Metformin treatment of juvenile mice potentiates innate immune response to bacterial lipopolysaccharide in males

Abstract

Objective: Metformin is a widely used drug, with a good safety profile, for treatment of type 2 diabetes. Recently, it has been found to also exhibit immunomodulatory potential. We aim to preliminarily explore the potential role of metformin in regulating innate immunity using UM-HET3 mice.

Methods: In this study, we treated juvenile UM-HET3 mice with metformin and investigated the alteration of the innate immune response. Peripheral blood of HET3 mice was treated with ex-vivo lipopolysaccharide (LPS) stimulation to induce a leukocyte inflammatory response, which was detected by analyzing the expression of LPS receptors TLR4 and CD14 on leukocytes with flow cytometry. Furthermore, cytokine release induced by LPS was measured in isolated cell culture supernatants with ELISA.

Results: In this study LPS treatment triggered a downregulation of TLR4 as well as an upregulation of CD14 expressed on the cell surface. Besides, it enhanced the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. The results showed that metformin treatment led to a significant (p < .05) increase in the expression of CD14 and pro-inflammatory cytokines, including IL-1β and IL-6, as well as increased proportion of TLR4 downregulation on the cell surface in response to LPS, specifically in males.

Conclusions: This study indicates that treating juvenile mice with metformin enhances the innate immune response to bacterial endotoxin in males, but not in females.

Keywords

Metformin innate immunity toll-like receptor 4 sex difference

Impact Statement

Use of metformin as a classic antidiabetic drug is approved by the FDA for use in obese children aged 10 years and older. Its pharmacological effect on younger children is still unclear. In this innovative study of the effect of metformin on the juvenile innate immune response in a heterogenous mouse model, we found that unlike the immunoregulatory effect that many other studies have shown, metformin treatment at early age potentiates leukocyte immune response mediated by Toll-like receptor 4, a crucial pathogen pattern recognition receptor in innate immunity. Additionally, this study found differences in the innate immune response between males and females, suggesting that sex is an important factor to consider in metformin treatment with respect to beneficial and adverse drug reactions. This study in the mouse model gives a clue about the effect of metformin treatment in innate immunity at early age, though further mechanism researches into NF-κB activity and the expression of downstream cytokines specific to TLR4 endocytosis pathway, are still to be implemented.

Introduction

Metformin has been widely used in diabetic children and adolescents older than 10 years to improve insulin sensitivity.¹ Moreover, metformin has been reported to exhibit immune-mediated effects in various diseases.^2,3,4 There have been limited evidence on the effectiveness and safety after its off-label use in children younger than 10 years old. We aim to explore the effect of metformin treatment at early age on the innate immune response.

Toll like receptor 4 (TLR4) is expressed on the cytomembrane of cells from the myelomonocyte lineage. It is activated through two different signaling pathways from different subcellular locations. The first signaling pathway is activated from the plasma membrane after TLR4 encounters LPS and is mediated by myeloid differentiation primary response 88 (MYD88), which results in the activation of NF-κB, a pro-inflammatory transcription factor.⁵ In MyD88-deficient macrophages, production of IL-1β, TNF-α, and IL-6 in response to LPS is completely suppressed.⁶ Upon activation on the cell membrane, TLR4 is then internalized into the endosomal network where the second signaling pathway is triggered through adaptor protein TIR-domain-containing adapter-inducing interferon-β (TRIF). TRIF mediates the activation of the transcription factor interferon regulatory factor-3 (IRF3) to regulate type I interferon (IFN) expression.⁷ These pathways also mediate late-phase activation of NF-κB, and play an important role in recognition of and host protection from LPS-mediated infection.⁸

CD14 is a glycolipid-anchored membrane glycoprotein and a key molecule in the activation of innate immune cells.⁹ Its expression progressively increases on myeloid cells as they mature toward monocytes and macrophages.¹⁰ In peripheral blood, CD14 is primarily expressed on monocytes.¹¹ CD14 acts as a co-receptor along with TLR4 and myeloid differentiation factor 2 (MD-2) for the detection of bacterial lipopolysaccharide (LPS). CD14 chaperones LPS molecules to the TLR4-MD-2 complex.^9,12 Also, CD14 delivers TLR4 to endosomes where TRIF-dependent signaling can occur.¹³ LPS-induced dimerization of IRF3 activated by TRIF was not detected in CD14-deficient bone marrow-derived macrophages (BMDM),¹³ suggesting the endocytosis of TLR4 is CD14-dependent. Kinetic analysis of LPS-induced CD14 endocytosis in immortalized BMDM¹⁴ showed that CD14 was rapidly endocytosed within minutes of LPS treatment and reappeared on the cell surface at 120 min. Co-administration of LPS and the translation inhibitor cycloheximide blocked the reappearance of CD14,¹⁴ suggesting resynthesis of this receptor, instead of recycling of previously internalized CD14, accounted for the reappearance. Furthermore, CD14 resynthesis is dependent on NF-κB activation, as the upregulation of CD14 expression induced by LPS can be canceled by blocking NF-κB. In response to LPS, the synthesis and expression of CD14 is often upregulated to compensate for the accelerated rate of CD14 endocytosis.^7,14

In contrast, TLR4 chaperoned by CD14 was rapidly internalized, but never reappeared on the cell surface, over the 120-min time course of detection in the presence of LPS.¹⁴ Tan et al. further detected the surface level of the TLR4 molecule in the presence or absence of protein synthesis inhibitor cycloheximide to clarify if alteration of TLR4 synthesis contributed to its downregulation on the cytomembrane. Unlike CD14, surface staining of TLR4 did not significantly change when protein synthesis was inhibited with cycloheximide in primary BMDMs treated with LPS, suggesting a very low or nearly undetectable level of TLR4 resynthesis in response to LPS. This is also supported by significantly downregulated gene expression of TLR4 in response to LPS. Moreover, a high dose of LPS drives a rapid monocyte differentiation and triggers an increase in the expression of CD14 ¹³, thus providing another potential explanation for the enhancement of TLR4 endocytosis and signaling by the maturation-induced upregulation of CD14 on the cell surface.

Materials and methods

Mice

The UM-HET3 mouse strain was used for this work, and the 10 breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, Maine 04,609) and housed in the animal facility of the Southern Illinois University School of Medicine, Springfield, IL. Each breeder pair was assigned a unique number and a random number generator was used to pick up litter of pups from breeder pairs for different treatment at the age of day 15. From day 15 to day 56 after birth, mice were given 200 mg/kg metformin (Cat. #151691, MP Biomedicals, Ohio, 44,139) or same volume of saline daily via i.p. Injection. The rationale for the dosage and approach of metformin treatment was elucidated in previous report.¹⁵ Peripheral blood was collected from the submandibular vein plexus of the mice for following experiments. A priori power analysis using Gpower estimated that the animal sample size would be at least 52 in total, and the Gpower computation details were as followed: effect size f=0.4; alpha error=0.05; power=0.8; numerator df=1; number of groups=2. In practice, 10 cages of female pups (25 saline- and 24 metformin-treated) and eight cages of male pups (17 saline- and 18 metformin-treated), aged 61 to 71 days, were randomly picked for the study. No animal was excluded from this study. The order of treatment and measurement of control and experimental groups was set in advance and not randomly. The experimenters were aware of the group allocation during the conduct of the experiment, the outcome assessment and the data analysis. The present study followed NIH guidelines and the policies of the Laboratory Animal Care and Use Committee at Southern Illinois University School of Medicine for humane animal treatment and complied with relevant legislation. In data analysis, data out of the range of mean+-SD were excluded.

Culture and LPS stimulation of peripheral blood

We established the culture method in reference to a published protocol.¹⁶ At least 150 μL of peripheral whole blood was collected from each mouse into heparinized tubes (Minicollect, Greiner One) and mixed well. Fifty microliters of blood were then transferred to a new eppendorf tube and used for flow cytometry analysis. Fifty microliters of whole blood were diluted with 450 μL RPMI 1640 medium containing 10% FBS. LPS solution (diluted in PBS, 2.5 μL) (or an equal volume of PBS as control) was then added so that the final concentration of LPS was 100ng/mL, a concentration used in previous report.¹⁷ Blood cells were mixed evenly with the culture medium and incubated at 37°C in 5% CO₂. After 4 hours of stimulation, cell culture supernatants were collected and frozen immediately at -20°C for further use.

