IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential 'inflammatory' type 2 innate lymphoid cells.

Innate lymphoid cells (ILCs) are lymphocyte-like cells that lack T cell or B cell antigen receptors and mediate protective and repair functions through cytokine secretion. Among these, type 2 ILCs (ILC2 cells) are able to produce type 2 cytokines. We report the existence of an inflammatory ILC2 (iILC2) population responsive to interleukin 25 (IL-25) that complemented IL-33-responsive natural ILC2 (nILC2) cells. iILC2 cells developed into nILC2-like cells in vitro and in vivo and contributed to the expulsion of Nippostrongylus brasiliensis. They also acquired IL-17-producing ability and provided partial protection against Candida albicans. We propose that iILC2 cells are transient progenitors of ILCs mobilized by inflammation and infection that develop into nILC2-like cells or ILC3-like cells and contribute to immunity to both helminths and fungi.

The Pat1-Lsm Complex Stabilizes ATG mRNA during Nitrogen Starvation-Induced Autophagy.

Macroautophagy/autophagy is a key catabolic recycling pathway that requires fine-tuned regulation to prevent pathologies and preserve homeostasis. Here, we report a new post-transcriptional pathway regulating autophagy involving the Pat1-Lsm (Lsm1 to Lsm7) mRNA-binding complex. Under nitrogen-starvation conditions, Pat1-Lsm binds a specific subset of autophagy-related (ATG) transcripts and prevents their 3' to 5' degradation by the exosome complex, leading to ATG mRNA stabilization and accumulation. This process is regulated through Pat1 dephosphorylation, is necessary for the efficient expression of specific Atg proteins, and is required for robust autophagy induction during nitrogen starvation. To the best of our knowledge, this work presents the first example of ATG transcript regulation via 3' binding factors and exosomal degradation.

Antigen titers in cryptococcal meningitis: what determines how fast they fall?

Follow-up of previously healthy patients surviving cryptococcal meningitis found that cryptococcal antigen could be detected for more than one year in serum from 38 of 44 (86%) patients and in CSF from 20 of 31 patients (67%), far beyond the time of culture conversion. The speed of titer decline, measured as the number of days for a two fold drop in titer to occur, was slower in serum than in CSF. Speed of decline of antigen titers was much slower in serum and CSF for patients infected with C. gattii than C. neoformans. The speed of decline in CSF and serum titers was also much slower in patients who had received a ventriculoperitoneal shunt for increased intracranial pressure. The variable and extraordinarily slow rate of clearance in our patients did not appear to reflect differences in disease control but rather differences in species and shunting for increased intracranial pressure.

Farnesol remodels the peritoneal cavity immune environment influencing Candida albicans pathogenesis during intra-abdominal infection.

Candida albicans is a lifelong member of the mycobiome causing mucosal candidiasis and life-threatening, systemic, and intra-abdominal disease in immunocompromised and transplant patients. Despite the clinical importance of intra-abdominal candidiasis with mortality rates between 40% and 70%, the contribution of fungal virulence factors and host immune responses to disease has not been extensively studied. Secretion of the quorum-sensing molecule, farnesol, acts as a virulence factor for C. albicans during systemic infection, while inducing local, protective innate immune responses in oral models of infection. Previously, we reported that farnesol recruits macrophages to the peritoneal cavity in mice, suggesting a role for farnesol in innate immune responses. Here, we expand on our initial findings, showing that farnesol profoundly alters the peritoneal cavity microenvironment promoting innate inflammation. Intra-peritoneal injection of farnesol stimulates rapid local death of resident peritoneal cells followed by recruitment of neutrophils and inflammatory macrophages into the peritoneal cavity and peritoneal mesothelium associated with an early increase in chemokines followed by proinflammatory cytokines. These rapid inflammatory responses to farnesol significantly increase morbidity and mortality of mice with intra-abdominal candidiasis associated with increased formation of peritoneal adhesions, despite similar rates of fungal clearance from the peritoneal cavity and retro-peritoneal organs. C. albicans ddp3Δ/ddp3Δ knockout and reconstituted strains recapitulate these findings. This indicates that farnesol may be detrimental to the host during intra-abdominal infections. Importantly, our results highlight a need to understand how C. albicans virulence factors modulate the host immune response within the peritoneum, an exceedingly common site of Candida infection.

