The Immunopathogenesis of Cryptococcal Immune Reconstitution Inflammatory Syndrome - Understanding a Conundrum

Abstract

Purpose of Review

Cryptococcal meningitis (CM) causes significant mortality among HIV-infected patients, despite antifungal therapy and use of antiretroviral therapy (ART). In patients with CM, ART is often complicated by immune reconstitution inflammatory syndrome (IRIS), manifesting as unmasking of previously unrecognized subclinical infection (unmasking CM-IRIS) or paradoxical worsening of symptoms in the central nervous system after prior improvement with antifungal therapy (paradoxical CM-IRIS). We review our current understanding of the pathogenesis of this phenomenon, focusing on unifying innate and adaptive immune mechanisms leading to the development of this often fatal syndrome.

Recent Findings

We propose that HIV-associated CD4⁺ T cell depletion, chemokine-driven trafficking of monocytes into cerebrospinal fluid (CSF) in response to CM, and poor localized innate cytokine responses lead to inadequate cryptococcal killing and clearance of the fungus. Subsequent ART-associated recovery of T cell signaling and restored cytokine responses, characterized by Interferon-γ production, triggers an inflammatory response. The inflammatory response triggered by ART is dysregulated due to impaired homeostatic and regulatory mechanisms, culminating in the development of CM-IRIS.

Summary

Despite our incomplete understanding of the immunopathogenesis of CM-IRIS, emerging data exploring innate and adaptive immune responses could be exploited to predict, prevent and manage CM-IRIS and associated morbid consequences.

Introduction

Cryptococcal meningitis (CM) causes 20–25% of AIDS-related mortality worldwide [1]. In sub-Saharan Africa, Cryptococcus is the most common cause of meningitis in adults, accounting for 26% of cases in Malawi, 45% in Zimbabwe, 30% in South Africa, and 60% in Uganda [2–6]. Despite antiretroviral therapy (ART) scale up, many patients with a new HIV diagnosis still present with very low CD4⁺ T cells (<200/μL) [7] and opportunistic infections, such as Cryptococcal meningitis, due to failure to initiate ART in a timely manner and poor linkage to HIV care.

Initiation of ART can be complicated by Immune reconstitution inflammatory syndrome (IRIS), which contributes to the mortality following ART. IRIS develops in up to 30% of patients with CM, manifesting as clinical deterioration a few weeks or months after initiating ART [8, 9]. This syndrome is thought to be an aberrant inflammatory response to persistent cryptococcal antigens during recovery of the host immune system [10]. IRIS may manifest as unmasking of previously unrecognized subclinical infection (unmasking CM-IRIS) or paradoxical worsening of CM-associated symptoms in the central nervous system after improvement with antifungal therapy (paradoxical CM-IRIS). Occurring within the closed central nervous system space, CM-IRIS can be fatal, and has been associated with a high mortality (Table 1).

Table 1.

Characteristics of Studies related to Cryptococcal Immune Reconstitution Inflammatory Syndrome in Patients starting Antiretroviral Therapy.

Type of Study	Number of subjects	Baseline CD4/μL	Frequency of CM-IRIS (%)	Timing after ART start	CM-IRIS related mortality (%)	Country	Reference
Retrospective	10	39	50	2–11 months	..	USA	Jenny-Avital et al (2002) [11]
Prospective	434	86	2	4 weeks	67	South Africa	Lawn et al (2005)[12]
Retrospective	59	72	31	4 weeks	2	USA	Shelburne et al (2005) [13]
Retrospective	59	32	38	18 weeks	32	South Africa	Jenkin et al (2006) [14]
Retrospective	52	26	19	9.9 months	0	Thailand	Sungkanuparph et al (2007)[15]
Prospective	65	28	17	29 days	36	South Africa	Tihana B et al (2009) [16]
Retrospective	120	13	8.3	8 months	30	France	Lortholary A et al (2005) [8]
Retrospective	154	..	17.5	27 days	11	China	Yan et al (2014) [17]
Prospective	44	20	16	12 weeks	40	Uganda	Kambugu et al (2010) [18]
Prospective	101	19	45	8.8 weeks	36	Uganda	Boulware et al (2010) [19]
Prospective	40	..	22.5	10 weeks	..	Brazil	Colombo et al (2011) [20]
Prospective	90	27	14	8.6 weeks	..	South Africa	Jarvis et al (2012) [21]
Prospective	13	16	25.5	..	19.8	South Africa	Chang et al (2013) [22]

Role of Primary Immune or Host response

Primary immune deficiencies of CD4⁺ T cells, impaired phagocytic function of macrophages, and variants in major histocompatibility complex molecules I/II are associated with development of serious cryptococcal infections [23, 24]. Similarly, patients with X-linked hyper-IgM and hyper-IgE syndromes, and those with secondary immune disorders, such as malignancies, solid organ transplants, use of immunosuppressant drugs, and with HIV infection show a similar predisposition [25, 26]. In healthy humans, the initial immune response involves fungal recognition by lung innate immune cells and generation of signals that lead to expansion of Cryptococcus-specific CD4⁺ T cells to control the infection [27]. Minimal trafficking of immune cells to the CSF occurs in severely immunosuppressed HIV-infected patients with CM [28*]. The primary immune response to cryptococcal infection may determine the mechanisms that eventually cause cryptococcal IRIS.

