A cross-sectional study evidences regulations of leukocytes in the colostrum of mothers with obesity

We conducted a cross-sectional study of leukocyte subpopulations in blood and colostrum of mothers with BMI <25 and BMI >30. This study was approved by the Ethics Committee of the Hospital Regional Materno Infantil, Servicios de Salud de Nuevo León, Mexico, and the Institutional Review Board at Escuela de Medicina y Ciencias de la Salud, TecSalud, in Monterrey, Mexico, with the ID CarMicrobioLHum-2018. All samples were collected and used following signed informed consent and anonymization, between October 2020 and March 2021 at the Hospital Regional Materno Infantil, in Nuevo León, Mexico. Briefly, adult mothers between 18 and 34 years of age were invited to participate during the first obstetric consultation occurring during the first trimester of gestation. Participants were allocated to the obese cohort (BMI >30) or lean cohort (BMI <25), according to declared pre-pregnancy weight during the first visit and in accordance with the World Health Organization classification guidelines [26]. Eligibility to participate in the study was determined based on (1) mother’s age between 18 and 34 years, (2) adequate prenatal visits without any adverse event during pregnancy, (3) pre-pregnancy BMI <25 or >30, (4) term infant, and (5) willingness to participate. Exclusion criteria included (1) having received antibiotics anytime during the 3-month period before birth, or having received a prolonged antibiotic treatment (>3 months) anytime during pregnancy; (2) having received immunosuppressive doses of steroids during pregnancy; (3) previous monoclonal antibody treatment; (4) history of chronic disease (outside of obesity); (5) suffering from any dietary disease; (6) episodes of diarrhea during the last 2 weeks of pregnancy; (7) history of surgery within 12 months prior to pregnancy; and (8) history of antineoplastic treatment. Elimination criteria included (1) having received antibiotics for >24 h post-birth, (2) necessity of intensive care unit admission of the neonate, and (3) any additional cause impeding sample collection. Oxytocin was not used during the first stages of labor. However, oxytocin was prescribed in 34 of 41 subjects (84%), during the first 8 h after delivery, as per international recommendations [27].

Regarding the variables of the study, the main independent variable and hypothesized predictor was the BMI, calculated from self-reported pre-pregnancy weight and size. Additional variables collected or measured in this work included participant’s age, primiparity (yes/no), infant gender, gestational age at birth, type of delivery (vaginal/C-section), weight of infant at birth, volume of colostrum obtained, and frequency of leukocyte subpopulations in blood and colostrum samples. Additional details are included in the study’s STROBE statement (Sup. Table 1) [26, 28‐31].

Participants provided blood and colostrum samples on a single occasion, within 2 days of giving birth. Briefly, 3–4 ml of peripheral blood was collected in K2-EDTA vacutainers and placed on ice until processing. Following washing of the breast and nipple area using soap and water, 1–3 ml of colostrum per donor was obtained through pump-assisted milk extraction and immediately stored on ice. All samples were processed and acquired on the flow cytometer within 3 h of collection.

Around 1 ml of colostrum was processed for cell enrichment prior to staining for flow cytometry. Briefly, samples were centrifuged at 400 g for 15 min at 4°C. The supernatant was discarded, and the cell pellet was washed twice with PBS/2% FBS. Cells were counted using trypan blue for viability assessment and aliquoted for flow cytometry staining.

Depending on availability, between 200,000 and 1 × 10⁶ cells were used for staining, and the same number of cells per sample was kept as unstained control. The same antibody lots were used to stain both types of tissues and antibody titration optimizations were performed for each tissue type to optimize resolution of fluorescence intensity over background. Cells were resuspended in the antibody master mix, which consisted of 2.5 μL mouse anti-human CD2-APC (BD® cat. 560642), 5 μL mouse anti-human CD16-APC-H7 (BD® cat. 560195), 5 μL mouse anti-human CD19-V450 (BD® cat. 560353), 2.5 μL mouse anti-human CD36-PE (BD® cat. 555455), 5 μL mouse anti-human CD45-V500 (BD® cat. 560777), and 1.25 μL rat anti-human CD294-Alexa Fluor 647 (BD® cat. 558042), in a final 100-μL staining volume with PBS + 2% FBS per 10⁶ cells. Samples were then incubated for 30 min on ice in the dark, then washed and resuspended in PBS/2% FBS. Ten min before acquisition, propidium iodide (BD® cat. 556463) was added to the tube as per the manufacturer’s recommendations.

