CYTOSPOT: Intracellular Staining and Detection of Cytokines by Flow Cytometer, an Alternative to LYMESPOT.

Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. This methodology has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation, and functional parameters pertaining to responding T cells. This methodology applied after short-term culture of cells, followed by fixation and permeabilization make this technique ideal for the assessment of T-cell immune responses induced by different challenges. Here we describe an intracellular staining method followed by flow cytometry after cell stimulation with immune-relevant antigens for Lyme disease.

Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.

CD163L1 and CLEC5A discriminate subsets of human resident and inflammatory macrophages in vivo.

Macrophages (Mϕ) can be differentiated and polarized in vitro from human CD14(+) monocytes under the influence of GM-CSF (GM-Mϕ) and M-CSF (M-Mϕ). GM-Mϕs are proinflammatory and M-Mϕs have an anti-inflammatory phenotype. We found selective expression of the lectin C-type lectin domain family 5 member A (CLEC5A) transcripts in GM-Mϕs and the scavenger receptor CD163 molecule-like 1 (CD163L1) in M-Mϕs by microarray assay. In vitro, CD163L1 expression was induced by IL-10 and M-CSF and CLEC5A by inflammatory cytokines and cell adherence. In secondary lymphoid organs, their respective expression was restricted to CD68(+)/CD163(+) Mϕs that preferentially produced either TNF (CLEC5A(+)) or IL-10 (CD163L1(+)). Mϕs from healthy liver and colon tissue were mostly CD163L1(+), and CLEC5A(+) cells were scarce. In contrast, CLEC5A(+) Mϕs were abundant in the intestinal lamina propria from patients with inflammatory bowel disease (IBD), with higher numbers of CLEC5A(+)CD163L1(+) found compared with those in secondary lymphoid organs. CLEC5A(+) cells were CD14(+)CD209(-)CD11b(+)CD11c(+)TNF(+)IL-10(+), and single positive CD163L1(+) cells were CD14(-)CD209(+)CD11b(-)CD11c(-)TNF(-)IL-10(+) in healthy donors and had lost the ability to produce IL-10 and to express CD209 in those with IBD. In melanomas, CLEC5A(+) tumor-associated Mϕs (TAMs) were not detected in 42% of the cases evaluated, but CD163L1(+) TAMs were found in 100%. Similar to IBD, CD163L1(+) TAMs expressed high levels of CD209 and produced significant amounts of IL-10, and CLEC5A(+) TAMs were CD14(hi) and produced enhanced levels of TNF in metastases. Overall, these results suggest that CD163L1 expression is associated with tissue-resident Mϕs with an anti-inflammatory or anergic phenotype and that CLEC5A(+) Mϕs exhibit TNF-producing ability and might display a proinflammatory effect.