Comprehensive Evaluation of 11 Cytokines in Premature Infants with Surgical Necrotizing Enterocolitis

<div><p>Objective</p><p>A prospective study to investigate the pattern of pro- and anti-inflammatory cytokine responses in neonates with surgical necrotizing enterocolitis (NEC) and identify those cytokines being the most promising for future research.</p> <p>Methods</p><p>A panel of 11 different cytokines were measured in 9 infants with proven NEC and compared with 18 age-matched healthy neonates.</p> <p>Results</p><p>The serum concentrations of the interleukins (IL)-6, IL-8, and IL-10 were significantly (32–fold to 56-fold) higher in NEC infants compared with controls. In contrast, IL-5, IFN gamma, IL-4 and IL-2 showed slightly (1.4-fold to 5.9-fold) lower levels in the NEC samples. However, these cytokines showed a very low absolute concentration in infants with NEC and in controls. The sum of the serum concentrations of IL-6, IL-8 and IL-10 was able to clearly separate infants with NEC from control samples. IL-1 beta and TNF-alpha showed no statistically different levels. The serum levels of TNF-beta and IL-12p70 were below the detection limit in more than 50% of all samples per group.</p> <p>Conclusion</p><p>In spite of strong local inflammation only three out of eleven cytokines (IL-6, IL-8, and IL-10) showed strongly increased serum levels indicating an important role of them in the pathogenesis of NEC. At least two of these three cytokines were elevated in every single NEC patient. Thus, longitudinal monitoring of combined IL-8, IL-6, and IL-10 levels could reveal their potency in being clinical relevant markers in NEC.</p> </div

Clinical characteristics of infants with surgical NEC recruited for cytokine analysis.

<p>Patient nr.4 did not receive surgical treatment and was excluded.</p

Neonatal characteristics of infants with NEC versus age matched controls.

a<p>median values; ^b at the time of diagnosis.</p

Heat map and unsupervised hierarchical clustering of 9 NEC patients and 18 healthy age matched controls according to the serum levels of IL-6, IL-8, and IL-10.

<p>The colored heat map indicates the relative serum level of the respective interleukin: red represents the 0 percentile, black the 50% percentile and green the 100% percentile. The clustering was based on a median linkage analysis of the non-transformed serum levels using the Euclidean distance between cases (vertical cluster) or parameters (horizontal cluster). The numbers beneath the heat map indicates the summation of the serum concentration of all three cytokines (in pg/ml).</p

Serum concentrations of different interleukins in NEC patients (n = 9) and healthy age matched controls (n = 18).

<p>The median values of the respective group are indicated in the graph. Serum concentrations of Interleukin-12 (NEC and control group) and TNF-beta (NEC group) were below detection limit. The p-value was calculated using a Student's t-test.</p

In Situ Characterization of Tissue-Resident Immune Cells by MALDI Mass Spectrometry Imaging

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain <i>m</i>/<i>z</i> values have the same spatial distribution as CD3⁺ T lymphocytes in the lymphoid follicular structures or as CD206⁺ macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M₁ and anti-inflammatory M₂ phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the <i>m</i>/<i>z</i> values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells