Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation

ACKNOWLEDGMENTS We acknowledge Jennifer Lodge, Woei Lam, and Rajendra Upadhya for developing and sharing the chitin and chitosan MTBH assay. We thank Todd Brennan of Duke University for providing MyD88-deficient mice. We acknowledge Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We also acknowledge Connie Nichols for critical reading of the manuscript. These experiments were supported by an NIH grant to J.A.A. and F.L.W., Jr. (R01 AI074677). C.M.L.W. was supported by a fellowship provided through the Army Research Office of the Department of Defense (no. W911NF-11-1-0136 f) (F.L.W., Jr.). J.W., L.W., and C.M. were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the MRC, Centre for Medical Mycology (MR/N006364/1). FUNDING INFORMATION MRC Centre for Medical MycologyMR/N006364/1 Carol A. Munro HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060R01 AI074677J. Andrew Alspaugh Wellcome https://doi.org/10.13039/100010269097377 Carol A. Munro DOD | United States Army | RDECOM | Army Research Office (ARO) https://doi.org/10.13039/100000183W911NF-11-1-0136 f Chrissy M. Leopold WagerPeer reviewe

Macrophage nuclear receptors: Emerging key players in infectious diseases.

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a variety of cells, including macrophages. For decades, NRs have been therapeutic targets because their activity can be pharmacologically modulated by specific ligands and small molecule inhibitors. NRs regulate a variety of processes, including those intersecting metabolic and immune functions, and have been studied in regard to various autoimmune diseases. However, the complex roles of NRs in host response to infection are only recently being investigated. The NRs peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptors (LXRs) have been most studied in the context of infectious diseases; however, recent work has also linked xenobiotic pregnane X receptors (PXRs), vitamin D receptor (VDR), REV-ERBα, the nuclear receptor 4A (NR4A) family, farnesoid X receptors (FXRs), and estrogen-related receptors (ERRs) to macrophage responses to pathogens. Pharmacological inhibition or antagonism of certain NRs can greatly influence overall disease outcome, and NRs that are protective against some diseases can lead to susceptibility to others. Targeting NRs as a novel host-directed treatment approach to infectious diseases appears to be a viable option, considering that these transcription factors play a pivotal role in macrophage lipid metabolism, cholesterol efflux, inflammatory responses, apoptosis, and production of antimicrobial byproducts. In the current review, we discuss recent findings concerning the role of NRs in infectious diseases with an emphasis on PPARγ and LXR, the two most studied. We also highlight newer work on the activity of emerging NRs during infection

The activation status of the macrophage directly influences cryptococcal killing.

<p>In the presence of Th1-type cytokine IFN-γ, macrophages polarize to a classically activated (M1) phenotype. These macrophages produce reactive oxygen species (ROS) and NO, which contribute to their anticryptococcal activity. However, when the Th2-type cytokines IL-4 and/or IL-13 are more prevalent, macrophages polarize toward an alternatively activated (M2) phenotype. M2 macrophages do not have anticryptococcal activity and are permissive to intracellular proliferation of <i>C</i>. <i>neoformans</i>.</p

Cryptococcus and Phagocytes: Complex Interactions that Influence Disease Outcome

Cryptococcus neoformans and C. gattii are fungal pathogens that cause life-threatening disease. These fungi commonly enter their host via inhalation into the lungs where they encounter resident phagocytes, including macrophages and dendritic cells, whose response has a pronounced impact on the outcome of disease. Cryptococcus has complex interactions with the resident and infiltrating innate immune cells that, ideally, result in destruction of the yeast. These phagocytic cells have pattern recognition receptors that allow recognition of specific cryptococcal cell wall and capsule components. However, Cryptococcus possesses several virulence factors including a polysaccharide capsule, melanin production and secretion of various enzymes that aid in evasion of the immune system or enhance its ability to thrive within the phagocyte. This review focuses on the intricate interactions between the cryptococci and innate phagocytic cells including discussion of manipulation and evasion strategies used by Cryptococcus, anti-cryptococcal responses by the phagocytes and approaches for targeting phagocytes for the development of novel immunotherapeutics

Dectin-3 Is Not Required for Protection against Cryptococcus neoformans Infection.

