Rats were terminated with chloral hydrate (10%) and perfused transcardially with ice cold 0.01M phosphate-buffered saline (PBS). The brain was rapidly dissected; one hemisphere was used for immunostaining and the other for western blot. The hemisphere used for immunostaining was postfixed in 4% paraformaldehyde overnight at 4°C. After dehydrating with 15% and 30% sucrose, the brains were embedded in OCT (SAKURA Tissue-Tek, USA), and cryostat transverse sections of the brain (20 μm) were obtained. Four sections of hippocampus were randomly picked from 4 sets of serial sections from each rat at −3.6 to −4.16 mm anteroposterior to the bregma for immunostaining. Sections were washed three times in 0.01M PBS and incubated in 3% H2O2 for 10 min. After three washes, sections were blocked with 5% BSA in 0.01M PBS plus 0.1% Triton X-100 for 1 h at room temperature and then incubated with primary antibodies (1:1000, Iba-1, HMGB1, p-CREB) at 4°C overnight. Full details on the antibodies used are presented in **Table 1**. After washing three times in 0.01M PBS, the sections were incubated with secondary antibodies (Biotinylated Goat Anti-Rabbit IgG, 1:200, Vector, USA) for 1 h at room temperature, subsequently washed three times in 0.01M PBS and finally covered with permount containing DAPI (Vector, USA, H-1200). For each staining, pictures of Cornu Ammonis (CA)1 and Dentate Gyrus (DG) areas were taken under the same magnification (40× objective lens) and constant light intensity with a microscope (DS-Ri1, Nikon, Japan) by an author blinded to treatments.

#### Western Blot

Western blotting was used to assess the expression of *N*-methyld-aspartate receptor (NMDAR) subunits NR2A and NR2B, HMGB1, and GAPDH in the hippocampus. Briefly, frozen hippocampus was homogenized in lysis buffer containing protease inhibitors cocktails (Roche, Germany, cat num P8340) and phenylmethanesulfonylfluoride (PMSF, Sigma, USA, cat num p7626). The quantity of protein of samples was determined using a BCA protein assay kit (CWBio, China) according to the manufacturer's instructions. Equal amounts of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 10% skim milk in TBST buffer for 1 h and then incubated with primary antibodies, HMGB1 (1:1000 Abcam, USA), NR2A (1:2000 Epitomics, USA), NR2B (1:1000 Abcam, USA), GAPDH (1:2000 Proteintech, China), and GAPDH (1:2000 Proteintech, China), anti-His antibody (1:5000 Sigma) overnight at 4°C (**Table 1**). After three washes, membranes were incubated with the secondary antibodies (Goat Anti-Rabbit IgG, HRP Conjugated, 1:2000, CWBIO, China, catalog number CW0103, Goat Anti-Mouse IgG, HRP Conjugated, 1:2000, CWBIO, China, catalog number CW0102) at room temperature for 2 h. Finally, visualization of the proteins was accomplished by enhanced chemiluminescence detection kit (Pierce; Thermo Scientific, Shanghai, China), and the intensity of each band was quantified by densitometry. Relative expression levels of protein were normalized by the ratio of target protein (HMGB1, NR2A, and NR2B) to GAPDH.

#### Enzyme-Linked Immunosorbent Assay

Under anesthesia, blood was collected from the right auricle in EDTA coated tubes and then centrifuged for 10 min at 3000 rpm. Plasma was collected and frozen at −80°C for further analyses. Human plasma was collected under an approved protocol by the Ethics Committee of the Third Xiangya Hospital of Central South University (protocol number: 2015-S165). Six patients identified with POD by The Confusion Assessment Method (CAM) (21) daily testing were compared to age-matched surgical controls (non-delirious). Blood was centrifuged at 3000 rpm for 10 min at room temperature, and the plasma samples were immediately frozen at −80°C. Concentration of HMGB1 in plasma was measured using ELISA (IBL International, Germany, catalog number ST51011) according to the manufacturer's protocol.

#### Statistical Analysis

Data are shown as mean ± SEM. Two-way repeated measures ANOVA was used to analyze the data from Barnes Maze test. ANOVA was used to analyze the data from open field test, Western blot, and immunostaining. Bonferroni Multiple Comparison Test was performed to compare selected groups when ANOVA showed significance. Statistical analysis was performed using Prism 5 (Graph Pad Software Inc., La Jolla, CA, USA). Significance was set at *p* < 0.05.

### RESULTS

#### Blocking HMGB1 Improves Postoperative Cognitive Decline and Anxiety in Aged Rats

All rats were included in the study protocol, and no mortality was reported after the surgical procedure. We evaluated learning

and memory by error number in the Barnes maze and detected levels of anxiety in the open field. In this test, repeated measures ANOVA showed that the error number was significantly affected by treatment [*F*(2,69)= 5.73, *p*= 0.01], measure time [*F*(3,69)= 36.79, *p* < 0.001], and the interaction of treatment and measure time [*F*(6,69) = 3.41, *p* = 0.005]. Compared to the control group, the errors of the surgery group treated with control antibodies (S + IgG group) were significantly increased on days 2 and 3 after surgery, indicating an impairment in learning and memory of aged rats after surgery (*p* = 0.001, respectively) (**Figure 1A**). Compared to the S + IgG group, errors in S + anti-HMGB1 group were significantly decreased on days 2 (*p* = 0.035) and 3 after surgery (*p* = 0.023), denoting a protective effect by perioperative anti-HMGB1 treatment (**Figure 1A**).

In addition, in the open field test, the percentage of square crossing in the central area in the S + IgG group was significantly lower than that of control group on day 3 after surgery (7.88 ± 1.74 vs. 16.68 ± 0.79%, *p* < 0.05) (**Figure 1B**). Percentage square crossing in the central area in S + anti-HMGB1group significantly increased relative to the S + IgG group (16.79 ± 4.53 vs. 7.88 ± 1.74%, *p* < 0.05) (**Figure 1B**).

#### Effects of Anti-HMGB1 on Memory Marker p-CREB

To further investigate the mechanisms of anti-HMGB1 in preventing surgery-induced memory decline, we assessed the transcription factor CREB, which is closely involved in synaptic plasticity and memory function (22). Compared to the control group, the level of p-CREB in the DG area of the S + IgG group was distinctly decreased on day 3 (13.19 ± 1.05 vs. 2.85 ± 0.43, *p* < 0.05) (**Figures 2A,B**). Rats treated with anti-HMGB1 mAb had significantly higher p-CREB in the DG and CA1 areas than the S + IgG group (14.04 ± 0.80 vs. 2.85 ± 0.43 and 9.66 ± 0.47 vs. 4.68 ± 0.50, *p* < 0.05, respectively), which was similar to control rats (**Figures 2B,C**).

We also assessed NMDAR expression in hippocampal lysate. Compared to control, expression of NMDAR subunits NR2A and NR2B was increased at day 3 after surgery (1.00 ± 0.09 vs. 2.04 ± 0.21 and 1.00 ± 0.04 vs. 1.54 ± 0.08, *p* < 0.05, respectively) (**Figures 3A,B**). Treatment with anti-HMGB1 mAb significantly reduced the upregulation, with levels returning to baseline at day 3 as compared to the S + IgG group (2.04 ± 0.21 vs. 1.24 ± 0.07 and 1.54 ± 0.08 vs. 1.09 ± 0.15, *p* < 0.05, respectively) (**Figure 3**).

#### Microglia Activation Is Attenuated by Anti-HMGB1 Treatment

Neuroinflammation and microglia activation have been implicated in the pathophysiology of cognitive decline (23). Compared to control, the percentage of activated microglia, defined by pleomorphic and bigger cell body with de-ramification and shortening of cell processes, was significantly higher in the CA1 and DG hippocampal areas after surgery (59.64 ± 2.54 vs. 85.30 ± 0.90 and 57.23 ± 3.25 vs. 82.25 ± 1.36, *p* < 0.05, respectively) (**Figure 4**). Notably, treatment with anti-HMGB1 mAb decreased microglia activation in CA1 and DG at day 3 after surgery (71.10 ± 3.30 vs. 85.30 ± 0.90 and 65.92 ± 0.95 vs. 82.25 ± 1.36, *p* < 0.05, respectively) (**Figure 4**).

#### Peripheral HMGB1 Contributes to Postoperative Neuroinflammation

To characterize the role of HMGB1 in neuroinflammation after surgery, we measured the cellular distribution and expression of HMGB1 in the hippocampus. Nuclear translocation of HMGB1 to the cytoplasm has been reported as a critical pathological mechanism in a number of different conditions (24, 25). Notably, we found no evident change in cellular localization of HMGB1 in the hippocampus after surgery (**Figure 5A**).

This prompted us to evaluate if peripheral HMGB1 was responsible for this neuroinflammatory response. We used His-tagged HMGB1 injected into the tail vein of aged rats to

FIGURE 1 | Anti-HMGB1 improves memory dysfunction and anxiety in aged rats. Spatial memory was evaluated in the Barnes maze. Rats that had undergone liver surgery had significant memory impairments as detected by error numbers in reaching the correct target box (A). Treatment with anti-HMGB1 mAb significantly improved postoperative memory dysfunction on both days 2 and 3 after surgery. (B) Anxiety was measured in an open field on postoperative day 3. Treatment with anti-HMGB1 mAb prevented postoperative anxiety as compared to the S + IgG group. Results are expressed as mean ± SEM (*n* = 12). \**p* < 0.05 vs. C; #*p* < 0.05 vs. S + IgG group by two-way repeated measures ANOVA for Barnes Maze and one-way ANOVA for open field. Abbreviations: C, control; S, surgery.

detect blood-derived HMGB1 and found a significant increase in His-tagged HMGB1 in the hippocampus of both control and surgery groups (1.00 ± 0.13 vs. 4.69 ± 0.61 and 5.41 ± 0.95 vs. 1.10 ± 0.32, *p* < 0.05, respectively) (**Figure 5B**) suggesting that peripheral HMGB1 could directly enter into the CNS. Plasma levels of HMGB1 were significantly increased in aged rats at days 1 and 3 after surgery (322.20 ± 45.25 vs. 1098.00 ± 82.64 and 322.20 ± 45.25 vs. 918.30 ± 140.80, *p* < 0.05, respectively) (**Figure 5C**). We also evaluated human plasma levels of HMGB1 from patients diagnosed with POD on day 3 and found a significant elevation compared to age-matched surgical subjects without delirium (627.1 ± 160.2 vs. 1418 ± 258.4, *p* < 0.05) (**Figure 5D**).

#### DISCUSSION

The current study aimed at investigating the role of HMGB1 signaling after surgery and the therapeutic potential of neutralizing anti-HMGB1 mAbs to prevent surgery-induced memory dysfunction. Our data indicate a key role for peripherally released HMGB1 causing neuroinflammation and memory dysfunction after abdominal surgery. Treatment with a neutralizing anti-HMGB1 mAb reduced microglial activation and prevented synaptic plasticity dysfunction. Herein, we uncovered a novel mechanism for HMGB1 in regulating p-CREB and synaptic plasticity after surgery.

HMGB1 is a multifunctional molecule with a critical role in sterile injury, including surgical manipulations (26). Passive and active release of HMGB1 may occur after injury: passive release occurs instantaneously as a result of disruption of cellular integrity and tissue necrosis; active secretion is a slower process due to the interaction with other cellular products (5, 27, 28). Release of HMGB1 as well as other DAMPs has been reported after surgery, with levels rapidly increasing within 30 min after orthopedic injury (10), suggesting that the release of HMGB1 may be critical in initiating an inflammatory cascade leading to CNS dysfunction. Furthermore, increased systemic levels of HMGB1 have been reported in elderly patients after gastrointestinal surgery, and these levels correlated with the development of POCD (12). Vacas et al. (29) showed a single dose of HMGB1 mAb effectively reduce circulating levels of HMGB1 and IL-6 after orthopedic surgery in younger mice. In our model, we found a more prolonged upregulation of systemic HMGB1. This may be related to a direct surgical insult to the liver, which contains a large pool of HMGB1, and greater cellular senescence promoting ageassociated inflammation (30, 31). Thus, we treated animals twice with mAb to prevent both the initial, passive, release of HMGB1 from the direct tissue trauma, and the secondary increase due to the ensuing inflammatory response.

The mechanism whereby peripheral inflammation contributes to neuroinflammation, neurodegeneration, and cognitive dysfunction remains unclear. Intracellular translocation of HMGB1 from the nucleus to the cytoplasm is critical for extracellular release by a non-classical secretory mechanism (24, 32). In our model, we did not observe evident translocation of HMGB1 from the nucleus to the cytoplasm in the CNS. The subcellular localization of HMGB1 in the studied hippocampal areas after surgery was similar to normal expression in the rat brain (33). It is possible that the protective effects in our model are mediated by an overall dampening of systemic DAMPs and cytokines, which in turn prevent endothelial dysfunction and neuroinflammation, and also a direct CNS effect or vagal regulation (34). Neuroinflammation has been reported in several models of POCD (10, 11, 35), and here we could confirm microglia activation in the hippocampal formation after surgery. The important discovery of the present study is that neuroinflammation was reduced by treatment with anti-HMGB1 mAb. Notably, HMGB1 is necessary and sufficient to trigger memory dysfunction (29), microglia activation (36), and prime the immune system (37) as demonstrated by administering recombinant HMGB1 in otherwise healthy animals. Thus, in POCD models preventing systemic HMGB1 increase may be critical in limiting BBB opening (11, 38, 39), subsequent monocyte chemoattractant protein (MCP-1) expression in the hippocampus (29), microglial activation, and memory deficits. The role of the systemic milieu in triggering CNS complications after surgery is further demonstrated by the experiments with His-tagged HMGB1, suggesting that peripheral HMGB1 can directly access the brain parenchyma of aged rats.

