According to the World Health Organization, infectious diseases are primary contributors to the global burden of disease currently estimated to result in hundreds of millions of disability-adjusted life years worldwide [1
]. Infectious diseases are amongst the leading causes of mortality, responsible for approximately 16% of total global deaths each year [3
]. While much research has been carried out to understand the relationship between infectious disease patterns and human behavior [4
], the role that environmental contaminants play as agents that alter infectious disease susceptibility is under-recognized.
There is increasing evidence that exposure to environmental contaminants influences or disrupts the host defense responses to infectious agents, namely the innate and adaptive immune systems and their associated inflammatory response pathways. Alterations in the expression or activity of proteins within the immune system can influence susceptibility to infection from viral, bacterial, or parasitic diseases. As specific environmental examples, cigarette smoke [6
], mercury [7
], and organophosphates [8
] are established immunomodulators that can increase susceptibility to infectious diseases under conditions of chronic exposure in adults.
In addition to the deleterious health outcomes associated with chronic exposure of adults to environmental contaminants, in utero
exposures can harm the developing fetus disrupting host defenses and altering appropriate responses to infectious agents. There is mounting evidence related to developmental toxicity of in utero
exposures to the world-wide poisons inorganic arsenic (iAs) and cadmium (Cd), currently ranked amongst the highest prioritized hazardous substances in the U.S. [9
]. Current estimates suggest that more than 100 million individuals worldwide are exposed to iAs at levels associated with adverse health outcomes [10
]. This is of concern as chronic exposure to iAs, as well as Cd, has been associated with both cancer and non-cancer endpoints in adults [11
]. Additionally, in utero
and early life exposure to iAs can cause detrimental impacts on fetal and childhood development and increase the risk for certain diseases/disorders later in life, including respiratory problems, cardiovascular disease, and cancer [13
]. The evidence supporting these relationships was largely informed by population studies of prenatal iAs exposure in Antofagasta, Chile. Specifically, increased incidence of morbidity and mortality from cancer and non-cancer endpoints were observed up to forty years after prenatal/early childhood exposures to iAs [13
]. These data highlight the long-lasting health consequences of this early life exposure to iAs. In addition to the later life health consequences of exposure, prenatal exposure to both iAs or Cd have been associated with detrimental health effects at birth including risk of low birth weight [21
]. Being born at lower birth weight puts infants at subsequent increased risk for diseases including those that are associated with infectious agents [24
Directly relevant to the research presented here, prenatal iAs exposure has also been associated with increased susceptibility to infectious diseases in infants. Several studies of Bangladeshi infants have demonstrated that elevated levels of prenatal iAs increases the risk of diarrhea and upper and lower respiratory infections [25
]. Such findings have also been replicated in areas where iAs levels are lower than in Bangladesh. For instance, infants in New Hampshire, U.S., who experienced elevated exposure to iAs in utero
displayed increased severity of respiratory tract infections [27
]. While a specific mechanism for the prenatal iAs-associated increased susceptibility to infection is not established, it has been shown that such exposure reduces fetal and childhood thymic function through a reduction in naive T cells [28
] potentially resulting in immunodeficiency. While studies have yet to investigate the influence of prenatal iAs exposure on infectious disease susceptibility later in adulthood, it is likely that iAs-associated increased risk for infection extends beyond infancy. While human studies on risk of infection associated with prenatal Cd have not been carried out, rodent studies have supported that prenatal Cd exposure alters the immune system [29
To begin to fill the knowledge gap of specific genes/pathways that underlie prenatal iAs or Cd exposure-induced susceptibility to infectious disease, we developed and employed the Comparative Genomic Enrichment Method (CGEM). Using this approach, a set of exposure and infectious disease-related genes that were enriched for pathways of high priority were identified. Given their toxicological role in responses to infectious agents and their modulation across numerous studies, these pathways are likely important contributors to environmental exposure-induced immunomodulation underlying response to infectious agents.
Current estimates suggest that millions of pregnant women are exposed to harmful levels of toxic metalloids/metals such as iAs and Cd putting their developing fetuses at risk [10
]. Such exposures are associated not only with detrimental health effects observable at birth, but also with increased risk for infant susceptibility to diseases caused by infectious agents. As a result, in areas with high iAs/Cd exposure, endemic patterns of infectious disease are likely greatly influenced by the presence and exposure to these environmental contaminants. Nevertheless, specific genes/pathways altered in their cellular signaling capacities that may underlie these associations are currently unknown, thus hindering opportunities for prevention or clinical treatment of disease. To prioritize signal transduction pathways that likely mediate these relationships, the CGEM was developed to integrate information from an infectious disease database and an exposure responsive database compiled from a compendium of pregnancy cohort studies. For analysis, the pregnancy cohort studies were required to have assessed gene-specific information on iAs or Cd-associated changes in DNA methylation levels, mRNA expression levels, and/or protein levels. This CGEM approach resulted in the identification of 82 genes associated with prenatal iAs/Cd exposure and infectious disease responses. Amongst these genes were 19 genes that are involved in the GR signal transduction pathway (p
= 1.58 × 10−18
) as well as enriched for the presence of specific binding elements for the GR transcription factor. These data support the novel finding that the GR signal transduction pathway is a likely contributor to prenatal iAs/Cd-associated infectious disease susceptibility.
