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At Home with Mastomys and Rattus: Human-Rodent Interactions and Potential for Primary Transmission of Lassa Virus in Domestic Spaces

The multimammate mouse (Mastomys natalensis) is the reservoir for Lassa virus (LASV). Zoonotic transmission occurs when humans are directly or indirectly exposed to fluids of the multimammate mouse, such as urine, saliva, and blood. Housing characteristics and domestic organization affect rodent density in and around households and villages, and are likely to be a risk factor for Lassa fever in humans where the reservoir exists. We use semi-structured interviews (N = 51), a quantitative survey (N = 429), direct observations, and a rodent ecology study to provide new insights into how the organization of domestic spaces brings together humans and rodents and creates pathways for infection in rural settlements in Bo District, Sierra Leone. Rodents were frequently reported inside houses (92.4% of respondents), in which we predominantly trapped M. natalensis (57% of trapped rodents) and Rattus rattus (38% of trapped rodents). Building design and materials provide hiding and nesting places for rodents and lead to close proximity with humans. Patterns of contact are both unintentional and intentional and research participants reported high levels of contact with rodents (34.2% of respondents) and rodent fluids (52.8% of respondents). Rodents are also perceived as a serious threat to food security. These results present detailed knowledge about how humans live with and come into contact with rodents, including the LASV reservoir. Our results argue for further collaborative research in housing and environmental modification such as ceiling construction, food storage, and sanitation as prevention against zoonotic LASV transmission.

INTRODUCTION
Lassa fever (LF) is a viral zoonotic illness and a significant cause of morbidity and mortality in countries across West Africa, namely Benin, Guinea, Liberia, Nigeria, and Sierra Leone. [1][2][3] LF is estimated to affect between 250 and 300,000 people and cause between 5,000 and 10,000 fatalities annually across the region, 3 but many cases are likely to go unreported due to a lack of diagnostic facilities.
The main reservoir for Lassa virus (LASV) is the multimammate mouse, Mastomys natalensis. Other rodent reservoirs (Mastomys erythroleucus and Hylomyscus pamfi) have been recently identified 4 but their relative contribution to human infections is unknown. Transmission from rodents to humans occurs through direct exposure to rodent fluids such as urine, saliva, and blood or indirect exposure via surfaces and foodstuffs contaminated by these fluids. 5,6 Urine may present a particular risk for human infections as M. natalensis can shed LASV in urine at any age 7 and LASV has been shown to be aerosolized under laboratory conditions. 8 Secondary human-to-human transmission follows contact with human bodily fluids in the household or healthcare facilities, and is estimated to occur in 20% of LF cases. 9 Risk factors for primary (zoonotic) transmission are unclear and possibly linked to housing 10 and hunting and consumption of rodents. [11][12][13] No licensed vaccine exits but the antiviral ribavirin can improve prognosis if administered early after symptoms appear. Current recommendations for the prevention of primary transmission focus on reducing rodent abundance in houses and surrounding spaces, improving sanitation (rodent proofing houses and/or stored food), and avoiding direct contact with rodents as occurs during hunting and consumption. 14 Preventing primary transmission in this way requires detailed knowledge about how humans live with and come into contact with M. natalensis. 15 In West Africa, the prevalence of LASV in M. natalensis ranges between 5% and 20%. [16][17][18][19] In Upper Guinea, M. natalensis comprises between 95% and 98% of rodents captured in houses. 20 In coastal Guinea, the black rat Rattus rattus enters into houses and tends to evict M. erythroleucus. 21 In Sierra Leone, both species are present, with R. rattus already recorded in 1972 in Panguma, 22 and in 1978-1980 in many other localities (J. Krebs in GBIF database; http://www.gbif.org/species). Houses, kitchens, and stores built with mud and wattle provide rodents with increased opportunities to burrow and food stores attract and support rodent populations. 3,23 A conclusive causal link between housing quality and human LASV infection has yet to be determined, the principal difficulty residing in the fact that the existence of other potential risk factors in the domestic environment makes it difficult to disentangle various risks. In a study of refugee camps in Sierra Leone, Bonner and others 10 found that the presence of rodent burrows, and external hygiene around the house in particular, was directly associated with a history of LF in the household. The presence of rodent burrows in turn was directly associated with housing quality (defined as construction material used and current state of maintenance). In Nigeria, there was no statistical difference between LASV-positive and LASV-negative households with regard to housing quality, but there was an association between housing hygiene (defined as waste disposal and food storage) and a (self-reported) history of LF in the household. 24 In Sierra Leone, Moses and others 25 found a correlation between M. natalensis trapping success and rodent burrows in the home; however, trapping success was not correlated with wall or roof type, and only weakly with floor construction. Seroprevalence of LASV antibodies was not associated with presence of rodents in households in Guinea. 12 Nevertheless, housing characteristics that lead to an increased rodent density in and around households and villages are likely to be a risk factor for LF in humans 10 and warrant further investigations. [26][27][28][29] However, there is little information describing the specificities of rodent-human interaction inside homes and facilitators and barriers such as construction methods and domestic organization. This study seeks to address this gap by describing how household organization creates the conditions for contact between humans and rodents and provides insights on how these interactions may form pathways for infection.

METHODS AND MATERIALS
We combined qualitative and quantitative surveys to capture a finely grained picture of rodent-human interactions. We place our observations into perspective by presenting results from our rodent ecology survey. Ethical clearance was received from the ethics committee of the Government of Sierra Leone, Charité-Universitätsmedizin, Berlin, and the Royal Veterinary College, London. Written consent was obtained from all participants.
Study sites. In Bo District, the Mende form the majority ethnic group (79%) followed by the Temne (7%). Islam (72%) and Christianity (27%) are the two principle religions. 30 The main economic activities are crop farming, diamond mining, and construction work. 30 A majority of the population (60%) is rural. Fishing, hunting, and farming (rice, cassava, yam, and sweet potato) serve as means of subsistence or as income generating activities with pineapple, mango, coffee, cacao, and palm oil as main cash crops. 31 We conducted anthropological fieldwork in Bo District (southern province) over a period of 4 months (May-June 2014 and October-December 2015). Rodent ecology investigations took place between April 2014 and February 2015. Making use of the long-standing presence of our local research team in the area since 2010, we identified 17 villages of varying size (500-1,500 inhabitants) and distance from main transport axes (from 4.5 to 40 km from the outskirts of Bo Town) ( Figure 1).
Anthropological investigations. Qualitative survey. In all 17 study villages, we applied common methods to collect qualitative data until saturation was achieved: in-depth interviews (IDIs, N = 51), spontaneously occurring focus groups discussions (N = 4), and observations (over the entire duration of the study period). Potential study participants were identified through our local researchers' previous work in the area and were purposefully selected to achieve representation from various groups (socioeconomic status, profession, religion, ethnicity, age, and sex).
The principal topics included in our interview and observation guides covered contact with rodents and their fluids inside homes, perceptions of rodent behavior and ecology (e.g., feeding, nesting), materials, design and maintenance of dwelling spaces, food security and storage (damage caused by rodents to foodstuffs), types of rodent control measures, and knowledge of LF (transmission routes, symptoms, and prevention strategies). Patterns of contact that occurs during hunting and consumption of rodents were also explored as part of this study but are described in a separate paper. 13 Assuming that the presence of peridomestic rodents is related to the physical set up of  domestic spaces, we paid particular attention to the construction and spatial organization of houses. Qualitative protocols are usually divided into two phases, which are iterative and complementary: the first one is informed by a literature survey to design the principal lines of research, in our case corresponding to biomedical risk factors for disease transmission (e.g., direct and indirect contact with rodents and their fluids) and factors that affect rodent ecology (e.g., feeding and nesting). The second phase occurs during fieldwork, where the daily preliminary narrative analysis of transcripts and field notes helps adapt the interviews and observations guides to the emergent lines of investigations.
Discussions were carried out in Mende, Krio, or English and facilitated by a translator. Formal discussions were recorded and transcribed. Informal discussions and observations were documented with field notes and photographs. Interviews lasted on average for 1 hour and were conversational and open-ended, treated as occasions for a mutual exchange of information with as much time as possible to informal interactions with the communities to establish trust.
Recordings and field notes were immediately transcribed using MS Word 2011 (Microsoft Corp., Redmond, WA). Individual and village identifiers were removed and coded to ensure anonymity. The transcripts were reviewed using a thematic analysis and segments of interests were color coded according to the topics described earlier. Analysis was done on a daily basis so that questions and observation guides could be refined in an iterative fashion. Reflective notes were made daily, compared with published literature, and regularly shared with the research group.
Quantitative survey. A cross-sectional questionnaire survey was carried out midway during the first fieldwork period (May-June 2014). We purposefully selected nine villages out of the 17 study villages to represent different population sizes and distance from main transport axes. Selection of individuals was carried out according to the World Health Organization Expanded Programme on Immunization Coverage Survey method. 32 In total, 524 subjects were recruited (see details in Bonwitt and others, 2016). Fiftyseven records were excluded because respondents lived in a major city, 21 because respondents lived in a village other than the study villages, and seven because the village name was not indicated on the questionnaire.
The questions were based on findings from a first set of IDIs and covered all forms of contact with rodents (contact in homes and farms, contact during hunting, butchering, and consumption) as well as food security and knowledge of LF. A total of 55 questions were asked. The answer format relevant to the questions described in this study was either single or multiple choices. Questions were in English and administered by local staff trained to translate the questions in Krio and Mende.
Records with answers stating "unknown" or "don't know" were not included in the analysis for that particular question. The final number of respondents varies according to question because skip logic was used to avoid asking redundant or irrelevant questions based on the respondent's previous answers. Data were collated and analyzed with STATA 13 (StataCorp. 2013; StataCorp LP, College Station, TX) and MS Excel 2011 (Microsoft Corp., Redmond, WA). We estimated proportions of subjects with contact with rodents, control measures, and food security. The adjusted Wald method was used to calculate 95% confidence intervals.
Rodent survey. Of 17 villages investigated for this study, six were chosen for rodent sampling ( Figure 1). These villages were chosen according to criteria that limit colonization of R. rattus, in villages and which could lead to displacement of other rodent species. The criteria included: village population between 500 and 1,000 people, village surrounded by forest or wooded savanna, absence of paved road access to the village, absence of weekly markets, and location within 45 minutes driving distance from Bo Town. The commensal rodents were sampled in April 2014, July 2014, October 2014, and February 2015. Usually, 100 large folding aluminum Sherman traps were set inside houses, kitchen, and stores if separate from the main house, along a transect crossing the village. Two to 12 traps per house (depending on the size of the house) were set during three consecutive nights of each trapping session. In July 2014, the trapping session was reduced because of challenges brought by the Ebola virus disease outbreak. The total trapping effort for the four sessions reached 5,868 trap-nights. Traps were checked each morning, and animals were necropsied in a safe location near the village, according to BSL3 procedures. 33,34 Morphological identification was done in situ by weighing and measuring the animals. As several species of Mastomys can live in the area, further molecular identification based on the cytochrome b was done in the laboratory. 35