In the brain, Glucuronoxylomannan, (GXM), the principal component of the cryptococcal polysaccharide capsule, binds to macrophage FcγRIIIB, which inhibits the Fcγ receptor and proinflammatory cytokine expression [29, 30], whereas mannoprotein induces antigen-specific T cell responses [31]. C. neoformans promotes an imbalance toward a Th2 response by suppressing T-helper (Th) 1 responses [32–34]. Uncontrolled murine cerebral cryptococcosis results from IL-4/IL-13-dependent alternative activation of macrophages [35]. Consistent with these observations, patients who later develop CM-IRIS show higher baseline serum levels of Th2 cytokines (IL-4 and IL-13), lower levels of Tumor Necrosis Factor (TNF)-α [19] and lower CSF levels of IFN-γ [36] at CM diagnosis compared with those who do not develop CM-IRIS.

Cryptococcus inhibits T cell migration into the CSF by increasing L-selectin shedding from T cells, limiting their ability to extravasate into the CNS [37, 38]. The imbalance of these functional immune constituents may underlie the pathologic clinical outcome of ART-induced IRIS during cryptococcal infection. In patients with AIDS, Cryptococcus establishes a chronic infection. Upon ART-associated immune recovery, the persistent fungal antigen burden is proposed to trigger an exaggerated inflammatory response due to HIV-associated impairment of homeostatic regulatory mechanisms [39].

Immune Reconstitution with Antiretroviral Therapy

During early HIV infection, HIV-specific immune responses limit viral replication and the destruction of CD4⁺ T cells is balanced by the generation of new CD4⁺ T cells [40]. Over time, ongoing viral replication, immune cell depletion and dysregulation are associated with chronic inflammation, resulting in scarring and destruction of the normal lymph node architecture [41]. ART suppresses HIV replication and CD4⁺ T cell apoptosis, allowing immune regeneration. During immune reconstitution, CD4⁺ T cells increase rapidly (26 cells/mm³/month) [42], some pathogen-specific immune responses recover [43*], a decreased susceptibility to OIs is observed, and genes encoding innate antiviral responses, immune activation, cellular proliferation and apoptosis are downregulated [44]. HIV infection also affects the innate immune axis, with expansion of monocytes [45], whereas immune reconstitution decreases the activation of monocytes/macrophages [46].

Epidemiology of Cryptococcal Meningitis Immune Reconstitution Inflammatory Syndrome (CM-IRIS)

An estimated 10%–25% of persons without a known OI who start ART experience IRIS [40, 47–49]. IRIS has been associated with >20 infectious pathogens (Mycobacterium avium complex, M. tuberculosis, Cryptococcus neoformans, Cytomegalovirus, JC virus, Pneumocystis jirovecii, Herpes zoster (VZV), and hepatitis B and C), malignancy, (Kaposi’s sarcoma) and occasionally non-infectious autoimmune syndromes and malignancies [40, 41, 47, 48]. The majority of IRIS events will occur within the first eight weeks of ART, however, delayed IRIS occurring years later has also been described, particularly for cryptococcal-IRIS and CMV-IRIS [11, 50].

Approximately 25% of CM patients treated with antifungals and ART [8, 9, 16] will experience paradoxical deterioration, despite decreasing fungal burden [8, 16, 19, 51] (Table 1). Cryptococcal IRIS may manifest as relapsing aseptic meningitis, increased intracranial pressure, new focal neurologic signs, intracranial cryptococcomas, lymphadenopathy, and/or development of abscesses [11, 19, 52–54] which can present as multifocal or multiphasic disease [55*]. Mortality associated with cryptococcal IRIS ranges from 0 to 67% [19, 20, 50].

In the absence of HIV coinfection, cryptococcal IRIS has also been described with reversal of iatrogenic immunosuppression. Cutaneous cryptococcal IRIS was reported in a patient with T cell pro-lymphocytic leukemia [56] following use of alemtuzumab, a monoclonal antibody that blocks CD52, a surface ligand involved in immune cell mobility [57]. Tumor necrosis factor (TNF)-α inhibitors have also been associated with an increased risk of cryptococcal infections [58–60], highlighting the important role played by TNF-α in the immune response against Cryptococcus [61].

