Landmark clinical observations and immunopathogenesis pathways linked to HIV and Cryptococcus fatal central nervous system co‐infection

Summary Cryptococcal meningitis remains one of the leading causes of death among HIV‐infected adults in the fourth decade of HIV era in sub‐Saharan Africa, contributing to 10%–20% of global HIV‐related deaths. Despite widespread use and early induction of ART among HIV‐infected adults, incidence of cryptococcosis remains significant in those with advanced HIV disease. Cryptococcus species that causes fatal infection follows systemic spread from initial environmental acquired infection in lungs to antigenaemia and fungaemia in circulation prior to establishment of often fatal disease, cryptococcal meningitis in the CNS. Cryptococcus person‐to‐person transmission is uncommon, and deaths related to blood infection without CNS involvement are rare. Keen to the persistent high mortality associated with HIV‐cryptococcal meningitis, seizures are common among a third of the patients, altered mental status is frequent, anaemia is prevalent with ensuing brain hypoxia and at autopsy, brain fibrosis and infarction are evident. In addition, fungal burden is 3‐to‐4‐fold higher in those with seizures. And high immune activation together with exacerbated inflammation and elevated PD‐1/PD‐L immune checkpoint expression is immunomodulated phenotypes elevated in CSF relative to blood. Lastly, though multiple Cryptococcus species cause disease in this setting, observations are mostly generalised to cryptococcal infection/meningitis or regional dominant species (C neoformans or gattii complex) that may limit our understanding of interspecies differences in infection, progression, treatment or recovery outcome. Together, these factors and underlying mechanisms are hypotheses generating for research to find targets to prevent infection or adequate therapy to prevent persistent high mortality with current optimal therapy.


| INTRODUC TI ON
The central nervous system (CNS) (brain, cerebrospinal fluid (CSF) and spinal cord) provides a formidable niche for disseminated fatal cryptococcal meningitis. 1,2 Cryptococcal meningitis is an acute fungal disease caused by an encapsulated yeast of the genus Cryptococcus. 3 Cryptococcus emerged from the environment to cause disease in man and the species that cause fatal infection have high preference to infect the CNS to cause meningoencephalitis in individuals with ensuing immunosuppression. 2,4-6 Several species of Cryptococcus exist with Cryptococcus neoformans complex and Cryptococcus gattii complex leading infection in those with an underlying immunosuppression. 3,5,[7][8][9][10][11][12][13] The C neoformans species complex comprise of C neoformans sensu stricto that causes 60%-90% of HIV-associated cryptococcal meningitis together with C deneoformans and hybrids between both species. [8][9][10][11]14,15 Prior to individual species characterisation, C gattii complex was known to cause disease mostly among individuals without HIV infection. 6,8 But to date increasing reports of different species of C gattii complex are being documented to cause disease among HIV immunocompromised patients globally. 8 The C. gattii species complex includes C gattii sensu stricto (AFLP4/VGI), C deuterogatii (AFLP6/VGII), C bacillisporus (AFLP5/VGIII), C tetragattii (AFLP7/ VGIV) and C decagattii (AFLP10/VGIII and VGIV). 7,10,[16][17][18] Thus, the limitations of existing diagnostic tools in common clinical use that are unable to identify species-related infection may limit our understanding of information related to species-specific infection, pathogenesis and disease outcome. The species-related information may be relevant in designing treatment strategies amidst high residual cryptococcal meningitis-related deaths with optimal use of antifungal drugs to treat those co-infected with HIV.
Among HIV-infected adults (>18 years of age), cryptococcal meningitis is diagnosed in CSF 7-28 days from onset of symptoms. [19][20][21][22][23][24] However, early symptoms including fever and headache may complicate early diagnosis and delay antifungal treatment in regions with other endemic pathogens that present with similar symptoms. 20,21,25,26 Cryptococcosis results in 20%-40% HIVrelated deaths worldwide. 23,27 Among HIV-infected adults, CD4 T-cell count < 100 cell/µL is one of the risk factors for cryptococcosis. 20,23,28 However, despite attempts to restore and maintain immune response with early antiretroviral therapy (ART) among HIV-infected individuals with higher CD4 T cells, some persons still present with high incidence of HIV-associated cryptococcosis. 23,[29][30][31][32] Surprisingly, among regions with a high incidence of HIV-associated cryptococcosis, one would relate incidence of infection with frequent yeast exposure from the environment. But rare infection among healthy individuals in this regions alters this speculation. 33 Hence, the challenges in the pathogenesis of human Cryptococcus infection have led to the incidence of cryptococcosis remaining significant over the last three to four decades of the HIV/AIDS epidemic especially in sub-Saharan Africa. 21,23,30,34,35 Indeed, persistent high mortality from HIV-associated cryptococcosis occurs with the use of optimal antifungal drugs and HAART for the treatment of those co-infected. [20][21][22][23] It remains unclear what constitutes a translational mechanism to attenuate this high mortality among those with HIVassociated cryptococcosis. Of note, in large cryptococcosis cohorts, factors associated with treatment failure have been inconsistently reported. [20][21][22][23][24]36,37 Hence, this review aims to point the field to the cryptococcosis immunopathogenesis factors and pathways to be targeted in further investigations to prevent infection or to alter persistent poor treatment outcome.

