Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes

Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes View ORCID Profile Emily J. Illingworth , View ORCID Profile Sylvia S. Sanchez , View ORCID Profile Kristal A. Rychlik , View ORCID Profile Andre Kleensang , View ORCID Profile Gabriel A. Lopez-Cecetaite , M. Carolina Rodriguez Steube , Aakriti Mathur , View ORCID Profile Pritam Sadhukhan , View ORCID Profile Daniel A. Medina-Cleghorn , View ORCID Profile Daniel Nomura , View ORCID Profile Martyn T. Smith , View ORCID Profile Mohammad O. Hoque , View ORCID Profile Fenna C.M. Sillé doi: https://doi.org/10.1101/2025.07.14.663593 Emily J. Illingworth 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Emily J. Illingworth Sylvia S. Sanchez 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sylvia S. Sanchez Kristal A. Rychlik 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA 2 Public Health Program, School of Health Professions, University of Mary Hardin-Baylor , Belton, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kristal A. Rychlik Andre Kleensang 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andre Kleensang Gabriel A. Lopez-Cecetaite 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriel A. Lopez-Cecetaite M. Carolina Rodriguez Steube 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Aakriti Mathur 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pritam Sadhukhan 3 Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pritam Sadhukhan Daniel A. Medina-Cleghorn 4 Department of Chemistry, University of California Berkeley , Berkeley, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel A. Medina-Cleghorn Daniel Nomura 4 Department of Chemistry, University of California Berkeley , Berkeley, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel Nomura Martyn T. Smith 5 Department of Environmental Health Sciences, University of California Berkeley , Berkeley, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martyn T. Smith Mohammad O. Hoque 3 Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine , Baltimore, MD, USA 6 Department of Oncology, Johns Hopkins University School of Medicine , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mohammad O. Hoque Fenna C.M. Sillé 1 Johns Hopkins University, Bloomberg School of Public Health, Department of Environmental Health and Engineering , Baltimore, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fenna C.M. Sillé For correspondence: fsille1{at}jhu.edu Abstract Full Text Info/History Metrics Preview PDF Abstract In many regions around the world, including the United States, inorganic arsenic (iAs) contaminates groundwater used for drinking, food production, and irrigation. Although the World Health Organization has set a safety limit of 10 µg/L for arsenic in drinking water, an estimated 200 million people worldwide are still exposed to arsenic concentrations above this threshold. Eliciting a broad range of adverse health effects, arsenic is a known carcinogen classified by the International Agency for Research on Cancer (IARC) and causes increased susceptibility to infectious diseases, highlighting its role as an immunotoxicant. The purpose of this study is to elucidate the effects of arsenic on the innate immune system, namely macrophages, using in vitro exposure models. Bone marrow-derived macrophages (BMDMs) were cultured from adult male and female C57/BL6 mice. These naïve macrophages (“M0” BMDMs) were exposed in vitro to a non-cytotoxic dose of iAs (0.1 µM sodium (meta)arsenite) during the 7 day period of macrophage differentiation and stimulated for 24 hrs with LPS and IFNγ (to induce “M1” pro-inflammatory activation) or IL-4 and IL-13 (to induce “M2” anti-inflammatory activation). In a parallel chronic exposure model, RAW 264.7 (RAW) macrophages were cultured in vitro with iAs for 70 days. Culture supernatant analysis for nitric oxide and cytokine secretion revealed sex-dependent differences in immune response between exposure models, as well as between iAs-exposed and nonexposed macrophages, with and without stimulation. Additionally, iAs-exposed macrophages exhibited increased lipid droplet formation and altered lipidomic and metabolomic profiles, as determined by LC/MS. Flow cytometric analysis further revealed changes in macrophage polarization markers in a sex- and stimulation-dependent manner, with M2-related markers being upregulated in iAs-exposed conditions. Finally, to assess the effects of iAs on macrophages in the context of cancer, we demonstrated that iAs-exposed macrophages displayed increased migration toward cancer cell-conditioned media, and promoted cancer cell proliferation. These results suggest that dysregulated macrophage polarization due to iAs exposure could impact susceptibility to diseases. This research contributes to our understanding of the full spectrum of adverse health effects of iAs exposure and may aid in the development of therapeutics for iAs-induced diseases, including cancer. Introduction Inorganic arsenic (iAs) is a naturally occurring metalloid in the earth’s crust and remains an urgent public health concern due to its widespread contamination of drinking water and crop irrigation systems ( WHO, 2006 ). Despite the World Health Organization (WHO) setting the drinking water safety standard of 10 µg/L (10 ppb), more than 200 million people worldwide continue to be exposed to arsenic at levels exceeding this standard ( Podgorski & Berg, 2020 ; Shaji et al., 2021 ; WHO, 2006 ). In addition to being a potent carcinogen affecting the lung, skin, and bladder ( IARC, 2012 ), both epidemiological studies ( Banerjee et al., 2009 ; A. Rahman et al., 2011 ; Smith et al., 2011 ) and experimental findings support arsenic’s role as an immunotoxicant ( Bishayi & Sengupta, 2006 ; Kozul et al., 2009 ; Lemaire et al., 2015 ; Lemarie et al., 2006 , 2008 ; Medina et al., 2020 ; Sengupta & Bishayi, 2002 ). For example, individuals exposed to arsenic at levels above the WHO threshold show increased mortality from infectious diseases such as tuberculosis ( Smith et al., 2011 ) and are more susceptible to lower respiratory tract infections ( Rahman et al., 2011 ). Several reviews have broadly summarized evidence supporting arsenic’s role as an immunotoxicant ( Bellamri et al., 2018 ; Dangleben et al., 2013 ) during pregnancy and early life ( Attreed et al., 2017 ) and, specifically, on professional antigen presenting cells ( Bahari & Salmani, 2017 ; Giles & Mann, 2022 ; Raqib et al., 2017 , 2023 ). Among these professional antigen-presenting cells, macrophages are a key target of arsenic toxicity. Macrophages are innate immune cells that recognize, phagocytose, process, and present antigens in the context of infectious disease and cancer ( Hajishengallis & Russell, 2015 ; Hirayama et al., 2017 ; Parameswaran & Patial, 2010 ; Zhou et al., 2021 ). Macrophages are highly plastic and can polarize along a spectrum of activation ( Mills et al., 2000 ; Orecchioni et al., 2019 ). For simplicity, we refer to these activation states as M0 (homeostatic or resting macrophage that has not seen a stimulus), M1 (“pro-inflammatory” macrophage, typically responding to infection), and M2 (“anti-inflammatory,” tissue remodeling macrophages). The activation states are determined by signals from their microenvironment ( Mills et al., 2000 ; Orecchioni et al., 2019 ). For example, if macrophage toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, this will trigger an M1 response characterized by pro-inflammatory cytokines such as TNF and IL-6 ( Martinez & Gordon, 2014 ; Mosser & Edwards, 2008 ). Conversely, within the tumor microenvironment, for example, macrophages can be polarized toward an M2 phenotype by IL-10, IL-4, TGFβ, and metabolites of cancer cells like prostaglandins ( Cui et al., 2017 ; Liu et al., 2021 ; Rőszer, 2015 ). These tumor-associated macrophages (TAMs) are often pro-tumorigenic and indicative of poor prognosis for many cancers due to their secretion of cytokines, such as IL-10 and TGFβ ( Chen et al., 2019 ; Fiorentino et al., 1991 ; Zhang et al., 2016 ) and other metabolites (e.g., lipids) that drive tumor growth, angiogenesis, and promote an immunosuppressive microenvironment ( Jayasingam et al., 2019 ; Liu et al., 2021 ). Understanding how toxicants affect macrophage differentiation and polarization is critical for uncovering immune-mediated mechanisms of disease susceptibility. Disruption of the M1/M2 balance required for a normal immune response by environmental exposures, like arsenic, may compromise immune homeostasis. For example, Lemaire et al. (2011) demonstrated that arsenic-enhanced atherosclerosis is mediated by dysregulated lipid homeostasis of arsenic-exposed macrophages within atherosclerotic plaques ( Lemaire et al., 2011 ). Further, in infectious disease models, decreased macrophage adhesion and chemotaxis were associated with reduced bacterial clearance of Staphylococcus