RESULTS
We provide a statistical description of the study participants from all nine villages chosen for the quantitative survey (Table 1).
Domestic spaces. The supporting structures of houses in the study villages are built from various materials, including cement brick, earth/clay brick, or from earth/clay and wattle over a supporting skeleton built of wooden poles woven with smaller branches (these latter two structures have a lifespan of several years). Walls are sometimes plastered with cement. Roofs are either made of thatch (from palm trees) that require re-thatching every 1-3 years, or corrugated metal that usually requires little repair over a lifetime. Floors are either dried mud or cemented. Houses and other structures (schools, religious edifices, and place for community meetings) built with cement are rare. Indoors, ceilings are built to create a lower boundary under the roof and storage for rarely used objects. Ceilings are typically formed by an alignment of dried branches (Figure 2), which may be covered with mats made from plant fibers. Ceilings made from other materials such as corrugated metal or wood planks are uncommon. Houses generally consist of multiple rooms with a single room serving many purposes: bedroom, storage or, sometimes, for small businesses. Most houses and kitchens have a veranda for cooking and eating, but people also cook indoors during rainy or cold periods. A kitchen consists of an open fire on the ground with three stones supporting the cooking pot. Spilled raw and cooked food is swept aside but not removed at night. Corridors are used for storing various objects such as cooking utensils (mortars, pots) and agricultural products. Cupboards or trunks are rare and possessions (clothes, cooking utensils, and agricultural and fishing equipment) can often be found heaped on the floor, stored in plastic buckets with lids, or hung from the ceiling or walls. Storerooms and corridors are usually devoid of windows. Bedroom windows (without glass) are invariably small, and, in the absence of the owner, shutters are kept closed during the day. The little light that penetrates inside houses does so through cracks in shutters, doors, and holes in the roof. Electricity is nonexistent save for an occasional generator often shared among village members, and the only commonly available light sources in villages are battery-powered torches.
Outdoors, villages have well-trodden earth in areas immediately around and between houses with occasional shrubs or bushes, sometimes interspersed with abandoned and crumbling homes invaded by grasses and shrubs, which are regularly cut to flush out rodents. Latrines, where these exist, are placed at some distance from the house, often at the junction with the bush. Garbage (notably food leftovers and rice husk) is disposed of in pits or more commonly openly thrown on the ground on the outer limit of the village.
Farmhouses serve as simple second homes and are located away from people's main homes close to their agricultural land. They constitute an individual unit of domestic space in the "bush" and are used to facilitate agricultural work (including resting, cooking, and storage). In essence, farmhouses in the bush mirror houses in villages, with similar but simpler and more temporary structures.
Food stock and cooking uses. Grains, leguminous crops, and fruit are stored on the floor in covered buckets or large flour bags. Food left over from the evening meal is kept for the following morning. Such food, termed "sleep rice" or "cold rice," is usually stored overnight in covered pots and eaten for breakfast. Wealthier people have better quality containers for storing both cooked and raw food (e.g., pots with fitting lids, wooden trunks for food, and other possessions). Bowls and utensils are not always washed immediately after use because of the lack of running water and lighting, especially after the evening meal. Younger female household members are traditionally expected to wash these in the morning. Grain (principally rice) is stored on ceiling rafters, inside the home, or in designated grain stores outside the main dwelling area made of thatch, which sometimes double as kitchens. For subsistence farmers, the stored rice harvest is meant to last the whole year for household consumption, sale, gifts, and ceremony contributions, and to provide the next year's seeds. Storing foods indoors, in particular rice, was reported to be a major source of attraction to rodents.
Contact with rats. Small-to medium-sized rodents are collectively termed "rats" in English ("arata" in Krio), a terminology that we continue in the result section when referring to the word "rat." Our research participants reported both unintentional (and generally undesired) forms of rodent-human contact as well as intentional contact with M. natalensis and other rodent species. These forms of interaction sometimes involved direct or indirect contact  with rodent urine, feces, or blood. Our rodent survey in six villages during a 1-year period showed that M. natalensis shared the domestic space (defined here as houses within the study villages) with R. rattus (Table 2). Our quantitative survey indicates that a large portion of people have contact with live rats (34.2%, 150/439) or rat urine (52.8%, 232/439) ( Table 3). In the morning, evidence of nocturnal activity was found through the presence of feces and rice husks around dishes and grain stores. Another undesirable form of unintentional direct contact occurred at night, with people describing having the soles of their feet occasionally nibbled by rats during their sleep, which was considered an omen of death in the family by some. The most frequently discussed form of unintentional contact with fluids from rats occurred at night, when the hut becomes alive with activity indicated by the incessant sounds of soft-footed movement. Showing little respect for their host, rats urinate down from the interspersed rafters onto the household members. Even though this does not necessarily interrupt the residents' sleep, the pungent smell of rat urine and yellow stains in the morning served as a reminder of the nightly visit. Informants discussed this casually as an unpleasant event but part of daily life ( Figure 3).
Informants reported that intentional contact between humans and rats within villages was mostly restricted to children. This was corroborated with observational data. It is common for children to keep young animals of various species, including small rats, as pets. Neonate rats are caught when a nest is discovered, and children described playing with older rats when they are found "drunk" with poison.
Attempts to control rats inside homes are common, with a majority of informants (85.0%, 373/439) using some form of rat control including poison (76.8%, 337/439), cats (28.5%, 125/439), and traps (23.0%, 101/439) ( Table 3). Trapping and poisoning are done in a reactive rather than preventive fashion and is mainly undertaken through individual rather than collective initiative. Other measures against rats include storing prepared and raw food in covered pans with lids. People of all age and gender will also opportunistically kill rats using whatever is at hand (e.g., sticks, stones, machetes). For example, rat abundance is considered so high that dismantling old thatch roofs during repairs is considered an opportunity to kill rats as they are dislodged and people will prepare to catch rats that flee on these occasions.
Rats as a threat to food security. A frequently recurrent theme discussed spontaneously by informants was the material damage caused by rats in homes and on farms. Informants overwhelmingly reported that rats ate leftover food, destroyed grain stores and even other possessions such as clothes, bags, and bank notes (Figure 3). It is common to see container bags eaten through and harvests can be completely lost if the damage is not spotted early enough. In this respect, rats are considered voracious animals. Many people regularly reported rats contaminating food that could not be stored safely and the need to make the difficult decision of throwing cooked food away, although some informants claimed that they could not afford to do so, or they would forfeit the next meal. In addition, rats destroy grains that are needed to plant the next year's crop. Table 3 provides further evidence of the widespread negative impact of rats with 90.0% (395/439) and 85.0% (373/439) of individuals, respectively, reporting damage to food stores and crop plantations. Steps are taken to minimize damage caused by rats, such as hanging bags from rafters, but even these are not always effective.

