Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1β, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFβ) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

A 17-year-old female patient with hereditary spherocytosis (HS) presented with fatigue, nausea, headache, and fever for 2 days. On physical examination, she had high fever, pallor, icterus, and mild splenomegaly. Peripheral blood examination demonstrated a Hb level of 8.2 g/dL, WBC 1.9 Â 10 9 /L, platelets 74 Â 10 9 /L, MCV 88.9 fL, MCH 33.9 pg, MCHC 38.1 g/dL, and reticulocyte 0.7%. The peripheral blood smear revealed 70% neutrophils, 18% lymphocytes, 10% monocytes and 2% band neutrophils, vacuolization in neutrophils and monocytes; many spherocytes. Bone marrow aspirate showed hypocellularity with many hemophagocytic histiocytes, giant proerythroblasts, and absence of mature erythroblasts, suggesting a parvovirus infection (Fig. 1). On the second day of hospitalization, her Hb level decreased to 5.6 g/dL. Hepatomegaly developed and splenomegaly progressed. Serum ferritin level was 7,478 ng/mL, triglyceride 1.17 nmol/L and fibrinogen 2.31 g/L. Prothrombin and partial thromboplastin time were within normal limits, but the D-dimer level was very high (3,004 mg/L). The serum folic acid level was normal. Parvovirus B19 IgM and IgG was found to be positive by indirect immunofluoroscent assay. Secondary hemophagocytic lymphohistiocytosis (HLH) and aplastic crisis due to Parvovirus B19 infection was diagnosed. She received red blood cell transfusions and intravenous immunoglobulin was given for the HLH. She was discharged from the hospital after a marked clinical and hematological improvement on day 6 with a Hb level of 9.9 g/dL, WBC 4.8 Â 10 9 /L and platelets 198 Â 10 9 /L. Ten days later, her 13-year-old brother, who also had HS, presented with similar clinical features and Parvovirus B19 infection. He improved with red blood cell transfusions and supportive treatment. In patients with hemolytic anemia, with shortened red cell survival time and expanded marrow erythropoiesis, Parvovirus B19 infection can lead to a transient aplastic crisis. Giant proerythroblasts and absence of mature erythroblasts are characteristic findings of parvovirus infection [1][2][3].

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Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αβ T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations (approx. 1.8 mM). This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence the survival of the lymphoid cells. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ concentrations can boost cytokine production by both mouse and human lymphoid cells.