TLR4-MyD88 signaling pathway is responsible for acute lung inflammation induced by reclaimed water

TLR4-MyD88 signaling pathway is responsible for acute lung inflammation induced by reclaimed water

Journal of Hazardous Materials 396 (2020) 122586 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.else...

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Journal of Hazardous Materials 396 (2020) 122586

Contents lists available at ScienceDirect

Journal of Hazardous Materials journal homepage:

TLR4-MyD88 signaling pathway is responsible for acute lung inflammation induced by reclaimed water


Gang Liua, Yun Lua,*, Liangliang Shia, Yunru Rena, Jiayang Konga, Mengyu Zhangb, Menghao Chena, Wanli Liub a b

State Key Joint Laboratory of Environmental Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084, China School of Life Science, Tsinghua University, Beijing, 100084, China




Editor: D. Age

Previous research found that inhalation exposure of reclaimed water could cause severe pulmonary inflammation, and the endotoxin was proposed to be the key risk factor. To further support this view, the toxic effects of different reclaimed water induced by acute inhalation exposure were compared between wildtype C57BL/6J and TLR4 signaling pathway defect mice. It was found that reclaimed water with high levels of endotoxin could induce strong inflammation in wildtype mice, but not in Tlr4−/− and MyD88−/− mutants. The mixed bacterial culture from the reclaimed water showed very weak response in wildtype mice and no response in TLR4-signaling pathway deficient mice, which further suggested that the cell-bound endotoxins contribute little in the inflammation induced by reclaimed water. In addition, conditional knockout of the Tlr4 gene in myeloid cells resulted in a significant reduction of sensitivity to the reclaimed water in mutants, which indicates that myeloid cells play the most important role in the defensive immune system against the pollutants in the water. In general, this study demonstrated that the TLR4-MyD88 signaling pathway is responsible for the acute lung inflammation induced by reclaimed water, which excludes the possibility of other signaling pathway dependent inflammation inducers in reclaimed water.

Keywords: Reclaimed water Endotoxin Inhalation exposure TLR4 MyD88

1. Introduction The effective utilization of reclaimed water helps to alleviate the

problem of water scarcity and can produce substantial economic and environmental benefits (Anderson, 2003). The reclaimed water can replace freshwater in traditional practices such as landscape irrigation,

Corresponding author at: Room 724, School of Environment, Tsinghua University, Beijing, 100084, China. E-mail address: [email protected] (Y. Lu). Received 23 December 2019; Received in revised form 28 February 2020; Accepted 24 March 2020 Available online 02 April 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. The TLR signaling pathway plays a crucial role in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. MyD88 and TRIF are two canonical adaptors for inflammatory signaling pathway downstream of members of TLRs (Gay et al., 2014).

surface water replenishment, street cleaning, firefighting and urban construction (Levine and Asano, 2004). However, risk factors present in reclaimed water are abundant, such as bacteria and their derivatives, viruses, protozoa, helminths, heavy metals, and micropollutants such as disinfection by-products, endocrine disrupting chemicals, pharmaceutically-active compounds, microplastic (Paranychianakis et al., 2014; Salgot et al., 2006; Toze, 2006). One of the common exposure routes of reclaimed water to human is the inhalation of water aerosols (Toze, 2006). However, the health risks originating from reclaimed water aerosols have not been well studied. Among the risk factors, endotoxin is ignored in reclaimed water quality control, though it has caused significant diseases in contaminated aerosolized water (Anderson et al., 2007). Endotoxin, also known as lipopolysaccharide (LPS), is an amphiphilic glycolipid of the outer membrane of gram-negative bacteria (Rietschel et al., 1994). In water, there are two different forms. Free endotoxin dissolves in water as small aggregates, and cell-bound endotoxin is a component of the bacterial cell wall. Both of these are associated with toxicity and immunogenicity in diverse eukaryotic species (Alexander and Rietschel, 2016). Endotoxins in aerosols are likely to be the etiological agent behind outbreaks of a transient, flu-like syndrome (fever, malaise, muscle pains, tightness of the chest and respiratory-tract symptoms) after taking a bath or shower (Annadotter et al., 2005). Airway inflammation can also occur if humidifier reservoirs are filled with water with high endotoxin levels (Anderson et al., 2007). Long-term exposure to endotoxin has been proven to correlate to interstitial lung disease, lung injury and airways disease, such as chronic obstructive pulmonary disease (COPD) and asthma (Arora et al., 2019; Reed and Milton, 2001). Therefore, the International Commission on Occupational Health (ICOH) proposed that the concentration of endotoxin in the air should be lower than 10 ng/m3 (about 100EU/m3) in order to avoid respiratory

inflammation (Rylander, 1997). Unfortunately, endotoxin has a wide distribution and there can be considerable amounts in reclaimed water. The median concentrations of endotoxin in secondary effluent and tertiary effluent were 1440 endotoxin units per mL (EU/mL) and 422EU/mL (Huang et al., 2013). The removal efficiency of endotoxin is different with various treatment techniques. The coagulation and precipitation filtration process can reach about 80% removal (Rapala et al., 2006). The biological activated carbon method cannot remove endotoxin and even causes an increase of endotoxin concentration (Rapala, 2002). The membrane bioreactor (MBR) system can remove a significant amount (about 80%) of endotoxin (Mokhtar, 2012). In addition, disinfection with chlorine and ozone cannot remove the endotoxin activity, but can significantly reduce its toxicity (Ren et al., 2019; Zhang et al., 2016). Advanced oxidation can effectively remove both the activity and toxicity of endotoxin (Xue et al., 2019). Thus, the endotoxin levels of reclaimed water vary dramatically, depending on the treatment processes. A recent study proposed that free endotoxin is the critical risk factor of reclaimed water during acute inhalation exposure through fractionating the reclaimed water by particle size, comparing the dose-effect relationship between reference LPS with water samples, and specifically removing the free endotoxin in the water (Xue et al., 2016). All the efforts of the study focused on the sample preparation. However, it still cannot exclude the possibility of inflammation induction by other pollutants through different mechanisms, since polymyxin B could not completely remove the endotoxin and toxicity from the water and it might non-specifically absorb something else. The canonical mechanism of the endotoxin-induced innate immune response is through the toll-like receptor 4 (TLR4) signaling pathway (Khan et al., 2018). The LPS molecule first binds to LPS binding protein (LBP), which is produced mainly by the liver. The LPS-LBP forms a


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larger complex with cluster of differentiation 14 (CD14), and then activates the host cell receptor, TLR4/myeloid differentiation factor 2 (MD2) complex (Kim and Kim, 2017). After the ligand binding, the intracellular part of TLR4 activates the myeloid differentiation primary response protein 88 (MyD88) dependent pathway and/or TIR-domaincontaining adapter-inducing interferon-β (TRIF) dependent pathway, which transduce the signal to the nucleus and induce the expression of pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 (IL-1), IL-5 and IL-6 etc.) (Arora et al., 2019). This signaling pathway mainly presents on the cells of innate immunity (monocytes, macrophages, dendritic cells, neutrophils etc.) and non-immune cells like fibroblasts, endothelial cells, epithelial cells (Arora et al., 2019). Therefore, TLR4 plays a central role in endotoxin-induced immune responses (Miller et al., 2005; Raetz and Whitfield, 2002). However, in addition to endotoxins, diacyl and triacyl lipopeptides, flagellin, dsRNA, ssRNA, CpG DNA, et al. can all induce inflammation through different toll-like receptors (TLRs) as shown in Fig. 1 (Akira and Takeda, 2004). And the reclaimed water contains hundreds of pollutants, most of which are not identified. It is unknown whether there are more potential inflammation inducers in reclaimed water. More evidence can be derived from the genetically modified host. In this work, the possibility of non-TLR4 pathway dependent inflammation induction by reclaimed water was tested by TLR4 signaling pathway deficient mice by hypothesizing that these mice have no or weaker response to the endotoxin-containing reclaimed water, which further supports the view that endotoxin is the dominant toxicity inducer from the host perspective.

Table 1 The primers used for genotyping*. Mouse


Length (bp)

Tlr4 knockout

Mutant F: 5′-gcaagtttctatatgcattctc-3′ Mutant R: 5′-cctccatttccaataggtag-3′ Wildtype F: 5′-atatgcatgatcaacaccacag-3′ Wildtype R: 5′-tttccattgctgccctatag-3′ Mutant F 5′-gttggctacccgtgatattgctga-3′ Wildtype F: 5′-tggcatgcctccatcatagttaacc-3′ Common R: 5′-gtcagaaacaaccaccaccatgc-3′ F: 5′-tgaccacccatattgcctatac-3′ R: 5′-tgatggtgtgagcaggagag-3′ Mutant F: 5′-cccagaaatgccagattacg-3′ Wildtype F: 5′-ttacagtcggccaggctgac-3′ Common R: 5′-cttgggctgccagaatttctc-3′

Tlr4-/-: 140 Wildtype: 390

Myd88 knockout

Tlr4flox LysMcre

Myd88-/-: 700 Wildtype: 550 Tlr4fl/fl: 285 Wildtype: 234 LysMcre: 700 Wildtype: 350

* The primers were from the genotyping protocol of the Jackson Laboratory.

electrophoresis on a 2% agarose gel. All the genotypes were identified as shown in Figure S1. 2.3. Water samples Secondary effluent, denitrification biofilter (DNBF) and membrane bioreactor (MBR) effluent samples were obtained from three sewage treatment plants in Beijing, China (Table 2). The secondary effluent was from a conventional activated sludge treatment process. The carbon source of DNBF was sodium acetate. Meanwhile, ultrapure water (MilliQ, C9185) was used as exposure water for the control group. The reclaimed water samples were centrifuged at 1,500 rpm for ten minutes to remove large particles, which might clog the nebulizer. The water quality was analyzed immediately after centrifugation. The heterotrophic bacteria were counted after being cultured in a nutrient agar (peptone 10 g/L, beef extract 3 g/L, NaCl 5 g/L, and agar 15 g/L, catalogue number HB0109, Qingdao HopeBio Technology Co., Ltd. Qingdao, Shandong, China) plate for 48 h at 37 °C. The dissolved organic carbon (DOC) of water samples and stroke-physiological saline solution (SPSS) were measured using the total organic carbon analyser (Shimadzu, Japan). The total nitrogen (TN) of samples and SPSS were detected by the total nitrogen analyser (Shimadzu, Japan). In addition, the ammonia nitrogen (NH3-N) levels of the water samples were tested though Nessler's reagent colorimetric method (ammonia nitrogen detection agent, catalogue number LH–N2N3–100, Beijing Gaoxin Lianhua Technology Co., Ltd. Beijing, China).

