**3.2. Strain differences and associated mechanisms**

Knowledge of differences among strains of animals in disease models can provide ideal tools for the discovery of mechanisms of disease development [83]. A strain is defined as a group of genetically identical animals. Laboratory mice are often very diverse in behavior and physiology due to a large variety of inbred, outbred, and transgenic strains produced. In laboratory mice, this is developed through inbreeding. Different mouse strains show different responses to lung infection and environmental exposures, and these can also be affected by sex and age [84]. The most common mice strains used for the study of human pneumonia are BALB/c, C57BL/6, DBA/2, 129/Sy, CBA/Ca, C3H, SJL, and A/J. In addition, a recently developed strain, collaborative cross (CC) is derived from an eight-way cross using several founder strains [85].

A study comparing susceptibility to lung infection in mice reported that, after inducing pneumococcus infection in the respiratory track of various strains, BALB/c mice, which have the ability to produce monoclonal antibodies, show no bacteremia and no lethality. Contrarily, C57BL/6 and DBA/2 mice, which are widely used inbred strains with opposite genetic susceptibility, showed 50% lethality and an intermediate response to bacteremia. Moreover, strains, such as CBA/Ca, C3H, and SJL which are highly susceptible to infection, developed acute bacteremia with 100% lethality [86]. In a similar experiment, following pulmonary *Klebsiella* sp*.* infection, C57BL/6 strain exhibited more susceptibility to bacterial dissemination and lethality than 129/Sy mice, a strain widely used in the production of targeted mutations [87]. When exposed to *Yersinia pestis*, BALB/c and C57BL/6 mice succumbed to disease, whereas C3H mice were significantly more resistant with 80% survival [88]. Other studies of *Pneumocystis carinii-*induced pneumonia revealed severe effects in C3H mice; moderate effects in BALB/c, C57BL/6, B1O.A(2R), AKR/J, and Swiss Webster mice; and mild effects in DBA/2 and DBA/IJ mice [89]. Furthermore, most strains were unable to get infected by *L. pneumophila.* Finally, it was discovered that A/J mice are susceptible to *L. pneumophila*-induced lung infection because of the lack of cells specific to the adaptive immune system in these mice [90, 91]*.*

In all these species, innate immune mechanisms defend the airways from a wide array of infections that enter the lungs and cause pneumonia. Inbred laboratory mouse strains highly differ in their immune response patterns as a result of mutations and polymorphisms. As an overall rule, toll-like receptor 4 (TLR4) mutant mice, such as C3H/HeJ are more susceptible to Gram-negative infections (e.g. *K. pneumoniae*) than other strains [92]. Moreover, some strains including A/J, DBA/2, DBA/1, FVB/NJ, and SWR are more prevalent to develop pneumonia after infection with microorganisms, such as *Bacillus anthracis*, *Aspergillus fumigatus*, and *Candida albicans.* The latter is due to a mutation in the complement component 5 (C5), which plays a role in the pathogenesis of autoimmune diseases [93, 94]. However, AKR/J mice, which also carry a C5 mutation, are less susceptible to *C. albicans* infection due to the *C. albicans* resistance loci that modifies host responses in these mice. In addition, studies of the *Klra* natural killer (NK) receptors have demonstrated that *Klra15 is* expressed in 129/J mice, and *Klra12* is expressed in CBA/J and C3H/He mice. None of these, however, are expressed in C57BL/6 mice [95].

Inbred strains are preferred to investigate genetically identical groups by facilitating the use of molecular approaches to understand the mechanisms of diseases. Since studies in mice have become popular in scientific research, the creation of new studies benefit from the extensive literature available regarding genetic engineering, immunological responses to pathogens and

Knowledge of differences among strains of animals in disease models can provide ideal tools for the discovery of mechanisms of disease development [83]. A strain is defined as a group of genetically identical animals. Laboratory mice are often very diverse in behavior and physiology due to a large variety of inbred, outbred, and transgenic strains produced. In laboratory mice, this is developed through inbreeding. Different mouse strains show different responses to lung infection and environmental exposures, and these can also be affected by sex and age [84]. The most common mice strains used for the study of human pneumonia are BALB/c, C57BL/6, DBA/2, 129/Sy, CBA/Ca, C3H, SJL, and A/J. In addition, a recently developed strain, collaborative cross (CC) is derived from an eight-way cross using several founder strains [85]. A study comparing susceptibility to lung infection in mice reported that, after inducing pneumococcus infection in the respiratory track of various strains, BALB/c mice, which have the ability to produce monoclonal antibodies, show no bacteremia and no lethality. Contrarily, C57BL/6 and DBA/2 mice, which are widely used inbred strains with opposite genetic susceptibility, showed 50% lethality and an intermediate response to bacteremia. Moreover, strains, such as CBA/Ca, C3H, and SJL which are highly susceptible to infection, developed acute bacteremia with 100% lethality [86]. In a similar experiment, following pulmonary *Klebsiella* sp*.* infection, C57BL/6 strain exhibited more susceptibility to bacterial dissemination and lethality than 129/Sy mice, a strain widely used in the production of targeted mutations [87]. When exposed to *Yersinia pestis*, BALB/c and C57BL/6 mice succumbed to disease, whereas C3H mice were significantly more resistant with 80% survival [88]. Other studies of *Pneumocystis carinii-*induced pneumonia revealed severe effects in C3H mice; moderate effects in BALB/c, C57BL/6, B1O.A(2R), AKR/J, and Swiss Webster mice; and mild effects in DBA/2 and DBA/IJ mice [89]. Furthermore, most strains were unable to get infected by *L. pneumophila.* Finally, it was discovered that A/J mice are susceptible to *L. pneumophila*-induced lung infection because

of the lack of cells specific to the adaptive immune system in these mice [90, 91]*.*

In all these species, innate immune mechanisms defend the airways from a wide array of infections that enter the lungs and cause pneumonia. Inbred laboratory mouse strains highly differ in their immune response patterns as a result of mutations and polymorphisms. As an overall rule, toll-like receptor 4 (TLR4) mutant mice, such as C3H/HeJ are more susceptible to Gram-negative infections (e.g. *K. pneumoniae*) than other strains [92]. Moreover, some strains including A/J, DBA/2, DBA/1, FVB/NJ, and SWR are more prevalent to develop pneumonia after infection with microorganisms, such as *Bacillus anthracis*, *Aspergillus fumigatus*, and *Candida albicans.* The latter is due to a mutation in the complement component 5 (C5), which plays a role in the pathogenesis of autoimmune diseases [93, 94]. However, AKR/J mice, which also carry a C5 mutation, are less susceptible to *C. albicans* infection due

host defenses.

10 Contemporary Topics of Pneumonia

**3.2. Strain differences and associated mechanisms**

Following infection, both human and mouse lungs produce immune mediators, such as cytokines, chemokines, and other components of the immune system. A regulator of IL-1β that is also highly expressed in mouse and human lungs after infection is prostaglandin E (PGE<sup>2</sup> ), and its precursor enzyme cyclooxygenase-2 (COX2) [52]. Studies using depletion of alveolar macrophages have demonstrated that these contribute largely to the stimulation of pro-inflammatory cytokines, such as IL-6 and TNFα [48]. Moreover, interleukin-1β (IL-1β) is induced only by strains containing the cholesterol-dependent cytolysin, pneumolysin (PLY), a major virulence factor of pneumococci infection [51]. In addition, the levels of tolllike receptor 2 (TLR2) and toll-like receptor 4 (TLR4) increase after *S. pneumoniae* infection in the Crl:CD1 mouse strain [96]. Both BALB/c mice and human lungs liberate hydrogen peroxide leading to DNA damage and apoptosis in lung cells [50, 97].

