T-cell reconstitution during murine acquired immunodeficiency syndrome (MAIDS) produces neuroinflammation and mortality in animals harboring opportunistic viral brain infection
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Highly active antiretroviral therapy (HAART) restores inflammatory immune responses in AIDS patients which may unmask previous subclinical infections or paradoxically exacerbate symptoms of opportunistic infections. In resource-poor settings, 25% of patients receiving HAART may develop CNS-related immune reconstitution inflammatory syndrome (IRIS). Here we describe a reliable mouse model to study underlying immunopathological mechanisms of CNS-IRIS.
Utilizing our HSV brain infection model and mice with MAIDS, we investigated the effect of immune reconstitution on MAIDS mice harboring opportunistic viral brain infection. Using multi-color flow cytometry, we quantitatively measured the cellular infiltrate and microglial activation.
Infection with the LP-BM5 retroviral mixture was found to confer susceptibility to herpes simplex virus (HSV)-1 brain infection to normally-resistant C57BL/6 mice. Increased susceptibility to brain infection was due to severe immunodeficiency at 8 wks p.i. and a marked increase in programmed death-1 (PD-1) expression on CD4+ and CD8+ T-cells. Both T-cell loss and opportunistic brain infection were associated with high level PD-1 expression because PD-1-knockout mice infected with LP-BM5 did not exhibit lymphopenia and retained resistance to HSV-1. In addition, HSV-infection of MAIDS mice stimulated peripheral immune cell infiltration into the brain and its ensuing microglial activation. Interestingly, while opportunistic herpes virus brain infection of C57BL/6 MAIDS mice was not itself lethal, when T-cell immunity was reconstituted through adoptive transfer of virus-specific CD3+ T-cells, it resulted in significant mortality among recipients. This immune reconstitution-induced mortality was associated with exacerbated neuroinflammation, as determined by MHC class II expression on resident microglia and elevated levels of Th1 cytokines in the brain.
Taken together, these results indicate development of an immune reconstitution disease within the central nervous system (CNS-IRD). Experimental immune reconstitution disease of the CNS using T-cell repopulation of lymphopenic murine hosts harboring opportunistic brain infections may help elucidate neuroimmunoregulatory networks that produce CNS-IRIS in patients initiating HAART.
KeywordsHerpes Simplex Virus Adoptive Transfer Immune Reconstitution Immune Reconstitution Inflammatory Syndrome Brain Infection
Central nervous system
Chemokine (C-X-C motif) ligand
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Highly active antiretroviral therapy
Hank’s balanced salt solution
Hypoxanthine guanine phosphoribosyl transferase-1
Herpes resistance locus
Herpes simplex virus
Inducible nitric oxide synthase
Immune reconstitution or restoration disease
Immune reconstitution inflammatory syndrome
Murine acquired immunodeficiency syndrome
Immune reconstitution syndrome
Murine acquired immunodeficiency syndrome
Major histocompatability complex
Murine leukemia virus
Peripheral blood mononuclear cells
Red blood cells
Quantitative polymerase chain reaction
Roswell Park Memorial Institute
Highly active antiretroviral therapy (HAART) has transformed HIV-induced disease from a fatal infection to a chronic yet manageable condition. HAART has led to dramatic reductions in plasma viral load, improvement in CD4+ T-cell counts, and partial restoration of overall immune function. These immunological changes correlate with reduced frequency of opportunistic infections (OI) and prolonged survival [1, 2]. However, a subgroup of patients experience paradoxical clinical deterioration as a consequence of rapid, dysregulated restoration of antigen-specific immune responses following the initiation of antiretroviral treatment . This was first noted following introduction of zidovudine monotherapy in the early 1990s, when localized forms of Mycobacterium avium-intracellulare infection were observed in association with recovery rather than failure of cellular immune responses . Over the past two decades, symptomatic deterioration in patients initiating HAART has been described in relation to a number of pre-existing subclinical infections, inflammatory disorders, and autoimmune diseases. This phenomenon is known by a multitude of names including, immune reconstitution inflammatory syndrome (IRIS), immune reconstitution or restoration disease (IRD), and immune reconstitution syndrome (IRS), however, all these names refer to a disease process produced by the reemergence of functional immune responses, as opposed to disorders due to immune suppression .