2. Experimental 2.1. Animals The Tlr4 knockout mouse (The Jackson Laboratory, JAX, stock number 007227), Myd88 knockout mouse (JAX stock number 009088), Tlr4fl mouse (JAX stock number 024872) and LysMcre mouse (JAX stock number 018956) were from JAX (Hou et al., 2008; Liu et al., 2017; Poltorak et al., 1998; Vogel et al., 1979). C57BL/6J mice were purchased from the Vital River (Beijing, China). Male mice aged 6–8 weeks were used in this study. Body weight information of the mice is shown in the supplement (Table ST1−5). The animals were bred in the Laboratory Animal Research Centre of Tsinghua University (TH–LARC). TH–LARC has been issued by Beijing municipal science and technology commission with an experimental animal use license (SYXK (Beijing), 2019−0037) and an experimental animal production license (SCXK (Beijing), 2019−0012). The Public Health Service (PHS) approved animal welfare assurance number is F16−00228 (A5916−01) from the Office of Laboratory Animal Welfare (OLAW). In addition, Tsinghua University has passed the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) international certification. The experimental protocol of this study was approved by the institutional Animal Care and Use Committee (IACUC) of Tsinghua University (17–LY1). All efforts were made to minimize the suffering of the test animals.

2.4. Standard endotoxin and endotoxin activity assay Lipopolysaccharides from Escherichia coli O55:B5 (L2637, SigmaAldrich) were used to prepare standard LPS solutions. The endotoxin powder was dissolved with ultrapure water (Milli-Q, C9185) and the endotoxin activity was detected by a limulus amebocyte lysate (LAL) test kit (catalogue number EC64405S, Xiamen Bioendo Technology Co., Ltd. Xiamen, Fujian, China). 250EU/mL, 500EU/mL, 750EU/mL, 1,000EU/mL and 1,250EU/mL LPS were exposed to wildtype mice, 1,000EU/mL, 1,250EU/mL, 1,750EU/mL and 2,500EU/mL LPS were exposed to genetically defective mice.

2.2. Genotyping 2.5. Indigenous bacterial culture from reclaimed water The genomic DNA of every mouse was isolated from the tail with hot sodium hydroxide and tris (Truett et al., 2000). The components of the polymerase chain reaction (PCR) reaction included DreamTaq Green PCR Master Mix (2X, Thermo Scientific) 10 μL, forward primer 1 μM, reverse primer 1 μM (see Table 1 for the sequences), genomic DNA 1 μL, and ultrapure water to 20 μL. The reaction used the touchdown method: 94℃ 2 min; 94℃ 20 s, 65℃ 15 s (subtract 0.5℃ per cycle decrease), 68℃ 10 s, repeat for 10 cycles; 94℃ 15 s, 60℃ 15 s, 72℃ 10 s, repeat for 28 cycles; 72℃ 2 min. The PCR products were separated by

Lysogeny broth (LB, tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g for 1 L) medium was sterilized by high pressure steam (121℃, 30 min). One millilitre of water mixture from five water samples (SE-1 0.2 mL, SE-2 0.2 mL, DNBFs 0.2 mL, MBR-1 0.2 mL and MBR-2 0.2 mL) was inoculated to LB medium and cultivated overnight at 37℃. The culture was centrifuged at 3,500 g for five minutes to remove the medium, and washed with an equal volume of SPSS five times to remove the free endotoxin. The endotoxin level of final wash buffer was less than 1EU/ 3

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Table 2 Reclaimed water and quality*. Reclaimed water

treatment process

treatment plant

Endotoxin (EU/mL)

DOC (mg/L)

TN (mg/L)

NH3-N (mg/L)

HPC (× 103 CFU/mL)




2202 ± 33 2313 ± 49 2663 ± 149 26.3 ± 0.10 32.8 ± 0.60 1075 ± 56 2278 ± 97

11.33 ± 0.36 13.51 ± 1.29 16.73 ± 1.50 5.92 ± 0.16 8.79 ± 0.24 8.99 ± 0.71 9.90 ± 1.26

17.12 ± 0.15 19.25 ± 0.29 10.27 ± 0.04 24.05 ± 0.61 8.74 ± 0.10 18.37 ± 1.20 19.82 ± 0.83

4.53 4.42 4.75 0.60 0.27 4.11 4.46

5.9 4.8 4.2 0.8 0.8 5.1 6.5

± ± ± ± ± ± ±

0.07 0.30 0.01 0.01 0.05 0.61 0.31

± ± ± ± ± ± ±

0.2 0.9 0.2 0.1 0.1 0.7 0.2

* The water quality was tested three times for each sample.

the BALF were detected using ELISA kits made by R&D Systems (Minneapolis, MN, USA) following the manufacturer’s instructions.

mL. The total bacteria number was calculated with 4′,6-diamidino-2phenylindole (DAPI) staining on polycarbonate black filtration membranes (Whatman, 110656), and the cultured bacteria were then diluted into different concentrations (4.0☓105, 4.0☓106, 4.0☓107 and 4.0☓108cells/mL) with SPSS immediately before the exposure (Xue et al., 2016).

