Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas aeruginosa

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. population. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.


Introduction
According to the World Health Organization (WHO) and the Agency for Toxic Substances and Disease Registry (ATSDR) arsenic is the number one contaminant of concern for human health worldwide [1]. Hundreds of millions of people worldwide are exposed to arsenic via their drinking water, many at doses higher than the WHO maximum contaminant level of acid (MMA III ) and dimethylarsenic acid (DMA V ), at concentrations relevant to blood levels measured in the U.S. population. Exposure of bronchial epithelial cells to ingested arsenic occurs via the blood in vivo. Primary HBEC from several individuals were exposed to arsenic concentrations relevant to blood levels in cell culture media.

Chemicals and Bacterial Strains
DMA V and iAs III were purchased from Sigma (St. Louis, MO). MMA III was synthesized at the Synthetic Chemistry Facility Core at University of Arizona according to previously published methods [28,29]. Fresh concentrated stocks of iAs III , DMA V and MMA III (10 ppm) were made in distilled, ultrapure water. Concentrated stocks were diluted to working stock solutions (1 ppm) in cell culture media with fresh dilution stock for each experiment. To minimize degradation of MMA III , stocks were maintained at -20°C and fresh dilutions were used for each experiment per standard protocols from the synthetic chemistry facility core at University of Arizona [29]. Purified CXCL2 was purchased from R&D Systems (Minneapolis, MN) and diluted in distilled, ultrapure water. P. aeruginosa (PAO1) was grown in rich medium (Luria broth, LB, Invitrogen Grand Island, NY) at 37°C. Overnight cultures were washed three times and then added to the HBEC cells at a multiplicity of infection (MOI) of 25 as previously described [30].

Cells
Primary cultures of human bronchial epithelial cells (HBEC) were purchased from Lonza (Hopkinton, MD). Cells from four individual donors were used for these studies. All donors were Caucasian males between the ages of 32-40 years old. HBEC cells were passaged a maximum of two times. All experiments were repeated with each individual donor a minimum of twice, each with a different passage. Control and P. aeruginosa exposure were repeated with each cell passages along with arsenic exposure. HBEC were maintained at 37°C with 5% CO 2 in bronchial epithelial growth media (BEGM) supplemented with bovine pituitary extract, insulin, hydrocortisone, human epithelial growth factor, epinephrine, transferrin, retinoic acid, triiodothryonine and gentamycin from Lonza, according to the manufacturers instructions. Cells were plated in coated 6 well tissue culture plates at 5x10 5 cells per well. HBEC were exposed to either iAs III , MMA III or DMA V in cell culture media at concentrations from 0.5-10 ppb for 6 days, with media renewal every 2 days. Media contained either iAs III , MMA III or DMA V as appropriate at each change. Dose ranges were chosen based on blood levels measured in those with drinking water containing 90 ppb in Bangladesh and US blood levels [3,31]. In some experiments HBEC were exposed to a combination of iAs III (1.25 ppb), MMA III (1.25 ppb) and DMA V (2.5 ppb) or iAs III , (2.5 ppb), MMA III (2.5 ppb) and DMA V (5 ppb), to mimic blood levels of 5 ppb and 10 ppb total arsenic. These ratios reflect levels measured in blood [31]. After 6 days, cells were exposed to vehicle or P. aeruginosa at a MOI of 25 for 1 hour. After PAO1 exposure, HBEC were washed to remove P. aeruginosa using cell culture media containing 75 μg/mL gentamycin to kill any adherent P. aeruginosa as P. aeruginosa exposure for longer than 1h caused significant cell death [30]. After washing, HBEC were incubated without P. aeruginosa for 5 hours to allow cells to elaborate an immune response. Supernatant was collected to measure cytokine release and cells were lysed to isolate total RNA. All experiments were repeated a minimum of two times with each individual donor. In all donors examined the responses were similar in direction (i.e., increase, decrease or no change), but varied in the absolute changes in cytokine secretion.
To determine if the arsenic induced changes in cytokine secretion by HBEC were biologically relevant, THP-1 cells, a monocyte cell line, purchased from ATCC (Manassas, VA), were exposed to purified CXCL2 at concentrations secreted by HBEC or conditioned media from HBEC experiments. After exposure to CXCL2 or HBEC conditioned media, IL-1β production was measured by ELISA. THP-1 cells were grown in RPMI-1640 with 10% FBS and penicillin and streptomycin. Cells were plated at 1x10 6 cells per well in 6 well plates and differentiated to macrophages with PMA (20 ng/mL for 48h, Sigma St. Louis, MO) [32]. Cells were then exposed to 150, 500 or 1000 pg/mL purified CXCL2 (R&D Systems, Minneapolis, MN) in standard cell culture media for 24h. In conditioned media experiments, THP-1 cells were plated at 5x10 5 cells per well in 12 well plates and differentiated to macrophages with PMA as described above. THP-1 cells were then exposed to conditioned media from HBEC experiments diluted 1:3 in standard cell culture media for 24h to ensure that THP-1 cells remained healthy and cytokine response levels were in the linear range. Conditioned media from a minimum of 3 different HBEC donors exposed to 10 ppb MMA III , 10 ppb iAs III or 10 ppb total arsenic with and without P. aeruginosa were used in two replicate THP-1 wells per treatment.

