Linking Endotoxins, African Dust PM10 and Asthma in an Urban and Rural Environment of Puerto Rico

Mediators of Inflammation, Volume 2015, Article ID 784212, 14 pages, 




Mario G. Ortiz-Martínez,1,2,3 Rosa I. Rodríguez-Cotto,1,2 Mónica A. Ortiz Rivera,2,4 Cedric W. Pluguez-Turull,2,5 and Braulio D. Jiménez-Vélez1,2 

1Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, San Juan, PR 00935, USA

2Center for Environmental and Toxicological Research, University of Puerto Rico-Medical Sciences Campus, San Juan, PR 00935, USA

3Department of Biology, University of Puerto Rico at Humacao, Humacao, PR 00792, USA

4School of Medicine, University of Puerto Rico-Medical Sciences Campus, San Juan, PR 00931, USA

5Department of Radiology, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX 78229, USA



African Dust Events (ADE) are a seasonal phenomenon that has been suggested to exacerbate respiratory and proinflammatory diseases in Puerto Rico (PR). Increases in PM10 concentration and the effects of biological endotoxins (ENX) are critical factors to consider during these storms. ENX promote proinflammatory responses in lungs of susceptible individuals through activation of the Toll-like receptors (TLR2/4) signaling pathways. The objective of the study was to evaluate the toxicological and proinflammatory responses stimulated by ADE PM10 ENX reaching PR using human bronchial epithelial cells. PM10 organic extracts from a rural and urban site in PR (March 2004) were obtained from ADE and non-ADE and compared. A retrospective data analysis (PM10 concentration, aerosol images, and pediatric asthma claims) was performed from 2000 to 2012 with particular emphasis in 2004 to classify PM samples. Urban extractswere highly toxic, proinflammatory (IL-6/IL-8 secretion), and induced higher TLR4 expression and NF-κB activation compared to rural extracts. ENX were found to contribute to cytotoxicity and inflammatory responses provoked by urban ADE PM10 exposure suggesting a synergistic potency of local and natural ENX incoming from ADE. The contribution of ADE PM10 ENX is valuable in order to understand interactions and action mechanisms of airborne pollutants as asthma triggers in PR.



The Caribbean region has been receiving the impact of a natural phenomenon known as African Dust Storms or African Dust Events (abbreviated as ADE) since ages. While crossing the Atlantic Ocean more than 170 years ago, Charles Darwin described dust storms in his notes as fine dust reducing visibility and falling over the HMS Beagle [1,2]. The effects of these dust storms from Africa (three billion metric tons annually) have impacted the Caribbean inhabitants since its colonization by the Indians (4,000 years ago) latest being the Taíno and modern European (>500 years ago) [3]. Today, the high prevalence of asthma in Puerto Rico has focused on the people complaint (mostly asthmatics) of allergies and exacerbations caused by the contribution of particulate matter (PM) from dust storm particles. Therefore, it is of utmost importance to study the constituents found in particles arriving with ADE and determining the health impact of specific constituents to the Caribbean. Particulate matter with 10 µm or less in diameter (PM10) is a complex mixture of inorganic and organic compounds that derive from natural and anthropogenic sources. Our laboratory studies the in vitro toxic and pro-inflammatory effects of PM using human bronchial epithelial cell (BEAS-2B) line as a model. This study focuses on the cytotoxic and pro-inflammatory capacity of endotoxins (ENX), one of the biological constituents of African dust, as well as local PM sources. These endotoxins or lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria, which are known to be abundant in African Dust. In order to mediate an inflammatory response, these ENX have to be recognized by Toll-like receptors (TLR2/4) at the bronchial epithelial cell surface and activate a signaling cascade leading to the activation of specific transcription factors such as nuclear factor kappa B (NF-κB). This activation directs the transcription and release of pro-inflammatory mediators (e.g. cytokines/chemokines: IL-6 and IL-8).

Therefore, we hypothesized that ENX found in PM10 derived from ADE (2004) were toxic and induced pro-inflammatory responses (cytokine secretion, TLR gene expression and NF-κB activation) in BEAS-2B. We compared PM10 from 2 different locations: Guaynabo (an urban San Juan site) and Fajardo (a rural reference site (Figure 1).