In this study, we measured CREB expression in the hippocampus, which is critical in memory and synaptic plasticity (22). Phosphorylated CREB was significantly decreased 3 days after surgery and treatment with anti-HMGB1 mAb reversed this impairment in the hippocampus. Since CREB is implicated in hippocampal-dependent memory function and synaptic plasticity, the rescuing effect of the anti-HMGB1 mAb treatment is consistent with the improved behavioral outcomes demonstrated in this study. Although we can describe a novel effect of anti-HMGB1 mAb to modulate neuroplasticity, the effects of peripheral surgery on neuronal function and p-CREB regulation require further elucidation. POCD has been associated with changes in synaptic plasticity, including long-term potentiation (LTP), and this may be dependent on the neuroinflammatory response after trauma (40). In this study, we found changes in NMDAR expression after surgery, which was restored by anti-HMGB1 mAb treatment. NR2A and NR2B are critical for sustaining LTP and are prominently expressed in hippocampal pyramidal excitatory cells (41). The increase in NR2A and NR2B after surgery may relate to acute neurotoxicity as higher expression of NR2A in the hippocampus has been associated with poorer cognitive outcomes (42). Furthermore, HMGB1 has been demonstrated as an excitatory and neurotoxic signaling molecule in the CNS (43) and exerts a prominent role in conditions like epilepsy (44).

The anti-inflammatory effects of HMGB1 neutralization have been described in several models, including POCD (7–9, 29). Here, we provide additional evidence for HMGB1 modulation of neuronal molecules of relevance to synaptic plasticity and memory function.

In the present study, we have not determined the redox states of HMGB1. This is critical to a better understating of the role of HMGB1 signaling after trauma as posttranslational modifications regulate receptor usage and thus functionality (45). The HMGB1-receptors TLR4 and RAGE have been demonstrated to be upregulated in POCD models (11, 35). No HMGB1 redox isoform restriction is known for RAGE signaling, while disulfide-HMGB1 is required for interaction with TLR4 and TLR4-mediated neuroinflammation and disruption of memoryrelated signaling in the hippocampus. Moreover, TLR4 activation modulates NMDAR subunits through HMGB1 signaling (46),

suggesting that neuroinflammation may be critical in mediating the cognitive deficits.

Postoperative neurocognitive disorders are multifactorial conditions with several factors contributing to overall changes in mental status (**Figure 6**). Recent evidence suggests women are at greater risk for cognitive decline after surgery than men (47, 48). Although we did not include age-matched males or younger females for comparison in this study, several studies have focused on POCD using males and younger rodent models. Herein, we attempted to bridge a critical gap in translation by applying a clinically relevant model to study the therapeutic effects of HMGB1 mAb in aged females. Given HMGB1 is a protein highly conserved across species (6), these findings may have implications beyond specific sex, age, and strain differences.

In conclusion, blocking HMGB1 during the perioperative period attenuated postoperative neuroinflammation and memory dysfunction. Based on these findings, we propose a key role for HMGB1 in mediating POD and provide new evidence for a protective role of neutralizing HMGB1 treatment in the perioperative setting.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

NT, TY, WO, and JT conceived of the study, analyzed the data, and wrote the manuscript. XW, JF, and MC performed the research. UA and HEH participated in the design of the study and contributed key reagents. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by grants from the National Natural Science Foundation of China (No. 81371216; No. 81471107; No. 81541028), the Swedish Research Council (No. 2012-1870), and the Anesthesiology Department, Duke University Medical Center.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2016.00441

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Terrando, Yang, Wang, Fang, Cao, Andersson, Erlandsson Harris, Ouyang and Tong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Inflammatory Regulation by Driving Microglial M2 Polarization: Neuroprotective Effects of Cannabinoid Receptor-2 Activation in Intracerebral Hemorrhage

*Li Lin1† , Tao Yihao2† , Feng Zhou2 , Niu Yin2 , Tan Qiang2 , Zheng Haowen3 , Chen Qianwei2 , Tang Jun2 , Zhang Yuan1 , Zhu Gang2 , Feng Hua2 , Yang Yunfeng4 and Chen Zhi2 \**

#### *Edited by:*

*Jixin Zhong, Case Western Reserve University, USA*

#### *Reviewed by:*

*Ying Ding, University of Miami, USA Ping Chen, Cincinnati Children's Hospital Medical Center, USA Zhi-Qiang Wang, BC Cancer Agency, Canada*

*\*Correspondence:*

*Chen Zhi<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 22 November 2016 Accepted: 24 January 2017 Published: 14 February 2017*

#### *Citation:*

*Lin L, Yihao T, Zhou F, Yin N, Qiang T, Haowen Z, Qianwei C, Jun T, Yuan Z, Gang Z, Hua F, Yunfeng Y and Zhi C (2017) Inflammatory Regulation by Driving Microglial M2 Polarization: Neuroprotective Effects of Cannabinoid Receptor-2 Activation in Intracerebral Hemorrhage. Front. Immunol. 8:112. doi: 10.3389/fimmu.2017.00112*

*1Department of Neurosurgery, Nanchong Central Hospital, Nanchong, China, 2Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, China, 3Department of Neurosurgery, Southwest Medical University Affiliated Hospital, Southwest Medical University, Luzhou, China, 4Department of Neurosurgery, Sichuan Provincial Corps Hospital, Chinese People's Armed Police Forces, Leshan, China*

The cannabinoid receptor-2 (CB2R) was initially thought to be the "peripheral cannabinoid receptor." Recent studies, however, have documented CB2R expression in the brain in both glial and neuronal cells, and increasing evidence suggests an important role for CB2R in the central nervous system inflammatory response. Intracerebral hemorrhage (ICH), which occurs when a diseased cerebral vessel ruptures, accounts for 10–15% of all strokes. Although surgical techniques have significantly advanced in the past two decades, ICH continues to have a high mortality rate. The aim of this study was to investigate the therapeutic effects of CB2R stimulation in acute phase after experimental ICH in rats and its related mechanisms.  The neuroprotective effects were prevented by SR144528, a selective CB2R inhibitor. Additionally, JWH133 suppressed neuroinflammation and upregulated the expression of microglial M2-associated marker in both gene and protein level. Furthermore, the expression of phosphorylated cAMP-dependent protein kinase (pPKA) and its downstream effector, cAMP-response element binding protein (CREB), were facilitated. Knockdown of CREB significantly inversed the increase of M2 polarization in microglia, indicating that the JWH133-mediated anti-inflammatory effects are closely associated with PKA/CREB signaling pathway. These findings demonstrated that CB2R stimulation significantly protected the brain damage and suppressed neuroinflammation by promoting the acquisition of microglial M2 phenotype in acute stage after ICH. Taken together, this study provided mechanism insight into neuroprotective effects by CB2R stimulation after ICH.

Keywords: intracerebral hemorrhage, cannabinoid receptor-2, neuroinflammation, microglial polarization, CREB

#### INTRODUCTION

Intracerebral hemorrhage (ICH) is a subtype of stroke with high morbidity and mortality, accounting for about 15% of all deaths from strokes (1). Pronounced inflammatory reactions play an important role in secondary brain injury following ICH (2, 3); various stimuli, including thrombin or glutamate, activate microglia and initiate an inflammatory response, and subsequently release pro-inflammatory cytokines or chemokines to enhance neuroinflammation (3, 4).

Microglia are the resident macrophages of the brain and the first responders of the immune system (5). They are highly plastic cells that can assume diverse phenotypes and engage different functional programs in response to specific microenvironmental signals. In particular, stimulation with interferon-γ promotes classically activated microglia/macrophage (M1 phenotype) that release destructive pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), causing damage to healthy cells and tissues (6). In contrast, cytokines such as interleukin-4 and interleukin-10 (IL-10) induce an alternative activated microglia (M2 phenotype) that generates anti-inflammatory cytokines, such as transforming growth factor beta (TGF-β) and IL-10, which possess neuroprotective properties (7, 8). Microglia activation and M1/ M2 polarization have been reported in several types of acute central nervous system (CNS) injury, such as traumatic brain injury, spinal cord injury, and ischemic stroke (9, 10). Microglia activation and polarization also occur in hemorrhagic stroke (11, 12); however, their underlying mechanisms after ICH remain unclear.

The cannabinoid receptor-2 (CB2R) signaling pathway plays an important role in CNS injury via regulation of microglial activities (13, 14). In addition, the expression of CB2R on microglia mainly depends on the activation state of microglia (15). CB2R on activated microglia have been shown to modulate properties of microglial migration and infiltration into the CNS during active neuroinflammation and degeneration (16). In our previous study, we showed that CB2R stimulation attenuated microglial accumulation after germinal matrix hemorrhage (GMH) in neonatal rats (17). However, whether CB2R has effects on microglia polarization following ICH remains unknown.

In this study, we investigated the therapeutic effects of CB2R stimulation in acute phase following experimental ICH and its related mechanisms. We found that selective stimulation of CB2R significantly protected brain injury and suppressed neuroinflammation by promoting M2 polarization of microglia. In addition, we also demonstrated that PKA/CREB signaling pathway was involved in, at least partially, the suppression of neuroinflammation. These results indicated that CB2R may be a potential therapeutic target for ICH.

#### ANIMALS AND METHODS

#### Animals

Excluding 6 rats that died of an overdose of anesthetic before modeling, total 213 adult male Sprague-Dawley rats (250–300 g) were used for this study. Rats were housed under specific pathogen-free conditions and had free access to food and water. Animals were sacrificed at the endpoint under deep anesthesia using an overdose of intraperitoneal pentobarbital. All efforts were made to minimize suffering and animal numbers according to the *Guide for the Care and Use of Laboratory Animals*; the study was approved by the Animal Care and Use Committee at the Third Military Medical University.

#### Experimental Design Experiment I

To determine the expression time course of CB2R after ICH, 18 rats were randomly assigned into three groups: ICH 0 h (*n* = 6), ICH 24 h (*n* = 6), and ICH 72 h (*n* = 6). Perihematomal tissue (shown as the white quadrangle in **Figure 1A**) was collected to detect the protein expression of CB2R.

#### Experiment II

A total of 168 rats were randomly divided into four groups for the mechanism study: sham-operated (Sham group, *n* = 42), ICH + Vehicle (ICH + Vehi group, *n* = 42), ICH + JWH133

(ICH + JWH group, *n* = 42), and ICH + JWH133 + SR144528 (ICH + JWH + SR group, *n* = 42). Sham group received only a needle insertion. The ICH + Vehi group received an equal volume of vehicle, and the ICH + JWH group received an intraperitoneal injection of a selective CB2R agonist, JWH133 (1.5 mg/kg, Tocris Bioscience, Minneapolis, MN, USA) at 1 h after surgery. The ICH + JWH + SR group was treated with a selective CB2R antagonist, SR144528 (3 mg/kg, Santa Cruz Biotechnologies, Dallas, TX, USA) 3 min before JWH133 (1.5 mg/kg) intraperitoneally. Brain water content (*n* = 6 in each group) and neurological scores (*n* = 6 in each group), including the modified Neurological Severity Score (mNSS) and forelimb placing test, were tested at 24 and 72 h after ICH. Gene levels of microglial M1/M2 markers and inflammatory cytokines in perihematomal tissue were evaluated at 6, 12, 24, and 72 h after ICH (*n* = 3 in each group). Western blots (*n* = 6 per group) and immunofluorescence staining (*n* = 6 per group) for protein expression of the tissues around the hematoma were conducted at 24 h post-ictus. The use of JWH133 and SR144528 was according to the publication (18).

#### Experiment III

For further study of the mechanism, 27 rats were randomly assigned into three groups: ICH + JWH133 + Vehicle group (JWH + Vehi, *n* = 9), ICH + JWH133 + CREB-1 siRNA group (JWH + si-CREB, *n* = 9), and ICH + JWH133 + scrambled siRNA group (JWH + scr-siRNA, *n* = 9). siRNA dilution buffer (Santa Cruz Biotechnologies, Dallas, TX, USA), CREB-1 siRNA (Santa Cruz Biotechnologies, Dallas, TX, USA), or scrambled siRNA (Santa Cruz Biotechnologies, Dallas, TX, USA) was intracerebroventricularly injected at 24 h before ICH modeling, and JWH133 was injected 1 h after surgery. PCR for gene levels (*n* = 3 in each group) and western blots for protein levels (*n* = 6 in each group) were performed at 24 h after ICH in each group.

#### ICH Model

To induce ICH, rats were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). A feedback-controlled heating pad was used to maintain body temperature at 37.0°C. A cranial burr hole (about 1 mm) was drilled, and a 29-gauge needle was inserted stereotaxically into the right basal ganglia (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 4.0 mm lateral to the bregma) (19, 20). A total of 100 µl autologous arterial blood was infused at a rate of 10 µl/min using a microinfusion pump in each rat. The sham groups received only a needle injection into the right basal ganglia.

#### Brain Water Content Measurement

Brain water content was examined 24 and 72 h after surgery. As previously described (21), animals were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). Brains were removed, and the tissue was sliced coronally (4 mm thickness) around the hematoma. Samples were divided into four parts: ipsilateral basal ganglia, ipsilateral cortex, contralateral basal ganglia, and contralateral cortex. Cerebellum was regarded as the internal control. Sample weights were determined immediately after removal and after drying for 24 h in a 100°C oven using an electric analytical balance. Brain water content (%) was calculated as (wet weight − dry weight)/wet weight × 100%.

#### Assessment of Behavioral Outcome

Behavioral outcomes were assessed in a blinded fashion at 24 and 72 h after surgery. The neurological abnormalities were assessed by the mNSS method. The evaluation was performed by an investigator blinded to the experimental scheme. The mNSS is a composite test of motor, sensory, and balance functions. Neurological function was graded on a scale of 0–18 (normal score, 0; maximal deficit score, 18) (22). For the forelimb placing test, each rat was tested 10 times for each forelimb; the percentage of trials that the rat placed the appropriate forelimb at the edge of the countertop in response to the vibrissae stimulation was determined. Testers were experienced and blind to the condition of the animal. The mean neurological score was evaluated by two blinded observers (23).