Cytotoxicity
Lactate dehydrogenase (LDH) release by HBEC was used to assess cytotoxicity of all treatments and was measured using the Promega CytoTox 96 Non-Radioactive Cytotoxicity assay per manufacturers instructions (Madison, WI).

Measurement of intracellular arsenic
To determine if HBEC metabolize arsenic and to measure the intracellular concentration of iAs, MMA or DMA, HBEC were exposed to 10 ppb or 50 ppb of iAs III , MMA III or DMA V for either 2 hours (50 pbb) or 7 days (10 ppb), times that are adequate to metabolize arsenic [33]. Thereafter, HBEC were washed on ice with PBS five times. Cells were lysed with 0.1% Triton-X, and spun at 14,000xg for 20 minutes to pellet cells debris. Speciation analysis of arsenic was done by anion exchange chromatography coupled to ICP-MS and detected iAs III , iAs V , but only oxidized organic species MMA V and DMA V [34].

RNA Isolation
Total RNA was isolated from HBEC after exposure to MMA or a combination to all three species with and without exposure to P. aeruginosa as described above. Total RNA was isolated with the miReasy kit (Qiagen, Valencia, CA) according to manufacturers instructions. Briefly, cells were lysed in phenol and then chloroform was added for phase extraction. The aqueous phase was mixed with ethanol to precipitate RNA. RNA was cleaned up on a glass fiber filter, and washed three times prior to elution. RNA was eluted in nuclease free water and stored at -80°C until time of use. RNA integrity and concentration was assessed using micro-capillary electrophoresis on an Agilent 2100 Bioanalyzer. RNA was compared to a RNA ladder with 6 RNA transcripts of varying sizes and known concentration of 150 ng/mL. RNA quality was verified by observation of corresponding 18 S and 28 S peaks on the electropherogram. Only intact RNA was used for further analysis. qPCR For quantitative PCR (qPCR) cDNA was synthesized from1 μg of total RNA using Retroscript Reverse Transcriptase (Ambion, Austin, TX) with random decamers. TaqMan Gene Expression Assays for human IL8 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00174103), human IL-6 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00985639), human CXCL1 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00605382) and human CXCL2 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00601975) were purchased from Applied Biosystems (ABI, Foster City, CA). Amplicons were sequenced to verify products. Triplicate reactions containing 100ng cDNA from each sample were amplified with an initial denaturing at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Transcript abundance was calculated based on serial dilution of a standard curve. The standard curves showed a correlation coefficient close to 1 (R 2 > 0.95) and were linear over a 4-log range.