Elisa

Frozen aliquots of supernatants were thawed immediately before use. The level of TNFɑ, IL6, and IL1β in the blood cell supernatant was measured according to the manufacturer's protocols using commercially available kits (Invitrogen, 88-7324-22, 88-7064-22, 88-7013-22). Assay range for IL-1β and TNF-α were 8-1000 pg/mL with an analytical sensitivity of 8 pg/mL, and for IL6 4-500 pg/mL with a sensitivity of 4 pg/mL. Duplicate measurements were performed for each sample. The OD value of samples in comparison to a provided standard was measured with a colorimetric microplate reader (Multiskan spectrum, Thermo).

Flow cytometry

FITC-conjugated anti-CD14 (1:800 dilution, Biolegend) and APC-conjugated anti-TLR4 (1:50 dilution, Biolegend) were added to each tube of 50 μL whole blood, mixed well, and placed on ice in the dark for 30 min. Control cells were not stained with either antibody. Then, blood cells were treated with RBC lysis buffer (Santa Cruz) for 15 min to remove RBC and stop staining, followed by centrifugation and washed two times with cell staining buffer (PBS, 2%FBS, 2 mM EDTA, 2 mM NaN₃). The RBC lysis step was repeated if red pellet was still observed. Cells were resuspended in cell staining buffer and kept on ice until ready for FACS. For cells in incubation with or without LPS, a similar staining, RBC lysis, and washing process was applied. Cells were run on flow cytometer (BD Accuri C6) and the same gates were applied to blood samples collected from different days. The method of cell surface flow cytometry staining of whole blood was based on the technical protocol provided by Biolegend.

Statistical Analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) (SPSS, Inc., Chicago, IL, USA). The assumptions of normality and homoscedasticity were respectively checked by Shapiro-Wilk test and Levene’s test. Means were compared by Student’s t tests (between two groups) or one-way ANOVA (among three or more groups). Data are presented as means ± standard deviation (SD). The graphs were created using SPSS software and Figure 6 was created from BioRender.com.

Results

Metformin treatment downregulated the proportion of TLR4-positive leukocytes, but did not alter the proportion of CD14-positive cells.

In peripheral blood, CD14 is highly expressed on cells of the myelomonocyte lineage, including monocytes and some granulocytes. Some leukocytes also express a certain level of TLR4. CD14 and TLR4 both play important roles in the recognition of and protection from bacterial endotoxin. Cells expressing both TLR4 and CD14 (TLR4+CD14+) act as the primary cells responsive to LPS. In the current study, CD14+ and TLR4+ leukocytes were determined as shown in S.Figure 1.

This study utilized 24 male and 18 female metformin-treated mice, along with 25 male and 17 female untreated mice as controls. Before the LPS treatment, two-way ANOVA did not show a significant effect of sex, metformin, or the interaction between sex and metformin on the proportion of CD14+ cells (Table 1, Figure 1(a)). However, sex and metformin treatment significantly altered the proportion of the TLR4+ cells (Table 1, Figure 1(b)). ANOVA showed that metformin treatment significantly reduced (p=0.034) the proportion of TLR4+ cells, although, within either sex, the differences were not significant (t test, p=0.131, and 0.146). Between sexes, male mice had a significantly higher (t test, p=0.001) proportion of TLR4+ cells. Furthermore, in both metformin-treated and control groups, the differences between sexes were significant (t test, p=0.014 and 0.014, respectively, Figure 1(b)3). Additionally, male mice had a significantly higher (p = .001) average expression level of TLR4, calculated as the Median Fluorescence Intensity (MFI) of TLR4, than females (S.Table.1), and the differences between sexes were significant in both the control and metformin-treated groups (t test, p = 0.021 and 0.016 respectively, S.Figure 2). Metformin treatment also significantly (ANOVA, p = .029) reduced the TLR4+CD14+ leukocytes (Table 1), although no significant difference was found within either sex by t test (Figure 1(c)).