### Fluoro-Jade C and Terminal dUDP Nick End Labeling (TUNEL) Staining and Cell Counting

Fluoro-Jade C staining was used to assess neuronal degeneration. Briefly, sections were rinsed for 5 min in basic alcohol, followed by a 2-min rinse in 70% alcohol. Then, the sections were rinsed in distilled water and incubated in 0.06% KMnO4 for 10 min. They were rinsed in distilled water to remove excess KMnO4 and incubated in 0.0001% Fluoro-Jade C stain (Millipore, Boston, MA, USA) stain in 0.1% acetic acid for 10 min. Following Fluoro-Jade C labeling, the sections were rinsed three times in distilled water, air dried for 10 min, cleared in xylene, and covered with DPX. TUNEL staining was performed using an *in situ* cell death detection kit-POD (Roche, Switzerland) to reveal DNA damage according to the manufacturer's instruction (24). High-power images (×40 magnification) were taken around the hematoma using a digital camera. Fluoro-Jade C and TUNEL-positive cells were counted. Counts were performed on four areas in each brain section.

#### Real-Time PCR

PCR was performed and analyzed as previously described (25). Total RNA from brain tissue around the hematoma was extracted using Qiagen RNeasy mini kits. One microgram of RNA was reverse-transcripted and cDNA was synthesized using the PrimescriptTM RT kit (Takara, Dalian, China), substituting DNase and RNase-free water for no-RT controls. qPCR reactions were set up in 25 µl using SYBR Premix Ex TaqII kit (Takara, Dalian, China) and conducted on a CFX-96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The running procedure was 30 s at 95°C, 40 cycles of 5 s at 95°C, and 30 s at 60°C, following a melt curve. The qPCR primers were listed in Table S1 in Supplementary Material. Gene expression was quantified with standard samples and normalized with GAPDH. The data are expressed as normalized messenger RNA (mRNA) expression (fold mRNA increase).

#### Immunofluorescence Staining

Immunofluorescence staining was performed as previously described (21). Briefly, free-floating slices were incubated with primary goat anti-Iba1 (1:200, Abcam, Cambridge, United Kingdom) at 4°C overnight, followed by Alexa 555-labeled rabbit anti-goat IgG (H + L) (1:500; Beyotime, Wuhan, China) secondary antibody (1 h, 37°C). Sections were washed and blocked with 10% normal goat serum for 1 h, then incubated overnight with mouse anti-CD68 (1:200, 1:500, AbD Serotec, Oxford, UK) or rabbit anti-CD206 (1:400, Santa Cruz Biotechnologies, Dallas, TX, USA). Finally, sections were incubated with the appropriate secondary antibodies for 1 h at 37°C. Colocalization was examined using a fluorescent microscope (Zeiss, LSM780).

#### Western Blot Analysis

Western blot assays were performed as described previously (26). A total of 50 µg of prepared protein was loaded into each lane of SDS-PAGE gels. Gel electrophoresis was performed, and protein was transferred to a nitrocellulose membrane. The membrane was blocked in Carnation® non-fat milk and probed with primary and secondary antibodies.  Then, membranes were incubated in the appropriate HRP-conjugated secondary antibody (diluted 1:1,000 in secondary antibody dilution buffer) for 1 h at 37°C. Protein bands were visualized using a nickel-intensified DAB solution, and the densitometric values were analyzed using Image J software. The housekeeping proteins β-tubulin and β-actin were used as internal controls.

#### Intracerebroventricular Infusion

Intracerebroventricular infusion was performed as described previously (27). Rats were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). The needle of a 10-µl Hamilton syringe (Microliter No. 701; Hamilton Company) was inserted through a burr hole in the skull into the left lateral ventricle, according to the following coordinates: 1.5 mm posterior, 4.2 mm ventral, and 0.8 mm lateral to the bregma. CREB-1 siRNA or an irrelevant scrambled siRNA [500 pmol each in 1 µl siRNA dilution buffer (Santa Cruz Biotechnology)] was injected (0.5 µl/ min) using a microinfusion pump at 24 h before ICH induction. The needle was removed 10 min later to prevent backflow. The burr hole was sealed with bone wax, and skin incisions were closed with sutures after the needle was removed. All rats received JWH133 (1.5 mg/kg) intraperitoneally 1 h after ICH. CREB-1 siRNA consists of three different siRNA duplexes to improve the knockdown efficiency. All CREB-1 siRNA sequences were provided in 5′–3′ orientation as shown in Table S2 in Supplementary Material.

#### Statistical Analysis

Data are reported as the mean ± SD. Data were analyzed using one-way analysis of variance tests followed by Student–Newman– Keuls tests. A non-parametric test (Kruskal–Wallis H) was used if the data were not normally distributed, followed by a Nemenyi test when a two-group comparison was necessary. Differences were considered statistically significant at *P* < 0.05.

#### RESULTS

#### CB2R Was Upregulated after ICH Injury

We first investigated whether CB2R alterations would respond to brain injury after ICH. Data showed that CB2R levels around hematoma (**Figure 1A**) were significantly increased at 24 h after ICH when compared with the ICH 0 h group (*P* < 0.05) and remained at a high level until 72 h (**Figures 1B,C**).

#### JWH133 Reduced Brain Water Content and Improved Neurobehavioral Outcomes both at 24 and 72 h after ICH

To assess brain edema and neurobehavioral outcomes after ICH, brain water content and behavioral testings, including mNSS test and forelimb placing test, were used. Data showed that both at 24 and 72 h after surgery, rats subjected to ICH had increased perihematomal edema in the ipsilateral basal ganglia (*P* < 0.01 vs. Sham; **Figures 2A,B**). The animals also had significantly worse mNSS test performance (*P* < 0.01 vs. Sham; **Figures 3A,B**) and forelimb placing scores (*P* < 0.01 vs. Sham; **Figures 3C,D**). In ICH + JWH groups, the perihematomal brain edemas were significantly reduced (*P* < 0.05 vs. ICH + Vehi; **Figures 2A,B**), and the neurobehavioral outcomes were significantly improved (*P* < 0.05 vs. ICH + vehi; **Figures 3A–D**). All of these JWH133 effects were reversed by SR144528.

#### JWH133 Attenuated Neuronal Death and DNA Damage after ICH

To investigate the neuronal degeneration after ICH, Fluoro-Jade C and TUNEL staining were used. At 24 h after ICH, the treatment of JWH133 significantly decreased the number of Fluoro-Jade C-positive cells (497 vs. 720/mm2 in ICH + Vehi group, *P* < 0.01) (**Figures 4A,B**). Similarly, the number of TUNEL-positive cells were significantly decreased in the ICH + JWH group (524 vs. 325/mm2 in ICH + Vehi group, *P* < 0.01) (**Figures 4A,C**). The result indicated that JWH133 could significantly attenuate neuronal death and DNA damage after ICH.

### Microglia Primarily Polarized to Classic M1 Phenotype during the ICH Acute Phase, and JWH133 Promoted M2 Polarization

To investigate the characteristics of microglial activation during the acute phase following ICH induction, gene levels of M1/M2

markers were investigated time dependently. After ICH, M1-associated markers CD32, CD68, and CD86 increased immediately and peaked at 6 h, and remained a high level to 72 h (**Figures 5A,C,E**). M2-associated markers Arg-1, Ym-1, and CCL-22 increased slowly and peaked at 24 h, and kept a low level from 6 to 72 h (**Figures 5B,D,F**). However, the use of JWH133 significantly inhibited the increase of M1-associated mRNA levels (**Figures 5A,C,E**) and promoted M2-associated mRNA levels (**Figures 5B,D,F**), with the most obvious differences at 24 h, and all the effects could be significantly reversed by SR144528 (**Figures 5A–E**). Consistent with the qPCR results, immunofluorescent double-labeled staining and western blot results showed that the M1-associated marker CD68 was also more significantly upregulated than M2-associated marker CD206 at 24 h after ICH (**Figures 6A,B**). Treatment with JWH133 promoted the protein expression of CD206 and inhibited CD68 expression

(**Figures 6A–C**). These results indicated that CB2R stimulation could drive the acquisition of M2 polarization in microglia and reduce M1 phenotype.

### Inflammatory Responses during ICH Were Inhibited by JWH133 Treatment

Inflammatory cytokine secretion plays an important role in brain injury after ICH. In this study, the mRNA expression levels of both pro-inflammatory (IL-1β, TNF-α, and iNOS) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) in perihematomal brain tissue were detected at different time points after ICH. Expression levels of pro-inflammatory cytokines increased immediately and IL-1β peaked at 6 h (**Figure 7A**), while TNF-α and iNOS (**Figures 7C,E**) peaked at 24 h post-ICH. The gene levels of IL-4, IL-10, and TGF-β were upregulated at 6 h after ICH and reached a peak at 24 h (**Figures 7B,D,F**). However, treatment with JWH133 prevented the increase of IL-1β, TNF-α, and iNOS at 6 h after ICH induction, and the effect persisted to 72 h, with the most significant changes occurring at 24 and 72 h (**Figures 7A,C,E**). In contrast, IL-4, IL-10, and TGF-β were significantly increased at 6 h, with the most significant changes occurring at 24 h (**Figures 7B,D,F**). All of the JWH133-mediated effects were prevented by treatment with SR144528.

## pPKA–CREB Signaling Pathway Was Upregulated by JWH133 Treatment

To further explore the underlying mechanisms, we tested the expression of pPKA–CREB signaling pathway. Results of western blot showed that pPKA and pCREB expression was significantly lower in ICH + Vehi group when compared with Sham group (*P* < 0.01), and the ICH + JWH group had significantly higher expression of pPKA (*P* < 0.01) and pCREB (*P* < 0.05) when compared with the ICH + Vehi group (**Figures 8A,B**), indicating that pPKA–CREB signaling pathway was significantly upregulated by JWH133 treatment. The pretreatment of SR144528 prevented the upregulation effects.

### pPKA–CREB Signaling Pathway Was Involved in the JWH133-Induced Increase of Microglial M2 Polarization

cAMP-response element binding protein (CREB) *in vivo* knockdown was performed to investigate the potential role of CREB in the effects of JWH133 induced the increase of microglial M2 polarization and the decrease of M1 phenotype. Pretreatment with CREB-1 siRNA sufficiently downregulated the expression of CREB both at gene and protein levels (Figures S1A,B in Supplementary Material). Data showed that CREB-1 siRNA abolished the JWH133-induced increase of M2-associated markers

CD206 and Ym-1 at protein or gene level (**Figures 9B,D,F**), as well as the decrease of M1-associated markers CD68 and CD32 in protein or gene levels (**Figures 9A,C,E**). These results demonstrated that PKA/CREB signaling pathway plays a crucial role in the JWH133-mediated acquisition of M2 phenotype of microglia.

 We also demonstrated that JWH133 suppresses neuroinflammation by driving microglial M2 polarization through PKA/CREB pathway.

Increasing evidence indicates that inflammatory mechanisms are involved in stroke-induced brain injury, and microglia/ macrophage activation is thought to play a pivotal pathophysiological role (1, 4). Overactivation of microglia/macrophages can exacerbate the inflammatory response after a stroke, resulting in blood–brain barrier disruption and neuronal damage, but also represents a promising target for stroke treatment. However,

broad suppression of microglia/macrophages may deprive the normal physiological defense mechanism of the CNS and lead to unintended side effects. Therefore, an improved understanding of the dual beneficial and detrimental roles of microglia in CNS injury and recovery is critical for improving stroke treatment (11).

Once activated, microglia/macrophages serve as a doubleedged sword during the battle between neurological damage and protection. Microglia can be activated to two polarization states: the M1, classically activated phenotype, and the M2, alternatively activated phenotype (28). Upon stimulation with lipopolysaccharide or interferon gamma, microglia can be activated to the M1 phenotype and produce pro-inflammatory mediators, chemokines, redox molecules, costimulatory proteins, and major histocompatibility complex II (29, 30), exacerbating inflammatory damage. Alternatively, microglia can also be activated to the M2 polarization state, which produces anti-inflammatory mediators that promote brain recovery by scavenging cell debris, resolving local inflammation, and are involved in tissue remodeling (31, 32). There is no doubt that it is very important to understand the status and function of polarized microglia activation for assessing inflammation progression and optimizing treatment.

&&*P* < 0.01 compared with ICH + JWH group.

CD206 (D) and Ym-1 (F) (*n* = 3 in each group). The values were mean ± SD. \**P* < 0.05, \*\**P* < 0.01 compared with JWH + Vehi group.

TBI was reported to create a severe cortical lesion, which led to chronic and persistent M1-primed activation that lasted for months to years. On the other hand, ICH often occurs after TBI. However, characteristic of microglial polarization and inflammatory response after ICH remains unclear (33, 34). Xi et al. (11) found that ICH induces microglia activation and polarization in mice. They reported that M1 phenotypic markers were increased and reached a peak as early as 4 h, remained high at 3 days, and decreased 7 days after ICH, whereas M2 phenotypic markers were upregulated later than M1 markers, reaching a peak at day 1 and declined on day 7 after ICH, which is largely consistent with the findings of this study. The results confirmed our hypothesis that in the ICH acute phase, microglia/macrophage were largely activated toward the classical M1 phenotype, and JWH133 influenced the polarization process by promoting the anti-inflammatory M2 phenotype.

We further investigated the intracellular molecular switches that regulate microglia/macrophage polarization. A recent breakthrough in research on microglia/macrophages has revealed that several transcriptional regulators may serve crucial roles in M2-associated marker expression (5, 35). Interferon regulatory factor 4 serves as a key transcriptional factor modulating M2 polarization, whereas the interferon regulatory factor 5 and interferon regulatory factor 8 control macrophages toward M1 polarization. The nuclear hormone receptor peroxisome proliferator-activated receptor γ is an important transcriptional factor that mediates macrophages primed toward M2 polarization (36, 37). CB2R can couple to Gi proteins, and CB2R activation has been shown to activate cAMP/PKA (38). Additionally, it has been demonstrated that CB2R stimulation enhances CREB activation after cerebral ischemia through phosphorylation of AMPK (39). JWH133 was also found to attenuate apoptosis by activation of phosphorylated CREB-Bcl-2 pathway after subarachnoid hemorrhage in rats (27). cAMP signaling is spatially and temporally regulated, allowing for the selective activation of a subset of targets. A-kinase anchoring proteins provided the platform for the assembly of signalosomes, which consist of cAMP effectors and their substrates. Among them, the role that cAMP plays in maintaining both microglia and monocyte homeostasis to prevent M1 activation has been reported. PKA was considered to be the cAMP receptor and seem to be closely associated with inflammation (40). Although CREB could be activated by various signaling pathways, including cAMP/ PKA, ERK1/2, and PI3K/Akt (41), in this study, we selectively detected PKA/CREB signaling pathway. CB2R agonist JWH133 significantly enhanced the phosphorylated expression of PKA and CREB and knockdown of CREB significantly inversed the JWH133-induced increase of M2 polarization in microglia, indicating that PKA/CREB pathway participates in the effects of CB2R stimulation on microglia polarization during ICH. The results are in agreement with our previous study that the promotion of microglial M2 polarization through the cAMP/PKA pathway participates in the CB2R-mediated anti-inflammatory effects during GMH (26).

