HDL suppresses dendritic cell maturation and modulates CD4+ T cell subtypes against inflammatory in a mouse model of rheumatoid arthritis

doi:10.21203/rs.3.rs-3012228/v1

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HDL suppresses dendritic cell maturation and modulates CD4+ T cell subtypes against inflammatory in a mouse model of rheumatoid arthritis

https://doi.org/10.21203/rs.3.rs-3012228/v1

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Background: To investigate the mechanism of anti-inflammatory effect of High-density lipoprotein (HDL) in collagen-induced arthritis (CIA) mice.

Methods: The CIA animal model was constructed, and the mice were separated into three groups: control, CIA and CIA + HDL. In the CIA + HDL group, HDL was given on day 40, and an additional injection was administered every 3 days for a total of five doses. The levels of mouse HDL were measured by using ELISA kits, and liver, heart, kidney and colon tissue sections were stained with H＆E. Additionally, the splenic CD4+ T cell phenotypes in each group were analyzed. Immature DCs were stimulated with LPS in the presence or absence of HDL, and the expression of surface markers was investigated by FACS.

Results: HDL decreased the ratio of Th1/Th2 cells and increased the Th1/Th17 cell ratio in the CIA + HDL group compared with those of the CIA one (P<0.05). In vitro, the expression of MHC-II, CD86, and CD40 in CD11c+ BMDCs was suppressed by HDL. The level of HDL in the CIA group was significantly reduced compared with that of the control one (P<0.05), and there were no significant pathological changes in the liver, heart, kidney and colon tissues of the mice among the three groups.

Conclusion: HDL exerts anti-inflammatory effect in CIA mice by suppressing DCs maturation and modulating CD4+ T cell subtypes.

high-density lipoprotein

collagen-induced arthritis

dendritic cells

CD4+ T cells

Rheumatoid arthritis (RA) is a multifactorial polygenic disease with 1% prevalence in the industrialized world, and its etiology is unknown. The disease causes articular destruction, functional decline, and substantial comorbidity in the cardiovascular disease (CVD) and metabolic systems. Disability and premature death occur if this condition is insufficiently treated ^1,2. Abnormal blood lipids are associated with inflammatory burden, which increase cardiovascular (CV) events elevating the mortality rate in RA patients ^3,4. In recent years, the outcome of treatment of patients with RA has revolutionary improved due to new drugs and targeted therapeutic approaches. However, there remains an urgent need for more effective and durable remedies with low toxicity profiles to therapy RA along with amelioration in CV events ⁵.

HDL is a heterogeneous mixture of lipoprotein particles that differ in size, shape, and lipid and apolipoprotein composition. HDL is well characterized as having protective effects against CVD. In addition, HDL alleviates inflammation and suppresses immune activation ^6–8. There is growing evidence that the quantity, composition and function of HDL is altered in patients with RA ⁹. There is a search for standard and novel anti-inflammatory treatments, such as methotrexate (MTX) and infliximab, which not only achieve rapid remission but also improve HDL function in RA patients to reduce CV events ¹⁰. A previous study of our group demonstrated that HDL has potential therapeutic effects in RA model of mice, which was associated with reduced inflammatory conditions in vivo, including decreased expression of inflammatory factors and reduced activity of inflammatory signaling pathways. However, the mechanism by which HDL affects RA remains elusive ¹¹. Therefore, we sought to investigate the role of HDL in the development of the CIA mouse model and assess the effect on the activation and maturation of the immune cells.

2.1 Reagents and antibodies

Bovine collagen type II was obtained from Chondrex, Inc. Freund’s complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were purchased from Sigma. Human HDL was obtained from ProSpec-Tany Technogene Ltd. ELISA kits and antibodies were purchased from Elabscience.

2.2 Induction of CIA and administration of HDL

Male DBA/1 mice aged 8 weeks were provided by the Shanghai SLRC Laboratory Animal Technology Co. and housed at the animal facility of the Laboratory Animals Center of the Affiliated Hospital of Qingdao University. The temperature was maintained at approximately 23°C. The mice were separated into three groups: control (treated with saline), CIA and CIA + HDL. In DBA/1 mice, CIA was induced by intradermal injection of bovine type II collagen in the presence of FCA on day 0 and reinjection on day 21 with bovine type II collagen in an emulsion with FIA as a booster. The immunization protocol was performed as previously described by Pietrosimone et al ¹². In the CIA + HDL group, HDL (10 mg/kg body weight) was administered by tail vein injection on day 40; an additional injection was administered every 3 days for a total of five doses.

2.3 Measurement of mouse HDL levels

Serum was isolated from all mice at the end of the study. HDL levels were determined by using an ELISA kit according to the manufacturer’s instructions.

2.4 Histopathological analysis

The mice were sacrificed by cervical dislocation on day 75. To compare histological changes in the liver, heart, kidney and colon in the different groups, the tissue sections were stained with H༆E after routine formalin fixation and paraffin embedding. Images were captured with a Nikon TE2000-S (Nikon, Tokyo, Japan) and analyzed.

2.5 Flow cytometry

Flow cytometric analysis of the T helper (Th) cell subtypes was performed using FITC anti-mouse CD4, PE anti-mouse IFN-γ, PE anti-mouse IL-4, APC anti-mouse IL-17A, PE anti-mouse CD25 and APC anti-mouse Foxp3.

For intracellular cytokine staining, cells at a concentration of 1×10⁶ cells/ml were stimulated with leukocyte activation cocktail containing PMA, Lonomycin and Brefeldin (BD GolgiPlug) and incubated for 5 hours. Then, the stimulated cells were collected and resuspended in staining buffer, preincubated with Fc Block reagent and then stained with FITC anti-mouse CD4 for 30 min at 4°C in the dark. The cells were then washed with staining buffer and fixed in Fix/Perm Buffer for 20 min at 4°C protected from the light. Subsequently, the cells were washed with Perm/Wash Buffer, followed by centrifugation. The cells were then stained with PE anti-mouse IFN-γ, PE anti-mouse IL-4, and PE anti-mouse IL-17A for 30 min at 4°C in the dark. After washing, the cells were resuspended in staining buffer and stored at 4°C in the dark for subsequent detection.

For regulatory T cells (Tregs) staining, cells at a concentration of 1×10⁶ cells/ml were collected and resuspended in staining buffer and stained for 30 min at 4°C in the dark with FITC anti-mouse CD4 and PE anti-mouse CD25. The cells were then washed with staining buffer and fixed in Fix/Perm Buffer for 40 min at 4°C in the dark. Subsequently, the cells were washed with Perm/Wash Buffer. The cells were then stained with APC anti-mouse Foxp3 for 50 min at 4°C in the dark. After washing, the cells were resuspended in staining buffer and stored at 4°C in the dark for subsequent detection.

The cells were analyzed by using a Beckman Coulter FC500 flow cytometer (Beckman, Miami, FL, USA). A total of 40,000 CD4 + T cells were counted in the Treg sample, and 10,000 CD4 + T cells were counted in the Th1, Th2 and Th17 samples. A gate was set on the lymphocytes using characteristic forward scatter (FSC) and side scatter (SSC) parameters. Isotype-matched FITC mouse IgG1 antibody, PE mouse IgG1 antibody, and APC mouse IgG1 antibody were used as controls.

2.6 Preparation of mouse bone marrow-derived dendritic cells(BMDCs)

BMDCs were generated as described previously with minor modifications ¹³. DBA mice, 6–8 weeks old, were euthanized and immersed in 75% ethanol for 10 min. Bone marrow was obtained from femurs and tibias of male mice by removing and purifying the surrounding muscle tissue by rubbing with sterile gauze. Both ends were cut with scissors, and the marrow was flushed with HBSS (Procell) using a disposable syringe. The bone marrow was centrifuged at 1300 rpm at room temperature for 10 min, and the supernatant was discarded. Bone marrow was treated with RBC lysis buffer (Beijing Solarbio Science & Technology Co.) for 3 min, passed through a 40µm aperture nylon mesh to remove small pieces of bone and debris, and plated at 2.5×10⁶ cells per 100 nm bacteriological petri dish in 10 ml IMDM medium supplemented with 10% FBS (BI, Logan, UT), 0.1% β-mercaptoethanol (Sigma), 20 ng/ml recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF; PeproTech EC Ltd, London, UK) and 10 ng/ml interleukin (IL-4, PeproTech EC Ltd, London, UK). On day 3, half of the culture medium was replaced, and immature BMDCs were harvested on day 6.

2.7 Culture of BMDCs

The cells were cultured in 24-well tissue culture plates (1×10⁶ cells/well) in the presence of 1 µg/ml E. coli LPS (Escherichia O111:B4, Sigma) and various concentrations of HDL for 48 hours. After 48 hours, non-adherent cells were used for fluorescence-activated cell sorting (FACS) analyses. Culture supernatants were collected at 48 hours for cytokine analysis by ELISA.

