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. 2022 Apr;66(8):e2100852.
doi: 10.1002/mnfr.202100852. Epub 2022 Feb 26.

Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function

Nandini Ghosh  1 , Amitava Das  1 , Nirupam Biswas  1 , Sanskruti P Mahajan  1 , Amit K Madeshiya  1 , Savita Khanna  1 , Chandan K Sen  1 , Sashwati Roy  1
Affiliations

Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function

Nandini Ghosh et al. Mol Nutr Food Res. 2022 Apr.

Abstract

Scope: Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened host-defense results in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolsters respiratory burst activity and improves wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation.

Methods and results: In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. In addition, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerged as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid.

Conclusion: These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.

Keywords: ROS; dietary supplement; macrophage; myo-inositol; phagocytosis.

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Figures

Figure 1.
Figure 1.. FPP treatment increases intracellular myo-inositol.
(A-E) Peripheral blood mononuclear cells (PBMCs) were treated with FPP (3mg/ml) for 24h. Intracellular sugar and sugar alcohol contents of the cell lysates were quantified using liquid chromatography and mass spectrometry. (A) Representative LC-MS/MS chromatogram (B) myo-inositol (C) glucose (D) sorbitol and (E) arabitol/ribitol/xylitol levels. Data are expressed as mean ± SEM (n = 4-5). (F) Intracellular myo-inositol levels in FPP-treated (3mg/ml) differentiated THP-1 human macrophages after 24h. Data are expressed as mean ± SEM (n = 6).
Figure 2.
Figure 2.. Myo-inositol treatment improves PMA-induced ROS and phagocytosis.
(A) Intracellular myo-inositol levels in FPP-equivalent myo-inositol (myo-inositolFPP-E; 3μg/ml) treated differentiated THP-1 human macrophages following 6h-24h post-treatment. Data are expressed as mean ± SEM (n = 6-7). (B) Differentiated THP-1 human macrophages were treated with FPP (3mg/ml) or FPP-equivalent myo-inositol (myo-inositolFPP-E; 3μg/ml) for 24h. Inducible ROS production was measured after stimulation with phorbol ester (PMA, 1μg/mL) for 30 min. Data are presented as fold change as compared to vehicle treated cells. Data are expressed as mean ± SEM (n = 6). (C) THP-1 macrophages were treated with FPP (3mg/ml) or FPP-equivalent myo-inositol (3μg/ml) for 24h and subjected to phagocytosis assay. Data are presented as fold change as compared to vehicle treated cells. Data are expressed as mean ± SEM (n = 6).
Figure 3.
Figure 3.. Primary myo-inositol transporters play a key role in regulating myo-inositol levels in human macrophages.
(A) Schematics illustrating myo-inositol transport inside macrophages (B) Relative expression of primary myo-inositol transporters (HMIT, SMIT1, and SMIT2) in differentiated THP-1 macrophages. The mRNA expression of HMIT, SMIT1, SMIT2 was determined using qPCR. Data are expressed as mean ± SEM (n = 6). (C) Intracellular myo-inositol levels in FPP (3mg/ml) treated THP-1 macrophages following knockdown of myo-inositol transporters or ISYNA1 using target specific siRNA or siControl (scrambled sequence control). Intracellular myo-inositol levels were measured 24h post-FPP-treatment. Data are expressed as mean ± SEM (n = 4-7). (D) Intracellular ISYNA1 levels in FPP-treated (3mg/ml) THP-1 macrophages after 24h. Data are expressed as mean ± SEM (n = 7-8). (E) Intracellular ISYNA1 levels in myo-inositol-treated (3μg/ml) THP-1 macrophages after 24h. Data are expressed as mean ± SEM (n = 5). (F) ISYNA1 mRNA expression in FPP-equivalent myo-inositol (3μg/ml) treated THP-1 macrophages after 24h. Data are expressed as mean ± SEM (n = 5). (G) Intracellular ISYNA1 levels in myo-inositol (3μg/ml) treated THP-1 macrophages following knockdown of myo-inositol transporters using target specific siRNA or siControl (scrambled sequence control). Intracellular ISYNA1 levels were measured 24h post-FPP myo-inositol-treatment. Data are expressed as mean ± SEM (n = 3-5).