| PATHOG ENE S IS OF H UMAN CRYP TO CO CCOS IS INFEC TI ON
The initial Cryptococcus infection is postulated to occur in the lungs where alveolar macrophages, (the primary cells to encounter Cryptococcus) together with activated Th1 and Th17 cells form the cornerstone of protection. 2,27 In theory, recruited activated immunocytes surround infected primary macrophages to form a granuloma. 27,38 During this process, the primary infection is contained through phagocytosis leading to complete resolution of infection or through evasion of phagocytosis leading to establishment of latency. The evidence for cryptococcosis resolution without disease onset (aborted infection) and/or latency is based on the presence of Cryptococcus-positive binding antibodies in children that demonstrate early exposure to the fatal yeast. 39 Among children and adults who develop active cryptococcosis, progression follows an onset of HIV and other immunosuppressive disease condition including HIV advanced disease. 6,14,40 Hence, latent Cryptococcus infection may persist for a lifetime among Cryptococcus exposed individuals without an underlying immunosuppression trigger. 23,41,42 It is proven by molecular diagnostics (eg MLST on cultures) that Cryptococcus tetragattii (known to only occur in Africa/India) can be dormant or latent for 20-30 years after exposure, for example in immigrants who develop cryptococcal meningitis with strains from country of origin after acquiring HIV in the country of residence. 6 Conversely, whether fatal systemic cryptococcosis is due to new infection or reactivated latent infection is not clearly understood. 34,59 However, Cryptococcus genotyping studies from clinical isolates of emigrants with Cryptococcus strains from endemic regions support both the latent and new environmental acquired Cryptococcus infection causality theories. 5,60 That, individuals who travel and developed cryptococcosis infection with strains endemic to their region support the latent infection theory. Clearly, from European multilocus sequence typing (MLST)-studies, as above mentioned with immigrants from, for example Africa who developed C tetragattii infection ~26 years after immigrated from Zambia to Sweden where the patient acquired HIV followed by cryptococcal meningitis, the isolated strains were identical to the C tetragattii lineage from Southern Africa. 40 And that, similar MLST studies in Africa, Americas and Asia among adult travellers and immigrants who develop cryptococcosis infection with strains endemic to regions they travelled or settled support the new infection theory. 6,8,9 Moreover, person-to-person transmission of Cryptococcus to cause infection is rare. 6,8,9,11,43

| CRYPTOCOCCUS CENTR AL NERVOUS SYS TEM INFEC TI ON
Recent findings indicate several Cryptococcus transmission mechanisms are used by the yeast to infect the CNS. The trojan horse model, where Cryptococcus-infected macrophages, traverse the blood-brain barrier (BBB) to infect the central nervous system. 62 This trojan horse mechanism is also observed to facilitate HIV transmission across the BBB in response to intrathecal CCL2 chemokine stimulation. 63 And, restriction of adhesion molecules (JAM-A and ALCAM and chemokine receptors (CCR2 and CCR5) with inhibitory antibodies restricted monocytes trafficking to the CSF lowered CSF HIV viraemia. 63 Transcytosis is another mechanism, where Cryptococcus, with the help of cellular binding motifs and adhesion molecules, manoeuvres through interstitial cellular spaces of the BBB and the brain parenchyma to infect the CNS. Dendritic cells are also associated with the transcytosis mechanism of compartmentalised pathogen transmission. Lastly, paracytosis is another mechanism where Cryptococcus with the help of secreted proteases digest its way through the cells lining the BBB to gain access of the CNS. 62,64 Thus, interruption of the pathways that aid Cryptococcus access to the CNS where it establishes fatal disease could alter clinical outcomes. 23,45,65 The evidence that Cryptococcus is predominant in the CSF compartment of individuals with cryptococcal meningitis (Figure 1), 24,66 suggests that the fungus influences host immunological and treatment outcomes via its effects on the central nervous system (brain, spinal cord, and CSF). Thus, the predominance of the facultative extracellular Cryptococcus in the closed CSF interacting with immunocytes may be a translational target to model innate and adaptive immune factors and mechanisms of infection and treatment outcome. But the perturbing question is the elusive role and the lack of clinical relevance of the CSF immunocytes in influencing infection and treatment outcome. Evidence from a Ugandan cohort suggests that some individuals with asymptomatic cryptococcosis present with measurable cryptococcal antigen in blood but not in CSF. 23,29 And another observation suggests that Cryptococcus is unable to proliferate in CSF supernatant from healthy individuals without apparent immunosuppression. 38 The above two pieces of observations contradict our understanding of the role of CSF in merely supporting establishment of fatal cryptococcosis.