2.9. Polymorphonuclear (PMN) cell percentage counting PMN plays a central role in the endotoxin induced innate immune response by destroying foreign particles either intracellularly in phagosomes or extracellularly by releasing neutrophil extracellular traps, and promoting acute lung inflammation (Selders et al., 2017). The percentage of PMN is a sensitive biomarker of inflammatory intensity in pulmonary endotoxin toxicity (Michel et al., 1997). The cells from the BALF were dried on slides at room temperature and fixed with methanol for 15 min. The cells were stained with Wright stain solution (catalogue G1040, Solarbio, Beijing, China) for six minutes and Giemsa stain solution (catalogue G1015, Solarbio, Beijing, China) for ten minutes. A deionized water wash was performed between and after the two stainings. The cells were cleared in xylene for five minutes. The slide was then sealed with neutral balsam (catalogue G8590, Solarbio, Beijing, China), and dried at room temperature. The white blood cells, including PMN, were identified under a light microscope (Olympus BX43, Tokyo, Japan), and counted in randomly selected fields. All of the observed white cells were counted without overlapping until the total cell number was more than 300 in each slide. The percentage of PMN = PMN number / total white blood cells.

2.6. Inhalation exposure The exposure was performed in inhalation exposure devices which were made according to the characteristics of mouse inhalation exposure by our research team. The volume of the exposure cabinet is about 35 L, and the bottom area is 15 dm2. Each cabinet was used for the exposure of five mice (one experimental group). Carrier gas was filtered and dried air with a flow rate of 8 L/min. A compressor nebulizer (Strong health, MCN-S600MD) was used to treat the water samples. The relative humidity increase in the cabinet was about 50% in the experiment. The mice were exposed for three hours at 25℃. The exposure dose was calculated based on the following equations (Xue et al., 2016). IWQ=C × V×DI×T


where IWQ is the inhaled water quantity (mg/kg), C is the water aerosol concentration (mg/L), V is the volume of air that animal inspired in 1 min (L/(min∙kg)), DI is the deposition index (for mice, DI = 0.3 (Patlolla et al., 2010)), and T is the duration of exposure (180 min in this study). ED=IWQ×EC / 1000

2.10. Statistical methods The data were analyzed using the Student's t-test. Statistical significance was determined at P < 0.05. All data were shown as means ± standard deviation (SD). The benchmark dose (BMD) approach is the US EPA’s preferred dose-response assessment method. It can be used to derive the lower 5% confidence limit of benchmark dose (BMDL) for human health guidance values such as reference dose (RfD) or derived no-effect level (DNEL) or acceptable daily intake (ADI). The free software BMDS (version 2.4, U.S. Environmental Protection Agency) was used, and the calculation of threshold endotoxin concentration from BMDL used Eqs. (1) and (2).


Where ED is the endotoxin dose (EU/kg), EC is the endotoxin concentration in water (EU/mL). 2.7. Bronchioalveolar lavage fluid (BALF) BALF analysis is a powerful method for examining inflammatory responses, like cytokine secretion and neutrophil infiltration (Reynolds, 1987). The mice were euthanized by cervical dislocation at certain time points after exposure. Part of the blood was immediately taken out by cardiac puncture to reduce the blood leakage in BALF. Then bronchioalveolar lavage (BAL) was performed gently with 2 mL PBS/BSA buffer (NaCl 136.89 mM, KCl 2.67 mM, Na2HPO4 8.1 mM, KH2PO4 1.76 mM and 0.5% bovine serum albumin, pH 7.4). Then the BALF was centrifuged at 800 g for five minutes at 4℃. The supernatant of BALF was stored at -80℃ for cytokine detections, and the cells were carefully coated onto adhesive microscope slides (Citotest Scientific Co., Ltd, Haimen, Jiangsu, China). The BALF quality was tested as in the previous description (Xue et al., 2016).

3. Results 3.1. The quality of water samples The DOC of secondary effluents and DNBF were between 11.3 mg/L and 16.7 mg/L (Table 2). The DOC content of the biofilter was slightly higher than secondary effluents. Two MBR effluents had relatively lower DOC than secondary effluents and DNBF. The total nitrogen in DNBF was significantly lower than secondary effluent compared with SE-2 (which was the inflow water of DNBF), which indicated that the biofilter worked well in denitrifying. However, the MBR-1 water sample had the highest total nitrogen of all water samples. That may be caused by the simple AO pre-treatment process. The ammonia nitrogen concentrations in secondary effluents and biofilter were lower than 5 mg/L, and MBR effluent was under 1 mg/L (Table 2).

2.8. Cytokine detection Interleukin 1β (IL-1β, catalogue MLB00C), interleukin 6 (IL-6, catalogue M6000B), tumour necrosis factor α (TNF-α, catalogue MTA00B) and transforming growth factor beta 1 (TGF-β1, catalogue MB100B) in 4

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Fig. 2. Time curve of PMN percentage and cytokines in C57BL/6J mice BALF. The asterisks mean that the values are significantly higher than that of the nonexposure group (p < 0.05). (a, b, c, d) The percentage of PMN and levels of IL-1β, IL-6, TNF-α in BALF respectively within 24 h of exposure.


In addition, the total heterotrophic plate count (HPC) of secondary effluents and biofilter effluent was between 4,800 and 6,000CFU/mL. The HPCs of two MBR effluents were below 1,000CFU/mL. The endotoxin activities of secondary effluent samples reached more than 2,200EU/mL. And the endotoxin level increased by about 15% (compared with SE-2) after DNBF treatment. The endotoxin levels of MBR effluents were much lower than those of the secondary effluents and biofilter (Table 2).

3.3. The dose-effect relationship of reference LPS exposure in wildtype and mutants To test the reliability of gene knockout mice, three strains of mice, wildtype (C57BL/6J), Tlr4−/− and MyD88−/−, were exposed to reference LPS. The PMN% of wildtype had a nice “S” shaped dose-effect curve, and its BMDL level was calculated as 394EU/kg (Fig. 3a). The cytokines, IL-1β, IL-6, TNF-α, all had similar dose-effect relationship as PMN% (Fig. 3b-d). TNF-α was the most sensitive cytokine, which showed significant increase at the lowest dose. It is necessary to mention that the levels of cytokines should be higher at one hour after exposure according to the results of Fig. 2. To minimize the use of animals, for Tlr4 knockout and Myd88 knockout mice, only two concentrations of reference LPS, 1,000 and 2,500EU/mL (dose group 621 and 1,552EU/kg), were tested. The concentration of 1,250EU/mL (largest dose 776EU/kg) induced a nearly saturated response in wildtype mice, and the reported medium endotoxin level of reclaimed water is 422EU/mL (Huang et al., 2013). Therefore, 2,500EU/mL was considered high enough for the test. Neither of the knockout mice had an inflammatory response to the reference LPS, which demonstrated that the TLR4 signaling pathway was completely deficient and the LPS did not induce detectable immune response through other possible pathways at the tested dose range. The lung deposition efficiency in the dose calculation was based on a literature value (Patlolla et al., 2010), which is not specific for endotoxin. However, deposition efficiency will only

3.2. Immune response dynamics of acute exposure in wildtype Before exposure, the dynamics of inflammatory responses were measured, since the various gene backgrounds of different mice might cause an earlier or delayed immune response between C57BL/6J and ICR. One thousand EU/mL standard endotoxin was used to expose the C57BL/6J mice. The percentage of PMN in BALF rose up to the highest level three hours after exposure, and quickly returned to the background level within nine hours (Fig. 2a). However, IL-1β, IL-6 and TNFα all reached the peak levels at one hour after exposure (Fig. 2b-d). Then IL-6 and TNF-α dropped close to the control level immediately, while IL-1β stayed significantly high for 24 h. TGF-β1 was not detected in all the BALFs (data not shown), which might be due to the much lower LPS dose (62 ng/kg) compared with other studies (100 μg/kg to 15 mg/kg) (Luo et al., 2020; Yang et al., 2018; Dong and Yuan, 2018; Zhao et al., 2017). Here 10EU LPS was assumed to equal 1 ng (Anderson et al., 2007). Since the key testing endpoint (PMN%) peaked at three hours, this detection time point was chosen for the following 5

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Fig. 3. Inflammatory responses induced by the reference LPS in wildtype (C57BL/6J), Tlr4−/− and MyD88−/− mice. The 0, 155, 310, 465, and 776EU/kg of reference LPS were prepared for exposure of wildtype, and 621 and 1,552EU/kg of reference LPS were prepared for the knockout mice. The asterisks mean the values are significantly higher than that of the unexposed group (p < 0.05). (a, b, c, d) The percentage of PMN and levels of IL-1β, IL-6, TNF-α in BALF respectively three hours after exposure.

in this study, and free endotoxin was washed away before exposure. The PMN and IL-1β did not respond at all. Only the expression of IL-6 and TNF-α in BALF increased at 4.0☓108cells/mL (Fig. 5). In reality, the highest bacterial concentration of recycled water is around 107cells/mL (Garner et al., 2018; Xue et al., 2018,).