aureus infection in mice ( Bishayi & Sengupta, 2003 ). Finally, in the context of cancer, arsenic-exposed macrophages co-cultured with lung ( Cui et al., 2017 ) or liver ( Wang et al., 2021 ; Xue et al., 2021 ) cancer cells have been shown to enhance cancer cell proliferation compared to co-cultures with unexposed control macrophages. In some cases ( Cui et al., 2017 ; Xue et al., 2021 ), this proliferative effect was accompanied by increased expression of M2 polarization markers, suggesting a shift toward an immunosuppressive phenotype. Arsenic also impacts the expression of genes related to hematopoiesis, which encompasses monocytes and myeloid-derived suppressor cells (MDSCs)--myeloid progenitors that are precursors to mature macrophages in the presence of macrophage colony-stimulating factor (M-CSF). These cell types play crucial, predominantly immunosuppressive, roles in the pathology of many arsenic-associated diseases, including cancers ( Veglia et al., 2021 ). Together, these data implicate macrophage differentiation and polarization as key mechanistic targets of arsenic exposure and underline the need for further characterization of arsenic-exposed macrophages in the context of various polarization stimuli. In this study, we utilized an in vitro exposure model of murine bone marrow-derived macrophages (BMDMs). BMDMs were exposed to noncytotoxic, human-relevant concentrations of inorganic arsenic to assess its effects on macrophage differentiation and polarization. We evaluated a range of M1- and M2-relevant endpoints, including nitric oxide (NO) production, differentiation and expression of M1- and M2-surfacemarkers via flow cytometry, cytokine secretion, lipid droplet formation, and comprehensive lipidomic and metabolomic profiling. Given the limited culture lifespan (∼1 week) of BMDMs, we also compared select findings in BMDMs to RAW 264.7 macrophages that were chronically exposed (70 days) to arsenic in vitro ( Berghaus et al., 2010 ). Finally, to assess the effects of iAs on macrophages in the context of cancer, we determined whether iAs-exposed macrophages would increase migration toward cancer cell-conditioned media, and promote cancer cell proliferation. Methods Animals Adult (8–10-week-old) male and female C57BL/6CR mice (five per sex) were purchased from Charles River Laboratories (Frederick, MD) and acclimated for 1 week prior to experimental procedures. Mice were cohoused five/cage and maintained in a temperature- and humidity-controlled facility on a 14:10 light:dark cycle and provided food and drinking water ad libitum . Mice received low arsenic chow (AIN-93M: Research Diets, New Brunswick, NJ, CAT# D10012Mi) and arsenic-free water (Crystal Geiser, Calistoga, CA). ARRIVE Statement This study was conducted with approval by the Johns Hopkins University Institutional Animal Care and Use Committee (Protocols # MO19H465 and # MO20H283), following the National Research Council’s Guide for the Care and Use of Laboratory Animals. All animals were housed in the Johns Hopkins School of Public Health vivarium, which is in compliance with the Animal Welfare Act regulations and Public Health Service (PHS) Policy (PHS Animal Welfare Assurance #: D16-00173 (A3272-01)), and is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International (# 000503). In addition, we have been following the ARRIVE guidelines 2.0 ( Percie du Sert et al., 2020 ) for the entire study. Isolation, Differentiation, and Polarization of Bone Marrow-derived Macrophages Bone marrow from C57BL/6CR mice was isolated from the femurs and tibias of 8–10-week-old male and female mice, processed separately to maintain sex stratification for this study. Bones were flushed with phosphate buffered saline (1x PBS, pH 7.2, Gibco, Waltham, MA) containing 10% fetal bovine serum (FBS) using a 23G needle, and single cell suspensions were prepared using a 70 µm cell strainer. Red blood cells were lysed with ACK lysis buffer (Quality Biological, Gaithersburg, MD) and pelleted cells were resuspended and cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) media containing 10% FBS, 200 µM L-glutamine, 100 µM HEPES (Gibco, Waltham, MA), 100 µM MEM Non-Essential Amino Acids (NEAA) (Gibco, Waltham, MA), 100 µM sodium pyruvate (Gibco, Waltham, MA), and 25 ng/mL of macrophage colony stimulating factor (M-CSF) (complete media) as previously described ( Assouvie et al., 2018 ). Cells from each biological replicate were not pooled. During the 7-day differentiation period, half the cells (∼2-4 x 10 7 ) from each animal were maintained in media supplemented with inorganic arsenic (iAs) in the form of 0.1 µM sodium (meta) arsenite (Millipore Sigma, St. Louis, MO, CAT# S7400-100G). Cultures were maintained at 37 °C with 5% CO 2 for 7 days with media refreshment (half of the media was replaced with 2x growth factors) +/- 0.1 µM sodium (meta) arsenite after 4 days. After 7 days of culture, complete and arsenic-containing media was removed and adherent cells were incubated with ice-cold PBS, harvested by manual cell scraping, and plated at 2-5 x 10 6 cells per well in a clear, U-bottom 96-well plate. Cells were then polarized overnight to the M0 (complete medium alone), the M1 (complete media containing 300 ng/mL LPS and 6.25 ng/mL IFNγ), or the M2 (complete media containing 20 ng/mL each of IL-4 and IL-13) phenotype. Cells were not exposed to sodium (meta) arsenite during polarization. For lipidomics analysis, female M0 BMDMs were cultured as previously described, then treated with 1 µM monomethylarsonous acid (MMA(III)), a methylated metabolite of sodium arsenite, for 6 hours post-differentiation. Cell Line Cultures For a chronic exposure macrophage model, RAW 264.7 macrophages (catalog TIB-71 ™ , ATCC, Manassas, VA) were cultured in complete DMEM media as described above, without M-CSF, at 37°C with 5% CO 2 . Chronic exposure was modeled by culturing RAW macrophages with or without continual passage with 0.1 µM sodium (meta) arsenite for 70 days (12 passages). For cancer cell cultures, we used lung (MLE-15, LLC1, KLN205) and bladder (MB49) tumorigenic cancer cell lines. MLE-15 tumorigenic lung cells (catalog T0495, Applied Biological Systems Inc., Waltham, MA) were cultured according to manufacturer’s protocol with RPMI medium with 2% FBS, 5 µg/mL insulin-transferrin-sodium selenite supplement, and 10 µM HEPES. LLC1 lung cancer cells (catalog CRL-1642, ATCC, Manassas, VA) and KLN205 cells (catalog CRL-1453, ATCC, Manassas, VA) were cultured according to manufacturer’s protocol with DMEM and 10% FBS. All cell lines were maintained at 37°C with 5% CO 2 . Cell Viability Assay Cell viability was measured using a crystal violet staining assay as previously described ( Feoktistova et al., 2016 ). BMDMs isolated from culture as described above, were seeded at 2 x 10 6 cells in 200 µL volumes into flat-bottom 96-well plates and incubated overnight at 37°C with 5% CO 2 . The next day, plates were centrifuged at 1600 rpm for 10 minutes and the culture media was gently aspirated to remove non-adherent (presumed dead) cells. The remaining cells were washed once with PBS and adherent cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature. Once PFA was removed with aspiration, the cells were stained with 100 µL of 0.05% crystal violet solution for 30 minutes. Plates were then rinsed twice with tap water, drained, and dried inverted. The crystal violet stain was solubilized with 200 µL/well of methanol, the resultant supernatant was transferred to a new flat-bottom 96-well plate, and absorbance was measured at 570 nm using a microplate reader (Cytation 5, Biotek, Winooski, VT). Methanol-only wells served as blanks. Data are reported as fold-change in cell viability, comparing cancer cell proliferation in coculture with untreated versus arsenic-treated macrophages. Griess Assay Nitric oxide (NO) was quantified in macrophage culture supernatant using the Griess assay ( Amano & Noda, 1995 ). Briefly, 100 µL cell supernatant was collected from each well of stimulated macrophages in a new flat-bottom 96-well plate format. Griess reagents -- sulphanilamide and N-1-napthylethylenediamine dihydrochloride (NED) -- (50 µL each) were added consecutively to each well incubated at room temperature for 10 minutes. Absorbance was read at 540 using a microplate reader (Cytation 5, Biotek, Winooski, VT). Nitric oxide concentrations were determined using a sodium nitrite standard curve (with a range 0-200 µM NO) and expressed as µM of NO released in culture supernatant. Cytokine and Chemokine Multiplex Assay The concentrations of 36 cytokines and chemokines were measured in macrophage culture supernatants using the R&D Systems Mouse Premixed Multi-Analyte Kits (LXSAMSM-30 and LXSAMSM-06). A complete list of all cytokines/chemokines measured is provided in Supplemental Table 1 . Assays were performed following the manufacturer’s instructions and protocols optimized for use with the DropArray™ microplates and the Curiox plate washer system (Curiox, Woburn, MA) ( Versteeg et al., 2017 ). All analytes were measured in technical duplicate and results were acquired using a Luminex MagPix system (Bio-Rad, Hercules, CA). Data was analyzed using xPONENT software. Cytokine concentrations were calculated from standard curves generated using five-parameter logistic regression and are reported in pg/mL. Flow Cytometric Analysis Following the 7-day differentiation of bone marrow-derived macrophages, cells were suspended in phenol-free DMEM supplemented with 10% FBS, 200 µM L-glutamine, 100 µM HEPES, 100 µM MEM Non-Essential Amino Acids (NEAA), and 100 µM sodium pyruvate. Cells were counted using a hemocytometer with 0.4% trypan blue staining to determine cell viability. Viable cell counts were adjusted to a concentration of 2-10 x 10 6 cells/ml and plated in a round-bottom 96-well plate. Macrophage polarization was performed as described above over a 24 hour period. Post stimulations, cells were harvested by centrifugation at 1,600 rpm for 10 minutes at room temperature, supernatant was removed and cells were washed with 1X PBS. After another round of centrifugation and PBS removal, the cells were first stained with zombie aqua viability dye (Biolegend, San Diego, CA, CAT# 423102) according to manufacturer’s instructions. Upon removal of the Zombie dye buffer, the Fc receptors were blocked with antibodies (Biolegend, San Diego, CA, CAT# 101320) prior to surface staining. Cells were then stained for 30 minutes at 4°C with fluorochrome-conjugated antibodies targeting specific surface markers (see Supplementary Table 4 ). After surface staining, cells were washed, fixed and final preservation was performed in 1X BD FACS-lysing buffer (BD Biosciences, San Jose, CA, CAT# 349202) diluted in milli-Q ultrapure water (Milli-Q® IQ 7000 Ultrapure Lab Water System, MilliporeSigma, Burlington, MA). Intracellular staining was then performed to stain cytokines of interest using the BD Cytofix/Cytoperm (Catalog BD554714, BD Biosciences, San Jose, CA) protocol. Samples were individually run on a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ) utilizing FACSDiva software v6 and analyzed using FlowJo v10 (BD Biosciences, San Jose, CA). Compensation and gating strategies were applied using fluorescence minus one (FMO) and unstained controls. Lipid Droplet Assay Lipid droplet formation was assessed using the Lipid Droplets Fluorescence Assay Kit (Cayman Chemical, Ann Arbor, MI, Item# 500001) ( Listenberger & Brown, 2007 ) according to manufacturer’s instructions. Briefly, cultured macrophages were plated in a black, clear-flat-bottom 96-well plate at a density of 5 x 10 4 cells/well in 200 µL volumes. As a positive control, the oleic acid supplement was added to culture media to a final dilution of 1:2000. The next day (∼18 hours later), cells were fixed and stained with 1 µg/mL Nile Red Solution. Relative fluorescence intensity was measured at FITC filter excitation/emission wavelengths of 485/535 nm on a microplate reader (Cytation 5, Biotek, Winooski, VT). Data are presented as relative fluorescence units (RFU), with background fluorescence subtracted from all readings. Fluorescent imaging of lipid droplets After fluorescence intensity was quantified using the microplate reader, representative images of lipid droplets were acquired for each experimental condition using the R4 ECHO Revolve microscope (Discover Echo Inc., San Diego, CA). Stained cells were imaged directly in black, clear-flat-bottom 96-well formats, which was covered with a black box during imaging to minimize photobleaching from ambient light. A FITC filter cube (excitation/emission 485/535 nm) was used with a 10X objective lens and Ph1 phase ring condenser. Images were captured at a fixed exposure time of 565 ms. Metabolomics and Lipidomics Mass Spectrometry Analysis For lipidomics analysis, BMDMs from female mice were cultured as described previously and treated with 1 µM monomethylarsonous acid (MMA(III)), a methylated metabolite of sodium arsenite, for 6 hours after differentiation. Macrophages (1 x 10 6 macrophages) were pelleted, cell lysates were snap-frozen, and lipids were extracted using 40:40:20 acetonitrile:methanol:water for polar lipids ( Kopp et al., 2010 ) or 2:1:1 chloroform:methanol:PBS for nonpolar lipids ( Benjamin et al., 2013 ). Internal standards were included in all extractions. Lipid extracts were analyzed by LC/MS following established protocols. For metabolomics analysis, BMDMs derived from both male and female mice were cultured with 0.1 µM iAs. Snap frozen cell pellets were resuspended in 50 µL of Milli-Q ultrapure water (Milli-Q® IQ 7000 Ultrapure Lab Water System, MilliporeSigma, Burlington, MA), washed with methanol, and extracted twice with methyl tert-butyl ether (MTBE), with speed vacuum drying between each extraction. Samples were vortexed, sonicated for 2 minutes, then centrifuged at 25,000 g for 30 minutes at 4°C. The lipid fraction (organic phase) was retained for future lipidomics comparison to MMA(III) treated samples. Metabolites were collected from the lower fraction (aqueous phase), subjected to another speed vacuum dry, then resuspended in 150 µL 1:1 acetonitrile:Milli-Q water for LC/MS analysis. Migration Toward Cancer Cell-conditioned Media Macrophage migration in response to cancer cell secreted signals was assessed using a transwell migration assay. Conditioned medium was collected from cultures of murine lung carcinoma cells (LLC1 or KLN205) once they reached >85% confluency, as previously described ( Guigni et al., 2020 ). To reduce potential contamination of any nonadherent cancer cells, the media was first centrifuged at 1,600 rpm for 10 minutes, and the supernatant was carefully removed without disturbing the pellet. Migration assays were conducted using 24-well plates with 8.0 µm pore size transwell inserts (Corning, Corning, NY). Conditioned media (600uL) were added to the bottom wells, and BMDMs, cultured as described above, were seeded at 2 x 10 5 per insert in 100uL of serum-free media. BMDMs were allowed to migrate overnight in a humidified incubator with 5% CO 2 , then the following day the media was aspirated from both top and bottom chambers. Cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature. Once PFA was removed with aspiration, the apical surface of the insert was swabbed with a cotton applicator to remove nonmigratory cells. The bottom of the insert was stained with 0.05% crystal violet solution for 30 minutes in the well to fully account for cells stuck to the basolateral transwell and those that migrated fully to the bottom. The inserts were rinsed twice with tap water, drained and dried inverted. The crystal violet stain was solubilized with 200 µL/transwell of methanol, and the methanol supernatant was transferred to a clean plate. Absorbance was measured at 570 nm using a microplate reader (Cytation 5, Biotek, Winooski, VT), with methanol-only wells as blanks. Results reflect relative migration efficacy and are presented as fold-changes compared to control conditions. Proliferation of Cancer Cells Cocultured with iAs-exposed Macrophages (MTT Assay) To evaluate the impact of arsenic-exposed macrophages on cancer cell proliferation, indirect coculture experiments were conducted using 0.4 µm pore size transwell inserts (Corning, Corning, NY). Murine lung (MLE-15 and KLN205) and bladder (MB49) cancer cell lines were cultured, as previously described, and seeded at 1 × 10 4 cells per well in the bottom chamber of the transwell plate in 600uL of complete mediim. In parallel, 1 × 10 5 M0 BMDMs or RAW 264.7 macrophages that were previously exposed to iAs (in the form of 0.1 µM sodium (meta) arsenite), were seeded on the apical side of the transwells. The coculture was allowed to incubate for 6 days at 37°C with 5% CO 2 . After the coculture period, the transwell inserts were removed and the media was aspirated. To assess cell proliferation, a 300 µL volume of 1X MTT labeling reagent (diluted in PBS) was added to the bottom well. After an incubation of 24 hours, the MTT solution was solubilized with DMSO, and the plate was incubated for an additional 2 hours. Color change due to metabolic activity as a measure of proliferation of the cancer cells was measured using a microplate reader (Cytation 5, Biotek, Winooski, VT) at 550 nm using DMSO-only wells as blanks. Absorbance values were used as a proxy for cancer cell proliferation in response to macrophage derived signlaing. A subset of technical replicates of cocultured cancer cells were stored in DNA/RNA Shield (Catalog R1100, Zymo Research, Irvine, CA) for downstream RNA isolation and gene expression analysis. Gene Expression Analysis by Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNA was extracted from polarized murine bone marrow-derived macrophages using the Quick-RNA™ MiniPrep Kit (Zymo Research, Irvine, CA). RNA concentration was assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific™, Waltham, MA), and RNA quality was confirmed with the Ribogreen assay (Quant-it™ RiboGreen RNA Assay Kit, Invitrogen™, Waltham, MA). Complementary DNA (cDNA) was synthesized using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific™, Waltham, MA), with total RNA diluted to a final concentration of 7.5 ng/μL. RT-qPCR was performed in triplicate for all experimental groups--male- and female-derived BMDMs across each stimulation and arsenic treatment group--using the Applied Biosystems™ QuantStudio™ 3 Real-Time PCR System (Thermo Scientific™, Waltham, MA). Primers for target genes involved in macrophage polarization and metabolism were selected based on literature; sequences along with working annealing temps, are provided in Supplemental Table 5 . Each 10 μL reaction included SsoFast™ EvaGreen ® Supermix (Bio-Rad Laboratories, Hercules, CA) and forward and reverse primers at 400 nM. Amplification curves were generated using an Applied Biosystems™ QuantStudio™ 3 System software (Thermo Scientific™, Waltham, MA) with the cycle conditions as follows: a hold stage (95°C for 2 minutes), 40 cycles at a PCR stage (95°C for 15 seconds, 56°C for 1 minute, 60°C for 1 minute), followed by a continuous melt curve analysis (95°C for 15 seconds, 60°C for 1 minute, and dissociation at 95°C for 15 seconds). Relative mRNA expression was calculated using the ΔΔCt method ( Livak & Schmittgen, 2001 ), normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were excluded if they did not meet the inclusion criteria of transcript detection at <33 cycles. Statistical Analysis Nitric oxide production, lipid droplet content, and gene expression by RT-qPCR, were analyzed using unpaired Student’s t-tests. Multiplex cytokine, lipidomic, metabolomic, and flow cytometric data were analyzed using one-way ANOVA with Sidak’s multiple comparisons correction. When appropriate, analyses were stratified by sex, and results were normalized to cell viability. Unless otherwise indicated, significance is stated as compared to the sex-matched M0 untreated control. Statistical significance was defined as p -value <0.05. Statistical analyses were performed using GraphPad Prism 9 (Graphpad Software Inc, La Jolla, CA). Results Arsenic-induced Cytotoxicity to BMDMs To establish a non-cytotoxic exposure dose for downstream analyses, we conducted a dose-response assessment of of sodium (meta) arsenite (0-1 µM) applied to BMDM cultures during the 7 day differentiation period. Briefly, to determine the cytotoxicity of iAs to BMDMs at each dose, we employed the crystal violet assay to measure cell viability. No significant cytotoxicity was observed at concentrations of 0.0001, 0.001, 0.01, or 0.1 µM iAs doses; however, exposure to 1 µM resulted in a significant ∼18% reduction in viability compared to the control ( Fig. 1 ). To avoid off-target effects of BMDMs responding to significant amounts of dead and/or dying cells, we proceeded with the highest non-cytotoxic concentration of iAs, 0.1 µM, for subsequent experiments. It is important to note that this concentration equates to ∼13 ppb, slightly above the WHO drinking water guideline of 10 µg/L, and is therefore relevant to represent human exposure levels ( WHO, 2006 ). Download figure Open in new tab Figure 1. Cytotoxicity of iAs to BMDMs M0 BMDMs were treated with a dose range of iAs (0-1 µM) and viability was measured by crystal violet staining and absorbance. Plots shows mean ± SD with % viability normalized to nonexposed macrophages and dose range across the x-axis. * p -value<0.05. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. iAs Exposure Reduces Nitric Oxide Production in iAs-treated Macrophages Macrophage nitric oxide (NO) production is a hallmark of M1 polarization in response to inflammatory signals, including cytokines and/or microbial pathogen-associated molecular patterns (PAMPs) ( Tripathi, 2007 ). Since this is an important M1 endpoint, we evaluated NO production in culture supernatant from both iAs-exposed BMDMs and RAW 264.7 macrophages using the Griess assay. No significant differences in NO production were observed in either male- or female-derived BMDMs exposed to 0.1 µM iAs during differentiation following stimulation with 300 ng/mL LPS plus 6.25 ng/mL IFNγ ( Fig. 2A, 2B ). However, RAW 265.7 macrophages chronically exposed to the same dose of arsenic exhibited a significant reduction in NO release following M1 stimulation ( p<0.05 ; Fig. 2C ). As expected and previously known ( Mills, 2001 ), NO production was negligible in both M0- and M2-stimulated BMDMs and RAW 264.7 macrophages (data not shown). Download figure Open in new tab Figure 2. NO release by iAs-exposed M1 macrophages NO release was measured by the Griess assay on culture supernatant obtained from female (A) or male (B) M1-stimulated BMDMs exposed to 0.1 µM iAs for 1 wk during differentiation, and from M1-stimulated RAW 264.7 macrophages (C) exposed to iAs for 70 d. n=5 biological replicates for BMDMs and n=3 technical replicates for RAW 264.7 macrophages. M0- and M2-stimulated macrophages from both culture models had negligible production of NO (data not shown). Plots show mean ± SD. * p -value <0.05. NO=nitric oxide; iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. Cytokine and Chemokine Profiles of iAs-treated BMDMs In response to signals they receive from their microenvironment, macrophages produce cytokines and chemokines that signal in autocrine, paracrine, and endocrine fashion to help orchestrate an immune response ( Mantovani et al., 2004 ; Rostam et al., 2017 ). To determine the effects of arsenic exposure on cytokine profiles of M0-, M1-, and M2-stimulated macrophages, we performed multiplex cytokine and chemokine analysis on cell supernatant harvested from BMDMs 24 hours post-stimulation. A total of 36 analytes were measured and we found significant changes in cytokine/chemokine profiles of iAs-treated BMDMs that were sex- and stimulation-dependent. Female M0 (unstimulated) BMDMs that were iAs-treated showed a decrease in TIMP-1 secretion ( p <0.01) and an increase, although not statistically significant ( p =0.14), in CCL5, compared to unexposed matched control BMDMs, whereas iAs-treated male M0 (unstimulated) BMDMs had a significant reduction in CHI3L1 and CXCL2 ( p <0.001 and p <0.0001, respectively), compared to unexposed male control BMDMs ( Fig. 3A, 3B ). Under M1 stimulation, iAs exposure significantly decreased CCL5 in females (p<0.05) and CCL4 in males (p<0.0001). A similar but nonsignificant trend was oberseved in CCL4 signaling in female BMDMs (p=0.053) ( Fig. 3C, 3D ). Male and female M2-stimulated BMDMs that were iAs-treated similarly showed a decrease in chemokine secretion, with iAs-treated females demonstrating significantly decreased expression of CCL4 ( p <0.01), CCL5 (p <0.001) and CXCL2 (p <0.001), while iAs-treated males showed significantly reduced CXCL2 expression ( p <0.001) compared to untreated controls ( Fig. 3E, 3F ). Collectively, these data demonstrate that iAs exposure suppresses chemokine (CCL4, CCL5, and CXCL2) and cytokine (CHI3L1 and TIMP-1) secretion in both male and female macrohphages across stimulation conditions. Download figure Open in new tab Figure 3. Changes in cytokine profiles of iAs-exposed macrophages Using a multiplex assay, cytokine concentrations for 36 analytes were measured from culture supernatant of M0-(A, B), M1-(C, D), or M2-stimulated (E, F) female and male BMDMs exposed to 0.1 µM iAs for 1 wk during differentiation. n=5 biological replicates. Only analytes reaching or approaching significance are shown. Plots show mean ± SD. * p -value<0.05; ** p -value<0.01; *** p -value<0.001; **** p -value<0.0001. BMDMs=bone marrow-derived macrophages; iAs=inorganic arsenic. Flow Cytometric Characterization of iAs-treated BMDMs To assess the effects of arsenic exposure on macrophage differentiation and polarization, we designed a multicolor flow cytometry panel ( Supplemental Table 4 ) including markers of monocyte-macrophage differentiation (Ly6C, CD11b, F4/80), polarization status (iNOS, Arg1, CD163), and functional capacity (CD86, MHCII, PD-L1, TGFβ, IL-6) across M0-, M1-or M2-polarization. For these studies, we defined mature macrophages as CD11b + F4/80 + ( Zajd et al., 2020 ), while CD11b + Ly6C + F4/80 - MHCII - cells were included to capture monocytic precursors and myeloid-derived suppressor cells (MDSCs) ( Bronte et al., 2016 ; Damuzzo et al., 2015 ) (for a representative gating strategy, see Supplemental Fig. 3 ). In female BMDMs, arsenic exposure during differentiation led to a nonsignificant increase in Arg1-expressing mature macrophages after M2 stimulation ( p = 0.119, Fig. 4A ). No significant changes in monocyte to macrophage differentiation were observed in either male-or female-derived BMDMs as evidenced by similar percents of mature macrophages (CD11b+F4/80+), although the iAs-exposed male-derived M2 group showed a slightly reduced percentage of mature macrophages compared to… ( p =0.111, Fig. 4D ). TGFβ production was decreased nonsignificantly in iAs-treated M0 male-derived BMDMs ( p = 0.127, Fig. 4C ), while CD163 expression increased in M1 stimulated iAs treated male BMDMs, though this trend was also not statically signicant ( p = 0.120, Fig. 4B ). Download figure Open in new tab Figure 4. Flow cytometric characterization of iAs-exposed female and male BMDM polarization and functional markers as a frequency of parent gates Mature macrophages (CD11b+F4/80+ live cells) were analyzed from BMDM cultures for expression of select polarization and functional markers Arg1 (A), CD163 (B), TGFβ (C), and for culture homogeneity as a percent of live cells (D). Only plots with p -values <0.15 are included (complete panel with all markers analyzed can be found in Supplemental Table 4 ). Plots show % expression of parent gate on the y-axis by stimulation condition on the x-axis, with the mean ± SD. n=4 biological replicates. * p -value<0.05. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages Among mature macrophages (CD11b+F4/80+), the level of protein production (reflected as mean fluorescent intensity, or MFI) was altered for a variety of markers. In iAs-treated M2-stimulated female- and male-derived BMDMs, Arg1 expression was increased, though only significantly in males ( p = 0.109 and p <0.05, respectively, Fig. 5A, 5C ). CD86 followed the same trend of upregulation with only the male-derived group reaching significance ( p= 0.113 and p <0.05, for females and males, respectively, Fig. 5B, 5D ). In contrast, the expression of iNOS was significantly reduced in M1-stimulated male BMDMs ( p <0.01, Fig. 5E ) and the expression of MHCII was decreased in both M1 and M2 groups (p=0.056 and p <0.01, respectively, Fig. 5F ). Download figure Open in new tab Figure 5. Flow cytometric characterization of iAs-exposed female and male BMDM polarization and functional markers as MFI expression levels Mature macrophages (CD11b+F4/80+ live cells) were analyzed from BMDM cultures for expression of select polarization and functional markers Arg1 (A, C), CD86 (B, D), iNOS (E), and MHCII (F). Only plots with p -values <0.15 are included (complete panel with all markers analyzed can be found in Supplemental Table 4 ). Plots show MFI on the y-axis by stimulation condition on the x-axis, with the mean ± SD. n=4 biological replicates. * p -value<0.05; ** p -value<0.01. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. MFI= mean fluorescence intensity. There were no differences in the frequency of MDSCs in our iAs-exposed cultures, though differences were observed for frequency and expression levels of certain markers. The frequency of CD163-expressing MDSCs was increased in the iAs-treated female M1-stimulated group and the iAs-treated male M0 group upon iAs exposure, though only the females reached statistical significance ( p <0.05 and p = 0.102, respectively, Supp. Fig. 1A, 1B ). Similar to our findings in BMDMs, the frequency of live MDSCs expressing iNOS was significantly reduced in the iAs-treated male M1-stimulated group ( p <0.05, Supp. Fig. 1D ) and CD86 expression trended lower in the same group ( p = 0.122, Supp. Fig. 1C ). Male M2-stimulated MDSCs with iAs treatment had a nonsignificant increase in the frequency of PD-L1 expression compared to controls ( p = 0.079, Supp. Fig. 1E ). iAs exposure also affected the MFI of certain markers on MDSCs. CD86 expression decreased in M1-stimulated cells in both females and males, though not significantly ( p = 0.107 and p = 0.121, respectively, Supp. Fig 2B, 2C ). In M2 female MDSCs only, iAs significantly increased the MFI of CD163 ( p <0.05, Supp. Fig. 2A ). In M1 male MDSCs only, iAs significantly increased the MFI of PD-L1 ( p <0.05, Supp. Fig. 2D ). As shown by the decreased expression of pro-inflammatory M1 markers and the increased expression of M2 markers in arsenic-treated BMDM cultures across stimulation groups, these data collectively suggest that chronic arsenic exposure skews macrophages and MDSCs toward the M2 phenotype. Lipid Droplet Formation in iAs-treated BMDMs and RAW 264.7 Macrophages Macrophages have different modes of lipid metabolism based on their polarization state. M1 macrophages favor aerobic glycolysis and fatty acid synthesis, while M2 macrophages rely on fatty acid oxidation and oxidative phosphorylation ( Kelly & O’Neill, 2015 ; Morgan et al., 2021 ; Rodríguez et al., 2025 ). Because of the metabolic reprogramming that coincides with macrophage polarization, we investigated whether arsenic exposure skewed the metabolic signature of M0 (homeostatic) macrophages. First, we assessed whether iAs treatment affected the formation of lipid droplets in either M0 BMDMs or M0 RAW 264.7 macrophages. Briefly, BMDMs or RAW 264.7 macrophages were cultured, plated, and stained with Nile Red, a lipid indicator. Fluorescence intensity was measured, and representative images were captured using the ECHO Revolve fluorescence microscope. No significant differences were observed between iAs-treated and control male or female BMDMs ( Fig. 6A, 6B ). However, chronic iAs exposure significantly increased lipid droplet accumulation in M0 RAW 264.7 cells compared to controls ( Fig. 6C, 6D ). These findings demonstrate that chronic arsenic treatment enhances lipid droplet biogenesis in M0 RAW 264.7 macrophages. Download figure Open in new tab Download figure Open in new tab Figure 6. Lipid droplet formation in iAs-exposed M0 macrophages Lipid droplet formation was measured using a lipid droplet fluorescence assay in female (A) or male (B) BMDMs exposed to 0.1 µM iAs for 1 wk during differentiation, and from RAW 264.7 macrophages (C) exposed to iAs for 70 days from which representative images are shown (D). n=5 biological replicates for BMDMs and n=3 technical replicates for RAW 264.7 macrophages. Plots show mean ± SD. *p-value<0.05. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. Lipidomic Profiles of iAs-treated BMDMs Since increased lipid droplet formation was observed in chronic iAs-exposed M0 RAW 264.7 macrophages ( Fig. 6C, 6D ), it was of particular interest to detect any alterations in the levels of lipid metabolites of iAs-treated macrophages using a more sensitive method. Female BMDMs were M0 stimulated and cultured as previously described, then treated with monomethylarsonous acid (MMA(III)), a methylated metabolite of sodium arsenite, for 6 hours after differentiation. M0 macrophages were pelleted, snap frozen, and polar and nonpolar lipids were extracted ( Benjamin et al., 2013 ; Kopp et al., 2010 ) and analyzed by LC/MS. Of the 30 lipids measured across 6 lipid classes ( Supplemental Table 2 ), 11 were significantly altered following MMA(III) exposure ( Fig. 7 ). Acylcarnitines were dysregulated with arsenic exposure, with lauroyl carnitine (C12:0) significantly downregulated (p <0.0001) while palmitoyl L-carnitine (C16:0) showed a trending but nonsignificant upregulation ( p = 0.0968). Other lipid targets that were significantly altered and upregulated by arsenic exposure were as follows (lipid classes bolded): eicosanoids prostaglandin E2 and D2 (PGE2/PGD2, p <0.0001), ether lipids lysophosphatidic acid-platelet activating factor (lyso-PAF [C18:1], p <0.0001) and phosphatidic acid ether (PAe [C16:0/C20:4], p <0.001), lysophospholipids lysophosphatidic acid (LPA [C20:4], p <0.0001), lysophosphatidylcholine (LPC [C20:4], p <0.01), lysophosphoethanolamines (LPE [C18:1], p <0.0001; LPE [C18:0], p <0.0001; LPE [C20:4], p <0.05; LPS [C18:0], p <0.05) and one analyte from the sphingolipids, sphingosine 1-phosphate (S1P [C16:0], p <0.001) ( Fig. 7 ). These results suggest that female-derived BMDMs exposed to MMA(III), a toxic metabolite of sodium (meta) arsenite, have enhanced production of lipid mediators associated with inflammation and tumorigenesis. In the future, we will repeat this analysis with sex- and stimulation-stratified BMDMs treated with sodium (meta) arsenite. Download figure Open in new tab Figure 7. Lipid profiles of MMA(III)-exposed female BMDMs Polar and nonpolar relative lipid levels from BMDMs exposed to 1 µM MMA(III) for 6 hrs were determined by LC/MS. n=3-4 biological replicates. Plots show mean ± SD. * p -value<0.05; ** p -value<0.01; *** p -value<0.001; **** p -value<0.0001. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. Metabolomic Profiles of iAs-treated BMDMs In addition to lipid analytes, we also measured the metabolic profiles of iAs-treated (0, 0.01, or 0.1 µM iAs) BMDMs using LC/MS. Of the 23 metabolites measured ( Supplemental Table 3 ), only 3/23 and 6/23 were approaching or at significance in iAs-treated female- and male-derived BMDMs, respectively ( Fig. 8 ). At 0.1 µM iAs exposure in females, norepinephrine levels were higher, at near statistical significance ( p =0.0503). At the highest exposure dose, L-arginine was significantly decreased ( p <0.01) and S-adenosylmethionine (SAM) was decreased, albeit nonsignificantly ( p = 0.0933) ( Fig. 8A ). In male BMDMs, significant decreases were observed for L-ascorbic acid ( p <0.05 at 0.1 µM iAs), norepinephrine ( p <0.01 for 0.01 µM iAs; p <0.05 at 0.1 µM iAs), succinic acid ( p <0.01 for 0.01 µM iAs; p <0.05 at 0.1 µM iAs), L-arginine ( p <0.01 for 0.01 µM iAs; p <0.0001 at 0.1 µM iAs) and γ-Aminobutyric acid (GABA) approaching significance ( p =0.085 for 0.01 µM iAs) ( Fig. 8B ). These results suggest that iAs treatment suppresses metabolite availability in BMDMs and helps to support the known effects of iAs as a methyl scavenger (as shown by decreases in SAM) and as an activator of Arg1 (as shown by decreases in L-arginine). Download figure Open in new tab Figure 8. Metabolite profiles of iAs-exposed BMDMs Polar and nonpolar relative lipid levels from BMDMs exposed to 0, 0.01, or 0.1 µM iAs 1 wk during differentiation were determined by LC/MS. n=2 biological replicates per dose for both males and females, with only significant (or approaching significant) analytes plotted (females: 3/21 analytes vs. males: 6/21 analytes). Plots show mean ± SD. * p -value<0.05; ** p -value<0.01; *** p -value<0.001; **** p -value<0.0001. iAs=inorganic arsenic; BMDMs=bone marrow-derived macrophages. Migration of iAs-treated Macrophages Toward Cancer Cell-conditioned Media To contextualize our phenotypic findings within the framework of iAs-associated disease, we next assessed how chronic iAs exposure influenced macrophage responses to cancer cell signals. First, we evaluated how iAs exposure affected macrophages’ ability to migrate toward cancer cell conditioned media. Conditioned media from two murine lung cancer cell lines, LLC1 and KLN205, was used to test the migratory capacity of male and female BMDMs by transwell migration assay. Migration toward LLC1 conditioned media was significantly increased in both male- and female-derived iAs-exposed M0 (unstimulated) BMDMs compared to unexposed M0 controls ( p <0.01, Fig. 9A, 9B ). In contrast, migration toward KLN205 conditioned media was not significantly altered in neither male-nor female-derived BMDMs, although iAs-exposed female-derived BMDMs exhibited a noteworthy increase in migration compared to controls ( p =0.0662, Fig. 9A ). These results indicate that iAs-treated macrophages migrate more readily toward lung cancer cell-conditioned media compared to controls. Download figure Open in new tab Figure 9. Migration of iAs-exposed BMDMs toward cancer cell conditioned media Transwell migration assay was used to measure migratory capacity of exposed M0 female and male BMDMs toward LLC1 and KLN205 lung cancer cell conditioned media. n=3-5 biological replicates. Plots show mean ± SD of percent migration measured with crystal violet staining. **p<0.01. iAs= inorganic arsenic; BMDMs= bone marrow-derived macrophages. Proliferation of Cancer Cells Cocultured with iAs-treated Macrophages To test the tumor-promoting potential of iAs-exposed macrophages, we cocultured chronic iAs-exposed RAW 264.7 macrophages with three different cancer cell lines: MLE-15 (lung), LLC1 (lung), or MB49 (bladder). After the coculture period, we determined how the microenvironment created by iAs-exposed RAW 264.7 macrophages drove cancer cell proliferation by MTT assay. MB49 bladder cancer cells cocultured with iAs-exposed RAW 264.7 macrophages exhibited significantly increased proliferation compared to those cocultured with nonexposed macrophages ( p <0.05, Fig. 10A ). LLC1 cells also showed increased proliferation in response to coculture with exposed macrophages, though nonsignificant ( p =0.081, Fig. 10A ). MLE-15 cells cocultured with iAs-exposed RAW 264.7 macrophages showed no significant proliferation differences compared to those cocultured with nonexposed RAW 264.7 macrophages. However, MLE-15 cells cocultured with female-derived iAs-exposed BMDMs demonstrated a significant increase in proliferation compared to control cocultures ( p <0.05, Fig. 10B, 10C ). These results suggest that iAs-treated M0 macrophages create a tumor microenvironment that promotes cancer cell growth. Download figure Open in new tab Figure 10. Proliferation of cancer cells cocultured with iAs-exposed macrophages Proliferation of cancer cells was measured by MTT assay after coculture with exposed M0 RAW 264.7 macrophages (A), female (B) or male (C) BMDMs. n=5 replicates. Plots show mean ± SD of proliferation normalized to control . *p<0.05. iAs= inorganic arsenic; BMDMs= bone marrow-derived macrophages. Expression of Cancer Stem Cell Markers in Cells Cocultured with iAs-treated RAW 264.7 Macrophages A subset of replicates of MLE-15, LLC1, and MB49 cancer cells cocultured with iAs-exposed or nonexposed RAW 264.7 macrophages were used for targeted gene expression analysis of cancer stem cell markers. Genes associated with arsenic exposure that were selected for analysis included Yes-Associated-Protein 1, Cyclooxygenase-2 ( YAP and SOX2 , respectively) ( Michel-Ramirez et al., 2020 ; Ooki et al., 2018 ) and self-renewal Notch Receptor 1 ( NOTCH1 ) ( Tokar et al., 2010 ). Gap Junction Protein beta 1 ( GJB1) , a gene involved in cell-cell communication and reported to be downregulated in bladder cancer ( Zaravinos et al., 2011 ) but which has dual roles in other cancer types ( Aasen et al., 2019 ; Yang et al., 2021 ), was also examined. NOTCH1 expression was unchanged across all cancer cell lines; however, changes were observed for YAP , SOX2 , and GJB1 . MB49 bladder cancer cells cocultured with iAs-exposed RAW 264.7 macrophages had significantly upregulated YAP expression compared to those cocultured with controls ( p <0.01, Fig. 11A ). SOX2 expression was elevated in both MLE-15 and LLC-1 cells following coculture, but only the increase in MLE-15 cells reached statistical significance ( p <0.001, Fig. 11B ; LLC1 p =0.123). GJB1 expression was significantly decreased in LLC1 cells cocultured with iAs-exposed RAW 264.7 macrophages ( p <0.05, Fig. 11C ). These results suggest that iAs exposure modulates how M0 macrophages respond to tumor cell conditioned media and that iAs-treated macrophages create a microenvironment supportive of cancer cell growth and cancer stem cell marker expression. Download figure Open in new tab Figure 11. Gene expression analysis of cancer targets in cancer cell lines after coculture with iAs-exposed RAW 264.7 macrophages RT-qPCR was used to measure expression of select cancer targets YAP (A), SOX2 (B), GJB1 (C), and Notch (D). n=3 replicates. Plots show mean ± SEM of expression normalized to control using GAPDH. Discussion In this study, we report sex- and stimulation-dependent phenotypic changes in macrophages chronically exposed to inorganic arsenic (iAs). The decrease in nitric oxide (NO) production observed in our study is consistent with prior findings from chronic arsenic exposure in humans ( Banerjee et al., 2009 ), as well as murine peritoneal macrophages ( Arkusz et al., 2005 ; Liu et al., 2008 ), splenic macrophages ( Bishayi & Sengupta, 2006 ; Sengupta & Bishayi, 2002 ), and RAW 264.7 macrophages ( Chakravortty et al., 2001 ). To our knowledge, however, we are the first to report a decrease in NO production in RAW 264.7 macrophages following chronic exposure to iAs at a dose as low as 0.1 µM ( Fig. 2C ). Given that NO is a major mediator of inflammation and plays an essential role in killing of infectious pathogens and tumor cells ( Nath & Kashfi, 2020 ; Tripathi, 2007 ), our findings suggest that chronic arsenic exposure dampens the proinflammatory response of macrophages in response to M1 stimuli. In this study, we demonstrate that, under the conditions of our iAs exposure model, there are significant reductions in the expression of chemokines CCL4, CCL5, and CXCL2. CCL4 and CCL5 were downregulated in M1 and M2 iAs-treated female BMDMs, respectively. These chemokines signal through chemokine receptors (CCRs) CCR1 and CCR5, which are expressed on a wide range of immune cells, including monocytes, macrophages, dendritic cells, T cells, B cells, and natural killer cells ( Mantovani et al., 2004 ), and function to increase immune cell adhesion to endothelial cells and elicit a pro-inflammatory response ( Gunawardana et al., 2021 ; Quandt & Dorovini-Zis, 2004 ). Similarly, CXCL2 signals via CXCR2 on neutrophils to trigger adhesion and migration through the vascular endothelium ( Wang et al., 2021 ), and its reduction may compromise the ability to mount a pro-inflammatory immune response. Decreases in TIMP-1 and CHI3L1 were observed in iAs-treated M0 female and male macrophages, respectively. TIMP-1 is an endogenous inhibitor of matrix metalloproteinases ( Winkler et al., 2020 ) which may be relevant in iAs-induced diseases like cancer, where macrophages act as major remodelers of the extracellular matrix to promote angiogenesis and modulate T cell