Oral Lipid Nanocrystal Amphotericin B for Cryptococcal Meningitis: A Randomized Clinical Trial.

BACKGROUND: Amphotericin B is the gold standard treatment for severe mycoses. A new orally delivered, less-toxic formulation of amphotericin has been developed. METHODS: In our randomized clinical trial, we tested oral lipid nanocrystal (LNC) amphotericin B (MAT2203, Matinas Biopharma) vs intravenous (IV) amphotericin for human immunodeficiency virus-associated cryptococcal meningitis in 4 sequential cohorts. Two pilot cohorts assessed safety and tolerability (n = 10 each), and 2 cohorts assessed efficacy with/without 2 IV loading doses (n = 40 each). The experimental arm received 1.8 g/d oral LNC amphotericin through 2 weeks with 100 mg/kg/d flucytosine, then 1.2 g/d LNC amphotericin through 6 weeks. The randomized control arm (n = 41) received 7 days of IV amphotericin with flucytosine, then 7 days of fluconazole 1200 mg/d. The primary end point was cerebrospinal fluid (CSF) early fungicidal activity (EFA). RESULTS: We randomized 80 participants to oral LNC amphotericin + flucytosine with (n = 40) and without (n = 40) 2 IV loading doses and 41 control participants to IV amphotericin + flucytosine. Mean EFA was 0.40 log10 colony-forming units (CFU)/mL/d for all-oral LNC amphotericin, 0.42 log10  Cryptococcus CFU/mL/d for oral LNC amphotericin with IV loading doses, and 0.46 log10 CFU/mL/d for IV amphotericin controls. LNC amphotericin groups achieved 2-week CSF sterility in 63% (44 of 70) vs 68% (23 of 34) of controls. The 18-week survival was 85% (34 of 40) with all-oral LNC amphotericin, 90% (36 of 40) with oral LNC amphotericin given IV loading doses, and 85% (35 of 41) with IV amphotericin.Grade 3-4 laboratory adverse events occurred less frequently in LNC amphotericin groups (41%) than the IV amphotericin group (61%, P = .05), particularly for anemia (21% vs 44%; P = .01) and potassium (5% vs 17%; P = .04). CONCLUSIONS: This new oral amphotericin B LNC formulation appears promising for cryptococcal meningitis with antifungal activity, similar survival, and less toxicity than IV amphotericin. CLINICAL TRIALS REGISTRATION: NCT04031833.

Tocilizumab as a Potential Adjunctive Therapy to Corticosteroids in Cryptococcal Post-infectious Inflammatory Response Syndrome (PIIRS): a Report of Two Cases.

PURPOSE: Non-HIV cryptococcal meningoencephalitis (CM) in previously healthy individuals is often complicated by a post-infectious inflammatory response syndrome (c-PIIRS) characterized by neurologic deterioration after appropriate antifungal therapy with sterilization of CSF fungal cultures. c-PIIRS results from an excessive inflammatory response to fungal antigens released during fungal lysis, mediated by IFN-γ, IL-6, and activated T-helper cells, leading to immune-mediated host damage that responds to pulse-corticosteroid taper therapy (PCT). Typically, oral steroids may take up to a year to taper, and occasionally, patients will be refractory to steroid therapy or may demonstrate high-risk lesions such as those involving intracranial arteries. Also, patients can have problematic side effects from prolonged corticosteroids. Hence, appropriate adjunctive agents are needed to reduce corticosteroid doses in the treatment of c-PIIRS. Due to a possible role of IL-6 in pathogenesis, IL-6 receptor blockade by tocilizumab may be useful in the treatment of c-PIIRS. METHODS: Two previously healthy patients with non-HIV cPIIRS were seen at the NIH. Due to concerns for intracranial vascular rupture in an area of inflammation (Patient 1) and intractable symptoms on high-dose oral corticosteroids (Patient 2) with evidence of persistent CSF inflammation, patients were treated with 4-8 mg/kg tocilizumab every 2 weeks while maintained on a constant dose of prednisone. RESULTS: Two patients exhibited rapid immunological improvement following treatment with tocilizumab. Patient 1 remained vascularly stable, and Patient 2 had near resolution of headaches with improvement in mental status as evidenced by improved MOCA score. The two had improved CSF inflammatory parameters and no significant side effects. Both CSF cultures remained negative throughout treatment. CONCLUSIONS: Tocilizumab may be a safe adjunctive treatment for CM-related PIIRS suggesting further study.

