In this prospective cohort study of patients with AIDS and recent CM in Uganda, we characterized the incidence and clinical features of CM-IRIS and identified serum biomarkers that can diagnose IRIS and can stratify the risk for IRIS or death. CM-IRIS is a common complication of ART in sub-Saharan Africa, with an incidence reported in the literature ranging from 10% to 31% 
. In our cohort with <1% loss to follow up, the incidence of CM-IRIS was 45%, two-thirds of whom experienced characteristic CNS manifestations (30%). Unlike many other forms of IRIS, which produce less dramatic consequences, CM-IRIS is exceptional for its substantial morbidity and mortality 
. The 36% CM-IRIS mortality in our study is consistent with other CM-IRIS studies 
. Such dire outcomes may derive from the increased inflammation that accompanies CM-IRIS in the anatomically constrained compartment of the CNS.
Before this study, it was not possible to predict risk for CM-IRIS. Epidemiologic factors, that were identified in retrospective studies, were not predictive of CM-IRIS in our study, and have not been found consistently across other studies 
. Similar to a recent ACTG (AIDS Clinical Trial Group) a5164 trial, in our study timing of ART initiation was not associated with IRIS 
. In contrast, however, we identified specific patterns of biomarkers that were predictive of the development of IRIS and death at initiation of ART, after initiation of ART, and at IRIS event.
Based on our identification of biomarkers that predict CM-IRIS, our theoretical model is one whereby persons predisposed to IRIS have more dysfunctional immune systems that fail to effectively clear antigens from cryptococcus before ART-associated immune restoration. Indeed, persons with IRIS had higher median serum CRAG titers when initiating ART than controls without IRIS (p
0.006), but they had identical 1
1024 median CSF CRAG titers at their initial CM diagnosis 
. This result may have implications for customization of timing of ART based on initial induction antifungal therapy prescribed with a longer delay required for less active antifungal therapy (e.g., fluconazole monotherapy).
Further failures of antigen clearance may result from an imbalance in immune regulation, initially characterized by increased production of Th2 cytokines (IL-4, IL-13) and a lack of the proinflammatory mediators (VEGF, TNF-α, G-CSF, GM-CSF) that are required to recruit lymphocytes and activate macrophages and other innate cells to clear C. neoformans
. Specifically, in neutrophils and macrophages, G-CSF and GM-CSF promote phagocytosis, up-regulation of reactive oxygen species, and intracellular destruction of cryptococcus 
. Decreased pre-ART levels of G-CSF, GM-CSF, and TNF-α should result in decreased phagocytosis and promotion of a fertile intracellular environment within macrophages for cryptococcus 
. Based on our model of CM-IRIS pathogenesis, this inappropriate or ineffective macrophage activation leads to failure to clear cryptococcal organisms and predisposes toward the development of CM-IRIS. In this respect, CM-IRIS pathogenesis may parallel the proposed mechanism of TB-IRIS 
A failure of antigen clearance promotes generalized inflammatory signaling to then occur, specifically with IL-6. Compared with time-matched control participants, persons who developed IRIS had elevated levels of CRP before ART initiation, and elevated levels of CRP and IL-6 before and at the time of IRIS events. Our results indicate that the inflammatory signaling response involving IL-6 progressively increased on ART until the development of IRIS. Since IL-6 is the primary stimulant of hepatic CRP production, it is unsurprising that these biomarkers had similar patterns. French and colleagues reported similar increases in plasma IL-6 after a variety of IRIS events or before herpesvirus-related IRIS events, and they verified by flow cytometry experiments that activated macrophages were the primary source of IL-6 
We hypothesize that the abnormal cytokine profiles we observed before CM-IRIS, including elevations of Th2 cytokine (e.g., IL-4, IL-13) and lack of proinflammatory cytokines such as TNF-α, indicate ineffective or inappropriate alternative macrophage activation. In contrast to Th1 responses, which promote IFN-γ–mediated killing of cryptococcus by macrophages 
