Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Although some degree of inflammation is required for protection, progressive inflammation may worsen disease and ultimately prevents pathogen eradication. To define molecular pathways leading to or diverting from pathogenic inflammation in infection, we resorted to dendritic cells (DCs), known to activate distinct signaling pathways in response to pathogens. We found that distinct intracellular pathways mediated the sensing of conidia and hyphae by lung DCs in vitro, which translate in vivo in the activation of protective Th1/Treg responses by conidia or inflammatory Th2/Th17 responses by hyphae. In vivo targeting inflammatory (PI3K/Akt/mTOR) or anti-inflammatory (STAT3/IDO) DC pathways by intranasally delivered small interfering RNA (siRNA) accordingly modified inflammation and immunity to infection. Thus, the screening of signaling pathways in DCs through a systems biology approach may be exploited for the development of siRNA therapeutics to attenuate inflammation in respiratory fungal infections and diseases.     

Interleukin-17 in host defence against bacterial,mycobacterial and fungal pathogens

The mammalian immune system is intricately regulated, allowing for potent pathogen-specific immunity to be rapidly activated in response to infection with a broad and diverse array of potential pathogens. As a result of their ability to differentiate into distinct effector lineages, CD4 T cells significantly contribute to pathogen-specific adaptive immune responses. Through the production of effector cytokines, CD4 T helper (Th) cells orchestrate the precise mobilization of specific immune cells to eradicate infection. The protective effects of the newly identified lineage of Th17 cells against pathogens like Klebsiella pneumoniae, Citrobacter rodentium and Candida albicans indicate the capacity of Th17 cells to confer protection against extracellular bacterial and fungal pathogens, filling a critical void in host immunity not covered by the classically described Th1 lineage that activates immunity to intracellular pathogens or the Th2 lineage that is important in protection against mucosal parasitic pathogens. Host defence by Th17 cells extends beyond protection against extracellular bacterial and fungal pathogens, as demonstrated in infections against intracellular bacteria like Listeria monocytogenes and Salmonella enterica, as well as Mycobacterium tuberculosis. Herein, we summarize both experimental data from mouse infection models and epidemiological studies in humans that demonstrate the protective effects of interleukin-17 and Th17 CD4 T cells in immunity to bacterial, mycobacterial and fungal pathogens.     

The C-Type Lectin Receptor MCL Mediates Vaccine-Induced Immunity against Infection with Blastomyces dermatitidis

C-type lectin receptors (CLRs) are essential in shaping the immune response to fungal pathogens. Vaccine-induced resistance requires Dectin-2 to promote differentiation of antifungal Th1 and Th17 cells. Since Dectin-2 and MCL heterodimerize and both CLRs use FcRγ as the signaling adaptor, we investigated the role of MCL in vaccine immunity to the fungal pathogen Blastomyces dermatitidis. MCL^−/− mice showed impaired vaccine resistance against B. dermatitidis infection compared to that of wild-type animals. The lack of resistance correlated with the reduced recruitment of Th17 cells to the lung upon recall following experimental challenge and impaired interleukin-17 (IL-17) production by vaccine antigen-stimulated splenocytes in vitro. Soluble MCL fusion protein recognized and bound a water-soluble ligand from the cell wall of vaccine yeast, but the addition of soluble Dectin-2 fusion protein did not augment ligand recognition by MCL. Taken together, our data indicate that MCL regulates the development of vaccine-induced Th17 cells and protective immunity against lethal experimental infection with B. dermatitidis.     

Harnessing local and systemic immunity for vaccines against tuberculosis

The lung is the portal of entry for Mycobacterium tuberculosis (Mtb) and animal experimental evidence indicates that local immune defense mechanisms are crucial for protective immunity. Immunization via the lower respiratory tract efficiently induces a dividing, activated, antigen-dependent, lung-resident, memory T-cell population, which is partly recoverable by bronchoalveolar lavage. These cells can inhibit the growth of Mtb in the lungs immediately after infection. Delivery of appropriate signals to the lung innate immune system is critical for induction of effective local immunity. In contrast after parenteral immunization, antigen-specific cells may be found in lung tissue but few are recoverable by lavage and inhibition of mycobacterial growth is delayed. Harnessing both local and systemic immunity can provide highly effective protection in animal models and the evidence suggests that taken in aggregate, multiple animal models may predict the success of novel vaccine strategies in humans.     

