Exploitation of Scavenger Receptor, Macrophage Receptor with Collagenous Structure, by Cryptococcus neoformans Promotes Alternative Activation of Pulmonary Lymph Node CD11b+ Conventional Dendritic Cells and Non-Protective Th2 Bias

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

Induction of the epithelial polarizing cytokine interleukin-33 by the fungus «Cryptococcus neoformans» in genetically susceptible mice

With the progression of the AIDS epidemic, understanding the host responseto the opportunistic fungal pathogen Cryptococcus neoformans, responsible forapproximately 650,000 deaths in Sub-Saharan Africa each year, has become animportant topic of research. Current knowledge suggests that susceptibility tocryptococcal pneumonia in both mice and humans proceeds via an allergic (TH2)pattern of lung inflammation, while resistance is attributable to a TH1 response in thelungs. The epithelial polarizing cytokines thymic stromal lymphopoietin (TSLP),interleukin-25 (IL-25) and IL-33 have been implicated in TH2 mucosal andrespiratory inflammation caused by allergens and helminths; however, their rolesduring C. neoformans infection are not known. We demonstrated that in vitrostimulation of the mouse lung epithelial cell line (MLE-12) with both the acapsularmutant C. neoformans CAP64 and the highly virulent C. neoformans H99, resulted indose- and time-dependent increases in both Il25 and Il33 mRNA expression.Correspondingly, intranasal infection of susceptible Balb/c mice with C. neoformansH99 showed time-dependent IL-33 mRNA and protein in the lungs. Furthermore,moderately virulent C. neoformans 52D induced differential Il33 mRNA expressionamong susceptible and resistant strains of mice, with susceptible C57BL/6 micedeveloping a significant increase in lung Il33 mRNA compared to resistant CBAmice. Finally, Balb/c mice lacking the IL-33 receptor T1/ST2 had significantlyreduced lung, spleen and brain fungal burdens following intratracheal instillation ofC. neoformans. These observations support a role for IL-33 in polarization of the hostinflammatory response that facilitates progressive pulmonary C. neoformansinfection.Avec la prevalence de l'épidémie du SIDA, comprendre la reaction de l'hôte au champignon opportuniste pathogène Cryptococcus neoformans, responsable de 650,000 décès chaque année en Afrique sub-saharienne, est devenu un sujet de recherche profonde. Les connaissances actuelles suggèrent que la susceptibilité à la pneumonie de cryptocoque chez les souris et les humains se produit par l'intermédiaire des réponses allergiques de type TH2 d'inflammation pulmonaire, alors que la résistance est attribuableau développent d'une réponse TH1 dans les poumons. Les cytokines épithéliales polarisatrices comme thymic stromal lymphopoietin (TSLP), l'interleukine-25 (IL-25) et IL-33 sont impliqués dans l'inflammation allergique TH2 respiratoire, mais leur rôle pendant l'infection C. neoformans reste inconnu. Nous démontrons que la stimulation invitro des cellules épithéliales de poumons de souris (MLE-12) par la souche C. neoformans sans-capsule CAP64 ou la souche C. neoformans très virulente H99, révèleune augmentation sélective de doses de l'expression d'ARNm d'Il25 et d'Il33.Corrélativement, l'infection intranasale de souris susceptible Balb/c au H99 a montré l'induction en fonction du temps de l'ARNm et du protéine de l'IL-33 dans les poumons.Par ailleurs, l'utilisation de la souche de C. neoformans modérément virulente 52D, adémontré une expression de l'Il33 différente entre les souches de souris susceptibles et résistantes. La souche C57BL/6 a connu une augmentation significative de l'ARNm de l'Il33 dans les poumons comparée à la souche résistante CBA. Enfin, les souris Balb/cknockout qui manquaient le récepteur T1/ST2 pour l'IL-33, avaient une charge fongique réduite dans les poumons, les spleens et les cerveaux suite à l'infection par C. neoformans. Ces observations suggèrent un role désavantageux de l'IL-33 dans la polarisation de la réponse inflammatoire pendant l'infection C. neoformans pulmonaire

IL-33 signaling regulates innate and adaptive immunity to Cryptococcus neoformans.

Susceptibility to progressive infection with the fungus Cryptococcus neoformans is associated with an allergic pattern of lung inflammation, yet the factors that govern this host response are not clearly understood. Using a clinically relevant mouse model of inhalational infection with virulent C. neoformans H99, we demonstrate a role for IL-33-dependent signaling in host immune defense. Infection of BALB/c mice with 10(4) CFU of C. neoformans H99 caused a time-dependent induction of IL-33 with accumulation of type 2 pulmonary innate lymphoid cells and alternatively activated macrophages in the lungs as well as Th2-polarized CD4(+) T cells in draining lymph nodes. IL-33R subunit T1/ST2-deficient (T1/ST2(-/-)) mice infected with C. neoformans H99 had improved survival with a decreased fungal burden in the lungs, spleen, and brain, compared with wild-type mice. Signaling through T1/ST2 was required for the accumulation and early production of IL-5 and IL-13 by lung type 2 pulmonary innate lymphoid cells. Further analysis of T1/ST2(-/-) mice revealed increased fungicidal exudate macrophages in the lungs and decreased C. neoformans-specific Th2 cells in the mediastinal lymph nodes. T1/ST2 deficiency also diminished goblet cell hyperplasia, mucus hypersecretion, bronchoalveolar lavage eosinophilia, alternative activation of macrophages, and serum IgE. These observations demonstrate that IL-33-dependent signaling contributes to the expansion of innate type 2 immunity and subsequent Th2-biased lung immunopathology that facilitates C. neoformans growth and dissemination

A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro

Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4+ TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4+ TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the host’s immune system (e.g., cancer)

The Fusion Protein rFlaA:Betv1 Modulates DC Responses by a p38-MAPK and COX2-Dependent Secretion of PGE2 from Epithelial Cells

Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins

Susceptibility to progressive Cryptococcus neoformans pulmonary infection is regulated by loci on mouse chromosomes 1 and 9.

Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation

Targeting of immune cells by dual tlr2/7 ligands suppresses features of allergic th2 immune responses in mice

Background. TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim. To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods. Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. Results. CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions. Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment

Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice

Background. TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim. To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods. Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. Results. CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions. Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment