Priming Inflammation through Type I Interferon Pathway: The Aggravating Role of ADAR1 Down-Regulation in Sepsis-Induced Lung Injury

doi:10.21203/rs.3.rs-2948514/v1

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Priming Inflammation through Type I Interferon Pathway: The Aggravating Role of ADAR1 Down-Regulation in Sepsis-Induced Lung Injury

https://doi.org/10.21203/rs.3.rs-2948514/v1

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Objectives and design:

The objective of this prospective experimental study is to investigate the role and underlying mechanism of ADAR1 knockdown in regulating inflammation and lung injury in mice with sepsis.

Subjects:

ADAR1 TM-inducible knockout mice(ADAR1^{flox/flox;ER−Cre+}).

Treatment:

To achieve ADAR1 knockdown, intraperitoneal injections of tamoxifen or vehicle corn oil were administered to 6- to 8-week-old mice on day 1 and day 3. After 2 days of TM treatment, sepsis was induced in mice by administering intraperitoneal injections of lipopolysaccharide (LPS).

Methods

ADAR1 inducible knockout mice and bone marrow-derived macrophages (BMDMs) were used for this study. Tamoxifen treatment was administered to induce ADAR1 knockdown, and LPS was injected to induce sepsis in mice. The cecal ligation and puncture model was also used.

Results

ADAR1 increase worsened sepsis in mice and patients. Knockdown led to increased mortality and lung injury, as well as elevated levels of inflammatory cytokines.

Conclusions

ADAR1 is crucial in regulating inflammation and lung injury in sepsis. Decrease of ADAR1 expression level activates type I interferon signaling pathways and increase inflammatory response, reducing LPS resistance. This study highlights ADAR1's essential role in maintaining immune inflammatory response.

ADAR1

Macrophage

Sepsis

Lung injury

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a common critical care disease with a high incidence rate of 81/10,000. It is an urgent and rapidly progressing condition with a high fatality rate [1]. Despite advances in drug treatment and respiratory support technology, the fatality rate of ALI/ARDS remains high at 40–45% in current times [2]. Sepsis, trauma, and shock are common causes of ALI, with sepsis being the leading cause of approximately 40% of ALI cases [3]. Sepsis is the leading cause of death in ALI, resulting in multiple organ dysfunction [4]. Alveolar macrophages secrete inflammatory factors in response to bacterial infection, causing an uncontrolled inflammatory cascade reaction and cell death. The activation of inflammatory signaling pathways is essential in sepsis regulation, yet many regulatory mechanisms remain unclear [5]. Therefore, exploring inflammation-activated signal paths is crucial in sepsis research.

Given the extensive RNA editing functions in the body and their involvement in many inflammatory and immune responses, it has been increasingly recognized that ADAR1 may have a significant relationship with sepsis. ADAR1, a member of the RNA specificity adenosine deaminase gene family, is present in organisms ranging from worms to mammals. As an RNA editing enzyme, ADAR1 has two isoforms, P150 and P110, due to alternative usage of exon1a or 1b during RNA splicing [6, 7]. ADAR1 enzymes bind to double-stranded RNA (dsRNA) and convert adenosine (A) to inosine (I) through deamination, thereby regulating a vast array of protein expression and RNA signaling pathways. This allows ADAR1 to play a broad regulatory role in vivo, including in inflammation [8].

However, it remains largely unknown whether ADAR1 is an inflammatory injury protein or an anti-infective protein. Previous studies have demonstrated that LPS and E. coli can increase alveolar macrophage ADAR1 expression, accompanied by upregulated interferon expression and increased lung injury add reference. Researchers have suggested that interferon-induced ADAR1 upregulation may be involved in lung injury processes [9]. Another study showed that inhibiting P150 expression might reduce LPS-induced lung injury [10]. However, we have recently discovered that ADAR1 is essential for maintaining normal intestinal and liver physiological function, and deletion of ADAR1 leads to cell injury and inflammatory activation [11, 12]. We also found that ADAR1 can inhibit the downstream RIG-I like receptors (RLRs) dependent RNA sensing signaling pathway to suppress interferon secretion [13]. Moreover, it has been reported that ADAR1 can inhibit interferon secretion by suppressing the MDA-5 signaling pathway and reversing the lethality caused by ADAR1 knockout in mice [14, 15]. Thus, ADAR1 plays a key role in inflammatory regulation, potentially closely linked to many inflammatory pathogenic conditions. However, the role of ADAR1 in the pathogenesis of sepsis and sepsis-induced lung injury remains unclear.