Fifty microliters of anticoagulated peripheral blood was stained using 2.5 μL CD2-APC, 1.25 μL CD16-APC-H7, 5μL CD19-V450, 2.5 μL CD36-PE, 1.25 μL CD45-V500, and 1.25 μL CD294-Alexa Fluor 647, in a final 100-μL staining volume with PBS + 2% FBS for 30 minutes on ice, in the dark. Samples were then subjected to erythrocyte lysis using BD® Pharm Lyse solution (BD® cat. 555899) as per the manufacturer’s instructions. Ten minutes before acquisition propidium iodide was added to tubes as per the manufacturer’s recommendations.

Samples were analyzed on a BD® FACSCelesta flow cytometer fitted with 405-nm, 488-nm, and 633-nm lasers and operated through the BD® FACSDiva software v.8. Cytometer settings were checked prior to all acquisition using CS&T beads (BD® cat. 642412) according to manufacturer’s instructions. Compensation controls were prepared using compensation beads (anti-mouse Ig, K Neg Control compensation, BD® cat. 552843) following the manufacturer’s recommendations. At least 30,000 uncompensated events were recorded from every sample, with the forward scatter (FSC) event threshold adjusted to 35,000 for peripheral blood and 28,000 for colostrum samples.

Cytometry data were analyzed using FlowJo software v.10 (Treestar LLC). Automatic compensation was performed prior to analysis, with a compensation matrix generated at each acquisition. A strict quality control workflow was established to ensure the exclusion of suboptimal quality samples that may artificially skew the final analysis (Fig. S1) [32]. Briefly, samples had to exhibit a stable flow stream (Side Scatter (SSC) vs. time), debris was excluded on SSC/FSC plot, sample viability >85% from the singlet gate, and >10,000 leukocytes (CD45⁺ cells) acquired, to be included in the final analysis and comparisons [33]. The gating strategy applied to discriminate the leukocyte populations has been previously described and is summarized in Table 1 [31, 34]. Fluorescence minus one (FMO) controls of colostrum and blood samples were used to adjust gates, which were then applied to all samples.

Proportions of leukocyte subsets were calculated as % of CD45⁺ live cells per sample. Shapiro-Wilk tests were used to investigate data normality with α=0.05. Wilcoxon matched-pairs tests were used to compare intra-individual leukocyte proportions in paired blood-colostrum samples. Mann-Whitney U tests were used to compare leukocyte % and median fluorescence intensity (MFI) of surface markers in colostrum between study groups. Student t-tests were used to compare leukocyte proportions in blood samples. All statistical analyses were performed using GraphPad Prism v. 9, or SPSS® v. 26, IBM Corporation, Armonk, NY, USA. Graphs are showing discrete data and mean with SD and p values in the APA format.

Ten leukocyte subpopulations were confidently identified in peripheral blood using the gating strategy presented in Fig. 1. For all leukocyte proportions measured, there was no difference between the lean and obese cohorts (Fig. 2). Median percentages for each subpopulation, and results of statistical comparisons are presented in Table 3.

The flow cytometry plots from colostrum samples highlighted consistent differences compared to blood, such as increased debris and doublets proportion, and overall decreased viability (Fig. 3). The B lymphocyte fraction was significantly reduced in the obese cohort compared to the lean cohort (Fig. 2 panel I and Table 3, median 0.17% vs. 0.41% in the lean cohort, p = 0.029). The remaining 9 leukocyte subpopulations exhibited similar proportions in colostrum between cohorts. Median % of all leukocyte subtypes for both cohorts are found in Table 3.

Neutrophils were the most abundant leukocytes in both tissue types, as expected [31, 35]. There was no difference in neutrophil proportions between groups in either tissue (Fig. 2, Table 3). However, there were significantly less neutrophils in lean mothers’ colostrum compared to lean mothers’ blood, while this difference was not recapitulated in the obese cohort (p=0.007 vs. p=0.51, respectively, Table 3).

