Titan cell production in Cryptococcus neoformans reshapes the cell wall and capsule composition during infection

This work was supported by the National Institutes of Health (R01AI080275 and R21AI22352), the NIH Fogarty International Center (R25TW009345), the University of Minnesota Center for Translational Science Institute (UL1TR000114), Wellcome Trust (086827, 075470, 097377, 101873 & 200208) and MRC Centre for Medical Mycology (N006364/1). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection

Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection

Sputum Concentration Improves Diagnosis Of Pulmonary Tuberculosis Cases In Children At A Tertiary Care Institution In Rwanda

Background: Pulmonary tuberculosis diagnosis by direct sputum smear microscopy is still questionable because of its low sensitivity and this technique is not sufficient to diagnose pulmonary tuberculosis especially in people who are not able to spit out properly like children and old people; therefore another more sensitive conventional technique like concentrated sputum smear microscopy is needed in patients suspected of having pulmonary tuberculosis. We aimed to find out if the sputum concentration technique can be more sensitive than direct smear microscopy in the diagnosis of pulmonary tuberculosis according to age groups. Methods: This study was a cross-sectional, conducted on 70 participants at CHUK. The sputa were examined microscopically on direct and concentrated smears and result compared to culture. Finally the data were analyzed with MS excel and SPSS. Results: A total of 210 sputa were analyzed by direct and concentration methods with culture as a gold standard. In patients under 15 years both methods were different in sensitivity (25% vs. 75%, CI= 95%, P= 0.047), in patients of 15 years of age and more, both methods had the same sensitivity (75% vs. 75%, CI= 95%, P = 0, 87). Regardless of age groups both methods were different in sensitivity (80% vs.90.9%, C.I= 95%, P = 0.001). Conclusion: Sputum concentration is more sensitive than direct technique especially in children under 15 years. We would recommend all researchers involved in tuberculosis to increase the sample size and use different study sites to validate this method before its implementation universally.Introduction: Le diagnostic de la tuberculose pulmonaire par examen microscopique des frottis direct est encore discutable en raison de sa faible sensibilité et ne permet pas elle seule de poser le diagnostic de la tuberculose pulmonaire. Ceci est surtout le cas chez les malades qui ne sont pas en mesure de trouver le crachat à savoir les enfants et les personnes âgées. Par conséquent, une autre technique classique plus sensible comme le frottis concentré est nécessaire dans ce groupe des patients. Ce travail avait pour but de déterminer l’apport réel de la technique de concentration des crachats comparé à celui de l’examen microscopique de frottis direct dans le diagnostic de la tuberculose pulmonaire selon les groupes d’âge. Méthodes: Il s’agissait d’une étude prospective, transversale portant sur 70 patients suspect de tuberculose pulmonaire vue au CHUK. Les résultats de l’examen microscopique direct; et après concentration du crachat ont été comparés aux résultats de la culture. Les données ont été saisies et analysées avec MS Excel et SPSS. Résultats: Sur un total de 210 crachats examinés, l’approche d’examen microscopique basées sur la concentration du crachat a détecté un nombre significativement plus élevé chez les patients de moins de 15 ans (25% vs 75%, IC = 95%, P = 0,047). Chez les patients de 15 ans et plus, les deux méthodes ont détectée de façon égale avec une même sensibilité (75% contre 75%, IC = 95%, P = 0, 87). Quel que soit e le groupe d’âge, la méthode d’examen microscopique après concentration du crachat aune sensibilité légèrement supérieur qu’ à celle dont l’approche utilise un examen direct (80% vs.90.9%, IC = 95%, P = 0,001). Conclusion: L’examen microscopique basé sur la concentration du crachat est plus sensible que la technique directe surtout chez les enfants de moins de 15 ans. Nos résultats font appel à des recherches portant sur grande taille de l’échantillon et impliquant différents sites d’étude pour valider cette méthode avant sa mise en oeuvre

Polyploid Titan Cells Produce Haploid and Aneuploid Progeny To Promote Stress Adaptation

Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans

Contains fulltext : 96420.pdf (publisher's version ) (Open Access)In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1beta in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans

Chitotriosidase Promotes Chitin Recognition and Th2 Cell-mediated Disease.