IRIS occurs in approximately 20 to 35% of HIV patients treated with HAART , of which an estimated 1% develop central nervous system (CNS)-related IRIS . Up to 28% of patients starting therapy in resource-limited settings may develop CNS-IRIS . The immunopathogenesis of CNS-IRIS is unclear and there is a paucity of literature due to lack of available animal models. Although a wide variety of opportunistic pathogens have been associated with CNS-IRIS, common immunopathological mechanisms are suspected of driving the disproportionate neuroinflammation defining this disease. Proposed mechanisms of CNS-IRIS suggest that dysregulated neuroimmune responses to a variety of antigenic stimuli following initiation of therapy produce disease. The antigenic stimulus in infectious conditions may be either intact viable organisms or dead organisms along with their residual antigens, whereas autoimmune responses to innate antigens are involved in non-infectious causes. The pathophysiology is believed to involve a combination of factors, including reconstitution of immune cell numbers and function, redistribution of lymphocytes, defects in regulatory function, changes in T-helper (Th) cell profiles, underlying antigenic burden, and host genetic susceptibility .
Diagnosis of CNS-IRIS is challenging due to varying severity of clinical presentation as well as limited access to the CNS. The ideal criteria for diagnosing CNS-IRIS include a history of HIV infection, worsening of clinical neurological status, either new neuroradiological findings or deterioration of previous findings unexplainable by previous illness or therapy, a log-fold or greater decrease in viral load, and HAART often with increasing CD4+ cell counts. If available, histopathologic findings demonstrating T-cell infiltrates into the CNS confirm a CNS-IRIS diagnosis .
Because humans are the only natural hosts for HIV, a limited number of approaches are available to study CNS-IRIS. Susceptible strains of mice inoculated as adults with the LP-BM5 murine leukemia virus (MuLV) mixture develop a syndrome termed murine acquired immunodeficiency syndrome (MAIDS). Although not completely analogous to AIDS, many features of MAIDS resemble those observed in individuals infected with HIV, including polyclonal B-cell activation, hypergammaglobulinemia, enhanced susceptibility to infection, profoundly decreased T- and B-cell responses, increased susceptibility to opportunistic pathogens, and the development of terminal B-cell lymphomas [11, 12]. Despite definite differences between LP-BM5 and HIV-1, decades of research have established that LP-BM5 infection in susceptible C57BL/6 mice induces an HIV-like immunodeficiency syndrome, hence the term murine AIDS.
Immunopathogenic mechanisms resulting in CNS-IRIS are difficult to address through clinical studies. In the current study, we explored the possibility of developing a small-animal model to address these mechanisms. We found that C57BL/6 mice infected with LP-BM5 displayed profound lymphopenia and exhausted immune responses as measured by programmed death-1 (PD-1) expression on T-cells, and became sensitive to opportunistic herpes simplex virus (HSV)-1 brain infection. Opportunistic viral infection of the brain was associated with T-cell exhaustion, as PD-1 knockout (KO) mice infected with LP-BM5 retained resistance to opportunistic infection. Further, T-cell reconstitution was found to be lethal in MAIDS mice harboring OI of the brain. This mortality was associated with increased microglial activation and elevated levels of proinflammatory cytokine expression within the brain. This model will be useful in further elucidating the mechanisms responsible for HIV-associated CNS-IRIS.
This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol Number: 1105A99494) of the University of Minnesota.
Viruses and animals
The LP-BM5 retrovirus mixture was procured from the NIH AIDS reagent program (Germantown, MD, USA). LP-BM5 viral stocks were prepared as described previously . Virus stocks used for infection were produced as cell-free supernatants of SC-1 cells. Titers were determined by a standard retroviral XC plaque assay for the BM5eco virus. C57BL/6 (Charles River Laboratories, Wilmington, MA, USA) or PD-1 KO (provided by Sing-Sing Way, University of Minnesota, Minneapolis, MN, USA) female mice were inoculated via the intraperitoneal (i.p.) route with two doses (2 × 104/PFU dose) in 250 μl, with 3 d between doses .
HSV-1 strain 17syn+, a neurovirulent strain of HSV-1, provided by LT Feldman (University of California, Los Angeles, CA, USA) was used in all experiments. The virus was propagated in rabbit skin fibroblasts (CCL68; American Type Culture Collection), sucrose purified, and titered using standard plaque assay. MAIDS mice were challenged intranasally (i.n.) with 2.0 × 105 PFU/mouse at various time points to examine their susceptibility to HSV brain infection.