Persistent neurological symptoms and elevated intracranial pressures in a previously healthy host with cryptococcal meningitis.

Cryptococcal meningoencephalitis can occur in both previously healthy and immunocompromised hosts. Here, we describe a 55 year-old HIV-negative male with no known prior medical problems, who presented with three months of worsening headaches, confusion, and memory changes without fever. Magnetic resonance imaging of the brain demonstrated bilateral enlargement/enhancement of the choroid plexi, with hydrocephalus, temporal and occipital horn entrapments, as well as marked periventricular transependymal cerebrospinal fluid (CSF) seepage. CSF analysis yielded a lymphocytic pleocytosis and cryptococcal antigen titer of 1:160 but sterile fungal cultures. Despite standard antifungal therapy and CSF drainage, the patient had worsening confusion and persistently elevated intracranial pressures. External ventricular drainage led to improved mental status but only with valve settings at negative values. Ventriculoperitoneal shunt placement could thus not be considered due to a requirement for drainage into the positive pressure venous system. Due to this persistent CSF inflammation and cerebral circulation obstruction, the patient required transfer to the National Institute of Health. He was treated for cryptococcal post-infectious inflammatory response syndrome with pulse-taper corticosteroid therapy, with resultant reductions in CSF pressures along with decreased protein and obstructive material, allowing successful shunt placement. After tapering of corticosteroids, the patient recovered without sequelae. This case highlights (1) the necessity to consider cryptococcal meningitis as a rare cause of neurological deterioration in the absence of fever even in apparently immunocompetent individuals and (2) the potential for obstructive phenomena from inflammatory sequelae and the prompt response to corticosteroid therapy.

Ocular Findings of Cryptococcal Meningitis in Previously Healthy Adults.

BACKGROUND: Patients with cryptococcal meningitis (CM) often have ocular manifestations; although data are describing these findings in nonimmunosuppressed, previously healthy individuals are scarce. METHODS: A retrospective chart review was performed for previously healthy patients with CM who underwent a complete ophthalmological examination within a 5-year period at the National Institutes of Health. Demographics, CSF parameters, findings on initial ophthalmological examination, and MRI abnormalities were analyzed. RESULTS: Forty-four patients within a median of 12 weeks after CM diagnosis were included in our study; 27 patients (61%) reported abnormal vision on presentation. Seventy-one percent of patients were not shunted at the time of their initial eye examination. The most common ocular abnormalities were visual field defects in 21 (66%), decreased visual acuity in 14 (38%), and papilledema in 8 (26%) patients. Intraocular pressure was within normal range in all patients. Cranial nerve defects were identified in 5 patients and optic neuropathy in 2 patients. Patients who had hydrocephalus or did not receive a ventriculoperitoneal shunt were not noted to have worse ocular abnormalities. CONCLUSIONS: The most common ocular findings in our cohort of nontransplant, non-HIV cryptococcal meningitis patients were visual field defects, decreased visual acuity, and papilledema. Our results emphasize the need for a comprehensive eye examination in patients with CM who may not always report a change in vision on presentation.

Neutralizing GM-CSF autoantibodies in pulmonary alveolar proteinosis, cryptococcal meningitis and severe nocardiosis.