<p>(<b>A</b>) Chitinase enzyme activity of recombinant enzymes or lung homogenates from 14 days post-infected or naïve mice. (<b>B</b>) Flow cytometry plots of CD4+, Foxp3-, CD44+ Cda2+ Th cells expressing Th2 cytokines, IL-5 & IL-13, at 14 days post-infection and (<b>C</b>) the quantification of these plots. (<b>D&E</b>) Survival of mutant or wildtype mice infected with KN99α either with or without IL-2 complex treatment. Logrank test comparing each survival curve relative with 10 mice for each group to: Chit1 −/− vs. wildtype, <i>P</i><0.005; Chit1 −/− vs. Chit1 −/− + IL-2 complex, <i>P</i><0.0005; Chit1 −/− + IL-2 complex vs. wildtype, <i>P</i> = 0.07. (<b>F</b>) Cytokines from lung homogenates of naïve or wildtype and Chit1−/− mice 14 days post-infection with KN99α or age-matched, naïve Chit1−/− mice. (<b>G</b>) IL-5+ IL-13+ antigen-specific Th2 cells from lungs of Chit1−/− mice 14 days post-infection and mice treated with intranasal chitin particles or chitin heptamer fragments (C7). (<b>H</b>) Chitinase enzyme activity of human plasma without (LEFT) or with (RIGHT) cryptococcal lysate antigen stimulation. Data are presented as the mean +/- standard error with at least 2 independent experiments per group. * = <i>P</i> < 0.05, ** = <i>P</i> < 0.005, *** = <i>P</i> < 0.0005 by Mann-Whitney <i>U</i> or Kruskal Wallis ANOVA. AMCase = Acidic Mammalian Chitinase, C7 = chitin heptamer, CCL = chemokine ligand, Cda = chitin deacetylase, Chit1 = Chitotriosidase, IFN = interferon, IL = interleukin, TNF = tumor necrosis factor.</p

Differences in total chitin due to cell morphology.

<p>^a Proportion of cryptococcal titan cells in the lungs, as reported by Okagaki et al. 2011[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004701#ppat.1004701.ref034" target="_blank">34</a>].</p><p>^b Mean fluorescence intesity of calcofluor white in size-fractionated cryptococcal cells analyzed by flow cytometry. GMFI = geometric mean fluorescence intensity</p><p>^c Estimated total chitin calculated uising the folowing equation: Relative Total Chitin = [(Proportion Titan Cells × Titan GMFI) + (Proportion Typical Cells × Typical GMFI)]/KN99α Typical Cell GMFI</p><p>Differences in total chitin due to cell morphology.</p

Model of Th2 Cell-mediated Disease during Pulmonary Fungal Infection.

<p><b>1</b>) Chitotriosidase is released by the host and degrades fungal chitin to generate small chitin fragments, such as chitin heptamers. <b>2</b>) Chitin recognition occurs by an unknown mechanism that could involve either epithelial cell production of alarmins, such as thymic stromal lymphopoietin (TLSP) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004701#ppat.1004701.ref009" target="_blank">9</a>] or antibodies that bind chitin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004701#ppat.1004701.ref009" target="_blank">9</a>]. <b>3</b>) These chitin-based signals are recognized by lung-resident CD11b+ conventional dendritic cells that are capable of producing IL-4, an essential Th2 cell differentiation factor. <b>4</b>) The adaptive Th2 cell response results in enhanced disease in the absence of significant changes in pulmonary fungal burden.</p

CD4+ T cell Cda2-MHCII tetramer peptide sequence and putative cross reactive peptides from other <i>C. neoformans</i> chitin deacetylases.

<p>The <b>bold</b> sequences indicate regions that align with the P1-P9 residues within the Cda2-MHCII tetramer core. The <u>underlined</u> sequences correspond to the total peptide included in the Cda2-MHCII tetramer.</p><p>^a Homologous amino acid sequences within the catalytic domain of chitin deacetylases.</p><p>^b Peptide loading score on MHCII I-A^B (74).</p><p>^c Cda2-MHCII tetramer made from this gene and amino acid sequence.</p><p>CD4+ T cell Cda2-MHCII tetramer peptide sequence and putative cross reactive peptides from other <i>C. neoformans</i> chitin deacetylases.</p

Type-2 Helper T Cells Accumulate in the Lungs of Mice Infected with <i>C. neoformans</i>.

<p>(<b>A</b>) Cda2-MHCII tetramer identifies <i>C. neoformans</i>-specific helper T (Th) cells from mice 14 days post-infection with strain KN99α, <i>cda2Δ</i>, or age-matched, naïve mice. Flow cytometry plot (<b>B</b>) or graphs (<b>C&D</b>) from lung digests showing CD4+, Foxp3-, CD44+ Cda2+ Th cells expressing Th1 (IFNγ), Th2 (IL-5 & IL-13), or Th17 (IL-17A) cytokines. (<b>E</b>) Cytokines from lung homogenates 14 days post-infection with KN99α or age-matched, naïve mice. Flow cytometry plot (<b>F</b>) and graphs (<b>G&H</b>) from mediastinal lymph node suspensions of Th cells expressing Th1, Th2, or Th17 cytokines. Data are presented as mean +/- standard error with 2 independent experiments of at least 5 mice per group. Cda = Chitin deacetylase, CCL = chemokine ligand, IFN = interferon, IL = interleukin, MLN = mediastinal lymph node, TNF = tumor necrosis factor.</p