affect the dose calculation, but not the conclusion. 3.4. Effects of reclaimed water exposure in wildtype and knockout mutants To demonstrate that the TLR4 signaling pathway is crucial to reclaimed water induced inflammatory responses, five reclaimed water samples (Table 2) were used for the inhalation exposure to wildtype, Tlr4−/− and MyD88−/− mice. The proportion of PMN cells in wildtype mice BALF was higher than 85% when exposed with secondary and DNBF effluents. However, the PMN percentage in wildtypes exposed to MBR effluents was close to zero (Fig. 4a), which indicates that the inflammatory response was not induced due to the low endotoxin levels. From the acute BMDL value above (394EU/kg), the sample endotoxin threshold to induce PMN-dominant response is 634EU/mL, which is much higher than that of the MBR effluents. Additionally, Tlr4−/− and Myd88−/− mice showed no significant response in PMN percentage regardless of the endotoxin levels of the samples (Fig. 4a). Similar results were observed in the secreted cytokines, IL-1β, IL-6, and TNF-α (Fig. 4b-d), which were consistent with the PMN response. The reclaimed water samples contained both bacterial cell-bound endotoxin and free endotoxin, and the bacteria were also an important health risk. More than 98% of bacteria in secondary effluent were found to be gram-negative (Xue et al., 2016). The previous study used isolated strains to represent the general gram-negative population. But different bacteria have different activities. Therefore, to overcome the weakness, mixed indigenous bacterial culture from reclaimed water was prepared

3.5. LPS exposure of myeloid cells Tlr4 conditional knockout mice Macrophages (the major myeloid cells in healthy lungs) play the role of sentinel to recognise LPS, and induce a series of biological responses required for shaping both the innate and adaptive immunological activities (Janeway and Medzhitov, 2002). However, the Tlr4 gene is expressed not only in immune cells (mainly macrophages in healthy lungs) but also in the epithelia of the lung (Akira et al., 2001). To identify the role of immune cells in the reclaimed water induced inflammatory response, Tlr4 was conditionally knocked out in the myeloid cells of Tlr4fl/flLysMcre mice. First, the reference LPS was tested, and the PMN response was significantly attenuated in the conditional knockout mice. The PMN percentage did not rise at a 776EU/ kg LPS dose. At the same dose, the PMN percentage already reached the top level in the wildtype. And the percentage of PMN in Tlr4fl/flLysMcre mice started to increase at 1,086EU/kg, and was close to saturation at 1,552EU/kg (Fig. 6a). The increases of IL-1β, IL-6 and TNF-α were significantly lower even though the dose was twice the highest exposure of the wildtype (Fig. 6b-d). These results fit the hypothesis that macrophage is the main responder to LPS. 6

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Fig. 4. PMN percentage and cytokine secretion in BALF after exposure to reclaimed water. Secondary effluent (SE), effluent from biofilter (DNBF) and membrane bioreactor (MBR) were exposed to wildtype (C57BL/6J), Tlr4 knockout (Tlr4−/−), and Myd88 knockout (Myd88−/−) mice. The endotoxin activities of all samples are listed in (a). Asterisks in wildtype groups mean that their values are significantly higher than those of the Tlr4−/− and Myd88−/− mice (p < 0.05). (a) The percentage of PMN in BALF. (b-d) The concentrations of IL-1β, IL-6 and TNF-α in BALF.

Then two secondary effluences with different endotoxin concentrations (Table 2), SE-M1 and SE-M2, were exposed to wildtype and Tlr4fl/flLysMcre mice. After relatively low-dose exposure (SE-M1), all four inflammatory response indicators rose up in the wildtype, but there was no change in the Tlr4fl/flLysMcre mice (Fig. 7). In the highdose group (SE-M2), the PMN percentage was similar in both the wildtype and mutants (Fig. 7a). However, the conditional knockout mice showed significantly lower cytokine levels than the wildtype (Fig. 7b-d), which was similar to the results from reference LPS (Fig. 6). 4. Discussion This study attempted to demonstrate that acute inflammation induced by reclaimed water is TLR4 signaling pathway dependent using genetically modified mice. Seven types of reclaimed water with different levels of endotoxin were used. Secondary effluent (except SE-M1, 1,075EU/mL) and DNBFs had higher endotoxin levels than 2,200EU/ mL. While the concentrations of MBR effluents were lower than 35EU/ mL (Table 2), these levels were similar to a previous report (Huang et al., 2013). Various treatment processes have differing endotoxin removal ability. The MBR treatment system was effective at endotoxin removal, because the membrane usually has a high retention rate to endotoxin (Huang et al., 2011; Mokhtar, 2012; Rapala, 2002). The pore sizes of most commercial membranes used in MBR are tens of nanometres (Jeon et al., 2016), which are comparable with endotoxin aggregates. The DNBF was effective at removing total nitrogen, but it

Fig. 5. The response of C57BL/6J to reclaimed water bacterial exposure. C57BL/6J mice were exposed with 4.0☓105, 4.0☓106, 4.0☓107 and 4.0☓108cells/mL cultured indigenous bacteria from reclaimed water. The corresponding dose was calculated as described in a previous study (Xue et al., 2016). The saline was set as the control. The endotoxin concentrations of the five samples were 0.11EU/mL, 0.11EU/mL, 2.81EU/mL, 42.82EU/mL and 431.87EU/mL from low to high. The asterisks mean that the values are significantly higher than that of the control group (p < 0.05).


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Fig. 6. The response of myeloid cell-Tlr4 conditional knockout mice to LPS exposure. a, b, c, d denote the PMN% and the levels of IL-1β, IL-6, TNF-α in lavage respectively. The 0, 621, 776, 1,086 and 1,552EU/kg of reference LPS were used for exposed to myeloid cells Tlr4 knockout (Tlr4fl/flLysMcre) mice. An asterisk means the value is significantly higher than that of the control group (p < 0.05).

inducers in the reclaimed water, or they had too low concentrations to induce significant inflammation, such as lipoprotein, flagellin, viral RNA et al. And the endotoxins in the reclaimed water induced inflammation mainly through TLR4. The negative response in MyD88−/− mice suggests that reclaimed water induced inflammation is MyD88 dependent, and the alternative TRIF pathway was not significantly activated. For the extremely weak responses from bacteria exposure (Fig. 5), one consideration is that bacteria and free endotoxin may have different deposition efficiencies in the lung. The deposition of aerosols depends on the size and surface properties (Darquenne, 2012). Since the surface of gram-negative bacteria is covered by endotoxins, the only difference between free endotoxin aerosols and bacterial aerosols is the size. Free endotoxin is about tens of nanometers in water, and the diameter of bacteria is about 500 nm. Therefore, both of them can reach the alveoli (Xing et al., 2016), but bacteria might have more deposition in the upper airways. This could be one reason that bacteria did not induce significant inflammation. Another consideration is that the response dynamics of cell-bound endotoxin might be different from free endotoxin. In other words, bacteria might induce a PMN peak at another time point. However, this assumption does not fit the observation that the whole secondary effluent induced the top PMN level at three hours and induced the cytokines peak before three hours within an eighteen-hour period (Xue et al., 2016). Furthermore, it was reported that mice exposed to gram negative bacteria demonstrated significantly greater PMN infiltrations two to four hours after exposure (Pierce et al., 1977; Xue et al., 2016). Seventy-seven percent of Proteus mirabilis (gram

caused a 15% increase in endotoxin. The increased endotoxin was likely derived from the biofilm in the DNBF. The mouse strain used in this study was C57BL/6J (B6), because the two knockout strains, Tlr4−/− and MyD88−/−, were all bred on the B6 background. This strain had a different cytokine response than ICR mice. IL-1β was detected in exposed B6 (Fig. 2–4,6,7), but not in ICR (Xue et al., 2016). Additionally, the dose-effect relationship curve of ICR was relatively flat compared with C57BL/6J. The PMN percentage was up to 19.8% in C57BL/6 J mice at 465EU/kg LPS (Sigma, L2637), while the PMN percentage in ICR mice was about 30% at 458EU/kg (Sigma, L2637) (Xue et al., 2016). Therefore, there are differences in BMDL between these two types of mice. Mammals have about ten functional TLRs, which can be triggered by pathogen associated molecular patterns (PAMPs) and lead to TLRassociated signaling events involved in host defence (Arora et al., 2019). Different TLRs recognise different microbial products such as bacterial DNA, LPS, peptidoglycan, teichoic acids, flagellin, and viral dsRNA (Fig. 1). For example, TLR2 is essential in the recognition of microbial lipopeptides, TLR4 is the receptor for LPS, and flagellin is recognized by TLR5. MyD88 is the canonical adaptor for inflammatory signaling pathways downstream of many TLR receptor family members. TLR2/1, TLR2/6, TLR4, TLR5, TLR7, TLR8, and TLR9 recruit MyD88 in signaling transduction. TRIF is an adapter in responding to the activation of TLR3 and TLR4 (Hou et al., 2008). So, the downstream of TLR4 has two pathways, MyD88-dependent and TRIF-dependent (Arora et al., 2019). Therefore, the negative response in Tlr4−/− mice indicates that there were no other signaling pathway dependent acute inflammation 8

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Fig. 7. The response of myeloid cell-Tlr4 conditional knockout mice to reclaimed water exposure. SE-M1 and SE-M2 denotes two secondary effluents. The exposure doses of the two water samples were calculated as 668EU/kg and 1,415EU/kg respectively. An asterisk means the value of wildtype is significantly higher than that of the Tlr4fl/flLysMcre group (p < 0.05).

Alenghat, 2014). All the data in this work points to the conclusion that the TLR4 signaling pathway is extremely important to the inflammation induced by the reclaimed water, and the response pattern is similar to that of endotoxin. However, many other pollutants besides endotoxin can also induce inflammation through TLR4, such as mannan of fungi, structure proteins of some viruses, and taxol of plants (Akira and Takeda, 2004; Arora et al., 2019). Therefore, the above results do not necessarily determine the critical role of endotoxin. Fortunately, two important pieces of evidence have been provided by the previous work mentioned in the introduction (Xue et al., 2016). One is that the endotoxin level in the reclaimed water had a good positive correlation with the inflammatory responses. The other is that specific reduction of free endotoxin by polymyxin B column could decrease the inflammation inducing ability of the water simultaneously. Combining these pieces of evidence, endotoxin is probably the major inflammation inducer in the reclaimed water after acute exposure.