infiltration ( Afik et al., 2016 ; Gunawardana et al., 2021 ). CHI3L1 (chitinase 3 like 1) is a member of the glycosyl hydrolase 18 family that lacks chitinase activity. CHI3L1 is expressed in both M1 and M2 macrophages depending on stimulation context ( Zhao et al., 2020 ) and signals to regulate growth and differentiation of macrophages and other cell types. Like TIMP-1, there is also evidence that CHI3L1 regulates remodeling of the extracellular matrix ( Kzhyshkowska et al., 2006 ; Li et al., 2021 ). These findings suggest that arsenic exposure may impair macrophages’ ability to remodel the extracellular matrix. Future experiments could explore this in vitro with biological scaffold techniques ( Valentin et al., 2009 ) and in vivo in the context of disease with histological analysis targeting markers involved in remodeling ( Rahman et al., 2020 ). Because arsenic can also disrupt cell differentiation ( Lemarie et al., 2006 ; Liu et al., 2019 ; Sakurai et al., 2006 ), we also wanted to characterize MDSCs that were derived from our bone marrow cultures. MDSCs are potent immunoregulators and precursors of macrophages that are also elicited from bone marrow. They have implicated roles in arsenic-relevant diseases, including cancers ( Yang et al., 2020 ) and atherosclerosis ( Foks et al., 2016 ). However, to our knowledge, no direct link between arsenic exposure and changes in MDSC phenotype or function has been reported. Unique to our MDSCs, we observed a decrease of PD-L1 expression on M1 MDSCs and an increase in cells expressing PD-L1 in our M2-stimulated MDSCs ( Supp. Figs. 1 and 2 ). PD-L1 is a common marker of an immunosuppressive microenvironment in cancer and is expressed by macrophages to dampen the inflammatory response and inhibit T cell effector function ( Jiang et al., 2019 ; Liu et al., 2020 ). These findings suggest that chronic iAs exposure reprograms not only macrophages but also their myeloid precursors toward a less inflammatory, more immunosuppressive phenotype. Intracellular lipid droplets provide sources of fatty acids for macrophages, and fatty acid metabolism is tightly linked to metabolic rewiring of M1 and M2 macrophages. For example, M1 macrophages have greater metabolic flux through aerobic glycolysis, the pentose phosphate pathway, and increased fatty acid synthesis to fuel the energetic demand of M1-associated functions such as phagocytosis and rapid generation of inflammatory mediators ( Batista-Gonzalez et al., 2019 ). M2 macrophages, on the other hand, rely heavily on glutaminolysis, the TCA cycle, oxidative phosphorylation (OXPHOS), and fatty acid oxidation ( Ren et al., 2019 ; Sadiku & Walmsley, 2019 ). We first observed an increase in lipid droplet formation in chronic iAs-esposed RAW 264.7 macrophages using a fluorescent lipid staining assay ( Fig. 6C, 6D ). To identify which specific lipids were upregulated in iAs-exposed macrophages, we performed LC/MS lipidomics analysis on BMDMs treated with MMA(III), a toxic metabolite of iAs. We identified several lipid classes that were upregulated, including prostaglandins, ether lipids, lysophospholipids, and sphingolipids ( Fig. 7 ). These findings suggest that arsenic exposure disrupts the arachidonic acid metabolism cascade, with potential implications for macrophage function in disease. For example, prostaglandins are derived from arachidonic acid through enzymatic activity of COX2 and signal in an autoregulatory fashion to downregulate proinflammatory (e.g., TNF) cytokine production and upregulate anti-inflammatory (e.g., IL-10) cytokine production in macrophages ( Saleh et al., 2020 ). Aditionally, prostaglandins participate bi-directionally in cancer cell-macrophage crosstalk as prostaglandins secreted from macrophages can also serve as ligands to G-protein coupled receptors (GPCRs) on cancer cells to promote survival, migration, and further inflammation and immunosuppression ( Inaba et al., 2003 ; Kashiwagi et al., 2018 ; Thompson, 2020 ). Phospholipids have additionally been shown to regulate macrophage phenotype. This has been characterized especially in obesity ( Morgan et al., 2021 ; Poledne et al., 2019 ; Serbulea et al., 2018 ) and in cardiovascular diseases like atherosclerosis ( Nishikawa et al., 2015 ; Rosenblat et al., 2006 ). Macrophage-derived phospholipids are also upregulated in macrophage-cancer cell coculture systems ( Rabold et al., 2020 ). We observed elevated sphingosine-1-phosphate (S1P), which is tightly linked to eicosanoid metabolism and has been shown to promote differentiation of monocytes to macrophages and tumor cell signaling ( Weigert et al., 2019 ). However, increased S1P ( Fig. 7 ) does not does not coincide with any significantly increased monocyte-macrophage differentiation based on our flow cytometric analysis ( Fig. 4D ). Importantly, S1P may produce paracrine signaling to other cells to regulate migration, proliferation, and differentiation ( Weigert et al., 2019 ). Interestingly, COX2, a major regulator of arachidonic acid metabolism and the rate-limiting step in prostagladin synthesis, is regulated by S-nitrosylation and may interact directly with iNOS ( Salvemini et al., 2013 ). Production of metabolites downstream of COX2 can suppress NO and NOS isoform production through negative feedback mechansims ( Cheng et al., 2002 ; Mollace et al., 2005 ). In line with this, we observed reduced NO in our chronic iAs-exposed RAW 264.7 macrophages ( Fig. 2 ), and decreased iNOS in our flow cytometric analysis of iAs-exposed BMDMs ( Fig. 5E ). This connection represents one plausible explanation for these collective phenotypic findings. We also observed distinct effects on iAs-exposed male-derived BMDMs, which had significant reductions in L-ascorbic acid, norepinephrine, and succinic acid ( Fig. 8B ). While succinic acid is associated with M2 polarization and flux through the TCA cycle ( Trauelsen et al., 2021 ), ascorbate has dual effects on macrophage polarization and function; it has been shown to attenuate LPS-induced activation and cytokine production in monocytes ( Schmidt et al., 2022 ), partially explaining the iAs-induced cytokine and chemokine profiles we observed ( Fig. 3 ). Nevertheless, it also diminishes expression of certain M2 markers including CD163, which contradicts our flow cytometric analysis ( Fig. 5B ), further supporting the mixed polarization phenotype. In addition to determining migratory capability of iAs-exposed macrophages toward lung cancer cell conditioned media, we cocultured either chronic exposed RAW 264.7 macrophages in a transwell system (without cell-cell contact) with MLE-15, LLC1, or MB49 cells to further understand arsenic’s effects on macrophages in the context of cancer promotion. Interestingly, both LLC1 lung cancer cells and MB49 bladder cancer cells displayed increased proliferation after coculture with iAs-exposed macrophages compared to controls, with MB49 proliferation reaching statistical significance ( Fig. 10A ). Of note, while we have not tested their ability to promote proliferation of other cancer cell types, iAs-exposed female BMDMs cocultured with the tumorigenic lung epithelial cell line MLE-15 promoted their proliferation in a statistically significant manner ( Fig. 10B ). Because this coculture does not allow for cell-cell contact, we can infer that the presence of signaling molecules derived from iAs-exposed macrophages in the shared transwell media drove the proliferation of cancer cells. This is not surprising, given that macrophage-derived soluble mediators such as metabolites, chemokines, and cytokines ( Ge & Ding, 2020 ), as well as non-soluble mediators such as lipids and the content of exosomes ( Liu et al., 2020 ), have been shown to influence tumor cell growth. We next sought to determine which cancer-related genes might be driving the increased proliferation after coculture with arsenic-exposed macrophages. We harvested cancer cells after coculture and performed gene expression analysis for a panel of PCR primers ( Supplemental Table 5 ). Subsequent qPCR analysis revealed cancer stem cell markers YAP and SOX2 significantly increased in MB49 and MLE-15 cells, respectively, after coculture with iAs-exposed macrophages compared to controls ( Fig. 11A, 11B ). Together, these results imply that iAs-exposed macrophages create a microenvironment conducive to cancer cell growth and cancer stem cell promotion. Indeed, many of the properties we have identified in our analysis of iAs-exposed macrophages have been characterized in TAMs, including expression of M2 markers ( Figs. 4 and 5 ) as negative prognostic factors, as well as enhanced lipid biosynthesis ( Rabold et al., 2020 ) ( Figs. 6 and 7 ). Our study has several strengths. First, our exposure model uses a human-relevant low-dose arsenic exposure level. Even though we show that this dose is noncytotoxic to M0 BMDMs ( Fig. 1 ), we also normalize to cell viability and cell number whenever