 pg/mL, in 6/10 participants. Despite two rounds of PZQ treatment, 4/13 participants showed signs of persistent infection. Additional one- or three-day PZQ treatment (1 × 60 mg/kg and 3 × 60 mg/kg) or artemether did not result in cure, but over time three participants self-cured. Antibody, cellular, and cytokine responses peaked at week 4 post infection, with a mixed Th1, Th2, and regulatory profile. Cellular responses were (most) discriminative for symptoms.INTERPRETATIONFemale-only infections exhibit similar clinical and immunological profiles as male-only infections but are more resistant to PZQ treatment. This limits future use of this model and may have important implications for disease control programs.FUNDINGEuropean Union's Horizon 2020 (grant no. 81564).
Allergic diseases and parasitic infections, such as asthma and schistosomiasis, are characterised by type-2 inflammation. It is understood that dendritic cells (DCs) are critical to drive type-2 ...inflammatory responses in allergic and helminthic diseases, via induction of T helper 2 (Th2) cells. In addition to inflammatory responses, regulatory responses arise during allergic airway inflammation (AAI), with dampening of inflammation by T regulatory cells (Tregs) critical. DCs have been shown to promote Treg expansion. However, the interaction between DCs and Tregs during ongoing AAI is not well understood, particularly with respect to AAI driven by fungal allergens. In this thesis we begin by elucidating Treg responses during fungal, Aspergillus fumigatus (A.f.) driven AAI, revealing an expansion of pulmonary Tregs alongside the development of the inflammatory response. We go on to show that Tregs specifically expand at the site of AAI – lung tissues and the airways – whilst increasing their expression of the suppressive cytokine IL-10. Previous reports have shown Tregs to be unstable during inflammation, gaining an inflammatory phenotype or losing expression of the characteristic transcription factor Foxp3. During A.f. driven AAI we found Tregs to be stable, however a small proportion of these cells began to express the Th2 associated cytokine IL-4. By sorting and performingin vitro suppression assays on Tregs from control or A.f. driven AAI we found that IL-4 expressing Tregs had reduced suppressive capacity. We next moved on to ask how Treg expansion was controlled during A.f. driven AAI, focusing on the role of DCs due to their published importance in this role. By systematically depleting DC subsets we reveal that type-2 conventional DCs (cDC2s) that express the lectin MGL2 are required for expansion of Tregs during A.f. driven AAI. In addition, we reveal DC expression of the integrin avb8, critical for activating the suppressive cytokine TGFb, is also required for lung Treg expansion. Moving on from AAI, we go on to investigate the regulation of pulmonary type-2 immune responses in parasite infection, focusing on the trematode Schistosoma mansoni (S. mansoni). We provide novel insights into lung immunological responses to S. mansoni at both lung migratory and adult, egg producing (patent) stages of its life cycle in both a mouse model and during human infection. During murine pulmonary schistosomiasis we reveal type-2 dominated inflammatory responses at both lung migratory and patent stages of infection, in addition to an expansion of cDC2s. Utilising sputum as a proxy for lung responses in human S. mansoni infection we observe trends for an increase in inflammatory cytokines during lung migratory infection, which are not observed in endemic patent S. mansoni infection. With the exception of an increase in Th cells during endemic patent S. mansoni infection, there were no significant changes in sputum granulocytes or lymphocytes measured. In line with our murine studies, we reveal consistent increases in cDCs, and in particular cDC2s during human lung migratory and endemic patent infection. Going back to the murine model, we reveal cDC2s to contribute to driving type-2 inflammatory responses to S. mansoniinfection. Taken together this work provides novel insights into the regulation of pulmonary type-2 inflammation, centring on the role of cDC2s in promoting both Th2 and Treg responses.
T-bet is the lineage-specifying transcription factor for CD4+ TH 1 cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional ...characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.
Summary
HIC-Vac is an international network of researchers dedicated to developing human infection challenge studies to accelerate vaccine development against pathogens of high global impact. The ...HIC-Vac Annual Meeting (3rd and 4th November 2022) brought together stakeholders including researchers, ethicists, volunteers, policymakers, industry partners, and funders with a strong representation from low- and middle-income countries. The network enables sharing of research findings, especially in endemic regions. Discussions included pandemic preparedness and the role of human challenge to accelerate vaccine development during outbreak, with industry speakers emphasising the great utility of human challenge in vaccine development. Public consent, engagement, and participation in human challenge studies were addressed, along with the role of embedded social science and empirical studies to uncover social, ethical, and regulatory issues around human infection challenge studies. Study volunteers shared their experiences and motivations for participating in studies. This report summarises completed and ongoing human challenge studies across a variety of pathogens and demographics, and addresses other key issues discussed at the meeting.
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