Table 1.

The two-way ANOVA table for flow cytometry results of peripheral blood cells.

		Source	Type III sum of squares	df	Mean square	F	Sig.
No LPS treatment	CD14+ %	MET treatment	0.002	1	0.002	0.000	0.984
		Sex	0.556	1	0.556	0.092	0.763
		Sex × MET treatment	0.841	1	0.841	0.139	0.711
	TLR4+%	MET treatment	1.095	1	1.095	4.629	0.034*
		Sex	3.089	1	3.089	13.062	0.001**
		Sex × MET treatment	0.014	1	0.014	0.057	0.811
	TLR4+ CD14+/CD14+ (%)	MET treatment	10.330	1	10.330	4.961	0.029*
		Sex	1.387	1	1.387	0.666	0.417
		Sex × MET treatment	0.687	1	0.687	0.330	0.567
LPS treated	CD14+ %	MET treatment	39.964	1	39.964	4.132	0.048*
		Sex	35.284	1	35.284	3.648	0.063
		Sex × MET treatment	9.332	1	9.332	0.965	0.332
	TLR4+%	MET treatment	10.816	1	10.816	8.159	0.007**
		Sex	6.788	1	6.788	5.121	0.029*
		Sex × MET treatment	4.217	1	4.217	3.181	0.082
	TLR4+ CD14+/CD14+ (%)	MET treatment	22.269	1	22.269	7.842	0.008**
		Sex	18.926	1	18.926	6.665	0.013*
		Sex × MET treatment	8.432	1	8.432	2.969	0.092

MET: Metformin; ×interaction; *p < 0.05, **p < 0.01.

Figure 1.

Metformin treatment downregulated the proportion of TLR4+ cells in leukocytes, but did not alter the proportion of CD14+ cells. Both the expression of CD14 and TLR4 on murine peripheral blood nucleated cells were analyzed by FACS. No significant difference in the proportion of CD14+ cells was found between sexes or between control and metformin-treated groups (a). The proportion of TLR4+ leukocytes was significantly increased in males compared to females (p = 0.001) (b). Metformin treatment significantly decreased the proportion of TLR4+ cells (p = 0.034) (b), and TLR4+CD14+ cells (p = 0.029) (c). (*: p< 0.05, t test).

Under LPS treatment, metformin increased the proportion of CD14-positive cells and decreased the proportion of TLR4-positive cells in leukocytes more significantly in males.

Pairwise t test showed that LPS treatment significantly increased the percentage of CD14+ cells (p = 0.014) and their expression level of CD14 (p < 0.001), as shown by the right-shifted peak of CD14+ cells (Figure 2(a) & S.Figure 3A). Meanwhile, LPS treatment significantly decreased the percentage of TLR4+ cells (t test, p < 0.001) and the expression level of TLR4 (p < 0.001), as shown by the left-shifted distribution of TLR4 intensity (Figure 2(b) & S.Figure 3(b)).

Figure 2.

LPS treatment significantly increased the proportion of CD14+ cells and decreased the proportion of TLR4+ cells in leukocytes. The treatment of LPS significantly increased the percentage of CD14+ cells in leukocytes (t test, p = 0.014), and decreased the percentage of TLR4+ cells (t test, p < 0.001) (b).

Under LPS treatment, ANOVA showed that metformin significantly increased the proportion of CD14+ cells (p = 0.048), though within either sex the differences were not significant (t test, p = 0.430 and 0.116) (Figure 3(a)). Metformin significantly decreased the proportion of TLR4+ cells and the expression level of TLR4 (ANOVA, p = 0.007 and 0.011 respectively) (Table 1 & S.Table.1), and further comparison between control and metformin-treated groups within both sexes showed that the decrease was significant in males (t test, p = 0.019 and 0.018, respectively), but not in females (t test, p = 0.332 and 0.414, respectively) (Figure 3(b) & S.Figure 4). Likewise, ANOVA showed that metformin treatment decreased the proportion of TLR4+CD14+ cells in CD14+ cells (p = 0.008), and further comparison within the sex shows that the difference between the metformin-treated and control groups was significant in males (t test, p = 0.027) but not in females (t test, p = 0.174) (Figure 3(c)).

Figure 3.

Under LPS treatment, metformin increased the proportion of CD14+ cells and decreased the proportion of TLR4+ cells in leukocytes. The expression of CD14 and TLR4 on murine leukocytes cultured in the presence of 100ng/mL LPS were analyzed by FACS. Under LPS treatment, metformin increased the proportion of CD14+ cells (p = 0.048), with a higher increase rate in males compared to females (27.75% vs 10.57%) (a). Metformin significantly decreased the proportion of TLR4+ cells in leukocytes (p = 0.007), and further comparison between control and metformin-treated groups in both sexes showed that the decrease was significant in males, but not females (p = 0.019 and 0.332 respectively), with a decrease rate of 37.87% in males versus 13.07% in females (b). Likewise, TLR4+CD14+/CD14+ % was also decreased in metformin-treated groups (p = 0.008), and more significantly in males (p = 0.027) than in females (p = 0.174) (c). *p < 0.05, **p < 0.01, t test.

Metformin treatment amplified LPS-induced alteration of the proportion of TLR4+ cells and TLR4 expression level.

The change rate of the proportion of TLR4+ cells in leukocytes induced by LPS treatment is derived from the formula: (TLR4+%_LPS-treated - TLR4+%_non-treated)/TLR4+%_non-treated. The metformin-treated group exhibited a higher change rate of TLR4+ cells % than the control group (ANOVA, p = 0.076), which was significant in males (t test, p = 0.005), but not in females (t test, p = 0.760) (Figure 4(a)). We also compared the LPS-induced alteration of the TLR4 expression level between the control and metformin-treated groups, using the change rate of the log-transformed MFI of TLR4. The result show that metformin treatment contributed to a higher change rate of TLR4 expression level induced by LPS than the control groups (ANOVA, p = 0.016) (Figure 4(b) & S.Table.2), but within the sex the difference is not significant either in females (t test, p = 0.261), or males (t test, p = 0.058).

Figure 4.

Metformin amplified LPS-induced alteration of the proportion of TLR4+ cells and TLR4 expression level. The change rate of the proportion of TLR4+ cells in leukocytes induced by LPS treatment is derived from the formula: (TLR4+%_LPS-treated - TLR4+%_non-treated)/TLR4+%_non-treated. The metformin-treated group exhibited a higher change rate of TLR4+ cells % than the control group (ANOVA, P=0.076), which was significant in males (t test, P=0.005), but not in females (t test, P=0.760) (a). Likewise for the alteration of the MFI of TLR4 (b), metformin treatment contributes to a higher change rate of MFI-TLR4 induced by LPS than the control groups (ANOVA, P=0.016), but within sex the difference is not significant either in females (t test, P=0.261), or males (t test, P=0.058).**p < 0.01 t test.

Metformin treatment increased the secretion of pro-inflammatory cytokines IL-1β and IL-6 by leukocytes under LPS treatment.

The level of pro-inflammatory cytokines TNF-α、IL-1β and IL-6 in the supernatant of peripheral blood without LPS treatment was barely detectable. LPS stimulation boosted the level of these cytokines, which are regulated by NF-κB.¹⁸ Importantly, we found that under LPS treatment, metformin treatment, sex, and the interaction between metformin treatment and sex, all have a significant effect on the concentration of IL-1β and IL-6. ANOVA showed that metformin significantly upregulated (Table 2) the secretion level of IL-1β and IL-6 (p = 0.002 and 0.026 respectively), and that blood cells from males had significantly higher secretion level of these two cytokines (p = 0.034 and 0.001 respectively). The way that metformin treatment affects IL-1β levels was significantly different between sexes (ANOVA, p = 0.030). Metformin treatment significantly upregulated the secretion level of IL-1β in males (t test, p < 0.001), but not in females (t test, p = 0.429). Likewise, metformin treatment affected the secretion level of IL-6 differently between sexes (ANOVA, p = 0.049). Metformin significantly upregulated the IL-6 level in males (t test, p = 0.035), but did not significantly change it in females (t test, p = 0.767) (Figure 5(b) & (c)). Neither metformin treatment (ANOVA, p = 0.395), nor sex (ANOVA, p = 0.825) significantly changed the secretion level of TNF-α (Figure 5(a)).

Table 2.

The two-way ANOVA table for Elisa results under LPS treatment.

Cytokines	Source	Type III sum of squares	df	Mean square	F	Sig.
TNF-α	MET treatment	804.935	1	804.935	0.750	0.395
	Sex	53.377	1	53.377	0.050	0.825
	Sex×MET treatment	84.446	1	84.446	0.079	0.782
IL-1β	MET treatment	97.981	1	97.981	14.434	0.002**
	Sex	36.938	1	36.938	5.442	0.034*
	Sex×MET treatment	39.096	1	39.096	5.760	0.030*
IL-6	MET treatment	218.487	1	218.487	6.101	0.026*
	Sex	616.000	1	616.000	17.201	0.001**
	Sex×MET treatment	164.843	1	164.843	4.603	0.049*

Dependent variable: Concentration (pg/ml); MET: Metformin; ×interaction; *p < 0.05, **p < 0.01.

Figure 5.

Metformin treatment increased the secretion of pro-inflammatory cytokines IL-1β and IL-6 by leukocytes in response to LPS. The supernatant of peripheral blood cells cultured under LPS treatment was detected by ELISA. In both sexes, no significant difference in TNF-α was detected between the control and metformin-treated groups (a). In males, IL-1β and IL-6 were significantly increased in the metformin-treated group compared to the control group; however, no significant difference was detected in the females (b), (c). *p < 0.05, ***p < 0.001, t test.

Discussion

In the current study, using young UM-HET3 mice, we investigated if metformin treatment could alter the immune function at early age. Heterogeneous UM-HET3 mice were treated by metformin (i.p. Injection, 200 mg/kg daily) from age of 15 days through 56 days. Metformin has been reported to decrease TLR4 expression on adult human blood monocytes and cytokine production.¹⁹ Similarly, our results show that metformin treatment in juvenile UM-HET3 mice decreased the proportion of TLR4-positive cells in leukocyte. However, we also found the downregulation of TLR4 expression on the cell surface in response to LPS, mostly due to the TLR4 endocytosis, was amplified by metformin treatment. Since TLR4 must rely on CD14 to be endocytosed, it is safe to conclude that in response to LPS, CD14 on the cell surface was also internalized by an increased proportion in the metformin-treated group (Figure 6). Meanwhile, flow cytometry results showed that at 4 hours after LPS treatment, the frequency of CD14-positive cells in the metformin-treated group was actually higher than in the control group. According to the report of Tan et al. regarding the kinetic analysis of LPS-induced CD14 expression,¹⁴ we speculate that LPS stimulation at this timepoint already induced a stronger activation of the NF-κB signaling pathway in the metformin-treated group (Figure 6), which led to increased expression of CD14. This speculation has also been proofed by our obsevation that metformin increased LPS-induced secretion of IL-6 and IL-1β, two pro-inflammatory cytokines reportedly dependent on NF-κB activation.¹⁸

Figure 6.

The possible mechanism of metformin treatment in regulating LPS-induced leukocyte responses: It is well established that TLR4, chaperoned by CD14, binds with LPS to initiate MyD88- and TRIF-dependent signaling pathways respectively, localized on cytomembrane and endosome, which thereafter activates transcription factors Nuclear factor kappa B (NF-κB) and Interferon regulatory factors (IRFs). It is speculated that metformin treatment potentiates LPS-induced leukocyte responses mediated by TLR4. In support of this, we have detected that the expression of CD14 and inflammatory cytokines IL-1β and IL-6, the product of the first signaling pathway, as well as the TLR4 internalization related to the second signaling pathway, are both enhanced by metformin treatment. The activity of NF-κB, IRFs, and the downstream product type Ⅰ interferons are yet to be studied to further confirm the hypothesis.