Figure 1 (adapted from the article featured here): Map of Puerto Rico depicting the sites where PM10 was collected. The urban site, Guaynabo (Station #24: 18˚:26:22/66˚:06:54) is represented by a black dot and the rural reference site, Fajardo (Station #22: 18˚:23:00/65˚:37:10) as a black square. Retrieved and modified from


We performed a retrospective analysis from years 2000 to 2012 on PM10 concentrations collected at each of the sampling sites and gathered all pediatric asthma cases reported by a government insurance agency for 2004. These analyses revealed a peak in ADE duration during 2004 particularly during March; and PM10 concentration increases in the same season. Asthma case distribution also showed an increase during March (0-5 years category in particular at San Juan). A significant correlation was found between asthma cases at the rural site during ADE days, PM10 concentration and increment. All findings using ADE (2004) in our studies demonstrate its relevance as a potent environmental trigger for asthma in Puerto Rico. Adding to our work, we evaluated the ENX content of PM10 organic extracts and found higher concentration of ENX in the ADE extract from the urban site as compared to our reference site. Another goal of our study was to assess the contribution of ENX towards eliciting toxicological and pro-inflammatory responses using human lung cells (in vitro), BEAS-2B. We reported that ADE urban extract was efficient in inducing cytotoxicity at a concentration of 10 µg/ml and demonstrating that this cytotoxicity was mainly due to ENX. We demonstrated the cytotoxic role of ENX by repeating the toxicity assays using an ENX inhibitor Polymyxin B (PMB). The inhibitor under the same experimental conditions reversed most of the cytotoxic effect of the ambient PM10 extract, linking ENX with its toxicity. The same ambient PM10 extracts (after 24 hr exposure) were shown to induce pro-inflammatory cytokines IL-6 and IL-8 in human lung cells. Again the use of ENX inhibitor showed that both interleukins (IL-6 and IL-8) were significantly reduced, linking pro-inflammatory mediators with ambient ENX exposure. Then, we evaluated the expression of two cell membrane receptors, which ENX bind to (TLR2 and TLR4) after exposure to ambient PM10 extracts at different time points (7 and 12 hr). We found induction of both TLR2-4 however; TLR4 was significantly reduced upon ENX inhibition.


Figure 2 (also adapted from the article featured here): Proposed schematic model of the in vitro toxic and inflammatory responses stimulated in the bronchial epithelium.


Finally, we followed the activation of a transcription factor (NF-κB) that is activated downstream from the TLR receptors and which is responsible for the release of the pro-inflammatory mediators measured (IL-6, IL-8) after ambient PM10 exposure (Figure 2). We found that only the ADE urban extract stimulated NF-κB activation (after 4 hr exposure) and it was decreased with the use of PMB supporting the importance of ENX in the extract. Natural ENX (arriving in ADE) appear to have significant effects when combined with the local input at the urban site, suggesting the influence of anthropogenic sources of ENX. A summary of what we observed is represented in Figure 2 (adapted from Figure 9 of the article featured here): Proposed schematic model of the in vitro toxic and inflammatory responses stimulated in the bronchial epithelium. In this diagram we have modeled all the findings of our work, red arrows indicate all the parameters measured and their outcome. The use of ENX inhibitors was shown to greatly reduce those effects.

The results of our work demonstrate the importance of ENX as a PM biological constituent, which is able to induce cytotoxicity and pro-inflammatory responses in human bronchial epithelial cells. This is the first study that demonstrates a direct link of ADE, PM10 concentration and pediatric asthma cases in Puerto Rico. We proved that the general believe of the Puerto Rican population linking African dust storms to adverse respiratory health outcome is not a myth but a reality. Furthermore, it reveals the first link of ENX found in ADE to health outcomes in vitro. Our findings highlight the necessity to pinpoint the effects of particular constituents in the complex cloud of ADE. This study recommends preventive measures to our asthmatic population during African Dust episodes in order to avoid exacerbations of the condition. It also highlights TLRs as potential targets of future asthma drug therapy that in combination with asthma genetics (e.g. genetic variations) could modulate asthma manifestations and guide towards the current field of pharmacogenomics and personalized medicine.



[1] Garrison VH, Shinn EA, Foreman WT, Griffin DW, Holmes CW, Kellogg CA, Majewski MS, Richardson LL, Ritchie KB, Smith GW. African and Asian dust: from desert soils to coral reefs. BioScience. 2003 May 1;53(5):469-80.

[2] Darwin C. An account of the Fine Dust which often falls on Vessels in the Atlantic Ocean. Quarterly Journal of the Geological Society. 1846 Jan 1;2(1-2):26-30.

[3] Monge JT. Puerto Rico: The trials of the oldest colony in the world. Yale University Press; 1999 Feb 1.



  Mario G. Ortiz Martínez, Ph.D. (right)               


Assistant Professor

Department of Biology

University of Puerto Rico at Humacao

Humacao, PR


Braulio D. Jiménez Vélez, Ph.D. (left)

Full Professor

Department of Biochemistry

University of Puerto Rico

Medical Sciences Campus

San Juan, PR