Brief report: alternative activation of laser-captured murine hemophagocytes.

OBJECTIVE: Hemophagocytes (HPCs) are activated macrophages that have engulfed other hematopoietic cells. Although HPCs are rarely identified in normal spleen tissue and bone marrow, an excess of these macrophages characterizes many cytokine storm syndromes, particularly macrophage activation syndrome and hemophagocytic lymphohistiocytosis. This study was undertaken to assess the functions of HPCs and their significance in acute inflammatory conditions. METHODS: HPCs were generated in wild-type mice using repeated stimulation with Toll-like receptor 9 (TLR-9) and interleukin-10 receptor blockade. RNA was extracted from HPCs that had been isolated by lasercaptured microdissection. Transcriptional profiles of the HPCs were then compared to those of resting splenic macrophages. In addition, bone marrow samples were obtained from a diverse cohort of patients in whom excess hemophagocytosis was identified by clinical bone marrow biopsy or aspiration. The bone marrow samples were analyzed by immunohistochemistry for markers of classic (CD64) or alternative (CD163 and CD206) macrophage activation. RESULTS: Differential gene expression and gene set enrichment analyses of murine HPCs identified upregulation of genes and gene sets associated with alternative activation of HPCs. Immunohistochemical analyses of HPCs in human bone marrow samples showed universal staining of HPCs for CD163, but rarely for CD206 or CD64. CONCLUSION: Laser-captured murine TLR-9­ induced HPCs had a transcriptional profile similar to that of alternatively activated macrophages. In addition, HPC expression of CD163 was confirmed in a uniquely diverse cohort of patients with hemophagocytic syndromes. Collectively, these data support the hypothesis that HPCs have both immunoregulatory and clean-up functions.

Stress-induced changes in gene interactions in human cells.

Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.

Defective actin accumulation impairs human natural killer cell function in patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. OBJECTIVES: We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. METHODS: A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. RESULTS: DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. CONCLUSIONS: DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.