Monitoring of LP-BM5 infection
Mice infected with LP-BM5 were monitored by physical examination for cervical lymphadenopathy and by bleeding at various time points. Blood collected through facial vein puncture was lysed using RBS lysis buffer (ACK lysing buffer, Life Technologies, Grand Island, NY, USA). Peripheral blood mononuclear cells (PBMC) thus prepared were stained with Mabs CD4-eflour450, CD8-PE-Cy7, PD-1-FITC, CD3-APC-Cy7 (ebioscience, San Diego, CA, USA) and analyzed using flow cytometry.
Isolation of brain leukocytes and fluorescence-activated cell sorting (FACS)
Leukocytes were isolated from different groups of mice infected with either LP-BM5 or HSV, or dual infection using a previously described procedure with minor modifications [14, 15]. In brief, brain tissues harvested from four to six animals were minced finely in Roswell Park Memorial Institute (RPMI) medium 1640 (2 g/L D-glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) and digested in 0.0625% trypsin (in Ca/Mg-free Hank’s balanced salt solution (HBSS)) at room temperature for 20 minutes. Single cell preparations from infected brains were resuspended in 30% Percoll and banded on a 70% Percoll cushion at 900 × g at 15°C. Brain leukocytes obtained from the 30 to 70% Percoll interface were treated with Fc Block (anti-CD32/CD16 in the form of 2.4G2 hybridoma culture supernatant with 2% normal rat and 2% normal mouse serum) to inhibit nonspecific antibody (Ab) binding and were stained with anti-mouse immune cell surface markers for 45 minutes at 4°C (anti-CD45-PE-Cy5, anti-CD11b-AF700, anti-CD4-eflour450, anti-major histocompatability complex (MHC) class II-allophycocyanin (APC), anti-CD8-PE-Cy7, and anti-CD3-APC-Cy7 (ebioscience, San Diego, CA, USA) and analyzed by flow cytometry. Control isotype Abs were used for all isotype and fluorochrome combinations to assess nonspecific Ab binding. Live leukocytes were gated using forward scatter and side scatter parameters on a BD FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Spleen and lymph nodes (cervical, lumbar, mesenteric and inguinal) from HSV-primed (1 × 104 PFU/mouse, i.p. injection) donor animals were collected aseptically at 7 d post-priming. Single cell suspensions of immunocytes were depleted of red blood cells (RBC) by treatment with 0.87% ammonium chloride and washed twice, and cell viability was confirmed using trypan blue. CD3+ lymphocytes were enriched by negative selection using a CD3+ cell purification kit, as per the manufacturer’s instructions (R&D systems, Minneapolis, MN USA). Immune cells were transferred (2 × 106 or 5 × 106 cells/mouse) into MAIDS mice via the tail vein 7 d post-infection (p.i.) with HSV.
Total RNA was extracted from brain tissue homogenates using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). One μg RNA was DNase (Ambion, Applied Biosystems, Austin, TX, USA) treated, and reverse transcribed to cDNA with SuperScript™ III (Invitrogen), dNTP (GE Healthcare, Piscataway, NJ, USA) and oligo (dT)12–18 (Promega, Madison, WI, USA). Real-time PCR was performed in an Mx3000p (Stratagene, La Jolla, CA, USA) with SYBR Advantage qPCR Premix (Clontech, Mountain View, CA, USA), primers and cDNA according to the manufacturer’s protocol. Reaction conditions for quantitative PCR (qPCR) were as follows: initial denaturation at 95°C for 15 sec, amplification for 40 cycles at 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec followed by dissociation curve analysis (one cycle at 95°C for 60 sec, 55°C for 30 sec and 95°C for 30 sec) to verify product specificity. After normalizing to hypoxanthine guanine phosphoribosyl transferase-1 (HPRT-1) expression (Δ cycle threshold (Ct) = target gene Ct - HPRT Ct) and then to the control group (ΔΔCt = treatment ΔCt - C ΔCt), relative quantification using 2∧−ΔΔCt was calculated as fold change of target mRNA expression versus control. Primer sequences for HPRT, HSV glyD, IL-2, IFN-γ, inducible nitric oxide synthase (iNOS), chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 will be available upon request. For HSV glyD expression, samples with Ct values below 35 cycles were identified as positive. Expression was validated by running all samples on a 2% agarose electrophoresis to confirm presence of the PCR product (208 bp).
Brains isolated from HSV-infected MAIDS mice at 7 d p.i. were divided into cortex, sub-cortex, cerebellum, and brain stem. Tissue samples were homogenized in DMEM with 5% FBS and stored at −80°C until they were processed. Fifty μL of tissue homogenate was then inoculated onto previously prepared rabbit skin fibroblast cultures in a 24-well plate. The cultures were maintained in DMEM supplemented with 10% FBS and antibiotics. The cultures were analyzed microscopically for the occurrence of cytopathic effect (CPE) and maintained for a period of 7 d before reporting.