Case Definition- Cryptococcal IRIS

The International Network for the Study of HIV-associated IRIS (INSHI) has developed a consensus case definition for CM-IRIS [51] (Supplementary Table 1). IRIS remains a diagnosis of exclusion, after considering adverse drug reactions, new OIs, and microbiologic treatment failure (whether due to drug-resistance or malabsorption). Differentiation of IRIS from these other diagnoses has important implications that may impact clinical management and outcomes.

Clinical Risk factors for Cryptococcal IRIS

The immune response to Cryptococcus requires both the innate and adaptive immune axes to be functionally coupled to control the infection [62*, 63]. During HIV infection, the adaptive immune response is severely compromised and fungal dissemination to the meninges occurs in the absence of antifungal therapy. ART alone is insufficient to prevent cryptococcal disease when subclinical infection is present [64]. Microbiologic, virologic, immunologic and therapeutic factors may affect the incidence of CM-IRIS (Table 2).

Table 2.

Risk Factors for Cryptococcal IRIS

Microbiologic	Virologic	Immunologic	Therapeutic
Initial Fungal Burden - Higher fungal burden or cryptococcal antigen titer at CM diagnosis [13, 19, 21, 65]	Baseline and IRIS HIV RNA - Higher viral load prior to ART initiation - A robust virologic response to ART with a rapid decline in VL [16]	Baseline CD4 and Change with ART ±Lower pre-ART CD4 count [21, 66]. ±A robust immunologic responses to ART with rapid CD4 T cell increase [16] (excluding virologic failure).	ART Timing - Very early ART initiation in relation to diagnosis and therapy of CM [67–70]
Persistent Positive Cryptococcal CSF cultures. - Positive residual culture at the end of induction therapy when starting fluconazole 400mg [21, 22]		CSF WBC and protein - Poor initial CSF immune response as suggested by CSF white cell count (<5μL) cells/and protein (<50 mg/dl) when Cryptococcus disseminates to the CNS [36]
		Cytokine responses - Low CSF IFN-γ, TNF-α, IL-2, IL-6, IL-8, and IL-17 [21, 22, 36]

Abbreviations: ART- Antiretroviral therapy; CM- cryptococcal meningitis; WBC- white blood cells; CSF- cerebrospinal fluid; IL- Interleukin; IFN- Interferon; TNF- tumor necrosis factor; IRIS- immune reconstitution inflammatory syndrome.

Proposed immunologic mechanisms for CM-IRIS

We highlight three stages of CM-IRIS pathogenesis (Table 3): 1) pre-ART paucity of inflammation, immune system dysfunction, and/or anergy resulting in poor macrophage killing and clearance of cryptococcal organisms; 2) macrophages are poised to receive missing T cell signals, such as from IFN-γ, which are restored after ART initiation and induce cryptococcal killing triggering an inflammatory response; and 3) dysregulated homeostatic and regulatory mechanisms leading to exaggerated responses.

Table 3.

Summary of Paradoxical Cryptococcal-IRIS pathogenesis framework [19].

Phase	Immunologic Activity	Evidence in CM-IRIS Patients
Before ART	Paucity of appropriate inflammation for cryptococcosis and/or	↓ TNF-α, G-CSF, GM-CSF, VEGF in serum ↓ IFN-γ, G-CSF, TNF-α, IL-6 in CSF [21, 36]
	Inappropriate (Th2) responses resulting in:	↑IL-4 pre-ART
	Poor antigen clearance, pre-ART	Similar CSF CRAG at initial infection [36] Higher CRAG pre-ART
	Elevated CSF chemokine expression	↑Expression of CCL2, CCL3 in CSF[71]
After Starting ART	Increasing proinflammatory signaling from antigen presenting cells (APCs) due to persisting antigen burden and failure to clear antigen	↑ IL-6 from macrophages [72] then downstream ↑ CRP production; ↑ IL-7 from APCs
	Secondary activation of coagulation cascade	↑ D-dimer
At IRIS	Effective response of innate and adaptive immune systems	Th1 ↑IFN-γ, VEGF; TH17 ↑ IL-17 Innate: ↑ IL-8, G-CSF, GM-CSF ± Elevated CSF White blood cells ± Elevated CSF Protein Negative CSF culture ↑ IL-6+, TNF-α+ monocytes[73*]
	Activation of coagulation cascade	↑ D-dimer
	Neuronal cell activation and damage	↑ CSF FGF-2
	Aberrant innate cell trafficking	Trafficking of pro-inflammatory monocytes and CD4+ T cells into CSF [28].