Until now, most studies on microglial polarization have focused on TBI and cerebral ischemia, but relatively little was known about the polarization of microglia/macrophages following ICH (31). In this study, we demonstrated the neuroprotective effects of a CB2R agonist via microglial polarization and the underlying molecular mechanisms that involved in microglial polarization, which may represent a new target for ICH therapy.

In summary, our results reveal microglia/macrophage activation and polarization after ICH in rats. Our data showed that the CB2R agonist JWH133 lessened the brain water content, improved neurobehavioral outcomes, and ameliorated DNA damage and neuron death, which resulted in alleviating brain injury after ICH. We also found that JWH133 inhibited the pro-inflammatory cytokine release and promoted microglia M2 polarization, leading to a beneficial anti-inflammatory cytokine release. JWH133 also facilitated the phosphorylation of PKA and its downstream effector, CREB. Moreover, knockdown of CREB *in vivo* abolished the effects of JWH133 on microglial polarization, indicating that the PKA/CREB signaling pathway plays a critical role in the JWH133-mediated effects. Therefore, CB2R may provide a new target to modulate microglia/macrophage-mediated inflammatory injury and recovery after ICH.

### ETHICS STATMENT

All institutional and national guidelines for the care and use of laboratory animals were followed.

## AUTHOR CONTRIBUTIONS

LL, TY, FZ, NY, TQ, ZH, CQ, TJ, and ZY contribute to the implementation of the experiment. ZG, FH, YY, and CZ contribute to the design and paper writing.

## ACKNOWLEDGMENTS

We gratefully thank Professor Guohua Xi for his generous suggestions regarding the experimental processes. This work was supported by Grant Nos. 81571130 (CZ), 81271281 (CZ), and 81371440 (YY) from the National Natural Science Foundation of China, the Science and Technology project of Nanchong (KY-15A0020), and Grant No. 2014CB541606 (FH) from the National Key Basic Research Development Program (973 Program) of China.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu. 2017.00112/full#supplementary-material.

#### REFERENCES

hemorrhage with ventricular extension. *Transl Stroke Res* (2015) 6(2):125–32. doi:10.1007/s12975-014-0367-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Lin, Yihao, Zhou, Yin, Qiang, Haowen, Qianwei, Jun, Yuan, Gang, Hua, Yunfeng and Zhi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Mitochondria-Derived Damage-Associated Molecular Patterns in Neurodegeneration

*Heather M. Wilkins1,2 , Ian W. Weidling2,3, Yan Ji1,2 and Russell H. Swerdlow1,2,3,4 \**

*1Department of Neurology, University of Kansas Medical Center, Kansas City, KS, USA, 2University of Kansas Alzheimer's Disease Center, Kansas City, KS, USA, 3Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA, 4Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA*

Inflammation is increasingly implicated in neurodegenerative disease pathology. As no acquired pathogen appears to drive this inflammation, the question of what does remains. Recent advances indicate damage-associated molecular pattern (DAMP) molecules, which are released by injured and dying cells, can cause specific inflammatory cascades. Inflammation, therefore, can be endogenously induced. Mitochondrial components induce inflammatory responses in several pathological conditions. Due to evidence such as this, a number of mitochondrial components, including mitochondrial DNA, have been labeled as DAMP molecules. In this review, we consider the contributions of mitochondrial-derived DAMPs to inflammation observed in neurodegenerative diseases.

#### *Edited by:*

*Walter Gottlieb Land, University of Strasbourg, France*

#### *Reviewed by:*

*R. Clinton Webb, Georgia Health Sciences University, USA Florence Burté, Newcastle University, UK*

#### *\*Correspondence:*

*Russell H. Swerdlow<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 26 October 2016 Accepted: 12 April 2017 Published: 26 April 2017*

#### *Citation:*

*Wilkins HM, Weidling IW, Ji Y and Swerdlow RH (2017) Mitochondria-Derived Damage-Associated Molecular Patterns in Neurodegeneration. Front. Immunol. 8:508. doi: 10.3389/fimmu.2017.00508*

Keywords: damage-associated molecular pattern, mitochondria, neuroinflammation, neurodegeneration, sterile inflammation

#### INTRODUCTION

Inflammatory pathways are activated through either pathogen-initiated or damage-initiated events. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate similar inflammatory cascades and are therefore difficult to distinguish. Inflammation stimulated by DAMPs is an area of study that has recently gained notice. In particular,

**Abbreviations:** 3NP, 3-nitropropionic acid; ACS, apoptosis-associated speck-like protein containing a CARD; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; APOE, apolipoprotein E; APP, amyloid precursor protein; ATP, adenosine triphosphate; BBB, blood–brain barrier; CD33, Siglec-3; CD38, cluster of differentiation 38; CLU, clusterin; CNS, central nervous system; COX, cytochrome oxidase; CR1, complement rector 1; CSF, cerebral spinal fluid; CSF1R, colony-stimulating factor 1 receptor; CXCR31, CX3C chemokine receptor 1 or fractalkine receptor; cybrid, cytoplasmic hybrid; DAMP, damage-associated molecular pattern; EPHA1, ephrin type A receptor 1; ER, endoplasmic reticulum; FCγRII, Fc receptor or CD32; FPR or FPRL1, formyl peptide receptors; FTLD, frontotemporal lobar degeneration; GFAP, glial fibrillary acidic protein; GWAS, genomewide association study; HLA-DRB5/DRB1, major histocompatibility complex, class II, DR beta 5; HMGB1, high mobility group box 1; IFN-γ, interferon-γ; IgG, immunoglobulin G; IL-1, interleukin-1; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8; INPPD5, inositol polyphosphate-5-phosphatase; IP, intraperitoneal; LPS, lipopolysaccharide; LRRK2, leucine-rich repeat kinase 2; MCI, mild-cognitive impairment; MCP-1, monocyte chemotactic protein 1; M-CSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; MIP-1α, macrophage inflammatory protein-1α; MMP-8, matrix metalloproteinase 8; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; mtDNA, mitochondrial DNA; NLRP3, NLR family pyrin domain containing 3; NO, nitric oxide; NOD, nucleotide-binding oligomerization domain-like receptors; NSAID, non-steroidal anti-inflammatory drug; PAMP, pathogen-associated molecular pattern; PD, Parkinson's disease; PET, positron emission tomography; PRR, pattern-recognition receptor; RAGE, receptor for advanced glycation end products; RANTES, regulated on activation, normal T cell expressed and secreted; ROS, reactive oxygen species; SNP, single-nucleotide polymorphism; SOD1, superoxide dismutase 1; SRA, microglial scavenger receptor A; TBI, traumatic brain injury; TFAM, transcription factor A; TGF-β, transforming growth factor β; TNFα, tumor necrosis factor α; TREM2, triggering receptor expressed on myeloid cells 2.

DAMPs derived from mitochondrial components are interesting due to the prokaryotic origin of this organelle. Furthermore, mitochondrial-derived DAMP molecules may play a role in heart disease, arthritis, liver disease, trauma, and sepsis (1–5).

Neuroinflammation and mitochondrial dysfunction are observed across numerous neurodegenerative diseases (6–8). Mitochondrial dysfunction can induce inflammation and *vice versa*. Mitochondrial dysfunction may modulate the release of mitochondria-derived DAMP molecules (9, 10). Here, we discuss the relationship between neuroinflammation, mitochondrial dysfunction, and mitochondriaderived DAMP molecules in the context of neurodegenerative diseases.

#### NEUROINFLAMMATION IN NEURODEGENERATIVE DISEASES

Neuroinflammation is classically defined as proliferation and activation of microglia (microgliosis), and/or astrocytes (astrogliosis). Microglia are macrophage cells of mesenchymal origin, which monitor the central nervous system (CNS) for pathogens. Astrocytes are derived from the ectoderm and have numerous functions including metabolic support for neurons, regulating synapses, brain structure, and repair. Further evidence of neuroinflammation can encompass activation of inflammatory pathways, increased expression of cytokines or chemokines, and in some cases disruption of the blood–brain barrier (BBB) accompanied by infiltration of peripheral immune cells (such as T cells). Neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) all have evidence of neuroinflammation pathology.

#### Alzheimer's Disease

Initial interest in neuroinflammation as a causative factor in AD centered on the link between reduced risk for AD and longterm non-steroidal anti-inflammatory drug (NSAID) use. Early evidence from epidemiological studies suggested that long-term use of NSAIDs led to a decreased risk of developing AD (11, 12). Subsequent studies found a correlation between apolipoprotein E (APOE, a genetic risk factor for AD) genotype and the protective effects gained from NSAID use. The correlation between decreased AD risk and NSAID use is greatest in individuals who harbor an APOE ε4 allele (13, 14). Furthermore, the age of the individual taking NSAIDs and the type of NSAID administered affect the association of AD risk reduction (15).

Clinical trials investigating whether NSAIDs might benefit AD subjects, though, were disappointing. Early trials experienced high attrition rates among participants due to adverse effects and thus did not provide clear answers (16, 17). A larger trial of one NSAID, tarenflurbil, showed largely negative results (18). However, a recent publication that showed positive memory and brain inflammation outcomes for a different NSAID, fenamate, in an AD mouse model has led to renewed interest (19).

Genome-wide association studies (GWAS) have also renewed interest for neuroinflammation as a potential causative factor for AD. In particular, a rare variant of triggering receptor expressed on myeloid cells 2 (TREM2), R47H, is associated with an increased risk for late onset Alzheimer's disease (LOAD) (20). TREM2 is a membrane protein in myeloid cells (such as microglia), which modulates inflammatory pathways by inhibiting cytokine production. TREM2 is likely responsible for determining microglial phenotypes (i.e., M1 activation versus M2 activation). Furthermore, TREM2 is also important for lipid sensing, providing a further link between neuroinflammation and bioenergetic pathways (21). The R47H TREM2 variant leads to a reduction in microglial phagocytosis (22). Soluble TREM2 levels are increased in cerebral spinal fluid (CSF) of AD subjects, a parameter that positively correlates with gray matter volume but negatively correlates with diffusivity (sometimes considered a function of cell integrity) (23). Other single-nucleotide polymorphisms (SNPs) associated with AD risk also affect inflammatory pathways. These include SNPs in complement receptor 1, clusterin (CLU), major histocompatibility complex (MHC), class II, DR beta 5, ephrin type A receptor 1, inositol polyphosphate-5-phosphatase, and or Siglec-3 (24–29). The relationship of each of these genes to inflammatory signaling has been reviewed elsewhere (30).

 The quantity of interleukin-1 (IL-1) reactive microglia is increased sixfold in AD brain (33). Furthermore, microglial IL-1 expression correlates with plaque distribution (34). Levels of macrophage colony-stimulating factor, an activator of macrophages, was found to be increased in plasma and CSF from AD subjects compared to those with mild-cognitive impairment (35). A number of cytokines and chemokines, which can be released by macrophages or other immune cells, are increased in AD, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), interleukin-8 (IL-8), transforming growth factor β, and macrophage inflammatory protein-1α (36).

#### Parkinson's Disease

The discovery of reactive microglia in the substantia nigra of PD brain tissue provided early evidence for the role of neuroinflammation in PD (37). Further evidence for microglial activation in PD brain came from positron emission tomography (PET) imaging studies. PET studies showed increased microglial activation as evidenced by increased levels of peripheral benzodiazepine sites, a selective marker of activated microglia (38). Increased levels of glial cells expressing TNFα, IL-1β, and interferon-γ (IFN-γ) have also been observed in the substantia nigra of deceased PD subjects (39). Furthermore, microglial activation is observed in animal models of PD (36, 40).

Studies on astrogliosis in the PD brain have generated conflicting results. Early studies suggested increased astrogliosis in the PD brain. More recent studies have shown minimal levels of nigral astrogliosis, although astrocytic accumulation of α-synuclein was observed. The authors speculated that α-synuclein accumulation may cause astrocytes to be less reactive (41). A subsequent report showed minimal astrogliosis and an inverse correlation between levels of α-synuclein and astrogliosis in the PD brain, suggesting α-synuclein may suppress astrogliosis (42).

Similar to AD, PD risk is reduced with regular or chronic NSAID use. Long-term aspirin use is associated with less PD risk; however, non-aspirin-based NSAID use afforded better risk reduction. Furthermore, women who used aspirin in a chronic (greater than 24 months) or regular manner had a lower risk for PD than men (43).

Genome-wide association studies have identified the gene that encodes leucine-rich repeat kinase 2 (LRRK2) as a risk factor for sporadic PD. LRRK2 mutations are also associated with autosomal dominant forms of PD (44). Similar to TREM2 in AD, LRRK2 mediates microglial function (45, 46). LRRK2 facilitates vesicle trafficking and cytoskeletal remodeling and may skew microglia toward a pro-inflammatory phenotype (45–47).

Changes in peripheral inflammation may contribute to PD. Studies of peripheral inflammation showed an increased ratio of CD8+ T cells to CD4+ T cells in the blood of PD patients (48). Peripheral inflammation may affect the brain through disruption of the BBB, an event that is observed in PD subjects (49). Furthermore, peripheral inflammatory changes correlate with PD risk. Increased plasma IL-6 levels correlated with disease risk, although with a small sample size (50). CD8+ and CD4+ T cell reactivity is increased in the substantia nigra of PD patients. Furthermore, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin used to generate a commonly utilized PD animal model, was found to increase T cell infiltration into the substantia nigra (51).

