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1.
mBio ; 11(1)2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911495

RESUMO

Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that overexpresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformans IMPORTANCE Cryptococcus neoformans is a fungal pathogen that is found throughout the environment and can cause life-threatening infections of the lung and central nervous system in severely immunocompromised individuals. Caspase recruitment domain-containing protein 9 (CARD9) is a critical molecule that is activated after interactions of C-type lectin receptors (CLRs) found on the surfaces of specific immune cells, with carbohydrate structures associated with fungi. Patients with defects in CARD9 are significantly more susceptible to a multitude of fungal infections. C. neoformans contains several carbohydrate structures that interact with CLRs on immune cells and activate CARD9. Consequently, these studies evaluated the necessity of CARD9 for the induction of protective immunity against C. neoformans infection. These results are important, as they advance our understanding of cryptococcal pathogenesis and host factors necessary for the induction of protective immunity against C. neoformans.

2.
Sci Adv ; 5(12): eaaw9051, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31840058

RESUMO

Epigenetic modifications play critical roles in inducing long-lasting immunological memory in innate immune cells, termed trained immunity. Whether similar epigenetic mechanisms regulate dendtritic cell (DC) function to orchestrate development of adaptive immunity remains unknown. We report that DCs matured with IFNγ and TNFα or matured in the lungs during invasive fungal infection with endogenous TNFα acquired a stable TNFα-dependent DC1 program, rendering them resistant to both antigen- and cytokine-induced alternative activation. TNFα-programmed DC1 had increased association of H3K4me3 with DC1 gene promoter regions. Furthermore, MLL1 inhibition blocked TNFα-mediated DC1 phenotype stabilization. During IFI, TNFα-programmed DC1s were required for the development of sustained TH1/TH17 protective immunity, and bone marrow pre-DCs exhibited TNFα-dependent preprogramming, supporting continuous generation of programmed DC1 throughout the infection. TNFα signaling, associated with epigenetic activation of DC1 genes particularly via H3K4me3, critically contributes to generation and sustenance of type 1/17 adaptive immunity and the immune protection against persistent infection.

3.
Dev Comp Immunol ; 104: 103534, 2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31689452

RESUMO

Expression of scavenger receptor A (SRA) in macrophages plays key role in macrophage mediated uptake of microbes. However, little is known about the role of porcine scavenger receptor A (pSRA) in phagocytic function of macrophages in swine species. In this study, polyclonal antibody against pSRA was generated by using recombinant proteins to study expression and function of pSRA. We report broad expression of pSRA in different tissues. In the lungs, pSRA is mainly expressed by alveolar macrophages. Blockade of class A scavenger receptor by fucoidan treatment demonstrates that pSRA has role in bacterial phagocytosis by macrophages. Furthermore, importance of SRA-mediated bacterial phagocytosis has been shown using CHO cell line expressing pSRA. In summary, these findings reveal that pSRA, which is predominantly expressed in alveolar macrophages is likely to be an important receptor mediating recognition and uptake of bacteria in pig lungs.

4.
Front Immunol ; 10: 1725, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31404168

RESUMO

Tumor necrosis factor alpha (TNF-α) plays a critical role in the control of cryptococcal infection, and its insufficiency promotes cryptococcal persistence. To explore the therapeutic potential of TNF-α supplementation as a booster of host anti-cryptococcal responses, we engineered a C. neoformans strain expressing murine TNF-α. Using a murine model of pulmonary cryptococcosis, we demonstrated that TNF-α-producing C. neoformans strain enhances protective elements of host response including preferential T-cell accumulation and improved Th1/Th2 cytokine balance, diminished pulmonary eosinophilia and alternative activation of lung macrophages at the adaptive phase of infection compared to wild type strain-infected mice. Furthermore, TNF-α expression by C. neoformans enhanced the fungicidal activity of macrophages in vitro. Finally, mice infected with the TNF-α-producing C. neoformans strain showed improved fungal control and considerably prolonged survival compared to wild type strain-infected mice, but could not induce sterilizing immunity. Taken together, our results support that TNF-α expression by an engineered C. neoformans strain while insufficient to drive complete immune protection, strongly enhanced protective responses during primary cryptococcal infection.