DISCUSSION
Overall, there was consensus between the quantitative and quantitative results regarding contact with rodent and rodent control measures: contact with rodents and their body fluids was found to be widespread, and damage to food stores was significant However, study participants may have over reported the impact of rodents in the hope of receiving benefits such as interventions to decrease rodent abundance or improve food security.
Building use, materials and design, and peri-domestic rodents. In Bo District (excluding urban Bo City), most houses are thatched (20.9%) with mud/mud and wattle walls (77%) and earth floors (59.2%). A majority of these are deemed to require minor (66.1%) or major repairs (20.8%). 30 These natural building materials are obtained from the surrounding bush (bamboo, wood, thatch), are friable, and provide opportunities for burrowing. The clutter lining walls and floors allow for furtive movements suitable to rodent behaviors and can provide habitats for rodents without the need for burrows. The high abundance of rodents within homes reported by household members (92.4%, 404/437) is in line with previous surveys in the eastern province of Sierra Leone (86%) 36 and is likely to be linked to building materials and modes of domestic organization in the region. One study in urban Sao Paulo, Brazil, found that environmental characteristics similar to the ones described in this study were strongly correlated with rodent infestation. The odds of urban premises to be infested by rodents was 4.5 times higher when there were access facilities (defined by building structure or sewage), 3.2 times higher with harborage sources (dense bush, derelict materials, ceiling, and wall cracks), and 1.6 times higher with the presence of various food sources. 37 Similar environmental determinants for rodent infestation (based on observations by villagers) were observed in villages in Lao People's Democratic Republic, notably housing structure (open ceilings), presence of rubble, and access to food. 38 In hindsight, it would have been worth-while for our survey to include housing infrastructure (such as wall, ceiling, and roof materials) and a measure of the status of repair to determine a possible correlation with rodent infestation.
Ecology studies suggest that rodent abundance in houses doubles during the dry season indoors, possibly as a result of restricted food supply outdoors and increase food supply indoors. 19 This may be due to storing harvests on ceilings that may attract rodents, whose movements are facilitated by roof and ceiling constructions and where it is harder to instigate rodent control measures.
In the bush, many daily activities such as cooking, resting, and certain agricultural activities occur in farmhouses. Their structure (e.g., thatch roofs and grain stores) echoes those of houses and encourages commensality between rodents and humans similar to those described in villages. However, the site of these rodent-human interactions occurs in different ecotones (farmhouse/agricultural land/ forest), where the species richness may differ from those in villages. Further, the location determines how humans perceive rodents, and in contrast to villages, contact with rodents outside of villages is often intentional and motivated FIGURE 3. Reported interactions between humans and rats (excerpts from qualitative survey). by various factors related to rodents as agricultural pests and a source of food. 13 Direct risks for zoonotic transmission. Research participants reported high levels of contact with rodents and rodent fluids, particularly at nighttime when levels of rodent activity in houses were highest and when rodents moved around domestic spaces in close proximity to humans. The permanently dark conditions created indoors probably extend the crepuscular activity of M. natalensis 28,39 and provide increased opportunities for environmental contamination. Further, the absence of ultraviolet light indoors may also prolong virus survival on surfaces 40 contaminated by rodents.
Our quantitative survey indicates that a large portion of people report contact with live rodents or rodent urine, the latter being facilitated by the roof and ceiling structure that favor rodent activity. We identify this as a possible transmission route given that infected rodents secrete arenaviruses and Morogoro virus in urine and feces, 7,41,42 and that LASV has been shown to be aerosolized under laboratory conditions. 8 We cannot conclude that the respondents of the quantitative survey who reported exposure to urine were exposed specifically to urine from ceilings because the questionnaire did not specify the urine source. However, we can infer from our qualitative data that urine contamination from ceilings is widespread and common. Further, we did not specifically trap in ceilings so we cannot conclude that M. natalensis is the specific culprit of ceiling urination. Our rodent ecology data show that the two main species cohabiting with people are R. rattus (38% of rodents trapped) and M. natalensis (57% of rodents trapped). Colonization of ceilings is more likely due to R. rattus (commonly termed the roof rat), which is the most agile climber among the species caught during the rodent survey. [43][44][45] Colonization of ceilings by this species is especially likely in villages in proximity to Bo Town (nine out of 17 villages for our anthropological investigation) because R. rattus is more abundant than M. natalensis near urban areas and major transport axes. 46 Future research could determine the spatial distribution of different species within houses. For example, preferential colonization of ceilings could pose a risk for LF and other urine-borne zoonotic diseases, whereas ground floor colonization could pose a risk through food contamination. Finally, our data might underestimate the abundance of R. rattus because we used large folding aluminum Sherman traps. These traps are smaller than the full length (rostrum to tail) of an adult R. rattus and might have discouraged them from entering our traps.
We previously reported that rodents found outside of villages ("bush rats") are hunted for food but that rodents found in villages ("town rats") are not eaten because of their association with disease. 13 Herein, we describe forms of contact with rodents found in villages that are generally unintentional and unwanted. However, many adult informants have been unwilling to admit to intentional contact with rodents (e.g., for consumption) within villages. Although our data suggest that most people differentiate between these two categories of rodents for the purpose of consumption, there is likely to be a degree of overlap depending on personal degrees of tolerance for eating rodents that are deemed to carry diseases. Intentional contact with rodents within villages was described as being restricted to children, which places them at risk through bites and contact with fluids of adults and neonates rodents, which can shed LASV at any age. 47 Contact with rodents in and around houses was frequent, intimate, generally undesired, and possibly associated with specific features of the structure of dwellings and the organization of domestic space. Thus, the behavior of rodents and humans and ways in which they overlap have relevance for the eco-epidemiology of LF and other rodent-borne diseases (e.g., plague, hemorrhagic fever with renal syndrome, relapsing fever, rickettsiosis, toxoplasmosis), including those transmitted through urine (e.g., leptospirosis). 48 This is of particular importance considering the role of rodents in emerging infectious diseases 49,50 and the recent discovery of new reservoirs for LASV 4 that have a different ecology to M. natalensis.
Aspects of rodent control. The majority of study participants used some forms of rodent control. Trapping and poisoning are done in a reactive rather than preventive fashion and mainly undertaken through individual rather than collective initiative. This is likely to have minimal effects due to rapid recolonization as opposed to preventive and coordinated control at household, compound, or village level. [51][52][53] The frequent requests for help or advice on rodent control received during fieldwork was an indication of the overwhelmingly pernicious influence rodents had on everyday life and the difficulty of controlling them. Rodent damage contributes significantly to food wastage poses a threat to food security, which is of particular concern in a country where more than half of the population lives below the poverty line 54 and malnutrition is the second leading cause of death. 31 Reducing the frequency and intensity of contact between M. natalensis and humans remains the sole prevention measure against LF infection. Our research suggests that a different rationale toward rodent prevention is needed depending on spatial locations. In swidden and forests, contact with rodents is often motivated or intended, notably during hunting and consumption of rodents 13 ; prevention strategies are best focused on sensitization. In domestic spaces, however, contact with rodents are usually unintended or undesired; prevention strategies are best focused on improving rodent control measures including through building materials, structures, and maintenance.
It is unlikely that rodent control alone is sufficient to reduce LF incidence. 28,55 There is little published evidence on the efficacy of rodent proofing of houses in tropical settings. Two studies in rural United States suggest that relatively inexpensive rodent proofing measures can decrease the frequency and intensity of rodent activity inside houses. 56,57 Our observations suggest possibilities for additional targeted forms of environmental modification that could improve the reduction of rodent abundance and the frequency of contact with humans. These include improving ceiling construction, doors, windows, junctions between walls and roofs, and removing sources of attraction by improving methods of food storage.
Further, people should be encouraged to avoid direct contact that occurs when dead or dying rodents are removed from the house following trapping or poisoning. In this instance, communities do not consider contact with dead rodents a risky activity, yet disposing of dead rodents may serve as an additional risk for LF exposure, which needs to be taken into account by intervention strategies favoring rodent control.

CONCLUSIONS
Domestic settings are hypothesized to be important sites for instances of primary transmission. 19,39 This study opens the black box of zoonotic transmission within domestic spaces and provides a description of the frequent and intense patterns of rodent-human interactions, drawing on data collected in rural settlements in Bo District, Sierra Leone. Our data show the value of social scientific and observational methodologies for gaining detailed understanding of potential pathways of zoonotic transmission. At the root of rodent-human interactions lies structural poverty-poor housing infrastructure and lack of basic amenities encourage colonization by rodents and increase the frequency and intensity of rodent-human contact.
We support the call for further collaborative research in housing improvement (building materials and design) and environmental modification to make houses less attractive to rodents as tools against LF. 27 These are likely to have high levels of acceptance because they address the concerns of community members. Such interventions can be further justified as they are likely to impact other rodent-borne and poverty-related diseases while at the same time contributing to food security.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Spectrum of pathogen- and model-specific histopathologies in mouse models of acute pneumonia

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.

