Rubella Virus Infected Macrophages and Neutrophils Define Patterns of Granulomatous Inflammation in Inborn and Acquired Errors of Immunity

Abstract

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.

Keywords: granuloma treatments; granulomatous inflammation; inborn errors of immunity; macrophages; neutrophils; primary immunodeficiency; skin lesion; vaccine-derived rubella viruses.

Figure 1

Characteristics of the patient cohort. Underlying immunodeficiencies (A). Genetic causes (B). Frequency of chronic infections (C). Organs involved in inflammation (D). Granuloma location at onset (E). Age of the patients at the granuloma onset by the IEI type (F).

Figure 2

A structure of M-type RuV-associated cutaneous granuloma and its cellular elements. Histological double immunofluorescent staining of sequential tissue sections (P18) for RVC (red) and one of the cell type markers (green) shows the presence of RVC predominantly in CD206⁺ M2 macrophages (A) and infrequently in CD163⁺ M2 macrophages (B), CD14+ monocytes (C) and in sporadic MPO+ neutrophils (D). Layers of many RVC^- CD3⁺ T cells surround RVC⁺/CD206⁺ granuloma centers (E). Scale bar: 200 µm. Schematic of M-type RuV-associated granuloma pattern (F).

Figure 3

A progression of cutaneous granuloma from M-type to M(n)-type. Histological double staining of sequential tissue slides (P18) for either RVC (red) and one of the cell type markers (green). Non-necrotizing M-type granuloma contains RVC⁺CD206⁺ M2 macrophages in the center with infrequent, mainly RVC^- neutrophils and abundant surrounding RVC^-CD3⁺ T cells (A). Necrotizing M(n)-type granulomas with MPO⁺ and CD3 ⁺ staining of the necrotic centers surrounded by RVC⁺CD206⁺ macrophages (B, C). Notice a rim CD3⁺ T cells located between acellular necrotic center and RVC⁺CD206⁺ macrophages (C). Scale bar: 100 µM. Schematic of M(n)-type RuV-associated granuloma pattern (D).

Figure 4

A structure of N-type RuV-associated cutaneous granuloma and its cellular elements. Histological double immunofluorescent staining of sequential tissue sections of skin biopsy (P21) for RVC (red) and one of the cell type markers (green) shows predominant RVC staining of MPO⁺ neutrophils (A), infrequent RVC staining of CD206⁺ M2 macrophages (B), CD163⁺ M2 macrophages (C), and CD14⁺ monocytes (D). Numerous RVC^-CD3⁺ T cells surround the RVC⁺ neutrophil core (E). Scale bars: 200 µm and 20 µm (inlet). Schematic of N-type RuV-associated granuloma pattern (F).

Figure 5

Biopsy of a newly developed cutaneous lesion (P1). Double histological immunofluorescent staining shows small clusters of RVC⁺CD206⁺ M2 macrophages under the epidermis. Scale bars: 100 µm (top panel) and 20 µm (bottom panel).

Figure 6

A structure of DNI-type RuV-associated granuloma and its cellular elements. Histological double immunofluorescent staining for RVC and either MPO (A), CD206 (B, D), or CD3 (C) showing RVC⁺MPO⁺ neutrophils, RVC⁺CD206⁺ macrophages with globular RVC likely in a phagosome (yellow arrow) and abundant RVC^-CD3⁺ T cells in the inflamed GI tissue of P16 (A–C). RVC⁺ neutrophil phagocytized by CD206⁺ macrophage (D). Scale bars: 100 µm (A-C), 20 µm (inlets), and 2 µm (D). Schematic of DNI-type RuV- associated granuloma pattern (E).

Figure 7

RuV in brain neutrophils. Histological double immunofluorescent staining for RVC (A–D) and either MPO (A, D), vWF (B) or CD206 (C), showing RVC⁺ neutrophils in the lumen of blood vessels, perivascular cuff, and around the area of hemorrhage (yellow arrows) in P12 brain (A, B). Numerous RVC⁺ neutrophils surrounding RVC^-CD206⁺ granuloma in P27 brain (C, D). Notice a colocalization of MPO granules with RVC (Dii). Scale bars: 20 µm (A, B, Ci, Di). 50 µm (C, D), and 2 µm (Dii).

Figure 8

RuV in bone marrow. Histological immunofluorescent staining of bone marrow core biopsies of P4 (A) and P21 (B) for RVC (A, B) and either for CD206 (A) or MPO (B) shows the presence of RuV in M2 macrophages (A) and neutrophils (B). Scale bars: 20 µm (A) and 100 µm (B).

Figure 9

Neutrophil recruitment to inflamed tissues and formation of different patterns of RuV associated inflammation (a proposed model). After maturation, RuV infected mature neutrophils leave the bone marrow where RuV subclinically persisted in myeloblasts before weakening of the immune control mechanisms. The circulating neutrophils recognize signs of ongoing inflammation and migrate into the tissue. The location of the inflammatory signal and microenvironment at the site influence the formation of a particular pattern of RuV-associated granulomas. In skin, tissue macrophages become infected with RuV either by extracellular virions released by neutrophils or after ingesting infected neutrophils. They subsequently differentiate into epithelioid CD206⁺ macrophages. T cells, which are recruited to the infected tissues but cannot eradicate RuV infection, form a structure of a mature granuloma around the RuV⁺ macrophage core together with migrating monocytes and macrophages. Presence of B lymphocytes and neutrophils in the mature M-type RuV-associated granulomas is negligible. Some individuals develop N-type necrotizing granulomas with RuV neutrophil cores, but the underlying mechanism is unclear. Influx of RuV infected neutrophils into inflamed internal organs and tissues attracts immune cells of the innate and adaptive immunity but does not result in the formation of compact granuloma structures. Instead, diffuse inflammation pattern dominated by RuV positive neutrophils intermixed with other immune cells (DNI-type) is formed.