Clinical Significance and Molecular Characterization of Nonsporulating Molds Isolated from the Respiratory Tracts of Bronchopulmonary Mycosis Patients with Special Reference to Basidiomycetes

Nonsporulating molds (NSMs), especially basidiomycetes, have predominantly been reported as human pathogens responsible for allergic and invasive disease. Their conventional identification is problematic, as many isolates remain sterile in culture. Thus, inconclusive culture reports might adversely affect treatment decisions. The clinical significance of NSMs in pulmonary mycoses is poorly understood. We sequenced the internal transcribed spacer (ITS) region and D1/D2 domain of the larger subunit (LSU) of 52 NSMs isolated from respiratory specimens. The basidiomycetes were the predominant NSMs, of which Schizophyllum commune was the most common agent in allergic bronchopulmonary mycosis (ABPM), followed by Ceriporia lacerata in invasive fungal disease. Porostereum spadiceum, Phanaerochaete stereoides, Neosartorya fischeri, and Marasmiellus palmivorus were the other molds observed. Application of ITS and LSU region sequencing identified 92% of the isolates. The antifungal susceptibility data revealed that all basidiomycetes tested were susceptible to amphotericin B and resistant to caspofungin, fluconazole, and flucytosine. Except for 3 isolates of S. commune and a solitary isolate of M. palmivorus, all basidiomycetes had low MICs for itraconazole, posaconazole, and voriconazole. Basidiomycetes were isolated from patients with ABPM, invasive pulmonary mycosis/pneumonia, or fungal balls. In addition, the majority of the basidiomycetes were isolated from patients with chronic respiratory disorders who were sensitized to one of the basidiomycetous fungi and demonstrated precipitating antibodies against the incriminating fungi, indicating an indolent tissue reaction. Thus, isolation of basidiomycetes from the lower respiratory tract could be significant, and it is important to monitor these patients in order to prevent subsequent lung damage.     

Immunity to pathogenic fungi in the eye

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production.     

Immunosurveillance of Candida albicans commensalism by the adaptive immune system

The fungal microbiota (mycobiota) is an integral part of the microbial community colonizing the body surfaces and is involved in many key aspects of human physiology, while an imbalance of the fungal communities, termed fungal dysbiosis, has been described in pathologies ranging from infections to inflammatory bowel disease. Commensal organisms, such as the fungus Candida albicans, induce antigen-specific immune responses that maintain immune homeostasis. Adaptive immune mechanisms are vital in this process, while deficiencies in adaptive immunity are linked to fungal infections. We start to understand the mechanisms by which a shift in mycobiota composition, in particular in C. albicans abundance, is linked to immunopathological conditions. This review discusses the mechanisms that ensure continuous immunosurveillance of C. albicans during mucosal colonization, how these protective adaptive immune responses can also promote immunopathology, and highlight therapeutic advances against C. albicans-associated disease.     

The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection

Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined.     

The role of B cells and humoral immunity in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis remains a major public health burden. It is generally thought that while B cell- and antibody-mediated immunity plays an important role in host defense against extracellular pathogens, the primary control of intracellular microbes derives from cellular immune mechanisms. Studies on the immune regulatory mechanisms during infection with M. tuberculosis, a facultative intracellular organism, has established the importance of cell-mediated immunity in host defense during tuberculous infection. Emerging evidence suggest a role for B cell and humoral immunity in the control of intracellular pathogens, including obligatory species, through interactions with the cell-mediated immune compartment. Recent studies have shown that B cells and antibodies can significantly impact on the development of immune responses to the tubercle bacillus. In this review, we present experimental evidence supporting the notion that the importance of humoral and cellular immunity in host defense may not be entirely determined by the niche of the pathogen. A comprehensive approach that examines both humoral and cellular immunity could lead to better understanding of the immune response to M. tuberculosis.     