Introduction
As one of the most frequent infectious diseases, pneumonia causes a tremendous socioeconomic burden in industrialized countries [1] and is the leading infectious cause of death in children worldwide [2]. Numerous classes of pathogens can cause acute pneumonia [3] and the risk of pneumonia is greatly enhanced under conditions of impaired pulmonary host defense, including preceding viral infections [4], mechanical ventilation [5] and sepsis [6]. The leading causative pathogen of community acquired pneumonia (CAP) is the Gram-positive bacterium Streptococcus (S.) pneumoniae [7,8] which accounts for the majority of bacterial upper and lower respiratory tract infections and is responsible for millions of deaths annually [9,10]. As another cause of CAP influenza A virus (IAV) infection leads to rapid progression of lung failure with limited treatment options and frequent fatal outcome [3,11,12]. Moreover, IAV infections are commonly complicated by bacterial superinfection, mostly caused by S. pneumoniae, resulting in severe progressive pneumonia associated with increased mortality [13]. In contrast, the Gram-negative and facultatively intracellular bacterium Legionella (L.) pneumophila is the causative agent of the severe CAP Legionnaires' disease, and the second most commonly detected pathogen in pneumonia in patients admitted to intensive care units (ICU) in industrialized countries [14,15]. However, in addition to CAP, ventilator-associated pneumonia (VAP) is also a major cause of hospital morbidity and mortality in ICUs [16] and the spectrum of pathogens is shifted in these forms of pneumonia. Here Staphylococcus (S.) aureus, Klebsiella (K.) pneumoniae, Acinetobacter (A.) baumannii, and Escherichia (E.) coli have been isolated with varying prevalences [17][18][19]. More specifically, the Gram-negative K. pneumoniae is a significant opportunistic pathogen causing severe life-threatening hospitalacquired respiratory tract infections [20][21][22] while S. aureus, a Gram-positive bacterium, is one of the most prevalent pathogens of community-and hospital-acquired lower respiratory tract infections in humans and accounts for a significant health and economic burden [23][24][25]. A. baumannii and E. coli are ubiquitous Gram-negative bacteria which have recently emerged as major causes of community-associated, nosocomial [26,27] and ventilator-associated pneumonia [19,28] as well as septicemia induced acute lung injury (ALI) [29,30].
In addition, more recently discovered pulmonary pathogens indicate that novel emerging diseases may add to the list of highly relevant pneumonias that may also be of interest to be studied in animal models. For example, the Middle East respiratory syndrome coronavirus (MERS-CoV) which is transmitted by dromedary camels as vectors [31] has emerged as the cause of severe human respiratory disease worldwide [32,33] with elderly and immunocompromised individuals particularly in Saudi Arabia being at highest risk [34][35][36].
The various forms of pneumonia have been successfully reproduced in specific murine models of experimentally-induced acute pneumonia [37][38][39]. These models have substantially contributed to our understanding of the pathogenesis of community-and hospital-acquired pneumonia as well as emerging lung infections worldwide and are indispensable for the development of novel therapeutic strategies [40][41][42].
Histopathology has been a powerful, reliable, and reproducible read-out tool for the evaluation of morphological changes in animal lung infection experiments for many decades [43,44]. Qualitative diagnoses are based on a summation of microscopically observable changes in the morphology and cellular composition of the tissue and cell types involved. For a more comparative inclusion of histopathologic information in biomedical research, scoring systems have been widely applied which allow for a first semiquantitative assessment of lesions compared to controls [44,45]. Moreover, all preclinical models used for the development of novel treatment strategies and acceptance by regulatory agencies need to be assessed histopathologically by board certified pathologists as gold standard for qualitative and semiquantitative evaluation of tissue alterations in experimental animals [46][47][48].
Previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs [38,41,42]. However, scoring schemes for acute murine pneumonia existing to date are very superficial, addressing only a few, rather unspecific parameters [45,49,50]. More importantly, they hardly allow for a differentiating perspective between distinct pathogens or for group comparisons, e.g., infections of wild type versus genetically modified mice. Clearly, there is a strong need for more precise and pathogen-as well as model-specific parameters to allow for an accurate description and semiquantification of the inflammatory phenotype for reliable and reproducible comparisons between experimental groups within each model. Therefore, we have recently adapted more specific scoring criteria for S. pneumoniae and S. aureus-induced pneumonia [38,42]. However, such pathogenspecific scoring criteria have not been employed for other lung pathogens in mice.
Here, we systematically describe and compare the histopathologies at their peaks of inflammation and injury of nine previously established acute lung infection models induced by S. pneumoniae, S. aureus, K. pneumoniae, A. baumannii, L. pneumophila, E. coli, MERS-CoV, IAV and superinfection with IAV and pneumococci. We provide model-specific criteria that can be used for appropriate histological quantitative comparisons, e.g., when different therapeutic interventions are evaluated within these established models. Whole mouse lung sections were used to obtain complete overviews, particularly of the distributions of lesions and inflammatory patterns. On the basis of the different and oftentimes quite pathogen-and model-specific changes we identified the most suitable evaluation criteria for each model that will allow for more accurate semiquantitative assessments of the severities and distributions of pneumonic lesions.
All mice, except for MERS-CoV infected mice, were monitored at 12 hour intervals throughout the experiment to assess appearance, behavior, grooming, respiration, body weight, and rectal temperature. Humane endpoints were defined (body temperature <30˚C, body weight loss = 20%, cumbersome breathing, accelerated breathing in combination with staggering, pain or paleness) but not reached by any of the mice at the indicated time points of termination of the experiments. MERS-CoV infected mice were monitored once daily and appearance, behavior, grooming, respiration and body weight were protocolled. Here, a single humane endpoint (loss of body weight of >15%) was defined but not reached by any of the mice employed due to their favourable clinical outcome at the infection dose used.

Mice
For all experimental infection models, except of K. pneumoniae, female mice (aged 8-12 weeks and weighing 17-22 g) were randomly assigned to groups (n = 2-4) per cage whereas in the K. pneumoniae model female and male mice (aged 23-25 weeks and weighing 22-24 g) were used for model specific reasons [41]. Furthermore, for all experimental infection models, specificpathogen-free (SPF) mice on C57BL/6 (all except for MERS-CoV) or BALB/c (as previously used for the MERS-CoV model [52,54]) background were used and housed in individually ventilated cages under SPF conditions with a room temperature of 22 ± 2˚C and a relative humidity of 55 ± 10%. A 12 hour light/ 12 hour dark cycle was maintained and the animals had unlimited access to standard pellet food and tap water. All experimental details of the infection models compared here were applied following previously published and well established protocols that partly vary in terms of infection doses, routes of infection and time point of examination due to pathogen-or model specific reasons, as given below.
E. coli (ATCC 25922) from -80˚C glycerol stock was added to LB broth (Carl Roth, Karlsruhe, Germany) and incubated for 12 hours at 200 rpm and 37˚C with 5% CO 2 . Optical density of 0.03 was adjusted in LB broth followed by incubation until midlogarithmic phase for 1.5 hours at 200 rpm and 37˚C. After centrifugation, the pellet was resuspended in sterile 0.9% NaCl at 8 x 10 5 CFU E. coli / 200 μl and administered intraperitoneally (n = 10).

Viral infections and superinfection
For initial transduction of human DPP4 for subsequent infection of BALB/c mice with MERS-CoV (hCoV EMC) viruses were cultured and prepared as described [52,54,60]. Mice were transduced transnasally with 20 μl of an adenovirus vector encoding human DPP4 and mCherry with a final titer of 2.5 x 10 8 PFU per inoculum (AdV-hDPP4; ViraQuest Inc.), resulting in hDPP4 expression in the epithelial compartment of the lung [60] and transnasally infected with a final titer of 7 x 10 4 TCID 50 of MERS-CoV in 20 μl DMEM (n = 17) under isoflurane anesthesia (Forene; Abbott, Wiesbaden, Germany).
For superinfection experiments, the IAV infection procedure was applied as described above with 40 PFU PR8 in 50 μl PBS. 8 days after viral infection, S. pneumoniae was cultured as described [37] and resuspended in sterile PBS. Mice were anesthetized intraperitoneally and transnasally inoculated with 1 x 10 3 CFU S. pneumoniae in 20 μl PBS (n = 4).

Histopathology
Mice were humanely euthanized at model-specific time points as indicated ( Table 1). Between 2 and 6 repetitions of the entire experimental procedures were performed in each model with similar group sizes in each repetition. Lungs were carefully removed after ligation of the trachea to prevent alveolar collapse, immersion-fixed in formalin pH 7.0 for 24 to 48 hours (MERS-CoV for 7 days), embedded in paraffin, cut in 2 μm sections and stained with hematoxylin and eosin (HE) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. Bacteria were visualized using the Giemsa and Gram (modified by Brown and Brenn) stains. For the display of whole lung overviews, HE stained slides of entire lung sections were automatically digitized using the Aperio CS2 slide scanner (Leica Biosystems Imaging Inc., CA, USA) and image files were generated using the Image Scope Software (Leica Biosystems Imaging Inc.).
Three evenly distributed whole-organ horizontal sections throughout the entire lungs were microscopically evaluated to assess the distribution and character of pathologic alterations, generating a modified panel of specific lung inflammation parameters adapted to each pathogen used (Table 1 and Table 2). All examinations were performed by trained veterinary experimental pathologists.

Immunohistochemistry
For immunohistochemical detection of S. pneumoniae and IAV (H1N1), antigen retrieval was performed with microwave heating (600 W) in 10 mM citric acid (750 ml, pH 6.0) for 12 minutes (min). Lung sections were then incubated with a purified rabbit antibody polyclonal to S. pneumoniae (1:2,000, kindly provided by S. Hammerschmidt) or with a purified goat antibody polyclonal to IAV H1N1 (1:4,000, OBT155, Bio-Rad, Puchheim, Germany) at 4˚C overnight. Incubation with an immuno-purified rabbit or goat antibody at the same dilution served as negative controls. Subsequently, slides were incubated with a secondary, alkaline phosphataseconjugated goat anti-rabbit (1:500, AP-1000, Vector, Burlingame, CA) antibody for 30 min at room temperature. The alkaline chromogen triamino-tritolyl-methanechloride (Neufuchsin) was used as phosphatase substrate for color development. All slides were counterstained with hematoxylin, dehydrated through graded ethanols, cleared in xylene and coverslipped.

S. pneumoniae
Transnasal infection of mice with S. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. Specific for this model, lesions widely expanded down to the periphery of the lung lobes (Fig    1A) with inflammation closely surrounding the airways and blood vessels. Pneumococcal spread led to an early immune response which was mainly characterized by predominantly intrabronchial ( Fig 1B) and intraalveolar ( Fig 1C) infiltrations of neutrophils provoking a lobular, suppurative bronchopneumonia with consolidation of affected lung areas. Large areas of coagulation and liquefaction necrosis (Fig 1D, arrowhead) as indicated by cellular fragmentation, decay, and loss of cellular details, accumulation of cellular and karyorrhectic debris as well as karyorrhexis, karyopyknosis, and karyolysis with consecutive hemorrhage were also present. The perivascular interstitium was widely expanded by edema due to vascular leakage [53] with massive extravasation of neutrophils recruited into perivascular spaces ( Fig 1E). Furthermore, suppurative and necrotizing vasculitis accompanied by hyaline thrombi within small-sized blood vessels were occasionally present, indicating early histological evidence of incipient septicemia. Increased pulmonary vascular permeability [53] also led to expanded areas of protein-rich alveolar edema which presented as homogenous, lightly pink material  within the alveolar spaces in the HE stain (Fig 1F, asterisk). A distinctive histopathological feature of pneumococcal pneumonia was the occurrence of massive suppurative to necrotizing pleuritis (Fig 1G, arrowhead) and steatitis ( Fig 1H) with widespread dispersion of bacteria into the thoracic cavity, likely accounting for the painful and morbid clinical behavior with rapid progression in affected mice. Myriads of pneumococci were clearly visible as bluish to purple dots of approximately 1 μm in size in the standard HE stain, mostly located on the pleural surface, in the mediastinal adipose tissue or within perivascular spaces in the lungs.