possible to minimize off-target results from dead or dying cells because macrophages are also potent responders to Damage-associated molecular patterns (DAMPs) ( Blander, 2017 ) and plating density alone can influence their differentiation states ( Lee & Hu, 2013 ). We also conducted sex stratified analyses when using BMDMs since there are differences between female and male biotransformation of iAs ( Lindberg et al., 2008 ; Muhetaer et al., 2022 ; Pierce et al., 2013 ) and sex differences in morbidity and mortality of iAs-induced diseases, especially cancers ( Ferreccio et al., 2000 ; Parvez et al., 2013 ; M. Rahman et al., 2006 ; Smith et al., 2006 ). In addition, macrophage phenotype is influenced by sex through mechanisms such as hormone signaling ( Barcena et al., 2021 ; Klein & Flanagan, 2016 ) and sex-linked genes ( Schurz et al., 2019 ; Syrett et al., 2019 ). Combined with these sex differences reported in the literature, the sex-dependent changes in iAs-exposed macrophages that we observed in this study across cytokine profiles ( Fig. 3 ), expression of polarization markers ( Figs. 4 and 5 ), and influence on cancer cell growth ( Fig. 10 ) support the need for further investigation into sex-informed toxicology. Another strength of our study design is that when characterizing our arsenic-exposed macrophages, we utilized M0, M1, and M2 stimulation conditions for multiple endpoints. This allowed us to contextualize changes in NO production, chemokine and cytokine production, and flow cytometric characterization of polarization markers, as many of these changes are stimulation-dependent ( Figs. 2 – 5 ). Still, we recognize that this contextualization is limited compared to the mixed stimulation conditions BMDMs experience in vivo. In the future, experiments should include cancer cell conditioned media as its own stimulation control. One limitation of this study is that thus far, we have only explored the tumor-promoting effects of shared conditioned media on cancer cell promotion. Future work will continue to pursue the mechanism by which exposed macrophages drive cancer cell proliferation with targeted agonists and inhibitors based on our phenotypic findings and knowledge of upstream ligands that signal to regulate cancer stem cell marker expression. For example, we will target enzymes involved in lipid synthesis (specifically to upregulated targets we observed in our lipidomics analysis), such as COX2, a key enzyme in prostaglandin synthesis (e.g., with inhibitor Celecoxib) ( Zhang et al., 2016 ), phospholipase A2, a key enzyme regulating phospholipid production (e.g., with inhibitor Varespladib) ( Arsenault et al., 2011 ) and S1P sphingosine kinase, a key enzyme regulating S1P production (e.g., with inhibitor SKI-II) ( Pitman et al., 2022 ). Another limitation of this study is that we were only to run lipidomics/metabolomics analyses on macrophages under a single stimulation condition (M0 female BMDMs) with MMA(III) exposure rather than with exposure to sodium (meta) arsenite, which was used in our exposure model for other functional endpoints. Since macrophages have distinct modes of lipid metabolism upon receiving polarization queues and in many disease statuses ( Batista-Gonzalez et al., 2019 ; Kelly & O’Neill, 2015 ), we need to further explore the effects of arsenic under additional M1 and M2 polarization conditions across sexes. In summary, iAs does not drive unidirectional polarization shift but induces a hybrid macrophage phenotype with sex dependent metabolic, phenotypic, and functional changes. These macrophages contribute to tumor-promoting microenvironment and represent a key immunotoxicological target in arsenic-associated diseases. Overall, understanding the relationship between iAs-relevant diseases and macrophage biology may offer insight toward novel strategies for macrophage-associated disease therapy. Funding This work was supported by the National Institutes of Environmental Health Sciences (NIEHS) grants R00ES024808 (F.C.M.S.), Ruth L. Kirschstein Institutional National Research Service Award from NIEHS T32ES07141 (appointees: E.I. and K.A.R.), Ruth L. Kirschstein Institutional National Research Service Award from the National Heart, Lung, and Blood Institute, USA, award number T32HL007534 (appointee: S.S.), as well as a Johns Hopkins Bloomberg School of Public Health 2019 Ho-Ching Yang Memorial Faculty Innovation Award (F.C.M.S.), and a 2021 Maryland Cigarette Restitution Fund Research Grant at Johns Hopkins (F.C.M.S.). SUPPLEMENTAL FIGURES Download figure Open in new tab Supplemental Figure 1. Flow cytometric characterization of iAs-exposed female and male MDSCs as a frequency of parent gates MDSCs (Ly6C+CD11b+F4/80-MHCII-live cells) were analyzed from BMDM cultures for expression of select polarization and functional markers CD163 (A B), CD86 (C), iNOS (D), and PD-L1 (E). Only plots with p -values <0.15 are included (complete panel with all markers analyzed can be found in Supplemental Table 4 ). Plots show % expression of parent gate on the y-axis by stimulation condition on the x-axis, with the mean ± SD. n=4 biological replicates. * p -value<0.05. iAs=inorganic arsenic; MDSCs=monocytic myeloid derived suppressor cells. Download figure Open in new tab Supplemental Figure 2. Flow cytometric characterization of iAs-exposed female and male MDSC polarization and functional markers as MFI expression levels MDSCs (Ly6C+CD11b+F4/80-MHCII-live cells) were analyzed from BMDM cultures for expression of select polarization and functional markers CD163 (A), CD86 (B, C), and PD-L1 (D). Only plots with p -values <0.15 are included (complete panel with all markers analyzed can be found in Supplemental Table 4 ). Plots show MFI on the y-axis by stimulation condition on the x-axis, with the mean ± SD. n=4 biological replicates. * p -value<0.05; ** p -value<0.01. iAs=inorganic arsenic; MDSCs=monocytic myeloid derived suppressor cells. MFI= mean fluorescence intensity. Download figure Open in new tab Supplemental Figure 3. Representative flow cytometry gating strategy SUPPLEMENTAL TABLES View this table: View inline View popup Supplemental Table 1. Cytokine targets View this table: View inline View popup Supplemental Table 2. Lipid targets View this table: View inline View popup Supplemental Table 3. Metabolite targets View this table: View inline View popup Download powerpoint Supplemental Table 4. Flow cytometric panel design View this table: View inline View popup Supplemental Table 5. Primers for RT-qPCR validation Funder Information Declared National Institutes of Environmental Health Sciences (NIEHS) , R00ES024808 , T32ES07141 the National Heart, Lung, and Blood Institute , T32HL007534 Johns Hopkins Bloomberg School of Public Health 2019 Ho-Ching Yang Memorial Faculty Innovation Awar 2021 Maryland Cigarette Restitution Fund Research Grant at Johns Hopkins Footnotes Emily J. Illingworth: emily52695{at}gmail.com Sylvia S. Sanchez: ssanch22{at}jhu.edu Kristal A. Rychlik: krychlik{at}umhb.edu Andre Kleensang, kleensang{at}web.de Gabriel A. Lopez-Cecetaite: glopezc1{at}jhu.edu M. Carolina Rodriguez Steube: mrodri67{at}jhmi.edu Aakriti Mathur: aakritimathur2013{at}gmail.com Pritam Sadhukhan: pritamsadhukhan.bi{at}gmail.com Daniel A. Medina-Cleghorn: d.medinacleghorn{at}gmail.com Daniel Nomura: dnomura{at}berkeley.edu Martyn T. Smith: martynts{at}berkeley.edu Mohammad O. Hoque: mhoque1{at}jhmi.edu Fenna C.M. Sillé: fsille1{at}jhu.edu References ↵ Aasen , T. , Sansano , I. , Montero , M. Á. , Romagosa , C. , Temprana-Salvador , J. , Martínez-Marti , A. , Moliné , T. , Hernández-Losa , J. , & Ramón y Cajal , S. ( 2019 ). Insight into the Role and Regulation of Gap Junction Genes in Lung Cancer and Identification of Nuclear Cx43 as a Putative Biomarker of Poor Prognosis . Cancers , 11 ( 3 ), 320 . doi: 10.3390/cancers11030320 OpenUrl CrossRef PubMed ↵ Afik , R. , Zigmond , E. , Vugman , M. , Klepfish , M. , Shimshoni , E. , Pasmanik-Chor , M. , Shenoy , A. , Bassat , E. , Halpern , Z. , Geiger , T. , Sagi , I. , & Varol , C . ( 2016 ). 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Share Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes Emily J. Illingworth , Sylvia S. Sanchez , Kristal A. Rychlik , Andre Kleensang , Gabriel A. Lopez-Cecetaite , M. Carolina Rodriguez Steube , Aakriti Mathur , Pritam Sadhukhan , Daniel A. Medina-Cleghorn , Daniel Nomura , Martyn T. Smith , Mohammad O. Hoque , Fenna C.M. Sillé bioRxiv 2025.07.14.663593; doi: https://doi.org/10.1101/2025.07.14.663593 Share This Article: Copy Citation Tools Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes Emily J. Illingworth , Sylvia S. Sanchez , Kristal A. Rychlik , Andre Kleensang , Gabriel A. Lopez-Cecetaite , M. Carolina Rodriguez Steube , Aakriti Mathur , Pritam Sadhukhan , Daniel A. Medina-Cleghorn , Daniel Nomura , Martyn T. Smith , Mohammad O. Hoque , Fenna C.M. 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