Inhibition of Microbicidal Activity of Canine Macrophages DH82 Cell Line by Capsular Polysaccharides from Cryptococcus neoformans.

Cryptococcus neoformans is a lethal fungus that primarily affects the respiratory system and the central nervous system. One of the main virulence factors is the capsule, constituted by the polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomanogalactan (GXMGal). Polysaccharides are immunomodulators. One of the target cell populations for modulation are macrophages, which are part of the first line of defense and important for innate and adaptive immunity. It has been reported that macrophages can be modulated to act as a "Trojan horse," taking phagocytosed yeasts to strategic sites or having their machinery activation compromised. The scarcity of information on canine cryptococcosis led us to assess whether the purified capsular polysaccharides from C. neoformans would be able to modulate the microbicidal action of macrophages. In the present study, we observed that the capsular polysaccharides, GXM, GXMGal, or capsule total did not induce apoptosis in the DH82 macrophage cell line. However, it was possible to demonstrate that the phagocytic activity was decreased after treatment with polysaccharides. In addition, recovered yeasts from macrophages treated with polysaccharides after phagocytosis could be cultured, showing that their viability was not altered. The polysaccharides led to a reduction in ROS production and the mRNA expression of IL-12 and IL-6. We observed that GXMGal inhibits MHC class II expression and GXM reduces ERK phosphorylation. In contrast, GXMGal and GXM were able to increase the PPAR-γ expression. Furthermore, our data suggest that capsular polysaccharides can reduce the microbicidal activity of canine macrophages DH82.

A Comparative Analysis of Innate Immune Responses and the Structural Characterization of Spike from SARS-CoV-2 Gamma Variants and Subvariants.

The SARS-CoV-2 P.1 variant, responsible for an outbreak in Manaus, Brazil, is distinguished by 12 amino acid differences in the S protein, potentially increasing its ACE-2 affinity and immune evasion capability. We investigated the innate immune response of this variant compared to the original B.1 strain, particularly concerning cytokine production. Blood samples from three severe COVID-19 patients were analyzed post-infection with both strains. Results showed no significant difference in cytokine production of mononuclear cells and neutrophils for either variant. While B.1 had higher cytopathogenicity, neither showed viral replication in mononuclear cells. Structural analyses of the S protein highlighted physicochemical variations, which might be linked to the differences in infectivity between the strains. Our studies point to the increased infectivity of P.1 could stem from altered immunogenicity and receptor-binding affinity.

Pulmonary Fibrosis and Hypereosinophilia in TLR9-/- Mice Infected by Cryptococcus gattii.

Cryptococcus gattii is a worldwide-distributed basidiomycetous yeast that can infect immunocompetent hosts. However, little is known about the mechanisms involved in the disease. The innate immune response is essential to the control of infections by microorganisms. Toll-like receptor 9 (TLR9) is an innate immune receptor, classically described as a non-methylated DNA recognizer and associated with bacteria, protozoa and opportunistic mycosis infection models. Previously, our group showed that TLR9-/- mice were more susceptible to C. gattii after 21 days of infection. However, some questions about the innate immunity involving TLR9 response against C. gattii remain unknown. In order to investigate the systemic cryptococcal infection, we evaluated C57BL/6 mice and C57BL/6 TLR9-/- after intratracheal infection with 104C. gattii yeasts for 21 days. Our data evidenced that TLR9-/- was more susceptible to C. gattii. TLR9-/- mice had hypereosinophilia in pulmonary mixed cellular infiltrate, severe bronchiolitis and vasculitis and type 2 alveolar cell hyperplasia. In addition, TLR9-/- mice developed severe pulmonary fibrosis and areas with strongly birefringent fibers. Together, our results corroborate the hypothesis that TLR9 is important to support the Th1/Th17 response against C. gattii infection in the murine experimental model.

X-linked immunodeficient (XID) mice exhibit high susceptibility to Cryptococcus gattii infection.

Cryptococcosis is an opportunistic disease caused by the fungus Cryptococcus neoformans and Cryptococcus gattii. It starts as a pulmonary infection that can spread to other organs, such as the brain, leading to the most serious occurrence of the disease, meningoencephalitis. The humoral response has already been described in limiting the progression of cryptococcosis where the B-1 cell seems to be responsible for producing natural IgM antibodies, crucial for combating fungal infections. The role of the B-1 cell in C. neoformans infection has been initially described, however the role of the humoral response of B-1 cells has not yet been evaluated during C. gattii infections. In the present study we tried to unravel this issue using XID mice, a murine model deficient in the Btk protein which compromises the development of B-1 lymphocytes. We use the XID mice compared to BALB/c mice that are sufficient for the B-1 population during C. gattii infection. Our model of chronic lung infection revealed that XID mice, unlike the sufficient group of B-1, had early mortality with significant weight loss, in addition to reduced levels of IgM and IgG specific to GXM isolated from the capsule of C. neoformans. In addition to this, we observed an increased fungal load in the blood and in the brain. We described an increase in the capsular size of C. gattii and the predominant presence of cytokines with a Th2 profile was also observed in these animals. Thus, the present study strongly points to a higher susceptibility of the XID mouse to C. gattii, which suggests that the presence of B-1 cells and anti-GXM antibodies is fundamental during the control of infection by C. gattii.

The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection.

Toll-like receptor 9 (TLR9) is crucial to the host immune response against fungi, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, but its importance in Cryptococcus gattii infection is unknown. Our study aimed to understand the role of TLR9 during the course of experimental C. gattii infection in vivo, considering that the cryptococcal DNA interaction with the receptor could contribute to host immunity even in an extremely susceptible model. We inoculated C57BL/6 (WT) and TLR9 knock-out (TLR9-/-) mice intratracheally with 104 C. gattii yeast cells. TLR9-/- mice had a higher mortality rate compared to WT mice and more yeast cells that had abnormal size, known as titan cells, in the lungs. TLR9-/- mice also had a greater number of CFUs in the spleen and brain than WT mice, in addition to having lower levels of IFN-γ and IL-17 in the lung. With these markers of aggressive cryptococcosis, we can state that TLR9-/- mice are more susceptible to C. gattii, probably due to a mechanism associated with the decrease of a Th1 and Th17-type immune response that promotes the formation of titan cells in the lungs. Therefore, our results indicate the participation of TLR9 in murine resistance to C. gattii infection.