5.
Nucleic Acids Res ; 47(D1): D693-D700, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30365026

RESUMO

Virulence factors (VFs) are molecules that allow microbial pathogens to overcome host defense mechanisms and cause disease in a host. It is critical to study VFs for better understanding microbial pathogenesis and host defense mechanisms. Victors (http://www.phidias.us/victors) is a novel, manually curated, web-based integrative knowledge base and analysis resource for VFs of pathogens that cause infectious diseases in human and animals. Currently, Victors contains 5296 VFs obtained via manual annotation from peer-reviewed publications, with 4648, 179, 105 and 364 VFs originating from 51 bacterial, 54 viral, 13 parasitic and 8 fungal species, respectively. Our data analysis identified many VF-specific patterns. Within the global VF pool, cytoplasmic proteins were more common, while adhesins were less common compared to findings on protective vaccine antigens. Many VFs showed homology with host proteins and the human proteins interacting with VFs represented the hubs of human-pathogen interactions. All Victors data are queriable with a user-friendly web interface. The VFs can also be searched by a customized BLAST sequence similarity searching program. These VFs and their interactions with the host are represented in a machine-readable Ontology of Host-Pathogen Interactions. Victors supports the 'One Health' research as a vital source of VFs in human and animal pathogens.

6.
Front Microbiol ; 9: 2958, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564211

RESUMO

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation, and virulence of Cryptococcus neoformans, which causes fatal meningoencephalitis. A putative membrane sensor protein, Sho1, has been postulated to regulate HOG pathway, but its regulatory mechanism remains elusive. In this study, we characterized the function of Sho1 with relation to the HOG pathway in C. neoformans. Sho1 played minor roles in osmoresistance, thermotolerance, and maintenance of membrane integrity mainly in a HOG-independent manner. However, it was dispensable for cryostress resistance, primarily mediated through the HOG pathway. A mucin-like transmembrane (TM) protein, Msb2, which interacts with Sho1 in Saccharomyces cerevisiae, was identified in C. neoformans, but found not to interact with Sho1. MSB2 codeletion with SHO1 further decreased osmoresistance and membrane integrity, but not thermotolerance, of sho1Δ mutant, indicating that both factors play to some level redundant but also discrete roles in C. neoformans. Sho1 and Msb2 played redundant roles in promoting the filamentous growth in sexual differentiation in a Cpk1-independent manner, in contrast to the inhibitory effect of the HOG pathway in the process. However, both factors contributed independently to Cpk1 phosphorylation during vegetative growth and endoplasmic reticulum (ER) stress response. Finally, Sho1 and Msb2 play distinct but complementary roles in the pulmonary virulence of C. neoformans. Overall, Sho1 and Msb2 play complementary but distinct roles in stress response, differentiation, and pathogenicity of C. neoformans.

7.
Gut Pathog ; 10: 37, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30214488

RESUMO

Background: Culture-based diagnostic methods cannot achieve rapid and precise diagnoses for the identification of multiple diarrhoeal pathogens (DPs). A high-throughput multiplex genetic detection system (HMGS) was adapted and evaluated for the simultaneous identification and differentiation of infectious DPs and a broad analysis of DP infection aetiology. Results: DP-HMGS was highly sensitive and specific for DP detection compared with culture-based techniques and was similar to singleplex real-time PCR. The uniform level of sensitivity of DP-HMGS for all DPs allowed us to remap the aetiology of acute diarrhoeal infections in Shanghai, correcting incidences of massively underdiagnosed DP species with accuracy approaching that of sequencing-based methods. The most frequent DPs were enteropathogenic Escherichia coli, rotavirus and Campylobacter jejuni. DP-HMGS detected two additional causes of infectious diarrhoea that were previously missed by routine culture-based methods: enterohemorrhagic E. coli and Yersinia enterocolitica. We demonstrated the age dependence of specific DP distributions, especially the distributions of rotavirus, intestinal adenovirus and Clostridium difficile in paediatric patients as well as those of dominant bacterial infections in adults, with a distinct "top 3" pattern for each age group. Finally, the multiplexing capability and high sensitivity of DP-HMGS allowed the detection of infections co-induced by multiple pathogens (approximately 1/3 of the cases), with some DPs preferentially co-occurring as infectious agents. Conclusions: DP-HMGS has been shown to be a rapid, specific, sensitive and appropriate method for the simultaneous screening/detection of polymicrobial DP infections in faecal specimens. Widespread use of DP-HMGS is likely to advance routine diagnostic and clinical studies on the aetiology of acute diarrhoea.

8.
J Immunol ; 201(7): 2004-2015, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30097531

RESUMO

The substantial morbidity and mortality caused by invasive fungal pathogens, including Cryptococcus neoformans, necessitates increased understanding of protective immune responses against these infections. Our previous work using murine models of cryptococcal lung infection demonstrated that dendritic cells (DCs) orchestrate critical transitions from innate to adaptive immunity and that IL-10 signaling blockade improves fungal clearance. To further understand interrelationships among IL-10 production, fungal clearance, and the effect of IL-10 on lung DCs, we performed a comparative temporal analysis of cryptococcal lung infection in wild type C57BL/6J mice (designated IL-10+/+) and IL-10-/- mice inoculated intratracheally with C. neoformans (strain 52D). Early and sustained IL-10 production by lung leukocytes was associated with persistent infection in IL-10+/+ mice, whereas fungal clearance was improved in IL-10-/- mice during the late adaptive phase of infection. Numbers of monocyte-derived DCs, T cells, and alveolar and exudate macrophages were increased in lungs of IL-10-/- versus IL-10+/+ mice concurrent with evidence of enhanced DC type-1, Th1/Th17 CD4 cell, and classical macrophage activation. Bone marrow-derived DCs stimulated with cryptococcal mannoproteins, a component of the fungal capsule, upregulated expression of IL-10 and IL-10R, which promoted DC type-2 activation in an autocrine manner. Thus, our findings implicate fungus-triggered autocrine IL-10 signaling and DC type-2 activation as important contributors to the development of nonprotective immune effector responses, which characterize persistent cryptococcal lung infection. Collectively, this study informs and strengthens the rationale for IL-10 signaling blockade as a novel treatment for fungal infections.


Assuntos
Criptococose/imunologia , Cryptococcus neoformans/fisiologia , Células Dendríticas/imunologia , Inflamação/imunologia , Interleucina-10/metabolismo , Pneumopatias Fúngicas/imunologia , Pulmão/imunologia , Animais , Comunicação Autócrina , Modelos Animais de Doenças , Humanos , Interleucina-10/genética , Pulmão/microbiologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Células Th2/imunologia
10.
MBio ; 8(6)2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29162707