Viruses can also lead to pneumonia. Influenza A and B viruses are the most common causes of pneumonia in adults, but other viruses can contribute to the disease development. The susceptibility of mice models to influenza viruses depends on the strain of virus used. The most commonly used strains in research are A/Puerto Rico/8/1934 (H1N1, PR8) or A/WSN/1933 (H1N1, HSN). Researchers also use several pandemic viruses, such as the 1918 H1N1 pandemic strain, highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype, certain H7 subtype viruses, a subset of low pathogenic avian influenza viruses, and the 2009 H1N1 pandemic strains. After viral infection in mice, several immunomodulatory mediators are released including IL-1β, IL-6, IL-8, MCP-1, MIP-1α/β, interferon-gamma inducible protein (IP-10) and interferon-beta (IFN-β) in a somewhat strain-specific manner [98–100].

Animal research in viral pneumonia employs either BALB/c or C57BL/6 mice [68]. The majority of laboratory mice are vulnerable to disease and death after infection, whereas, wild mice are resistant to exposure. This is due to the lack of the antiviral factor Mx1 protein in inbred strains [72]. On the other hand, it is possible for researchers to adapt strains to mouse models. DBA/2J and A/J mice are more susceptible to diseases, even with viral isolates that were not adapted to mice, than the more frequently used BALB/C and C57BL/6 strains. Even though mouse-adapted strains are important to model seasonal H1N1 and H3N2 virus infections, certain influenza viruses cause disease in mice without prior adaptation [101]. Therefore, the interpretation of research outcomes in a particular strain may not be applicable in other strains and molecular pathways in pneumonic mouse lungs may differ.

Typically, Th1 cells are important in the clearance of intracellular pathogens, whereas Th2 cells are associated with responses to parasites. C57BL/6 mice display a typical Th1-type bias to pathogens, whereas other strains, such as BALB/c, A/J, and DBA/2 mice, tend toward a Th2 response [102]. These variations may also be reflected in the M1 and M2 macrophage responses to antigen stimulation. In addition, the region *D7Mit341* to *D7Mit247* on mouse chromosome 7 has been reported to be a survival trait against illness associated with *S. pneumoniae*. Susceptibility to experimental pneumococcal infection is strain dependent. In this regard, strains from least to most sensitive include BALB/c, DBA/2, C57BL/6, NIH, AKR, FVB/N, CSH/He, SJL, and CBA/Ca [103]. The majority of inbred mouse strains are resistant to infection with *L. pneumophila*, however, A/J mice carry the Lgn1-s allele, making them susceptible to infection [104].

higher levels of inflammatory molecules than male mice. In this context, IL-10-deficient male mice exhibited elevated levels of bacteria when compared to C57BL/6 male mice. These results confirm that both C57BL/6 and IL-10-deficient male mice are more resistant to *P. aeruginosa* infection than female mice [115]. Moreover, external administration of estrogen to adult male mice infected with *P. aeruginosa* resulted in an extreme progression of inflammation and fluid

Understanding the Intersection of Environmental Pollution, Pneumonia, and Inflammation: Does...

http://dx.doi.org/10.5772/intechopen.69627

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Currently, there is limited understanding of the molecular processes that lead to either immune-suppression or stimulation during pneumonia pathogenesis in males and females. In general, females display strong humoral immune responses after infection or vaccination when compared to males [114]. This is partially due to high levels of CD4+ T cells and variations in regulatory T cells (Treg) that regulate immune responses during the menstrual cycle in women [117]. It is known that estrogen influences transcription of specific genes that alter host immunity and promotes the proliferation of Treg during the follicular phase of the ovarian cycle [89, 118]. Because estrogen regulates CD4+ T cell subsets, there is a direct effect on Th1/Th2 equilibrium known to be crucial against bacterial and viral infections. On the other hand, studies indicate that negative outcomes from infectious pulmonary diseases in males is associated with testosterone-induced immunosuppression causing a decrease in T and B cell proliferation, and immunoglobulin and cytokine production after puberty [14]. These alterations in the adaptive immune system could help explain why men are more susceptible than women to some pulmonary diseases caused by infectious agents. However, treatments for pneumonia are standardized for both men and women indicating a general lack of under-

