Darin L. Wiesner

Clinical Features and Serum Biomarkers in HIV Immune Reconstitution Inflammatory Syndrome after Cryptococcal Meningitis: A Prospective Cohort Study

David Boulware and colleagues investigate clinical features in a prospective cohort with AIDS and recent cryptococcal meningitis after initiation of antiretroviral therapy to identify biomarkers for prediction and diagnosis of CM-IRIS (cryptococcal meninigitis-related immune reconstitution inflammatory syndrome).

Paucity of Initial Cerebrospinal Fluid Inflammation in Cryptococcal Meningitis is associated with subsequent Immune Reconstitution Inflammatory Syndrome

BACKGROUND Cryptococcal meningitis (CM)-related immune reconstitution inflammatory syndrome (IRIS) complicates antiretroviral therapy (ART) in 20%-40% of ART-naive persons with AIDS and prior CM. Pathogenesis is unknown. METHODS We compared initial cerebrospinal fluid (CSF) cultures, inflammatory markers, and cytokine profiles in ART-naive patients with AIDS who did or did not subsequently develop IRIS after starting ART. We also compared results obtained at IRIS events or CM relapse. RESULTS Of 85 subjects with CM, 33 (39%) developed CM-related IRIS and 5 (6%) developed culture-positive CM relapse. At CM diagnosis, subjects subsequently developing IRIS had less inflammation, with decreased CSF leukocytes, protein, interferon-gamma, interleukin-6, interleukin-8, and tumor necrosis factor-alpha, compared with subjects not developing IRIS (P<.05, for each). Initial CSF white blood cell counts < or =25 cells/microL and protein levels < or =50 mg/dL were associated with development of IRIS (odds ratio, 7.2 [95% confidence interval, 2.7-18.7]; P<.001). Compared with baseline levels, we identified CSF elevations of interferon-gamma, tumor necrosis factor-alpha, granulocyte colony-stimulating factor, vascular-endothelial growth factor, and eotaxin (CCL11) (P<.05, for each) at the time of IRIS but minimal inflammatory changes in those with CM relapse. CONCLUSIONS Patients who subsequently develop CM-related IRIS exhibit less initial CSF inflammation at the time of CM diagnosis, compared with those who do not develop IRIS. The inflammatory CSF cytokine profiles observed at time of IRIS can distinguish IRIS from CM relapse.

Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay

ABSTRACT CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3′ untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3′ UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3′ UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.

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.

Cryptococcus: from environmental saprophyte to global pathogen

Cryptococcosis is a globally distributed invasive fungal infection that is caused by species within the genus Cryptococcus which presents substantial therapeutic challenges. Although natural human-to-human transmission has never been observed, recent work has identified multiple virulence mechanisms that enable cryptococci to infect, disseminate within and ultimately kill their human host. In this Review, we describe these recent discoveries that illustrate the intricacy of host–pathogen interactions and reveal new details about the host immune responses that either help to protect against disease or increase host susceptibility. In addition, we discuss how this improved understanding of both the host and the pathogen informs potential new avenues for therapeutic development.

Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

ABSTRACT Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.

Cryptococcal Genotype Influences Immunologic Response and Human Clinical Outcome after Meningitis

ABSTRACT In sub-Saharan Africa, cryptococcal meningitis (CM) continues to be a predominant cause of AIDS-related mortality. Understanding virulence and improving clinical treatments remain important. To characterize the role of the fungal strain genotype in clinical disease, we analyzed 140 Cryptococcus isolates from 111 Ugandans with AIDS and CM. Isolates consisted of 107 nonredundant Cryptococcus neoformans var. grubii strains and 8 C. neoformans var. grubii/neoformans hybrid strains. Multilocus sequence typing (MLST) was used to characterize genotypes, yielding 15 sequence types and 4 clonal clusters. The largest clonal cluster consisted of 74 isolates. The results of Burst and phylogenetic analysis suggested that the C. neoformans var. grubii strains could be separated into three nonredundant evolutionary groups (Burst group 1 to group 3). Patient mortality was differentially associated with the different evolutionary groups (P = 0.04), with the highest mortality observed among Burst group 1, Burst group 2, and hybrid strains. Compared to Burst group 3 strains, Burst group 1 strains were associated with higher mortality (P = 0.02), exhibited increased capsule shedding (P = 0.02), and elicited a more pronounced Th2 response during ex vivo cytokine release assays with strain-specific capsule stimulation (P = 0.02). The results of these analyses suggest that cryptococcal strain variation can be an important determinant of human immune responses and mortality. IMPORTANCE Cryptococcus neoformans is a common life-threatening human fungal pathogen that is responsible for an estimated 1 million cases of meningitis in HIV-infected patients annually. Virulence factors that are important in human disease have been identified, yet the impacts of the fungal strain genotype on virulence and outcomes of human infection remain poorly understood. Using an analysis of strain variation based on in vitro assays and clinical data from Ugandans living with AIDS and cryptococcal infection, we report that strain genotype predicts the type of immune response and mortality risk. These studies suggest that knowledge of the strain genotype during human infections could be used to predict disease outcomes and lead to improved treatment approaches aimed at targeting the specific combination of pathogen virulence and host response. Cryptococcus neoformans is a common life-threatening human fungal pathogen that is responsible for an estimated 1 million cases of meningitis in HIV-infected patients annually. Virulence factors that are important in human disease have been identified, yet the impacts of the fungal strain genotype on virulence and outcomes of human infection remain poorly understood. Using an analysis of strain variation based on in vitro assays and clinical data from Ugandans living with AIDS and cryptococcal infection, we report that strain genotype predicts the type of immune response and mortality risk. These studies suggest that knowledge of the strain genotype during human infections could be used to predict disease outcomes and lead to improved treatment approaches aimed at targeting the specific combination of pathogen virulence and host response.

