Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies.
To the Editor: Cutaneous granulomas are a well recognized pathologic feature in patients with various primary immunodeficiency diseases (PIDs) and may be self limited or can progress to a persisting granulomatous disorder. Rubella virus (RV) vaccine strain RA27/3 has been recently detected in disseminated cutaneous granulomas of 2 patients with ataxia telangiectasia (AT) and a patient with Simpson Golabi Behmel syndrome (who had combined immunodeficiency [CID] However, a more detailed study of a larger series of granuloma cases in patients with different PIDs was required to confirm and extend this observation. Patients with cutaneous granulomas and with diverse PIDs were selected from the United States Immune Deficiency Network registry. Additional cases (cases 1 and 11) were recruited from the Clinical Immunology Society immune deficiency Listserv. Presence of RV in granuloma containing tissue samples (Fig 1, A C) was examined by immunofluorescence staining with 2 different RV capsid specific antibodies (see this article's Methods section in the Online Repository at www.jacionline.org) by a reader blinded to the diagnosis. Seven out of 14 patients (50% exhibited positive RV antigen staining (Table I) whereas the tissue samples of 5 non PID granuloma patients were negative. Staining intensity varied substantially between patients and did not correlate with the severity of granulomatous disease. RV immunostaining was typically observed in both epidermis and granulomas in dermis (Fig 1, D F) however, staining only in granulomas (case 3) was seen (Table I) Multiple granulomas within a sample contained RV antigen with typically a few positive cells in the middle except cases 2 and 6, in which virtually all cells in the granulomas were positive. RV was found exclusively in patients with CIDs: CID cause unknown (n 5 2) AT (n5 4) and cartilage hair hypoplasia (n5 1) The immune deficiencies in which granulomas were not found to be positive for rubella were common variable immune deficiency (n 5 2) AT (n 5 2) X linked agammaglobulinemia (n 5 1) MardenWalker syndrome (n 5 1) and nuclear factor kappa B essential modulator (n 5 1) None of the nonimmune deficient samples was positive. RV positive cells in granulomas were positive for CD14 and CD68, markers of monocyte/macrophage cell lineage, and CD206 and CD163, activation markers for M2 macrophages, but negative for iNOS, an M1 macrophage marker (see this article's Methods section; see Table E1 in this article's Online Repository at www.jacionline.org; Fig 1, G H) Endothelial cells (vWF) T cells (CD3) B cells (CD20) dermal Langerhans (CD1a) and dendritic (CD11c) cells were negative for RV antigen (see this article's Results section in the Online Repository at www.jacionline.org; Table E1) These results demonstrate that M2 macrophages were the cell type harboring RV antigen in granulomas. There was a high production of cytokeratin in many RV positive keratinocytes, suggesting that RV replication in keratinocytes can lead to dysregulation of keratin synthesis (Fig 1, I) Overexpression of keratin is known to alter the architecture of the epidermis and impact healing of ulcers. The RV immunostaining patterns were unchanged in the 3 skin samples obtained within a 7 year period (case 1) indicating longterm persistence of RVantigen. Antigen persistence is a hallmark of granulomas. Biopsy specimens collected from different body sites contained both RV positive and RV negative specimens (case 3) indicating focal distribution of RVantigen positive cells in patient tissues. In addition to the skin samples from case 6, a bone periosteum tissue (collected 5 years later) contained RVpositive M2 macrophages in the granulomas. PCR fragments covering the entire genome were amplified and sequenced from 1 patient. Phylogenetic analysis revealed that rubella virus of genotype 1a was present in the patient skin (RVs/ Oulu.FIN/22.15/PID; see Fig E1 in this article's Online Repository at www.jacionline.org) The sequence was similar (97.4% identity) to that of the RA27/3 vaccine virus. In RVs/Oulu.FIN/ 22.15/PID, 2 out of 7 RA27/3 specific amino acid residues had reverted to the wild type (see this article's Results section; see Table E2 in this article's Online Repository at www.jacionline.org) There were 69 amino acid substitutions in RVs/Oulu.FIN/22.15/ PID compared with RA27/3; 52 of them were not found in wild type RV genomes (see Table E3 in this article's Online Repository at www.jacionline.org) Most neutralizing epitopes, which are located in the E1 protein, were conserved including the immunodominant epitope E1214 233, whereas each of 3 known CD8 T cell epitopes (largely predicted to be A2 binding) all located in the capsid, contained single mutations (see this article's Results section; see Table E4 in this article's Online Repository at www.jacionline.org) suggesting a role for CD8 T cell selective pressure in viral evolution during chronic RA27/3 infection. Similar to the previous report, ATwas the most common single diagnosis among RV positive patients. We have also identified additional PIDs (cartilage hair hypoplasia and CID) in which RV was found in granulomas. Thus, current data have clearly shown an association between defects in T cell immunity, granulomas, and RV. One of the important findings of this study is the identification of RV positive cells in granulomas as M2 macrophages. ProinflammatoryM1macrophages play an essential role in eliminating pathogens, whereas anti inflammatory M2 macrophages are crucial for maintaining tissue homeostasis. RV can infect most cell types and can persistently infect a macrophage like cell line, but it is currently unknown whether this occurs in vivo. Another novel finding is persisting RVantigen in epidermal keratinocytes, in all epidermal layers except the basal layer. In contrast, in acute postnatal rubella cases, rubella antigen was found only in the deep dermis (cell types not defined) in skin biopsies from rubella rashes, whereas the epidermis was negative. Wild type RV can establish persistent infections and cause disease in immunologically normal individuals if infection occurs in immune privileged sites, for example, Fuchs' uveitis or fetal development. A role for vaccine virus in Fuchs' uveitis is also suspected. Granulomas have been reported only once in CRS. We hypothesize that the immune deficiency allows persistence of the attenuated virus, polarization of macrophages to M2 occurs,
The Journal of allergy and clinical immunology