Non-canonical IL-22 receptor signaling remodels the mucosal barrier during fungal immunosurveillance.

Mucosal barrier integrity is vital for homeostasis with commensal organisms while preventing pathogen invasion. We unexpectedly found that fungal-induced immunosurveillance enhances resistance to fungal outgrowth and tissue invasion by remodeling the oral mucosal epithelial barrier in mouse models of adult and neonatal Candida albicans colonization. Epithelial subset expansion and tissue remodeling were dependent on interleukin-22 (IL-22) and signal transducer and activator of transcription 3 (STAT3) signaling, through a non-canonical receptor complex composed of glycoprotein 130 (gp130) coupled with IL-22RA1 and IL-10RB. Immunosurveillance-induced epithelial remodeling was restricted to the oral mucosa, whereas barrier architecture was reset once fungal-specific immunity developed. Collectively, these findings identify fungal-induced transient mucosal remodeling as a critical determinant of resistance to mucosal fungal infection during early stages of microbial colonization.

Focus on fungi.

Fungi play critical roles in the homeostasis of ecosystems globally and have emerged as significant causes of an expanding repertoire of devastating diseases in plants, animals, and humans. In this Commentary, we highlight the importance of fungal pathogens and argue for concerted research efforts to enhance understanding of fungal virulence, antifungal immunity, novel drug targets, antifungal resistance, and the mycobiota to improve human health.

A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Successful Treatment of Refractory Cutaneous Protothecosis With MAT2203, an Oral Lipid Nanocrystal Formulation of Amphotericin B.

Prototheca wickerhamii is a rare cause of cutaneous and systemic infection that requires long treatment courses with potentially toxic medications. We describe a patient with cutaneous protothecosis refractory to triazole monotherapy who experienced clinical and radiographic improvement with the novel oral lipid nanocrystal formulation of amphotericin B without experiencing toxicity.

Lymphocyte-Directed Immunomodulation Remits Thymoma-Associated Autoimmune Pneumonitis.

BACKGROUND: Thymoma presents with several autoimmune manifestations and is associated with secondary autoimmune regulator (AIRE) deficiency. Pneumonitis has recently been described as an autoimmune manifestation associated with thymoma presenting with similar clinical, radiographic, histological, and autoantibody features as seen in patients with inherited AIRE deficiency who suffer from Autoimmune PolyEndocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) syndrome. OBJECTIVES: To treat two patients with biopsy-proven thymoma-associated pneumonitis with lymphocyte-directed immunomodulation. METHODS: Two patients with thymoma were enrolled on IRB-approved protocols at the NIH Clinical Center. We performed history and physical examination; laboratory, radiographic, histologic and pulmonary function evaluations; and measurement of the lung-directed autoantibodies KCNRG and BPIFB1 prior to and at 1- and 6-months following initiation of lymphocyte-directed immunomodulation with azathioprine with or without rituximab. RESULTS: Combination T- and B-lymphocyte-directed immunomodulation resulted in improvement of clinical, functional, and radiographic parameters at 6-month follow-up evaluations in both patients with sustained remission up to 12-36 months following treatment initiation. CONCLUSION: Lymphocyte-directed immunomodulation remitted autoimmune pneumonitis in two patients with thymoma.

Longitudinal analysis of the lung proteome reveals persistent repair months after mild to moderate COVID-19.

In order to assess homeostatic mechanisms in the lung after COVID-19, changes in the protein signature of bronchoalveolar lavage from 45 patients with mild to moderate disease at three phases (acute, recovery, and convalescent) are evaluated over a year. During the acute phase, inflamed and uninflamed phenotypes are characterized by the expression of tissue repair and host defense response molecules. With recovery, inflammatory and fibrogenic mediators decline and clinical symptoms abate. However, at 9 months, quantified radiographic abnormalities resolve in the majority of patients, and yet compared to healthy persons, all showed ongoing activation of cellular repair processes and depression of the renin-kallikrein-kinin, coagulation, and complement systems. This dissociation of prolonged reparative processes from symptom and radiographic resolution suggests that occult ongoing disruption of the lung proteome is underrecognized and may be relevant to recovery from other serious viral pneumonias.

Yeast and filamentous Candida auris stimulate distinct immune responses in the skin.