In this study, we examined changes in sepsis tolerance and sepsis-induced lung injury by down-regulating ADAR1 expression in mice using TM-induced ADAR1 knockout mice. The primary outcome measures included mouse survival rates, levels of inflammatory activation, and lung injury.

Mice and BMDM

ADAR1 inducible Knockout (iKO) mice were prepared by crossing floxed ADAR1 mice [16] and Cre-ER transgenic mice[17] as described previously [12]. Mouse Bone Marrow Derived Macrophage (BMDM) was prepared from ADAR1 flox/flox;CreER⁺ (ADAR1^f/f;Cre+) and ADAR1 flox/flox;CreER⁻ (ADAR1^f/f) male mice. PCR genotyping was used to identify the mice genotype. ADAR1 gene deletion responding to TM induction is effective in ADAR1^f/f;Cre+ genotyping but no for ADAR1^f/f. Procedures for all animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

TM Treatment

For ADAR1 knockout, tamoxifen (cat# T5648, Sigma, St. Louis, MO, USA;200 mg/kg, TM) was injected intraperitoneally (i.p.) into 6- to 8-week-old mice on day 1, 2, 3. For ADAR1 knockdown, TM (100 mg/kg) was injected on day 1, 3. LPS was

injected with 1 mg/kg i.p. after 1 day of TM treatment. Mice were killed at various times to collect the tissues and lung macrophage and peritoneal macrophage.

BMDM Isolation and Culture

Bone marrow was flushed out with pre-chilled Dulbecco's Modified Eagle Medium (DMEM) from femurs and tibias, which were harvested from WT and the above-mentioned gene knockout mice following the previously described method [18, 19]. In brief, cell pellets were collected by centrifugation at 4°C, and erythrocytes were lysed with RBC lysis buffer (eBioscience, San Diego, CA, USA). The resultant cells were then washed two times with PBS and suspended in BMDM culture medium (DMEM containing 10% fetal bovine serum complemented with 50 µg/ml penicillin/streptomycin and 10 ng/ml recombinant macrophage-colony stimulating factor (M-CSF; Sigma-Aldrich, St Louis, MO, USA)) at a concentration of 106 cells/ml and seeded into 6-cm ultra-low attachment surface plates (Corning Costar, Corning, NY, USA). The BMDM culture medium was changed on day 3 and day 6. BMDM were fully differentiated and ready for use at day 7.

Inducible Gene Deletion in BMDM

BMDM were cultured in DMEM with 10% FBS and penicillin-streptomycin at 37°C, 5% CO₂. Once the cells were prepared, BMDM were split into two parallel groups: one for TM induction and another as a non-TM treatment control. A total of 200 nM 4-hydroxytamoxifen were added to the cells on the second day. Gene deletion and ADAR1 protein depletion were monitored each day after TM addition. As ADAR1 protein nearly disappeared at 48 h, the cells were stimulated with LPS at 72 h after TM induction.

Cecal Ligation and Puncture Model

Cecal ligation and puncture was performed as previously described [20] with some modifications. Isoflurane anesthesia (2.5% in 100% O2) was initiated in an induction chamber and maintained by delivery through a face mask. After shaving and cleaning the ventral abdominal wall with alcohol, a midline incision was made and the cecum was exteriorized. Cecal contents were massaged out of the tip and towards the base of the cecum and the distal 0.5 cm of the apex was ligated with 3 − 0 silk suture. A 25-gauge needle was used to perforate the ligated portion of the cecum once in a through-and-through manner. Sham CLP mice, in which the cecum was exteriorized but neither ligated nor punctured, were performed as controls. The cecum was returned to the abdominal cavity, the abdominal wall and skin were closed with 4 − 0 Vicryl suture.