Abbreviations: APC- Antigen presenting cell; IL- Interleukin; CRP- C reactive protein; IFN- Interferon; CSF- Cerebrospinal fluid; TNF- Tumor necrosis factor; CCL- C-C chemokine; ART- Antiretroviral Therapy; GM-CSF- Granulocyte macrophage colony stimulating factor; TH- T helper; CRAG- Cryptococcal Antigen; FGF-Fibroblast growth factor.

Phase 1: Paucity of inflammatory response

Neutrophil migration and fungistatic activity is impaired during HIV infection [74, [Walenkamp, 2003 #299]]. Poor CSF cytokine responses predispose to poor antigen clearance and increase the risk of IRIS. At CM diagnosis, 3 of 5 studies suggest a paucity of inflammation increases the risk of developing CM-IRIS (Table 4a). Significant elevation of cells and protein is not consistently demonstrated. HIV-uninfected subjects with CM show a more robust immune response in CSF. Patients with HIV infection had higher cryptococcal antigen titers at baseline, with a significant decrease at CM-IRIS. It remains unclear whether the changes that occur at CM-IRIS suggest a recovering immune system responding normally or reflect exaggerated immune responses to residual cryptococcal antigen. The recurrence of symptoms/signs of meningitis associated with CM-IRIS in the setting of negative CSF cultures, however, appears to fit the latter.

Table 4a.

Profiles of Inflammation in Blood and CSF among patients with Cryptococcal Meningitis and Cryptococcal IRIS by HIV Serostatus.

Reference	Patient Description	CSF WBC/μL Median (range)	CSF Protein (mg/dL) Median (range)	CRAG Titer/Colony forming Units
Boulware, D et al, 2010 [36]	HIV Positive at CM-IRIS (n=33)	31 (5–85)	80 (57.5–122.5)	1:64 (CSF)
	HIV Positive at CM diagnosis (n=85)	5 (2.5–25)	40 (20–80)	1:1024 (CSF)
Bicanic, et al, 2009 [16]	HIV Positive at CM-IRIS (n=11)	23 (4–37)	72 (50–100)	1:256 (CSF)
	HIV Positive at CM diagnosis (n=65)	4 (0–85)	60 (40–130)	1:2048 (CSF)
Chang C et al, 2013 [22]	HIV Positive with CM-IRIS (baseline) (n=27)	10 (0–24)	70 (49–82)	≥ 1:1024 (Serum)
	HIV Positive without CM-IRIS (baseline) (n=63)	34 (8–144)	91 (61–154)	≥ 1: 1024 (Serum)
Shelburne, SA, et al, 2005 [13]	HIV Positive at CM-IRIS (n=18)	56 (27–93)	98 (54–202)	1:128 (CSF)
	HIV Positive without CM-IRIS (n=97)	12 (3–62)	91(46–141)	1:2048 (CSF)
Chau, T et al, 2010 [75]	HIV Negative (n=57)	287 (2–1080)	128 (35–263)	1:256 (Serum)
Lee SJ, et al, 2011 [76]	HIV Positive (n=11)	285 (1–286)	-	1: 256 (CSF)
	HIV Negative (n=9)	150 (6–150)	-	1:16 (CSF)
Lee YC, et al, 2011 [77]	HIV Positive (n=37)	26 (mean)	88	≥ 1:512 (77%) (Serum)
	HIV Negative (n=51)	86 (mean)	149	≥ 1:512 (50%) (Serum)
Mora, DJ, et al 2015 [78]	HIV Positive at CM diagnosis (n=30)	<20 (67%)	92 (61–119)	≥ 1:1024 (CSF)
	HIV Positive No CM (n=56)	<20 (67%)	34	-
	HIV Negative (n=48)	<20 (53%)	42	-
Jarvis et al 2012 [21]	HIV Positive with CM-IRIS (baseline) (n=9)	13	-	5.53 log10CFUs
	HIV Positive without CM-IRIS (baseline) (n=9)	28	-	5.03 log10CFUs

Comparison of different studies describing elements of the inflammatory response and antigen burden in CSF and serum for HIV negative patients with cryptococcal meningitis, HIV positive patients with and without IRIS. Percent in brackets indicates proportion of patients.