#### Amyotrophic Lateral Sclerosis

Inflammatory changes have been observed in the CNS of ALS patients. Early studies in ALS spinal cord and motor cortex discovered accumulation of immunoglobulin G and complement (52). Subsequent studies found T cell infiltration and increased levels of MHC I and II antigens on macrophages and dendritic cells (53, 54). Immunohistochemical studies in postmortem brain tissue showed increased levels of phagocytic and leukocyte surface proteins on microglia, as well as evidence to support the infiltration of activated lymphocytes into the precentral gyrus (55). PET studies using PK11195, a ligand that binds microglia, have provided further evidence of microglial activation in ALS (56).

Astrogliosis is observed within the spinal cord (ventral and dorsal horns) and brain (cortical gray matter and subcortical white matter) in postmortem ALS subjects (57). Microgliosis is evident in the spinal cord ventral horn, the corticospinal tract, and the motor cortex (55). The role of microglia and astrocytes in ALS disease progression is further supported in transgenic mutant superoxide dismutase 1 (SOD1) ALS mouse models. Selective deletion of mutant SOD1 from microglia (which also removes expression from peripheral myeloid cells) slowed disease progression (58). Similar results were observed when mutant SOD1 expression was deleted from astrocytes (59). Conversely, deleting mutant SOD1 expression from motor neurons had no effect on disease progression or survival, but did delay disease onset (58). These studies emphasize the potential role of neuroinflammation in ALS disease progression.

Genome-wide association studies in ALS have linked inflammatory genes with disease outcome. A SNP in CX3C chemokine receptor 1 (CXCR31, fractalkine receptor) is associated with reduced survival in sporadic ALS subjects; however, this SNP fails to associate with ALS risk (60). CXCR31 is important for the migration of leukocytes and may play a role in microglial migration (61). RNA seq and ingenuity pathway analysis of postmortem ALS spinal cord samples found upregulation of inflammatory pathways, with TNFα being a predicted upstream regulator of inflammation in these ALS samples (62).

Neuroinflammation is evident in AD, PD, and ALS. However, whether or not neuroinflammation contributes to disease onset, progression, and risk requires further study. To this end, a growing appreciation of a relationship between mitochondrial dysfunction and inflammatory pathways may provide insight into this important question. Below, we discuss evidence for mitochondrial dysfunction in AD, PD, and ALS.

#### Primary Mitochondrial Diseases

Primary mitochondrial diseases are caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA genes that encode mitochondrial proteins. Diseases caused by mtDNA mutations include Leber's hereditary optic neuropathy, myoclonic epilepsy and ragged red fiber, neuropathy ataxia and retinitis pigmentosa, Kearns–Sayre syndrome, and Leigh's syndrome. Diseases caused by a nuclear DNA mutation of which affects a mitochondrial localized protein include Friedreich's ataxia, Wilson's disease, and Mohr–Tranebjaerg syndrome. Commonly, these diseases manifest in neurological symptoms. A few of these diseases are associated with neuroinflammation pathology.

Friedreich's ataxia is caused by autosomal recessive inheritance of a mutant *Frataxin* gene. The product of the *Frataxin* gene is responsible for iron homeostasis within mitochondria, and loss of this gene in Schwann cells leads to reduced mitochondrial respiration, inflammation, increased mitochondrial iron concentrations, and cell death (63–65). COX2 expression is elevated in both animal models and Friedreich's ataxia patient lymphocytes, an indicator of increased inflammation (66).

For subjects with Leigh's syndrome, mtDNA mutations occur in several genes including ATPase 6, ND 1–6 (NADH dehydrogenase), and CO3 [cytochrome oxidase (COX)] (67, 68). These subjects often have deficient ETC enzyme activities (67). In a mouse model of Leigh's syndrome, evidence of neuroinflammation is abundant (69). However, inflammatory markers have not been measured from human subject tissues.

Wilson's disease is caused by a mutation in the *ATP7B* (ATPase copper transporting β polypeptide) gene and is characterized by liver disease, ataxia, parkinsonism, seizures, and reduced cognition (70, 71). This gene encodes a copper transporting ATPase that localizes to mitochondria and affects mitochondrial copper levels (70, 72). Subjects with this mutation have reduced ETC function (73, 74). Pentraxin 3, a marker of inflammation, is elevated in the serum of Wilson's disease subjects (75).

Despite the association of mitochondrial dysfunction and neuroinflammation or inflammation (discussed below), these processes have not been extensively studied in primary mitochondrial diseases. Future research endeavors into this area would likely benefit our understanding of these diseases.

#### MITOCHONDRIAL DYSFUNCTION IN NEURODEGENERATIVE DISEASES

The Krebs cycle and oxidative phosphorylation occur in the matrix and inner mitochondrial membrane, respectively. Oxidative phosphorylation requires the mitochondrial respiratory chain. These bioenergetic pathways generate the high energy compound adenosine triphosphate (ATP) (76). Mitochondria and bioenergetic intermediates generated within mitochondria regulate cell signaling pathways (including pro-inflammatory responses, as discussed below).

The brain comprises approximately 2% of the body's weight yet consumes about 20% of its oxygen uptake. The brain requires high amounts of energy for numerous processes, including neurotransmitter production and synaptic activity. Therefore, the brain is highly susceptible to mitochondrial dysfunction, which has been observed in several neurodegenerative diseases including (but not limited to) AD, ALS, and PD (77–80). Furthermore, mitochondrial dysfunction declines with age, and age is the greatest risk factor for these neurodegenerative diseases (78, 80). Mitochondrial dysfunction can lead to increased reactive oxygen species (ROS) production, decreased ATP production, alterations in mitochondrial membrane potential, damage to mtDNA, and activation of cell death pathways (81).

#### Alzheimer's Disease

In postmortem AD brains, decreased COX function, reduced intact mitochondrial number, and increased mitochondrial autophagy have been reported (78, 82–86). Mitochondrial dysfunction appears to be systemic in AD, as deficits in COX activity are apparent in AD patient fibroblasts and platelets (83, 87–89). Changes in mtDNA may drive cell signaling changes, bioenergetic pathway deficits, and histopathological hallmarks of AD. Cytoplasmic hybrid (cybrid) studies in which mtDNA from human AD subjects is transferred into a donor cell line that lacks its own mtDNA provides a system in which mtDNA-derived biochemical and molecular consequences can be assessed. The cybrid model system controls for nuclear DNA alterations, as patient mtDNA is transferred into the context of a consistent nuclear DNA background (90). Cybrid cells generated using AD patient mtDNA have reduced COX activity, increased ROS production, and increased Aβ deposition (90, 91).

Evidence of mtDNA mutations, deletions, and oxidative modifications are present in AD subjects (92–97). mtDNA is inherited from the mother, and interestingly a maternal inheritance pattern for AD has been noted. This maternal inheritance pattern is associated with early changes in brain atrophy and mitochondrial biomarkers (98–103). Finally, mitochondrial haplotypes are associated with increased AD risk (104–106). These studies suggest changes in mitochondrial function, possibly at the level of mDNA maintenance and inheritance, are important in AD pathology.

#### Parkinson's Disease

The most studied respiratory chain aberration in PD is a deficit in complex I activity. Initial insight into this deficit stems from cases of recreational drug users exposed to MPTP. After MPTP exposure, individuals developed parkinsonian symptoms and at autopsy were found to have degeneration in the substantia nigra, similar to that observed with PD. This degeneration occurred in the absence of Lewy bodies (or aggregated α-synuclein). Following its accidental discovery, MPTP was adapted to produce monkey and rodent models of PD (107–109). MPTP is oxidized to MPP+, which accumulates in neurons and is a potent complex I inhibitor (107, 110).

Complex I deficits are observed in postmortem brain, platelets, and fibroblasts from PD subjects (111, 112). The observed deficits in complex I activity could be driven by oxidative damage to its catalytic subunits or altered mtDNA (90, 113, 114). Cybrid cell lines generated from PD subject mtDNA recapitulate the complex I deficit. Sporadic PD cybrid cell lines also show reduced mtDNA copy number, reduced ATP, cell death pathway activation, and a relatively depolarized mitochondrial membrane potential (113–115).

Parkinson's disease risk is associated with mtDNA haplotype, similar to AD (116–118). Changes to mtDNA are also observed in PD, such as mutations and deletions (119–121). Polymorphisms of mtDNA polymerase γ influences PD risk as well (122). Overall, mitochondrial function and mtDNA inheritance and maintenance are important to the pathology of PD.

#### Amyotrophic Lateral Sclerosis

ALS subjects have evidence of mitochondrial dysfunction within the CNS and periphery. CNS mitochondrial abnormalities include altered mitochondrial morphology, mitochondrial inclusions/aggregates, reductions in COX activity, and lower mtDNA levels with increased levels of mtDNA point mutations and deletions (123–128). COX deficits, lowered mitochondrial number, altered mitochondrial calcium levels, and mtDNA deletions, are also observed in ALS subject muscle (129–131). Lymphocytes from ALS subjects have reduced mitochondrial maximum respiration, liver mitochondria appear swollen, and platelet mitochondria manifest a depolarized mitochondrial membrane potential with increased apoptosis (132–135). Cybrid cells generated from ALS patient mtDNA have alterations in antioxidant enzyme activity and reduced complex I activity (136). The association of ALS with distinct mtDNA haplotypes is under investigation (137).

### MITOCHONDRIAL DYSFUNCTION AND INFLAMMATION

Mitochondria are important modulators of innate immunity pathways. Mitochondria-derived ROS, calcium, and ATP are signaling molecules that activate inflammatory responses. Under conditions in which mitochondria are damaged, such as accumulated mtDNA mutations or mitochondrial dysfunction (possibly through aberrant ROS production), a sustained inflammatory response and downstream pathological inflammation could ensue. A relationship between mitochondrial dysfunction and inflammatory signaling is discussed below.

Mitochondria can directly activate inflammasome signaling. Mitochondria-derived ROS activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. NLRP3 is normally associated with the endoplasmic reticulum membrane, but upon activation is redistributed to nuclear and mitochondrial membranes, where it oligomerizes with apoptosis-associated speck-like protein containing a CARD (ACS) and pro-caspase 1 to form the NLRP3 inflammasome (138–140). Mitochondria also mediate inflammatory pathway activation through redox sensitive proteins (140).

Mitochondrial dysfunction initiates inflammation across various models. *In vitro* and *in vivo* experiments demonstrate complex II inhibition using 3-nitropropionic acid (3NP) induces microglial activation and reduces the ability of microglia to undergo alternate activation (141). Human microglial cells treated with 3NP became activated and showed increased ROS production and cell death rates. Similar results were observed following intrastriatal 3NP injection in adult rats, where microglial activation, ROS production, and cell death were increased (142). Chronic subcutaneous injections of rotenone in rats (a complex I inhibitor that is used to model PD in rodents) increases IL-1β within the hypothalamus. These rats also displayed a decreased number of tyrosine hydroxylase positive neurons in the substantia nigra, perturbed locomotion, and sleep abnormalities (143).

Conversely, pro-inflammatory cytokines also modulate mitochondrial function. For example, TNFα can decrease complex I activity, reduce ATP production, depolarize the mitochondrial membrane potential, increase ROS, and lower activities of complexes II and IV (depending on the cell type). In hepatocytes, TNFα uncouples mitochondria and increases ROS production. This ROS production is generated from complex I and III of the respiratory chain and initiates NFĸB activation (144, 145). Similar effects have been reported in L929 cells (a mouse fibroblast cell line), a leukemia cell line, and 3T3-L1 adipocytes (146–148). In a mouse hippocampal cell line (HT22), TNFα induces mitochondrial respiration deficits and a loss of mitochondrial membrane potential (149). IL-1β also yields similar effects on mitochondrial function (150). Nitric oxide (NO), another inflammatory signaling molecule, disrupts mitochondrial membrane potential and inhibits COX activity. In human retinal pigment cells, TNFα, IL-1β, and IFN-γ increase the production of both mitochondrialand NADPH oxidase-derived ROS (151).

*In vivo* studies also suggest pro-inflammatory molecules influence mitochondrial function. Intraperitoneal injection of lipopolysaccharide in rodents increases brain cytokine and inflammatory receptor expression [including IL-1β, toll like receptor 2 and 4, microglial scavenger receptor A (SRA), and Fc receptor (FCγRII)] in a region-specific manner. The authors also reported increased microglial number and mitochondrial functional alterations, including decreased glutathione (an antioxidant) and increased complex II/III activity (152).

A clear relationship between mitochondrial dysfunction and inflammatory cascades exists. Both of these pathological hallmarks are present across multiple neurodegenerative diseases. Mitochondria-derived DAMP molecules could provide a further link between these pathologies. Below, we discuss mitochondriaderived DAMP molecules, potential routes of release, and evidence for their role in neuroinflammation.

#### MITOCHONDRIA-DERIVED DAMPs: INDUCERS OF NEUROINFLAMMATION?

Previously identified mitochondria-derived DAMP molecules include mtDNA, cardiolipin, ATP, mitochondrial transcription factor A (TFAM), cytochrome *c*, and formyl-methionine-labeled peptides (30). Formyl-methionine-labeled peptides may also include mitochondrial protein-derived cryptides. Cryptides are endogenous, fragmented functional peptides. Pro-inflammatory cryptides from mtDNA and nuclear DNA-encoded mitochondrial proteins were recently described (153–155). As reviewed in **Table 1**, each of these molecules has been observed to initiate a pro-inflammatory phenotype under various disease and pathological states.

Damage-associated molecular pattern molecules activate inflammatory signaling in a manner similar to PAMPs. The danger molecule is recognized by a pattern-recognition receptor (PRR), and adaptor molecules initiate intracellular signaling cascades and cytokine production. Similar to PAMPs, mitochondria-derived DAMPs are recognized by various PRRs. These include TNF receptor, NLRP3, IL-1 receptor, nucleotide-binding oligomerization domain-like receptor, receptor for advanced glycation end products, formyl peptide receptors (FPR or FPRL1), and purogenic receptors. With the increasing recognition of mitochondria-derived DAMP molecules as pathogenic instigators in various diseases (**Table 1**), recent studies have begun to explore their contribution to neuroinflammation.