Candida auris, an emerging multidrug-resistant fungal pathogen, predominately colonizes the human skin long term leading to subsequent life-threatening invasive infections. Fungal morphology is believed to play a critical role in modulating mucocutaneous antifungal immunity. In this study, we used an intradermal mouse model of C. auris infection to examine fungal colonization and the associated innate and adaptive immune response to yeast and filamentous C. auris strains. Our results indicate that mice infected with a filamentous C. auris had significantly decreased fungal load compared to mice infected with the yeast form. Mice infected with yeast and filamentous forms of C. auris stimulated distinct innate immune responses. Phagocytic cells (CD11b+Ly6G+ neutrophils, CD11b+Ly6Chi inflammatory monocytes, and CD11b+MHCII+CD64+ macrophages) were differentially recruited to mouse skin tissue infected with yeast and filamentous C. auris. The percentage and absolute number of interleukin 17 (IL-17) producing innate lymphoid cells, TCRγδ+, and CD4+ T cells in the skin tissue of mice infected with filamentous C. auris were significantly increased compared to the wild-type of yeast strain. Furthermore, complementation of filamentous mutant strain of C. auris (Δelm1 + ELM1) strain exhibited wild-type yeast morphology in vivo and induced comparable level of skin immune responses similar to mice infected with yeast strain. Collectively, our findings indicate that yeast and filamentous C. auris induce distinct local immune responses in the skin. The decreased fungal load observed in mouse skin infected with filamentous C. auris is associated with a potent IL-17 immune response induced by this morphotype.IMPORTANCECandida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue resulting in outbreaks of systemic infections. Understanding the factors that regulate C. auris skin colonization and host immune response is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We identified that yeast and filamentous forms of C. auris induce distinct skin immune responses in the skin. These findings may help explain the differential colonization and persistence of C. auris morphotypes in skin tissue. Understanding the skin immune responses induced by yeast and filamentous C. auris is important to develop novel vaccine strategies to combat this emerging fungal pathogen.

A human STAT3 gain-of-function variant drives local Th17 dysregulation and skin inflammation in mice.

Germline gain-of-function (GOF) variants in STAT3 cause an inborn error of immunity associated with early-onset poly-autoimmunity and immune dysregulation. To study tissue-specific immune dysregulation, we used a mouse model carrying a missense variant (p.G421R) that causes human disease. We observed spontaneous and imiquimod (IMQ)-induced skin inflammation associated with cell-intrinsic local Th17 responses in STAT3 GOF mice. CD4+ T cells were sufficient to drive skin inflammation and showed increased Il22 expression in expanded clones. Certain aspects of disease, including increased epidermal thickness, also required the presence of STAT3 GOF in epithelial cells. Treatment with a JAK inhibitor improved skin disease without affecting local Th17 recruitment and cytokine production. These findings collectively support the involvement of Th17 responses in the development of organ-specific immune dysregulation in STAT3 GOF and suggest that the presence of STAT3 GOF in tissues is important for disease and can be targeted with JAK inhibition.

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

An enhanced IL17 and muted type I interferon nasal epithelial cell state characterizes severe COVID-19 with fungal coinfection.

Recent case reports and epidemiological data suggest that fungal infections represent an underappreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing data set characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our previous study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL17 stimulation and anti-fungal immunity. Further, we observe a significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggest that IL17 stimulation-in part driven by Candida colonization-and blunted interferon signaling represent a common feature of severe COVID-19 infection. IMPORTANCE: In this paper, we present an analysis suggesting that symptomatic and asymptomatic fungal coinfections can impact patient disease progression during COVID-19 hospitalization. By looking into the presence of other pathogens and their effect on the host immune response during COVID-19 hospitalizations, we aim to offer insight into an underestimated scenario, furthering our current knowledge of determinants of severity that could be considered for future diagnostic and intervention strategies.

Early recognition of the APECED rash can accelerate the diagnosis of APECED.

Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) is a monogenic autoimmune disease most often resulting from biallelic loss-of-function variants in the autoimmune regulator (AIRE) gene. Although typically characterized by the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, we have recently reported that the clinical spectrum of the syndrome is far broader that previously described and that incorporation of an adjunct triad of APECED rash, autoimmune enteritis-associated intestinal dysfunction, and enamel hypoplasia in the classic triad manifestations could lead to earlier diagnosis. Among the adjunct triad manifestations, APECED rash occurs in 66% of American APECED patients by age 3, most often developing in the first year of life. Here, we describe the clinical and histological features of protracted APECED rash manifesting together with recurrent mucocutaneous candidiasis as the first two disease components of APECED in a 10-month-old girl.