Western Blot

Cell or tissues lysates were separated by 8 and 12% SDS-PAGE, and then were transferred onto PVDF membranes. After blocking for 1 h at room temperature with blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), blots were incubated with the primary antibody at 4°C overnight, followed by incubation with the appropriate secondary antibodies (Santa cruz) for 1 h. Protein bands were detected using the HRP system.

Flow Cytometric Analysis

Single BMDM, lung and peritoneal macrophage and were analyzed by flow cytometry using a BD FACSAria II system (BD Biosciences, San Jose, CA).

Histopathologic Examination

Lung tissue sections were fixed in 4% paraformaldehyde and paraffin-embedded. The sections were stained with hematoxylin and eosin for histopathologic examination.

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of cytokines in the serum were measured using specific ELISA kits (R&D Systems, Minneapolis, MN).

Analysis of BALF

Bronchoalveolar lavage fluid sampling and cell counting were conducted as previously reported [21]. Briefly, at a predetermined time point, the tracheas were cannulated, and the lungs were lavaged with PBS three times, 3 mL for each after mice were anesthetized. The lavaged fluid was harvested by gentle aspiration, centrifuged at 300g for 10 min at room temperature. Aliquots of the cell suspension were put onto glass slides and stained using a modified giemsa's staining for differential cell counts or used for flow cytometry analysis.

Statistical Analysis

The Student t test or Kaplan-Meier survival analysis followed by log-rank tests was performed using GraphPad Prism VI software (Graph-Pad Software, San Diego, CA). Data are presented as mean ± SD. A p value < 0.05 was considered to be significant. The means ± SD are shown in the figures where applicable.

ADAR1 expression increases in lung tissues of sepsis mice and clinical sepsis patients

To elucidate the expression of ADAR1 in sepsis, we assessed ADAR1 expression using an LPS i.p. injection sepsis mouse model and collected lung tissue samples at various time points. ADAR1 expression in the LPS group was significantly higher after 6 hours compared to the PBS i.p. injection control group (P < 0.05) (Fig. 1A). We also investigated the classic CLP sepsis model, where mice exhibited typical septic symptoms 12 hours after the CLP procedure. The respiratory rate of the sham control group was significantly higher, and the body surface temperature was significantly lower (P < 0.05) (Fig. 1B) compared to the CLP group. ADAR1 protein expression significantly increased 12 hours after CLP establishment compared with the sham group (P < 0.05) (Fig. 1C). In the BALF of sepsis and non-sepsis patients in the ICU, ADAR1 P150 expression in sepsis patients was significantly higher compared to non-sepsis patients (P < 0.05) (Fig. 1D).

ADAR1 deletion in ADAR1-inducable knockout and knockdown mouse models

To examine the effect of ADAR1 on sepsis regulation in mice, we used an inducible ADAR1 knockout mouse model in which the exon E12-E15 was deleted upon tamoxifen induction(Fig. 2A). Mouse genotypes were confirmed via PCR. After TM 100 mg/kg i.p. injection on days 1 and 3, the loxp band of ADAR1flox/flox (ADAR1^f/f) mice was consistent, indicating ADAR1 gene knockout. ADAR1^f/f; Cre⁺ mice displayed a deleted band and loxp band, suggesting partial knockout of ADAR1 (Fig. 2B), resulting in down-regulated ADAR1 expression in the tested tissue or ADAR1 knockdown in the lungs. Lung tissue samples were collected on day 5 after 200 mg/kg TM i.p. injection for 3 consecutive days or 100 mg/kg on days 1 and 3. ADAR1 was deleted after i.p. injection of 200 mg/kg TM for 3 consecutive days, while ADAR1 was down-regulated after i.p. injection of 100 mg/kg TM on days 1 and 3, suggesting ADAR1 gene transcription knockdown (Fig. 2C).

Partial ADAR1 deletion increased mortality in mice under nonlethal dosage LPS challenge

To investigate the impact of ADAR1 knockdown on sepsis in mice, we observed mouse survival after TM knockdown of ADAR1 and i.p. injection of varying LPS concentrations. Intriguingly, ADAR1 knockout mice exhibited significantly higher mortality following 20 mg/kg, 5 mg/kg, and 1 mg/kg LPS challenges compared to the control group (P < 0.05) (Fig. 3A, B, and C). To confirm that mouse death was not caused by ADAR1 down-regulation, we used ADAR1^f/f; Cre⁺ mice as a control, administered TM 100 mg/kg to induce ADAR1 knockdown, and challenged with PBS 5 days later to observe mouse survival. Compared to ADAR1 non-knockdown mice, LPS at 1, 5, and 20 mg/kg significantly increased the mortality of ADAR1 knockdown mice (P < 0.05). No significant difference was observed among the three groups with different LPS doses (P > 0.05) (Fig. 3D).

Partial ADAR1 deletion increased lung injury and inflammatory activation and LPS stimulation

To investigate the causes of increased death and lung injury induced by LPS after ADAR1 knockdown, we detected serum interferon-α (IFN-α) and other inflammatory factors, as well as lung exudation. Serum IFN-α, IL-6, and MCP-1 levels in ADAR1 knockdown mice were significantly up-regulated compared to control mice (P < 0.05) (Fig. 4A). Cytokine levels were also significantly higher in ADAR1 knockdown mice 6 hours after LPS challenge (P < 0.05), compared to ADAR1 non-knockdown mice (P < 0.05) (Fig. 4B). Furthermore, lung pathological changes, including interstitial thickening, alveolar structural damage, and increased exudation, were observed in the lung HE staining section of ADAR1 knockdown mice after 1 mg/kg and 20 mg/kg LPS i.p. injection. These changes were not found in the ADAR1 non-knockdown group (Fig. 4C). Protein exudation in the BALF of ADAR1 knockdown mice was significantly higher than in non-knockdown mice after 1 mg/kg LPS pulmonary perfusion (P < 0.05) (Fig. 4D). Following ADAR1 knockdown and LPS i.p. injection, cells collected from BALF were Giemsa stained using the cytospin method. The counts of alveolar macrophages (AM) in ADAR1 knockdown mice were significantly higher than in non-knockdown mice (P < 0.05), accompanied by increased neutrophil cell exudation (Fig. 4E).

ADAR1 deletion Increased macrophage death in vivo and in vitro after LPS challenge

To study the effect of LPS on macrophage cell death after ADAR1 knockdown, we used flow cytometry to analyze apoptotic and necrotic AM and peritoneal macrophages (PM) by 7-AAD and Annexin V staining. The level of interferon-α in BMDM culture supernatant was detected by ELISA after ADAR1 knockdown. After 1 mg/kg LPS lung perfusion for 6 hours, AM death significantly increased in ADAR1 knockdown mice compared to ADAR1 un-knockdown mice (P < 0.05) (Fig. 5A). Additionally, PM death in ADAR1 knockdown mice was significantly higher than in ADAR1 non-knockdown mice after 1 mg/kg LPS i.p. injection for 6 hours (P < 0.05) (Fig. 5B).

Bone marrow-derived macrophages (BMDMs) were identified with the F4/80 marker by flow cytometry. The positive rate of F4/80 in cultured cells was over 90% (Fig. 5C). ADAR1 expression was significantly reduced after 48 hours by inducing knockout with 4-hydroxy TM 200 nM in vitro (Fig. 5D). BMDMs were stimulated by LPS in vitro for 24 hours following ADAR1 knockdown by 4-hydroxy, and macrophage death was analyzed by flow cytometry. Consistent with in vivo experiments, BMDM death in the ADAR1 knockdown group was significantly higher than in non-knockdown macrophages (P < 0.05) (Fig. 5E). The level of IFN-α in the culture medium of ADAR1 knockdown macrophages was significantly.

In this study, we confirmed that ADAR1 protein expression in lung tissue is up-regulated in sepsis mice, and for the first time, we discovered an increase in ADAR1 expression in the BALF of sepsis patients. Additionally, we investigated the role of ADAR1 in sepsis regulation using inducible ADAR1 knockout mice. The results demonstrated that ADAR1 knockdown in mice led to inflammation priming and decreased LPS challenge resistance, ultimately resulting in severe lung injury and death due to low-dose LPS exposure.