Table 4b summarizes studies that have measured cytokines/chemokines in blood and CSF among patients with CM or CM-IRIS. These data suggest that CM patients with decreased innate (IL-6, IL-8, TNF-α) and adaptive inflammatory cytokines (IFN-γ), at CM diagnosis are at higher risk of developing CM-IRIS. Elevation of the inflammatory cytokines, Th1 (IFN-γ), Th17 (IL-17) and the innate cytokines (TNF-α) occurs during CM IRIS. Notably, two studies suggest that increased expression of the chemokines, CCL2 and CCL3 at CM diagnosis is associated with risk of CM-IRIS [71, 80*]. Evaluating immunologic changes concurrently in CSF and blood sampled at CM diagnosis, after ART initiation, at CM-IRIS and after the IRIS event from patients with CM-IRIS compared with matched controls without CM-IRIS would identify the immunologic signature that defines the risk and immunopathogenesis of CM-IRIS.

Table 4b.

Cytokine/Chemokine Profiles in Blood and CSF among patients with Cryptococcal Meningitis and Cryptococcal IRIS by HIV Serostatus.

Reference	Patient Description	CSF Cytokines/Chemokines	Serum/Plasma Cytokines
Boulware et al, 2010 [19, 36]	HIV Positive at CM-IRIS (n=33)	↓IFN-γ, IL-6, IL-8, TNF-α at baseline ↑TNF-α, ↑IL-17, ↑VEGF, ↑IFN-γ at CM- IRIS event	↑IL-4, IL-17 at baseline ↓ TNF-α, G-CSF, GM-CSF, VEGF at baseline
	HIV Positive at CM diagnosis (n=85)	-	↓ G-CSF, ↓ IFN-γ, ↓ TNF-α, ↑MCP-1
Bicanic et al, 2009 [16]	HIV Positive at CM-IRIS (n=11)	-	↓IFN-γ, ↓TNF-α ↑IL-6 vs HIV Positive at CM diagnosis
	HIV Positive at CM diagnosis (n=65)	-	-
Chang et al, 2013 [22]	HIV Positive with CM-IRIS (baseline) (n=27)	↑CCL2, CCL3, CXCL10 in CSF compared to blood	-
	HIV Positive without CM-IRIS (baseline) (n=63)	-	-
Mora et al, 2015 [78]	HIV Positive at CM diagnosis (n=30)	↑IL-2, IL-4, IL-8, IL-10, IFN-γ, TNF-α, IL-17 compared to HIV negative ↑IL-2, IL-8, IL-17, IFN- γ, TNF-α vs HIV Positive no CM	↑IL-2, IL-8, IL-10, IFN-γ, TNF-α, IL-17 compared to HIV negative and HIV positive no CM
	HIV Positive No CM (n=56)	-	↑IL-4
	HIV Negative (n=48)	-	↑IL-12p40
Chaka et al, 1997 [34]	HIV Positive at CM diagnosis (n=16)	↑IL-1β, IL-6, IL-8, IL-10 compared to controls without CM	-
Jarvis et al [71]	HIV Positive at CM diagnosis (n=90)	↑IL-6, IL-8, IFN-γ, CCL2, CCL3 ↓IL-2, IL-4, IL-12	-
Meya, et al [73*]	HIV Positive at CM-IRIS (n=11)		↑IL-6, TNF-α-producing monocytes compared to controls without CM-IRIS
Naranbhai et al, 2014 [79*]	HIV Positive at CM Diagnosis (n=23)	↑CXCR3, CX3CR1, CXCL10, ↓TNF-α in CSF compared to blood	-

Comparison of different studies describing cytokine profiles in CSF and serum for HIV negative patients with cryptococcal meningitis, HIV positive patients with and without IRIS. Abbreviations: IL- Interleukin; IFN- Interferon; TNF- tumor necrosis factor; IRIS- immune reconstitution inflammatory syndrome; VEGF- Vascular endothelial growth factor, G-CSF- granulocyte colony-stimulating factor; GM-CSF- granulocyte-macrophage colony-stimulating factor; CCL- chemokine (C-C motif) ligand; CXCL- chemokine (C-X-C motif) ligand.

Naranbhai et al show during CM, natural killer (NK) cells are involved in CNS immune regulation during the primary response to cryptococcal infection via elevated expression of the chemokines CXCR3, CX3CR1 and diminished TNF-α in CSF [79*]. We have demonstrated elevated frequencies of TNF-α- and IL-6-producing monocytes at CM-IRIS compared with ART matched controls without CM-IRIS [73].

The timing of ART initiation in relation to diagnosis and therapy of CM may affect the incidence of CM-IRIS [8, 18, 19, 65, 67, 68*, 70], however, the increased incidence of cryptococcal IRIS with earlier ART initiation has not been consistent across cohorts. The excess deaths occurring with earlier ART initiation may be distinct IRIS events although clinically indistinguishable from deterioration due to cryptococcosis.