Regulatory T Cell Induction and Retention in the Lungs Drives Suppression of Detrimental Type 2 Th Cells During Pulmonary Cryptococcal Infection

Lethal disease caused by the fungus Cryptococcus neoformans is a consequence of the combined failure to control pulmonary fungal replication and immunopathology caused by induced type 2 Th2 cell responses in animal models. In order to gain insights into immune regulatory networks, we examined the role of regulatory T (Treg) cells in suppression of Th2 cells using a mouse model of experimental cryptococcosis. Upon pulmonary infection with Cryptococcus, Treg cells accumulated in the lung parenchyma independently of priming in the draining lymph node. Using peptide–MHC class II molecules to identify Cryptococcus-specific Treg cells combined with genetic fate-mapping, we noted that a majority of the Treg cells found in the lungs were induced during the infection. Additionally, we found that Treg cells used the transcription factor, IFN regulatory factor 4, to dampen harmful Th2 cell responses, as well as mediate chemokine retention of Treg cells in the lungs. Taken together, induction and IFN regulatory factor 4–dependent localization of Treg cells in the lungs allow Treg cells to suppress the deleterious effects of Th2 cells during cryptococcal infection.

Pulmonary neuroendocrine cells amplify allergic asthma responses

Finding a role for PNECs in asthma Pulmonary neuroendocrine cells (PNECs) are a rare cell type located in airway and alveolar epithelia and are often in contact with sensory nerve fibers. They have a wide phylogenic distribution and are found even in the relatively primitive lungs of amphibia and reptiles, suggesting a critical function. Sui et al. found that mice lacking PNECs have suppressed type 2 (allergic) immune responses. PNECs were observed in close proximity to group 2 innate lymphoid cells (ILC2s) around airway branch points. The PNECs enhanced ILC2 activity by secreting CGRP (calcitonin gene-related peptide). They also induced goblet-cell hyperplasia via the neurotransmitter GABA (γ-aminobutyric acid). Interestingly, human asthma patients were found to have increased PNEC numbers, suggesting a potential therapeutic target for the treatment of asthma. Science, this issue p. eaan8546 PNECs, a rare population of cells in the airways, are critical for amplifying the airway allergen signal into mucosal responses in the lungs. INTRODUCTION The lung, with its vast surface area, senses and responds to signals in inhaled air. Aberrant interactions between the lung and the environment underlie many diseases, including asthma. In vitro data show that pulmonary neuroendocrine cells (PNECs), a rare airway epithelial cell population, can act as chemosensors. Once stimulated in culture, they release dense core vesicles rich in neuropeptides, amines, and neurotransmitters. These bioactive molecules are capable of eliciting immune and physiological responses. A recent in vivo study by our group revealed that the proper development of PNECs into self-clustering units called neuroepithelial bodies is essential for restricting the number of immune cells in the naïve lung. However, whether PNECs can function in vivo to translate exogenous airway signals such as allergens into the cascade of downstream responses is unknown. RATIONALE To test the hypothesis that PNECs act as sensors in the lung, we generated mouse mutants that lack PNECs by inactivating Ascl1 in the airway epithelium—i.e., mutants that were depleted of PNECs starting at development. We exposed these mutants to either ovalbumin or house dust mites, following regimes of existing asthma models. We determined whether the mutants showed different asthmatic responses than controls. We elucidated the underlying mechanisms by identifying molecular effectors and cellular targets of PNECs. To complement the functional tests in mice, we investigated whether human asthma patients showed pathological changes in their PNECs. RESULTS Although normal at baseline, Ascl1-mutant mice exhibited severely reduced goblet cell hyperplasia and immune cell numbers compared with controls after allergen challenge. In investigating possible molecular effectors, we found that several PNEC products were decreased in mutants relative to controls after allergen challenge, including calcitonin gene-related peptide (CGRP) and γ-aminobutyric acid (GABA). In exploring possible cellular targets, we found that innate lymphoid group 2 cells (ILC2s) were enriched at airway branch points, similar to PNECs. The PNEC product CGRP stimulated ILC2 production of interleukin-5 in culture. Conversely, inactivation of the CGRP receptor gene Calcrl in ILC2s led to dampened immune responses to allergens. In contrast to CGRP, GABA did not increase ILC2 cytokine secretion. Rather, inactivation of GABA biogenesis led to defective goblet cell hyperplasia after allergen challenge, suggesting that GABA is required for this response in the airway epithelium. The instillation of a mixture of CGRP and GABA in Ascl1 mutants restored both immune cell increases and goblet cell hyperplasia after allergen challenge, indicating that these products are the primary molecular effectors of PNECs in vivo. Consistent with these results from mice, we found increased PNEC numbers and cluster sizes in human asthma patients, which may underlie the heightened response to allergens in these individuals. CONCLUSION Our results demonstrate that PNECs, despite being a rare population of cells in the airway, are critical for amplifying the airway allergen signal into mucosal type 2 responses. Specifically, PNECs act through their product GABA to stimulate airway epithelial mucus production. In parallel, PNECs act through another product, CGRP, to stimulate ILC2 production of cytokines, which in turn recruit downstream immune cells. PNECs and ILC2s form neuroimmunological modules at the airway branch points, which are also the sites where airway particles are enriched. Our findings indicate that the PNEC-ILC2 axis functions to sense inhaled inputs, such as allergens, and amplify them into lung outputs, such as the allergic asthma response. PNECs are preferentially localized at branch points. A mouse airway stained by antibody against CGRP, to label PNECs (magenta) and antibody against SCGB1A1 to label club cells (green) (200× magnification). PNECs often cluster into neuroepithelial bodies and are preferentially localized at branch points. Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells whose function is poorly understood. Here we show that Ascl1-mutant mice that have no PNECs exhibit severely blunted mucosal type 2 response in models of allergic asthma. PNECs reside in close proximity to group 2 innate lymphoid cells (ILC2s) near airway branch points. PNECs act through calcitonin gene-related peptide (CGRP) to stimulate ILC2s and elicit downstream immune responses. In addition, PNECs act through the neurotransmitter γ-aminobutyric acid (GABA) to induce goblet cell hyperplasia. The instillation of a mixture of CGRP and GABA in Ascl1-mutant airways restores both immune and goblet cell responses. In accordance, lungs from human asthmatics show increased PNECs. These findings demonstrate that the PNEC-ILC2 neuroimmunological modules function at airway branch points to amplify allergic asthma responses.

Different Lymphocyte Populations Direct Dichotomous Eosinophil or Neutrophil Responses to Pulmonary Cryptococcus Infection.

Many pulmonary infections elicit lymphocyte responses that lead to an accumulation of granulocytes in the lungs. A variety of lymphocytes are capable of directing eosinophils or neutrophils to the lungs, but the contribution of each subset remains enigmatic. In this study, we used a murine model to examine lymphocyte subsets that ultimately drive the eosinophil or neutrophil response to infection with the fungal pathogen Cryptococcus neoformans. We show that granulocytes are produced in the bone marrow, released into the blood stream, and accumulate in the lungs under the instruction of lung parenchymal lymphocytes. The eosinophils that populated the lungs of wild-type animals were highly dependent on Th cells or IL-5. Surprisingly, infected mice with Th cell impairment experienced a compensatory neutrophil response that required IL-17A. This unexpected swing in the response prompted us to investigate the ability of different lymphocyte subsets to produce this dichotomous eosinophilia or neutrophilia. We used mice with lymphocyte deficiencies to determine which of the remaining IL-5– or IL-17A–producing lymphocyte subsets dominated the neutrophil or eosinophil response. Finally, skewing the response toward neutrophil-inducing lymphocytes correlated with accelerated disease. Our data collectively demonstrate that the predominance of a lymphocyte subset determines the functional consequences of an immune response to pulmonary fungal infection that can ultimately affect disease.