#### Mitochondria-Derived DAMP Molecules in Neuroinflammation

In the periphery, TFAM functions as a potent DAMP molecule (**Table 1**). Recently, it was found that TFAM, in combination with IFN-γ, can activate human microglial cells, human peripheral blood monocytes, and THP1 monocytic cells (173). Cotreatment of THP1 monocytic cells with IFN-γ and TFAM or TFAM alone lead to an increase in cytokine expression (including IL-1β, IL-6, and IL-8). While treatment of SH-SY5Y neuroblastoma cells with IFN-γ and TFAM was not toxic, exposure to conditioned medium from monocytic cells activated with IFN-γ and TFAM-induced SH-SY5Y cell death. Finally, mitochondrial proteins extracted from THP1 monocytic cells produced effects similar to TFAM.

Degraded and oxidized mtDNA can initiate pro-inflammatory pathways in astrocytes. Exposure of mtDNA to hydrogen peroxide can induce its degradation, and these mtDNA degradation products are found in human CSF and plasma (9). Furthermore, transfection of mouse primary astrocytes with oxidant-

initiated, degraded mitochondrial polynucleotides caused a proinflammatory response. This inflammatory astrocyte phenotype was characterized by the upregulation of IL-6, monocyte chemotactic protein 1, TNFα, and IL-1β (9). It was also previously reported that mtDNA is degraded in response to hydrogen peroxide in HA-1 hamster ovarian cells, an effect that was not observed with nuclear DNA or cytoplasmic RNA. Degradation of the mitochondrial genome was apparent in both mtDNA and mitochondrial RNA species (10). These observations suggest a mechanism for mtDNA degradation, and also for the downstream activation of glial cell pro-inflammatory phenotypes.

Mitochondrial components induce inflammation in microglial (BV2) and neuronal (SH-SY5Y) cells (177). In the defining experiments, these cells were exposed to mitochondrial lysates prepared from SH-SY5Y cells containing mtDNA or alternatively SH-SY5Y cells lacking mtDNA. BV2 microglial cells exposed to mitochondrial lysates containing mtDNA had increased TNFα, IL-8, and matrix metalloproteinase 8 mRNA but decreased TREM2 mRNA. Furthermore, NFĸB nuclear localization was increased. These effects were not observed when BV2 microglial cells were exposed to mitochondrial lysates prepared from cells that lacked mtDNA. In SH-SY5Y neuronal cells exposed to mitochondrial lysates containing mtDNA, TNFα mRNA and NFĸB protein expression were elevated. In addition, mitochondrial lysate-exposed SH-SY5Y cells showed increased amyloid precursor protein (APP) mRNA and protein. Changes in APP expression or pro-inflammatory pathways did not occur when SH-SY5Y cells were exposed to mitochondrial lysates that lacked mtDNA.

Mitochondria-derived DAMP molecules induce neuroinflammation *in vivo*. We recently observed stereotactic injection of mitochondrial lysates or mtDNA into rodent hippocampi induced pro-inflammatory changes (178). Mitochondrial lysates increased hippocampal TNFα mRNA and decreased TREM2 mRNA expression. In addition, NFĸB phosphorylation was elevated in the cortex, while glial fibrillary acidic protein (GFAP) protein levels were elevated within the hippocampus. Hippocampal mtDNA injection lead to increased hippocampal TNFα mRNA but reduced hippocampal TREM2 mRNA, increased GFAP hippocampal protein expression, elevated cortical NFĸB phosphorylation, increased cortical colony-stimulating factor 1 receptor protein expression, and increased levels of phosphorylated AKT within the cortex. Beyond these inflammatory changes, whole mitochondria lysates increased protein and mRNA levels of endogenous rodent APP and Aβ1–42. These effects on APP and Aβ1–42 were not observed following injection of mtDNA. Overall, these studies provide evidence that mitochondria-derived DAMP molecules are capable of inducing neuroinflammation, as well as altering AD-related pathways.

While some studies suggest Aβ is pro-inflammatory and acts as a DAMP molecule, a recent study interestingly suggests Aβ has antimicrobial properties (179). In models that overproduce Aβ, including rodent, cell culture, and worm models, the severity of fungal and microbial infections was reduced. Injection of the bacterium *S. typhimurium* into the brain of an AD mouse model (5XFAD model) increased the propensity of Aβ to form plaques, and Aβ colocalized with the bacteria. In addition, the 5XFAD mice injected with *S. typhimurium* showed increased survival and reduced meningitis. This study further found that oligomerization of Aβ was required for this antimicrobial property to manifest. Earlier *in vitro* studies also showed Aβ initiated antimicrobial activity against several bacterial and fungal microorganisms. Aβ inhibited bacterial growth, and brain homogenates from AD subjects (containing Aβ) were also capable of inhibiting microbial growth (180). These studies suggest a possible connection between mitochondria-derived DAMP molecules, neuroinflammation, APP metabolism, and Aβ.

### Mitochondria-Derived DAMP Molecules As Biomarkers of Brain Integrity

Cell-free circulating mitochondrial components (DAMPs) such as mtDNA are altered in trauma. For example, in children with traumatic brain injury (TBI), CSF mtDNA levels are elevated. CSF mtDNA levels also correlate with TBI outcome. In children who survived a TBI, CSF mtDNA levels were in the lower range, while in children whose outcome included either death or severe disability CSF mtDNA levels were in the upper range. CSF levels of another DAMP molecule, high mobility group box 1, correlated with mtDNA levels in these subjects. Overall, mtDNA appears to represent a potential CSF mitochondrial DAMP biomarker for TBI and to have the potential to correlate with patient outcomes (3).

Circulating mtDNA is also associated with aging. In a study of 831 Caucasian subjects spanning ages 1–104 years, plasma mtDNA levels began to increase after the fifth decade of life. Plasma mtDNA levels further correlated with cytokine levels, specifically TNFα, IL-6, and regulated on activation normal T cell expressed and secreted (RANTES).

Circulating mtDNA may also potentially have the ability to serve as an AD or PD biomarker. Cell-free CSF mtDNA levels are reduced in clinically asymptomatic subjects with a genetically defined increased AD risk and clinically symptomatic AD subjects (157). However, no differences were observed for subjects with frontotemporal lobar degeneration. In a separate study, PD subjects were found to have lower levels of cell-free CSF mtDNA (158). While some may argue lower cell-free CSF mtDNA negates the possibility of a mitochondria-derived DAMP-induced neuroinflammation, other evidence contradicts this idea. It is important to note levels of another AD biomarker, Aβ, are elevated in the brain but significantly reduced in CSF from AD subjects (181, 182). Therefore, it is difficult to draw conclusions regarding CSF biomarker data and causation without sufficient knowledge of the mechanisms that underlie those biomarker changes.

### Specific Release of Mitochondria-Derived DAMP Molecules within the CNS

An important question relevant to the issue of mitochondriaderived DAMPs is how might these intracellular molecules access

FIGURE 1 | Hypothesized mechanism of mitochondrial-derived damage-associated molecular pattern molecule released in the central nervous system. (A) Neurons can release mitochondria (neuronal mitochondria are shown in red) to astrocytes, where they then undergo mitophagy (orange star shape). In addition, astrocytes can release mitochondria (astrocyte mitochondria are shown in blue) to neurons, under conditions of bioenergetic stress. (B) If the process of mitochondrial exchange between neurons and astrocytes malfunctions (X), then mitochondria and their components could be released into the extracellular space and initiate neuroinflammation.

the extracellular space? While cellular components are released during necrotic cell death, more specific process through which mitochondria are released to the brain's extracellular compartment have recently been described. One of these processes has been termed transcellular mitophagy. Neurons are large cells and can span long distances. Mitochondria continuously move between the cell body and dendrites, and between the cell body and axons. When peripheral mitochondria cease to function properly, they were believed to return to the cell body for disposal through the process of mitochondrial autophagy or mitophagy. Data now indicate neurons can also export mitochondria to surrounding glial cells, where they then undergo mitophagy. This process was first described to occur within the optic nerve and the cortex (183). In a separate study, it was found that astrocytes also transfer mitochondria to neurons (184). The transfer of mitochondria from astrocytes to neurons enhanced neuronal survival following ischemia reperfusion injury and required signaling from cluster of differentiation 38, cyclic ADP ribose, and calcium.

### CONCLUSION

In specific neurodegenerative diseases, if mitochondrial dysfunction overwhelmed the ability of neurons and astrocytes to adequately perform mitophagy, then mitochondria-derived DAMP molecules could predictably facilitate neuroinflammation (**Figure 1**). Clearly, a rational case can be made that mitochondria-derived DAMP molecules may contribute to neurodegenerative disease-associated neuroinflammation. However, important questions remain about how mitochondrial DAMPs contribute to neurodegenerative diseases and neurodegenerative disease-related pathologies. Future directions should focus on if and how specific mitochondrial-derived molecules initiate neuroinflammation. More specifically, how does mitochondrial dysfunction contribute to the release of DAMP molecules and is this process upstream or downstream of other disease pathologies? As this line of investigation moves forward, studies to address issues of cause versus consequence should help resolve these important knowledge gaps.

## AUTHOR CONTRIBUTIONS

HW was responsible for writing the article. IW and YJ contributed to sections of the article. RS edited and provided direction for the article.

## FUNDING

This project was supported by the University of Kansas Alzheimer's Disease Center (P30 AG035982), the Frank and Evangeline Thompson Alzheimer's Treatment Program fund, the Kansas IDeA Network for Biomedical Research Excellence (KINBRE, P20GM103418), the University of Kansas Medical Center Biomedical Research Training Program, and a Mabel Woodyard Fellowship award.

## REFERENCES

atrophy. *Neurobiol Aging* (2012) 33(5):856–66. doi:10.1016/j.neurobiolaging. 2010.07.020

in vitro and in vivo studies. *Neurobiol Dis* (2003) 12(2):121–32. doi:10.1016/ S0969-9961(03)00002-0

extracellular signaling molecule recognized by brain microglia. *Mol Cell Neurosci* (2014) 60:88–96. doi:10.1016/j.mcn.2014.04.003

**Conflict of Interest Statement:** The authors declare this review article was completed in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

*Copyright © 2017 Wilkins, Weidling, Ji and Swerdlow. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# IL-10-Producing CD1dhiCD5**<sup>+</sup>** Regulatory B Cells May Play a Critical Role in Modulating Immune Homeostasis in Silicosis Patients

*Ying Chen1 , Chao Li1 , Yiping Lu1 , Huiying Zhuang1 , Weijia Gu1 , Bo Liu1 , Fangwei Liu1 , Jinkai Sun2 , Bo Yan2 , Dong Weng1,3 \* and Jie Chen1 \**

*1 Division of Pneumoconiosis, School of Public Health, China Medical University, Shenyang, China, 2Department of Respiratory Medicine, Shenyang No. 9 Hospital, Shenyang, China, 3Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China*

#### *Edited by:*

*Pietro Ghezzi, Brighton and Sussex Medical School, UK*

#### *Reviewed by:*

*Frédéric Velard, Université de Reims Champagne-Ardenne, France Christopher Pattillo, LSU Health Sciences Center New Orleans, USA*

#### *\*Correspondence:*

*Jie Chen<EMAIL_ADDRESS>Dong Weng<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 07 October 2016 Accepted: 24 January 2017 Published: 13 February 2017*

#### *Citation:*

*Chen Y, Li C, Lu Y, Zhuang H, Gu W, Liu B, Liu F, Sun J, Yan B, Weng D and Chen J (2017) IL-10-Producing CD1dhiCD5+ Regulatory B Cells May Play a Critical Role in Modulating Immune Homeostasis in Silicosis Patients. Front. Immunol. 8:110. doi: 10.3389/fimmu.2017.00110*

Silicosis is characterized by chronic lung inflammation and fibrosis, which are extremely harmful to human health. The pathogenesis of silicosis involves uncontrolled immune processes. Evidence supports that regulatory B cells (Bregs) produce negative regulatory cytokines, such as IL-10, which can negatively regulate immune responses in inflammation and autoimmune diseases. Our previous study found that IL-10-producing B cells were involved in the development of silica-induced lung inflammation and fibrosis of mice. However, little is known about the role of Bregs in silicosis patients (SP). In this study, we found that serum concentrations of IL-10 were significantly increased in SP by using protein array screening. We further determined that the frequency of IL-10-producing CD1dhiCD5+ Bregs, not IL-10-producing non-B lymphocytes, was significantly higher in SP compared to subjects under surveillance (SS) and healthy workers (HW) by flow cytometry. We also found that regulatory T cells (Tregs) and Th2 cytokines (IL-4, IL-5, and IL-13) were significantly increased in SP. Th1 cytokines (IFN-γ, IL-2, and IL-12) and inflammatory cytokines (IL-1β, IL-6, and TNF-α) were not significantly different between SP, SS, and HW. Our study indicated that IL-10 producing CD1dhiCD5+ Bregs might maintain Tregs and regulate Th1/Th2 polarization in SP, suggesting that IL-10-producing Bregs may play a critical role in modulating immune homeostasis in SP.

Keywords: IL-10, regulatory B cells, regulatory T cells, Th1/Th2 polarization, immune homeostasis, silicosis patients

#### INTRODUCTION

Silicosis is a potentially fatal, but preventable, occupational lung disease caused by inhaling respirable crystalline silica (1). It is characterized by chronic lung inflammation and irreversible fibrosis (2). Progressive fibrosis leads to disability and mortality in patients, which results in a heavy burden to society. In China, there were 11,471 cases of silicosis reported in 2014, accounting for 42.69% of 26,873 new pneumoconiosis cases (Ministry of Health of the People's Republic of China, 2015). Silicosis is reported to be a special concern in young adults (aged 15–44 years) (3). Silicosis has become the most serious occupational disease in both developed and developing countries, yet little is known about the crucial cellular and molecular mechanisms that initiate and propagate the process of silicosis.