Phase 2: Innate cell activation

Wiesner and Boulware propose that in the early stages of ART immune recovery, in the absence of an effective T cell response, foreign antigens generate ongoing pro-inflammatory signaling by antigen presenting cells, demonstrated by increasing serum IL-6 and C reactive protein in the weeks prior to the IRIS event [19, 81]. The corresponding effector response is delayed until a cytokine storm predominated by a Th1 response occurs at the time of IRIS in blood and CSF [19, 36]. Similarly, Barber and colleagues suggest that, prior to ART, killing of intracellular pathogens by innate phagocytes is defective in the absence of IFN-γ signaling from CD4⁺ T cells [82**]. They further propose that partially primed myeloid cells accumulate with disease progression and the creation of a hyperresponsive state of the innate immune cells. Upon immune reconstitution of CD4⁺ T cell help with ART, broad activation of these myeloid cells results in the production of pro-inflammatory mediators, eliciting the immunopathology defining IRIS. Thus, IRIS is thought to result from uncoupling of the innate and adaptive immune axes following repletion of HIV-associated CD4⁺ T cell number and function with ART.

Differences in chemokine and cytokine expression occur in CSF and blood compartments pre-ART and during CM-IRIS with increased expression of CCL2 (monocyte chemo attractant protein-1, MCP-1) and CCL3 (macrophage inflammatory protein 1-α, or MIP 1-α) [80*]. These chemokines recruit myeloid cells into the CNS, suggesting monocyte recruitment into the CSF occurs during CM-IRIS. CCL2 and CCL3 were also higher in CSF of patients who remained culture positive following antifungal therapy, reflecting ongoing innate antigen presenting cell signaling among this culture-positive group with a higher risk of developing CM-IRIS. More recently, Jarvis et al have demonstrated that elevated CCL2 and CCL3 levels in CSF, low peripheral CD4⁺ T cells and low CSF white cell numbers at CM diagnosis were predictive of CM-IRIS [71]*. The authors suggest that over recruitment of myeloid cells into the CNS could lead to exaggerated immune responses that drive the immunopathology observed during CM-IRIS. Three subsets of monocytes are known to be present in blood depending on their degree of expression of the CD14 surface marker: classical monocytes (CD14⁺⁺ CD16⁻), intermediate monocytes (CD14⁺⁺CD16⁺) and the non-classical monocyte (CD14⁺CD16⁺⁺ monocyte) [83]. We have recently demonstrated differences in phenotype and activation of monocytes and CD4⁺ T cells in blood compared to CSF during CM-IRIS, with the more pro-inflammatory intermediate monocytes predominating in the CSF during CM-IRIS [28*]. Additionally, mortality in the early initiation of ART arm of the Cryptococcal Optimal ART Timing (COAT) trial was associated with elevated CCL2/CCL3 and macrophage/microglial activation in the CSF [84*]. We surmise that localized immune responses, particularly monocyte innate immune responses, are involved in the immunopathology during CM-IRIS.

Phase 3: Immune dysregulation

Almost every constituent of the Immune system is deranged during very advanced HIV infection, the time at which CM occurs (CD4+ T cells 15–30/uL).

Regulatory T cells

Regulatory T cells (Treg) downregulate immune responses to foreign antigens to prevent collateral damage from inflammatory responses [39]. However, a dampened immune response could also have the detrimental effect of delaying antigen clearance during acute infection [85]. A decrease in regulatory T cells at IRIS is thought to predispose to dysregulated effector responses. A higher proportion of regulatory T cells among subjects who developed CM-IRIS compared to healthy controls at baseline was found in one study [86] while substantial quantitative expansion of these cells with defective immunosuppressive function was found in another study during IRIS [87]. Several studies show no association between decreased proportions of regulatory T cells, including the T effector/T regulatory ratio, and IRIS [86, 88, 89] suggesting that these cells may have a limited role in the pathogenesis of CM-IRIS, however, anti-TNF-α molecules in cryptococcal IRIS enhance Treg and Th2 predominance over Th1 responses [90*]. Further studies focusing on the functional role of regulatory T cells during CM-IRIS are warranted.

Innate Immune Axis

Gene expression signatures related to infection and inflammation in circulating monocytes prior to ART initiation and at TB IRIS [91*, 92] demonstrate that increased numbers and activation of the classical monocyte subset appear to functionally predispose to and mediate the development of TB-IRIS with increased expression of IL-1β, IL-6 and TNF during TB-IRIS [93**]. Monocytes represent a smaller minority population in the CSF (8–10% of mononuclear cells [46, 94]) in healthy adults. However, during CM-IRIS, CSF monocyte subsets shift from classical to an intermediate/pro-inflammatory subset, with elevated programmed death ligand-1 (PD-L1) expression on monocytes and NK cells [28*]. The absence of non-classical monocytes in patients with CM pre-ART is associated with the development of CM-IRIS possibly due to poor fungal clearance. Notably, robust IL-6 and TNF-α responses to IFN-γ stimulation ex vivo occurred in patients experiencing CM-IRIS compared to controls [73*]. Two case reports showed improvement of cryptococcal IRIS in a solid organ transplant recipient and in an HIV-infected patient with an IRIS-related cryptococcoma after using adalimumab, which blocks human TNF-α [95*, 96]. Taken together, these data corroborate involvement of the innate immune axis in cryptococcal IRIS.