Sex and gender differences in clinical disorders are mostly driven by genetics and sex hormones. In order to understand hormonal effects not only in lung diseases, but also in other health conditions, it is essential to recognize their mechanisms of action, signaling pathways, and active metabolites. The major sex steroid hormones, such as estrogen, progesterone, and testosterone are derived from a common lipid precursor, cholesterol, by a complex series of reactions catalyzed by multiple enzymes [119]. In brief, cholesterol is converted to pregnenolone by the cytochrome P450 enzyme. Pregnenolone, which is a precursor and metabolic intermediate in the biosynthesis of the steroid hormones, can be transformed either to progesterone by the action of 3β-hydroxysteroid dehydrogenase (3β-HSD), or alternatively be converted to dehydroepiandrosterone (DHEA) via cytochrome P450c17 action. DHEA can turn into androstenedione via 3β-HSD and consequently testosterone or estrone via 17β-HSD and aromatase, respectively. Estrone may be further converted to estradiol via 17β-HSD.

infiltration in lung tissue [116].

standing of sex-based differences.

**4. Sex hormones and lung immunity**

**4.1. Sex hormones and mechanisms of action**

Testosterone can be also transformed into estradiol via aromatase.

**3.4. Sex-specific mechanisms of infection and immunity**

Currently, researchers are taking advantage of the phenotypic and genetic variations available in CC mice. The CC combines the genomes of eight genetically diverse founder strains, such as A/J, C57BL/6 J, 129S1/SvImJ, NOD/LtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/ EiJ [85]. This genetic combination is a significant element for the study on human-host susceptibility to major diseases, including infections, such as pneumonia [105]. In a recent study, scientists used the CC mouse model to determine whether the host genetic background could impact the risk of morbidity and mortality to pneumonia caused by infection with *P. aeruginosa*. In this study, the CC strain reproduced the responses of disease severity commonly observed in humans during infection, suggesting that variations in morbidity and mortality are highly affected by host genetic factors [105]. Whereas no significant gender differences on disease phenotypes were observed, it is important to note the sample size was small. Similar variations in morbidity and mortality were found in another study where scientists used CC animals to perform a quantitative trait locus mapping of host susceptibility to *Klebsiella* sp. infection in a study where females were found to be less susceptible to infection than males [106]. In summary, animal models with high genetic diversity, and large size and number of independent recombination are emerging as a powerful tool for genomic studies, helping scientists better understand and develop more effective therapies for pneumonia [107].

#### **3.3. Sex differences in pneumonia models**

It has been known for several years that sex is a contributing factor in the prevalence and development of a number of pulmonary diseases, such as pneumonia [11, 108]. Animal studies also suggest that there is a sexual dimorphism after puberty in innate and adaptive immune response genes in C57BL/6 mice, with innate immune response genes being highly upregulated in postpubertal male mice but not in female mice. In contrast, postpubertal female mice express high levels of adaptive immune response genes, and expression of these genes occurs at lower levels in postpubertal male mice [60].

Several studies in animals have reported that increase in circulating levels of estrogens may lead to reduced innate immunity, as measured by natural killer cell and macrophage activity, and a decrease of cytokine release [109–111]. Animal models of infection are the simplest tool available to study sex differences due to high availability of castrated animals and hormonal replacement therapies. Multiple studies have demonstrated that susceptibility to invasive viral, bacterial, fungal, and parasitic diseases is higher in males than in females in all age groups [57, 61, 112, 113]. The concept that males are more susceptible to lung infection is further sustained by data from mouse models of bacterial infection, such as *Pneumococcal pneumonia* and *Mycobacterium marinum*, where female mice display longer survival than male mice when exposed to severe sepsis [62, 114]. Infection of C57BL/6 mice with *K. pneumoniae* demonstrated a severe effect in male mice, but not in female mice [19]. In contrast, after infection with *P. aeruginosa*, C57BL/6 female mice showed greater weight loss, bacterial load, and higher levels of inflammatory molecules than male mice. In this context, IL-10-deficient male mice exhibited elevated levels of bacteria when compared to C57BL/6 male mice. These results confirm that both C57BL/6 and IL-10-deficient male mice are more resistant to *P. aeruginosa* infection than female mice [115]. Moreover, external administration of estrogen to adult male mice infected with *P. aeruginosa* resulted in an extreme progression of inflammation and fluid infiltration in lung tissue [116].