| PD -1 E XPRE SS I ON ON THE CEREB ROS PINAL FLU ID IMMUNO C Y TE S
The immunocytes in the CSF comprise of activated (HLA-DR/CD38) mature T cells (CD4 and CD8 T cells), monocytes (CD14/CD16; Classical monocytes, Intermediate monocytes and alternative monocytes) and natural killer (NK) cells (CD56/CD16; Bright, dim and negative NK cells). 66 In addition, the B cells (CD19) in the CSF comprise of activated (CD21 low) and differentiated memory (CD27) and plasmablasts/ plasma B cells. 24 Surprisingly, from the above studies, activation and differentiation of the CSF cellular phenotype seem to be altered by programmed death-1 (PD-1; CD279) immune checkpoint receptor and programmed death-1 ligand (PD-L1; CD274) expression. 24,66 The PD-1 is a ubiquitous immune checkpoint transmembrane molecule that is expressed on lymphoid cells particularly on T follicular helper cells 67

PD-1 expression on CSF B cells, T cells and monocytes is much
higher in the CSF compartment compared to the peripheral circulation during cryptococcosis. 24 Additionally, on monocytes, PD-L1 expression in human cryptococcosis is equally higher in CSF compared to its expression in peripheral circulation. 66 Moreover, the majority of CSF immune cells during human cryptococcosis are highly activated compared to those in circulation. 24

| PD -1 E XPRE SS I ON ON LUNG IMMUNOC Y TE S
The mucosal lining of the lung provides easy access by the scavenging cells, including the macrophages and dendritic cells to gain access to the inhaled pathogens to influence onset of infection. In mouse models of cryptococcosis, persistent lung infection sustains PD-1 expression on dendritic cells and on macrophages. 73 Moreover, sustained PD-1 expression on the macrophages on cryptococcosis brain mouse models promotes fungal growth through upregulated proliferation of Cryptococcus-infected macrophages, facilitating fungal dissemination. 73,78 The PD-1 upregulation further increases activation of microglial cells and promotes Th2 cellular-activated responses while downregulating Th1-activated responses. 78 Of note, application of anti-PD-1/PD-L1 antagonists (PD-1 blocking antibodies) altered PD-1-modulated responses by promoting fungal clearance, upregulating ICOS and XO40 on Th1, Th2, Th17 and regulatory T cells while downregulating IL-5 and IL-10 immune regulatory cytokines in the model of cryptococcosis. 73 In addition, similar responses were observed with IL-10 blockade in experimental cryptococcal infection. 79 The IL-10 modulates immune response in a similar manner as PD-1 by modulating immune activation, cellular proliferation and cytokine expression in addition to modulation of cellular differentiation. [80][81][82] Moreover, markers that induce PD-1 expression on B cells are found to induce IL-10 expression in B cells through Toll-like receptor-9-mediated mechanism. 72,83 The PD-1-binding antibodies (substitute for PD-L1) inhibit PD-1 interaction with its high-affinity ligand PD-L1, hence antagonising immune inhibitory activity of PD-L1 on effector cells and consequently restoring exhausted immune response as observed in the mouse model of lung cryptococcosis infection. 73 The similar interpretation of PD-1-blocking antibodies could be translated to IL-10 inhibitory (blocking) antibodies in application.  24,98 One study at cryptococcal meningitis diagnosis reported the PD-1 expression on circulating cellular lineages at 2% of B cells (CD19 + lymphocytes), 25% of T cells and 1% of monocytes. 24 Another study at the same timing reported PD-1 expression on T cells at 60% of CD4 + T cells and 30% of CD8 + T cells with persistant high PD-1 expression on circulating T cells beyond 12 weeks of follow-up. 99 In this case, it is not clear whether sustained PD-1 expression in cryptococcosis is due to persistent activation or another mechanism. But, whether PD-1 or its ligand expression is different with infecting Cryptococcus species is yet to the investigated.