Neutrophil extracellular traps impair fungal clearance in a mouse model of invasive pulmonary aspergillosis

Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4^-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4^-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. These findings suggest that release of NETs contributes to tissue damage and limits control of fungal outgrowth. Thus inhibition of NETosis might be a useful strategy to maintain neutrophil function and avoid lung damage in patients suffering from IPA, especially in those suffering from preexisting pulmonary disease.     

Eosinophils subvert host resistance to an intracellular pathogen by instigating non-protective IL-4 in CCR2−/− mice

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however, their influence on intracellular pathogens is less clear. We previously reported that CCR2^−/− mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2^−/− animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus, our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.     

Invasive fungal infections in congenital immunodeficiencies

Both acquired and congenital immunodeficiencies may be associated with increased susceptibility to invasive fungal infections (IFIs), depending on the type of immune deficit. IFIs frequently occur in patients with phagocytic and cellular immune defects, but are rarely observed in those with humoral or complement deficits. Among congenital immune disorders, chronic granulomatous disease and hyper-IgE syndrome are most frequently associated with IFIs; variable susceptibility to fungal pathogens is also seen in patients with severe combined immunodeficiency, X-linked hyper-IgM syndrome, Wiskott–Aldrich syndrome, DiGeorge syndrome, common variable immunodeficiency, defects in the interferon-γ–interleukin-12 axis, and myeloperoxidase deficiency. Aspergillus, Candida, Cryptococcus, Histoplasma and other fungal genera are variably implicated in causing invasive infections in these patients. Prompt diagnosis of IFIs in this patient population requires a high degree of suspicion, together with a knowledge of their clinical presentation and the limitations of diagnostic modalities. Apart from administration of appropriate antifungal agents, successful management often requires the addition of surgical intervention. Adjunctive immunotherapy may be considered, although this has not been systematically studied. Prophylactic interferon-γ and itraconazole administration have been shown to reduce the risk of IFIs in patients with chronic granulomatous disease; however, the possibility of infections with azole-resistant organisms following long-term itraconazole prophylaxis should not be overlooked.     

Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins

In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.     

Bromodomain inhibitor I‐BET151 suppresses immune responses during fungal–immune interaction

Changes in the epigenetic landscape of immune cells are a crucial component of gene activation during the induction of inflammatory responses, therefore it has been hypothesized that epigenetic modulation could be employed to restore homeostasis in inflammatory scenarios. Fungal pathogens cause a large burden of morbidity and even mortality due to the hyperinflammatory processes that induce mucosal, allergic or systemic infections. Bromodomain and extraterminal domain (BET) proteins are considered as one as the most tantalizing pharmacological targets for the modulation of inflammatory responses at the epigenetic level. Nothing is known of the role of BET inhibitors on the inflammation induced by fungal pathogens. In the present study, we assessed the in vitro efficacy of the small molecular histone mimic BET inhibitor I‐BET151 to modulate innate immune responses during fungal–immune interaction with the clinically relevant fungal pathogens Candida albicans and Aspergillus fumigatus. Our results prove that BET inhibitors (I‐BETs) represent an important modulator of inflammation induced by fungal pathogens: both direct production of proinflammatory cytokines and the induction of trained immunity were inhibited by I‐BET151. These modulatory effects are likely to have important potential implications in clinically relevant situations.     

