Innate phase production of IFN-γ by memory and effector T cells expressing early activation marker CD69 during infection with Cryptococcus deneoformans in the lungs.

Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.

Deficiency of lung-specific claudin-18 leads to aggravated infection with Cryptococcus deneoformans through dysregulation of the microenvironment in lungs.

Cryptococcus deneoformans is an opportunistic fungal pathogen that infects the lungs via airborne transmission and frequently causes fatal meningoencephalitis. Claudins (Cldns), a family of proteins with 27 members found in mammals, form the tight junctions within epithelial cell sheets. Cldn-4 and 18 are highly expressed in airway tissues, yet the roles of these claudins in respiratory infections have not been clarified. In the present study, we analyzed the roles of Cldn-4 and lung-specific Cldn-18 (luCldn-18) in host defense against C. deneoformans infection. luCldn-18-deficient mice exhibited increased susceptibility to pulmonary infection, while Cldn-4-deficient mice had normal fungal clearance. In luCldn-18-deficient mice, production of cytokines including IFN-γ was significantly decreased compared to wild-type mice, although infiltration of inflammatory cells including CD4+ T cells into the alveolar space was significantly increased. In addition, luCldn-18 deficiency led to high K+ ion concentrations in bronchoalveolar lavage fluids and also to alveolus acidification. The fungal replication was significantly enhanced both in acidic culture conditions and in the alveolar spaces of luCldn-18-deficient mice, compared with physiological pH conditions and those of wild-type mice, respectively. These results suggest that luCldn-18 may affect the clinical course of cryptococcal infection indirectly through dysregulation of the alveolar space microenvironment.

Role of Dectin-2 in the Phagocytosis of Cryptococcus neoformans by Dendritic Cells.

The cell walls and capsules of Cryptococcus neoformans, a yeast-type fungal pathogen, are rich in polysaccharides. Dectin-2 is a C-type lectin receptor (CLR) that recognizes high-mannose polysaccharides. Previously, we demonstrated that Dectin-2 is involved in cytokine production by bone marrow-derived dendritic cells (BM-DCs) in response to stimulation with C. neoformans. In the present study, we analyzed the role of Dectin-2 in the phagocytosis of C. neoformans by BM-DCs. The engulfment of this fungus by BM-DCs was significantly decreased in mice lacking Dectin-2 (Dectin-2 knockout [Dectin-2KO]) or caspase recruitment domain-containing protein 9 (CARD9KO), a common adapter molecule that delivers signals triggered by CLRs, compared to wild-type (WT) mice. Phagocytosis was likewise inhibited, to a similar degree, by the inhibition of Syk, a signaling molecule involved in CLR-triggered activation. A PI3K inhibitor, in contrast, completely abrogated the phagocytosis of C. neoformans. Actin polymerization, i.e., conformational changes in cytoskeletons detected at sites of contact with C. neoformans, was also decreased in BM-DCs of Dectin-2KO and CARD9KO mice. Finally, the engulfment of C. neoformans by macrophages was significantly decreased in the lungs of Dectin-2KO mice compared to WT mice. These results suggest that Dectin-2 may play an important role in the actin polymerization and phagocytosis of C. neoformans by DCs, possibly through signaling via CARD9 and a signaling pathway mediated by Syk and PI3K.

Dectin-2-mediated initiation of immune responses caused by influenza virus hemagglutinin.

Antigen-presenting cells express pattern recognition receptors (PRRs), which sense pathogen-associated molecular patterns from microorganisms and lead to the induction of inflammatory responses. C-type lectin receptors (CLRs), the representative PRRs, bind to microbial polysaccharides, among which Dectin-2 and Mincle recognize mannose-containing polysaccharides. Because influenza virus (IFV) hemagglutinin (HA) is rich in mannose polysaccharides, Dectin-2 or Mincle may contribute to the recognition of HA. In this study, we addressed the possible involvement of Dectin-2 and Mincle in the viral recognition and the initiation of cytokine production. Interleukin (IL)-12p40 and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) upon stimulation with HA was significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice whereas there was no difference between WT mice and Mincle KO mice. BM-DCs that were treated with Syk inhibitor resulted in a significant reduction of cytokine production upon stimulation with HA. The treatment of BM-DCs with methyl-α-D-mannopyranoside (ManP) also led to a significant reduction in cytokine production by BM-DCs that were stimulated with HA, except for the A/H1N1pdm09 subtype. IL-12p40 and IL-6 synthesis by BM-DCs was completely diminished upon stimulation with HA treated with concanavalin A (ConA)-bound sepharose beads. Finally, GFP expression was detected in reporter cells that were transfected with the Dectin-2 gene, but not with the Mincle gene, when stimulated with HA derived from the A/H3N2 subtype. These data suggested that Dectin-2 may be a key molecule as the sensor for IFV to initiate the immune response and regulate the pathogenesis of IFV infection.

Limited Role of Mincle in the Host Defense against Infection with Cryptococcus deneoformans.

Cryptococcus deneoformans is an opportunistic fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired cell-mediated immune responses such as AIDS. Caspase-associated recruitment domain 9 (CARD9) plays a critical role in the host defense against cryptococcal infection, suggesting the involvement of one or more C-type lectin receptors (CLRs). In the present study, we analyzed the role of macrophage-inducible C-type lectin (Mincle), one of the CLRs, in the host defense against C. deneoformans infection. Mincle expression in the lungs of wild-type (WT) mice was increased in the early stage of cryptococcal infection in a CARD9-dependent manner. In Mincle gene-disrupted (Mincle KO) mice, the clearance of this fungus, pathological findings, Th1/Th2 response, and antimicrobial peptide production in the infected lungs were nearly comparable to those in WT mice. However, the production of interleukin-22 (IL-22), tumor necrosis factor alpha (TNF-α), and IL-6 and the expression of AhR were significantly decreased in the lungs of Mincle KO mice compared to those of WT mice. In in vitro experiments, TNF-α production by bone marrow-derived dendritic cells was significantly decreased in Mincle KO mice. In addition, the disrupted lysates of C. deneoformans, but not those of whole yeast cells, activated Mincle-triggered signaling in an assay with a nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Mincle may be involved in the production of Th22-related cytokines at the early stage of cryptococcal infection, although its role may be limited in the host defense against infection with C. deneoformans.