Lymphopenia-induced proliferation

Space-driven homeostatic compensatory T cell proliferation occurring soon after ART initiation could link the innate and adaptive immune responses that are observed at CM-IRIS. Lymphopenia, such as occurs in HIV infection drives the homeostatic proliferation of memory-like T cells [97, 98]. Spontaneous proliferation could be induced by high-affinity major histocompatibility complex (MHC)/peptide interactions occurring in the presence of residual cryptococcal antigens and monocyte-derived IL-6 [99–102]. Elevated IL-6 and TNF-α-expressing monocytes pre-ART [73*] and an increase in IL-6 prior to development of CM-IRIS [19] appear to confirm a role for IL-6. Spontaneous proliferation causes naïve T cells to acquire the phenotypic and functional properties of effector memory cells [103] that upregulate IFN-γ expression and related Th1 responses that predominate during CM-IRIS as the state of immune suppression is reversed. Additionally, IL-2, IL-4, IL-7 and IL-15 favor memory T cell survival and expansion [87, 104, 105]. It is plausible that IL-6 produced by monocytes drives the proliferation and expansion of antigen specific CD4⁺ T cell memory cells which then provide signaling that fully activates macrophages primed by Cryptococcus in the brain parenchyma, with a resultant spike in inflammation in CSF during CM-IRIS.

That T cells play a pathogenic role in CM-IRIS is supported by both indirect evidence from cytokine studies both in blood and CSF, prior to ART initiation and at the time of IRIS [19, 36] as well as direct evidence of an influx of CD4⁺ T cells into the CSF at the time of IRIS [28]. The restoration of memory T cell responses coupled with dysregulated memory responses against existing residual antigen have been considered the source of this exuberant secretion of cytokines resulting in a ‘cytokine storm’ proposed to underlie CM-IRIS [106]. However, the degree of inflammation present in the CSF at time of CM-IRIS for such a ‘cytokine storm’ is not supported by the literature (Tables 4a and 4b). Rather, cellular and cytokine responses in CSF are actually quite similar to the cytokines levels present during initial CM among non- CM-IRIS patients [16, 36]. One potential confounding issue is that just as results in blood likely do not accurately reflect processes in the CSF, CSF may not be the best monitor of IRIS pathology but is merely the most accessible. IRIS pathology may be occurring primarily in the brain tissue, for which CSF is only a surrogate. Autopsy studies may help to clarify these compartmental differences, including the idea that during CM-IRIS, highly differentiated polyfunctional antigen-specific CD4⁺ T cells are expanded, and less so, the CD8⁺ T cells [106].

Effector T cells and Cytokine responses

Dysregulated expansion of activated antigen-specific T cells as severe immune suppression reverses following ART initiation is thought to drive CM-IRIS. A functionally impaired yet predominantly highly activated CD4⁺ T cell effector memory phenotype in blood is characteristic of patients with IRIS prior to and during IRIS episodes [89]. Pre-ART, programmed death-1 (PD-1), an inhibitory receptor that is up-regulated on T cells following antigen stimulation, was elevated on both CD4⁺ and CD8⁺ T cells among subjects who developed IRIS compared with non-IRIS controls [89], suggesting defects in T cell inhibitory control [89]. Further, a robust increase of polyfunctional, highly differentiated effector memory CD4⁺ T cell responses specifically against the underlying pathogen is a distinctive feature of IRIS [106], although T cells reactive with some pathogens may not recover with ART [43*].

Cytokines and chemokines measured in the CSF or blood provide insight into the mechanisms of CM-IRIS from patterns pre-ART or during IRIS events. IFN-γ largely defines Th1 responses with possible contributions from IL-2 and TNF-α whereas IL-4, IL-5, and IL-13 denote Th2 responses. A paucity of Th1 responses in CSF at CM diagnosis, thought to be associated with poor cryptococcal clearance, is considered a risk factor for developing future CM-IRIS [36]. However, an increase in the Th1 response in CSF coincides with an increase in CD4⁺ T cells in CSF with ART during CM-IRIS [28*, 36, 107]. These cytokine responses could be identified both in CSF and in peripheral blood [19, 108]. Of note, localized responses in CSF at the site of the effective response may not parallel those in blood [19, 36]. Understanding these cytokine responses could be useful in developing measurable biomarkers to stratify the risk of developing CM-IRIS and immunologic endpoints for therapeutic intervention studies to treat or prevent CM-IRIS. A proposed integrated model characterizing these features of CM IRIS pathogenesis is highlighted in Figure 1.

