Elevated CD40 ligand silences α interferon production in an HIV-related immune reconstitution inflammatory syndrome (2012)

The immune reconstitution inflammatory syndrome (IRIS) occurs in about 10% of HIV-1-infected patients at a median of 12 weeks upon initiation of antiretroviral therapy [1]. Low baseline CD4+ T-cell counts predispose for the occurrence of opportunistic infections like genital herpes, warts, herpes zoster, mycobacterial infections, and Kaposi's sarcoma [1]. Infections with herpes simplex virus type 2 (HSV-2) are most prevalent. The lesions can be treated with aciclovir and imiquimod, an agonist of Toll-like receptor (TLR) 7, but occasionally proliferate to an extensive verrucous mass [2–5]. Imiquimod targets plasmacytoid dendritic cells (PDC), the major producers of type I interferons (IFNs), which are recruited from the peripheral blood to the site of inflammation [6]. So far, it is unclear why IRIS patients develop opportunistic infections despite rapid CD4+ T-cell recovery. We report on a 47-year-old African woman who was diagnosed with HIV-1 in 1998. Details of the case history were reported previously [7]. Despite different antiretroviral regimen since 2000, the patient exhibited fluctuating viral loads between 20 and 23 000 HIV-1 RNA copies/ml, and CD4+ T-cell counts below 100/μl due to poor adherence (Fig. 1a). In April 2009, she was clinically diagnosed with progressive multifocal leukoencephalopathy, which improved upon a new antiretroviral regimen in June 2009. Good adherence resulted in rapid viral load decline and prompt CD4+ T-cell increase. In September 2009, she presented with previously unnoticed painful genital ulcerations (Fig. 1b). Several courses of aciclovir, topical application of imiquimod, and a radical bilateral vulvectomy only temporarily reduced the size and severity of the ulcers.Fig. 1: Clinical and immunological findings in the index patient suffering from an immune reconstitution inflammatory syndrome.(a) Dynamics of viral replication and CD4+ T-cell counts in response to highly active antiretroviral therapy (HAART). The dotted line indicates period of poor adherence. (b) Clinical aspect of the hyperproliferative genital lesions. (c) IFN-α production in PBMC of the P1 and a C1 upon stimulation with CpG-A (ODN6016; 0.75 μmol/l), a synthetic Toll-like receptor 7 agonist (S-27609; 5 μmol/l), and ultraviolet (UV)-inactivated or infected (inf) HSV (106 PFU/ml) for 20 h, analyzed by an IFN-α module set (Ebiosciences, Frankfurt, Germany). (d) Percentage of PDC, identified as cells expressing BDCA 2 and 4 within PBMC of the index patient. (e) Expression of surface markers for migration (CCR7), activation (CD80), and maturation (CD83) on the PDC of the index patient directly after PBMC isolation (baseline) and after stimulation of PBMC with the respective stimuli for 20 h. (f) Levels of sCD40L in consecutive plasma samples of the P1 and four other HIV-1-infected patients on HAART suffering from opportunistic infections. Statistics was performed using Tukey's HSD (honestly significant difference) test. BDCA, blood dendritic cell antigen; C1, control donor; HSV, herpes simplex virus; IFN, interferon; inf, infectious; IRIS, immune reconstitution inflammatory syndrome; P1, index patient; PBMC, peripheral blood mononuclear cells; PDC, plasmacytoid dendritic cells; sCD40L, soluble CD40 ligand.A swab from the hyperproliferative lesions was positive for HSV-2 DNA (RealArt HSV-1/2 PCR kit; Qiagen, Hilden, Germany), without evidence of drug-resistant variants, and human papillomavirus type 54 (HPV-54), using E1 consensus primers [8], sequencing, and GenBank alignement. HPV-54 was not diagnosed previously [7], as the formerly used in-situ hybridization probes could detect HPV strains 6, 11, 16, 18, 31, and 33 only. HPV-54 was first described in a patient suffering from giant verrucous penile carcinoma (Buschke–Löwenstein) [9]. To our knowledge, this is the first case of concomitant HSV-2 and HPV-54 infection. The cases reported so far describe verrucous lesions due to HSV but not HPV infection [2–5]. Abbo et al.