BACKGROUND: Anti GM-CSF autoantibodies (aAb) have been related to acquired pulmonary alveolar proteinosis (PAP) and described in cases of severe infections such as cryptococcosis and nocardiosis in previously healthy subjects. Whether there are different anti-GM-CSF autoantibodies corresponding to these phenotypes is unclear. Therefore, we examined anti-GM-CSF autoantibodies to determine whether amount or neutralizing activity could distinguish between groups. METHODS: Plasma samples gathered in the National Institute of Health from patients with anti GM-CSF aAb and either PAP (n = 15), cryptococcal meningitis (n = 15), severe nocardiosis (n = 5) or overlapping phenotypes (n = 6) were compared. The relative amount of aAb was assessed using a particle-based approach, reported as a mouse monoclonal anti-human GM-CSF as standard curve and expressed in an arbitrary Mouse Monoclonal Antibody Unit (MMAU). The neutralizing activity of the plasma was assessed by inhibition of GM-CSF-induced intracellular phospho-STAT5 (pSTAT5) in monocytes. RESULTS: Anti-GM-CSF aAb relative amounts were higher in PAP patients compared to those with cryptococcosis (mean 495 ± 464 MMAU vs 197 ± 159 MMAU, p = 0.02); there was no difference with patients with nocardiosis (430 ± 493 MMAU) nor between the two types of infections. The dilution of plasma resulting in 50% inhibition of GM-CSF-induced pSTAT5 (approximate IC50) did not vary appreciably across groups of patients (1.6 ± 3.1%, 3.9 ± 6% and 1.8 ± 2.2% in PAP patients, cryptococcosis and nocardiosis patients, respectively). Nor was the concentration of GM-CSF necessary to induce 50% of maximal GM-CSF-induced pSTAT5 in the presence of 10 MMAU of anti-GM-CSF aAb (EC50). When studying longitudinal samples from patients with PAP or disseminated nocardiosis, the neutralizing effect of anti-GM-CSF aAb was relatively constant over time despite targeted treatments and variations in aAb levels. CONCLUSIONS: Despite different clinical manifestations, anti-GM-CSF antibodies were similar across PAP, cryptococcosis and nocardiosis. Underlying host genetics and functional analyses may help further differentiate the biology of these conditions.

Identification of Antifungal Compounds against Multidrug-Resistant Candida auris Utilizing a High-Throughput Drug-Repurposing Screen.

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

Central Nervous System Histoplasma-Associated Post-infectious Inflammatory Response Syndrome (Histo-PIIRS).

We present a case of central nervous system (CNS) histoplasmosis in a previously healthy adult with hepatitis C (HCV) presenting with neurological symptoms refractory to antifungal therapy and ventriculoperitoneal (VP) shunting 4 months after initial diagnosis. Persistent symptoms were thought to be inflammatory rather than infectious given negative cerebrospinal fluid (CSF) and serum fungal antigens. The patient promptly improved after initiation of corticosteroid therapy. Elevated CSF cytokines and regional enhancement on brain MRI resolved with corticosteroid treatment. This is the first case of Histoplasma-associated post-infectious inflammatory response syndrome (Histo-PIIRS) documented by CSF cytokine reduction in response to corticosteroid therapy.

Prostaglandin E2 suppresses antifungal immunity by inhibiting interferon regulatory factor 4 function and interleukin-17 expression in T cells.

T helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4(+) T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.

Role of the ESCRT Pathway in Laccase Trafficking and Virulence of Cryptococcus neoformans.

The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect is not due to defects in actin-related localization. Extracellular vesicle maturation was also defective in the vps27Δ mutant, which had a larger vesicle size as measured by dynamic light scattering. Our data identify cryptococcal VPS27 as a required gene for laccase trafficking and attenuates virulence of C. neoformans in a mouse intravenous (i.v.) meningitis model.

A Multicenter, Longitudinal Cohort Study of Cryptococcosis in Human Immunodeficiency Virus-negative People in the United States.

BACKGROUND: Cryptococcosis is increasingly recognized in people without human immunodeficiency virus (HIV). METHODS: A multicenter, prospective cohort study was performed in 25 US centers. Consenting patients were prospectively followed for ≤2 years. Neurological morbidities were assessed with longitudinal event depiction and functional scores (Montreal Cognitive Assessment [MoCA]). Risks of death were analyzed using Cox regression. RESULTS: One hundred forty-five subjects were enrolled. Most were male (95; 65.5%) and had immunosuppression (120; 82.8%), including solid organ transplant (SOT; 33.8%), autoimmunity (15.9%), and hematologic malignancies (11.7%). Disease involved the central nervous system (CNS) in 71 subjects (49%). Fever was uncommon, documented in 40 (27.8%) subjects, and absence was associated with diagnostic delay (mean: 48.2 vs 16.5 days; P = .007). Abnormal MoCA scores (<26) were predictive of CNS disease; low scores (<22) were associated with poor long-term cognition. Longitudinal event depiction demonstrated frequent complications in people with CNS disease; 25 subjects (35.2%) required >1 lumbar puncture and 8 (11.3%) required ventriculostomies. In multivariable models, older age (>60 years) was associated with higher risks of death (hazard ratio [HR], 2.14; 95% confidence interval [CI], 1.05-4.38; P = .036), and lower risks were noted with underlying hematologic malignancy (HR, 0.29; 95% CI, 0.09-0.98; P = .05) and prior SOT (HR, 0.153; 95% CI, 0.05-0.44; P = .001). CONCLUSIONS: Despite aggressive antifungal therapies, outcomes of CNS cryptococcosis in people without HIV are characterized by substantial long-term neurological sequelae. Studies are needed to understand mechanism(s) of cognitive decline and to enable better treatment algorithms.