Statistics
Statistical significance was assessed by one-way ANOVA followed by Tukey's HSD post hoc test. All statistical analysis was done with Prism v5.0 (Graph Pad Software, San Diego, CA). All experiments were repeated a minimum of two times with different passages of each individual donor. Data are presented as the mean ± SEM.

HBEC Minimally Metabolize iAs III , MMA III or DMA V
In order to use HBEC to examine effects of individual species of arsenic, we first conducted studies to examine whether these cells metabolize arsenic. Measurements of intracellular arsenic by ICP-MS in HBEC exposed to 50 ppb iAs III , MMA III , or DMA V for two hours revealed that HBEC do not metabolize arsenic in this time frame (Table 1). Intracellular concentrations of arsenic varied slightly by donor, thus data in Table 1 are presented as the range of concentrations measured. Unexposed control cells showed very low levels of iAs V , which was present in all treatments except DMA V . In cells exposed to iAs III , both iAs III and iAs V were detected, as expected since these species readily interconvert in a pH dependent manner both extra-and intracellularly [13,35]. However, in cells exposed to MMA III only MMA V could be detected, and in cells exposed to DMA V , only DMA V could be detected (Table 1). These results agree with previous studies demonstrating that undifferentiated human bronchial epithelial cells minimally metabolize arsenic [36]. Additionally, HBEC were exposed to 10 ppb iAs III for seven days to more fully examine metabolism in longer exposure conditions. Unexposed HBEC and cells exposed to 10 ppb iAs III had iAs III and MMA V levels that were below detection limits (n = 7). Unexposed control HBEC had iAs V levels of 0.146 ± 0.08 ppb and DMA V levels of 0.034 ± 0.02 ppb (n = 7). HBEC exposed to iAs III had iAs V levels of 0.173 ± 0.09 ppb and DMA V levels of 0.054 ± 0.06 ppb (n = 7). Measured iAs V and DMA V were not significantly different between iAs III exposed and unexposed control cells. These longer exposures agree with the minimal metabolism of HBEC previously reported, and reveal that our model system is suitable for examining individual arsenic species [36].
iAs III , MMA III and DMA V are not cytotoxic Arsenic exposure at high levels can be cytotoxic but less is understood about low doses, in particular when combined with an additional stressor [35,37]. Although HBEC were exposed to very low levels (0.5 to 10 ppb) of iAs III , MMA III or DMA V , studies measuring LDH were conducted to determine if these arsenic species had cytotoxic effects alone or in combination with P. aeruginosa. As shown in Fig 1, neither iAs III , MMA III nor DMA V alone or in combination with P. aeruginosa were cytotoxic. P. aeruginosa alone had no effect on LDH release. This experiment eliminates cytotoxicity as a possible mechanism of action of arsenic and P. aeruginosa on cytokine secretion by HBEC. iAs III and DMA V have no effect on cytokine secretion by HBEC iAs III alone (0.5 to 10 ppb) had no effect on IL-6, IL-8, CXCL1 or CXCL2 secretion by HBEC, nor did iAs III alone affect P. aeruginosa stimulated cytokine secretion (Fig 2). Similarly, DMA V alone (0.5 to 10 ppb) did not significantly affect IL-6, IL-8, CXCL1 or CXCL2 secretion by HBEC (Fig 3).
MMA III increased P. aeruginosa stimulated cytokine secretion by HBEC MMA III alone (0.5 to 10 ppb) had no effect on IL-6, IL-8, CXCL1 or CXCL2 secretion (Fig 4). However, 5 ppb MMA III significantly increased P. aeruginosa stimulated IL-8 and IL-6 secretion in comparison to P. aeruginosa alone (Fig 4A and 4B). By contrast, 10 ppb MMA III had no effect on P. aeruginosa stimulated IL-8 and IL-6 secretion (Fig 4A and 4B). MMA III also had no effect on P. aeruginosa induced CXCL1 secretion at any concentration tested (Fig 4C). Although 0.5 and 5 ppb of MMA III had no effect on P. aeruginosa stimulated CXCL2 secretion, 10 ppb MMA III increased CXCL2 secretion (Fig 4D). Thus, taken together these data demonstrate that MMA III (5 ppb) enhances P. aeruginosa induced secretion of IL-8 and IL-6 and that MMA III (10 ppb) enhances P. aeruginosa induced secretion of CXCL2. A combination of iAs III , MMA III and DMA V reduced P. aeruginosa stimulated cytokine secretion Since blood of individuals who drink water contaminated with iAs III and iAs V typically contains mixtures of iAs, MMA and DMA, because inorganic arsenic is metabolized in the liver, we conducted studies to examine the effect of a combination of organic and inorganic arsenic at levels measured in blood samples obtained in the U.S. [3,31]. HBEC were exposed to 5 ppb or 10 ppb total arsenic, composed of 50% DMA V , 25% MMA III and 25% iAs III . Neither 5 ppb nor 10 ppb total arsenic alone had a significant effect on basal cytokine secretion compared to control (Fig 5). Both 5 ppb and 10 ppb total arsenic significantly reduced P. aeruginosa stimulated IL-8 cytokine secretion (Fig 5A). 5 ppb total arsenic had no effect on P. aeruginosa stimulated IL-6, CXCL1 or CXL2 secretion (Fig 5B, 5C and 5D), but 10 ppb total arsenic significantly reduced P. aeruginosa stimulated CXCL1 secretion (Fig 5C).