S. aureus
In contrast, transnasal infection with S. aureus resulted in multifocally extensive but non-expansive bronchopneumonia predominantly located near the lung hilus (Fig 2A), affecting the bronchi, alveoli and interstitium. The main inflammatory cell population consisted of neutrophils, leading to mainly suppurative (Fig 2B and 2C) lesions with a tendency towards abscess formation. In contrast to pneumococci, macrophages were also present albeit at lower numbers than neutrophils. (Fig 2C). Similar to Klebsiella and streptococci, large areas of necrosis and hemorrhage ( Fig 2D) were present. The perivascular areas were predominantly infiltrated by lymphocytes and fewer neutrophils (Fig 2E). Compared to the S. pneumoniae model mentioned above [53], vascular permeability seemed only slightly increased as reported before [38] and perivascular edema (Fig 2E) as well as protein-rich alveolar edema (Fig 2F, asterisk) were also present albeit to a lesser extent. Neither pleuritis nor steatitis were observed consistent with a rather favorable clinical outcome under the conditions used. Furthermore, staphylococci were largely undetectable by HE stain which was possibly due to the low bacterial spread within the lungs.

K. pneumoniae
Intratracheal infection of mice with K. pneumoniae resulted in severe widely expansive bronchopneumonia with increased lesion severity in the lung periphery ( Fig 3A). Recruited immune cells predominantly consisted of neutrophils, leading to suppurative ( Fig 3B) to abscessing ( Fig 3C) bronchopneumonia with hemorrhage and necrosis as well as neutrophilic interstitial pneumonia (Fig 3D) in less affected areas. Increased vascular permeability as reported [40] was associated with massive alveolar (Fig 3D, asterisk) and perivascular edema (Fig 3E), admixed with myriads of bacteria easily recognizable as purple dots in the HE stain. Suppurative to necrotizing vasculitis, pleuritis ( Fig 3F, arrowhead), and steatitis were also present and associated with marked bacterial spread and the rapid lethal clinical outcome.

A. baumannii
After transnasal infection with A. baumannii, mice developed a widely expansive ( Fig 4A) bronchopneumonia with predominantly infiltrating neutrophils causing a suppurative ( Fig  4B) to abscessing inflammation with areas of hemorrhage within alveoli and interstitium and large areas of parenchymal necrosis as well as alveolar edema. Perivascular spaces had mild to moderate edema and infiltration of lymphocytes and neutrophils ( Fig 4C). Vascular thrombosis was a common change (Fig 4D, arrowhead) in small-sized blood vessels. Similar to staphylococci, Acinetobacter was invisible by HE stain and neither pleuritis nor steatitis were present.

L. pneumophila
Transnasal infection of mice with L. pneumophila resulted in slightly different lesion patterns depending on the time point of examination after infection. At 48 hours after infection, nonexpansive interstitial pneumonia was found in close proximity to the hilus (Fig 5A) with   Comparative histopathology of mouse models of acute pneumonia prominent alveolar wall necrosis (Fig 5B). At the 6 day-time point, specifically the numbers of infiltrating macrophages were clearly increased, leading to accentuated perivascular granuloma formation (Fig 5C, arrowhead). Here, marked lymphocytic cuffing of most blood vessels as well as highly activated endothelial cells (Fig 5D, arrowhead) were observed. At both time points, neither pleuritis nor steatitis were present. Bacteria were invisible in the HE stained sections.

E. coli
After intraperitoneal infection, the hematogeneous spread of E. coli to the lungs had resulted in diffuse, interstitial suppurative pneumonia, diffusely affecting the entire lungs, modelling sepsis-induced ALI (Fig 6A). The interalveolar interstitium was heavily infiltrated with neutrophils ( Fig 6B) with most prominent aggregation around blood vessels (Fig 6C), consistent with bacterial entry via the circulation. Numerous hyaline thrombi were present within small-sized blood vessels (Fig 6D, arrowhead), suggestive of disseminated intravascular coagulopathy (DIC) due to bacterial septicemia. Large, rod-shaped bacteria were easily detectable only outside the lungs, mostly present in the adipose tissue surrounding the esophagus, possibly due to local spread of E. coli via the abdominal cavity.

MERS-coronavirus
Transnasal infection with MERS-CoV following adenoviral transduction of human DPP4 yielded an expansive, (Fig 7A) interstitial pneumonia with severe alveolar epithelial cell necrosis and infiltration of mainly macrophages, lymphocytes, and fewer neutrophils (Fig 7B). Only Comparative histopathology of mouse models of acute pneumonia moderate peribronchial ( Fig 7C) and perivascular (Fig 7D) lymphocytic infiltrations were present while most venous blood vessels had marked fibrinoid degeneration and necrosis of  Comparative histopathology of mouse models of acute pneumonia vascular walls (Fig 7D, asterisk). Additional hallmarks of MERS-CoV infection were large areas of protein-rich alveolar edema (Fig 7E, arrowhead), pronounced hemorrhage within perivascular and alveolar spaces, and interstitium (Fig 7F, arrowhead), and the formation of hyaline thrombi within small-sized blood vessels.

Influenza A virus
After transnasal infection with IAV, mouse lungs displayed a diffusely distributed bronchointerstitial pneumonia restricted to single lung lobes only (Fig 8A). Alveolar necrosis was prominent and alveolar septae were diffusely distended by infiltrating inflammatory cells (Fig 8B). Bronchial epithelial cells were markedly necrotic (Fig 8C, arrowhead) and extensively scaled off into the bronchial lumen. Alveoli and interstitium were filled with macrophages and lymphocytes as major effector cells ( Fig 8D) and prominent perivascular lymphocytic cuffing ( Fig  8E) was a characteristic change. Furthermore, large areas of alveolar edema (Fig 8F, asterisk) and, albeit to a much lesser extent, areas of hemorrhage within alveoli and interstitium were present, suggesting vascular damage and increased permeability.

Superinfection of influenza A virus pneumonia with S. pneumoniae
When mice had been infected with IAV prior to infection with S. pneumoniae, a combination and exponentiated phenotype of both models was observed 24 hours later. Lesions were widely expansive to the lung periphery but restricted to single lung lobes pre-damaged by IAV ( Fig  9A). The character of pneumonia included massive infiltration of neutrophils into alveoli ( Fig 9B) and bronchi, typical features of severe, suppurative bronchopneumonia. Bronchial epithelium was almost entirely necrotic and bronchi were filled up with pus ( Fig 9C). Perivascular spaces were edematous and infiltrated by neutrophils and lymphocytes (Fig 9D) whereas only mild lymphocytic perivascular cuffing (Fig 9E) was present. A severe proteinrich alveolar edema was seen, similar to that seen in the S. pneumoniae mono-infection (Fig Comparative histopathology of mouse models of acute pneumonia 9F). Pneumococci were difficult to visualize by HE stain, possibly due to the lower infectious dose used here when compared to the mono-infection.

Distributions, expansions and symmetry
Prior to processing for histopathology, small tissue samples from experimentally infected mouse lungs are commonly removed for molecular analyses of gene and/ or protein expression or other readout systems to receive additional information. To obtain representative data from such samples that can be correlated with the histological changes, it is crucial to know about the homogeneity of the distribution of lesions. Also, some experimental protocols recommend to use the left and right halves of the lungs, respectively, for different analytical procedures, again anticipating lesion homogeneity and symmetry. However, when we analyzed the distributions and bilateral symmetry of lung lesions for each of the infection models, we found a wide spectrum of distinct distributions and asymmetries (Fig 10). In principle, lesion distributions followed the route of pathogen entry into the lungs. However, the tendencies to spread towards the periphery of the lobes after aerogenous infection varied between different pathogens despite similar aerogenous infection routes. Mostly centrally focused lesions induced by S. aureus and L. pneumophila remained close to the hilus with no trend towards peripheral expansion. Infection with S. pneumoniae, A. baumannii and MERS-CoV resulted in lesions closely surrounding major airway segments with centrifugal expansion towards the periphery. In contrast, lesions induced by K. pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus despite aerogenous infection. Hematogenously-induced sepsis with E. coli was associated with an entirely diffuse distribution of lesions affecting the whole lung with myriads of inflammatory hot spots, commonly surrounding blood vessels. IAV-induced lesions were restricted to individual lung lobes only with a rather homogeneous distribution within affected lobes. Superinfection of S. pneumoniae into an IAV-pneumonia resulted in a pattern virtually identical to that seen after IAV infection alone. Except for blood borne E. coli pneumonia which was consistently and evenly distributed throughout the entire lungs, affected areas in all other models tested here were randomly distributed more or less asymmetrically between the right and left halves of the lungs and also between adjacent lobes (Fig 10).