, IL-4 produced in Th2 cells causes alternative activation of macrophages—typically a response to parasite infections—promoting fibrosis 
. Alternative activation of macrophages allows for intracellular growth, proliferation, and dissemination of C. neoformans
. In other fungal processes, IL-4 reverses GM-CSF–mediated macrophage killing of yeast in vitro 
. In our results, small quantitative increases in IL-4 were associated with large statistical increases in IRIS risk. Since Th1 and Th2 responses are often mutually exclusive, inappropriate increases in Th2 responses (e.g., IL-4, IL-10, IL-13) generate ineffective inflammation and inhibit the protective Th1 responses necessary for efficient antigen clearance. The association of a Th2 bias and risk for IRIS was evident with elevated IL-4 levels at baseline, elevated IL-13 levels on ART before IRIS events, and elevated IL-10 levels at IRIS events as well as an association between increased IL-10 levels at IRIS and subsequent death. The Th2 responses that limit clearance of C. neoformans
may be driven by preexisting Th2 biases to other concomitant coinfections, such as intestinal helminths or schistosomiasis. This phenomenon might underlie the higher overall incidence of IRIS in our cohort compared to cohorts from temperate regions 
. Unexplained absolute eosinophilia (>500 cells/µl) was present in over one-quarter of the cohort; however there were not statistical differences in eosinophils or total serum IgE between cases and controls. Overall, our data suggest that Th2 responses to cryptococcus are associated with poor outcomes, including increased risk for IRIS and death.
Risk factors for IRIS extend beyond the Th1:Th2 paradigm. Increased Th17 responses (i.e. IL-17) were a pre-ART risk for both IRIS and mortality on ART. Proinflammatory Th17 cells have been previously hypothesized as important in IRIS pathogenesis 
. In normal immune homeostasis, a balance between Th17 cells and regulatory T cells (Treg) is crucial. A Th17 imbalance can cause autoimmune diseases. In this balance, IL-6 plays a key role in naïve T cell differentiation inducing Th17 differentiation and inhibiting Treg differentiation in the presence of TGF-β 
. The copious IL-6 present before IRIS events may alter the balance between Treg and Th17 cells, suppressing Treg differentiation or function during ART-related immune reconstitution.
Additionally, other cytokines that we identified may play a role in IRIS pathogenesis by contributing to ineffective macrophage activation. For example, low pre-ART levels of TNF-α or VEGF were associated with increased IRIS risk. TNF-α, secreted by macrophages and T cells, is particularly important in activating antigen-presenting cells. Absence of TNF-α causes failure of mature dendritic cell activation and recruitment, thereby blunting further recruitment of T cells 
. Lack of TNF-α could indicate failure to present and/or process antigen and could contribute to ineffective macrophage activation in patients at risk for IRIS. In cryptococcosis, VEGF is secreted by a variety of leukocytes but especially by CD4+
T cells during antigen-specific responses to cryptococcal mannoprotein being presented by MHC-II molecules 
. Decreased VEGF may reflect greater immune dysfunction due to failure of CD4+
T cell antigen recognition from antigen-presenting cells. The downstream effects of decreased VEGF would further diminish both chemotaxis and the costimulatory activity of VEGF on IFN-γ–secreting Th1 memory T cells 
. Thus, decreased VEGF may then diminish the Th1 response and shift the Th1:Th2 balance toward Th2 responses. The decreased levels of TNF-α and VEGF in patients at risk for IRIS may be causes or reflections of impaired macrophage function. Overall, the inappropriate Th2 responses and IL-4 production, coupled with ineffective macrophage activation, suggest that a failure in pathogen recognition and clearance could set the stage for paradoxical IRIS by promoting antigen persistence. Upon eventual immune restoration of more appropriate antigen-specific responses, these responses are exaggerated because of the abundance of uncleared foreign antigen and promotion by IL-6 of a proinflammatory state.