IL-1β secretion by THP-1 cells is regulated by cytokines released by HBEC
Studies were conducted to determine if the MMA III induced changes in CXCL2 secretion by HBEC had a significant effect on IL-1β production by differentiated THP-1 cells, a model macrophage cell line (Fig 6A). IL-1β secretion by macrophages recruits additional macrophages and neutrophils into the lungs, and is an essential component of the innate immune response to bacterial infection [20]. Thus, studies were conducted to determine if the MMA III induced increase in CXCL2 secretion by P. aeruginosa exposed HBEC had a significant effect on IL-1β secretion by THP-1 cells. An increase in CXCL2 concentration from 500 to 1000 pg/ mL, the concentrations produced by HBEC exposed to P. aeruginosa and P. aeruginosa plus 10 ppb MMA III , respectively, significantly increased IL-1β secretion (Fig 6B). THP-1 IL-1β release was modest when stimulated with CXCL2 in comparison to stimulation with conditioned media from HBEC. To further examine the impact of altered HBEC cytokine secretion on macrophage IL-1β secretion, THP-1 cells were exposed to conditioned media from HBEC exposed to 10 ppb MMA III , 10 ppb iAs III or 10 ppb total arsenic with and without P. aeruginosa. Conditioned media from HBEC exposed to 10 ppb MMA III , 10 ppb iAs III or 10 ppb total arsenic in the absence of P. aeruginosa did not significantly alter THP-1 IL-1β production compared to conditioned media from control HBEC (Fig 6C). Conditioned media from HBEC exposed to P. aeruginosa significantly increased IL-1β secretion by THP-1 cells compared to control conditioned media. Conditioned media from HBEC exposed to P. aeruginosa plus 10 ppb MMA III significantly increased THP-1 production of IL-1β compared P. aeruginosa alone. It is important to note that in the studies presented in Fig 6C 10 ppb MMA III increased cytokine (IL-6 and IL-8) secretion by HBEC cells. 10 ppb iAs III did not alter cytokine secretion by HBEC in the presence of P. aeruginosa. While 10 pbb total arsenic reduced IL-8 and CXCL1 secretions by HBEC, these reductions were not sufficient to significantly alter IL-1β in macrophages. Taken together, these data suggest that the alteration of IL-6 and IL-8 secretion in P. aeruginosa stimulated HBEC by 10 ppb MMA III will significantly alter IL-1β production by macrophages.
Neither MMA III nor a combination of iAs III , MMA III and DMA V altered cytokine mRNA To examine whether changes in HBEC cytokine secretion were the result of transcriptional regulation or post-translational modification, cytokine mRNA levels were measured. As shown in Figs 7 and 5 ppb MMA III alone had no effect on IL-8 or on IL-6 mRNA levels (Fig 7A and 7B). In addition, 10 ppb MMA III alone had no effect on CXCL2 mRNA levels ( Fig 7C). P. aeruginosa significantly increased IL-8, IL-6 and CXCL2 transcript levels, but the presence of MMA III did not alter P. aeruginosa induced mRNA. Thus, the observed increases in IL-8, IL-6 and CXCL2 cytokine levels induced by MMA III were not related to an increase in cytokine mRNA. In addition, studies were conducted to determine if the inhibitory effect of a combination of iAs III , MMA III and DMA V (10 ppb) on IL-8 and CXCL1 secretion was mediated by a decrease in mRNA. However, as shown in Fig 8, the combination of iAs III , MMA III and DMA V alone had no effect on IL-8 or on CXCL1 mRNA levels (Fig 8A and 8B). P. aeruginosa stimulated IL-8 and CXCL1 mRNA, but 10 ppb total arsenic did not alter the P. aeruginosa induced mRNA. Thus, the observed decreased in IL-8 and CXCL1 cytokine levels induced by a combination of iAs III , MMA III and DMA V (10 ppb) were not related to decreased mRNA levels of these cytokines.