Antiviral cellular therapy for enhancing T-cell reconstitution before or after hematopoietic stem cell transplantation (ACES): a two-arm, open label phase II interventional trial of pediatric patients with risk factor assessment.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Anti-Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency.

BACKGROUND: Autoantibodies against interleukin-12 (anti-interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti-interleukin-23 and anti-interleukin-12 prompted further investigation. METHODS: Among the patients (most of whom had thymoma) who were known to have anti-interleukin-12, we screened for autoantibodies against interleukin-23 (anti-interleukin-23). To validate the potential role of anti-interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti-interleukin-12 who had unusual infections. RESULTS: Among 30 patients with anti-interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti-interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti-interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti-interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti-interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex. CONCLUSIONS: Among patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti-interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM.

Cryptococcosis is a major worldwide disseminated invasive fungal infection. Cryptococcosis, particularly in its most lethal manifestation of cryptococcal meningitis, accounts for substantial mortality and morbidity. The breadth of the clinical cryptococcosis syndromes, the different patient types at-risk and affected, and the vastly disparate resource settings where clinicians practice pose a complex array of challenges. Expert contributors from diverse regions of the world have collated data, reviewed the evidence, and provided insightful guideline recommendations for health practitioners across the globe. This guideline offers updated practical guidance and implementable recommendations on the clinical approaches, screening, diagnosis, management, and follow-up care of a patient with cryptococcosis and serves as a comprehensive synthesis of current evidence on cryptococcosis. This Review seeks to facilitate optimal clinical decision making on cryptococcosis and addresses the myriad of clinical complications by incorporating data from historical and contemporary clinical trials. This guideline is grounded on a set of core management principles, while acknowledging the practical challenges of antifungal access and resource limitations faced by many clinicians and patients. More than 70 societies internationally have endorsed the content, structure, evidence, recommendation, and pragmatic wisdom of this global cryptococcosis guideline to inform clinicians about the past, present, and future of care for a patient with cryptococcosis.

GM-CSF-mediated epithelial-immune cell crosstalk orchestrates pulmonary immunity to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening mold pneumonia in immune compromised patients, particularly in those with quantitative or qualitative defects in neutrophils. While innate immune cell crosstalk licenses neutrophil antifungal activity in the lung, the role of epithelial cells in this process is unknown. Here, we find that that surfactant protein C (SPC)-expressing lung epithelial cells integrate infection-induced IL-1 and type III interferon signaling to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) preferentially at local sites of fungal infection and neutrophil influx. Using in vivo models that distinguish the role of GM-CSF during acute infection from its homeostatic function in alveolar macrophage survival and surfactant catabolism, we demonstrate that epithelial-derived GM-CSF increases the accumulation and fungicidal activity of GM-CSF-responsive neutrophils, with the latter being essential for host survival. Our findings establish SPC + epithelial cells as a central player in regulating the quality and strength of neutrophil-dependent immunity against inhaled mold pathogens. HIGHLIGHTS: GM-CSF is essential for host defense against A. fumigatus in the lung IL-1 and IFN-λ promote GM-CSF production by lung epithelial cells in parallelEpithelial cell-derived GM-CSF increases neutrophil accumulation and fungal killing capacityEpithelial cells preferentially upregulate GM-CSF in local sites of inflammation.

Evaluation of murine renal phagocyte-fungal interactions using intravital confocal microscopy and flow cytometry.

Myeloid phagocytes are essential for antifungal host defense during systemic candidiasis. Here, we present a protocol for assessing phagocyte-fungal interactions in vivo in the kidney, the primary target organ of the murine systemic candidiasis model. We describe steps for intravital confocal microscopy and flow cytometry. We also detail a kidney tissue dissociation procedure to obtain highly pure functional phagocytes for utilization in downstream ex vivo fungal uptake and killing assays.

Differential skin immune responses in mice intradermally infected with Candida auris and Candida albicans.

IMPORTANCE: Candida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue, resulting in outbreaks of nosocomial infections. Understanding the factors that regulate C. auris skin colonization is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We established a model of intradermal C. auris inoculation in mice and found that mice infected with C. auris elicit less potent innate and adaptive immune responses in the infected skin compared to C. albicans. These findings help explain the clinical observation of persistent C. auris colonization in skin tissue.