Strengths of this study include its prospective design, careful and complete follow-up, and integration of pathophysiologic analyses in the context of characterizing clinical phenotypes. As such, we believe the results reported here are generalizable to cryptococcal IRIS as a pathophysiologic entity. In general, the diagnoses of other forms of IRIS are somewhat subjective, but in CM-IRIS, the identification of IRIS in the CNS is very objective due to the ability to examine the CSF, where the inflammation occurs. A problem arises with non-CNS manifestations of IRIS, which often represent diagnoses of exclusion and depend on the diagnostic capabilities available. In this cohort, we identified several (n
20) non-CNS IRIS events, of which 60% were associated with later CNS-IRIS events or with supportive/definitive histopathology (Text S1
). Eight cases (retinitis, optic neuritis, keratoconjunctivitis, pneumonitis; n
5) were probable IRIS diagnoses and unsupported by histopathology or culture (17% of total IRIS cases). Although cryptococcus most often causes meningitis, cryptococcosis is a disseminated disease with systemic CRAG measurable for months in peripheral blood 
. In our experience here, dead C. neoformans
causing IRIS reactions were identified in tissue by histopathology of brain, lymphatics, gut, skin, and tongue and were isolated via bronchoalveolar lavage. Thus, non-CNS cryptococcal IRIS events do occur, allowing these “probable events” as likely, but not definitely, attributable to IRIS. In separately considering these non-CNS IRIS events, there were no statistical differences in the inflammatory profiles observed at time of IRIS as compared to CNS-IRIS events, except for possibly D-dimer. In Uganda, there is likely high and ongoing environmental exposure to cryptococcus 
, thus pneumonitis-IRIS could equally be triggered by persisting original antigen or by environmental re-exposure manifesting effectively as an acute hypersensitivity pneumonitis reaction. Pulmonary cryptococcosis often goes unrecognized and/or is suspected as smear-negative TB 
This prospective observational study has allowed us to characterize the statistical association between cytokine profiles and IRIS. Of course, this does not prove causality; however, the anomalies we report are biologically plausible within the known immunology of responses to cryptococcus. A challenge in pathogenesis studies such as ours is that the inflammatory profiles associated with IRIS are heterogeneous, representing a continuum that is likely dependent on the duration of symptoms, the antigen, and the robustness of the event. In cytokine profiling, six of our participants had minimal immunologic perturbation in peripheral blood even though they clinically deteriorated, presenting with headache and elevated intracranial pressure several months into ART. Thus, some patients fulfilling the IRIS clinical case definition either have localized immune responses that were not detectable in peripheral blood, or they may not have had true immunologic IRIS but instead had delayed complications of CM that were clinically indistinguishable from CM-IRIS. This heterogeneity may have implications for IRIS management and the response to anti-inflammatory therapies. Although this prospective study of CM-IRIS is the largest to date, our biomarker data should be viewed as hypothesis-generating and will require validation in future cohorts. We plan to validate these biomarkers in subjects enrolled in the multi-site Cryptococcal Optimal ART Timing (COAT) trial (NCT 01075152; http://clinicaltrials.gov/ct2/show/NCT01075152
) and investigate these biomarkers with regard to timing of ART initiation after CM diagnosis. This validation will give insight on whether altering the timing of ART initiation is a potential intervention to alter high risk or whether other strategies should be pursued. Once validated, creating diagnostics such as multiplex ELISAs or bead-based techniques could move this into clinical use.
Given the high incidence, morbidity, and mortality associated with CM-IRIS, identifying patients at risk for IRIS may enable interventions to improve management. Three distinct phases of IRIS pathogenesis can be identified (). First, before ART, a paucity of innate inflammatory responses or inappropriate (Th2) responses promote ineffective antigen clearance. Second, after ART initiation, copious antigen presentation promotes proinflammatory signaling (e.g., IL-6, CRP, IL-7). Third, at the time of IRIS, a generalized cytokine storm occurs. The biomarkers identified here, although requiring validation, may help target interventions to decrease IRIS risk, such as (1) early adjunctive GM-CSF or IFN-γ to increase macrophage function, (2) antiparasitic therapy to eliminate coinfections that promote inappropriate Th2 bias, (3) anti-IL-6 receptor antibody (tocilizumab) therapy to blunt inflammatory signaling, or (4) delaying ART initiation. This study suggests that prediction of IRIS or death may be possible with measurement of pre-ART serum biomarkers.
Summary of paradoxical cryptococcal-IRIS pathogenesis hypothesis.