Cryptococcal meningitis is a cause for cross-reactivity in cerebrospinal fluid assays for anti-Histoplasma, anti-Coccidioides and anti-Blastomyces antibodies.

BACKGROUND/OBJECTIVES: Antibody detection is commonly used for diagnosis of histoplasmosis, and cross-reactions have been recognised due to endemic mycoses but not cryptococcosis. We observed cross-reactions in an anti-Histoplasma antibody enzyme immunoassay (EIA) in the cerebrospinal fluid (CSF) from a patient with cryptococcal meningitis and sought to assess the risk of cross-reactive anti-Histoplasma antibodies in persons with cryptococcal meningitis. METHODS: An anti-cryptococcal antibody EIA was developed to measure CSF antibody response in HIV-infected subjects from Kampala, Uganda and previously healthy, HIV-negative subjects at the National Institutes of Health (NIH) with cryptococcal meningitis. Specimens were tested for cross-reactivity in assays for IgG anti-Histoplasma, anti-Blastomyces and anti-Coccidioides antibodies. RESULTS: Among 61 subjects with cryptococcal meningitis (44 Kampala cohort, 17 NIH cohort), elevated CSF anti-cryptococcal antibody levels existed in 38% (23/61). Of the 23 CSF specimens containing elevated anti-cryptococcal antibodies, falsely positive results were detected in antibody EIAs for histoplasmosis (8/23, 35%), coccidioidomycosis (6/23, 26%) and blastomycosis (1/23, 4%). Overall, 2% (2/81) of control CSF specimens had elevated anti-cryptococcal antibody detected, both from Indiana. CONCLUSIONS: Cryptococcal meningitis may cause false-positive results in the CSF for antibodies against Histoplasma, Blastomyces and Coccidioides. Fungal antigen testing should be performed to aid in differentiating true- and false-positive antibody results in the CSF.

Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Spinal Arachnoiditis as a Complication of Cryptococcal Meningoencephalitis in Non-HIV Previously Healthy Adults.

BACKGROUND: Cryptococcus can cause meningoencephalitis (CM) among previously healthy non-HIV adults. Spinal arachnoiditis is under-recognized, since diagnosis is difficult with concomitant central nervous system (CNS) pathology. METHODS: We describe 6 cases of spinal arachnoiditis among 26 consecutively recruited CM patients with normal CD4 counts who achieved microbiologic control. We performed detailed neurological exams, cerebrospinal fluid (CSF) immunophenotyping and biomarker analysis before and after adjunctive immunomodulatory intervention with high dose pulse corticosteroids, affording causal inference into pathophysiology. RESULTS: All 6 exhibited severe lower motor neuron involvement in addition to cognitive changes and gait disturbances from meningoencephalitis. Spinal involvement was associated with asymmetric weakness and urinary retention. Diagnostic specificity was improved by MRI imaging which demonstrated lumbar spinal nerve root enhancement and clumping or lesions. Despite negative fungal cultures, CSF inflammatory biomarkers, sCD27 and sCD21, as well as the neuronal damage biomarker, neurofilament light chain (NFL), were elevated compared to healthy donor (HD) controls. Elevations in these biomarkers were associated with clinical symptoms and showed improvement with adjunctive high dose pulse corticosteroids. CONCLUSIONS: These data suggest that a post-infectious spinal arachnoiditis is an important complication of CM in previously healthy individuals, requiring heightened clinician awareness. Despite microbiological control, this syndrome causes significant pathology likely due to increased inflammation and may be amenable to suppressive therapeutics.

Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis.

The fungus Cryptococcus is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.