Interactions between human milk oligosaccharides, microbiota and immune factors in milk of women with and without mastitis

Irma Castro, Cristina García-Carral, Annalee Furst, Sadaf Khwajazada, Janneiry García, Rebeca Arroyo, Lorena Ruiz, Juan M Rodríguez, Lars Bode, Leónides Fernández, Irma Castro, Cristina García-Carral, Annalee Furst, Sadaf Khwajazada, Janneiry García, Rebeca Arroyo, Lorena Ruiz, Juan M Rodríguez, Lars Bode, Leónides Fernández

Abstract

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women.

Conflict of interest statement

The authors declare no competing interests.

© 2022. The Author(s).

Figures

Figure 1
Figure 1
Spearman rank correlations between individual HMOs concentrations in milk samples from healthy women (HW group; A) and mastitis cases (MW group; B) groups. Only statistically significant (p < 0.05) correlations between individual HMOs that were present in > 50% of the samples in each group are shown .The strength and colors indicate directionally (blue denotes positive; red denotes negative) of the association. DFLac, difucosyllactose; DFLNH, difucosyllacto-N-hexaose; DFLNT, difucosyllacto-N-tetrose; DSLNH, diasilyllacto-N-hexaose; DSLNT, diasilyllacto-N-tetraose; FDSLNH, fucodisialyllacto-N-hexaose; FLNH, fucosyllacto-N-hexaose; HMO, human milk oligosaccharide; LNFP, lacto-N-fucopentaose; LNH, lacto-N-hexaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LSTb, sialyl-lacto-N-tetraose b; LSTc, sialyl-lacto-N-tetraose c; 2’FL, 2’-fucosyllactose; 3FL, 3-fucosyllactose; 3’SL, 3’-sialyllactose; 6’SL, 6’-sialyllactose.
Figure 2
Figure 2
Spearman rank correlations between immunological compounds concentrations in milk samples from healthy women (HW group; A) and mastitis cases (MW group; B). Only statistically significant (p < 0.05) correlations between individual HMOs that were present in > 50% of the samples in each group are shown. The strength and colors indicate directionally (blue denotes positive; red denotes negative) of the association. G.CSF, granulocyte colony-stimulating factor; INF.g, interferon-γ; IL, interleukin; MCP.1, macrophage-monocyte chemoattractant protein-1; MIP1.b, macrophage inflammatory protein-1β; TGFb2, transforming growth factor-β2; TNF.a, tumor necrosis factor-α.
Figure 3
Figure 3
Spearman rank correlations between individual and grouped HMOs, microbial counts and immunological compounds concentrations in milk samples from healthy women (HW group; A) and mastitis cases (MW group; B). Only statistically significant (p < 0.05) correlations between those individual or grouped compounds or bacteria that were present in > 50% of the samples in each group are shown. The strength and colors indicate directionally (blue denotes positive; red denotes negative) of the association. DFLac, difucosyllactose; DFLNH, difucosyllacto-N-hexaose; DFLNT, difucosyllacto-N-tetrose; DSLNH, diasilyllacto-N-hexaose; DSLNT, diasilyllacto-N-tetraose; FDSLNH, fucodisialyllacto-N-hexaose; FLNH, fucosyllacto-N-hexaose; HMO, human milk oligosaccharide; LNFP, lacto-N-fucopentaose; LNH, lacto-N-hexaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LSTb, sialyl-lacto-N-tetraose b; LSTc, sialyl-lacto-N-tetraose c; 2’FL, 2’-fucosyllactose; 3FL, 3-fucosyllactose; 3’SL, 3’-sialyllactose; 6’SL, 6’-sialyllactose. Total HMO-bound sialic acid; total HMO-bound fucose; small HMO; type 1; type 2; α-1,2; α-1,3; and α-2,6 were calculated as the sum of all sialic acid moieties bound to each HMO; all fucose moieties bound to each HMO; 2’FL + 3FL + 3’SL + 6’SL; LNT + LNFP I + LNFP II + LSTb + DSLNT; LNnT + LNFP III + LSTc; LNFP I + 2’FL; LNFP III + 3FL; and LSTb + LSTc + 6’SL, respectively. GCSF, granulocyte colony-stimulating factor; INFγ, interferon-γ; IL, interleukin; MCP1, macrophage-monocyte chemoattractant protein-1; MIP1β, macrophage inflammatory protein-1β; TGFβ2, transforming growth factor-β2; TNFα, tumor necrosis factor-α.
Figure 4
Figure 4
Box-plots showing the concentrations (nmol/mL) of the individual HMOs. The boxes represent the values of the interquartile ranges, with the median represented as a line. Outliers are represented as dots. Samples colored in red belong to acute mastitis (AM) cases and those in green to subacute mastitis (SAM) cases. Wilcoxon rank sum tests or ANOVA tests (depending on the distribution of the data) were used to determine differences in HMOs concentration between samples from acute or subacute mastitis-suffering women. DFLac, difucosyllactose; DFLNH, difucosyllacto-N-hexaose; DFLNT, difucosyllacto-N-tetrose; DSLNH, diasilyllacto-N-hexaose; DSLNT, diasilyllacto-N-tetraose; FDSLNH, fucodisialyllacto-N-hexaose; FLNH, fucosyllacto-N-hexaose; HMO, human milk oligosaccharide; LNFP, lacto-N-fucopentaose; LNH, lacto-N-hexaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LSTb, sialyl-lacto-N-tetraose b; LSTc, sialyl-lacto-N-tetraose c; 2’FL, 2’-fucosyllactose; 3FL, 3-fucosyllactose; 3’SL, 3’-sialyllactose; 6’SL, 6’-sialyllactose.

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