RESUMO

Cryptococcus neoformans is a major fungal pathogen that disseminates to the central nervous system (CNS) to cause fatal meningoencephalitis, but little is known about immune responses within this immune-privileged site. CD4+ T cells have demonstrated roles in anticryptococcal defenses, but increasing evidence suggests that they may contribute to clinical deterioration and pathology in both HIV-positive (HIV+) and non-HIV patients who develop immune reconstitution inflammatory syndrome (IRIS) and post-infectious inflammatory response syndrome (PIIRS), respectively. Here we report a novel murine model of cryptococcal meningoencephalitis and a potential damaging role of T cells in disseminated cryptococcal CNS infection. In this model, fungal burdens plateaued in the infected brain by day 7 postinfection, but activation of microglia and accumulation of CD45hi leukocytes was significantly delayed relative to fungal growth and did not peak until day 21. The inflammatory leukocyte infiltrate consisted predominantly of gamma interferon (IFN-γ)-producing CD4+ T cells, conventionally believed to promote fungal clearance and recovery. However, more than 50% of mice succumbed to infection and neurological dysfunction between days 21 and 35 despite a 100-fold reduction in fungal burdens. Depletion of CD4+ cells significantly impaired IFN-γ production, CD8+ T cell and myeloid cell accumulation, and fungal clearance from the CNS but prevented the development of clinical symptoms and mortality. These findings conclusively demonstrate that although CD4+ T cells are necessary to control fungal growth, they can also promote significant immunopathology and mortality during CNS infection. The results from this model may provide important guidance for development and use of anti-inflammatory therapies to minimize CNS injury in patients with severe cryptococcal infections.IMPORTANCE CNS infection with the fungal pathogen Cryptococcus neoformans often results in debilitating brain injury and has a high mortality rate despite antifungal treatment. Treatment is complicated by the fact that immune responses needed to eliminate infection are also thought to drive CNS damage in a subset of both HIV+ and non-HIV patients. Thus, physicians need to balance efforts to enhance patients' immune responses and promote microbiological control with anti-inflammatory therapy to protect the CNS. Here we report a novel model of cryptococcal meningoencephalitis demonstrating that fungal growth within the CNS does not immediately cause symptomatic disease. Rather, accumulation of antifungal immune cells critically mediates CNS injury and mortality. This model demonstrates that antifungal immune responses in the CNS can cause detrimental pathology and addresses the urgent need for animal models to investigate the specific cellular and molecular mechanisms underlying cryptococcal disease in order to better treat treat patients with CNS infections.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Criptococose/imunologia , Meningoencefalite/imunologia , Meningoencefalite/patologia , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Criptococose/microbiologia , Criptococose/fisiopatologia , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Infecções por HIV/imunologia , Humanos , Inflamação , Interferon gama/imunologia , Meningite Criptocócica/microbiologia , Meningite Criptocócica/patologia , Meningoencefalite/microbiologia , Meningoencefalite/mortalidade , Camundongos , Células Mieloides
11.
Front Immunol ; 8: 1231, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29033946

RESUMO

Macrophage receptor with collagenous structure (MARCO) contributes to fungal containment during the early/innate phase of cryptococcal infection; however, its role in adaptive antifungal immunity remains unknown. Using a murine model of cryptococcosis, we compared host adaptive immune responses in wild-type and MARCO-/- mice throughout an extended time course post-infection. Unlike in early infection, MARCO deficiency resulted in improved pulmonary fungal clearance and diminished cryptococcal dissemination during the efferent phase. Improved fungal control in the absence of MARCO expression was associated with enhanced hallmarks of protective Th1-immunity, including higher frequency of pulmonary TNF-α-producing T cells, increased cryptococcal-antigen-triggered IFN-γ and TNF-α production by splenocytes, and enhanced expression of M1 polarization genes by pulmonary macrophages. Concurrently, we found lower frequencies of IL-5- and IL-13-producing T cells in the lungs, impaired production of IL-4 and IL-10 by cryptococcal antigen-pulsed splenocytes, and diminished serum IgE, which were hallmarks of profoundly suppressed efferent Th2 responses in MARCO-deficient mice compared to WT mice. Mechanistically, we found that MARCO expression facilitated early accumulation and alternative activation of CD11b+ conventional DC (cDC) in the lung-associated lymph nodes (LALNs), which contributed to the progressive shift of the immune response from Th1 toward Th2 at the priming site (LALNs) and local infection site (lungs) during the efferent phase of cryptococcal infection. Taken together, our study shows that MARCO can be exploited by the fungal pathogen to promote accumulation and alternative activation of CD11b+ cDC in the LALN, which in turn alters Th1/Th2 balance to promote fungal persistence and dissemination.