ADAR1 plays a crucial role in maintaining normal physiological functions of the bone marrow, small intestine, and skin [12, 22, 23]. ADAR1 deletion causes embryonic death in mice [16, 24]. Recently, significant discoveries have been made in the field of interferon signaling pathways and inflammation regulation. ADAR1 can inhibit the production of type I IFN and other inflammatory cytokines by blocking the MDA5/RIG-I signal pathway in the innate immune system [13–15, 25]. ADAR1 mRNA expression in animals with bacterial infections has been reported [26, 27]. Additionally, ADAR1 plays a ubiquitous regulatory role in the cellular immune system in response to viral infections [28]. However, the role of ADAR1 in sepsis regulation and organ damage is scarcely reported.

It has been reported that LPS can induce increased expression of ADAR1 mRNA in lung tissue, accompanied by increased interferon expression and lung injury. LPS and Escherichia coli stimulation resulted in ADAR1 mRNA up-regulation in the alveolar macrophage cell line (MH-S) in mice. This study suggested that increased ADAR1 expression induced by interferon might be involved in the lung injury process [9]. Another study demonstrated that LPS could induce P150 expression in rat alveolar macrophages and reduce inflammatory injury by down-regulating P150 expression in macrophages [10]. However, these studies did not clarify the mechanism of ADAR1 action, and the down-regulation of ADAR1 P150 reducing inflammatory damage was inconsistent with recent studies on ADAR1 maintaining physiological functions. Consequently, the regulatory role of ADAR1 in bacterial sepsis and sepsis-induced lung injury remains largely unknown.

We first determine the expression of ADAR1 in sepsis. We demonstrated increased expression of ADAR1 in lung tissue of the LPS i.p. injection and in the CLP model, which is a time-dependent manner. We also found that ADAR1 expression increased in the BALF of severe sepsis patients, as determined by Western Blot. These results suggested that ADAR1 may be involved in sepsis regulation.

For further investigate, we investigated the potential regulatory mechanismusing the ADAR1 knockout mouse model. Previous research has shown that ADAR1 deletion mice die in the embryonic phase, which is likely strongly associated with the high expression of type I IFN [14]. We constructed ADARl^f/f;cre+ mice and ADARl^f/f mice using the CreER-flox/flox system. The ADAR1 gene in ADARl^f/f;cre+ mice can be induced to knock out by TM, while ADAR1^f/f cannot. We found that adult mice died at 3 day after continuous TM 200 mg/kg i.p. injection (data not shown). We then reduced the TM does, allowing ADAR1 to be knocked down in ADAR1^f/f;cre+ mice without causing significant death. However, serum interferon level significantly increased. Interestingly, the resistance to LPS challenge in ADAR1 knockdown mice significantly reduced upon LPS stimulation. Even non-lethal doses of LPS led to rapid death in ADAR1 knockdown mice compared to controls. Cytokine levels in ADAR1 knockdown mice with LPS challenge were significantly higher than those without LPS, while the type I IFN levels in ADAR1 knockdown mice with or without LPS challenge were not significantly different. These results suggested that the change in interferon levels after ADAR1 knockdown is far greater than other inflammatory factors, which may be the key reason for inflammation priming.

Sepsis is a clinical course characterized by a systemic inflammatory response induced by bacterial infection. The lung is the most vulnerable target organ for sepsis [3]. Alveolar macrophage (AM) activation by sepsis triggers the release of inflammatory factors, leading to an uncontrolled inflammatory cascade reaction and cell death, which are at the core of the pathogenesis of sepsis-induced lung injury [5]. Studies have shown that macrophage migration inhibitory factor can improve the outcomes of ALI [29]. Therefore, the inflammatory response of alveolar macrophages is an important target for further study in sepsis-induced lung injury.