Visualization of pathogens using special stains and immunohistochemistry
For more than 100 years, a wide range of special stains have been used for the histological visualization of pathogens and other relevant structures, based on their more or less specific affinities to certain dyes. Here, Gram stain modified by Brown and Brenn was used for the visualization of Gram-positive bacteria, including S. pneumoniae (Fig 11A, arrowhead), as easily recognizable, dark blue cocci. In contrast, Giemsa stain was conducted predominantly for the detection of Gram-negative bacteria such as K. pneumoniae (Fig 11B, arrowhead) which then turned into light blue to greenish rods. For more specific pathogen detection on slides, particularly for viruses, immunohistochemistry is the method of choice. Here, S. pneumoniae and IAV were detected by immunohistochemistry using specific anti-S. pneumoniae or anti-IAV antibodies, respectively. S. pneumoniae-positive signals were obtained as myriads of red cocci predominantly in the perivascular interstitium (Fig 11C), within neutrophils in alveoli and interstitium, and on pleural surfaces as well as in mediastinal adipose tissue. In addition, pneumococci were also visualized in the marginal sinuses of tracheal lymph nodes, both in macrophages and extracellularly. IAV antigen was localized to the apical surface and cytosol of intact and necrotic bronchial epithelial cells (Fig 11D) and within alveolar macrophages.

Selection of criteria suitable for scoring systems for each pathogen
The diversity of lesions and in particular the presence or absence of specific patterns in several of the models used ( Table 1) strongly suggested that a uniform scoring scheme for the pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus. Hematogenous infection with E. coli was associated with entirely diffuse distribution of lesions affecting the whole lungs with myriads of inflammatory hot spots, commonly surrounding blood vessels. IAV-induced lesions were restricted to individual lung lobes with a rather homogeneous distribution within affected lobes. Which lobes were affected followed a rather random and inconsistent pattern. Superinfection of S. pneumoniae into an IAV-pneumonia resulted in a pattern virtually identical to that seen after IAV infection alone. Except for E. coli induced pneumonia, virtually all lung lesions were distributed asymmetrically between the left and right lung halves with no tendency of either half to be more often or more strongly affected. https://doi.org/10.1371/journal.pone.0188251.g010 Comparative histopathology of mouse models of acute pneumonia semiquantification of mouse pneumonia is inconceivable. Instead, scoring systems should take into account the more or less pathogen-specific lesion patterns that can be distilled from the comparative characterizations given above. For this purpose, we carved out the most characteristic lesion patterns that appear suitable for the development of specific scoring schemes for each model (Table 2).

Discussion
Different mouse models of acute pneumonia differ widely, with an obvious strong dependence on pathogen-specific features of virulence and spread, route of infection, infectious dose and other factors. Here, we provide a detailed descriptive overview of histopathological features and distributions of lesions within infected lungs and compare them between nine relevant and commonly used infection models at their peaks of injury and inflammation. The models employed here all represent well established protocols that have been optimized and successfully used in previous studies, with model-specific variations in infection doses, routes of pathogen administration and analyzed time points [37-42, 51, 52, 54-56]. Our model-specific description parameters (Table 2) provide a rational for the selection of histopathological quantification criteria, in order to best reflect the model-specific lesion and distribution characteristics, which appear to be most relevant. Clearly, the severity of lesions in terms of outcome of quantification systems will depend on several other factors that will have to be addressed separately in each model, such as the infection dose, time point of examination or therapeutic interventions. Comparative histopathology of mouse models of acute pneumonia The model-associated characteristics of tissue lesions and immune cell infiltrates are widely consistent with well-established properties of the different pathogens used. For example, the destructive tissue damage with mostly neutrophilic infiltrations as seen in S. pneumoniae, S. aureus, and A. baumannii, are typically seen with extracellular bacteria that express cytotoxic virulence factors, such as pneumolysin and hydrogen peroxide [61] from S. pneumoniae or immunogenic cell wall components such as lipoteichoic acid (LTA) from S. aureus [62,63]. On the other hand, the intracellular pathogen L. pneumophila which primarily infects macrophages [64] resulted in a histiocytic infiltrate at 48 h that developed into granulomatous inflammation at 6 days after infection, typical of a T H 1-response [65,66]. However, several of the pathogens used were associated with additional distinctive features. For example, histology revealed marked pleuritis and steatitis due to pathogen invasion into adjacent extrapulmonary tissues after infections with S. pneumoniae and K. pneumoniae. This massive bacterial spreading throughout the thoracic cavity was exclusively present in these two models and most likely associated with sepsis, responsible for the rapid clinical progression and unfavourable outcome [41,67]. However, only K. pneumoniae had the tendency of abscess formation which was not seen in pneumococcal pneumonia. Infection with S. aureus and A. baumannii also resulted in similar lesion patterns, except for Acinetobacter-induced lesions widely expanding to the lung periphery while Staphyloccocus-induced pneumonia was restricted to the lung hilus. A second difference between the two was the presence of prominent vascular thrombosis in A. baumannii-induced pneumonia which was absent from Staphylococcus pneumonia. The clinical outcome of mice infected with A. baumannii and S. aureus was more favourable when compared to infection with S. pneumoniae or K. pneumoniae [38] which may be explained by the lack of bacterial spreading throughout the thoracic cavity and adjacent tissues, and possibly sepsis. E. coli infection was included here as a model for sepsis-associated ALI [29, 30, 68] and consequently induced wide spread vascular thrombosis and vasculitis, most likely due to its blood borne entry into the lungs and concurrent septicemia with disseminated intravascular coagulopathy and associated vascular lesions. Vascular thrombosis with or without vasculitis was also observed in other models, including S. pneumoniae, A. baumannii and MERS-CoV, however, to a much lesser extent and only within the most strongly affected areas. MERS-CoV and IAV-associated lesions clearly reflected the known cellular tropisms of these viruses with necrosis of alveolar walls or bronchial epithelial cells, respectively, being the most characteristic histopathologic features [69][70][71][72]. Typical of virally induced lesions, the inflammatory cell infiltrates in MERS-CoV and IAV infections were dominated by lymphocytes with no or only few neutrophils. Nevertheless, the two viral models could be clearly distinguished from each other by additional histological characteristics. Only the MERS-CoV infection led to a marked vascular phenotype with necrosis and degeneration of blood vessels, vasculitis, and consecutive vascular thrombosis as well as pronounced hemorrhages [69,73]. In contrast, IAV-induced pneumonia did not display any of these features, but was dominated by marked perivascular lymphocytic cuffing and alveolar edema [42,74]. Subsequent superinfection with low-dose S. pneumoniae potentiated the severity of the IAV-induced lesions and aggravated the course of pneumonia. However, it did not alter the principal histological characteristics of IAV-pneumonia. The patterns seen after single infection with S. pneumonia were not repeated in this superinfection model, likely owing to the much lower inoculation dose which is usually rapidly cleared from virus-naive lungs.
When the distributions of lesions were compared among the 9 models tested, four distinct patterns could be clearly distinguished. The most common pattern, where lesions were focused around central airways and blood vessels close to the lung hilus with the periphery less or not affected can likely be explained by the aerogenous route of infection and pathogen entry. The opposite pattern characteristic of K. pneumoniae infection where the periphery of the lobes was more strongly affected than their hilus areas despite a similar aerogenous route of infection may be due to the aerosolic intratracheal application [75] of these bacteria which is typical of and necessary in this model [41,[57][58][59]. These differences are therefore more likely attributable to the model-specific route of infection rather than pathogen-specific properties. Similarly, the very homogenous distribution of E. coli induced pneumonia likely followed the diffuse blood borne entry of the pathogen into the lungs after intraperitoneal infection. Again unique among the pathogens tested here, the IAV-associated pattern affected entire but only select lung lobes with almost complete sparing of others. This distribution probably followed a random spread of the virus along major airways but why some lobes remained virtually unaffected after transnasal infection remains hard to explain. Apart from helping to understand differences in pathogen spread, the uneven and often quite asymmetrical distributions have a tremendous impact in practical terms when acute mouse pneumonia is sampled for molecular studies. When quantitative data on mRNA or protein expression levels or other biochemical information are to be compared with one another or with tissue lesions, it is imperative that only identically affected areas are compared. Since this is impossible to predict or recognize on the macroscopical level for most models, the practice of sampling different regions of such lungs for different readout systems appears highly problematic.
Another implication of the distinct lesion characteristics, immune cell reactions and distributions among the different models appears highly relevant for histological scoring systems that aim at first quantitative comparisons [45]. To narrow down the list of parameters appropriate for each pathogen and exclude features that are likely irrelevant for some of the models, we selected 23 single histopathologic criteria for the design of semiquantitative scoring systems suitable for each model. These criteria are partly composed of standard parameters such as the determination of the affected lung area, the distribution of lesions or the type of pneumonia induced. However, numerous other and more model-specific parameters were identified which precisely describe particular aspects and allow for a differentiation between the models, such as the presence of perivascular edema, vascular thrombosis, pleuritis or steatitis. Appropriate scoring systems may thus encompass more general parameters when different pathogens are compared to one another or more pathogen-specific parameters in case specific pathogen features are in the focus of an experiment. For example, some of the parameters selected here have proven helpful in the discovery and semiquantification of different phenotypes of mouse pneumonia following genetic engineering of pathogens or mice [38, 51,76]. However, scoring systems that claim universality for all mouse models of acute pneumonia seem neither generally applicable nor meaningful for all specific experimental goals. Even the list of 23 parameters selected here may become inappropriate or insufficient when genetic changes on the pathogen or host side may result in different types of lesions, immune cell responses, time courses or other relevant features. In those cases, the list selected here may have to be adjusted or extended to better meet the specific challenges of each new study.
As standard hematoxylin and eosin (HE) staining of tissue sections failed to visualize most pathogens, traditional special stains as well as immunohistochemical techniques were employed, depending on specific staining properties of the pathogens and the availability of appropriate antibodies. While S. pneumoniae, K. pneumoniae and E. coli were easily visible in HE stained tissue sections in areas with low density of inflammatory cells, e.g., in perivascular spaces, they were very difficult to identify in heavily infiltrated and consolidated lung parenchyma. In contrast, S. aureus, A. baumannii, L. pneumophila and both viruses were entirely invisible by HE staining and thus had to be visualized by appropriate histotechnical stains or immunohistochemistry. Both approaches will likely also allow for a rough quantification of pathogen numbers in tissues when appropriate image analysis tools are used.
In this first comparative study of its kind, we examined previously established models with their optimized routes of infection, time points, and infection doses and volumes specific for each model to reach peaks of lung injury and inflammation. Variations of such factors can be expected to result in different lesion severities, composition of the cellular infiltrates, and for some models in different expansions of lesions within the lung. Still, the conditions used here are all based on observations that have evolved during extensive previous establishment studies of these models [37-39, 41, 42, 51, 52, 54, 56-58, 60, 77-79]. Among the most important reasons, most human pathogens are not pathogenic for mice under non-experimental conditions and the decisive factor for obtaining a useful pneumonia model appears to be the determination of the appropriate infection dose and route of infection. In addition, the exact time points of tissue analysis after infection had to be determined for virtually all models with care to obtain a useful model, including a precise definition of the strain or variant of the pathogen used [51,53,80,81]. Another variable to consider is the mouse strain used. Except for BALB/c mice which were used in the MERS-CoV infection model here for model-specific reasons [60], all models were conducted with C57BL/6 mice which is among the most commonly used mouse strain in infection research and therefore allows for comparisons with similar studies. However, variations of the strain or genetic background may have a dramatic impact on the type, severity and outcome of inflammation, particularly in innate immune responses [82][83][84]. Again, the criteria suggested here for scoring procedures should allow to recognize and quantify such differences related to changes in infection dose and volume, time point of examination, strain and age of mice used, pathogen variant and other variables.
Histopathology of the lungs may be complex and requires fundamental knowledge in species-specific anatomy, physiology, organ-specific immunology, pathology, and histotechnical procedures. Furthermore, various background lesions in mice, including strain specific spontaneous degenerative or inflammatory conditions and the possibility of accidental infections unrelated to the experiment should not be confused with experimental outcome. Thus, despite our efforts to specify and simplify the criteria relevant for model-specific assessment and quantification of lesions, it appears crucial that trained histopathology experts be involved in the microscopical examination of mouse lungs [46,85].
Clearly, in addition to descriptive or semiquantitative histology, a number of other parameters may be useful for quantitative comparisons between experimental groups to determine the role of specific cell types, molecules, and therapeutic interventions, depending on the strategy and goal of the study [44]. Such parameters could include flow cytometric immune cell identifications and quantifications, ELISA or quantitative RT-PCR for the probing of cytokines, chemokines or matrix proteins involved in lung pathology and remodeling, and plaque/colony forming assays for the identification or quantification of pathogens, as previously published for most of the models used here [38, 41, 42, 51-53, 86, 87]. All of these methods, however, lack the spatial resolution that only histological assessments offer. Only the combination of these techniques will lead to a better understanding of the disease in the complex context of the entire lung pathology.
In conclusion, we have identified a spectrum of pathogen-and model-specific lesion characteristics in mouse models of acute pneumonia. Our findings underscore the necessity of model-specific criteria for the accurate histopathological characterization and quantitative assessments of experimental pneumonia. This comparative landscaping of acute mouse pneumonia histology provides a comprehensive framework for future studies on the role of individual pathogen or host factors, complex disease mechanisms, and novel therapeutic strategies that could help to treat pneumonia in human patients.


