Granzyme B ‐expressing neutrophils correlate with bacterial load in granulomas from M ycobacterium tuberculosis ‐infected cynomolgus macaques

Abstract

Summary The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M. tuberculosis bacilli, M. tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.

Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques

“…Data from mouse TB models indicated massive neutrophil influx into the lungs and elevated IL-10 in the absence of B cells, suggesting possible defects in inflammation control in B cell-deficient animals (4,13). To obtain a more complete picture of the cytokine environment in granulomas, ELISAs were performed on granuloma homogenates to assess the levels of proinflammatory cytokines IL-6, IL-17, and TNF, and for calprotectin, a marker for neutrophils (28). Granulomas from rituximab-treated animals had reduced amounts of IL-6 and IL-10 compared to those from control animals, but the IL-17 and TNF levels were not statistically different between the , although statistically significant, was only marginally lower in the rituximab-treated group.…”

“…At necropsy, single cell suspensions were derived from lung granulomas, uninvolved lung and thoracic draining lymph nodes. Cells from PBMCs and tissue samples were stained for T cells using anti-human CD3 (clone SP34-2; BD Biosciences), CD4 (clone L200; BD Biosciences), and CD8 (clone DK25; Dako), B cells using anti-human CD20 (clone 2H7; eBioscience) and CD79a (clone HM47; BD Pharmingen), neutrophils with CD11b (clone ICRF44; BD Pharmingen), and calprotectin (clone MAC387; Thermo Scientific) (28) and activation marker HLA-DR (clone LN3; eBioscience). Lymphocytes and neutrophils were identified based on size (forward scatter) and granularity (side scatter).…”

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4β1–Expressing Immune Cells

“…Immunohistochemistry (IHC) on 5 μm-thick FFPE tissue sections was performed as previously described (2, 3). For quantitative analysis of cell populations in granulomas, six granulomas were selected for imaging per animal: the three with the highest CPM and the three with the lowest CPM.…”

“…Granulomas are the pathologic lesions associated with TB and contain macrophages, neutrophils, T cells, and B cells in distinct microenvironments (2–4) that limit bacterial dissemination and facilitate bacterial killing. Individual lesions within a single infected host can vary substantially in terms of their ability to kill Mtb (5), and in their immunologic cell components and function (3, 5, 6).…”

Pathology and immune reactivity: understanding multidimensionality in pulmonary tuberculosis

“…Neutrophils have been detected in granulomas in multiple animal models [66,133,135] and in human lesions already in the pre-antibiotic era [61]. Heightened accumulation of neutrophils [82,83], which express granzyme B [136], parallels Mtb numbers and causes intra-granulomatous necrosis [136]. Involvement of neutrophils in lung injury and TB transmission will be discussed in the context of cavity formation in the following.…”