[2] reported defective IFN-α responses upon TLR9 stimulation in a case with hyperproliferative HSV-2 lesions. Approved by the ethical committee of the medical faculty, Friedrich-Alexander-Universität Erlangen-Nürnberg (Ref. no. 3375), we stimulated peripheral blood mononuclear cells of our patient in duplicates with TLR7 and TLR9 agonists, resulting in severely impaired IFN-α production (Fig. 1c). Notably, the response to CpG-A (oligodeoxynucleotide, ODN 6016) was below the 10th quantile of 23 untreated HIV-1-infected individuals [10]. Flow cytometry revealed a low percentage of PDC, defined as blood dendritic cell antigen (BDCA) 2+ BDCA4+ cells (0.11%) (Fig. 1d) and CD4+ lineage− CD11c− cells (0.05%); (data not shown). PDC surface markers for activation, maturation, and lymphocyte homing were upregulated on stimulated but also on mock-cultivated PDC, suggesting that PDC were prestimulated in vivo (Fig. 1e). Natural killer (NK) cells responded poorly to TLR stimulation (data not shown). As NK cells are indirectly activated by PDC-derived IFN-α, PDC and NK cell deficits contributed to the patient's disease. Recently, silencing of peripheral IFN-α responses in HIV-1 infection was associated with enhanced interaction of CD40 on the PDC with CD40 ligand (CD40L), a costimulatory molecule upregulated upon immune activation [11]. As CD40L levels transiently increase with the CD4+ T-cell recovery upon antiretroviral therapy, this mechanism may boost IFN-susceptible opportunistic infections in IRIS. In this respect, type I IFNs are important for the control of herpes virus infections [12]. Therefore, we retrospectively analyzed levels of soluble (s) CD40L in the plasma of our patient (P1) and four others suffering from opportunistic infections (P2, herpes zoster; P3, Kaposi's sarcoma; P4, atypical mycobacterial infection; P5, fungal eye infection). Notably, sCD40L levels in eight consecutive plasma samples of P1 after initiation of antiretroviral treatment (range, 12.5–25.3 ng/ml) were significantly higher than in cross-sectional samples of 52 untreated HIV-1 infected patients (range, 0.2–15.8 ng/ml) [11] (P < 0.001). In contrast, P2–P5 exhibited significantly lower sCD40L levels than P1 (P < 0.01) (Fig. 1f), similar to asymptomatic patients on antiretroviral therapy [11]. Thus, suppression of TLR7-induced and TLR9-induced IFN-α production by elevated CD40L levels does not account for all cases of opportunistic infections in IRIS but may have contributed to the unusual and treatment-refractory course in P1. Alternatively, enhanced sCD40L levels may reflect prior in-vivo stimulation caused by HSV-2 and HPV-54 infections. In any case, selected IRIS patients may profit from treatment with recombinant IFN-α. Acknowledgements K.T. and G.B. performed the experiments, K.K. provided diagnostic tools, L.S., E.H., and T.H. looked after the patients, and T.H., K.K., and B.S. wrote the study. All authors have read and approved the text. T.H. and B.S. contributed equally to this study. We thank Bernhard Fleckenstein for continuous support. The CpG-A ODN 6016 was kindly provided by Jörg Vollmer, Pfizer Oligonucleotide Therapeutics Unit – Coley Pharmaceutical GmbH (Düsseldorf, Germany), and S-27609 by Richard Miller, 3 M Pharmaceuticals (St Paul, United Kingdom). This work was supported by the graduate college 1071 (TP B5); the German Research Foundation (SCHM1702/3-1); the Interdisziplinäres Zentrum für Klinische Forschung (TP50); the ‘Akademie der Wissenschaften und Literatur zu Mainz’; and the German Competence Network HIV/AIDS. Conflicts of interest There are no conflicts of interest.