Previous evidence has shown that both innate and adaptive immune responses play a key role in the pathogenesis of silicosis (4, 5). Alveolar macrophages, the first defense against foreign substances, ingest inhaled silica and become activated to release a host of mediators, such as cytokines and chemokines, which initiate the influx of inflammatory cells and activation of immune cells (6, 7). The complex network of interactions between mediators and cells results in the onset of lung injury, inflammation, and potentially fibrosis. The inflammation mediators are involved in the causation of silicosis. Chemokines that primarily attract monocytes and macrophages induce inflammation mediators such as IL-1β, IL-6, and TNF-α, which initiate the activation of T and B immune cells and stimulate T-lymphocyte type switching (8, 9).

Previous studies have demonstrated that Th1 and Th2 cells participate in the pathogenesis of silicosis (10, 11). Regulatory T cells (Tregs) play a role of the maintenance of immune homeostasis, which limits inflammation and modulates Th1/ Th2 balance in silica-induced lung fibrosis (12). However, other regulatory subsets in silicosis have not been investigated in detail, and the mechanism of immune homeostasis modulation needs further exploration. Recent studies have demonstrated that another regulatory lymphocyte, specific B cell subsets, socalled regulatory B cells (Bregs), are potent immune response regulators and play important roles in regulating immune homeostasis (13, 14). A number of Breg subsets have been reported, of which the IL-10-producing B cell (B10) subset is among the best characterized (15, 16). Subsequent studies have shown that CD1dhighCD5<sup>+</sup> B cells were associated with the suppression of inflammation and autoimmune disease through IL-10 secretion (17, 18). B10 was also found to participate in regulating Th immune response by affecting the secretion of inflammatory cytokines such as TNF-α (19–21). In addition, B10 influences the proliferation of Tregs (22). Recent findings have shown that frequencies of peripheral Bregs increased in patients with lung cancer and B10s were significantly elevated in non-small cell lung carcinoma patients (23, 24). CD1d<sup>+</sup>CD5<sup>+</sup> B cell numbers were increased in patients with tuberculosis (25). Our previous study found that B10 was involved in the development of silica-induced lung inflammation and fibrosis of mice (26). However, little is known about the role of similar or equivalent regulatory B lymphocyte populations in silicosis patients (SP).

A protein microarray assay and Bio-Plex assay were used in the present study to find that the level of IL-10 was increased in the serum of SP. To explore whether the increased IL-10 was produced by B and/or T cell populations and to determine the role of Bregs in SP, we investigated peripheral B10 and CD19<sup>+</sup> IL-10<sup>+</sup>CD1dhiCD5<sup>+</sup> B cell subsets and Tregs and also determined the level of chemokines, inflammation mediators, and Th1 and Th2 cytokines in SP, subjects under surveillance (SS), and healthy workers (HW) that had prior exposure to silica dust. Our results may provide new insights into the role of Bregs in SP.

#### MATERIALS AND METHODS

#### Study Design

The study, conducted from May 2015 to March 2016, included 57 individuals. Nineteen first phase male SP were recruited from Shenyang No. 9 Hospital. Nineteen SS and nineteen HW with exposure to silica dust from Northern Heavy Industries Group Co., Ltd., matched by sex, age, year of exposure to silica dust, and ethnicity with SP were enrolled in this study. Criteria were based on the national diagnostic criteria of pneumoconiosis (GBZ70-2009). SS were workers with exposure to silica, who had no certain shadow changes visible in the chest X-ray films by health examination, and who needed periodic follow-ups within specified periods of time. The diagnosis of silicosis was based upon patient history of exposure to silica and available chest X-rays and was made by five qualified experts, who served as members of the Shenyang Municipal Pneumoconiosis Diagnosis Committee. Patients with chronic inflammatory diseases, respiratory infectious diseases, tuberculosis, asthma, diabetes, or cancer autoimmune disease were excluded.

Pulmonary function was measured from 2015 to 2016 by using spirometry. The data collected included forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1). Personal information, including sex, age, exposure period, and smoking status were obtain *via* questionnaire.

This study was carried out in accordance with the recommendations of the Ethics Committee of the China Medical University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol (CMU2100012006) was approved by the Ethics Committee of the China Medical University.

#### Blood Sample

Five milliliters of venous blood samples were collected into no additive vacutainer tubes. Serum was obtained after centrifugation and stored at −80°C for protein microarray analysis and the Bio-Plex assay.

Also, 6 ml venous blood samples were collected into vacutainer tubes containing EDTA. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by Ficoll density gradient centrifugation (TBD sciences, China) for Flow cytometric analysis.

#### Protein Microarray Analysis

RayBiotech Human Cytokine Antibody Array 440 (RayBiotech, Inc., Cat# QAH-CAA-440) was used to profile the variation of proteins in serum. The samples were detected by the Shanghai XY Biotech Company. The experimental procedure was carried out in accordance with the manufacturer's instructions. Briefly, pre-coated antibody array membranes were incubated with coating buffer for 30 min. After that, the blocking buffer was decanted and replaced with sample dilutions. Membranes were incubated overnight at 4°C with shaking. The next day, the membranes were incubated with biotin-conjugated antibody for 2 h after washing. Mixture of biotin-conjugated antibodies was removed and Streptavidin-Fluor was added to each subarray. The incubation chambers were covered with adhesive film and incubated for 2 h. After washing, signals were detected with a GenePix 4000B system (Axon Instruments, Foster City, CA, USA). GenePix Pro 6.0 software (Axon Instruments) was used for densitometric analysis of the spots. These values were normalized to the ratio of positive control values for each sample. Afterward, the total normalized fluorescence values of replicate spots were averaged and expressed as fold increase over the control samples.

#### Bio-Plex Assay

The levels of 27 cytokines in serum were measured by the Bio-Plex Pro™ human cytokine 27-plex assay (Bio-Rad Laboratories, Hercules, CA, USA; Cat# M500KCAF0Y). This multiplex assay detects: FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1)α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF. The experimental procedure was carried out according to the manufacturer's instructions. Briefly, beads coated with capture antibodies were incubated with premixed standards or sample supernatants for 30 min. Following incubation, premixed detection antibodies were added and incubated as before. After washing, streptavidin-PE was added and incubated for 10 min. After washing, the beads were resuspended in Bio-Plex cytokine assay buffer, and results were read on the Bio-Plex 200 system using Low PMT setting. Data were analyzed with Bio-Plex Manager™ software version 2.0.

#### Cell Preparation and Flow Cytometry Analysis

Peripheral blood mononuclear cells were isolated from blood samples by Ficoll density gradient centrifugation. After washing and addition of RPMI1640 medium, PBMCs were stimulated with Leukocyte Activation Cocktail (BD Pharmingen, San Jose, CA, USA) for 5 h, followed by blocking with Human BD Fc Block™ (BD Pharmingen) for 10 min at room temperature. PE-Cy7-conjugated CD4 (BD Pharmingen) and PerCP-Cy5.5 conjugated CD19 (BD Pharmingen) antibodies were used for cell surface staining. Cells were fixed and permeabilized using a fixation/permeabilization kit (eBioscience, San Diego, CA, USA) or BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution kit (BD Pharmingen) according to the manufacturer's instructions. The cells were then stained at 4°C with APC-conjugated anti-Foxp3 (BD Pharmingen), APC-conjugated anti-IL-10 (BD Pharmingen), FITC-conjugated anti-CD5 (BD Pharmingen), and PE-conjugated anti-CD1d (BD Pharmingen). After washing, stained cells were resuspended in 1% paraformaldehyde-PBS. Analysis of cell marker expression was performed using a FACSCanto II system (BD, Franklin Lakes, NJ, USA). Dead cells were gated out depending on forward scattering and side scattering. Cells were analyzed with FlowJo X software (Tree Star, OR, USA).

#### Statistical Analyses

Data were analyzed for statistical significance using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Means between two groups were analyzed by Student's *t*-test. The differences between values were evaluated through a one-way analysis of variance followed by pair-wise comparison with the Student–Newman– Keuls test. The chi-square test was used to compare categorical variables. Also, *p* < 0.05 was considered statistically significant. All values are means ± SEM.

### RESULTS

#### Study Subjects

The general characteristics of the study subjects are shown in **Table 1**. No difference in general characteristics, including median age, exposure period, and smoking status, were found between three groups. Pulmonary function (%FVC predicted, %FEV1 predicted, %FEV1/FVC radio) in SP was significantly decreased compared to SS and HW.

#### Multiple Cytokines Altered in Serum of SP

To profile the cytokines in the peripheral blood from patients with silicosis, a blinded screening using serum of SP, SS, and HW by protein microarray analysis was performed. As shown in the heat maps, 46 kinds of cytokines were different between SP and SS, and 18 kinds of cytokines were different between SP and HW. Fifty-six kinds of cytokines were different among HW, SS, and SP groups (data shown in Tables S1–S3 in Supplementary Material, respectively). Among those cytokines, there was a marked increase of IFN-γ, IL-7, TNF-α, IL-10, IL-13, IL-6, IL-5, and IL-8 in SP (*n* = 5) compared to SS (*n* = 5) (**Figure 1A**). There was also a marked increase of

*HW, healthy workers with exposure to silica dust; SS, subjects under surveillance; SP, silicosis patients; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s. Values are expressed as means* ± *SEM, # p* < *0.05 compared with HW, \*p* < *0.05 compared with SS.*

*a Calculated by one-way analysis of variance.*

*bCalculated by chi-square test.*

Image showed a marked increase of IFN-γ, IL-7, TNF-α, IL-10, IL-13, IL-6, IL-5, and IL-8 in SP (*n* = 5) compared to SS (*n* = 5). Red represents upregulated protein expression, and green represents downregulated protein expression. (B) Heat map analysis between SP and workers exposed to silica dust. Image showed a marked increase of GM-CSF, IL-10, and IL-6 in SP (*n* = 5) compared to healthy workers (HW) (*n* = 5). Red represents upregulated protein expression, and green represents downregulated protein expression.

GM-CSF, IL-10, and IL-6 in SP (*n* = 5) compared to HW (*n* = 5) (**Figure 1B**). The levels of IL-10 showed significance difference between SP and SS (*p* = 0.00001). The levels of IL-10 were significantly different between SP and HW (*p* = 0.00082). These results suggested that IL-10 might play a critical role in the pathogenesis of silicosis.

### Serum Level of IL-10 and IL-10-Producing CD1dhighCD5**+** Bregs Were Increased in SP

The sample size was enlarged (*n* = 19), and the Bio-Plex array was used to further confirm the variation of serum IL-10 in SP. The Bio-Plex suspension array system permitted the simultaneous measurement of 27 human cytokines, including interleukins, chemokines, colony-stimulating factors, growth factors, interferon, and tumor necrosis factor (data of 27 cytokines shown in Table S4 in Supplementary Material). The results showed that the serum concentrations of IL-10 were significantly increased in SP compared to SS and HW (*p* = 0.03, *p* = 0.017) (**Figure 2A**). To explore the possibility that the increased IL-10 was produced by B cell and/ or T cell populations, peripheral B10s and IL-10-producing non-B lymphocytes were investigated from all participants. The percentage of CD19<sup>+</sup>IL-10<sup>+</sup> B cells was observed to significantly increase in SP (2.02 ± 0.23%) compared to the SS group (1.46 ± 0.14%) and the HW group (1.21 ± 0.11%) (**Figures 2B,F**). However, there were no obvious differences in the percentage of CD19- IL-10<sup>+</sup> lymphocytes between the three groups (**Figures 2B,E**). These data suggested that increased serum IL-10 in SP might contribute to B10s, but not IL-10 producing non-B lymphocytes.

Emerging evidence indicates that CD1dhiCD5<sup>+</sup> B cells are responsible for most of the IL-10 production by B cells (17). We investigated peripheral IL-10-producing CD1dhiCD5<sup>+</sup> B cells in all participants. Our flow cytometry results showed that a proportion of cells highly expressed CD1d in CD19<sup>+</sup> B cells (**Figure 2D**). The percentage of IL-10<sup>+</sup>CD1dhiCD5<sup>+</sup> B cells significantly increased in SP (0.89 ± 0.18%) compared to the SS (0.39 ± 0.06%) and HW (0.24 ± 0.05%) groups (**Figures 2C,G**). These data suggested that IL-10<sup>+</sup>CD1dhiCD5<sup>+</sup> B cells might play a critical role in the pathogenesis of silicosis.

#### Serum Levels of Chemokines Were Increased in SS

Of the 27 cytokines detected by the Bio-Plex assay, some chemokines tended to increase in the early stages of silicosis.

FIGURE 2 | Level of IL-10 in serum, IL-10-producing B cell (B10), and IL-10**+**CD1dhighCD5**+** B cell subsets in the peripheral blood were assayed in all participants. (A) The level of IL-10 in serum was assayed by the Bio-Plex assay. (B,E,F) Percentages of CD19+IL-10+ B cells and CD19- IL-10+ cells in the peripheral blood were assayed by flow cytometry. (C,G) Percentage of IL-10+CD1dhighCD5+ B cells in the peripheral blood was assayed by flow cytometry. (D) The overlay graph of CD1d unstained and CD1d stained histograms [*n* = 19 per group, # *p* < 0.05 compared with healthy workers (HW); \**p* < 0.05 compared with SS]. Error bars indicate mean ± SEM.

Compared with HW, the serum concentration of MCP-1 in SS and SP was significantly increased (*p* = 0.008, *p* = 0.002) (**Figure 3A**). In addition, the serum concentration of interferon gamma-IP-10 was increased in SS and SP compared to HW (**Figure 3B**). The serum concentrations of MIP-1α and MIP-1β were increased in SS compared to HW and SP (**Figures 3C,D**), but these differences were not statistically significant. These results suggested that some chemokines might initially be secreted by activated macrophages.

#### Tregs, Not Inflammatory Cytokines, Were Increased in SP

To investigate whether Bregs could affect the production of inflammatory cytokines and maintain Tregs in SP, inflammatory cytokines were detected using the Bio-Plex assay, and Tregs in PBMCs were analyzed by flow cytometry. The results showed no significant difference in the serum concentrations of inflammatory cytokines IL-1β, IL-6, and TNF-α between the three groups (**Figures 4A–C**). The percentage of CD4<sup>+</sup> T cells expressing Foxp3, which were identified as Tregs, increased significantly in SP (5.92 ± 0.36%) compared with that of SS (3.88 ± 0.38%) and HW (3.91 ± 0.33%) (*p* < 0.001, *p* < 0.001) (**Figures 4D,E**). These results suggested that Bregs might inhibit the production of inflammatory cytokines and maintain Tregs in SP.

