Asthma is a heterogeneous disease with neutrophilic and eosinophilic asthma as the main endotypes that are distinguished according to the cells recruited to the airways and the related pathology. Eosinophilic asthma is the treatment-responsive endotype, which is mainly associated with allergic asthma. Neutrophilic asthma is a treatment-resistant endotype, affecting 5–10% of asthmatics. Although eosinophilic asthma is well-studied, a clear understanding of the endotypes is essential to devise effective diagnosis and treatment approaches for neutrophilic asthma. To this end, we directly compared adjuvant-induced mouse models of neutrophilic (CFA/OVA) and eosinophilic (Alum/OVA) asthma side-by-side. The immune response in the inflamed lung was analyzed by multi-parametric flow cytometry and immunofluorescence. We found that eosinophilic asthma was characterized by a preferential recruitment of interstitial macrophages and myeloid dendritic cells, whereas in neutrophilic asthma plasmacytoid dendritic cells, exudate macrophages, and GL7+ activated B cells predominated. This differential distribution of macrophage and dendritic cell subsets reveals important aspects of the pathophysiology of asthma and holds the promise to be used as biomarkers to diagnose asthma endotypes.

The clear and unequivocal identification of immune effector functions is essential to understand immune responses. The cytokine IL-10 is a critical immune regulator and was shown, for example, to limit pathology during various lung diseases. However, the clear identification of IL-10-producing cells is challenging and, therefore, reporter mouse lines were developed to facilitate their detection. Several such reporter lines utilize GFP, including the IL-10GFP (VeRT-X) reporter strain studied here. In line with previous reports, we found that this IL-10GFP line faithfully reports on the IL-10 production of lymphoid cells. However, we show that the IL-10GFP reporter is not suitable to analyse IL-10 production of myeloid cells during inflammation. During inflammation, the autofluorescence of myeloid cells increased to an extent that entirely masked the IL-10-specific GFP-signal. Our data illustrate a general and important technical caveat using GFP-reporter lines for the analysis of myeloid cells and suggest that previous reports on effector functions of myeloid cells using such GFP-based reporters might require re-evaluation.

ObjectiveA subset of patients with seropositive rheumatoid arthritis (RA) do not mount a C‐reactive protein (CRP) response during flares. We hypothesize that these patients are more likely to experience poor clinical care and less likely to respond to traditional therapy. This study questioned whether this presentation was associated with worse disease outcome and distinct immunological features.MethodsUsing Power Doppler ultrasound, 48 RA patients with active synovitis were recruited; 30 had normal (n)CRP (5 mg/L or less) and 18 had high (h)CRP (more than 5 mg/L) levels. All had equivalent disease burden assessed by other clinical and laboratory parameters.ResultsTime to diagnosis and time to first disease‐modifying antirheumatic drug were significantly longer in nCRP compared with hCRP patients (P < 0.05). Significantly more nCRP patients needed escalation to biologics after 2‐year follow‐up (P = 0.01). The inflammatory milieu was also different between the two subgroups. Synergy between inflammatory cytokines observed in hCRP patients was lost in nCRP patients, and nCRP patients had significantly increased regulatory T‐cell (Treg) frequencies that correlated positively with predictors of poor disease outcome. Conversely, hCRP but not nCRP patients demonstrated a significant upregulation of alternative complement pathway factors that correlated negatively with Treg frequency.ConclusionPatients with nCRP during flares of RA had an altered immunological profile compared with hCRP patients and experienced diagnostic delays and responded less favorably to conventional treatment.

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.

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. This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. 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+ levels can boost cytokine production by both mouse and human lymphoid cells.