Different from a previous in vitro study in mouse macrophages²⁰ and in vivo study in adult animals,²¹ in which the LPS induced pro-inflammatory cytokines were suppressed by the metformin treatment, our results suggest that metformin treatment of juvenile mice potentiates LPS-induced leukocyte responses mediated by TLR4. The underlying mechanisms may be related to upregulated NF-κB activity and/or enhanced endocytosis of TLR4. Further studies of NF-κB activity and the expression of typeⅠinterferon, the downstream cytokine specific to TLR4 endocytosis pathway, are needed to test this hypothesis.

Another interesting finding was that TLR4 levels differed between sexes, as others have reported.^22,23 Ian et al. reported that female-derived macrophages constitutively expressed lower levels of TLR4 and CD14 on their cell surface, although resting macrophage levels of mRNA encoding TLR4 and CD14 were not significantly different between sexes. Female-derived peritoneal macrophages produced significantly less IL-1β and the chemokine IP-10 following in vitro LPS challenge than their male counterparts.²³ Furthermore, female mice showed significantly lower plasma levels of pro-inflammatory cytokines IL-6 and TNF-α than males after i.p. Injection of bacterial LPS.²⁴ Consistent with these results, our data showed a significantly lower expression of TLR4 on leukocyte cytomembrane in females, regardless of metformin treatment. However, the CD14 level on the cell surface did not differ between sexes of juvenile mice. Importantly, our results showed that metformin treatment contributed to a sex-specific response to LPS. Leukocyte response to LPS stimulation in female mice was blunted compared to that of the males, as we found lower expression of CD14 and pro-inflammatory cytokines IL-1β and IL-6, as well as lower proportion of TLR4 internalization induced by LPS in the female mice compared to the males.

In the context of increasing incidence of childhood obesity and application of metformin, we aim to preliminarily explore the effect of this drug at early age in regulating immune responses. This study focuses on the TLR4-mediated innate immune reaction and offers a clue to the possible role of metformin in the innate immune system of juveniles, which needs to be established by further research into associated mechanism.

The study has some limitations. Firstly, the data of the study was obtained from ex-vivo experiments on a mouse model. Secondly, it lacked the analysis of the downstream cytokines of TLR4 endocytosis pathway, e.g. the expression of typeⅠinterferon. Moreover, we did not dig deeper into the mechanism of upregulated LPS response by detecting NF-κB activity. Finally, though our results indicate that metformin-treated male mice may have enhanced innate immune response to bacterial lipopolysaccharide compared with the females, the mechanism of this sex difference remains elusive, and the research into the molecular responses elicited by metformin and influenced by sex hormones, such as the activation of AMPK, and their underlying crosstalk with the signaling pathway mediating immune reactions, will be needed to determine its long-term effect on innate immune reactions.

Conclusions

In conclusion, the overall results indicate that metformin-treated male mice may have a more pronounced innate immune response to bacterial lipopolysaccharide than the females.

Supplemental Material

Supplemental Material - Metformin treatment of juvenile mice potentiates innate immune response to bacterial lipopolysaccharide in males

Supplemental Material for Metformin treatment of juvenile mice potentiates innate immune response to bacterial lipopolysaccharide in males by Yujie Sun, Yun Zhu, Andrzej Bartke, Lin Lin and Rong Yuan in European Journal of Inflammation

Footnotes

Acknowledgements

The Division of Laboratory Animal Medicine at Southern Illinois University School of Medicine provides an excellent environment for animal research. We thank Lisa Hensley for kindly editing the manuscript.

Authors’ contributions

RY designed the project; AB supplied critical reagents and mice; YS and YZ conducted the experiments; RY and YS analyzed experimental results; YS wrote the manuscript with approval from all authors; RY, LL and AB revised the manuscript.

Declaration of conflicting interests

The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The project was funded by NIH 1R21AG062985 and American Diabetes Association 119-IBS-126 (AB), SIU-SOM Team Seed Grant and SIU-SOM Research Seed Grant (RY), and the Geriatrics Research Initiative of the Department of Internal Medicine at SIU-SOM (AB, RY).

Correction (November 2023):

The article has been updated for minor changes in affiliations.

Ethical Statement

ORCID iD

Yujie Sun

Supplemental Material

Supplemental material for this article is available online.