### Th2 Cytokines, Not Th1 Cytokines, Were Increased in SP

To investigate effects of Bregs on the Th1/Th2 immune response, the levels of Th1/Th2 cytokines were assayed in all participants. There was no significant difference in the levels of Th1 cytokine IFN-γ, IL-2, and IL-12 (p70) in serum between the SP, SS, and HW groups (**Figures 5A–C**). In contrast, the serum concentration of IL-4 was significantly increased in SP compared to SS and HW (*p* = 0.015, *p* = 0.039) (**Figure 5D**). The serum concentration of IL-5 was significantly increased in SP compared to SS (*p* = 0.012) and obviously increased compared to HW (*p* = 0.098) (**Figure 5E**). The serum concentration of IL-13 was significantly increased in SP compared to HW (*p* = 0.011) and obviously increased compared to SS (*p* = 0.056) (**Figure 5F**). These results suggested that Bregs might inhibit the secretion Th1 cytokines and promote the secretion Th2 cytokines to modulate Th1/Th2 immune responses in SP.

### DISCUSSION

IL-10 is a pleiotropic cytokine produced by a number of leukocyte populations that impacts immune regulation and tissue homeostasis (27, 28). B10s play an important role in maintaining immune homeostasis (29). In this study, we found that the serum concentration of IL-10 was significantly increased in SP

FIGURE 4 | Levels of inflammatory cytokines in serum and regulatory T cells (Tregs) in the peripheral blood were assayed in all participants. (A) The level of IL-1β in serum was assayed by the Bio-Plex assay. (B) The level of IL-6 in serum was assayed by the Bio-Plex assay. (C) The level of TNF-α in serum was assayed by the Bio-Plex assay. (D,E) Percentage of CD4+Foxp3+ Tregs in the peripheral blood was assayed by flow cytometry [*n* = 19 per group, # *p* < 0.05 compared with healthy workers (HW); \**p* < 0.05 compared with subjects under surveillance (SS)]. Error bars indicate mean ± SEM.

FIGURE 5 | Levels of Th1/Th2 cytokines were assayed in all participants. (A) The level of IFN-γ in serum was assayed by the Bio-Plex assay. (B) The level of IL-2 in serum was assayed by the Bio-Plex assay. (C) The level of IL-12 (p70) in serum was assayed by the Bio-Plex assay. (D) The level of IL-4 in serum was assayed by the Bio-Plex assay. (E) The level of IL-5 in serum was assayed by the Bio-Plex assay. (F) The level of IL-13 in serum was assayed by the Bio-Plex assay [*n* = 19 per group, # *p* < 0.05 compared with healthy workers (HW); \**p* < 0.05 compared with subjects under surveillance (SS)]. Error bars indicate mean ± SEM.

compared to SS and HW by protein microarray analysis and the Bio-Plex assay. We compared the frequencies of IL-10 secreting B cells and IL-10-secreting non-B lymphocytes in all participants and determined that the frequency of B10s, not IL-10-producing non-B lymphocytes, was higher in SP than in SS and HW groups.

Regulatory B cells are important suppressors of inflammation and other autoimmune responses, producing the negative regulatory cytokines, IL-10 and TGF-β (30). IL-10 and TGFβ-producing B cells mediate tolerance in acute allergic airway inflammation (31). However, IL-10 is considered the hallmark cytokine of Bregs. B10s were the first Bregs to be recognized and were termed "B10" cells (32). Emerging evidence indicates that CD1dhiCD5<sup>+</sup> B cells are responsible for most IL-10 production by B cells (17, 33). The function of Bregs was mediated by IL-10 and dependent on the expression of CD1d (20, 34). A Bregs' deficiency leads to increased allergic airway Inflammation (35). Our results showed that the percentage of IL-10<sup>+</sup>CD1dhiCD5<sup>+</sup> B cells significantly increased in SP compared to SS and HW groups. The increase of B10s and IL-10<sup>+</sup>CD1dhiCD5<sup>+</sup> B cells in SP might be the results of effective immune response to lung injury. IL-10+CD1dhiCD5+ B cells might play a critical role in modulating immune homeostasis of SP (**Figure 6**).

The pathogenesis of silicosis involves alveolar cell injury and activation followed by release of cytokines and recruitment of cells into the areas of silica dust deposition (10, 36, 37). Chemokines such as MCP-1, MIP-1, and IP-10 are chemotactic cytokines that recruit specific groups of monocytes/macrophages and lymphocytes, which are involved in many chronic lung diseases (38–41). MCP-1 is a potent chemotactic factor for blood monocytes and produced by various inflammatory cells, which has been considered a potential biomarker for the progress of coal worker's pneumoconiosis (42). Our results showed that the levels of MCP-1 and IP-10, especially MCP-1, were significantly increased in SS and SP compared to HW. These results suggested that chemokines were involved in the initial stages of silicosis.

It is now widely appreciated that B cells can suppress inflammation and negatively regulate immune responses, in part through the secretion of anti-inflammatory IL-10 (19, 26, 43). The production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, by monocytes and macrophages is suppressed by IL-10 (39). In our previous study, we found that the levels of secreted TNF-α and IL-6 proteins in BALF clearly increased in B10-deficiency mice injected with an anti-CD22 antibody (26). Our data revealed that the serum concentrations of IL-1β, IL-6, and TNF-α were not significantly different between the SP, SS, and HW groups. B10s might partly restrain inflammation of silicosis by downregulating the production of pro-inflammatory cytokines. In addition, it has recently been shown that B10s are crucial for the maintenance of Tregs function (44–47). It was reported in mice that tumor-induced B10s could convert resting CD4<sup>+</sup> non-Tregs into Tregs (48). Another study showed that B10s promoted Tregs accumulation and function by stimulating Foxp3 expression (49). In our previous study, insufficient B10s reduced the frequency of Tregs, whereas Treg depletion did not influence B10 accumulation in silicosis of mice. B10s might act earlier than Tregs during silica-induced lung inflammation and fibrosis (26). Our present data showed that Tregs significantly increased in SP compared to SS and HW. These results indicated that Bregs might maintain Tregs function, which is involved in suppressing inflammation and regulating Th immune responses in SP (**Figure 6**).

Regulatory B cells have been found to have regulatory properties in inducing primary T cell proliferation and in generating and maintaining CD4<sup>+</sup> effect T cells (50). It was reported that B cell depletion therapy was associated with changes in T cell repertoire and function in patients with idiopathic thrombocytopenic purpura (51). Our previous studies had demonstrated insufficient B10 could exacerbate Th1 immune response and elevate levels of IFN-γ in silicosis of mice (26). Studies have shown B10s can influence the development of Th2 cells (52, 53). In the current study, the levels of Th1 cytokines IFN-γ, IL-2, and IL-12 (p70) in serum were found to have no significant difference among the three groups. The levels of Th2 cytokines IL-4, IL-5, and IL-13 in serum significantly increased in SP compared to SS and/or HW. These results suggested that increased levels of IL-10/IL-10-producing CD1dhiCD5+ Bregs might inhibit the

production of Th1 cytokines and maintain secretion of Th2 cytokines to regulate the Th1/Th2 balance toward a Th2 phenotype in SP (**Figure 6**).

The results of the present study show that increased level of inhibitory cytokine IL-10 and number of IL-10-producing CD1dhiCD5<sup>+</sup> Bregs may maintain Tregs and regulate Th1/Th2 polarization, suggesting that IL-10-producing Bregs may play a critical role in modulating immune homeostasis in SP. However, how B cells differentiate into IL-10-producing Bregs and what functions the cells play in silicosis progression remain to be further investigated.

### AUTHOR CONTRIBUTIONS

JC and YC conceived and designed the research; YC, CL, YL, HZ, WG, BL, JS, and BY performed experiments; YC, CL, FL, DW,

#### REFERENCES

and JC analyzed data and interpreted results; YC prepared figures and drafted the manuscript; YC, CL, FL, DW, and JC edited and revised the manuscript; all the authors approved the final manuscript version.

### FUNDING

This study was supported by the National Natural Science Foundation of China (No. 81273045) and the Program for Liaoning Innovative Research Team in University (LT2015028).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.00110/ full#supplementary-material.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Chen, Li, Lu, Zhuang, Gu, Liu, Liu, Sun, Yan, Weng and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Lipopolysaccharide Attenuates Induction of Proallergic Cytokines, Thymic Stromal Lymphopoietin, and Interleukin 33 in Respiratory Epithelial Cells Stimulated with PolyI:C and Human Parechovirus

*Tsang-Hsiung Lin1 , Chih-Chi Cheng2 , Hsing-Hao Su3 , Nan-Chieh Huang4 , Jih-Jung Chen5 , Hong-Yo Kang1,6 \* and Tsung-Hsien Chang2,7 \**

#### *Edited by:*

*Abdulraouf Ramadan, Indiana University – Purdue University Indianapolis, USA*

#### *Reviewed by:*

*Fabrice Cognasse, The Rhone-Alpes-Auvergne Regional Branch of the French National Blood System, France Niccolo Terrando, Duke University, USA*

#### *\*Correspondence:*

*Hong-Yo Kang<EMAIL_ADDRESS>Tsung-Hsien Chang<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 27 June 2016 Accepted: 06 October 2016 Published: 25 October 2016*

#### *Citation:*

*Lin T-H, Cheng C-C, Su H-H, Huang N-C, Chen J-J, Kang H-Y and Chang T-H (2016) Lipopolysaccharide Attenuates Induction of Proallergic Cytokines, Thymic Stromal Lymphopoietin, and Interleukin 33 in Respiratory Epithelial Cells Stimulated with PolyI:C and Human Parechovirus. Front. Immunol. 7:440. doi: 10.3389/fimmu.2016.00440*

*1Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Kaohsiung, Taiwan, 2Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 3Department of Otorhinolaryngology – Head and Neck Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 4Department of Family Medicine, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, 5Department of Pharmacy, Tajen University, Pingtung, Taiwan, 6Department of Obstetrics and Gynecology, Center for Menopause and Reproductive Medicine Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, 7Department of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan*

Epidemiological studies based on the "hygiene hypothesis" declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; toll-like receptor 4 ligand) on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP) and interleukin 33 (IL33) in H292 cells (pulmonary mucoepidermoid carcinoma cells). Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1) but not LPS-induced TSLP and IL33 through interferon regulatory factor 3 (IRF3) and NF-κB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082) and short hairpin RNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3, and NF-κB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin high-mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells *in vitro.* In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.

Keywords: TSLP, IL33, HMGB1, hygiene hypothesis, innate immunity

### INTRODUCTION

The hygiene hypothesis declares that a lack of early childhood exposure to environmental microorganisms and pathogens increases susceptibility to allergic diseases by suppressing the establishment of immune tolerance (1). Epidemiological data and experimental evidence showed that exposure to environmental pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), are associated with decreasing the incidence of allergic diseases in later life (2–4). Similarly, muramic acid, a constituent of peptidoglycan of bacteria in the environment was found inversely associated with respiratory wheezing in rural school children (5). Those reports suggest that environmental microbes modulate allergic response.

Innate immunity is a rapid host defense response against invading pathogens, this response is essential to establish antigen-specific adaptive immunity to further eradicate pathogens and instruct the immune memory (6). Human epithelial cells form the largest primary physical barrier against environmental microbes and provide protection to the host via TLR-mediated responses of innate immunity (7–9). TLR signaling in skin and airway epithelial cells promotes the expression of proallergic cytokines, such as thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor, interleukin-25 (IL25), and IL33, which are crucial for the initiation of the Th2 allergic immune cascade (7, 10–14). Epithelial cells command the innate and adaptive immune responses in atopic diseases (15). The implication of epithelial cells expressing the proallergic cytokines TSLP and IL33 in hygiene hypothesis is unknown.

Childhood exposure to environmental microorganism, such as viral infection can exacerbate asthma severity. Viral infection with rhinovirus, human metapneumovirus and respiratory syncytial virus can induce TSLP expression in airway epithelial cells (9, 16, 17). In particular, rhinovirus infection has been found associated with TSLP levels in the airways of young children (18). Rhinovirus infection can induce IL33 secretion in human bronchial epithelial cells to promote Th2 inflammation and exacerbate asthma severity in patients (19). This finding agreed with the observation of higher IL33 levels in the sera of patients with allergic rhinitis than normal controls (20). In addition, human parechovirus (HPeV), a small, round-structured, non-enveloped virus with a single-stranded and positive-sense RNA genome, belongs to the *Picornaviridae* (21). Nosocomial infection or outbreaks in neonate hospital departments seem to play a large role in HPeV infection (22, 23). Similar to rhinovirus, HPeV also causes respiratory disease in children, with high prevalence (24). It would be interested to understand whether HPeV1 acts like rhinovirus on prompting allergy.

Virus infection also can increase and activate TLR3 signal pathway (25). Among the various TLR ligands, only polyI:C (double-stranded RNA, TLR3 ligand) can stimulate high levels of TSLP expression, which is enhanced by the addition of IL4, IL13, or tumor necrosis factor α (26). Other TLR ligands, such as LPS (TLR4 ligand), CpG (TLR9 ligand), Pam3CSK4 (TLR2 ligand), and flagellin (TLR5 ligand), failed to induce TSLP expression in epithelial cells (16, 26). Similarly, IL33 mRNA expression could be induced by IFN-γ, the TLR9 ligand ODN2006, or polyI:C but not LPS in human nasal epithelial cells with allergic rhinitis (20, 27).

The immunoregulatory effect of the LPS/TLR4 axis in immune cells, such as dendritic cells and myeloid-derived suppressor cells was revealed in an animal model of asthma, which suggested that the dose of LPS is critical for the T helper 1 (Th1)/Th2 cell balance. Increased doses of LPS and antigens induce Th1 responses and inhibit allergic inflammation; however, reduced doses of LPS induce Th2 responses and promote airway inflammation (28–31). In addition to LPS, the TLR2 ligand Pam3CSK4 blocks the development of asthma (32). Therefore, TLRs in immune cells play roles during allergic airway responses.