Figure 1. Model integrating Innate and Adaptive immune Axes in Cryptococcal IRIS.

An integrated model showing events leading up to CM-IRIS. In the setting of high fungal burden, lack of effective type 1 T helper cell (Th1) response and/or a Th2 predominant response leads to poor cryptococcal clearance with residual cryptococcal antigen. Monocyte trafficking into the CSF occurs with increased expression of the chemokines CCL2 and CCL3.

In the absence of CD4⁺ T cells prior to ART initiation, the innate immune response consists mainly of primed macrophages/monocytes that take up antigen but do not have IFN-γ signaling from Th1 CD4⁺ T cells to become fully activated, thus antigen presenting cells accumulate and express suboptimal levels of innate signaling cytokines (e.g. IL-6). The confluence of factors including IL-6 from the myeloid cells and constant high-affinity MHC-peptide interactions resulting from partially cleared residual cryptococcal antigen in the setting of ART initiation could lead to lymphopenia-induced proliferation, expansion of CD4⁺ T cell memory cells and effective Th1 responses against residual cryptococcal antigen.

Occurring at the same time, IFN-γ signaling from the expanding CD4⁺ T cell population leads to the en masse activation of primed monocytes/macrophages leading to exaggerated innate cytokine responses. These combined responses lead to excessive inflammation, tissue destruction and within the closed CNS space, cryptococcal-IRIS involving the CNS can have morbid consequences.

Future Perspectives

Despite the benefits of ART in HIV-infected patients with CM, a dysregulated immune system after ART initiation poses clinical challenges. As advances are made in understanding the mechanistic pathways for paradoxical CM-IRIS, evidence of innate immune responses, specifically monocytes in the events leading up to paradoxical CM-IRIS is emerging. Our understanding of the effector immune responses involved in the pathogenesis of unmasking CM-IRIS remains to be elucidated.

Earlier initiation of ART soon after the diagnosis of CM when antigen levels are high is associated with worse outcomes [68*]. However, these excess early deaths were not directly related to the development of paradoxical IRIS, at least using current case definitions [51]. This clinical definition may be modified to include innate and adaptive biomarkers as we develop a more mechanistic understanding of the immunopathogenesis of CM-IRIS.

Because the dysregulated expansion of antigen-specific T cells [106] during paradoxical CM-IRIS occurs in the setting of residual cryptococcal antigen, decreasing the fungal burden more rapidly with more effective fungicidal agents in the induction phase of CM treatment is germane. Such treatments may include customized therapy using higher doses of fluconazole and a longer duration of Amphotericin or flucytosine for patients with high fungal burden, use of adjunctive interferon-γ [21], and other antifungal drugs such as sertraline [109*] to more rapidly reduce fungal burden. A more effective approach is to identify subclinical cryptococcal infection prior to ART and intervene preemptively with fluconazole, preventing CM and CM-IRIS, as recommended by the WHO [110]. Immunologic interventions to prevent and manage paradoxical CM-IRIS could inhibit monocyte migration into the CNS using CCR5 inhibitors, IL-6 blockade with siltuximab, inhibit TNF-α inhibitor with adalimumab and steroids.

Conclusions

Cryptococcal IRIS remains a conundrum, for effective prevention, diagnosis and management. A confluence of factors contribute to this syndrome, including initial HIV-associated CD4⁺ T cell depletion with attenuated localized inflammatory responses, followed by chemokine-driven trafficking of monocytes into the cerebrospinal fluid, the recovery and restoration of T cell signaling after initiating ART in the setting of dysregulated innate, adaptive and regulatory immune responses. Our current understanding of CM-IRIS provides further insight into how these cellular interactions could be exploited to predict, prevent and manage CM-IRIS.

Key Points.

• Poor cryptococcal antigen clearance is important in the development of cryptococcal IRIS.

• Monocyte recruitment into the CSF compartment during CM-IRIS may be essential for the subsequent cytokine-driven immunopathology that occurs during CM-IRIS.

• The confluence of residual cryptococcal antigen, chemokine-mediated trafficking of monocytes and ART-mediated CD4⁺ T cell redistribution into the CNS, may spawn exaggerated innate and adaptive immune responses that define cryptococcal IRIS.

• Future therapeutic interventions to prevent or treat cryptococcal IRIS could utilize our current understanding of the cellular kinetics of innate and adaptive cells in the CNS.