References

Masarwa

Brunetti

Aloe

, et al. Efficacy and Safety of Metformin for Obesity: A Systematic Review. Pediatrics 2021: 147.

Postler

Peng

Bhatt

, et al. Metformin selectively dampens the acute inflammatory response through an AMPK-dependent mechanism. Sci Rep 2021; 11: 1–13.

Ursini

Russo

Pellino

, et al. Metformin and Autoimmunity: A “New Deal” of an Old Drug. Front Immunol 2018; 9.

Nojima

Wada

. Metformin and Its Immune-Mediated Effects in Various Diseases. Int J Mol Sci 2023; 24: 755.

Kawasaki

Kawai

. Toll-like receptor signaling pathways. Front Immunol 2014; 5: 461.

Kawai

Takeuchi

Fujita

, et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001; 167: 5887–5894.

Ciesielska

Matyjek

Kwiatkowska

. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci 2021; 78: 1233–1261.

Kawai

Akira

. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 2007; 13: 460–469.

Zanoni

Granucci

. Role of CD14 in host protection against infections and in metabolism regulation. Front Cell Infect Microbiol 2013; 3: 32.

10.

Zamani

Zare Shahneh

Aghebati-Maleki

, et al. Induction of CD14 Expression and Differentiation to Monocytes or Mature Macrophages in Promyelocytic Cell Lines: New Approach. Adv Pharm Bull 2013; 3: 329–332.

11.

Kim

. Anti-CD14 antibody reduces LPS responsiveness via TLR4 internalization in human monocytes. Mol Immunol 2014; 57: 210–215.

12.

Zhang

Lei

, et al. CD14: Biology and role in the pathogenesis of disease. Cytokine Growth Factor Rev 2019; 48: 24–31.

13.

Zanoni

Ostuni

Marek

, et al. CD14 controls the LPS-induced endocytosis of Toll-like receptor 4. Cell 2011; 147: 868–880.

14.

Tan

Zanoni

Cullen

, et al. Mechanisms of Toll-like Receptor 4 Endocytosis Reveal a Common Immune-Evasion Strategy Used by Pathogenic and Commensal Bacteria. Immunity 2015; 43: 909–922.

15.

Zhu

Fang

Medina

, et al. Metformin treatment of juvenile mice alters aging-related developmental and metabolic phenotypes. Mechanisms of Ageing and Development 2022; 201: 111597.

16.

Thurm

Halsey

. Measurement of cytokine production using whole blood. Curr Protoc Immunol 2005; Chapter 7 :Unit 7 18B

17.

Jin

Conti

. Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-alpha responses. Proc Natl Acad Sci U S A 2002; 99: 7628–7633.

18.

Liu

Zhang

Joo

, et al. NF-kappaB signaling in inflammation. Signal Transduct Target Ther 2017; 2.

19.

Zwolak

Słabczyńska

Semeniuk

, et al. Metformin changes the relationship between blood monocyte Toll-like receptor 4 levels and nonalcoholic fatty liver disease—ex vivo studies. PLoS One 2016; 11: e0150233.

20.

Kim

Kwak

Cha

, et al. Metformin suppresses lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages via activating transcription factor-3 (ATF-3) induction. J Biol Chem 2014; 289: 23246–23255.

21.

Yang

Chen

, et al. Effects of metformin on lipopolysaccharide-induced depressive-like behavior in mice and its mechanisms. Neuroreport 2020; 31: 305–310.

22.

Kuo

S-M

. Gender difference in bacteria endotoxin-induced inflammatory and anorexic responses. PLoS One 2016; 11: e0162971.

23.

Marriott

Bost

Huet-Hudson

. Sexual dimorphism in expression of receptors for bacterial lipopolysaccharides in murine macrophages: a possible mechanism for gender-based differences in endotoxic shock susceptibility. J Reprod Immunol 2006; 71: 12–27.

24.

Robertson

Skinner

Care

. Essential role for IL-10 in resistance to lipopolysaccharide-induced preterm labor in mice. J Immunol 2006; 177: 4888–4896.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.45 MB