| CENTR AL NERVOUS SYS TEM IMMUNE AC TIVATI ON AND INFL AMMATI ON
In the HIV cohort, PD-1 expression persisted on T cells beyond 22-44 weeks of antiretroviral therapy. 65 In compartments, PD-1 is highly expressed in localised tissue infections that may be indicator altered immune response or pathogen immune escape mechanisms. HIV-infected host to arterial fibrosis. 65 Moreover, induced PD-1associated arterial stiffness persists for nearly a year. 65 The PD-1 pathway like the Th-2 predominant immune response modulates the immune response resulting in shutting down of immune activation and inflammation leading to tolerance (immune non-responsiveness) that may allow evading pathogen to thrive unchecked in the host.
Fibrosis may not be entirely bad, as may be used by the host as an attempt to limit the spread of infection. But fibrosis-inducing factors may work in synergy with other Cryptococcus host susceptible factors like anaemia to impair oxygen supply to the vital organs like the heart and the brain (hypoxic hypoxia) and consequently impairing tissue survival. Hence, pathogens that survive in a hypoxic microenvironment like Cryptococcus, which evades and thrives in the macrophage, could take advantage of the induced hypoxia in the brain to survive while leading to fatal outcomes. 65,102,104 Together, these host and pathogen factors that induce fibrosis could contribute to poor outcomes with cryptococcal meningitis.
In other studies, the PD-1/PD-L1 axis-induced tissue fibrosis is clearly demonstrated in the lungs 101 and brains of HIV-infected patients. 105 Together, brain tissue fibrosis (subarachnoid spaces and cerebellar) and arterial stiffness (anterior and posterior arteries) 102 could limit the effectiveness of antifungal and antiretroviral drug penetration into the CNS. The limited drug entry to the CSF could, in turn, limit antigen clearance in those with arterial stiffness and may influence fungal recrudescence. Further induced brain tissue fibrosis could influence intracranial pressure. And exacerbated arterial stiffness could influence brain hypoxia and subsequent brain tissue infarction. 37 It is plausible that targeted blood transfusion to increase blood oxygen carrying capacity especially to the brain, oxygen supplementation, 37

| IMMUNE RECONS TITUTION INFL AMMATORY SYNDROME (IRIS)
The IRIS is a fatal condition associated with an exaggerated immune response to recall or persistent antigen following immune restoration with antiretroviral therapy. This is common among HIV-infected patients with opportunistic co-infections like cryptococcosis after they initiate ART. 25 34 This implies that immune regulatory mechanisms are integral to a balanced immune response to infectious disease with less fatal effects (minimal host damage) prior to resolution of infection (with/ without treatment). 34 We postulate that B-cell immune modulatory mechanisms work in synergy with optimal treatment to regulate host damage responses to influence recovery.

| ANTIBODY RE S P ON S E IN CRYP TOCOCC AL MENING ITIS
In experiments to quantify Cryptococcus-specific GXM antibodies among subjects with confirmed cryptococcal meningitis co-infection by quantitative fungal culture, 45.7% of the subjects tested positive for Cryptococcus-specific GXM IgG antibody. 128 Interestingly, after acid treatment of the specimens to dissociate antibody from antigen-bound immune complexes to release bound antibodies for measurement, 97.1% of subjects had detectable Cryptococcusspecific GXM IgG antibody response. 128 Thus, HIV and cryptococcosis co-infected individuals demonstrate anti-Cryptococcus-specific antibody responses. 129 However, the magnitude and the quality of antibody response may be inadequate especially at the onset of primary cryptococcosis to control the fast replicating fungus. 129 In addition, the acute to early onset of cryptococcal meningitis from onset of symptoms could support the hypothesis that a protective antibody response in cryptococcosis occurs later (weeks to months following treatment) especially among index cases. 20,45 Additionally, the antibody response may be produced in later stages of infection when the host may be already overwhelmed with the infection. This may render antibody response less beneficial to the debilitated host as most of the produced antibody response may end up being bound to immune complexes and to the polysaccharide capsule. 128 Conversely, it could be interpreted that index cryptococcosis infection (without circulating protective antibodies) causes fatal disease in the susceptible host and not latent cryptococcosis (with circulating specific antibodies). This could be true since both the latent and new Cryptococcus infection cause cryptococcal meningitis. 5,60

| REG UL ATI ON OF B -CELL TR AFFI CK ING IN INFL AMMATI ON
The immune cells extravasate the peripheral and lymphatic circula-