2.8 Costimulatory molecule expression on DCs

To evaluate costimulatory molecule expression, DCs were stained in the dark at 4°C for 30 min with PE-labeled anti-CD11c antibody to identify DCs and FITC-conjugated anti-MHC-Ⅱ and APC-conjugated anti-CD40, -CD80, and -CD86 antibodies. The cells were preincubated with FC-block reagent for 5 min to prevent nonspecific staining via the FC receptor prior to adding the fluorescently labeled antibodies. As a control, APC-labeled, isotype-matched antibodies with irrelevant specificity were used to determine the unstained control levels. Costimulatory molecule expression in the gated CD11c + population were evaluated by FACS analysis.

2.9 Statistical analysis

The data are expressed as the mean ± SD. The analysis was performed by using GraphPad Prism 8.0. Student’s t-test was applied. A P value of less than 0.05 was considered statistically significant.

3.1 A model of CIA

In DBA/1 mice, CIA was established by intradermal injection of bovine type II collagen in the presence of FCA on day 0 and reinjection on day 21 with bovine type II collagen in an emulsion with FIA as a booster.

3.2 Analysis of mice HDL production

The levels of HDL in the peripheral blood the mice were measured. The HDL level in the CIA group was significantly reduced compared with that of the control one (P<0.05). However, there was no difference between the CIA and CIA + HDL groups (P>0.05) (Figure 1).

3.3 Effect of HDL on histopathological changes

The liver, heart, kidney and colon were collected and used determine the HDL. Histologic examination of the liver, heart, kidney and colon was carried out by two independent pathologists who were blinded to the experimental conditions. As shown in Figure 2, the liver, heart, kidney and colon tissues exhibited no significant pathological changes among the three groups. These results showed that HDL had no significant toxic effect on these organs.

3.4 HDL regulates Th cell subtypes

As shown in Figure 3 A, no significant differences among the three groups were found in the proportion of Th1, Th2, Th17 or Tregs among CD4+ T cells in splenic lymphocytes. As shown in Figure 3 B, the Th1/Th2 ratio of the CIA group was significantly increased and the Th1/Th17 ratio was decreased compared to those of the control one (P<0.05). However, HDL decreased the ratio of Th1/Th2 and increased the ratio of Th1/Th17 compared with those of the CIA group (P<0.05). These results indicate that HDL regulates both the Th1/Th2 and the Th1/Th17 balance. After treatment with HDL, the content of Treg cells increased, but the results were not significantly different compared with the CIA group (P>0.05).

3.5 Inﬂuence of HDL on DC costimulatory molecules

To obtain immature DCs, cells were harvested on day 6 after culture with GM-CSF and IL-4. To investigate the inﬂuence of HDL on DCs, immature DCs were stimulated with LPS in the presence or absence of HDL for 48 hours, and the expression of surface markers was investigated by FACS. As shown in Figure 4, the expression of costimulatory molecules in the LPS group was sharply upregulated compared with that in the control one. However, HDL inhibited the expression of costimulatory molecules, including MHC-II, CD86, and CD40, and this effect was positively related to the concentration of HDL. However, HDL did not affect the expression of CD80.

RA is a chronic inflammatory disease associated with intense immune activation within the synovial compartment of joints. The manifestations include swelling and pain in multiple joints, and inflammatory processes lead to cartilage and bone erosion ^14,15. Inflammation plays a critical role in different stages of RA. Moreover, numerous evident by data have demonstrated that inflammation is independently contributed to CV mortality and morbidity in RA ^16,17. Atherosclerosis and RA share common proinflammatory cytokines and inflammatory processes. The destructive proinflammatory cascade and the pathogenesis implicated in RA resemble those in the case of atherosclerosis. It is well known that atherogenicity occurs as a result of dyslipidemia in plasma. An inverse relationship has been noted between inflammation and lipid pattern in RA. HDL possesses athero-protective and anti-inflammatory actions. The level of HDL are curbed in active period RA patients and elevated following effective anti-rheumatic therapies, which improve stratification of CVD ^18,19. In our study, the HDL level in the CIA group was significantly lower than that in the control group, but there was no significant difference between the CIA and CIA + HDL groups. This trend is similar to that of patients with RA, who have lower HDL levels than normal individuals ¹⁸. Additionally, the liver, heart, kidney and colon tissues exhibited no significant pathological changes among the three groups. This means that human HDL has no significant toxic effect on these organs.