High-density lipoprotein metabolism and reverse cholesterol transport: strategies for raising HDL cholesterol

A key to effective treatment of cardiovascular disease is to understand the body’s complex lipoprotein transport system. Reverse cholesterol transport (RCT) is the process of cholesterol movement from the extrahepatic tissues back to the liver. Lipoproteins containing apoA-I [high-density lipoprotein (HDL)] are key mediators in RCT, whereas non-high-density lipoproteins (non-HDL, lipoproteins containing apoB) are involved in the lipid delivery pathway. HDL particles are heterogeneous; they differ in proportion of proteins and lipids, size, shape, and charge. HDL heterogeneity is the result of the activity of several factors that assemble and remodel HDL particles in plasma: ATP-binding cassette transporter A1 (ABCA1), lecithin cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), hepatic lipase (HL), phospholipid transfer protein (PLTP), endothelial lipase (EL), and scavenger receptor class B type I (SR-BI). The RCT pathway consists of the following steps: 1. Cholesterol efflux from peripheral tissues to plasma, 2. LCAT-mediated esterification of cholesterol and remodeling of HDL particles, 3. direct pathway of HDL cholesterol delivery to the liver, and 4. indirect pathway of HDL cholesterol delivery to the liver via CETP-mediated transfer There are several established strategies for raising HDL cholesterol in humans, such as lifestyle changes; use of drugs including fibrates, statins, and niacin; and new therapeutic approaches. The therapeutic approaches include CETP inhibition, peroxisome proliferator-activated receptor (PPAR) agonists, synthetic farnesoid X receptor agonists, and gene therapy. Results of clinical trials should be awaited before further clinical management of atherosclerotic cardiovascular disease.

ters and some triglycerides surrounded by a shell composed of phospholipids, free cholesterol, and proteins. HDL particles have different proportions of different lipids: TG, cholesterol esters (CE), free cholesterol (FC), and phospholipids (PL). Some HDL particles are lipid poor or lipid-free.
They range in size from 7.5 nm to 15 nm; they can range from size of albumin to size of LDL. They differ in shape. Many people have spherical HDL particles and some have discoidal HDL particles.
Density ranges from 1.063 g/mL to 1.25 g/mL. According to density, HDL can be divided in two subfractions: HDL 3 (small and dense particles) and HDL 2 (large and less-dense particles) (15)(16)(17)(18)(19)(20)(21). HDL particles have charge. Most of them have α electrophoretic migration, some of them have pre-β migration, and some of them have pre-α migration. Lipid poor apoA-I and discoidal particles have pre-β mobility, and spherical particles have α mobility on electrophoresis.
Several plasma proteins, enzymes, and transfer proteins are involved in HDL remodeling and metabolism, which results in HDL heterogeneity (17). Every component of HDL separately enters the plasma, and HDL particle is assembled in the plasma.

Protective properties of HDL
• Free apoA-I, pre-β HDL, and α-HDL can acquire cholesterol from peripheral cells and transfer it to the liver for excretion (23). • HDL has antioxidant and anti-inflammatory properties: it can inhibit the oxidation of LDL and the expression of endothelial adhesion molecules (24,25). • HDL has antithrombotic properties. • HDL can improve endothelial function, promote the repair of endothelial cells and promote angiogenesis (26). • HDL can improve glycemic control (27).

Assembling and remodeling of HDL in plasma
There are several factors that are involved in the process of HDL remodeling and metabolism:

Apolipoprotein A-I
ApoA-I is synthesized in the liver and intestine. After synthesis, it is secreted in the plasma. Intestinal apoA-I is incorporated into the chylomicrons and as soon as the chylomicrons are broken down by LPL, apoA-I falls off from the surface. Free apoA-I has a high affinity for cholesterol and it soon becomes lipidated (28)(29)(30).

ATP-binding membrane cassette transport protein A1
Lipid poor apoA-1 is released into the plasma from the liver or intestine. It interacts with cell membranes in the plasma throughout the body. ABCA1 translocates both FC and PL from the cell membrane on the lipid-free apoA-I (31). With a complex rearrangement, it converts apoA-I into a discoidal particle with two apoA-I molecules (32). This discoidal particle contains cholesterol and PL.

Lecithin cholesterol acyltransferase
LCT is highly reactive with discoidal HDL particles (33). It plays a role in the process of transesterification. It catalyzes the transfer of fatty acids from lecithin to FC, resulting in the formation of cholesteryl ester and lysophosphatidylcholine. The best substrate for LCAT is HDL, particularly discoidal form of HDL. ApoA-I is a potent activator, but it is not the only one. LCAT can also be activated by apoA-IV, apoE, and to some extent by apoC. It cannot be activated by apoA-II. The LCAT reaction is responsible for the majority of CE circulating in the plasma. About twothirds of the plasma cholesterol (about 70%) is esterified with fatty acids.
Discoidal HDL can accept more cholesterol from peripheral cells (or other lipoproteins), and more cholesterol is further esterified by LCAT. CE are less soluble than TG, and they cannot circulate when exposed to water; hence, they migrate from the surface of the particle to its core. This migration causes transition from discoidal to spherical form of HDL particle (34,35).
LCAT can increase the size of HDL in two ways. The first way is by combining two HDL particles, each having two apoA-I molecules, resulting in one big fusion product with four apoA-I molecules, which is very unstable. One apoA-I molecule falls off from this fusion product and a particle with three apoA-I proteins is formed.
There is another way of formation of a particle with three apoA-I molecules. A small HDL particle (with two apoA-I molecules) is reactive with LCAT, which esterifies cholesterol. This makes the particle bigger, and as it gets bigger, it needs more protein on the surface. LCAT can mediate incorporation of one apoA-I molecule into spherical HDL particle, resulting in the formation of an enlarged particle containing three apoA-I molecules.
These particles are not stable but are constantly remodeled in the plasma (34,35).