Figure 4.
Figure 4.. FPP induced ROS production and phagocytosis is via myo inositol transporters.
(A-C) THP-1 macrophages were transfected with (A) siSMIT1, (B) siHMIT or (C) siISYNA1 and then treated with FPP(3mg/ml). PMA inducible ROS was measured 24h post-FPP-treatment. Data are expressed as mean ± SEM (n = 6-8) (D-F) THP-1 macrophages were transfected with (A) siSMIT1, (B) siHMIT or (C) siISYNA1. Cells were then treated with FPP(3mg/ml) and subjected to phagocytosis. Data are expressed as mean ± SEM (n = 6-7).
Figure 5.
Figure 5.. FPP and Inositol-induced ROS and phagocytosis is via RAC2
(A) Schematic diagram representing the role of RAC2 in modulating ROS and phagocytosis (B-C) THP-1 macrophages were treated with FPP (3mg/ml) or FPP-equivalent myo-inositol (3μg/ml) for 24h. RAC2 protein expression was measured using immunocytochemistry. (B) Representative images. Scale bar, 20μm (C) Quantification. Data are expressed as mean ± SEM (n = 6). (D) THP-1 macrophages were treated with FPP (3mg/ml) or FPP-equivalent myo-inositol (3μg/ml) for 24h. RAC2 mRNA expression was measured using qPCR. Data are expressed as mean ± SEM (n = 4). (E) THP-1 macrophages were transfected with siRAC2 and then treated with FPP(3mg/ml). PMA inducible ROS was measured 24h post-FPP-treatment. Data are expressed as mean ± SEM (n = 7) (F) THP-1 macrophages were transfected with siRAC2 and then treated with FPP-equivalent myo-inositol (3μg/ml). PMA inducible ROS was measured 24h post-FPP-equivalent myo-inositol treatment. Data are expressed as mean ± SEM (n = 6). (G) THP-1 macrophages were transfected with siRAC2 and then treated with FPP(3mg/ml). Phagocytosis was measured 24h post-FPP-treatment. Data are expressed as mean ± SEM (n = 6). (H) THP-1 macrophages were transfected with siRAC2 and then treated with FPP-equivalent myo-inositol (3μg/ml). Phagocytosis was measured was measured 24h post-FPP-equivalent myo-inositol treatment. Data are expressed as mean ± SEM (n = 6).
Figure 6.
Figure 6.. Wound fluid from FPP-supplemented patients have increased levels of myo-inositol and induces ROS and phagocytosis in THP-1-differentiated macrophages
(A) study design and patient demographics. Orange arrow in study design denotes the study visit after supplementation from which wound fluids were used for analysis. Age and BMI are expressed as mean ± standard error of the mean. BMI: Body Mass Index. (B) myo-inositol levels were measured in wound fluids from FPP-supplemented patients at visit 3. The levels were normalized to the total albumin level. Data are expressed as mean ± SEM (n = 7). (C) THP-1 macrophages were treated with wound fluids from FPP-supplemented patients for 24h. Inducible ROS production was measured after PMA (1μg/mL) stimulation for 30 min. Data are expressed as mean ± SEM (n = 5). (D) THP-1 macrophages were treated with wound fluids from FPP-supplemented patients for 24h and subjected to phagocytosis assay. Data are expressed as as mean ± SEM (n = 6). (E-F) THP-1 macrophages were treated with wound fluids from FPP-supplemented patients for 24h. RAC2 protein expression was measured using immunocytochemistry. (E) Representative images. Scale bar, 20μm (F) Quantification. Data are expressed as mean ± SEM (n = 6). (G) THP-1 macrophages were transfected with siRAC2 and then treated with wound fluids from FPP-supplemented patients. PMA inducible ROS was measured 24h post-FPP-equivalent myo-inositol treatment. Data are expressed as mean ± SEM (n = 5) (H) THP-1 macrophages were transfected with siRAC2 and then treated with wound fluids from FPP-supplemented patients. Phagocytosis was measured 24h post-FPP- equivalent myo-inositol treatment. Data are expressed as mean ± SEM (n = 6).
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