We examined HE slices and the alveolar inflammation and observed alveolar inflammation and exudation. We found that low-dose LPS in ADAR1 knockdown mice can cause severe lung pathological damage and increased alveolar exudate compared to non-knockdown mice. As the core of inflammatory response, macrophage death caused by severe sepsis and inflammation challenge was the key reason of inflammation exacerbation and organism death. We examined macrophage death in the ADAR1 knockdown mice with LPS challenge and found the macrophage death significantly increased than the non-ADAR1 knockdown mice. Consistently, in vitro BMDM experiments revealed that ADAR1 knockdown led to up-regulation of type I IFN. The death of ADAR1 knockdown macrophage with LPS stimulation increased significantly compared with the non-knockdown mice, suggesting that ADAR1's role in sepsis inflammatory response is related to the type I IFN signaling pathway.

Low-doses of LPS challenge twice resulted in rapid mouse lethality due to the first LPS inflammation priming effect [30]. Therefore, combined with our results, we hypothesize that ADAR1 knockdown leads to increased type I IFN and other inflammatory factors, particularly type I IFN, which primes inflammation activation in vivo, making the organism more sensitive to LPS challenge. The organism rapidly suffers from sepsis and death when faced again with even non-lethal doses LPS challenge.

The two-hit hypothesis is common in clinical infection patients, where early onset of trauma or shock forms the first hit, followed by injury factors such as infection becoming the second hit, resulting in an amplification of the injury effect. This two-hit process severely affects the prognosis of sepsis patients; however, the specific regulatory mechanisms are not fully elucidated [31, 32]. Our study results suggest a possible important mechanism: abnormal expression of ADAR1, along with other inflammatory regulatory proteins, leads to inflammation priming in vivo, which sensitizes the organism to a second inflammatory hit. Additionally, we found that ADAR1 expression was down-regulated in aged mice and diabetic mice (data not shown). Our findings suggest that abnormal inflammation activation due to the IFN signaling pathway in response to ADAR1 down-regulation can reduce resistance to infection, which may be a potential mechanism to explain the susceptibility to infections in aging patients and those with diabetes. Moreover, as an up-regulated protein during infection and sepsis, ADAR1 plays a potentially important role in sepsis regulation. ADAR1 may serve as a promising marker for diagnosis, predicting prognosis, or as a potential therapeutic target for sepsis patients in future research.

In conclusion, our comprehensive in vitro and in vivo experiments have provided substantial evidence that the ADAR1 plays a significant role in the development and progression of sepsis and sepsis-induced lung injury. ADAR1 expression in lung tissue is up-regulated in sepsis; and suppression of ADAR1 expression results in tolerance to LPS challenge which can be attributed to inflammation priming that involves increased type I IFN expression and the release of other pro-inflammatory cytokines. These findings highlight the importance of understanding the complex regulatory mechanisms involved in sepsis and sepsis-induced lung injury and emphasize the potential of ADAR1 as a therapeutic target or diagnostic marker for sepsis patients. Further investigation into the role of ADAR1 in inflammation and its association with other factors contributing to sepsis susceptibility, such as aging and diabetes, may provide valuable insights into the development of novel therapeutic strategies and improve the clinical management of sepsis and sepsis-related complications.

DATA AVAILABILITY :

The data that supports the findings of this study are available upon reasonable request. Requests for data should be directed to

Hu Peng([email protected])

or Xiangyu Zhang([email protected])

CONFLICT OF INTEREST :

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

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Table 1．Descriptive baseline characteristics of the study population with measured ADAR1 expression of BALF.

Parameter	Non-Sepsis(n=7)	Sepsis(n=6)	P-value
Age, years	63.0(15.2)	65.6(13.1)	>0.05
Female gender	4(57.1%)	4(66.7%)	>0.05
Infections diagnosis	N/A	Severe pneumonia	N/A
SOFA score	2.2(1.2)	3.5(1.3)	<0.05

All continuous variables are presented as mean (SD) and ordinal variables are presented as n (%) except if stated otherwise.

No competing interests reported.

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Priming Inflammation through Type I Interferon Pathway: The Aggravating Role of ADAR1 Down-Regulation in Sepsis-Induced Lung Injury

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