Lymphopenia during chronic murine retroviral infection
Elevated levels of PD-1 on leukocytes during chronic infection
Murine retrovirus-induced acquired immunodeficiency conferred susceptibility to herpes virus brain infection onto resistant C57BL/6 mice
LP-BM5 disease progression increases susceptibility of C57BL/6 mice to herpes simplex virus brain infection
0 (n = 6)
2 (n = 3)
4 (n = 3)
6 (n = 6)
8 (n = 13)
LP-BM5-induced T-cell loss and susceptibility to herpes virus brain infection were mediated through PD-1
LP-BM5-infected programmed death-1 knockout animals retain resistance to herpes simplex virus brain infection
(8 weeks post-infection)
C57BL/6 (n = 8)
PD-1 KO (n = 7)
Dual infection promoted immune cell infiltration into the brain and its associated microglial cell activation
T-cell reconstitution was lethal to MAIDS mice with opportunistic brain infection
Lethal disease was associated with exacerbated neuroinflammation following immune reconstitution
CNS-IRD was independent of PD-1 expression on T-cells
CNS-IRIS is a major emerging health concern due to worldwide accessibility of HAART, along with the prolonged lifespan of HIV-infected individuals. However, the immunopathogenic mechanisms responsible are still poorly understood. Although human studies would be most relevant to address this disease, they are severely limited by numerous factors including availability of tissue samples and variability among patient cohorts. Unfortunately, our understanding of the molecular and cellular interactions driving destructive neuroimmune responses during CNS-IRIS is hampered due to lack of an appropriate animal model. Data presented here provide evidence that OI of MAIDS mice combined with T-cell immune reconstitution provides an accessible animal model for investigating neuropathologic immune reconstitution.
IRIS is most commonly seen in patients with severe CD4+ T cell lymphopenia (<100 cells/mL) at the initiation of treatment, often in the presence of an identifiable OI. In this study, we examined experimental immune reconstitution disease of the CNS using T-cell repopulation of lymphopenic murine hosts harboring opportunistic HSV-1 brain infection and assessed its potential to model CNS-IRIS. Murine models have been used extensively to study the pathogenesis of HSV-1 encephalitis and it is well-established that the genetic background of mice dictates disease outcomes. For example, HSV-1 17+ strain produces encephalitis in BALB/c mice when inoculated through the i.n. route, but the same virus does not produce brain disease in C57BL/6 mice when administered through an identical i.n. route. Resistance of C57BL/6 mice to HSV-1 encephalitis has been attributed to a number of factors including the Hrl on mouse chromosome 6 . Data presented here demonstrated that C57BL/6 MAIDS mice became susceptible to HSV-1 brain infection due to LP-BM5-induced immunodeficiency and an exhausted T-cell phenotype. Previous studies have shown that a lack of coordinated responses from CD8+ T-cells and natural killer cells facilitated HSV-1 spread into brains of C57BL/6 mice . MAIDS models have been extensively used to understand the pathogenesis of opportunistic infections [18, 20, 21, 32]. In our model of LP-BM5-induced immunodeficiency, otherwise resistant C57BL/6 mice became sensitive to opportunistic brain infection.
Although LP-BM5 infection also induces lymphoproliferation within secondary lymphoid organs, we detected a sharp decline in circulating CD4+ and CD8+ T-cells. This finding is similar to a previous result that utilized a Thy1.2 marker to demonstrate loss of T-cells in the blood . Interestingly, the remaining circulating T-cells were found to express high levels of PD-1, suggesting functional exhaustion. It has been demonstrated that inhibitory receptors, such as PD-1, are expressed at elevated levels on both CD4+ and CD8+ T-cells in subjects with chronic HIV-1 infection, and diminished function of these cells may contribute to ineffective control of HIV-1 replication [34, 35]. Furthermore, in a simian immunodeficiency (SIV) model, blockade of the PD-1 pathway in vivo increased SIV-specific T-cell function, decreased SIV viral loads (VLs), decreased opportunistic infections, and increased the life span of infected macaques . Previous studies using the LP-BM5 model have shown that blocking the PD-1 pathway substantially altered MAIDS progression . In this study, we demonstrated that following inhibition of the PD-1/PD-L1 pathway through the use of PD-1 KO animals, lymphopenia associated with LP-BM5 infection was not observed and these KO mice retained their normal resistance to HSV-1 brain infection.