Key regulators of RA inflammatory response are Th cells, which are subsets of T lymphocytes. Th cells polarize to Th1, Th2, Th17, and Tregs by the different cytokines. Th1 cells, which produce IFN-γ and IL-2, are involved in cellular immunity, and Th2 cells, which produce IL-4, IL-5 and IL-13 and are involved in humoral immunity, and these are two important subtypes of CD4 + T cells ^20,21. As a newly identified CD4 + Th cell subtype, Th17 cells produce IL-17, IL-6, IL-21, IL-22 and TNF-α, which induce inflammation and are important factors in the immunopathogenesis of RA ²². In contrast, Tregs are now known to limit immune-mediated pathology, which are essential in maintaining immune homeostasis and play central roles in immunoregulation and the induction of tolerance ²³. Th1/Th2 cell imbalance plays a pivotal role in the pathogenesis of RA, which is equivalent to proinflammatory/anti-inflammatory cytokine imbalance ^24,25. In CIA + HDL animals, the ratio of Th1/Th2 was decreased and the Th1/Th17 cell was increased compared with those of the CIA group (P < 0.05). The effect of HDL occurred by shifting the Th1/Th2 and Th1/Th17 balance, thus providing a balance in the proinflammatory and anti-inflammatory effects. In addition, although the differences in Treg levels among the three groups were not statistically significant, HDL may increase the level of Tregs. The reason may be that HDL decreases the Treg apoptosis, whereas HDL did not alter naïve or memory CD4 + T cell survival. Apart from this, HDL enhances Treg ATP concentration and mitochondrial activity, contributing to immunomodulatory effect ^26,27.

As professional APCs, DCs have the most potential to present antigens. It is well established that DCs play a central role in initiating both innate immune responses and highly specific acquired immunity. Pathogen recognition triggers DCs activation, and mature DCs that express high levels of costimulatory molecules and produce cytokines that provide a complete signal are required for full activation of T cells ²⁷. We observed that the expression of MHC-II, CD86, and CD40 in CD11c + BMDCs was suppressed by HDL in vitro. Costimulatory molecules other than CD80 in DCs was suppressed by HDL, which may lead to T cell unresponsiveness and promote tolerance. HDL is involved in interactions with ATP binding cassette transporter A1 (ABCA1) or ATP binding cassette transporter G1 (ABCG1) and removes cholesterol from lipid rafts in DCs ^28,29. The effect of HDL on surface marker expression may be relevant to the shifting composition of lipid rafts, particularly decreases in cholesterol. In addition, HDL inhibits the differentiation of monocytes to DCs by increasing monocyte secretion of prostaglandin E2 (PGE2) and IL-10 ³⁰. Our study demonstrated that HDL inhibited the expression of costimulatory molecules on DCs, but the mechanism is not clear.

In conclusion, HDL inhibits inflammation by suppressing DCs maturation and modulating CD4 + T cell subtypes. HDL might potentially modulate an aberrant immune response, which might be suitable therapeutic agents for inflammatory arthritis.

ABCG1 ATP binding cassette transporter G1

ABCA1 ATP binding cassette transporter A1

BMDCs marrow-derived dendritic cells

CIA collagen-induced arthritis

CV cardiovascular

CVD cardiovascular disease

DC dendritic cell

FCA Freund’s complete adjuvant

FACS ﬂuorescence-activated cell sorting

FIA Freund's incomplete adjuvant

GM-CSF granulocyte–macrophage colony-stimulating factor

HDL High-density lipoprotein

MTX methotrexate

RA Rheumatoid arthritis

Th T helper

Tregs regulatory T cells

Ethics approval and consent to participate

All experiments were approved by the Animal Use Committee of the affiliated hospital of Qingdao University.

Consent for publication

All authors gave their consent for publication.

Availability of data and material

The data that support the findings of this study are available from the corresponding author [ECM], upon reasonable request.

Competing interests

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in the manuscript.

Funding

This work was supported by these funds: Shandong provincial key research and development plan (2016GSF201040), Qingdao people's Livelihood Science and technology program (17-3-3-25-nsh), Shandong medical and health science and technology development plan (2016WS0264).

Authors' contributions

Conceived and designed the experiments: YW BL. Performed the experiments: XW SL DL JZ QD BL YW. Analyzed the data: XW DJ YW. Contributed reagents/materials/analysis tools: YW BL. Wrote the paper: XW YW.

Acknowledgements

We are grateful to Professor. JiGang Wang for providing helps in histopathological analysis.

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No competing interests reported.

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HDL suppresses dendritic cell maturation and modulates CD4+ T cell subtypes against inflammatory in a mouse model of rheumatoid arthritis

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Version 1

Abstract

Figures

1. Background

2. Methods

2.1 Reagents and antibodies

2.2 Induction of CIA and administration of HDL

2.3 Measurement of mouse HDL levels

2.4 Histopathological analysis

2.5 Flow cytometry

2.6 Preparation of mouse bone marrow-derived dendritic cells(BMDCs)

2.7 Culture of BMDCs

2.8 Costimulatory molecule expression on DCs

2.9 Statistical analysis

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

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