negative) and 91% Staphylococcus aureus (gram positive) could be eliminated by lung phagocytes four hours after inhalation exposure, mainly through phagocytosis (Green and Kass, 1964). The half time for phagocytosis and killing of Escherichia coli by neutrophils (PMN in the lung) were ten and two minutes, respectively, in vitro (Hampton et al., 1994). Phagocytosis is one important mechanism for controlling inflammation intensity (Silva, 2011). Combining the results in this study, the contribution of bacteria in reclaimed water is unlikely to be significant in acute inhalation exposure. The exposure of myeloid-Tlr4 conditional knockout showed significantly weaker inflammatory responses (Fig. 6,7). First, this suggests that macrophages play a leading role in the immune responses after reclaimed water exposure. TLR4 is highly expressed in alveolar macrophages, and macrophages are the main myeloid cells in the lung before the PMN infiltration (Arora et al., 2019). Second, other cells can also respond to the endotoxin and high endotoxin-containing reclaimed water, but are relatively insensitive. Lung airway epithelial cells also have high levels of TLR4 (Arora et al., 2019), which are the potential responders. It has been demonstrated that the pulmonary airway epithelial cells can secrete proinflammatory factors under LPS exposure (Elizur et al., 2007). However, the responses were much weaker in the conditional knockout mice (Fig. 6,7). This is because the epithelial cells express little MD2, which is necessary for optimization of TLR4 signaling transduction (Jia et al., 2004). The hyporesponsiveness of epithelial cells might serve as a means to modulate LPS responses, and MD2 expression can be upregulated after exposure in these cells to be a secondary defence line (Moldoveanu et al., 2009; Whitsett and

5. Conclusions The host TLR4-MyD88 signaling pathway is mainly responsible for acute lung inflammation induced by reclaimed water. The bacteria in reclaimed water alone could not induce a significant level of inflammation. Myeloid cells (mainly macrophages) are the main participants during the reclaimed water exposure. All these results support the hypothesis that free endotoxin is the major risk factor in reclaimed water after acute inhalation exposure. 9

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CRediT authorship contribution statement

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Gang Liu: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Visualization, Project administration. Yun Lu: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Funding acquisition. Liangliang Shi: Validation. Yunru Ren: Validation. Jiayang Kong: Validation. Mengyu Zhang: Visualization. Menghao Chen: Validation. Wanli Liu: Conceptualization, Methodology, Resources. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Fund (No. 21777084 and 51738005) and the National Key R&D Program of China for International Science & Innovation Cooperation Major Project between Governments (2016YFE0118800). We thank Dr. Feng Shao in the National Institute of Biological Sciences, Beijing (NIBS, Beijing) for sharing the Tlr4 knockout mice, and Dr. Jianwei Wang in School of Pharmaceutical Sciences, Tsinghua University for sharing the Tlr4fl mice. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi: References Akira, S., Takeda, K., 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511. Akira, S., Takeda, K., Kaisho, T., 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680. Alexander, C., Rietschel, E.T., 2016. Invited review: bacterial lipopolysaccharides and innate immunity. J. Endotoxin Res. 7, 167–202. 09680519010070030101. Anderson, J., 2003. The environmental benefits of water recycling and reuse. Water Sci. Technol: Water Supply. 3, 1–10. Anderson, W.B., George Dixon, D., Mayfield, C.I., 2007. Estimation of endotoxin inhalation from shower and humidifier exposure reveals potential risk to human health. J.Water Health 5, 553–572. Annadotter, H., Cronberg, G., Nystrand, R., Rylander, R., 2005. Endotoxins from cyanobacteria and gram-negative bacteria as the cause of an acute influenza-like reaction after inhalation of aerosols. EcoHealth 2, 209–221. Arora, S., Ahmad, S., Irshad, R., Goyal, Y., Rafat, S., Siddiqui, N., Dev, K., Husain, M., Ali, S., Mohan, A., Syed, M.A., 2019. TLRs in pulmonary diseases. Life Sci. 233, 116671. Darquenne, C., 2012. Aerosol deposition in health and disease. J. Aerosol Med. Pulm. Drug Deliv. 25, 140–147. Dong, Z.W., Yuan, Y.F., 2018. Juglanin suppresses fibrosis and inflammation response caused by LPS in acute lung injury. Int. J. Mol. Med. 41, 3353–3365. 10.3892/ijmm.2018.3554. Elizur, A., Adair-Kirk, T.L., Kelley, D.G., Griffin, G.L., deMello, D.E., Senior, R.M., 2007. Clara cells impact the pulmonary innate immune response to LPS. Am. J. Physiol. Lung Cell. Mol. Physiol. 293, 383–392. 2007. Garner, E., McLain, J., Bowers, J., Engelthaler, D.M., Edwards, M.A., Pruden, A., 2018. Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale reclaimed water distribution systems. Enviro. Sci. Technol. 52, 9056–9068. Gay, N.J., Symmons, M.F., Gangloff, M., Bryant, C.E., 2014. Assembly and localization of Toll-like receptor signalling complexes. Nat. Rev. Immunol. 14, 546–558. https://doi. org/10.1038/nri3713. Green, G.M., Kass, E.H., 1964. The role of the alveolar macrophage in the clearance of bacteria from the lung. J. Exp. Med. 119, 167–176. 119.1.167. Hampton, M.B., Vissers, M.C., Winterbourn, C.C., 1994. A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J. Leukoc. Biol. 55,


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