Characteristics of particular inciting pathogens may also affect immunopathology in IRIS. IRIS is most often associated with CD4+ Th1-mediated immune responses; however, both CD4+ and CD8+ effector T-cells are involved. In this study, we describe a new model of reconstitution disease that recapitulates the fundamental immunologic scenario of opportunistic infection-associated CNS-IRIS: T-cell reconstitution of lymphopenic hosts harboring active viral brain infection. Our data suggest that susceptibility to IRD may be a common property of microbial-infected immunodeficient hosts undergoing T-cell reconstitution. Indeed, induction of inflammatory disease has also been described following adoptive transfer of CD4+ T-cells into Pneumocystis carinii–exposed SCID mice  and Mycobacterium-infected lymphopenic mice . We used T-cells from HSV-1-primed mice in adoptive transfer experiments based on our previous findings that only activated T-cells infiltrate brains .
An essential feature of CNS-IRIS is infiltration of the brain with activated T-cells, which occurs in an attempt to control underlying CNS infection. While T-cell transfer into HSV-1–infected MAIDS mice triggers disease, no IRIS-like symptoms were seen in MAIDS mice that harbored herpesvirus brain infection without adoptive transfer despite having some cellular infiltration. These studies show that pathogen-driven neuroimmune responses are important in development of what is termed as “unmasking” CNS-IRIS. Although a wide variety of opportunistic pathogens have been associated with CNS-IRIS, the neuroimmunoregulatory networks involved remain to be elucidated using animal models because they are difficult to address through clinical studies.
Using a Mycobacterial model of IRIS in TCRα−/− mice, it was demonstrated that Ag-specific Th1 responses are deleterious. Rapid induction of serum nitric oxide was observed after transfer of Wt CD4 T-cells (but not IFNγ−/− CD4 T cells) into infected TCRα−/− recipients implying that IFN-γ-inducible downstream cytokines were produced in response to T-cell reconstitution. Similarly, in this study, we observed significantly elevated levels of Th1 proinflammatory mediators within brains of mice receiving T-cells. Indeed, previous studies from our laboratory investigating chronic neuroimmune responses following herpesvirus infections have revealed that brain-resident microglia respond to infiltrating T-cell produced IFN-γ [24, 25]. It has been documented in well-studied cohorts of cryptococcal CNS-IRIS that, at the site of inflammation in the CSF, numerous cytokines are increased, including IFN-γ (2.5-fold increase), TNF-α (threefold increase), and IL-6 (twofold increase) . Here, we also provided evidence of exacerbated microglial activation following T-cell reconstitution along with increased expression of Th1 cytokines in the brains of mice that received T-cells.
Hyperimmune activation, as determined by PD-1 expression on T-cells, has been shown to be a strong predictor of disease progression, and the reduction of hyperimmune activation following anti-PD-1 antibody treatment could contribute to enhanced survival. This phenomenon has been demonstrated in multiple studies in which blocking of the PD-1/PD-L1 pathway was shown to be advantageous, using both SIV  and humanized mouse  models. In the present study, MAIDS mice harboring HSV-brain infection were equally susceptible to immune reconstitution using T-cells obtained from PD-1 KO donors.
In recent years, the MAIDS system has fallen out of favor as a model of HIV-1 infection because B-cells were found to be infected with two of the three viruses in the LP-BM5 mixture, and a B-cell neoplastic disorder develops in the later stages of disease. The caveats of using this model are an inevitably fatal syndrome characterized by splenomegaly, lymphadenopathy, and a progressive loss of B- and T-cell responses to antigens and mitogens. Infected animals become sluggish with enlarged lymph nodes probably creating obstruction and discomfort. Although insights into the pathogenesis of CNS-IRIS emerging from this study may have important implications for the understanding of viral etiology-associated CNS-IRIS, the model itself does not fully explain all of the diverse manifestations of this disease.
Our results reveal that T-cell reconstitution of severely lymphopenic hosts harboring opportunistic viral brain infection can lead to adverse outcomes. Additionally, dual infection alone was not found to be lethal, but T-cell reconstitution produced exaggerated disease, neuroimmune responses, microglial activation, and increased expression of proinflammatory cytokines within the CNS. Taken together, this model of opportunistic viral brain infection is useful to investigate immune reconstitution disease of the CNS.
This project was supported by Award Number MH-066703 from the National Institute of Mental Health.
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