Immunotherapy Against Invasive Fungal Diseases in Stem Cell Transplant Recipients

Despite the availability of new antifungal compounds, morbidity and mortality of invasive fungal disease in allogeneic hematopoietic stem cell recipients are still unacceptably high. Over the past decade, one could witness an exciting improvement of the understanding of the molecular pathogenesis and of the complexity of host antifungal immune responses. This, in turn, provides critical information to augment host immunity against fungal pathogens. Strategies for enhancing the immune system include the administration of effector and regulatory cells (e.g., granulocytes, antigen-specific T cells, dendritic cells) as well as the administration of recombinant cytokines, interferons and growth factors (e.g., interferon-γ, keratinocyte growth factor, granulocyte- and granulocyte-macrophage colony stimulating factor). One has to recognize at the same time, however, that data of in vitro assays and animal models cannot necessarily be transferred into the clinical setting. In addition, meaningful clinical trials in allogeneic stem cell recipients suffering from invasive fungal disease require sufficiently large and homogenous cohorts of patients and can only be performed in international collaboration, but may ultimately improve the outcome of allogeneic transplant recipients with invasive fungal disease.     

Roles of Innate and Adaptive Immunity in Respiratory Mycoplasmosis

Current evidence suggests that host defense in respiratory mycoplasmosis is dependent on both innate and humoral immunity. To further delineate the roles of innate and adaptive immunity in antimycoplasmal defenses, we intranasally infected C3H/HeSnJ-scid/scid (C3H-SCID), C3H/HeSnJ (C3H), C57BL/6J-scid/scid (C57-SCID), and C57BL/6N (C57BL) mice with Mycoplasma pulmonis and at 14 and 21 days postinfection performed quantitative cultures of lungs and spleens, quantification of lung lesions, and histopathologic assessments of all other major organs. We found that numbers of mycoplasmas in lungs were associated with genetic background (C3H susceptible, C57BL resistant) rather than functional state of adaptive immunity, indicating that innate immunity is the main contributor to antimycoplasmal defense of the lungs. Extrapulmonary dissemination of mycoplasmas with colonization of spleens and histologic lesions in multiple organs was a common occurrence in all mice. The absence of adaptive immune responses in severe combined immunodeficient (SCID) mice resulted in increased mycoplasmal colonization of spleens and lesions in extrapulmonary sites, particularly spleens, hearts, and joints, and also reduced lung lesion severity. The transfer of anti-M. pulmonis serum to infected C3H-SCID mice prevented extrapulmonary infection and disease, while the severity of lung lesions was restored by transfer of naive spleen cells to infected C3H-SCID mice. Collectively, our results strongly support the conclusions that innate immunity provides antimycoplasmal defense of the lungs and humoral immunity has the major role in defense against systemic dissemination of mycoplasmal infection, but cellular immune responses may be important in exacerbation of mycoplasmal lung disease.     

DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12^−/− mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12^−/− mice. In contrast to WT NK cells, DAP12^−/− NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12^−/− macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.     

Early Innate Immunity to Bacterial Infection in the Lung Is Regulated Systemically by the Commensal Microbiota via Nod-Like Receptor Ligands

The commensal microbiota is a major regulator of the immune system. The majority of commensal bacteria inhabit the gastrointestinal tract and are known to regulate local mucosal defenses against intestinal pathogens. There is growing appreciation that the commensal microbiota also regulates immune responses at extraintestinal sites. Currently, however, it is unclear how this influences host defenses against bacterial infection outside the intestine. Microbiota depletion caused significant defects in the early innate response to lung infection by the major human pathogen Klebsiella pneumoniae. After microbiota depletion, early clearance of K. pneumoniae was impaired, and this could be rescued by administration of bacterial Nod-like receptor (NLR) ligands (the NOD1 ligand MurNAcTri_DAP and NOD2 ligand muramyl dipeptide [MDP]) but not bacterial Toll-like receptor (TLR) ligands. Importantly, NLR ligands from the gastrointestinal, but not upper respiratory, tract rescued host defenses in the lung. Defects in early innate immunity were found to be due to reduced reactive oxygen species-mediated killing of bacteria by alveolar macrophages. These data show that bacterial signals from the intestine have a profound influence on establishing the levels of antibacterial defenses in distal tissues.