The second highest frequency colostrum leukocytes were non-cytotoxic T cells (medians: 6.95% and 3.81% in lean and obese groups, respectively, Table 3). The frequency of non-cytotoxic T cells in peripheral blood was significantly higher compared to the frequency in colostrum, irrespective of the cohort (Fig. 2, Table 3).

For B lymphocytes, non-cytotoxic T cells, and eosinophils, relative proportions depended on the tissue of origin. Overall, there was a significantly higher fraction of B lymphocytes and non-cytotoxic T cells in peripheral blood compared to colostrum (Fig. 2 I, J and C, D, respectively). On the other hand, there was a significantly higher fraction of eosinophils in colostrum compared to peripheral blood (Fig. 2 S, T). These trends were pervasive across study groups (Table 3). Group-specific differences between tissue were also identified. There were significantly larger fractions of basophils and CD16⁺ monocytes in blood, only in the lean group, and a significantly larger fraction of immature granulocytes in colostrum from the obese group only (Table 3).

The relative size of various leukocyte populations varied significantly between tissues as estimated by FSC, and this was pervasive across both cohorts. B lymphocytes were significantly larger in colostrum compared to peripheral blood (Fig. 4A). On the other hand, both populations of T lymphocytes, basophils, and both populations of monocytes were significantly smaller in colostrum compared to blood (Fig. 4A). Of note, in blood, CD16⁺ monocytes were significantly smaller (Fig. 4A, p = 0.031) and significantly less internally complex (Fig. 4C, p < 0.0001) than classical CD16⁻ monocytes. These results together recapitulate well-described contrasts between these populations, further supporting the identity of these cells. In colostrum, the differences in size and internal complexity between both monocyte subpopulations were exacerbated (p < 0.0001 for both). Eosinophils and neutrophils did not exhibit a change in relative size between tissues.

There was a leukocyte-specific regulation of internal complexity between tissue, as determined by SSC of light, and this finding was pervasive across both cohorts. Lymphoid cells showed stable SSC in blood and colostrum (Fig. 4B), while the SSC of myeloid cells were significantly regulated between tissues (Fig. 4C). Basophils and classical CD16⁻ monocytes exhibited significantly higher SSC in colostrum, while neutrophils and CD16⁺ monocytes exhibited significantly higher SSC in peripheral blood. As seen for FSC properties, eosinophils did not exhibit a change in SSC properties between tissue types.

Overall, the relative expression of CD45 was higher on lymphocytes, with a mean MFI around 1000 (Fig. 5A) and lower on myeloid progenitors with a mean MFI <400 (Fig. 5C). There was no difference between tissues in the relative expression of CD45 on the surface of cells from the lymphoid lineage (Fig. 5A). All 3 granulocyte subtypes exhibited a significant increase in CD45 expression in colostrum compared to blood (Fig. 5B). Upregulation of CD45 was systematically more significant on granulocytes from the lean cohort. While early myeloid precursors did not exhibit changes in CD45 expression, with MFI consistently averaging around 150 in all groups, colostrum immature granulocytes exhibited twice the levels of CD45 expression observed in peripheral blood, with MFI averaging from 134 and 128 in blood to >300 in colostrum, irrespective of the study groups (Fig. 5C). Both monocyte populations downregulated CD45 expression in colostrum, irrespective of the study group, and the downregulation was systematically more significant in the obese cohort (Fig. 5D).

We observed stable, relatively low levels of CD16 on cytotoxic T/NK cells across tissues in both study groups (Fig. 6A). Blood neutrophils exhibited the highest expression of CD16 (mean MFI 5049 and 5584 for lean and obese study groups respectively, Fig. 6B) while the expression on blood-circulating monocytes and cytotoxic T/NK cells was overall 10-fold lower. There was a significant downregulation of CD16 on colostrum neutrophils compared to blood, irrespective of the study group, and the difference between tissues was more significant in the lean cohort (Fig. 6B). On the other hand, there was a significant upregulation of CD16 on the surface of colostrum CD16⁺ monocytes, in the lean group only (Fig. 6C). Interestingly, blood-circulating CD16⁺ monocytes expressed significantly more CD16 in the obese cohort than in the lean cohort (p = 0.0011, Fig. 6C).