Discussion
Arsenic exposure is a global health concern with a variety of deleterious health effects. The immunotoxicity of arsenic is poorly understood and represents an important area of study [17]. Alteration of inflammatory processes, in particular in TNFα and NFκB signaling, has been observed in infants exposed to arsenic in utero [38][39][40]. However nothing is known about the relative contributions of inorganic versus organic species of arsenic to immunotoxicity. To our knowledge this is the first study to examine the impacts of MMA III and DMA V , at concentrations that are relevant to the US population, on the innate immune response of HBEC to a bacterial pathogen. The major novel finding is that a combination of 10 ppb total iAs III , MMA III and DMA V , reflecting blood levels relevant to drinking water exposures, suppressed IL-8 and CXCL1 secretion by HBEC. In addition, MMA III alone exacerbated the immune response of HBEC to P. aeruginosa. Taken together, these data demonstrate that low levels of arsenic disrupt cytokine secretion by P. aeruginosa stimulated HBEC.
Results from this HBEC study are similar to research using a co-culture model of Caco-2 cells (a human colon epithelial cell line) and peripheral blood monocyte cells (PBMC) that showed 9 ppb MMA III enhanced LPS induced IL-6 and TNFα release [41]. The same study using Caco-2/PBMC in co-culture also showed that 105 ppb DMA III plus LPS reduced IL-8 and IL-6 release into the apical media in comparison to LPS alone [41]. Interestingly, in contrast to our findings, Caco-2 cells and the Caco-2/PBMC co-culture showed significant release of pro-inflammatory cytokines with exposure to iAs III , MMA III or DMA III alone [41,42]. By contrast, HBEC in this study showed low basal levels of pro-inflammatory cytokines regardless of arsenic exposure. These differences are potentially due to higher concentrations of iAs, MMA and DMA used in the Caco-2 study, or may simply represent tissue differences in response to arsenic species [42]. MMA III had no effect on cytokine mRNA levels. MMA III (5 ppb) had no effect on (A) IL-8 or on (B) IL-6 mRNA levels. In addition, MMA III (10 ppb) had no effect on (C) CXCL2 mRNA levels. Thus, the observed increases in IL-8, IL-6 and CXCL2 cytokine levels induced by MMA III were not related to increased mRNA levels. n = 4 donors. Pro-inflammatory cytokine secretion by bronchial epithelial cells is the first response to bacterial infection [23]. The initial increase in cytokine secretion by HBEC recruits professional immune cells, including macrophages and neutrophils, into the lungs, which secrete copious amounts of cytokines that mobilize additional immune cells to eliminate the bacterial infection [20]. Here we show that MMA III enhances the innate immune response of HBEC, which may increase the production of cytokines by macrophages, and potentially lead to excessive inflammation, which has been shown to produce lung damage [20]. We found that the change in cytokine secretion by HBEC induced by MMA III in this study had significant effects on IL-1β secretion by differentiated THP-1 cells, a model macrophage cell line. While in vivo prolonged production of cytokines can result in lung damage, initially this increased cytokine secretion may be a beneficial augmentation, resulting in enhanced recruitment of macrophages and more rapid clearance of pathogens [20]. Further study is required to determine which of these responses are seen with low level MMA III exposure.