The GR signal transduction pathway is known to respond to glucocorticoids released by the hypothalamic-pituitary-adrenal (HPA) axis [48
]. Transcription of target genes is modulated through direct binding of DNA sequences at glucocorticoid response elements (GRE) or negative glucocorticoid response elements (nGRE). Exogenous chemicals that disrupt the GR signal transduction pathway and alter homeostatic gene expression patterns may influence disease susceptibility through disruptions in immune-related pathways [49
]. As evidence for this, disruptions to the HPA axis or glucocorticoid levels increase rates of infection and toxicity in animal models [48
]. In the context of the environmental contaminants of interest here, both iAs and Cd have been previously shown to influence the GR [50
], where iAs in particular has been shown to alter GR function and GR-dependent gene expression [51
]. In support of the role that the GR pathway plays in mediating toxicity to iAs and Cd, we have recently demonstrated that chemical inhibition of GR signaling protects against iAs and Cd-induced cellular toxicity in vitro
, and that GR inhibition protects against iAs-induced teratogenesis [51
]. With the findings from the present study, we hypothesize that the GR pathway not only influences metal-induced birth defects, but may mediate the effects of iAs and Cd on infectious disease susceptibility. Future studies will test this hypothesis directly and evaluate the impact of GR signaling as a mediator of metals-associated changes in immune function.
Amongst the identified proteins that are known to interact with the GR pathway was IFNG [54
], a member of the 16 IDGs known to play a role in responding to viral, bacterial, and parasitic exposures. Prenatal iAs exposure has been associated with altered IFNG cytokine levels [39
] as well as IFNGR1
gene expression [42
], thus represented in two of the twelve studies of interest here. The ability of iAs and potentially other environmental contaminants to influence IFNG
expression is of concern as it is a critical regulator of a host’s immune and inflammatory response [56
]. IFNG plays a role in infectious disease pathogenesis where polymorphisms have been shown to increase risk for infectious diseases including tuberculosis [57
], malaria [58
], and Chagas disease [59
]. These polymorphisms are often correlated with varying IFNG
expression levels, where increased or decreased expression levels have been tied both to infectious disease risk and severity. Given the ties between iAs/Cd exposure, IFNG, and infectious disease, it is plausible that it represents a key player related to the GR pathway that underlies iAs/Cd exposure and disease susceptibility.
The GR signal transduction pathway is highly integrated in the cell, known to influence transcription indirectly through interactions with NF-κB, TLR, and TNF [48
], all of which were represented in the IER gene set. TNF signaling is a critical player in inflammatory response signaling and immune function [62
] with increased expression following exposure to lipopolysaccharides (LPS) [63
]. Interestingly, 49 of the 82 (60%) IER genes have known associations with LPS. In relationship to disease, TNF polymorphisms are associated with susceptibility to a range of infectious diseases including mucocutaneous leishmaniasis [64
], Chlamydia trachomatis
], cerebral malaria [66
], and lepromatous leprosy [67
]. Polymorphisms in TNF
can impact its expression directly altering the immune system impacting disease severity/susceptibility [68
]. Of relevance to the environmental metals/metalloid under study here, TNF polymorphisms have also been associated with iAs-associated skin lesions and respiratory disease [69
]. Similarly, altered TNF transcript and protein expression has been related to Cd-induced liver toxicity [70
]. Taken together, an important consideration is that environmental contaminants that impact TNF expression may influence infectious disease susceptibility. Thus, TNF represents an additional GR-related protein and gene target for future mechanistic investigations relating environmental contaminant and infectious disease relationships.
While supporting the novel finding that the GR pathway may be a key component underlying metals-induced immune dysfunction, this study is not without limitations. The endpoints that were selected for evaluation here (i.e.
, DNA methylation, mRNA abundance, and protein expression) can be transient in their response to toxic agents. Importantly, however, there is evidence that some of the mRNA expression changes associated with toxicant exposure have been identified as reversible but others were irreversible [71
]. As further support for permanent changes induced by metals during the in utero
period, adult mice that were exposed to iAs during gestation have shown sustained mRNA expression changes later in life related to carcinogenesis in the liver [72
]. DNA methylation changes associated with environmental stressors can also be permanent, persisting through multiple generations [73
]. Future research will establish whether the genes that are represented within the GR pathway are stably modified under conditions of in utero
There is evidence from this research that many of the iAs and Cd-associated genes are not solely responsive to these exposures as they are also perturbed by other metals present in the environment such as beryllium, chromium, cobalt, copper, lead, mercury, nickel, selenium, and zinc. Indeed, previous studies have reported that multiple metals, including iAs, beryllium, Cd, chromium, lead, mercury, selenium, and zinc, influence signaling of the GR pathway [51
]. Additionally the IER genes are also known to be impacted by a range of other environmental contaminants such as asbestos, benzene, benzo(a)pyrene, bisphenol A, estradiol, formaldehyde, nitric oxides, particulate matter, and silicon dioxide. Thus, the data highlight that in addition to prenatal iAs and Cd exposure in humans, many other environmental contaminants indeed impact signaling of the infectious disease-related genes. As data on prenatal exposure to these contaminants in human populations are currently limited, future studies will use CGEM to assess the relationship between prenatal exposure and IER genes pending the availability of genomic, epigenomic, and proteomic data from human cohorts. The current study’s findings are important given the extent of worldwide exposure to toxic substances and thus the potential for the GR pathway to mediate responses to infectious agents, representing a clear target for disease intervention.