| FAC TOR S LINKED TO FATAL CRYPTOCOCCUS CENTR AL NERVOUS SYS TEM INFEC TI ON
Among patients with HIV-associated cryptococcal meningitis where C neoformans sensu lato and interspecies hybrids cause most disease, [11][12][13] inconsistent variables ( Figure 3D) that influence disease outcome have been reported including early ART initiation among HIV and ART naïve subjects. 20 The high fungal burden and altered mental status at cryptococcal meningitis diagnosis coupled with the slow fungal clearance following antifungal therapy. 21,50 The prevalence of seizures at diagnosis and the incidence seizures during the course of treatment and follow-up among individuals with above 96 000 fungal colony-forming units that is 3-four fold higher in those with seizures compared to those without seizures. 36 Limited oxygen blood carrying capacity (anaemia) potentially leading to low brain oxygen saturation, hypoxia. 37,142 Although amphotericin B is known to suppress erythropoietin,a factor that may influence the extent of anaemia in cryptococcosis sequelae. 143 Surprisingly, amphotericin B-induced anaemia is not associated with cryptococcosis cause fatality in first two and half months of cryptococcosis diagnosis. 142 And fibrosis among cases of cryptococcal meningitis may influence onset and the extent of intracranial pressure build up, hypoxia, brain infarction and antifungal drug penetration. 102 Interestingly, these variables and factors associated with poor cryptococcosis outcome are reported independent of each other within similar large cohorts of HIV-associated cryptococcal meningitis. 20,21,36,37,50,102 This implies that multiple or slightly independent pathways drive host pathology.
Thus, advances to alter poor survival outcomes could require deliberate effects to combine multiple factors in combined interventions targeting multiple pathways in the host.
In addition, an ineffectual immune system with a skewed Th-2 immune response has been associated with poor disease outcomes. The Th-2 immune response is seen to inhibit activation of Cryptococcus-specific antifungal pro-inflammatory cytokine responses, thus impairing fungal clearance. 50 And the high frequency of pro-inflammatory cytokines responses (IFN-γ, TNF-α and IL-12) coupled with a high frequency of CSF infiltrating white blood cells after ART initiation is associated with a risk of cryptococcal meningitis-immune reconstitution inflammatory syndrome (CM-IRIS). 107 But, predominance of a pro-inflammatory cytokine response alone is postulated to work in synergy with antifungal treatment to enhance fungal clearance. 20

| CON CLUS I ON AND PER S PEC TIVE S
Despite four decades of HIV era and advances to treat those infected with optimal therapy, cryptococcal meningitis remains a significant contributor of death among HIV-infected adults especially in sub-Saharan Africa. Consolidated new insights show that the HIV and cryptococcal meningitis-associated poor survival outcome factors are associated with fungal induced neuropathies related to the inducers of seizures, altered mental status, brain tissue fibrosis and brain hypoxia and potentially the involved cryptococcal species. In addition, altered immune response involving PD-1/PD-L1 pathway may dysregulate immune activation to influence inflammation and impact on brain fibrosis and brain infarction. And dysregulated erythropoiesis associated with prevalent anaemia together with induced fibrosis may influence hypoxia and brain infarction. These insights come at a time of unknown immunological target to prevent HIV-associated cryptococcal meningitis and adequate therapy to prevent mortality. Together, the eluded factors and underlying mechanisms are hypotheses generating and will guide further studies to finding targets to prevent infection or adequate therapy to prevent mortality.
Interventions like the use of supplemental oxygen, blood transfusion to boost blood oxygen carrying capacity and the use of anti-PD-1/ PD-L1 blockers to restore appropriate immune reconstitution may be valuable advances to alter poor treatment outcome.

| LI M ITATI O N S
Our findings are without important limitations that our discussion is based on online published literature generalised to Cryptococcus in the setting of HIV-associated cryptococcosis. However, individual Cryptococcus species may influence infection or disease recovery differently requiring species identification to define species-specific pathologies and treatment outcome for clinical relevance. This is important because Cryptococcus still causes substantial morbidity and mortality that may require different treatment strategies to prevent residual 10%-20% of cryptococcosis-related deaths occurring with optimal use of antifungals. And that, although we conducted a wide literature search, we might have left out relevant literature. And some deductions are based on a few articles on the subject matter that may limit generalisation.

ACK N OWLED G EM ENTS
We thank Barbara Castelnuovo, Stephen Okoboi, Aidah Nanvuma, from Infectious Diseases Institute, Research Capacity Building Unit, for supporting the training programme.

CO N FLI C T O F I NTE R E S T
Nothing to disclose.

E TH I C A L CO N S I D ER ATI O N S
Nothing to disclose.