C-type lectin receptors (CLRs) are diverse, trans-membrane proteins that function as pattern recognition receptors (PRRs) which are necessary for orchestrating immune responses against pathogens. CLRs have been shown to play a major role in recognition and protection against fungal pathogens. Dectin-3 (also known as MCL, Clecsf8, or Clec4d) is a myeloid cell-specific CLR that recognizes mycobacterial trehalose 6,6'-dimycolate (TDM) as well as α-mannans present in the cell wall of fungal pathogens. To date, a potential role for Dectin-3 in the mediation of protective immune responses against C. neoformans has yet to be determined. Consequently, we evaluated the impact of Dectin-3 deficiency on the development of protective immune responses against C. neoformans using an experimental murine model of pulmonary cryptococcosis. Dectin-3 deficiency did not lead to increased susceptibility of mice to experimental pulmonary C. neoformans infection. Also, no significant differences in pulmonary leukocyte recruitment and cytokine production were observed in Dectin-3 deficient mice compared to wild type infected mice. In addition, we observed no differences in uptake and anti-cryptococcal activity of Dectin-3 deficient dendritic cells and macrophages. Altogether, our studies show that Dectin-3 is dispensable for mediating protective immune responses against pulmonary C. neoformans infection

Dectin-3 deficiency does not impact pulmonary leukocyte recruitment during immune response to <i>C</i>. <i>neoformans</i> infection.

<p>C57BL/6 (WT) and Dectin-3 KO mice were infected via intranasal inoculation with <i>C</i>. <i>neoformans</i> strain H99. Lungs were excised at days 7 and 14 post-inoculation and pulmonary infiltrates analyzed by flow cytometry. Leukocytes were labeled with anti-CD45 antibodies for total leukocytes (A) or dual labeled with anti-CD45 and antibodies specific for cell type (B-L) and were analyzed by flow cytometry. Data shown are the mean ± of SEM absolute cell numbers from three independent experiments performed using 5 mice per group per time point per experiment. Significant differences were defined as <i>P</i> < 0.05 (*), <i>P</i> < 0.01 (**) <i>P</i> < 0.001 (***), <i>P</i> < 0.0001(****).</p

Increased phagolysosomal fusion during CREB inhibition requires RIPK3 activity.

A) MDMs were plated on glass coverslips and pretreated with for 60 min with DMSO or CREB inhibitor 666–15 +/- Nec-1, GSK’872, or NSA, then infected with mCherry M.tb H37Rv (red) MOI 10. MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 4–5 donors. B) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are cumulative ± SEM of n = 4–5 donors. One-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

Dectin-3 is not necessary for survival or control of pulmonary fungal burden following infection with <i>C</i>. <i>neoformans</i>.

<p>C57BL/6 (WT) and Dectin-3 KO mice were given an intranasal inoculation with <i>C</i>. <i>neoformans</i> strain H99 (serotype A). Mice were observed up to Day 41 for survival (A) and pulmonary fungal burden (B) was analyzed at days 7 and 14 post-inoculation. Additionally, C57BL/6 (WT) and Dectin-3 KO mice were given an intranasal inoculation with <i>Cryptococcus</i> strain 52D (serotype D). Mice were observed up to Day 80 for survival (C). Survival data shown are representative of one study using 15 mice per group. Fungal burden data shown are mean ± SEM from three independent experiments performed using 5 mice per group per time point.</p

CREB inhibition induces activation of the necroptotic signaling pathway in <i>M</i>.<i>tb</i>-infected macrophages, but does not affect cell viability.

MDMs were pretreated with 666–15 or DMSO control for 60 min and subsequently infected with M.tb H37Rv at MOI 2 (A,D) or MOI 10 (B,C). A) Bright field images of the cells were taken at 40x; Data shown are representative of n = 10 donors. B,C) Cell lysates were collected and probed by WB blot for the indicated phosphorylated and total proteins. Densitometry was determined and graphed as fold change ± SEM of phosphorylated protein to total protein; A representative experiment is shown and graphed data are cumulative of n = 4 donors. One-way ANOVA with Tukey’s post-test. D) Membrane integrity and cell viability was determined by Cytotox Glo assay. Data are cumulative ± SEM of n = 3 donors. Two-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001.</p

<i>M</i>.<i>tb</i> induces CREB activation independent of cAMP in human macrophages.

MDMs were infected with M.tb H37Rv by synchronized phagocytosis at the indicated MOI. A) Western blot was performed to detect levels of phosphorylated and total CREB protein at the indicated time points. WB is representative of n = 4 donors. B) Densitometry analysis was performed and ratios of pCREB/total CREB were determined. Data are cumulative ± SEM of n = 4 donors. One-way ANOVA with Tukey’s post-test. C) MDMs were stimulated with cAMP agonists PGE2 or forskolin ± PDE inhibitor IBMX for 30 min. Data are representative ± SD of n = 4 donors. One-way ANOVA with Tukey’s post-test. D) cAMP levels in M.tb-infected (MOI 5) MDM lysates were determined. Graph is representative ± SD of n = 11 donors. One-way ANOVA with Tukey’s post-test. E) MDMs were infected with different strains of mycobacteria (MOI 5) or forskolin + IBMX as a control for cAMP production. Lysates were collected and analyzed for cAMP production. Dotted line indicates minimum level of detection for the assay. Data are representative ± SD of n = 2 donors; *p < 0.05, ***p < 0.001, ****p < 0.0001.</p