Cholesteryl ester transfer protein
CETP is a lipid transfer protein involved in the exchange and transfer of lipids among different classes of lipoproteins (36,37).
Cholesteryl esters are transferred from HDL to triacylglycerolrich lipoproteins (VLDL, IDL, and LDL) in reciprocal exchange for triacylglycerols in other direction. This produces triacylglycerolenriched HDL and cholesteryl-enriched LDL and VLDL.
The main sources of CETP in humans are the liver and adipose tissue.
CETP-mediated transfer of lipids occurs in humans, primates, and rabbits. Species such as rats and mice lack CETP and are very resistant to the development of atherosclerosis.
Since the molecular size of TG is greater than that of CE, the HDL size is increased. TG are the best substrate for enzyme HL. HL breaks down TG and releases FFA that can be taken up by tissues. It makes the particles smaller because they lose TG. CETP plays a major role in the remodeling of HDL, particularly when acting in the presence of HL (36)(37)(38).
When HDL particle becomes smaller, it does not need much protein and some apoA-I can dissociate. ApoA-I can be reincorporated in other HDL particles or it can be filtered in the kidneys and excreted through urine.
The role of CETP in the development of atherosclerosis is still controversial. Some studies indicate that CETP is atherogenic, as CETP decreases the level of HDL and transfers cholesterol to atherogenic apoB-containing lipoproteins. In contrast, other studies indicate that CETP has no effect on atherosclerosis development as it plays an important role in RCT by promoting the transport of CE to the liver by LDL or VLDL (36,37).

Hepatic lipase
HL is a lypolitic enzyme primarily synthesized and secreted by the liver. It can be found in the steroid hormone-producing glands (adrenal glands and ovaries). This enzyme is involved in the lipoprotein metabolism, particularly in HDL remodeling and metabolism. HL has triglyceridase and phospholipase activities. It is involved in the production of HDL 3 particles from HDL 2 particles (38).

Phospholipid transfer protein
PLTP efficiently catalyzes the transfer of different lipids, including phospholipids, diacylglycerol, cerebrosides, and lipopolysaccharides between lipoproteins. PLTP can cause conversion of HDL particles; it promotes the fusion of two HDL particles to form a very large unstable HDL. During HDL remodeling, lipid poor apoA-I is generated and it acts as suitable and efficient cholesterol acceptor. The physiological or pathological role of PLTP is still unknown (39).

Endothelial lipase
EL primarily hydrolyzes phospholipids in HDL and shows relatively little triglyceride lipase activity (40).
Its role in HDL remodeling remains to be determined.

Scavenger receptor class B type I
SR-BI is a cell membrane protein found in many tissues (liver, steroid hormone-producing tissues, monocytes, and macrophages). It also has an important role in HDL metabolism and remodeling action.
SR-BI promotes the selective uptake of HDL cholesterol and cholesteryl esters in the liver. When HDL binds to SR-BI receptors, it loses CE into the liver, dissociates from the liver, and circulates as a smaller particle (41).

Reverse cholesterol transport
RCT is the transport of cholesterol from peripheral cells, including macrophages in the arterial wall, back to the liver for further metabolism and excretion.

RCT comprises several steps:
Cholesterol efflux from peripheral tissues to plasma Lipid-free apoA-I is synthesized in the liver or intestine and secreted in the plasma. In bloodstream, it interacts with PL and forms nascent pre-β-HDL. The ABCA1 transporter operates as a harvester of FC in the cell and delivers it to the cell membrane. The efflux of cellular cholesterol onto lipid poor apoA-I creates discoidal HDL. The ABCA1 transporter transfers cholesterol from the macrophages to HDL.
LCAT-mediated esterification of cholesterol and remodeling of HDL particles LCAT esterifies FC on the surface of HDL particles. The discoidal particle is converted into a spherical particle. This particle is able to acquire more cholesterol via other pathways, such as diffusion, SR-BI, and ABCG1 transporter (which prefers big spherical particles, not discoidal or lipid-free particles). All forms of HDL particles, from lipid-free to large spherical HDL particles, have the ability to take cholesterol out of the cell. HDL participates in the collection of cholesterol from both caveoles and lipid rafts within the cell membrane (42). In these ways, HDL is involved in cholesterol movement (efflux) from the macrophages.
Direct pathway of HDL cholesterol delivery to the liver HDL cholesterol can be delivered directly to the liver via SR-BI for further metabolism, which is called as cholesterol transport in most species.
Indirect pathway of HDL cholesterol delivery to the liver via CETP-mediated transfer Alternatively, in some species, like humans, rabbits, and monkeys, cholesteryl ester present on HDL can be exchanged for triglycerides by CETP and transferred to VLDL and LDL. This CETP-mediated transfer results in an increased cholesterol concentration of apoB-containing lipoproteins and triglyceride accumulation in HDL. HDL triglycerides can be hydrolyzed by HL, converting them back to smaller HDL particles (HDL 3 ). HDL 2 particles can be delivered back to the liver via SR-BI. Cholesterolenriched lipoproteins deliver cholesterol to the liver via the LDL receptor for further metabolism and biliary excretion (Fig. 1).

Experimental and therapeutic strategies for raising HDL cholesterol
Rats and mice, naturally deficient in CETP, are resistant to the development of atherosclerosis. Expression of CETP gene in transgenic mice induces atherosclerosis (43). Rabbits have high levels of CETP and are naturally susceptible to atherosclerosis. CETP inhibition in rabbits leads to reduction of atherosclerotic lesions (44). In humans, CETP gene polymorphisms associated with lower CETP mass and/or lower activity are associated with increased levels of HDL, decreased levels of LDL, and reduced cardiovascular risk (45). Lifestyle changes (reduction of body weight, increased physical activity, and smoking cessation) are well-known strategies for raising HDL levels. There are several drugs, including fibrates, stains, and niacin, that have been shown to raise HDL.
Other approaches specifically targeting HDL: • Infusion of reconstituted HDL (rHDL) rHDL are complexes of PL and apoA-I. Their structure is very similar to the structure of nascent HDL particles secreted by the liver. They can accept cholesterol that is released from cells via ABCA1 (46). • HDL delipidation With the process of selective delipidation, spherical HDL particles can be converted to smaller discoidal particles, which can accept cholesterol from macrophages (47). • Development of apoA-I mimetic peptides They are synthetic peptides that mimic any potentially protective property of HDL particles (48). • CETP inhibition as therapeutic strategy for treatment of cardiovascular disease (49,50).

CETP inhibitors
Large randomized clinical trials with CETP inhibitors torcetrapib (ILLUMINATE, ClinicalTrials.gov ID NCT00134264), dalcetrapib (dal-OUTCOMES, ClinicalTrials.gov ID NCT00658515), and evacetrapib (ACCELERATE, ClinicalTrials.gov ID NCT01687998) failed to reduce atherosclerotic cardiovascular events. There are several possible explanations for the adverse events in the ILLUMINATE trial: CETP inhibition is proatherogenic, CETP inhibition produces dysfunctional HDL, and serious adverse events are not related to CETP (increased levels of serum sodium, bicarbonates, and aldosterone and decreased serum levels of potassium). The dal-OUTCOMES trial was stopped because there was no clinical efficacy. Possible explanations for the failure of dalcetrapib in reducing cardiovascular events are as follows: better protective properties of HDL were not seen with an increased HDL concentration and in patients who recently had an acute coronary syndrome event, CETP inhibition did not show effectiveness. The ACCELERATE trial was stopped because of insufficient efficacy of evacetrapid in the group of high-risk vascular patients, despite its impacts on cholesterol. The possible explanation why it failed is because the trial was too short to be able to detect benefit.
The REVEAL trial (ClinicalTrials.gov: NCT01252953) is an ongoing randomized trial investigating the effects of adding the CETP inhibitor anacetrapib to an effective LDL-lowering treatment with atorvastatin.
In November 2015 (after approximately 3 years' median follow-up), the independent Data Monitoring Committee (DMC) considered whether there were reasons to recommend early stopping for efficacy and futility. The DMC recommended to the Steering Committee that the trial should continue without any modification. The estimated study completion date is January 2017.

Future perspective targets
Peroxisome proliferator-activated receptor agonists Peroxisome proliferator-activated receptors (PPAR) are nuclear receptor proteins involved in the metabolism of lipids and glucose. These receptors influence the transcription of ABCA1 that facilitates cell-derived cholesterol efflux into nascent HDL. PPAR agonists (α, δ, and γ) are promising agents in future HDL raising strategies. Dual PPAR-α/γ agonists had positive effects on insulin sensitivity and dyslipidemia. But clinical trials were terminated due to increased ASCVD events. The potential of new selective PPAR agonists as therapeutic agents is still under investigation.
Synthetic farnesoid X receptor agonists Farnesoid X receptors (FXRs) are nuclear receptors expressed primarily in the liver and intestines. It is shown that they interact with PPAR. They induce HDL-derived excretion of cholesterol from the liver into feces. Preclinical studies suggested antiatherosclerotic effects of FXR activation. Gene therapy Application of microRNA can increase ABCA1/ABCG1 expression which can be a new genetic therapy to raise HDL-C. Over expression of antisense miR-33 is shown to increase hepatic ABCA1, resulting in a 25% increase in HDL-C. Thus, antagonists of miR-33 appear to be a potential therapeutic target for the prevention or treatment of CVD.

Conclusions
HDL particles are assembled and remodeled in the plasma by a variety of enzymes and proteins. Particle heterogeneity is a result of their diversity in size, shape, charge, and composition.
HDL particles have many properties with the potential to protect against atherosclerosis; they play a pivotal role in RCT, a multistep process of cholesterol movement from the peripheral tissues to the hepatocytes. HDL metabolism is influenced by genetic, metabolic, and environmental factors. Nonpharmacological approach that includes lifestyle changes (diet, reduction of body weight, regular aerobic exercise, smoking cessation, and moderate alcohol consumption) is a well-known strategy for raising HDL levels. Pharmacological therapy includes fibrates, stains, and niacin that have been shown to raise HDL. Future perspective targets include CETP inhibitors, PPAR agonists, synthetic FXR agonists, and gene therapy. Results of clinical trials should be awaited before further clinical management.