Interestingly, 10 ppb total arsenic, a combination of iAs III , MMA III and DMA V reflecting relative blood levels after exposure via drinking water, reduced cytokine secretion by HBEC. Reduced cytokine secretion by HBEC would be expected to reduce macrophage recruitment and pathogen clearance. However, the reduced cytokines produced by HBEC after exposure to 10 ppb total arsenic did not significantly reduce IL-1β in THP-1 cells, suggesting that the reduction in IL-8 and CXCL1 will not alter macrophage response. The differences between MMA III alone and the combinations of arsenic species will require further study to understand the specific mechanisms for each type of exposure.
Low dose inorganic arsenic has previously been shown to reduce clearance of pathogens in zebrafish and mouse lung, however similar results have not previously been reported in human cells [15,21]. The published animal studies used a variety of viral and bacterial pathogens including Influenza A (H1N1), snakehead rhabdovirus, and Edwardsiella tarda indicating that altered immune response with arsenic can happen with a variety of pathogens [15,21]. Studies related to arsenic in the human lung have shown that arsenic exposure compromises respiratory immune response through several mechanisms including decreased airway epithelial chloride secretion, altered activation of pulmonary alveolar macrophages and impaired wound response resulting in airway remodeling [43][44][45]. Thus, arsenic has many effects on the innate immune response to bacterial infection.
IL-8 and CXCL2 are transcriptionally regulated by NFκB, and other transcription factors [46,47]. Previous studies have indicated that one mechanism for immunotoxicity of arsenic is through interaction with NFκB; including enhanced NFκB activation by MMA III in uroepithelial cells, and activation of NFκB signaling in cord blood of newborns with in utero arsenic exposure [29,39]. However, we did not observe an effect of MMA III or the combination of iAs III , MMA III and DMA V on mRNA levels of IL-6, IL-8, CXCL1 or CXCL2, thus, the arsenic induced changes in cytokine production observed in the present study are likely to occur by post-transcriptional mechanisms.
One possible post-transcriptional mechanism is that MMA III may enhance secretion of these cytokines through a change in membrane fluidity. Studies have indicated that low levels of MMA III perturb cholesterol biosynthesis [14]. Additional studies are necessary to examine the distinct molecular mechanisms whereby MMA III and the combination of iAs III , MMA III and DMA V at levels found in blood alter P. aeruginosa induced cytokine secretion in HBEC.
Data from this study show dysregulation of proinflammatory cytokines in HBEC after organic arsenic exposure and that these altered cytokines have downstream effects, altering IL-1β secretion in THP-1 cells. Our data provide insight into the possible mechanisms whereby arsenic exposure increases the relative risk of respiratory infection and COPD, which is associated with chronic bacterial infections, and other non-malignant lung infections [16,48]. Moreover, this study provides important data demonstrating that organic forms of arsenic, at low doses, have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa infection in vitro.