12.
J Immunol ; 199(10): 3535-3546, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29038249

RESUMO

Activation of immunomodulatory pathways in response to invasive fungi can impair clearance and promote persistent infections. The programmed cell death protein-1 (PD-1) signaling pathway inhibits immune effector responses against tumors, and immune checkpoint inhibitors that block this pathway are being increasingly used as cancer therapy. The objective of this study was to investigate whether this pathway contributes to persistent fungal infection and to determine whether anti-PD-1 Ab treatment improves fungal clearance. Studies were performed using C57BL/6 mice infected with a moderately virulent strain of Cryptococcus neoformans (52D), which resulted in prolonged elevations in fungal burden and histopathologic evidence of chronic lung inflammation. Persistent infection was associated with increased and sustained expression of PD-1 on lung lymphocytes, including a mixed population of CD4+ T cells. In parallel, expression of the PD-1 ligands, PD-1 ligands 1 and 2, was similarly upregulated on specific subsets of resident and recruited lung dendritic cells and macrophages. Treatment of persistently infected mice for 4 wk by repetitive administration of neutralizing anti-PD-1 Ab significantly improved pulmonary fungal clearance. Treatment was well tolerated without evidence of morbidity. Immunophenotyping revealed that anti-PD-1 Ab treatment did not alter immune effector cell numbers or myeloid cell activation. Treatment did reduce gene expression of IL-5 and IL-10 by lung leukocytes and promoted sustained upregulation of OX40 by Th1 and Th17 cells. Collectively, this study demonstrates that PD-1 signaling promotes persistent cryptococcal lung infection and identifies this pathway as a potential target for novel immune-based treatments of chronic fungal disease.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Criptococose/terapia , Cryptococcus neoformans/imunologia , Imunoterapia/métodos , Pulmão/imunologia , Receptor de Morte Celular Programada 1/imunologia , Células Th1/efeitos dos fármacos , Animais , Contagem de Colônia Microbiana , Criptococose/imunologia , Cryptococcus neoformans/patogenicidade , Citocinas/metabolismo , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Células Th1/imunologia , Virulência
13.
Front Immunol ; 8: 1055, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28919893

RESUMO

Fas-associated death domain (FADD) and receptor interacting protein kinase 3 (RIPK3) are multifunctional regulators of cell death and immune response. Using a mouse model of cryptococcal infection, the roles of FADD and RIPK3 in anti-cryptococcal defense were investigated. Deletion of RIPK3 alone led to increased inflammatory cytokine production in the Cryptococcus neoformans-infected lungs, but in combination with FADD deletion, it led to a robust Th1-biased response with M1-biased macrophage activation. Rather than being protective, these responses led to paradoxical C. neoformans expansion and rapid clinical deterioration in Ripk3-/- and Ripk3-/-Fadd-/- mice. The increased mortality of Ripk3-/- and even more accelerated mortality in Ripk3-/-Fadd-/- mice was attributed to profound pulmonary damage due to neutrophil-dominant infiltration with prominent upregulation of pro-inflammatory cytokines. This phenomenon was partially associated with selective alterations in the apoptotic frequency of some leukocyte subsets, such as eosinophils and neutrophils, in infected Ripk3-/-Fadd-/- mice. In conclusion, our study shows that RIPK3 in concert with FADD serve as physiological "brakes," preventing the development of excessive inflammation and Th1 bias, which in turn contributes to pulmonary damage and defective fungal clearance. This novel link between the protective effect of FADD and RIPK3 in antifungal defense and sustenance of immune homeostasis may be important for the development of novel immunomodulatory therapies against invasive fungal infections.

14.
J Immunol ; 199(2): 643-655, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28615417

RESUMO

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans Inhibition of T cell-restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ-, IL-5-, and IL-13-producing CD4+ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8+ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.


Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Receptores Notch/metabolismo , Animais , Antígenos de Fungos/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Sistema Nervoso Central/parasitologia , Criptococose/microbiologia , Fator de Transcrição GATA3/metabolismo , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Pulmão/parasitologia , Ativação de Macrófagos , Camundongos , Receptores Notch/deficiência , Transdução de Sinais , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia
15.
J Immunol ; 198(9): 3548-3557, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28298522

RESUMO

The scavenger receptor macrophage receptor with collagenous structure (MARCO) promotes protective innate immunity against bacterial and parasitic infections; however, its role in host immunity against fungal pathogens, including the major human opportunistic fungal pathogen Cryptococcus neoformans, remains unknown. Using a mouse model of C. neoformans infection, we demonstrated that MARCO deficiency leads to impaired fungal control during the afferent phase of cryptococcal infection. Diminished fungal containment in MARCO-/- mice was accompanied by impaired recruitment of Ly6Chigh monocytes and monocyte-derived dendritic cells (moDC) and lower moDC costimulatory maturation. The reduced recruitment and activation of mononuclear phagocytes in MARCO-/- mice was linked to diminished early expression of IFN-γ along with profound suppression of CCL2 and CCL7 chemokines, providing evidence for roles of MARCO in activation of the CCR2 axis during C. neoformans infection. Lastly, we found that MARCO was involved in C. neoformans phagocytosis by resident pulmonary macrophages and DC. We conclude that MARCO facilitates early interactions between C. neoformans and lung-resident cells and promotes the production of CCR2 ligands. In turn, this contributes to a more robust recruitment and activation of moDC that opposes rapid fungal expansion during the afferent phase of cryptococcal infection.


Assuntos
Criptococose/imunologia , Cryptococcus neoformans/fisiologia , Células Dendríticas/imunologia , Pneumopatias Fúngicas/imunologia , Macrófagos/imunologia , Receptores Imunológicos/metabolismo , Receptores Depuradores/metabolismo , Animais , Células Cultivadas , Quimiocina CCL7/metabolismo , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interferon gama/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Receptores Imunológicos/genética , Receptores Depuradores/genética
16.
Microorganisms ; 4(4)2016 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-27973396

RESUMO

This review addresses specific regulatory mechanisms involved in the host immune response to fungal organisms. We focus on key cells and regulatory pathways involved in these responses, including a brief overview of their broader function preceding a discussion of their specific relevance to fungal disease. Important cell types discussed include dendritic cells and regulatory T cells, with a focus on specific studies relating to their effects on immune responses to fungi. We highlight the interleukin-10, programmed cell death 1, and cytotoxic T lymphocyte-associated protein 4 signaling pathways and emphasize interrelationships between these pathways and the regulatory functions of dendritic cells and regulatory T cells. Throughout our discussion, we identify selected studies best illustrating the role of these cells and pathways in response to specific fungal pathogens to provide a contextual understanding of the tightly-controlled network of regulatory mechanisms critical to determining the outcome of exposure to fungal pathogens. Lastly, we discuss two unique phenomena relating to immunoregulation, protective tolerance and immune reactivation inflammatory syndrome. These two clinically-relevant conditions provide perspective as to the range of immunoregulatory mechanisms active in response to fungi.

17.
Front Microbiol ; 7: 1652, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27833589

RESUMO

Cryptococcus neoformans is pathogenic yeast, responsible for highly lethal infections in compromised patients around the globe. C. neoformans typically initiates infections in mammalian lung tissue and subsequently disseminates to the central nervous system where it causes significant pathologies. Virulence genes of C. neoformans are being characterized at an increasing rate, however, we are far from a comprehensive understanding of their roles and genetic interactions. Some of these reported virulence genes are scattered throughout different databases, while others are not yet included. This study gathered and analyzed 150 reported virulence associated factors (VAFs) of C. neoformans. Using the web resource STRING database, our study identified different interactions between the total VAFs and those involved specifically in lung and brain infections and identified a new strain specific virulence gene, SHO1, involved in the mitogen-activated protein kinase signaling pathway. As predicted by our analysis, SHO1 expression enhanced C. neoformans virulence in a mouse model of pulmonary infection, contributing to enhanced non-protective immune Th2 bias and progressively enhancing fungal growth in the infected lungs. Sequence analysis indicated 77.4% (116) of total studied VAFs are soluble proteins, and 22.7% (34) are transmembrane proteins. Motifs involved in regulation and signaling such as protein kinases and transcription factors are highly enriched in Cryptococcus VAFs. Altogether, this study represents a pioneering effort in analysis of the virulence composite network of C. neoformans using a systems biology approach.

18.
Front Microbiol ; 7: 1401, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27656172

RESUMO

Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for H. pylori identification, quantification, virulence, and drug resistance by optimizing the singlet-PCR and multiple primers assay. Twenty-one pairs of chimeric primers consisted of conserved and specific gene sequences of H. pylori tagged with universal sequence at the 5' end were designed. Singlet-PCR assay and multiple primers assay were developed to optimize the HMGS. The specificity of HMGS assay was evaluated using standard H. pylori strains and bacterial controls. Six clinical isolates with known genetic background of target genes were detected to assess the accuracy of HMGS assay. Artificial mixed pathogen DNA templates were used to evaluate the ability to distinguish mixed infections using HMGS assay. Furthermore, gastric biopsy specimens with corresponding isolated strains were used to assess the capability of HMGS assay in detecting biopsy specimens directly. HMGS assay was specific for H. pylori identification. HMGS assay for H. pylori target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of H. pylori could be distinguished by HMGS assay. HMGS assay could efficiently diagnose H. pylori infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of H. pylori, analysis of virulence and drug resistance in both isolated strains and biopsy specimens. It could also be used to distinguish the mixed infection with different resistant genotype strains. Furthermore, HMGS could detect H. pylori infection in gastric biopsy specimens directly.

19.
Future Microbiol ; 11: 1521-1534, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27599152

RESUMO

AIM: We evaluated the direct high-throughput multiple genetic detection system (dHMGS) for Helicobacter pylori in gastric biopsies. MATERIALS & METHODS: One hundred and thirty-three specimens were concurrently analyzed by dHMGS, rapid urease test, culture and sequencing. RESULTS: dHMGS was highly sensitive and specific for H. pylori identification compared with culture and rapid urease test. The correlation coefficient of the quantitative standard curve was R2 = 0.983. A significant difference in the relative H. pylori DNA abundance was found in different gastroduodenal diseases. Concordance rates between dHMGS and sequencing for resistance mutations were 97.1, 100.0, 85.3 and 97.1%, respectively. Finally, dHMGS could efficiently distinguish mixed infection in biopsy specimens. CONCLUSION: The dHMGS could efficiently diagnose and quantify H. pylori burden in biopsies, simultaneously screening for virulence, antibiotic resistance and presence of the multistrain infections.


Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biópsia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Feminino , Infecções por Helicobacter/patologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Urease/genética , Urease/metabolismo , Adulto Jovem
20.
MBio ; 7(4)2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27406560

RESUMO

UNLABELLED: Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4(+) T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance. IMPORTANCE: Increased susceptibility to invasive fungal infections in patients on anti-TNF-α therapies underlines the need for understanding the cellular effects of TNF-α signaling in promoting protective immunity to fungal pathogens. Here, we demonstrate that early TNF-α signaling is required for classical activation and accumulation of DC in LALN of C. neoformans-infected mice. Subsequent transcriptional initiation of Th17 followed by Th1 programming in LALN results in pulmonary accumulation of gamma interferon- and interleukin-17A-producing T cells and effective fungal clearance. All of these crucial steps are severely impaired in mice that undergo anti-TNF-α treatment, consistent with their inability to clear C. neoformans This study identified critical interactions between cells of the innate immune system (DC), the emerging T cell responses, and cytokine networks with a central role for TNF-α which orchestrate the development of the immune protection against cryptococcal infection. This information will be important in aiding development and understanding the potential side effects of immunotherapies.


Assuntos
Criptococose/imunologia , Criptococose/prevenção & controle , Células Dendríticas/imunologia , Pneumopatias/imunologia , Pneumopatias/prevenção & controle , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Carga Bacteriana , Linfócitos T CD4-Positivos/imunologia , Cryptococcus neoformans/imunologia , Modelos Animais de Doenças , Pulmão/imunologia , Pulmão/microbiologia , Linfonodos/imunologia , Camundongos
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